MOLECULAR GENETICS

KEYWORDS
Gene structure and function, alleles and their types, the central dogma of molecular biology, replication,
transcription, translation, genetic code, types of gene mutations, changes in the primary structures of
gene products.

ESSENTIALS
G E N E – elementary unit of genetic function (genetic information) manifesting through phenotypic
expression. Defined simply, it is a stretch of a DNA-chain (or, in RNA-viruses, a stretch of an RNA-
chain), coding for a primary structure of a polypeptide as a translation product (in this case the gene is
usually called structural gene) or a stretch of a DNA-chain transcribed into the primary structure of
tRNA and other RNA species not intended for translation.

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A L L E L E – an alternative, particular form of a gene with a unique nucleotide sequence. MUTANT
ALLELE is an allele changed by mutation. STANDARD ALLELE = WILD-TYPE ALLELE is the allele
prevalent in the natural (wild) population. Different alleles of one gene are usually differentiated based
on their phenotypic expression, or by comparing their nucleotide sequences. The function of a

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DOMINANT ALLELE suppresses the expression of the RECESSIVE ALLELE of the same gene.
ALLELIC HETEROGENEITY is a phenomenon where a gene exists in several alternative forms

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(alleles), and each of them has its own characteristic phenotypic expression.
T H E C E N T R A L D O G M A O F M O L E C U L A R B I O L O G Y - a statement, according
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to which the flow of genetic information is possible from a nucleic acid to another nucleic acid or from a
nucleic acid to a protein, but not from a protein to a protein, nor from a protein to a nucleic acid.
R E P L I C A T I O N - duplication of DNA (or in some viruses RNA - see above), i.e. generation of
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identical copies (replicas) of nucleic acid molecules, ensuring the preservation of genetic information
during cellular or organismal reproduction.
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T R A N S C R I P T I O N - the process of transcribing the genetic information from DNA to RNA in the
form of a primary transcript.
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T R A N S L A T I O N - the process of translating the genetic information from mRNA to the primary structure
of a polypeptide.
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G E N E T I C C O D E determines the relationship between the individual codons of the mRNA and
the amino acids encoded by them (see table 1.1.).
C O D O N – a sequence of three nucleotides encoding for a corresponding standard amino acid in a
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polypeptide or determining a start or stop of the polypeptide synthesis on the ribosome.

G E N E M U T A T I O N – a change in the nucleotide sequence of the gene, generally having some
functional impact (see below).
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P O I N T M U T A T I O N is a change of a single nucleotide through substitution, deletion or insertion.

S U B S T I T U T I O N – an exchange of one nucleotide for another nucleotide in the DNA sequence.
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D E L E T I O N – loss of one or more nucleotides from the nucleotide sequence.

I N S E R T I O N – addition of one or more nucleotides into the nucleotide sequence.
M I S S E N S E M U T A T I O N leads to a change of the amino acid encoded by the mutated codon.
N O N S E N S E M U T A T I O N changes the codon for a certain amino acid into a stop codon.
F R A M E – S H I F T M U T A T I O N is a mutation caused by deletions or insertions that are not
multiples of three nucleotides and therefore change (shift) the reading frame and thus also the amino acid
composition of the synthetized protein from the mutation onwards. S I L E N T M U T A T I O N is a
mutation changing the sense of the codon, but not manifesting in the function of the polypeptide chain; in
modern genetic literature, such change of sequence is often called P O L Y M O R F I S M rather than
mutation. R E V E R S E M U T A T I O N or back mutation or reversion is a mutation through which
a mutant allele or a mutant regulatory region changes (partially or completely) into a standard one. This
should be clearly distinguished from a S U P P R E S S O R M U T A T I O N that describes a second
mutation, which negates consequences of a preceding mutation, with both mutations being detectable in
the mutant allele.

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L O S S - O F - F U N C T I O N M U T A T I O N is a mutational change rendering the mutated protein
unable to carry out its normal function. The loss of function or loss of expression can be partial
(HYPOMORPHIC MUTATION) or total (NULL MUTATION).
G A I N - O F - F U N C T I O N M U T A T I O N is a mutational change whereby the mutated protein
either gains a new additional function or becomes hyperactivated (the latter is sometimes called
HYPERMORPHIC MUTATION).
D O M I N A N T N E G A T I V E M U T A T I O N results in the loss of most functions of the
mutated protein; however, unlike the hypomorphic and null mutation, is capable of simultaneously
negating the function of the intact, non-mutated allele of the same gene.
N E G A T I V E D N A S T R A N D (- DNA, antisense) is the strand of double-stranded DNA that
serves as a template for RNA synthesis.
P O S I T I V E D N A S T R A N D (+ DNA, sense) is the strand of double-stranded DNA that has the
same nucleotide sequence as the RNA synthesized on the negative DNA strand (with the exception of

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containing uracils instead of thymines).

TASKS

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1.1 Genetic code. Following the base-pairing rule, fill in the complementary nucleotide

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sequences missing in the scheme below. Underline the first start codon and determine the number of
amino acids comprising the peptide coded by the mRNA nucleotide sequence, beginning from the start
codon.
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+ DNA 5'GAAACAGCTATGACC TAATGT

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TH

- DNA 3'CTTTGT TACCTTTCGCCCGTCACT ATTACA

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mRNA 5'GAAACA GCGCAACGCAAUUAAUGU

N
U

anticodon
3'CUU ACA
tRNA

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1.2 Changes due to mutation demonstrated in the polypeptide chain of tryptophan synthase in
E.coli.
In Fig.1.1, first fill in all the mRNA codons (see Tab. 1.1) corresponding to the changed amino acids
incorporated at the given positions as a result of the mutations. Then consider and fill in the most
likely mRNA codons for the original amino acids. Finally, write down the different effects these
changes had on the structure and function of the protein (see Tab. 1.2) Mark with crosses the changes
that influenced the protein structure and function the most. During this assessment, assume that in the
amino acid sequence of the protein, only a single change takes place at a time.
Follow the solved examples in the first two rows of Fig. 1.1.

Tab. 1.1 Genetic code

UUU - Phe UCU - Ser UAU - Tyr UGU - Cys
UUC - Phe UCC - Ser UAC - Tyr UGC - Cys

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UUA - Leu UCA - Ser UAA - STOP UGA - STOP, Sec*
UUG - Leu UCG - Ser UAG - STOP UGG - Trp
CUU - Leu CCU - Pro CAU - His CGU - Arg
CUC - Leu CCC - Pro CAC - His CGC - Arg

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CUA - Leu CCA - Pro CAA - Gln CGA - Arg
CUG - Leu CCG - Pro CAG - Gln CGG - Arg

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AUU - Ile ACU - Thr AAU - Asn AGU - Ser
AUC - Ile ACC - Thr AAC - Asn AGC - Ser
AUA - Ile ACA - Thr AAA - Lys AGA - Arg
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AUG+- Met ACG - Thr AAG - Lys AGG - Arg
GUU - Val GCU - Ala GAU - Asp GGU - Gly
GUC - Val GCC - Ala GAC - Asp GGC - Gly
GUA - Val GCA - Ala GAA - Glu GGA - Gly
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GUG - Val GCG - Ala GAG - Glu GGG - Gly

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* STOP = stop codons (UAA, UAG, UGA); codon UGA can also code for selenocysteine (Sec)
+
AUG = start codon
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TH

Tab. 1.2 Chemical properties of 21 amino acids occurring in proteins

AU

Hydrophobic non-polar amino acids

alanine - Ala phenylalanine - Phe
valine - Val isoleucine - Ile
leucine - Leu tryptophan - Trp
N

proline - Pro methionine - Met

glycine - Gly
U

Formation of disulphide bridges S-S

cysteine - Cys
Formation of diselenide bridges Se-Se
selenocysteine - Sec

Hydrophilic polar amino acids

neutral acidic basic
asparagine - Asn aspartic acid - Asp lysine - Lys
glutamine - Gln glutamic acid - Glu arginine - Arg
serine - Ser histidine - His
threonine - Thr
tyrosine - Tyr
Fig. 1.1. Mutant positions in the polypeptide chain of tryptophan synthase in
E.coli.

Fill in all possible

The original Fill in the most corresponding Describe the changes
Change
amino acids likely original mRNA codons in the protein due to
due to
and their mRNA codon(s) (underline the the mutation
mutation
positions nucleotide changed (see Tab. 1.2.)
by the mutation)

UAA,UAG,UGA substantial shortening

15. Lys AAA,AAG Stop
of the protein
exchange of a hydro-

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UUA,UUG,CUU,
22. Phe UUU,UUC Leu phobic AA for another
CUC,CUA,CUG
hydrophobic AA

Met

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49. Glu Gln

C
Val
ED
175. Tyr Cys
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177. Leu Arg

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183. Thr Met

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Gln
211. Glu
TH

Arg

213. Gly Val

AU

Asp
234. Gly
N

Cys
U

235. Ser Leu

243. Gln Stop

269. Stop Trp

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1.3 Mutations during evolution. A hypothalamic hormone was extracted from fish belonging to
the family Salmonidae. It was a decapeptide whose amino acid sequence matched that of a
decapeptide extracted from mammalian hypothalami, except for two amino acids. Both hormones
were cleaved by partial acidic hydrolysis, generating the below amino acid chains:

cleaved fish decapeptide cleaved mammalian decapeptide

1. Glu-His-Trp-Ser l. Trp-Ser-Tyr-Gly-Leu
2. Tyr-Gly-Trp-Leu-Pro 2. Arg-Pro-Gly
3. Ser-Tyr-Gly-Trp 3. Gly-Leu-Arg
4. Leu-Pro-Gly 4. Glu-His-Trp-Ser

Write down the amino acid sequence of the fish decapeptide:

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Write down the amino acid sequence of the mammalian decapeptide:

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ED
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Considering the similarity of the two decapeptides, the mammalian hormone can be assumed to have
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arisen from the fish decapeptide in the course of evolution.

Your task is to locate the positions of the mutations that changed the amino acid sequence of the fish
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decapeptide into that of the mammalian one. Mark the mutational changes in the nucleotide sequences
of the original (fish) mRNA as well as the mutated (mammalian) mRNA.
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Write down the original nucleotide sequence of the mRNA encoding the fish decapeptide:
TH
AU

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Write down the mutated nucleotide sequence of the mRNA encoding the mammalian decapeptide:
N
U

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

1.4 Basic types of mutations. With the aid of a computer program (Molecular Genetics), random
nucleotide sequences were generated of positive DNA strands (+DNA), in the length of 7 codons. To
each of the +DNA strands, the computer also provided the corresponding sequences of the
complementary negative DNA strand (-DNA), messenger RNA (mRNA), and the amino acid
sequence. In the following step, the program changed one to three nucleotide pairs within the original
nucleotide sequences. Your task is to compare the original sequence of +DNA strand with each of the
mutated +DNA strands, find the positions of the changes and mark them with arrows as is
demonstrated in the solved example. Determine and write down the type of mutation that caused the
change, and its consequences for the amino acid sequence. (End = stop codon)

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 S o l v e d e x a m p l e :

Original sequence: DNA+ TCA ATG GTA CTC GTC CCC TGT
DNA- AGT TAC CAT GAG CAG GGG ACA

UCA AUG GUA CUC GUC CCC UGU

Ser Met Val Leu Val Pro Cys

Substitution Deletion Insertion

  

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TCA ATG GTA CTC GTC ACC TGT CAA TGG TAC TCG TCC CCT GGT
AGT TAC CAT GAG CAG TGG ACA GTT ACC ATG AGC AGG GGA CCA

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UCA AUG GUA CUC GUC ACC UGU CAA UGG UAC UCG UCC CCU GGU

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Ser Met Val Leu Val Thr Cys Gln Trp Tyr Ser Ser Pro Gly

Deletion
ED Duplication
 
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TAA TGG TAC TCG TCC CCT GT TCA AAT GGT ACT CGT CCC CTG T
ATT ACC ATG AGC AGG GGA CA AGT TTA CCA TGA GCA GGG GAC A
R

UAA UGG UAC UCG UCC CCU GU UCA AAU GGU ACU CGU CCC CUG U
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End Trp Tyr Ser Ser Pro Ser Asn Gly Thr Arg Pro Leu
TH

T a s k s t o s o l v e :
AU

Original sequence A Original sequence B

TGT GTA ATA CCG GGT TTG ACC GCG TAC CAC TCC AGG TAG AAT
ACA CAT TAT GGC CCA AAC TGG CGC ATG GTG AGG TCC ATC TTA
N

UGU GUA AUA CCG GGU UUG ACC GCG UAC CAC UCC AGG UAG AAU
U

Cys Val Ile Pro Gly Leu Thr Ala Tyr His Ser Arg End Asn

Mutated sequences A1 - A7 Mutated sequences B1 - B7

TGT GTA ATA CCG GGT ATG ACC GCG TAC CAC TAC AGG TAG AAT
ACA CAT TAT GGC CCA TAC TGG CGC ATG GTG ATG TCC ATC TTA

UGU GUA AUA CCG GGU AUG ACC GCG UAC CAC UAC AGG UAG AAU

Cys Val Ile Pro Gly Met Thr Ala Tyr His Tyr Arg End Asn

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TGT GTA ATA CCG GTT TGA CC GCG TAC ACT CCA AGT AGA AT
ACA CAT TAT GGC CAA ACT GG CGC ATG TGA GGT TCA TCT TA

UGU GUA AUA CCG GUU UGA CC GCG UAC ACU CCA AGU AGA AU

Cys Val Ile Pro Val End Ala Tyr Thr Pro Ser Arg

TGT GTA ATA ACC GGG TTT GAC C GCG TAC CAT CCA GGT AGA AT
ACA CAT TAT TGG CCC AAA CTG G CGC ATG GTA GGT CCA TCT TA

UGU GUA AUA ACC GGG UUU GAC C GCG UAC CAU CCA GGU AGA AU

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Cys Val Ile Thr Gly Phe Asp Ala Tyr His Pro Gly Arg

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TGT TAA TAC CGG GTT TGA CCA GGT ACC ACT CCA GGT CAG AAT
ACA ATT ATG GCC CAA ACT GGT CCA TGG TGA GGT CCA GTC TTA

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UGU UAA UAC CGG GUU UGA CCA GGU ACG ACU CCA GGU CAG AAU
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Cys End Tyr Arg Val End Pro Gly Thr Thr Pro Gly Gln Asn
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TGC TGT AAT TAC ACG GGT TTG ACC GCG TAC CAC TCC AGG GTA GAA T
ACG ACA TTA ATG TGC CCA AAC TGG CGC ATG GTG AGG TCC CAT CTT A
R

UGC UGU AAU UAC ACG GGU UUG ACC GCG UAC CAC UCC AGG GUA GAA U
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Cys Cys Asn Tyr Thr Gly Leu Thr Ala Tyr His Ser Arg Val Glu
TH
AU

TGT GTA ATA CCG GGT TTG ACC GCG TAC CAC CTC CAG GTA GAT
ACA CAT TAT GGC CCA AAC TGG CGC ATG GTG GAG GTC CAT CTA

UGU GUA AUA CCG GGU UUG ACC GCG UAC CAC CUC CAG GUA GAU
N

Cys Val Ile Pro Gly Leu Thr Ala Tyr His Leu Gln Val Asp
U

TGT GTA ATA CCG GGA TTG ACC GCG TAA CAC TCC AGC GTA AAT
ACA CAT TAT GGC CCT AAC TGG CGC ATT GTG AGG TCG CAT TTA

UGU GUA AUA CCG GGA UUG ACC GCG UAA CAC UCC AGC GUA AAU

Cys Val Ile Pro Gly Leu Thr Ala End His Ser Ser Val Asn

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PRACTICAL 2
MONOHYBRIDISM, DETERMINATION OF
THEORETICAL GENOTYPIC AND PHENOTYPIC
SEGREGATION RATIOS
KEYWORDS
Gene, trait, allele, locus, genotype, phenotype, homozygote, heterozygote, dominance,
recessiveness, incomplete dominance, parental and filial generations, hybridization and backcross,
monohybridism, Mendel’s laws, genomic imprinting, χ2 test.

ESSENTIALS
L O C U S is a particular location on a chromosome where a specific gene is located

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G E N O T Y P E is the genetic constitution of an organism, represented by a set of specific alleles
contained in its genome. In a more narrow sense of the word, genotype means the written
representation of the allelic combination of a particular gene(s), e.g. “Aa“, “AaBB“ etc.

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P H E N O T Y P E means a set of traits and characteristics, resulting from the expression of the
genotype in a given environment.

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H O M O Z Y G O T E : a diploid individual carrying two identical alleles of a certain gene.
H E T E R O Z Y G O T E : a diploid individual carrying two non-identical alleles of a certain gene.
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C O M P L E T E D O M I N A N C E : the phenotypic expression of the dominant allele is the
same whether it is present in the homozygous or heterozygous state
I N C O M P L E T E D O M I N A N C E (semidominance): the phenotypic expression of the
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dominant allele differs dependent on whether it is present in a homozygous or heterozygous state.

The phenotype of the heterozygote is intermediate between both homozygotes regarding its intensity
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of manifestation.
C O D O M I N A N C E : in a heterozygote, both alleles maintain their individual phenotypical
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expression, undiluted and unmixed.

B A C K C R O S S : mating of a hybrid organism (offspring of genetically unlike parents) with one
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of its parents or a member of the parental generation (or with an organism genetically identical to
the parent. The first backcross generation (B1) is thus a result of mating an F1 heterozygote to the
homozygous parent of the P generation. In the case of T E S T C R O S S (see also practical Gene
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Linkage), only a homozygous recessive parent is used as a partner for the F1 heterozygote. In the
case of complete dominance, such testcross can distinguish between a heterozygote and a dominant
homozygote.
M E N D E L‘S L A W S
N

 Uniformity of F1 hybrids and identity of reciprocal crosses (sometimes

considered one of Mendel’s laws)
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In Mendelian traits, the first filial (F1) generation obtained by crossing homozygous parents (P), is
genotypically and phenotypically uniform. Reciprocal crosses always produce the same results, i.e.
mating a female AA parent with a male aa parent gives rise to an Aa hybrid identical to the Aa
hybrid arisen from the cross between a female aa parent and a male AA parent.
 Mendel’s first law: Law of segregation or law of purity of gametes
In a heterozygote, the two alleles in the allelic pair of a gene do not blend. The F2 offspring,
obtained by the cross of heterozygous individuals of the F1 generation, is heterogeneous, giving rise
to phenotype segregation. The frequencies of the individual phenotype categories follow exact
mathematical rules.
 Mendel’s second law: Law of independent assortment
For the alleles of genes, located on different chromosomes, F2 generation of dihybrid (or
multihybrid) involves all combinations of any of the allele of the first gene in question with any of
the alleles of the second gene in question (and any of the alleles of any other gene involved in a
particular cross). In the F2 generation, the number of zygotic phenotypic combinations will thus
correspond to that of mathematically independent quantities.
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L E T H A L G E N E S are genes whose expression results in the premature death of the
organism. A dominant lethal gene usually disappears from the population soon after its occurrence,
because the gene kills its bearer even in the heterozygous state. An exception is the case of diseases
with adult /(late) onset, where the onset of clinical symptoms is delayed until adulthood, sometimes
even to advanced age, so that the pathological lethal allele can be transmitted to the offspring.
Incomplete dominant lethal genes are usually only lethal for a homozygote, while a heterozygote is
affected to a lesser degree, enabling survival and, in some cases, reproduction; in these cases,
Mendelian segregation ratios change due to the lethality in the homozygous dominant offspring.
Recessive lethal genes kill their bearer only when in a homozygous state, and persist in the
population in the heterozygotes. Heterozygotes often have a selection advantage over both types of
homozygotes.
G E N O M I C I M P R I N T I N G is a special case of epigenetic modification where during
gametogenesis, certain gene loci are inactivated and, in this inactive state, they become part of the
zygote and of all the following somatic cell generations. Certain groups of genes become

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inactivated during spermatogenesis (referred to as paternally imprinted genes), and others during
oogenesis (maternally imprinted genes). Each type of gametogenesis abolishes the “opposite“
imprinting, i.e., during spermatogenesis, maternally imprinted genes become activated and during

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oogenesis, paternally imprinted genes become activated. As a consequence, although each
individual is diploid for the imprinted genes, only a single copy (the one that is not imprinted)

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remains active and thereby genetically expressed. A pathological phenotype will only occur when a
mutation is transferred from the parent who passes the gene in its active state, while a mutation
transferred by the parent who imprinted (inactivated) the gene during gametogenesis cannot
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manifest in the resulting phenotype. Thus, in this case, reciprocal crosses will not produce identical
results.
C H I - S Q U A R E T E S T (χ2 - test) is a statistical method used to determine the statistical
significance of the difference found between the observed (empirical) values and the expected
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(theoretical) values.
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We shall employ this test to verify the segregation ratios (see Task 2.1.).
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χ2 - test formula:
TH
AU

xi empirical value
ei expected theoretical value
N degrees of freedom = number of classes of the segregation ratio, reduced by one class
N

Compare the obtained χ2 value to the table value (see Appendix A1 for table), for N degrees of
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freedom, on the level of statistical significance α = 0.05. If our obtained χ2 value is lower than χ2
tab., then the difference between the empirically obtained and the theoretically expected values can
be considered statistically insignificant, which indicates a correspondence of the two compared
phenotype ratios.

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Example
Ex 2.1. Verification of segregation ratios using the χ2 test. Employing computer simulation of
random allele-pairing during the generation of zygotes (F2), the following empirical segregation
ratio of F2 generation was obtained: 97 (AA) : 200 (Aa) : 89 (aa). Does this segregation ration really
correspond to the F2 cross?

Solution: The theoretical segregation ratio of F2 generation is l (AA) : 2 (Aa) : l (aa), and
corresponds to the ratio of: 96,5 (AA) : 193 (Aa) : 96,5 (aa), since: 97 + 200 + 89 = 386, and 386 : 4
= 96,5.

Calculation of χ2:  

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In this case, χ2 = 0,839. Since the number of classes (genotypes) equals three (AA, Aa, aa), the

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value of degrees of freedom N = 3 – l = 2. In the table (see Appendix) for 2 degrees of freedom and
p = 0.05, we find the value of χ2 tab = 5.99. Since the χ2 value we have calculated (χ2 = 0.839) is lower

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than χ2 tab = 5.99, we can draw the following statistical conclusion: the empirical segregation ratio
97:200: 89 approximates the theoretical segregation ratio 1:2:1.
ED
TASKS
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2.1. Inheritance of the flower colour in snapdragon. In the scheme below, fill in all the genotypes
occurring when crossing two homozygous forms of snapdragon (Antirrhinum maius), one with red
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flowers and the other with white flowers. Flowers of the heterozygous snapdragon are pink. Fill in
the segregation ratios obtained in the F2 generation.
O
TH

red flower x white flower

AU

F1
N

pink flower
U

(hybrid)

Gametes of the hybrid:

F2 Punnett square:

Genotypic segregation ratio:

Phenotypic segregation ratio:

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Using the χ2 test, verify whether the following experimental segregation ratio of F2:
79 red-flowered : 170 pink-flowered : 95 white-flowered
corresponds to the theoretical segregation ratio you have determined.

χ2 =

What is the type of inheritance of the flower colour in snapdragon?

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Which parent line would produce a segregation ration 1 : 1 when crossed with an individual of the F1
generation? Write down the genotypes and phenotypes of all the plants concerned.

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C
ED
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2.2. Backcross. Write down the backcross to prove which of the individuals with a dominant
phenotype is homozygous (AA) and which is heterozygous (Aa), assuming complete dominance
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A>a. For this task, choose a specific monogenic trait with complete dominance.
O
TH
AU
N
U

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2.3. Monohybrid cross. A cross between a white-seeded bean and a black-seeded bean produced
the following offspring:

327 white-seeded plants

361 black-seeded plants

a) Write down the genotypes of the original plants:

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b) What is this kind of cross called?

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C
c) Perform the χ2 test to compare the obtained results to the expected theoretical segregation ratio.

χ2 =
ED
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R
O
TH
AU
N
U

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2.4. Inheritance of the MN blood groups. Humans do not have enough offspring for the
segregation ratios to be evaluated within individual families. To evaluate the segregation ratios, we
therefore need to analyse the expression of the trait in the offspring in a sufficiently large number of
families with each pair of parents having the same genotype. Mendelian inheritance in human has
been documented in classical studies of blood group inheritance. The table below shows the results
of the examinations of blood groups in children in families where both parents have blood group
MN. By evaluating the whole set of families, determine the type of inheritance of the MN blood
group antigens, and the expected segregation ratios. Verify by the χ2 test.
(Křenová, Otová, How to Practise Biological and Medical Genetics, Karolinum Praha 2009.)

family blood groups in children

1 son N, daughter M
2 daughter MN, son M

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3 two daughters MN, two sons M
4 two sons MN, daughter N

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5 son M, daughter N, son MN

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6 both son and daughter N
7 daughter MN, son N ED
8 son M, daughter MN
9 two daughters MN
10 son M, daughter MN
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11 son MN
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12 daughter MN
13 two daughters M, son N
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14 son MN, son N

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15 two daughters MN
16 son M, son N, daughter N
AU

17 son M and three daughters MN

18 son and daughter MN
19 two sons N, daughter MN
N

20 son MN, daughter M,

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two daughters N

2.5. Inheritance of achondroplasia. Achondroplasia represents a medium-severe form of nanism

(dwarfism), with its characteristically short limbs and normal fertility. This phenotype is dominantly
inherited, homozygous dominant constitution is lethal. What offspring, and in what segregation
ratios, can be expected from the marriage of two achondroplastic individuals?

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PRACTICAL 3
DIHYBRIDISM, POLYHYBRIDISM, THE FORKED LINE
METHOD (THE BRANCHING METHOD)
KEYWORDS
Dihybridism, polyhybridism, Mendelian segregation, segregation ratios and methods of their
determination, Punnett square for dihybridism and its rules, the forked line method, probability
rules.

ESSENTIALS
D I H Y B R I D I S M is tracking the inheritance of two traits.

P U N N E T T S Q U A R E F O R D I H Y B R I D I S M represents 16 zygotic combinations

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arisen as a result of a dihybrid cross (tab.3.1.).

F1 generation AaBb x AaBb

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gametes AB : Ab : aB : ab AB : Ab : aB : ab

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Tab. 3.1.: ED
F2 generation:

gametes AB Ab aB ab
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AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb
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aB AaBB AaBb aaBB aaBb

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ab AaBb Aabb aaBb aabb

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When writing the Punnett square, it is necessary to observe rules concerning the order of filling in
the gametic genotypes. Having filled in the square, you will find that one of its diagonals consists of
dihybrids (heterozygotes, AaBb) and the other diagonal consists of homozygotes (AABB, AAbb,
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aaBB, aabb). On the diagonal of homozygotes, the two middle genotype combinations (AAbb,
aaBB) give rise to „new“ phenotypes – phenotypes that did not exist in the P and F1 generations.
When writing the genotype and phenotype ratios, we start from the left hand side top corner and
proceed towards the right hand side bottom corner. Thus, the dihybrid genotype segregation ratio in
N

F2 generation is written as follows:

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l AABB : 2 AABb : l AAbb : 2 AaBB : 4 AaBb : 2 Aabb : l aaBB : 2 aaBb : l aabb

The dihybrid phenotype segregation ratio in F2 generation for complete dominance in both traits is:

9 A_B_ : 3 A_bb : 3 aaB_ : 1 aabb

The underscore in the genotypes can be replaced by either a dominant, or a recessive allele. In both
cases, the phenotypic expression is the same.

17
M E N D E L I A N SE G R E G A T I O N is a phenotype segregation occurring in case of
independent assortment of alleles.
P R O B A B I L I T Y R U L E S determine the probability of occurrence of a combined genotype
and phenotype comprised of several discrete constituent genotypes and phenotypes, based on the
knowledge of the probability of occurrence of the individual constituent genotypes and phenotypes.
According to these rules, the probability of a simultaneous combination of several constituent
genotypes or phenotypes is the product of their respective probabilities of occurrence. Probability of an
alternative occurrence of several constituent genotypes or phenotypes is the sum of their respective
probabilities of occurrence. These probability rules are applied in determining the segregation ratios in
polyhybrid crosses in the form of a so-called forked line method (branching method).
F O R K E D L I N E = B R A N C H I N G M E T H O D enables us to determine the segregation
ratios without using the Punnett square. This method is particularly suited for finding the segregation
ratios in the offspring of multiple hybrids (polyhybrids). Its principle is consecutive branching of the
alternatives of the individual allelic pairs. The method is suited both for finding the gametic genotypes

PY
of the polyhybrid (Ex.3.1.), and for determining the zygotic genotype and phenotype combinations
(Ex.3.2.).

O
Examples

C
Ex 3.1. Determine the gametic genotype combinations in a hybrid whose genotype is RrssTtUU:
ED
Solution:
IZ

Ratio of genotypes % Ratio

R
O

0,5 T 1 U 0,25 RsTU 25 1

0,5 R 1 s
TH

0,5 t 1 U 0,25 RstU 25 1

0,5 T 1 U 0,25 rsTU 25 1

0,5 r 1 s
AU

0,5 t 1 U 0,25 rstU 25 1

When using the branching method, we proceed stepwise according to the individual allelic pairs that
N

assort independently. After each alternative of the preceding allelic pair we put, one by one, all the
alternatives of the following pairs, including their frequencies. The final ideal frequency of each
U

segregated genotype is obtained by consecutive multiplication of all the constituent frequencies of the
respective branch.

18
Ex 3.2. Determine the genotypic composition in offspring of parents with the following genotypes:
RrssTtUU x RrSsTTuu

Solution: We proceed according to the individual allelic pairs and consider the segregation ratios. The
results are then used when constructing the branching scheme..

Rr x Rr ................... 0,25 RR + (0,25 Rr + 0,25 Rr) + 0,25 rr

ss x Ss ................... 0,5 Ss + 0,5 ss
Tt x TT .................. 0,5 TT + 0,5 Tt
UU x uu .................. l,0 Uu

PY
Ratio of
Ratio of genotypes % %
phenotypes

O
0,5 TT 1 Uu 0,0625 RRSsTTUu 6,25 0,375 R_S_T_U 37,5
0,5 Ss

C
0,5 Tt 1 Uu 0,0625 RRSsTtUu 6,25
RR ED
0,5 TT 1 Uu 0,0625 RRssTTUu 6,25 0,375 R_ssT_U_ 37,5
0,5 ss
0,5 Tt 1 Uu 0,0625 RRssTtUu 6,25
IZ

0,5 TT 1 Uu 0,125 RrSsTTUu 12,5

0,5 Ss
R

0,5 Tt 1 Uu 0,125 RrSsTtUu 12,5

Rr
O

0,5 TT 1 Uu 0,125 RrssTTUu 12,5

0,5 ss
TH

0,5 Tt 1 Uu 0,125 RrssTtUu 12,5

0,5 TT 1 Uu 0,0625 rrSsTTUu 6,25 0,125 rrS_T_U_ 12,5

AU

0,5 Ss
0,5 Tt 1 Uu 0,0625 rrSsTtUu 6,25
rr
0,5 TT 1 Uu 0,0625 rrssTTUu 6,25 0,125 rrssT_U_ 12,5
N

0,5 ss
0,5 Tt 1 Uu 0,0625 rrssTtUu 6,25
U

From the scheme, it is then very easy to determine e.g. a phenotype ratio in case of complete
dominance of the involved alleles. Genotypes with the same font and underline have the same
phenotypic expression. Their frequencies are simply added up, the lowest frequency (in our case 6%)
being unitary. We will arrive at the following phenotype segregation ratio:

6 R_S_T_Uu : 6 R_ssT_Uu : 2 rrS_T_Uu : 2 rrssT_Uu, which after reduction, corresponds to 3:3:1:1.

19
In the branching method, we arrive at the same results also in case we use segregation ratios in the
form of whole numbers, obtained from the Punnett squares for the individual genes:

R r s s T t U U

R RR Rr S Ss Ss T TT Tt u Uu Uu

r Rr rr s ss ss T TT Tt u Uu Uu

1 RR : 2 Rr : 1 rr 2 Ss : 2 ss 2 TT : 2 Tt 4 Uu

after reduction:

PY
1 RR : 2 Rr : 1 rr 1 Ss : 1 ss 1 TT : 1 Tt 1 Uu

O
C
Ratio of Ratio of After
genotypes phenotypes reduction
ED
1 TT 1 Uu 1 RRSsTTUu 6 R_S_T_U 3
1 Ss
1 Tt 1 Uu 1 RRSsTtUu
IZ

1 RR
1 TT 1 Uu 1 RRssTTUu 6 R_ssT_U_ 3
R

1 ss
1 Tt 1 Uu 1 RRssTtUu
O

1 TT 1 Uu 2 RrSsTTUu
TH

1 Ss
1 Tt 1 Uu 2 RrSsTtUu
2 Rr
AU

1 TT 1 Uu 2 RrssTTUu
1 ss
1 Tt 1 Uu 2 RrssTtUu
N

1 TT 1 Uu 1 rrSsTTUu 2 rrS_T_U_ 1
1 Ss
U

1 Tt 1 Uu 1 rrSsTtUu
1 rr
1 TT 1 Uu 1 rrssTTUu 2 rrssT_U_ 1
1 ss
1 Tt 1 Uu 1 rrssTtUu

20
TASKS
3.1. Dihybrid cross – two genes with complete dominance – Punnett square. Similar to Tab. 3.1., fill
in the dihybrid Punnett square below and determine all genotypes in the offspring of dihybrid parents
(RrBb).

F1 RrBb x RrBb

gametes:

F2

PY
O
C
ED
Let us assume that in this task, you followed guinea pig offspring. Rough coat (R) was dominant over
smooth coat (r) and black colour (B) was dominant over white (b). Determine the genotype and
phenotype segregation ratios in F2 generation and perform a backcross with the dihybrid.
IZ
R

Genotype segregation ratio in F2:

O
TH

Phenotype segregation ratio in F2:

AU
N

Backcross:
U

21
3.2. Dihybrid cross – branching method. Breeding a black rough coated guinea pig with a white
rough coated one gave rise to the following offspring:

32 : 33 : 12 : 9
rough coated rough coated smooth coated smooth coated
black white black white

a) What were the genotypes of the two crossed guinea pigs?

PY
genotypes:
black rough coated x white rough coated

O
b) Employing the branching method, find out the theoretical genotypic and phenotypic frequencies in
the offspring of the individuals identified in task a).

C
ED
IZ
R
O
TH

c) Verify the correspondence between the empirical segregation ratio (32:33:12:9) and the theoretical
segregation ratio found in task b).Use the χ2 test.
AU

χ2 =
N
U

3.3. Gametic genotype frequencies. Using the branching method, determine the ideal frequencies of
the individual genotypes in gametes produced by an individual with a genotype AaBBCcddEe.

22
3.4. Genotype and phenotype frequencies in case of complete and incomplete dominance. Using the
branching method, determine the theoretical frequencies of the zygotic genotypic combinations arisen
after crossing two individuals with genotypes KkLLMm x KkLlmm. Subsequently, determine the
phenotype segregation ratios assuming:
a) complete dominance for all genes
b) complete dominance for genes K and M and incomplete dominance for gene L.

PY
O
C
ED
IZ
R
O
TH
AU
N
U

23
3.5. The influence of lethal genes on phenotype segregation ratios. The inheritance of scale pattern in
common carp (Cyprinus carpio) is shown in Figure 3.1. There is a recessive lethal effect, inherited
along with the dominant allele N. The homozygote NN dies due to carrying the homozygous recessive
combination of the lethal gene.

a) Using the branching method, determine the phenotype segregation ratio in the offspring arisen after
breeding a carp with a linear scale pattern with a leather carp.

b) Ascertain the genotypes of the parents of scaly and mirror carps, occurring in 1 : 1 ratio.

PY
O
Fig. 3.1. Inheritance of scale patterns in common carp (Cyprinus carpio).

C
Reciprocal interaction with a lethal effect of the genotype combination NN.
ED
F1 linear x linear
IZ

SsNn x SsNn
R
O

F2 branching (forked-line) method:

Genotypes Phenotypes
TH

1 NN 1 SSNN __NN †
1 SS 2 Nn 2 SSNn S_Nn linear
AU

1 nn 1 SSnn S_nn scaly

1 NN 2 SsNN __NN †
2 Ss 2 Nn 4 SsNn S_Nn linear
N

1 nn 2 Ssnn S_nn scaly

U

1 NN 1 ssNN __NN †
1 ss 2 Nn 2 ssNn ssNn leather
1 nn 1 ssnn ssnn mirror

final ratio of phenotypes:

linear : scaly : leather : mirror

S_Nn S_nn ssNn ssnn
6 : 3 : 2 : 1

24
PRACTICAL 4
GENE LINKAGE
KEYWORDS
Principles of gene linkage and modification of segregation ratios, crossing-over, recombination
fraction, linkage strength, Morgan number, Bateson number, linkage phase, cis (coupling) and trans
(repulsion) allelic configurations, three-point test, chromosome map, linkage group, Morgan’s laws,
genetic and physical mapping, lod score

ESSENTIALS
G E N E T I C L I N K A G E occurs when particular genes are located on the same chromosome and
the particular alleles thereof cosegregate, i.e. they are coinherited more often than would be compatible
with the Mendel´s law of independent assortment. Genes located on one chromosome are physically

PY
connected to form a linkage group. A new allelic combination (i.e. loss of cosegregation) can only be
brought about by meiotic recombination (crossing-over) between the genes in question during the 1st
meiotic division. The probability of this event is directly proportional to the relative distance between
the respective loci. From this point of view, gene linkage is classically categorized as being either

O
complete (the two genes are so close to one another that the probability of crossing over between them
is negligible) or incomplete (crossing over between the genes in question occurs with a measurable

C
frequency)
L I N K A G E S T R E N G H T is a probability of crossing-over between alleles of different linked
ED
genes. The greater the distance between linked genes, the weaker linkage, and the greater the chance
that non-sister chromatids would cross over in the region between the genes. The linkage strength can
be expressed by the following numbers:
IZ
M O R G A N N U M B E R (p) is determined as a percentage of offspring with recombinant genotype (or
phenotype) from all offspring. Centimorgan (cM) is a unit of recombination frequency for measuring genetic
linkage and the distance between genes. One centimorgan is defined as the genetic distance between two loci
R

corresponding to recombination frequency of 1 %. In humans, 1 cM corresponds to a physical distance of

about one million base pairs (1 Mb). Theoretical range of Morgan number values is between 0 (complete
O

linkage) and 50 (no linkage – genes are located on different chromosomes and behave in compliance with the
Mendel´s law of independent assortment).
TH
AU

B A T E S O N N U M B E R (c) is a unit-less index giving how more often is the progeny (or
N

gametes) with nonrecombinant (parental) allelic combination over the recombinant new combination
resulting from crossing over between the respective genes.
U

MORGAN LAWS
1. Genes are arranged on the chromosome in linear order.
2. Number of linkage groups equals to the number of pairs of homologous chromosomes.
25
T H R E E – P O I N T C R O S S is a variant of B1 testcross (see also practical Monohybridism,
determination of genotypic and phenotypic segregation ratios), in which the phenotype ratios in the
offspring are used to find out the order of linked genes and their respective genetic distances. The correct way
of writing down a three-point trihybrid cross AaBbCc x aabbcc is

linkage phase cis (originally- 1911- called coupling)

linkage phase trans (originally- 1911- called repulsion)

PY
linkage phase trans (repulsion)

linkage phase trans (repulsion)

O
C
G E N E T I C M A P represents the relative order of genes or other defined loci like polymorphic genetic
markers (see also practical DNA diagnostics), with respective genetic distances between them in cM; in
ED
human genetics, a genetic distance between loci is marked by a symbol θ (theta). Genetic map is
assembled by repeated three-point crossings, whereby relative genetic distances reflect recombination
frequencies between the loci in question. P H Y S I C A L M A P represents the relative order of genes or
IZ

other defined loci along the chromosome, with distances between them given in base pairs of DNA (bp), or
higher units of DNA length, like kilobases (1Kb = 1000 pb) or megabases (1 Mb = 1 000 000 pb). In human,
1 cM of genetic distance corresponds approximately to 1 Mb of physical distance.
R

S O M A T I C H Y B R I D I Z A T I O N is a method how to assign one or more genes or other defined

O

loci to a particular human chromosome or a part thereof. The procedure is based on cell fusion between cells
in culture, most frequently between cells of different species, for example between human and mouse cells or
TH

human and hamster cells. The resulting fusion cells (called heterokaryons) are genetically unstable and tend
to spontaneously lose chromosomes. A panel of heterokaryons resulting from a single fusion event can thus
be used to decide, whether two genes are located on the same or on different chromosomes. If located on the
same chromosome, both genes would be either preserved or lost in heterokaryons. Genes located on different
AU

chromosomes will be distributed in a hererokaryon panel completely chaotically, i.e. there is the same
probability of both being lost, both being preserved, or either lost and the second preserved.
L O D S C O R E is a preferred way of expressing genetic distances of human genes. The Lod Score is
N

defined as the decimal logarithm of the ration of the probability that two genes are linked at a genetic distance
θ, over the probability that they are not linked at all (θ = 0.5).
U

Calculating Lod Score usually involves a computer simulation across a range of θ values, calculating the
Lod Score value for each of them. These Lod Score values give information about the reliability of the
respective results. The highest Lod Score value indicates the most probable θ values and hence the most
probable genetic distance between the loci. For example, if the maximal Lod Score of 3 have been found
for the θ values of 5 cM, the interpretation is that the most probable genetic distance between the two loci
is 5 cM and the probability of this being true is 1,000 times higher than the probability that these two loci
are not linked.

26
Examples
EX 4.1. Determine the absolute phenotypic segregation ratio of the progeny issued from the B1
cross (assuming complete dominance A>a, B>b):

providing that p(AB) = 16.6 cM and the size of the resulting B1 generation is n = 1152.

Solution: If the question is a segregation ratio, then the Bateson number (c) is a distinctly preferred way
how to express the linkage strength. Therefore, let’s start by converting the Morgan number (p) to the

PY
Bateson number (c):

O
The calculated value of c(AB) = 5 means that the gametic allelic combinations corresponding to the

C
allelic combinations carried by either chromosome of the heterozygote parent (AB or ab –
nonrecombinant combinations) would be 5 times more frequent than gametic allelic combinations that
ED
necessitate crossing-over to arise (Ab or aB - recombinant combinations). The most straightforward
way to determine theoretical phenotypic segregation ratio is to modify the B1 Punnett square
accordingly:
IZ

gametes 5AB 1 Ab 1 aB 5ab

1 ab 5 1 1 5
R
O

The theoretical genotypic and phenotypic segregation ratio of the B1 generation of the dihybrid cross
above is thus:
TH

5 AaBb : 1 Aabb : 1 aaBb : 5 aabb.

The absolute genotypic and phenotypic segregation ratio results from a re-counting of the theoretical
AU

ratio for the observed number of progeny n = 1152:

N
U

yielding the final ratio:

5 x 96(AaBb) : 96(Aabb) : 96(aaBb) : 5 x 96(aabb) = 480(AaBb) : 96(Aabb) : 96(aaBb) : 480(aabb).

27
EX 4.2. Determine the correct order of linked genes A, B, and C and genetic distances separating
them, based on the results of the three point B1 cross:

knowing the absolute numbers of progeny of defined genotypic (and phenotypic) classes:

Genotype Number of progeny %

A B C
ABC 1021 40.8
2021 80.8
abc 1000 40.0
a b c

PY
A B C
Abc 184 7.4
366 14.6

O
aBC 182 7.3
a b c

C
ABc 41 1.6 A B C
97 3.9
ED
abC 56 2.2
a b c
A B C
AbC 8 0.3
IZ

16 0.6
aBc 8 0.3
a b c
R

Total 2500 2500 0 0

O

Solution: The frequency of the different genotypic (and phenotypic) classes is inversely proportional
TH

to the number of crossing-over events necessary. The progeny brought about by a double crossing-over
(i.e. two crossing over events, always between the respective genes – last possibility) will thus be the
least frequent, the progeny resulting from the transmission of the unchanged parental allelic
AU

combinations (i.e. without any crossing-over – first possibility) will be the most frequent, and allelic
combinations resulting always from a single crossing-over event (the second and third possibility) will
be produced with an intermediate frequency, proportional to the genetic distance separating the
respective loci. Notably, the double crossing-over does not change the allelic forms at the two border
N

genes, only the alleles of the middle gene are exchanged (compare the uppermost and lowermost
variant). Comparing the most frequent and the least frequent progeny class gives thus the information
U

about the gene order (i.e. it identifies the middle gene). In the example above, only the gene B
underwent an allelic exchange in the double crossing-over class and therefore it is the gene B that is
located in the middle, with A and C constituting the two border genes. Having established the gene
order, we can now calculate the genetic distances separating both border genes and the middle gene,
respectively. The genetic distance is directly reflected by the proportion of progeny resulting from a
crossing-over event between the respective genes; the total frequency of crossing-over between the loci
under scrutiny (i.e. both the single crossing-over and the double-crossing over) should be taken into
account and added. The genetic distance between the A and B loci is thus given by the formula:
p(AB) = 14.6 + 0.6 = 15.2 cM
and likewise the genetic distance between the loci B and C:
p(BC) = 3.9 + 0.6 = 4.5 cM
Chromosome map:

28
TASKS
4.1. Changes of segregation ratios due to linkage. Calculate theoretical genotypic and phenotypic
segregation ratios of the F2 generation of the dihybrid cross bellow, assuming complete dominance in both
genes (A>a, B>b):

A and B are linked at c(AB) = 7.

PY
O
C
ED
IZ
R
O
TH
AU
N
U

29
4.2. Calculating ratios of allelic combinations of linked genes in gametes. Fill in the table below, by
calculating ratios, with which a trihybrid - heterozygote AaBbCc produces different allelic combinations
in his gametes, depending on the linkage phase as indicated. All the three genes are linked, with the
linkage strength between genes A and B c(AB) = 7 and between genes B and C c(BC) = 3.

Theoretical ratios
Gametic
Assuming the linkage phase trans
combinations Assuming the
linkage phase cis ABc/abC Abc/aBC AbC/aBc
ABC

PY
ABc
AbC
Abc

O
aBC

C
aBc
abC
ED
abc
IZ

Carry out the backcross of the heterozygote in the linkage phase cis and calculate genotypic and
phenotypic segregation ratios of the resulting B1 generation, assuming complete dominance in all the
R

three genes (A>a, B>b, C>c).

O
TH
AU
N
U

30
4.3. Completing gene map. The 5th chromosome of tomato carries 7 linked genes
(F,H,CH,K,L,N,S), each determining a discrete phenotypic characteristic (Fig. 4.1. – next page).
The three point B1 cross has been performed with all the gene triplets, with the progeny
proportions given bellow. For each three point cross, determine the gene order and calculate
genetic distances between the genes involved. By combining the results of all the three point
crosses, complete the gene map of the 5th chromosome of tomato.

CHnS+chNs = 0.4 % nSk+NsK = 0.8 % FchN+fCHn = 1.3 %

CHNs+chnS = 2.6 % Nsk+nSK = 2.2 % Fchn+fCHN = 7.7 %
CHns+chNS = 14.6 % NSk+nsK = 29.2 % FCHn+fchN = 13.7 %
CHNS+chns = 82.4 % NSK+nsk = 67.8 % FCHN+fchn = 77.3 %

HfCH+hFch = 1.8 % sKl+SkL = 8.6 %

HFch+hfCH = 7.2 % SKl+skL = 20.4 %
Hfch+hFCH = 20.2 % Skl+sKL = 21.4 %

PY
HFCH+hfch = 70.8 % SKL+skl = 49.6 %

O
Calculations:

C
ED
IZ
R
O
TH
AU
N
U

31
Fig. 4.1 Gene map of the 5th chromosome of tomato. Dominant phenotypes
are indicated on the left, recessive phenotypes on the right.

PY
O
C
ED
IZ
R
O
TH
AU
N
U

32
4.4. Drosophila genetics In the fruit fly Drosophila melanogaster, there has been a consensus to
mark only recessive alleles by a specific letter symbol, marking all the corresponding dominant
alleles simply by +, implying that recessive alleles represent specific mutants and + alleles represent
nonmutated alleles (wild type – wt); calling nonmutated alleles as wild type has since been
entrenched also in mouse or human genetics.
Based on the results of a three point cross bellow, determine the gene order and calculate respective
genetic distances (Drosophila melanogaster chromosome 2).

recessive phenotype dominant (wt) phenotype

cn cinnabar eyes + red eyes
dp dumpy wings + normal wings
vg vestigial wings + normal wings

PY
O
B1 cross results:

C
+ + + (cn dp vg) 676
cn dp + (+ + vg) 101
cn + + (+ dp vg) 32
ED
+ dp + (cn + vg) 591

Chromosome map:
IZ
R
O
TH
AU
N
U

33
4.5. Another Drosophila melanogaster three point cross:
recessive phenotype dominant (wt) phenotype
b black body + grey body
vg vestigial wings + normal wings
cn cinnabar eyes + red eyes

B1 cross results:

+ + + 699 cn b + 75

PY
+ + vg 70 cn b vg 675
+ b + 72 cn + vg 65
cn + + 7 + b vg 5

O
Determine the gene order, calculate respective genetic distances and draw the chromosome map:

C
ED
IZ
R
O
TH
AU
N
U

34
4.6. Somatic hybridization. Try to assign the gene H to a human chromosome using a panel of 10
human-hamster somatic hybrid cell lines. Each cell line has been characterized for the presence of
the protein H (encoded by the H gene) and for the retention of every individual human chromosome
(marked by +).

Clone/H H u m a n c h r o m o s o me
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Y X
1 + + - + + + - + + + - - - - - - - - + + + - - - +
2 - - - - - - - + - + - + - + + + - - - - - - - - +
3 + - - + + - - - - - - - - - - - + - - + - + - - +
4 + + - + + - + - + - + + + + + - - - - + + + + + +
5 + - + + + - - + + - + + + - + + + - - - + - - - +
6 + - - + - - + + - - + + - + - - - - - - + - + - +

PY
7 + + + + - - - + - + + + - + - - + - + - - + + - +
8 - - - - - - - - - - - + - + - - - - + - - - + - +
9 + - - + + + + - - - - + + - + + + + + - - + + - +

O
10 +D., Otová
(Křenová - +B: How
+ +to practice
+ + biological
- - and- medical
- + genetics,
- - Karolinum
- - +Praha
- 2009)
+ + - + - + +

C
ED
4.7. Lod score. Linkage between the locus for the Rhesus blood group (Rh) and gene(s), whose mutation(s)
IZ

result(s) in eliptocytosis (inherited erythrocyte damage – the presence of a predisposing allele is solely inferred by
the presence of the disease) has been analysed by calculating respective lod scores in two large unrelated families:
R

Family I Family II
O

θ Lod score Lod score

TH

0.00 -∞ -∞
0.05 4.74 -2.9
0.10 5.44 -1.44
AU

0.15 5.35 -0.73

0.20 4.89 -0.34
0.25 4.22 -0.12
0.30 3.39 -0.01
N

0.35 2.49 0.03

0.40 1.54 0.02
U

0.50 0.00 0.00

What conclusion could be drawn out of this analysis regarding the linkage of the Rh and
eliptocytosis loci? Explain the difference in both families.
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

35
PRACTICAL 5
SEX AND HEREDITY
KEYWORDS
Sex-linked inheritance, sex-limited and sex-influenced inheritance, pseudodominance, hemizygote,
localization of genes on heterochromosomes, holandric inheritance, inactivation of the X
chromosome.

ESSENTIALS
S E X – L I N K E D I N H E R I T A N C E is controlled by genes localized on sex chromosomes.
Genes and alleles are recorded in form of an upper index to the respective chromosome, e.g. XA or Xa
for the dominant or recessive allele of the X-chromosome located gene A.
Completely sex-linked inheritance – pertains genes localized on the heterologous portions of sex

PY
chromosomes and characterized by pseudodominance (see below) in the heterogametic sex.
 Inheritance completely linked to the heterologous portion of the X chromosome (X-
linked) can be called gonosomal recessive (GR) or gonosomal dominant (GD),
depending on the allelic interactions (see also practical Genealogy)

O
 Traits controlled by genes at the heterologous portion of the Y chromosome exhibit a so-
called holandric type of inheritance (see also practical Genealogy).

C
Incompletely sex-linked inheritance – inheritance controlled by genes localized on the homologous
portions of sex chromosomes.
ED
P S E U D O D O M I N A N C E is the phenotypic expression of the recessive allele caused by the absence
of the paired dominant allele, e.g. in a hemizygote (see below).
IZ

H E M I Z Y G O T E is a diploid individual, lacking the paired allele of a certain gene.

S E X – I N F L U E N C E D I N H E R I T A N C E is a type of inheritance where a heterozygous

R

combination of an allelic pair of an autosomal gene expresses phenotypically as dominant in one sex
and as recessive in the other sex.
O

S E X – L I M I T E D I N H E R I T A N C E applies when a gene localized on an autosome

TH

manifests only in one of the sexes. For instance, in the aquarium fish species Betta splendens, the size
of fins is inherited in this way; a so-called veil only occurs in males (fig. 5.1.). Inherited prostate cancer or
inherited ovarian cancer would be analogous examples in human genetics.
AU

D O S A G E C O M P E N S A T I O N applies to genes located on the X chromosome. In the

homogametic sex (XX), the dose is double compared to the heterogametic sex (XY). Only one of
the X chromosomes remains transcriptionally active, the second (and each additional) X
N

chromosome is inactivated during embryogenesis and takes a form of a dense nuclear substructure
called Barr body. Whether the maternal or the paternal X chromosome will be inactivated in a cell is
U

usually random; the same chromosome, however, will then always be inactivated in all cells arisen
from all the subsequent cell divisions of this original cell. Consequently, this usually leads to a
phenotypic mosaic in heterozygotes.

36
Obr. 5.1. Inheritance of fin size in the Betta splendens species. Sex-limited
inheritance.

Mails Females
Z>z No veil

PY
ZZ x zz

O
C
F1 ED
Zz x Zz
IZ
R

F2
O
TH
AU
N
U

ZZ, Zz, Zz, zz 4
ZZ, Zz, Zz 3 Uniform

zz 1

37
TASKS
5.1. Sex-linked inheritance – 1 gene. Determine the ideal segregation ratios in the offspring in F1
and F2 generations arisen from crossing a red-eyed Drosophila female with a white-eyed male.
Gene W for eye colour is X-linked. Mutated allele for white eye colour (w) is recessive in relation to
the allele for red eye colour (W). Sex determination in Drosophila is analogous to mammals, with
XX females and XY males.

PY
O
C
ED
IZ

5.2. Sex-linked inheritance – 2 genes. What offspring can you expect after crossing a white-eyed
R

(w) Drosophila male having a wild-type body colour, with a red-eyed female having a yellow body
colour (yy). Both genes W and Y are X-linked at p(WY) = 1.5 cM. Mutated recessive allele (y)
O

produces yellow body colour when in homozygous constitution (yy). Determine the segregation
ratios for F1, F2 and B1 generation.
TH
AU
N
U

38
5.3. Sex-linked inheritance in human. Haemophilia is completely sex-linked gonosomal recessive

PY
disease due to an inherited defect in the blood coagulation system, resulting in easy bruising,
inadequate clotting of traumatic injury or, in the case of severe haemophilia, a spontaneous
haemorrhage. Possible genotypes and phenotypes include: XhXh – affected woman, XhY – affected
man, XHXh – phenotypically normal carrier woman).

O
Calculate the probability of disease occurrence in children of all possible parental genotypic
combinations (separately for boys and girls).

C
ED
IZ
R
O
TH

5.4. Sex-linked inheritance in human – 2 genes. Genes for colour blindness (Gr) and haemophilia
AU

(H) are linked on the chromosome X at p(GrH) 10 cM. Both the disorders are gonosomal recessive.
A colour-blinded man married a phenotypically normal woman. Their first son is healthy, two next
sons are affected both with colour blindness and haemophilia. The first daughter is colour-blinded,
N

the second one is normal. Assuming that they both marry healthy men, what is the probability that
their sons would be affected with colour blindness and/or haemophilia?
U

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

39
 PY
O
C
5.5. Gonosomal dominant inheritance. The gene for the blood groupe system Xg is located on the
heterologous part of the X chromosome. The allele Xga codes for a functional antigenic product,
ED
whereas the recessive allele Xg is non-functional. Consequently, both the heterozygote female Xga Xg
and the homozygote female Xga Xga manifest the blood group Xg(a+), homozygote females XgXg are
devoid of the antigen (blood group Xg(a-) . Complete all the missing genotypes into the Table 5.2.
IZ

bellow.
Fig. 5.2. Blood group system Xg. Complete all the missing genotypes,
R

separately for: a) sons, b) daughters.

O

mother father children

TH

a)
Xga Xg Xg
b)
AU

a)
Xga Xga Xga
N

b)
U

a)
a
XgXg Xg
b)

a)Xga
Xg
b) XgXg, Xga Xg

a) Xga, Xg
Xga Xg
b) Xga Xga, Xga Xg

40
5.6. Sex-linked inheritance in human – 2 genes; combination of gonosomal dominant and
gonosomal recessive inheritance. Genes for the blood group system Xg (gonosomal dominant
inheritance – see above) and for the skin disease ichthyosis vulgaris (gonosomal recessive
inheritance) are linked at the X chromosome at p(XgIch) 17 cM.
Phenotypically heathy woman with the blood group Xg(a+), whose father has been affected by
ichtyosis vulgaris and whose mother has been of the blood group Xg(a-), married a phenotypically
healthy man with the blood group Xg(a-). They have two daughters. Both are phenotypically
healthy, the first with the blood group Xg(a+), the second Xg(a-). Assuming that they both marry
healthy men, what is the probability that their sons would be affected by ichthyosis?
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

PY
O
C
ED
IZ
R
O
TH
AU
N
U

41
5.7. X-linked inheritance and X-chromosome inactivation. Coat colour in cats is controlled by
several genes; for the sake of simplification, we will restrict our attention to one of them. Gene O for
the orange coat colour is X-linked (located on the heterologous portion of the X chromosome). In
female homozygotes and male hemizygotes, the dominant allele (O) is responsible for orange coat
colour and the recessive allele (o) for black coat colour. Heterozygous cats (XOXo) have a so-called
calico or tortoise coat , i.e. orange and black patches representing clones of cells in which either the
maternal or the paternal X chromosome was inactivated.
Solve the following tasks:
a) Black cat had a calico kitten and four black kittens. What genotype and coat colour had their
father? What gender were the black kittens of?

PY
O
C
b) A calico cat, whose father was yellow, was mated to a yellow male cat. What is the probability of
ED
the occurrence of yellow male and yellow female cats in the offspring?
IZ

5.8. Sex-influenced inheritance. Sex-influenced inheritance applies to coat colour in Ayrshire

R

cattle. Both cows and bulls with MM genotype are mahogany. Recessive homozygotes are red.
Heterozygous bulls are mahogany while heterozygous cows are red.
O

a) A red cow whose father was a mahogany bull was crossed with a red bull. List all possible
genotypes and phenotypes of their offspring.
TH

 
AU
N

b) Red cow gave birth to a mahogany calf. Can you determine the gender of the calf?
U

c) A mahogany-coloured cow gave birth to a red calf. Can you determine the gender of the calf?

d) What will be the phenotype segregation ratio in F2 generation?

42
5.9. Sex-influenced inheritance in human. Baldness is inherited as a sex-influenced trait. Men of
genotypes BB and Bb are bald, a bald woman is always of the genotype BB.
Calculate the probability of baldness occurrence in children of all possible parental genotypic
combinations (separately for boys and girls).

PY
O
C
ED
IZ
R
O
TH
AU
N
U

43
PRACTICAL 6
GENEALOGY
KEYWORDS
Pedigree, autosomal and gonosomal (sex-linked) types of inheritance, behaviour of dominant and
recessive traits in pedigrees, mitochondrial inheritance, genetic heterogeneity, fenocopy, incomplete
penetrance and variable expressivity

ESSENTIALS
P E D I G R E E provides a schematic illustration of kinship relations in a family, with graphical
indications of occurrence of a trait in question. A part of genetics dealing with pedigree analysis is called
genealogy. A series of specific symbols are used in pedigree depiction (Fig. 6.1). A person that a pedigree
is build from (i.e. this first person of the family drawing attention of clinical geneticists and thus giving

PY
impetus to the pedigree completion) is called proband and in the pedigree is marked by an arrow.
Generations are numbered by Roman numerals from the oldest to the youngest one, and within each
generation individual persons are numbered by Arabic numerals according to the same principle.
Consequently, using a two-number code composed from a generation number and personal number,

O
respectively, we can unambiguously identify any person in the pedigree.

C
In A U T O S O M A L D O M I N A N T (AD) traits is the responsible gene located on any autosome.
A single dominant allele is sufficient to result in phenotypic manifestation. The trait could be either
completely dominant, with both dominant homozygotes (AA) and heterozygotes (Aa) manifesting it in an
ED
undistinguishable manner (AA = Aa), or incompletely dominant (semidominant), with dominant
homozygotes manifesting the trait with higher intensity than heterozygotes (AA > Aa); in any case, only
recessive homozygotes (aa) are devoid of the trait. A typical pedigree with an autosomal dominant trait
(both completely and incompletely dominant) could be distinguished by a simultaneous presence of three
IZ

characteristics (provided the trait in question is fully penetrant – see below):

1. Both sexes are affected with the same probability
R

2. A person with the trait has at least one parent with the trait
3. Parents without the trait cannot have a child with the trait; the inverse situation (i.e. both parents
O

with the trait having a child without it) is possible, nevertheless.

In A U T O S O M A L R E C E S S I V E (AR) traits is the responsible gene located on any autosome,
TH

too, contrary to AD traits, nevertheless, any AR trait can only be manifested in recessive homozygotes
(aa). Parents without the trait can give birth to a child with the trait; in that case, both parent must be
heterozygote carriers (Aa). Both sexes can be equally affected. If both parents manifest the trait, they can
only have children with the trait. There are two exceptions to this last rule. First, there could be incomplete
AU

penetrance (in that case, some of the children born to affected parents might be unaffected), and second,
the trait can be genetically heterogeneous, and in that case neither of the children of two affected parents
would be affected (see below).
N

In C O D O M I N A N T traits each allele contributes independently to the resulting phenotype, with

a heterozygote expressing simultaneously both phenotypic characteristics, each allele underlying to one
U

of them. A typical pedigree is quite similar to AD traits.

In G O N O S O M A L D O M I N A N T (GD) traits is the responsible gene located on the
heterologous part of the X chromosome. Males and females are affected every time they inherit the
respective predisposing allele (possible genotypes XAY, and XA XA or XA Xa, respectively), males without
the trait are obligatorily of the genotype XaY and females of the genotype Xa Xa. A typical pedigree is
quite similar to AD traits, with three possible distinctions. First, there might be more females with the trait
than males (theoretically twice as much), but this aspect becomes apparent only in very large pedigrees.
Second, all the daughter of a man manifesting the trait must manifest it as well. Third, there cannot be any
father-to-son transmission. The penetrance and expressivity (including sex-dependent differences) might
be modulated by dosage compensation (see also practical Sex and Heredity).
In G O N O S O M A L R E C E S S I V E (GR) traits is likewise the responsible gene located on the
heterologous part of the X chromosome, the trait can be manifested either in hemizygous (XaY) or
recessive homozygous (Xa Xa) genotypes. Because a single recessive allele is sufficient to underlie the
trait only in men, the disease occurrence in men is distinctly higher than in women; for many of the GR
44
traits is a manifesting woman extremely rare and overwhelming majority of pedigrees shows only
manifesting men. Any father-to-son transmission is excluded. An affected man (XaY) has necessary a
healthy carrier mother (XA Xa) and all his unaffected daughters would carry the recessive allele (XA Xa).
The penetrance and expressivity might be modulated by dosage compensation (see also practical Sex and
Heredity).
In H O L A N D R I C traits would the responsible gene be located on the heterologous part of the Y
chromosome. Any such trait would only be manifested in men and transmitted in a father-to-son manner.
No such trait has been characterized in human yet.
M I T O C H O N D R I A L I N H E R I T A N C E gives a specigific pedigree pattern. In these traits,
the responsible gene is located on mitochondrial DNA. As the oocyte is the absolutely prevailing source
of mitochondria to the foetus, mitochondria of all the children are inherited from the mother only.
Consequently, all the children of affected mother will manifest the trait, irrespective of their sex, whereas
an affected father will not transmit the trait to any of his children.

PY
G E N E T I C (L O C U S) H E T E R O G E N E I T Y describes the situation, where more than one
gene can each independently underlie a defined phenotype. A genetically heterogeneous disease can thus
be alternatively caused by a mutation in different genes, either of them yielding the same phenotype.
P H E N O C O P Y leads to the same phenotype as can be observed due to a genetic cause, but in this

O
case entirely due to environmental influence.

C
P E N E T R A N C E gives the probability of phenotypic manifestation of a dominant allele or a
pair of recessive alleles. As a rule, a proportion of individuals carrying an appropriate genotype (i.e.
heterozygotes or, rarely, dominant homozygotes for dominant traits, recessive homozygotes for
ED
recessive traits) fails to manifest the corresponding trait. This situation is called incomplete
penetrance and its value gives a probability of phenotypic manifestation of a corresponding
underlying genotype. Occasionally, complete penetrance is observed for some traits, meaning that
every individual carrying an appropriate genotype will manifest the corresponding phenotype. A
IZ

typical pedigree of an autosomal dominant disease with incomplete penetrance will include healthy
individuals with both an affected parent or grandparent and affected child (the disease trait skips
R

one or more generations).

E X P R E S S I V I T Y gives a qualitative and quantitative degree, with which the trait is
O

manifested. In disease traits, expressivity involves severity of the disease, spectrum of particular
symptoms etc.; in a group of adult-onset diseases, an important expressivity-related aspect is the age
TH

of the onset of the disease. If there is a range of different severity grades, symptom spektra or ages
of onset, we speak about variable expressivity.
Both incomplete penetrance and variable expressivity may depend on both genetic and
AU

environmental factors. Genetic factors can involve specific alleles at modifier genes. Another
genetic mechanism operating in females includes variability of dosage compensation (see also
practical Sex and Heredity). We can consider two possibilities. There are gonosomal dominant
diseases showing a peculiar pedigree picture, with only women affected and an unusual abortus rate;
N

on closer inspection, all the miscarried foetuses are identified as males. A prevailing explanation is
that in XA Xa females, a proportion of cells in every female organism would inactivate the mutation
U

carrying X chromosome (XA), leaving the wild type X (Xa) as the only active; such cells will
develop normally, i.e. will be not influenced by the presence of the mutation. This compensatory
mechanism is missing in XAY males. Therefore, the males will be more severely affected, leading to
lethal phenotype. On the other hand, examples have been documented in gonosomal recessive
diseases, when heterozygous carrier women (XA Xa) manifested the disease. In these cases, unequal
X-chromosome inactivation might have taken place, leaving a disproportionate frequent cell
population with inactivated wild type X-chromosome (XA) and thus being directly affected by the
mutant X-chromosome (Xa). Nevertheless, even in these cases, some cells in the female body would
always inactivate the mutant X-chromosome (Xa) and hence will develop normally. Consequently,
these ”manifesting women” present usually milder disease than XaY men.

45
Fig. 6.1. Pedigree symbols.

Male, man Marriage (mating)

Consanguineous
Female, woman
marriage (mating)

Deceased male Couple not married

Deceased female Incest

PY
Probable consanguineous
Proband, propositus
marriage

O
Affected male Childless marriage

C
Affected female ED Siblings

Twice married
Heterozygote male,
female
IZ

Twins (dizygotic)
R

Abortion,
miscarriage
O

3 Three healthy men

TH

Twins monozygotic (one-

egg, identical)
Sex unspecified
AU

Two affected
2 women
N

Fig. 6.2. Example of a hypothetical pedigree.

U

46
 47
I
PY
II O
III Albert of Saxe-Coburg-Gotha
C Queeen Victoria
IV ED
IZ
V R
VI
O Alexei
VII Queen Elizabeth II TH
VIII Prince Charles AU
N
U
Examples
EX 6.1. Inheritance patterns. Draw hypothetical four-generation
pedigrees with transmission of AD, AR, GD, GR, holandric and sex-influenced traits,
respectively.

Solution:

Fig. 6.3. Autosomal dominant (AD) trait.

PY
O
C
ED
IZ
R
O
TH

Fig. 6.4. Autosomal recessive (AR) trait.

AU

I
1 2 3 4 5 6
N
U

II
1 2 3 4 5 6 7 8 9 10

III
1 2 3 4 5 6 7 8 9

IV
1 2 3 4 5 6 7 8 9

48
Fig. 6.5. Gonosomal dominant (GD) trait.

PY
O
C
ED
Fig. 6.6. Gonosomal recessive (GR) trait.
IZ
R
O
TH
AU
N
U

49
Fig. 6.7. Holandric trait.

I
1 2 3 4 5 6

II
1 2 3 4 5 6 7 8 9 10

PY
III
1 2 3 4 5 6 7 8 9

O
C
IV
1 2 3 4 5 6 7 8 9
ED
IZ

Fig 6.8. Sex-influenced trait

R
O
TH
AU
N
U

50
TASKS
6.1. Types of inheritance - 1. Carefully examine pedigrees bellow (Fig. 6.9. – 6.14.). Assign each
pedigree to a particular type of inheritance and substantiate, why your solution is the most probable.
Thereupon, fill in the pedigree symbols of the last generation in the way that would be compatible with the
particular type of inheritance.

Fig. 6.9.

PY
O
C
ED
IZ
R
O

Fig. 6.10.
TH
AU
N
U

51
Fig. 6.11.

PY
O
C
ED
Fig. 6.12.
IZ
R
O
TH
AU
N
U

52
Fig. 6.13.

PY
O
C
ED
IZ

Fig. 6.14.
R
O
TH
AU
N
U

53
6.2. Types of inheritance – 2. Fill in the pedigrees bellow (Fig. 6.15. – 6.17.) in the way that
would be compatible with the specified type of inheritance. Assume complete penetrance in all
cases.

Fig. 6.15. Autosomal dominant inheritance:

PY
O
C
ED
IZ
R

Fig. 6.16. Autosomal recessive inheritance:

O
TH
AU
N
U

54
Fig. 6.17. Gonosomal recessive inheritance:

PY
O
C
ED
6.3. Draw the pedigree of your own family, with as much generations as possible. Fill in the
occurrence of any disease or any other interesting trait transmitted in your family for multiple
generations.
IZ
R
O
TH
AU
N
U

55
PRACTICAL 7
GENE INTERACTIONS AND POLYGENIC
INHERITANCE
KEYWORDS
Gene interactions, classical crossing experiments and gene interactions in human disorders,
modification of phenotypic segregation ratios due to gene interactions, pleiotropy, polygenic
inheritance, quantitative and qualitative traits, additive interactions and epistasis, heritability, twin
studies.

ESSENTIALS
G E N E I N T E R A C T I O N applies to any phenotype that is brought about by a simultaneous
effect of two or more genes and proteins that they encode. This is to be clearly distinguished from

PY
allelic interactions that describe mutual relationship of alleles of the same gene.
R E C I P R O C A L I N T E R A C T I O N (interaction without any segregation ratio change)
results from discrete phenotypes each being determined by a discrete combination of dominant and

O
recessive alleles of interacting genes. Segregation ratios in F2 and B1 generations remain unchanged
from those observed in dihybrid (or polyhybrid) Mendelian crosses.

C
In D O M I N A N T E P I S T A S I S suppresses a dominant allele of one gene (called epistatic
gene) any phenotypic expression of another gene (called hypostatic), and F2 and B1 segregation
ED
ratios change accordingly.
In R E C E S S I V E E P I S T A S I S a recessive allele of an epistatic gene suppresses any
phenotypic expression of a hypostatic gene, and F2 and B1 segregation ratios change accordingly.
I N H I B I T I O N represents a type of gene interaction, in which a dominant allele of the
IZ

inhibitor gene suppresses any phenotypic expression of another gene without underlying any other
phenotypic effect. F2 and B1 segregation ratios change.
R

In C O M P L E M E N T A R I T Y dominant alleles of two (or more) genes cooperate in

realization of phenotype. The trait is expressed only if at least one dominant allele of all involved
O

genes is present at the same time, with a corresponding change of F2 and B1 segregation ratios.
TH

C O M P E N S A T I O N represents a type of gene interaction, where respective functions of

dominant alleles of two different genes are contradictory and their phenotype effects exclude each
other, with a corresponding change of F2 and B1 segregation ratios.
D U P L I C I T Y (or MULTIPLICITY in case of more than two genes) is interaction of two or
AU

more genes with the same effect on phenotype. The intensity of the phenotype and the corresponding
change of F2 and B1 segregation ratios depend on whether the effects of genes cumulate or not and
whether there is relationship of dominance between alleles of a particular gene.
N

P L E I O T R O P Y represents a phenomenon wherein one gene determines simultaneously several

different, frequently at first glance unrelated, phenotypic traits.
U

Q U A N T I T A T I V E T R A I T is any characteristic that could be readily and exactly

measured or otherwise quantified. Typical quantitative traits present in a population a continuum of
values with characteristic distribution, most frequently normal (Gaussian) distribution. Most of the
quantitative traits are multifactorial in aetiology, i.e. they are determined by simultaneous action of
many different genes, each with a relatively minor contribution, and by environmental influence.
The interactions between the underlying genes can be either additive or non-additive (epistatic).
In the case of A D D I T I V E G E N E I N T E R A C T I O N S represents the resultant
quantitative phenotype a summation of individual contributions of each underlying genes. Additive
gene interactions thus represent an extension of the duplicity cumulative without dominance (see
above).
N O N - A D D I T I V E G E N E I N T E R A C T I O N S are frequently modelled by the
resultant quantitative phenotype corresponding to the product of individual contributions of each
underlying genes. Non-additive gene interactions can be evidenced in qualitative traits as well and
are at present collectively called epistatic, not necessary implying that they are exactly identical to
56
cases of dominant of recessive epistasis described above.
R E L A T I V E R I S K (λ) gives how many times greater is the probability of contracting the
disease for a defined subpopulation compared to the general population. Very frequently, first degree
relatives (siblings, children, parents) of a diseased person are taken as a defined subpopulation and
their relative risk is marked as λs.
E D W A R D S F O R M U L A (also called empiric risk) is an approximation, according to
which could the absolute risk of a first degree relative of a diseased person be roughly estimated as a
root to population frequency of the trait in question. If more than one first degree relative is affected,
the resultant absolute risk would correspond to the root of the population frequency multiplied with
the number of affected first degree relatives, the final estimate being increasingly unprecise,
however.
H E R I T A B I L I T Y describes a relative contribution of genetic factors to a polygenic
quantitative phenotype. It could be expressed as a coefficient of heritability (h2) that gives relative

PY
contribution of genetic factors to total phenotypic variance in a population.
T W I N S T U D I E S could dissect the contribution of genetic factors (as opposed to
environment) to a given trait or condition of interest. The approach is most frequently based on

O
comparing trait’s congruence across large samples of monozygotic versus dizygotic twins.

C
Examples
ED
EX 7.1. F2 - phenotypic segregation ratios for selected classical gene interactions.
IZ

GENE INTERACTION A_ B_ A_ bb aa B_ aabb

R

Reciprocal interaction 9 3 3 1
O

Recessive epistasis 9 3 4
TH

Dominant epistasis 12 3 1
Inhibition 13* 3 *
AU

Complementarity 9 7
Compensation 10* 3 3 *
N

GENE INTERACTION A1_A2 A1_a2a2 a 1a 1A 2_ a 1a 1a 2a 2

U

Duplicity cumulative with dominance

9 6 1
(complete dominance)

Duplicity non-cumulative 15 1

A1A1A2A2 A1A1A2a2 A 1A 1a 2a 2 A 1a 1a 2a 2 a 1a 1a 2a 2
GENE INTERACTION A1a1A2A2 a 1a 1A 2A 2 a 1a 1A 2a 2
A 1a 1A 2a 2
Duplicity cumulative without
1 4 6 4 1
dominance (incomplete dominance)

57
TASKS
7.1. Phenotypic segregation ratios for selected classical gene interactions. Fill in the Punnett
squares bellow for gene interactions indicated, graphically distinguishing different phenotypes
(e.g. by using various coloured pens of using different modes of hatching). Derive the
respective B1 phenotypic segregation ratio and write it bellow each Punnett square.

Reciprocal interaction:

gametes AB Ab aB ab
AB
Ab
aB
ab

PY
B1 phenotypic segregation ratio:

Complementarity:

O
gametes AB Ab aB ab

C
AB
Ab
aB
ED
ab

B1 phenotypic segregation ratio:

IZ

Recessive epistasis:
R

gametes AB Ab aB ab
AB
O

Ab
aB
TH

ab

B1 phenotypic segregation ratio:

AU

Dominant epistasis:

gametes AB Ab aB ab
N

AB
Ab
U

aB
ab

B1 phenotypic segregation ratio:

Inhibition:

gametes AB Ab aB ab
AB
Ab
aB
ab

B1 phenotypic segregation ratio:

58
Compensation:

gametes AB Ab aB ab
AB
Ab
aB
ab

B1 phenotypic segregation ratio:

Duplicity cumulative without dominance:

gametes AB Ab aB ab
AB

PY
Ab
aB
ab

O
B1 phenotypic segregation ratio:

C
ED
7.2. Using phenotypic segregation ratios to determine the type of gene interaction. Let’s assume
that the feather colour is in budgerigar (a parrot species Melopsittocus undulatus) determined by
two interacting genes, F and O.
IZ

genotype feather colour (phenotype)

F_O_ green
R

F_oo yellow
ffO_ blue
O

ffoo white
TH

a) When green and blue parrots were crossed, a half of their progeny has been green and another
half blue. What genotypes had the parents?
AU

b) A yellow parrot has been crossed with a blue one. Their progeny involved all the possible
colours (green, blue, yellow and white). What genotypes had the parents and what would be the
N

theoretical phenotypic segregation ratio? Apply the branching method.

U

c) Identify the type of interaction between the genes F and O.

59
7.3. Fur colour in some rodents is determined by an interaction between genes A,B, and C. The
gene C is recessively epistatic towards the genes A and B and leads to albinism in recessive
homozygotes. The allele A conditions grey fur colour, recessive homozygotes aa are black. The
allele B determines a yellow pigmentation of hair endings, there is no phenotype in bb homozygotes.

a) Identify the type of interaction between the genes A and B.

b) After crossing a black - yellow hair ending animal with an albino animal, half of the progeny has
been albinotic, a quarter has been grey with yellow hair endings and a quarter black with yellow hair
endings. Deduce the genotypes of the parents.

PY
O
C
ED
IZ

c) With the aid of the branching method, derive the F2 genotypic and phenotypic segregation ratios.
R
O
TH
AU
N
U

60
7.4. Genetics of model polygenic qualitative trait. Let’s assume that the skin colour in humans is
determined duplicity cumulative without dominance between genes A1 and A2 (this should be
understood as an illustrative simplification, the skin colour in humans being actually genetically
much more complex). According to this model would the black people carry four dominant alleles
(A1 A1A2A2), the possession of any three dominant alleles would result in dark brown skin colour,
any two dominant alleles underlie brown skin pigmentation (mulatto), a single dominant allele is
associated with light brown skin colour and white people are double recessive homozygotes
(a1a1a2a2).

a) Write down all the possible genotypes of the following phenotypes:

Phenotype Genotypes

PY
dark brown:

mulatto:

O
light brown:

C
b) With the aid of the branching method, deduce skin colours of the children of the following
parental pairs:
ED
A1A1A2a2 x a1a1A2a2 children’s skin colour:
IZ
R
O

A1a1A2a2 x A1a1A2a2 children’s skin colour:

TH
AU

A1a1a2a2 x a1a1A2a2 children’s skin colour:

N
U

61
7.2 Genetics of model polygenic qualitative trait - 2. According to Hrubý, an interaction of six genes
underlies hair colour genetics in human. Analogically to the situation in rodents (see above), the gene
A controls the synthesis of a basic pigment molecule and it is therefore recessively epistatic to all the
other interacting genes, with recessive homozygotes aa being albino. The gene B controls the
production of brown pigmentation and it is dominantly epistatic towards the gene R, with recessive
homozygotes bb showing light hair colour. The gene R control the production of a red pigment,
recessive homozygotes are without any specific phenotype. D, F and V are modifier genes that
modulate the colour intensity; similarly to R, only respective dominant alleles can play this modifying
role. There is complete dominance in all interacting genes. The table 7.2 gives all the major genotypes
and phenotypes.

Tab. 7.2

PY
Genotype Genotype
Hair colour Hair colour
A_B_ _ _ A_bbR_

O
black D_F_V_ dark red D_F_V__

C
dark brown D_ F_vv red D_F_vv
D_ffV_ D_ffV_
ddF_V_ ddF_V_
ED
brown D_ffvv gold-red D_ffvv
ddF_vv ddF_vv
ddffV_ ddffV_
IZ

light brown ddffvv gold-fallow ddffvv

R
O

Genotype
Hair colour
TH

A_bbrr
dark fallow D_F_V__
AU

fallow D_F_vvD
ffV_
ddF_V__
N

light fallow D_ffvv

ddF_vv
U

ddffV_

super light ddffvv

fallow

a) Brown-haired parents had light-fallow and super light-fallow children. What were the genotypes of the
parents?

62
b) What hair colour(s) are to be expected in children of red-haired parents?

c) A fallow-haired mother has a brown-haired child. What hair colour could be expected in the father
of the child?

d) With the aid of the branching method, deduce hair colours of the children that are born to black-

PY
haired parents of the genotypes AaBBRrDDFfVv.

O
C
ED
IZ
R
O
TH
AU
N
U

63
 7.6. Genetics of model polygenic qualitative trait. Let’s assume that the body height is
modulated by an additive interaction (multiplicity cumulative without dominance) of 5 genes
(10 alleles). Basic body height if neither of the modulating alleles is active is 150 cm. Each
active allele of a modulating gene adds 5 cm to the basic body height. For simplicity, we dismiss
any environmental influence.
a) What height will have an individual with all modulating alleles active?
b) Derive the F2 genotypic and phenotypic segregation ratios.
c) What would be the height range in the progeny of parents with the body heights of 160 cm
both? What are the underlying genotypes?
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

PY
O
C
ED
IZ
R
O
TH
AU
N
U

64
7.7. Estimating absolute and relative risks. With the aid of the Edwards formula, calculate a risk
of disease for siblings and children of a proband affected by (the population frequencies given are
rounded-off approximations):
a) cleft lip (population frequency 1:900)
b) schizophrenia (population frequency 1:100)
c) diabetes mellitus (population frequency 1:16)
d) short-sightedness (population frequency 1:10)

Nobody else is affected in a respective family. Estimate a relative risk for each disease trait. Is
there any relationship between relative risk and population frequency? Advance a likely
explanation for high population frequency of some polygenic disorders.
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

PY
O
C
ED
IZ
R

7.8. Limitations of risk estimates based on Edwards formula. The proband is a girl suffering
O

from a special form of skin allergy – atopic eczema. Her older brother is affected by hay fever;
her mother suffers from asthma bronchiale. Her father is healthy, his father died from asthmatic
TH

complications. Current population frequency of allergic disorders is estimated to 16%; in young

population approaches 25%.
Draw the pedigree and perform risk calculation for siblings and children of the proband.
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)
AU
N
U

65
7.9. Twin studies and concordance calculation. Following values have been assessed in a large
Californian twin collection:

Tab. 7.3
Monozygotic twins Dizygotic twins
Disease
discordant (concordant) discordant (concordant)
Thyroid cancer 93 (18) 195 (18)
Infectious mononucleosis 428 (76) 711 (65)
Rheumatoid arthritis 377 (66) 952 (86)
Schizophrenia 29 (8) 102 (5)
Diabetes mellitus type I 125 (23) 336 (12)

PY
Asthma 1151 (487) 3272 (494)
Alcoholism 291 (140) 916 (170)

Calculate the respective concordance in both twin types. What conclusion can be drawn from

O
these calculations about a relative relevance of genetic factors versus environmental factors in the
aetiology of the diseases in the table above?

C
(Cockburn et al., The occurrence of chronic diseases and other conditions in a large population-based cohort of native
Californian twins. Twin Research 2002;5:460-467) ED
IZ
R
O
TH
AU
N
U

66
PRACTICAL 8
IMMUNOGENETICS AND MULTIPLE ALLELISM
KEYWORDS
Immunogenetics, humoral and cell-mediated immunity, innate and adaptive immunity, antigen,
immunogen, antibody, blood groups (ABO, Rhesus, MN – see above practical Monohybridism,
Determination of Genotypic and Phenotypic Segregation Ratios, Xg – see above practical Sex
and Heredity), alloantigen, histocompatibility antigens, HLA, haplotype, multiple allelism,
codominance.

ESSENTIALS
I M M U N O G E N E T I C S represents a part of genetics that has been established in the
course of studies on transplantation compatibility. Currently, it can be understood in broader

PY
term, as a genetic discipline focusing on genetic basis of immune response, and thus involving
besides the transplantation and transfusion immunology also issues dealing with proper
regulation of immune response and its disorders, like autoimmunity or immunodeficiency.

O
A N T I G E N is usually a macromolecular substance that is specifically recognized by a part of
adaptive immune system, i.e. by an antibody or a T-cell receptor. I M M U N O G E N is a

C
substance that lead to the synthesis of a particular antibody or differentiation of a particular T cell
clone upon immunization, i.e. after it has been introduced into an immunocompetent recipient.
Usually, antigen and immunogen are identical, but there examples where this they have to be
ED
distinguished, notably the ABO – blood group system. Here, in addition, antigens are called
agglutinogens, because they are evidenced by a specific immune reaction – erythrocyte
agglutination, upon mixing erythrocytes with an incompatible serum.
IZ
A N T I B O D Y (I M M U N O G L O B U L I N) is the result of humoral adaptive immune
response, which is executed by B-lymphocytes. After terminal differentiation into plasmatic cells,
they secrete antibodies into blood stream or other body fluids. In the ABO – blood group system,
R

the respective antibodies are called agglutinins (see above).

O

A B 0 B L O O D G R O U P S Y S T E M is the most extensively used blood group system

to guide the blood transfusion. For the most part, expression of the AB0 blood group antigens is
controlled by multiple alleles of a single gene (also known under the symbol I) located on human
TH

chromosome 9q34. Rarely, an additional gene, the Bombay gene (also known under the symbol
H), located on human chromosome 19q13.3, is also implicated. Functional alleles of both the I
and H genes code for glycosyltransferases that transfer various sugar residues on erythrocyte
AU

outer membrane. The synthesis of a basic structure (a complex sugar called fucose, also called
substance H) is under the control of the Bombay gene coding for the enzyme fucosyltransferase;
a loss-of function allele h does not code for any functional enzyme. The glycosyltransferases
encoded by the I gene modify the fucose further, either by grafting the N-acetyl-D-galactosamine
N

(glycosyltransferase A, encoded by the IA allele) or D-galactose (glycosyltransferase B, encoded

by the IB allele) on it. There are at least two IA alleles, IA1 and IA2, with IA2 encoding for a
U

catalytically less efficient enzyme resulting in a lower level of N-acetyl-D-galactosamine transfer

and also some qualitative deviation in the resulting glycosylation; IA2 thus represents a
hypomorphic allele. Complete loss-of-function allele i does not code for any functional enzyme.
The H allele is completely dominant over the h allele, the IA1 is completely dominant over the IA2
allele, both IA1 and IA2 alleles are codominant to the IB allele and IA1, IA2 and IB alleles are
completely dominant over the i allele. The h allele is recessively epistatic towards all the I alleles.
All the blood group traits have complete penetrance. Antibodies against the AB0 blood group
antigens occur as so called natural antibodies, i.e. without preceding immunization (e.g. in form
of an AB0-missmatched transfusion). The rule is that every person has antibodies in his/her
serum that recognize and bind all the AB0 blood group antigens except his/her own(s). Antigen
(=erythrocyte) – antibody reaction results is erythrocyte agglutination, i.e. forming of clusters
composed of multiple erythrocytes. (For the sake of simplification, the AB0 alleles are sometimes
recorded by the same symbols as the resulting blood groups, i.e. A1, A2, B and 0, omitting thus the I gene
from the record.)

67
R H E S U S (Rh) B L O O D G R O U P S Y S T E M is controlled by several closely linked
genes (at least three genes – C, D and E) located on 1p36.13–p34.3, the most important among
them being the gene D. Rh-positivity assumes the presence of at least one functional D allele, the
Rh-negativity results from dominant recessive genotype for a loss-of-function d allele (usually in
form of a complete deletion of the entire D gene). Anti-Rh antibodies are only induced after
immunization, e.g. in form of an Rh-mismatched transfusion (i.e. Rh¯ person receives blood
from an Rh+ donor) or the delivery of an Rh+ child by an Rh¯ mother. Antigen (=erythrocyte) –
antibody reaction results is erythrocyte lysis, which can lead to haemolytic anaemia of newborns,
if Rh¯ mother, after being immunized, is pregnant (anew) with an Rh+ child.
A L L O A N T I G E N is a transplantation antigen present on the cytoplasmic membrane of
cells of an allograft (transplanted tissue from the same biologic species as is the transplant
recipient, but genetically distinct and initiating an immune reaction after transplantation). Besides
blood group antigens, alloantigens must be specifically presented to the recipient’s immune
system, which is accomplished by the major histocompatibility complex (MHC). Allelic

PY
difference in MHC genes between the transplant donor and transplant recipient frequently results
in graft rejection or, in specific cases of bone marrow transplantation, in the graft-versus-host
disease. Transplantation antigens and blood group antigens are examples of isoantigens, being

O
defined as a type of antigen which is present only in subsets of a species.
H L A (Human Leukocyte Antigen) is the name for the MHC complex in human. It represents an

C
extensive gene cluster on the chromosome 6p21, consisting of several groups of genes. HLA
class I genes (HLA-A, HLA-B and HLA-C) code for proteins that are in charge of presenting
peptide fragments of endogenous proteins to cytotoxic T-lymphocytes (CTL), in a complex with
ED
β2-microglobulin, whose gene is not located within the HLA complex. HLA class II genes (HLA-
DP, HLA-DQ and HLA-DR, each represented by an α and β gene) code mainly for complexes
presenting peptide fragments of endocytosed proteins to helper T-lymphocytes (Th cells); each
presentation complex consists of a dimer of the respective α and β chains, both encoded within
IZ

the HLA complex. In addition, there are several dozens of other genes located within the HLA
complex, some of them coding for regulatory proteins implicated in immune response control,
R

others being in charge of antigen presentation in specific circumstances (called non-classical

HLA-genes). All the HLA genes within the HLA complex are tightly linked (almost complete
O

linkage - see above practical Gene Linkage). HLA genes are very polymorphic – there are
hundreds or even thousands of alleles in population (multiple allelism). Consequently, there are
TH

many heterozygotes for each HLA gene, and in this case, both alleles are expressed and
manifested, together contributing to antigen presentation (codominance). Due to the tight linkage,
an array of alleles for different HLA genes within the HLA complex located on one chromosome
(called haplotype) is coinherited as a single unit, only exceptionally undergoing crossing-over
AU

leading to a new allelic combination. The presentation capacity for each allelic variant differs
(different peptides will be presented to T-lymphocytes depending on the allele(s) of HLA genes).
Consequently, transplanting organs between individuals differing in alleles of HLA genes will
N

frequently result in completely different peptide spectra presented by the transplant cells on one
hand and by all the other cells of the recipient’s body on the other hand. This will result in
U

recognizing the transplant tissue as foreign and rejecting it by the recipient’s immune system.

68
TASKS
8.1. Genetic control of the ABO blood group system.
a) Complete all the possible genotypes that can underlie the indicated blood groups (phenotypes):

phenotype genotypes

A1

A2

A1B

PY
A2B

O
C
ED
b) Complete all the possible missing phenotypes (i.e. consider all possibilities):

mother father child

IZ

A2 B
R

0 A1
O
TH

0 A2

B A2B
AU

B 0

A2 A1
N

A1B B
U

0 A1

A1B A2B

A2B 0

69
c) Fill in missing genotypes into the pedigree bellow.
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

PY
O
C
ED
IZ

d) In this task, consider a possible involvement of the Bombay gene. A man with a blood group
R

A1 married a woman with the blood group 0. They had a son with the blood group A1B and a
daughter with the blood group 0. Complete the genotypes bellow:
O
TH

Father (A1):

Mother (0):
AU

Son (A1B):

Daughter (0):
N
U

What type of gene interaction corresponds this case with?

70
8.2. Secretion of ABO antigens. In some people, ABO antigens can be found in saliva and it
could be shown that this ability to secrete ABO antigens is controlled by an independent gene Se.
A single functional allele is sufficient to condition a secretory phenotype, recessive homozygotes
for a loss-of-function allele underlies the non-secretory phenotype.
The presence of A antigen in saliva has been analysed in children of parents sharing genotype
A0Sese. What proportion of children is to be expected to have A antigen in their saliva? What
type of gene interaction corresponds the ability to secrete blood group antigens with?
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

PY
O
C
ED
IZ
R
O
TH

8.3. Nail-patella syndrome. The pedigree bellow shows the inheritance of the nail-patella
syndrome (a rare condition syndrome characterized by abnormalities of the nails, knees, elbows,
and pelvis), together with AB0 blood groups genotypes (a simplified form of recording is used).
AU

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)
N
U

71
a) Assuming that the grandfather I/1 has not been a carrier of a nail-patella syndrome allele, what
type of inheritance conforms the nail-patella syndrome to (AD, AR, GD, GR)?

b) Is there any indication for linkage between the gene controlling the AB0 blood groups and the
gene underlying the nail-patella syndrome?

c) If yes, make a drawing of respective homologous chromosomes in both grandparents (I/1 and
I/2) with alleles of both the genes.

PY
d) Which child has been born after meiotic recombination?

O
e) Can we tentatively estimate genetic distance separating both the genes?

C
ED
IZ
R

8.3. Genetics of the Rhesus factor. Complete the table below (possible phenotypes are Rh+ or
O

Rh¯).
TH

Parental genotypes Genotypes of children Phenotypes of children

AU

CcDdee x CcDDEE
N
U

ccddEE x CCDDEe

CCDdee x ccDdee

72
8.4. Using blood groups for paternity testing. To increase the reliability of the test, multiple
blood group systems are frequently combined. Complete the table below (different combinations
of blood group systems AB0, Rhesus and MN - see above practical Monohybridism,
Determination of Genotypic and Phenotypic Segregation Ratios- are recorded always for a mother
and a child).
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

Blood groups of

mother child men possible as fathers men impossible as fathers

PY
0, M 0, MN

0, Rh+ 0, Rh-

O
C
0, Rh- A, Rh+

0, MN B, MN
ED
A, N 0, MN
IZ

A, MN A, N
R

A, Rh+ B,Rh-
O

A, Rh- A, Rh+
TH

A, N AB, MN
AU

B, MN 0, N

B, Rh+ B, Rh-
N
U

B, Rh- AB, Rh-

B, M 0, M

AB, N A, N

AB, Rh+ B, Rh-

AB, Rh- AB, Rh+

AB, MN AB, M

73
8.5. HLA haplotypes. Based on the allelic variants of HLA class I proteins identified in parents
and a child, deduce the respective HLA haplotypes and complete them into the table below.
Assume complete linkage.

HLA genotypes HLA haplotypes

solution:
father A1 A3 B8 Bwl5 Cwl Cwl A1 Bwl5 Cwl / A3 B8 Cwl
mother A1 Aw23 B7 B8 Cw2 Cw4 Aw23 B7 Cw4 / A1 B8 Cw2 A1
child A1 Aw23 B7 Bw15 Cw1 Cw4 Bwl5 Cwl / Aw23 B7 Cw4

PY
complete HLA haplotypes
father A2 A2 B5 Bw38 Cw3 Cw4
mother A11 Aw26 B12 B18 Cw1Cw2

O
child A2 Aw26 B18 Bw38 Cw2 Cw3

C
father A3 Aw24 B7 B12 Cw2 Cw4
ED
mother A2 A32 B7 Bw35 Cw4 Cw5
child A2 A3 B7 B7 Cw4 Cw4
IZ

father A28 Aw33 B14 Bw40 Cw3 Cw3

R

mother A11 A29 Bw15 Bw40 Cw3 Cw5

O

child A11 A28 Bw15 Bw40 Cw3 Cw3

TH

8.6. Using HLA haplotypes as an auxiliary criterion in paternity arguments. Due to tight linkage
AU

and correspondingly very low recombination frequency, as well as thanks to the extensive allelic
polymorphism of HLA genes and proteins, following the haplotype inheritance from father to a
child can provide additional information to resolve paternity arguments. In the example bellow,
N

only HLA-A and HLA-B proteins are followed. Deduce the corresponding haplotypes and decide,
whether the putative father can be the real biological father or not.
U

HLA phenotypes HLA genotypes HLA haplotypes

mother A3 A7 B7 A3 A7 B7 B7 A3 B7 / A7 B7
child A1 A3 B7 B8 A1 A3 B7 B8 A3 B7 / A1 B8

?father? A1 Aw24 B8 B12 A1 Aw24 B8 B12 A1 B8 / Aw24 B12

Conclusion: The putative father can be the real biological father of the child, as the child could
have inherited the respective haplotypes A3 B7 from the mother and A1 B8 from the father.

74
 HLA phenotypes HLA genotypes HLA haplotypes

mother A2 Aw32 B13 Bw35

child A1 Aw32 B8 Bw35

?father? A2 B7 B13

Conclusion:

PY
HLA phenotypes HLA genotypes HLA haplotypes

mother A11 Aw26 B8

O
child A3 A11 B5 B8
?father? Al A3 B5

C
Conclusion:
ED
IZ

HLA phenotypes HLA genotypes HLA haplotypes

R

mother A1 A2 B7
O

child Al A2 B7
TH

?father? A2 B7 B8
AU

Conclusion:
N

HLA phenotypes HLA genotypes HLA haplotypes

U

mother A2 B12
child A2 B7 B12

?father? A2 B8

Conclusion:

75
8.7. Base on the identified HLA phenotypes, deduce the corresponding HLA haplotypes in the
pedigree bellow and identify a person carrying a recombined haplotype.
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

A1 A2 B7 B27 A1 A3 B8 B22

PY
A1 B7 B8 A1 A3 B7 B22 A1 A2 B8 B27 A2 A3 B22 B 27 A1 A3 B22 B27

O
C
ED
IZ

8.8. Base on the identified HLA phenotypes, deduce the corresponding HLA haplotypes in the
pedigree bellow. The man in the second generation needs kidney transplantation – which of his
R

sisters would be a more convenient kidney doror for him?

O

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)
TH
AU

A2 A9 B5 B40 A3 A11 B5 B8
N
U

A2 A3 B5 A2 A11 B5 B8 A9 A11 B8 B40

76
PRACTICAL 9
POPULATION GENETICS, HARDY-WEINBERG LAW
KEYWORDS
Genetic composition of populations, gene pool, panmixia, Hardy-Weinberg law, Hardy-Weinberg
equilibrium, selection, selection advantage and disadvantage, selection coefficient, mutation,
mutation rate, migration, genetic drift, linkage disequilibrium, heterozygote advantage, fitness.

ESSENTIALS
P O P U L A T I O N involves all individuals of the same species inhabiting certain territory and
reproducing within it by mutual crossing, with a distinct gene pool discriminating it from other
populations.
G E N E P O O L represents all genes and alleles, as well as genotypes, carried by individuals in a

PY
population, with the respective gene, allele, and genotype frequencies.
As P A N M I X I A we call that way of reproduction, where parental pairs assemble solely on a
random basis.

O
H A R D Y - W E I N B E R G L A W is a mathematic expression of the relation between allelic,
genotypic and phenotypic frequencies in a population that is characterized by following attributes:

C
1. reproduction follows as panmixia
2. all the parental pairs have the same ability to contribute to the next generation – there is no
selection
ED
3. no new alleles are generated, i.e. there is no ongoing mutagenesis
4. there is no migration of individuals in and out of the population
5. the population is very large
IZ

Are these conditions met, the following relation between the allelic and genotypic frequencies can be
drawn (this basic form of the Hardy-Weinberg law assumes a biallelic system, i.e. a monogenic trait
R

with a dominant and a recessive allele and dominant and recessive homozygotes and heterozygotes,
respectively):
O

 
TH

where p = frequency of the dominant allele

q = frequency of the recessive allele
2pq = frequency of heterozygotes
AU

p2 = frequency of the dominant homozygotes

q2 = frequency of the recessive homozygotes
N

At the same time, it could be shown that under these conditions allelic and genotypic frequencies do
not change from one generation to the next – the population is in Hardy-Weinberg equilibrium.
U

S E L E C T I O N C O E F F I C I E N T (s) gives a relative intensity of selection against a phenotype.

Lethal phenotype has the selection coefficient s = 1.
S E L E K C T I O N P R E S S U R E describes the influence exerted by the selection on the respective
allelic frequency.
M U T A T I O N R A T E gives the frequency of new alleles due to mutations in a population.
M I G R A T I O N consists in inclusion of foreign (i.e. hitherto being part of different
population(s) individuals into a population in question, or exclusion of individuals out of it.
G E N E T I C D R I F T represents a mechanism of random fluctuation of allelic and genotypic
frequencies over generations. It consists in differences of allelic frequencies in a population and in
a sample of gametes actually implicated in the formation of the next generation. The magnitude of
such differences is inversely proportional to the population size. The impact of genetic drift is thus
most evident in small populations. As a result of genetic drift, alleles can be completely eliminated
from the population, with a single allele being fixed (i.e. achieving frequency of 100 %).
77
L I N K A G E D I S E Q U I L I B R I U M consist of preferential association of alleles in a
population, as a result of coinheritance (and thus a spread) of haplotypes, i.e. combinations of alleles of
tightly linked genes.
H E T E R O Z Y G O T E A D V A N T A G E represents a specific situation, where an allele
provides at the same time a selection advantage to heterozygotes and a selection disadvantage to
homozygotes. It results in equilibrium of allelic and genotypic frequencies that are achieved at
values different from those that would correspond to Hardy-Weinberg equilibrium. The equilibrium
frequencies due to heterozygote advantage are achieved if net increase in allelic frequency due to
positive selection of heterozygotes is exactly compensated by a net decrease of allelic frequency
due to negative selection of respective homozygotes.
F I T N E S S is the term to describe a complex characteristic that is subject to natural selection,
resulting in changes in allelic and genotypic frequencies. Fitness could be defined as an ability of an
individual organism to propagate its alleles in a population. It thus involves both general health and
survival abilities, a specific reproduction activity as well as characteristics determining social

PY
position in a population.

O
Examples

C
EX 9.1. Let us assume that eye colour in man is under the control of a single gene – the gene
ED
H, with the dominant allele determining brown colour and the recessive allele determining blue
colour. Assuming that the population is in Hardy-Weinberg equilibrium, what is its gene pool if 8%
of the population have blue eyes?
IZ

Solution: In a biallelic system, following two equations hold true:

R

 
O

 
TH

Knowing that the frequency of recessive homozygotes (hh) q2 = 8 % = 0.08, we can easily calculate
all the remaining allelic and genotypic frequencies:
AU

 
 
N

 
U

frequency of dominant homozygotes (HH)

frequency of heterozygotes (Hh)

The gene pool of the population in question thus consists of the dominant allele H (frequency 72%),
recessive allele h (frequency 28%), dominant homozygotes HH (frequency 52.84%), heterozygotes
Hh (frequency 40.32%) and recessive homozygotes hh (frequency 8%).

78
EX 9.2. Calculate genotypic and phenotypic frequencies of the AB0 blood groups in a panmictic
population that is in Hardy-Weinberg equilibrium. For the sake of this exercise, presume the existence
of a single A blood group, resulting in a triallelic system (IA, IB, i).

Let us assume that:

frequency of the allele (IA) p = not known
frequency of the allele (IB) q = 0.4
frequency of the allele (i) r = 0.4

Solution:

PY
O
In a triallelic system, frequency of the respective genotypes results, again, in an extension of the
formula

C
 
ED
resulting in

 
IZ

Calculations:
R

Genotypes Frequency calculation Phenotypes

O

A A 2 2
(I I ) frequency =p = 0,2 = 0,04 = 4% A
TH

A
(I i) frequency = 2pr = 2 x 0,2 x 0,4 = 0,16 = 16% A
B B 2 2
(I I ) frequency =q = 0,4 = 0,16 = 16% B
AU

B
(I i) frequency = 2qr = 2 x 0,4 x 0,4 = 0,32 = 32% B
A B
(I I ) frequency = 2pq = 2 x 0,2 x 0,4 = 0,16 = 16% AB
2 2
N

(ii) frequency = r = 0,4 = 0,16 = 16% 0

U

The blood group A is thus expressed in 20 % of population members, the blood group B in 48%,
blood group 0 in 16%, and the blood group AB is carried by 16% of the population’s individuals.

79
TASKS
9.1. Two alleles – complete dominance. Calculate respective genotypic and phenotypic
frequencies of a population in Hardy-Weinberg equilibrium, assuming a biallelic system (with
complete dominance A > a) and equilibrium allelic frequencies p(A) = 0.7 and q(a) = 0.3.

9.2. Autosomal recessive trait. Galaktosemia belongs to inborn metabolic diseases due to a defect

PY
in an individual's ability to metabolize the sugar galactose properly. Infants affected by galactosemia
typically present with symptoms of lethargy, vomiting, diarrhoea, failure to thrive, and jaundice. If
untreated, galactosemia may later lead to ovarian failure, developmental coordination disorder

O
(difficulty speaking correctly and consistently), and neurologic deficits. The disease is inherited as
an autosomal recessive trait, with recessive homozygotes (gg) affected. Calculate the frequency of

C
the mutated allele (g) and the frequency of heterozygote carriers (Gg) in a population where 1
affected child falls on 70 000 healthy individuals.
ED
IZ
R
O
TH

9.3. Autosomal dominant trait. Marfan syndrome is inherited connective tissue disorder resulting
from a mutation in fibrillin-1, a glycoprotein which forms elastic fibres in connective tissue and
contributes to cell signalling activity. Marfan syndrome features variable expressivity in form of
extensive variability of symptoms and severity, involving primarily the ocular, skeletal, and
AU

cardiovascular systems. According to last epidemiological search (Chiu HH et al., Mayo Clin Proc.
2014 Jan;89(1):34-42), 1 affected person falls to approximately 4 300 healthy individuals. Marfan
syndrome is inherited in autosomal dominant manner; let’s suppose that both heterozygotes (Mm)
N

and dominant homozygotes (MM) are equally affected (in the reality, the few described cases of
dominant homozygotes have been particularly severely affected). Calculate the respective allelic and
U

genotypic frequencies.

80
9.4. Gonosomal recessive trait. Ocular albinism type 1 is the X-linked recessive defect of the
colouring (pigmentation) of the iris, which is the coloured part of the eye, and the retina, which is
the light-sensitive tissue at the back of the eye, resulting in severely impaired sharpness of vision
(visual acuity) and problems with combining vision from both eyes to perceive depth (stereoscopic
vision). The mutated protein is G protein-coupled receptor 143, which participates in the control of
the growth and maturation of melanosomes. Affected women have to be recessive homozygotes
(XaXa), whereas affected men are hemizygotes (XaY). Calculate the frequency of heterozygous
carrier women (XaX) in a population where one affected man (XaY) falls on 60 000 men with
normal eye pigmentation (XY).

Genotype XX XaX XaXa XY XaY

Frequency p2 2pq q2 p q

PY
O
C
ED
IZ

9.5. Two alleles – incomplete dominance. Let’s suppose that the degree of earlobe attachment is
R

inherited as an incompletely dominant monogenic trait, with free form being dominant over attached
O

form. Phenotypic evaluation performed in a random sample of 100 individuals resulted in following
phenotypic distribution:
1. Attached earlobe (aa): 17 individuals
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2. Semi-attached earlobe (Aa): 45 individuals

3. Free earlobe (AA): 38 individuals
Calculate the gene pool of the population.
AU
N
U

81
PRACTICAL 10
GENETIC PROGNOSIS
KEYWORDS
Genetic prognosis and genetic counselling, risk prediction, genetic prevention, penetrance,
expressivity, coefficient of kinship, coefficient of inbreeding.

ESSENTIALS
G E N E T I C PROGN O SIS is the maximal calculated probability that a child
(conceived or intended) will be affected with a particular genetic disease. The genetic prognosis
depends especially on type of inheritance and family history of the disease in question.
G E N E T I C C O U N S E L I N G represents one of major subjects in clinical genetics. It deals
with informing a patient or a relative thereof about the genetic prognosis and advising of the

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consequences and nature of the disorder, the probability of developing or transmitting it, and the
options open to them in management and family planning.
P E N E T R A N C E is the probability of phenotypic expression of a genotype. For genetic

O
disorders, it gives proportion of individuals carrying a corresponding pathologic genotype (e.g.
heterozygote for a disease allele in dominant diseases, recessive homozygote in recessive diseases)

C
that are actually affected. Penetrance values inferior to 100% imply that some individuals inheriting
a pathological phenotype will be spared of the disease, without compromising their ability to
transmit disease alleles to the next generation. Such cases are collectively called incomplete
ED
penetrance (see also practical Genealogy).
E X P R E S S I V I T Y gives degree and quality of phenotypic manifestation. In the case
of variable expressivity, affected persons can differ e.g. in disease severity, age of onset or a
IZ

particular spectrum of symptoms (see also practical Genealogy).

C O E F F I C I E N T O F K I N S H I P (r) is the probability that two related persons carry an
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identical allele originating from a common ancestor.

C O E F F I C I E N T O F I N B R E E D I N G (F) is the probability of homozygosity for an allele
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originating from one ancestor.

TH
AU
N
U

82
Examples

EX 10.1. A woman suffering from the congenital cataract of the autosomal dominant type
married a healthy man, whose sister and parents are healthy as well. Calculate the genetic prognosis
of their children, assuming that the congenital cataract has 80% penetrance.
Solution:
1. Drawing the pedigree of the family (left) and putting known genotypes and constituent partial
risks of transmission and/or manifestation (right).
In autosomal dominant diseases, there is a consensus that for the purposes of risk calculation,
affected individuals are automatically taken as heterozygotes; the possibility of dominant
homozygotes is neglected, unless there is a clear epidemiological evidence for their existence.
Healthy persons are by consensus taken as recessive homozygotes; in addition, in this example,

PY
the disease-free family history in the father of the child under calculation (II-2) practically
excludes the possibility that he would be carrier of the cataract allele and healthy due to
incomplete penetrance. There is a risk of 50% that the heterozygote mother (II-1) passes the

O
cataract allele to her child, and, should this be the case, there is 80% probability that the child
would be affected.

C
ED
IZ
R
O
TH
AU
N
U

2. Total risk calculation.

Total risk is the product of all component partial risks that have to be materialized in
order for the child under calculation to become affected, i.e. 0.5 x 0.8 = 0.4. The
genetic prognosis that the child would be affected with the congenital cataract is thus
40 %

83
EX 10.2. A healthy married couple (mother and father of an intended child) ask about the risk
of the child to be affected with phenylketonuria, an autosomal recessive inborn metabolic disease due
to a homozygous loss-of function mutation in the gene encoding for phenylalanine hydroxylase,
leading to accumulation of phenylalanine to toxic concentrations and featuring brain damage,
intellectual disability, seizures, and other serious medical problems. There is a family history of the
disease – the mother’s uncle has been affected with phenylketonuria, his two sons are healthy,
nevertheless. According to available epidemiological data, we can assume that phenylketonuria
population frequency is approximately 1 : 10 000.
Solution:
1. Drawing the pedigree of the family (left) and putting known genotypes and constituent partial
risks of transmission and/or manifestation (right).
As there is no indication of any other affected person in the family except the mother’s uncle, we

PY
can assume that the mother’s grandparents (not shown in the pedigree) have been healthy. In this
case, they both had to be heterozygous carriers. The probability that a parental couple of
heterozygous carriers would have a heterozygous carrier child (we know from the family history
that the mother’s mother has been healthy) is ⅔. There is a consensus that for calculating the risk

O
of autosomal recessive diseases, all the persons coming into a family from outside and whose
family history is not known, are taken as dominant homozygotes, except the very last generation

C
(i.e. the father himself in this case). The probability that a heterozygous carrier would pass the
disease allele is thus ½. As we have no family information about the father, his probability of
being heterozygous carrier has to be calculated based on the population frequency of the disease,
ED
using the Hardy-Weinberg equations (see also practical Population Genetics). Should both the
mother and the father be heterozygous carriers, then the risk of their child being affected with an
autosomal recessive disease is ¼.
IZ
R
O
TH
AU
N
U

2. Calculation of the father’s probability of being heterozygous carrier.

84
3. Total risk calculation.

The probability of the intended child of being affected with phenylketonuria is 1/600, i.e. 0.17%.

EX 10.3. Becker muscular dystrophy is an X-linked recessive inherited disorder characterized

PY
by slowly progressive muscle weakness of the legs and pelvis. It is caused by hypomorphic mutations
(usually in-frame deletions) in the dystrophin gene; the encoded protein serves as a part of a protein
complex that connects the cytoskeleton of a muscle fibre to the surrounding extracellular matrix
through the cell membrane. Muscle weakness starts usually at puberty and progresses slowly, with

O
life expectancy averaging around 40 years, with notable variability. Null mutation (usually in form of
frame-shift causing deletions) in the same gene results in a more severe Duchenne muscular

C
dystrophy (see also practical Molecular Genetics). In the family seeking a genetic consultation, a
healthy woman whose brother and grandfather suffered from the Becker muscular dystrophy married
ED
a healthy man. She asks about the risk of her children.
Solution:
1. Drawing the pedigree of the family (left) and putting known genotypes and constituent partial
IZ

risks of transmission and/or manifestation (right).

For a gonosomal recessive disease, all affected men are hemizygotes for the underlying
R

mutation. A healthy daughter of an affected man has to be an obligate carrier. The probability
that a carrier woman would have a carrier daughter is ½. The same probability holds true that
O

she passes her mutation-bearing X-chromosome to her child, resulting in either an affected son
or a carrier daughter.
TH
AU
N
U

2. Total risk calculation.

For X-linked traits, genetic prognosis has to be calculated separately for boys and girls (see also practical Sex
and Heredity). For boys, the genetic prognosis is P = ½ x ½ = ¼ = 25%. For girls, the genetic prognosis is
zero, but there is a probability of 25% that they will be carriers.
85
TASKS
10.1. Alkaptonuria is a rare inherited autosomal recessive disorder in which the body cannot
process the amino acids phenylalanine and tyrosine. It is caused by a mutation in the HGD gene for
the enzyme homogentisate 1,2-dioxygenase. In recessive homozygotes the body accumulates an
intermediate substance called homogentisic acid in the blood and tissues, which is further oxidized to
alkapton and in this form excreted in the urine, giving it an unusually dark colour. The accumulating
homogentisic acid causes damage to cartilage (ochronosis, leading to osteoarthritis) and heart valves
as well as precipitating as kidney stones and stones in other organs.
Genetic consultation has been asked by a healthy man that married a healthy woman, whose sister is
affected by alkaptonuria. According to available epidemiological data, we can assume that alkaptonuria
occurs in the Czech population with the frequency 1 : 100 000.
(Interestingly, there us a dramatically larger frequency in Slovakia, estimated to 1: 19 000)

PY
O
C
ED
IZ
R

10.2. Retinoblastoma is a rare paediatric cancer that rapidly develops from the immature cells of a
O

retina, the light-detecting tissue of the eye. As for all inherited cancer syndrome, the disease
predisposition is inherited as autosomal dominant trait, necessitating somatic mutation of the wild
TH

type allele for the tumour to be initiated (see also Optional practical Selected Issues in Cancer
Genetics below).
A man suffering with the retinoblastoma in his childhood, whose mother had been affected as well,
married a healthy woman and asks about the risk of his children. Retinoblastoma features incomplete
AU

penetrance; the penetrance is estimated to 80%.

N
U

86
10.3. Red-green colour blindness is a genetically heterogonous gonosomal recessive disease
resulting most often from a mutation in L- or M-cone opsin genes, leading to a defective eye pigment
that is no more capable to register light wavelengths properly. The disease is highly prevalent, with
as much as 8% of males affected.
An red-green colour-blind man married a healthy woman. The couple asks for help at a department
of clinical genetics, wishing to know genetic prognosis for their children.

PY
O
C
ED
10.4. Cystic fibrosis is an autosomal recessive disease caused by loss-of-function mutations in a
IZ

chloride transporter called cystic fibrosis transmembrane conductance regulator (CFTR). Due to low
chloride export activity, there is a compensatory drop in cation export as well. Lower salt
R

concentration outside cells necessitates diminished water transport to keep isotonic environment.
O

This results in dense mucus in many tissues, with subsequent obstruction of gut in newborns
(meconium ileus), pancreatic ducts (absence of pancreatic enzymes in the gut and pancreatitis, i.e.
pancreas inflammation), vas deference (infertility) and lung colonization with numerous bacterial
TH

infections.
Calculate genetic prognosis for a child resulting from a marriage of cousins, whose grandmother’s
sister had been affected by cystic fibrosis. The estimated carrier frequency as much as 1:25, making
AU

the cystic fibrosis the most prevalent autosomal recessive disease in the white population.
N
U

87
10.5. Calculate the genetic prognosis for proband’s children in the pedigrees 6.9. – 6.14. analysed
previously within the context of the practical Genealogy. Use the pedigrees (pp. 49 – 51) to note the
genotypes and constituent partial risks of transmission and/or manifestation.

Pedigree 6.9. - genetic risk:

Pedigree 6.10. - genetic risk:

PY
O
Pedigree 6.11. - genetic risk:

C
ED
Pedigree 6.12. - genetic risk:
IZ
R
O
TH

Pedigree 6.13. - genetic risk:

AU
N

Pedigree 6.14. - genetic risk:

U

88
10.6. Adult-onset diseases. Huntington chorea (HC) is an autosomal dominant progressive
neurodegenerative adult-onset disease. It results from a gain-of function mutation in the Huntington
gene due to amplification of CAG codons, entailing a polyglutamine tract in the Huntington protein
(see below practical DNA diagnostic II). The children having inherited the mutation are born normal,
the disease can start at any age, by the age of 70 are all mutation carriers ill. This age dependency can
be expressed as an age-dependent penetrance, as summarized in the table bellow:

Age 10 20 30 40 50 60 70
Penetrance 0% 5% 20% 50% 80% 95% 100%

Proband is a healthy 31 years old man, whose mother has is affected with the Huntington chorea and whose
grandfather motherside succumbed to the disease. The proband’s mother has two healthy siblings – a
brother (55 years of age) and a sister (42 years). The mother’s brother has a healthy daughter (22 years),

PY
and the mother’s sister has a healthy daughter as well (12 years). Calculate genetic prognosis for all healthy
family members and their potential children.
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

O
C
ED
IZ
R
O

10.7. Genetic heterogeneity. Galaktosemia is an autosomal recessive disease due to a defect in an

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individual's ability to metabolize the sugar galactose properly (see above – Task 9.2). Galactosemia is
a genetically heterogenous disease. Three genes coding for key enzymes that are involved in
galactose metabolism can underlie galactosemia if mutated, namely galactose-1-phosphate uridyl
AU

transferase (GALT), galactokinase (GALK1) and UDP galactose epimerase (GALE). In each case,
homozygosity or compound heterozygosity for a loss-of-function mutation undelie the disease
phenotype, resulting in autosomal recessive mode of inheritance that confers a deficiency in an
enzyme responsible for adequate galactose degradation. Mutations in galactose-1-phosphate uridyl
N

transferase are the most frequent, with the population frequency of affected individuals averaging at
1 : 50 000, whereas mutations in galactokinase and UDP galactose epimerase are twice less frequent.
U

The overall population frequency 1 : 70 000 (see above – Task 9.2) is thus an average frequency of
three molecularly defined syndromes, each due to mutation in a different gene, but with an largely
identical phenotype.
A genetic consultation was sought by a healthy woman, whose uncle motherside suffered from
galactosemia due to a homozygous galactokinase mutation. She married a healthy man without a
family history of galactosemia. Calculate genetic prognosis for their children.

89
 PY
10.8. Coefficient of kinship and coefficient of inbreeding. Calculate the coefficient of kinship for
parental couples of related individuals in pedigrees 6.9 and 6.14. analyzed previously within the
context of the practical Genealogy and calculate the coefficient of inbreeding of their children.

O
Pedigree 6.9. - coefficient of kinship (r) for the individuals III-5 and III-6 :

C
ED
IZ

Coefficient of inbreeding (F) of their children:

R
O
TH
AU

Pedigree 6.14. - coefficient of kinship (r) for the individuals IV-4 and IV-5 :
N
U

Coefficient of inbreeding of their children:

90
PRACTICAL 11.
DNA-DIAGNOSTICS I
KEYWORDS
Genetic marker, restriction endonucleases, electrophoresis, Southern blotting, nucleic acid
denaturation and hybridization, hybridization probes, genomic probes and cDNA probes,
autoradiography, restriction fragment length polymorphism (RFLP), direct and indirect DNA-
diagnostics, marker and family informativeness.

ESSENTIALS
D N A D E N A T U R A T I O N is a type of treatment of DNA molecules that breaks hydrogen
bonds between complementary strands, consequently resulting in their separation from each other,
under generation of two separated single strands. The reverse process is called

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R E A S S O C I A T I O N if the original DNA molecule is exactly restored, or
H Y B R I D I Z A T I O N if unrelated single strands, e.g. issued from denaturation of two different
DNA molecules, or one resulting from denaturation of a natural DNA molecule and the second being
a synthetic single-stranded polynucleotide, align and re-establish hydrogen bonds between

O
complementary bases. Hybridization may even result in DNA-RNA doublestranded molecules. If
performed within the context of DNA-diagnostics, hybridization usually takes place between a DNA

C
molecule isolated from a diagnosed person, and an unrelated DNA (or, rarely, RNA) molecule
deliberately chosen to unravel a variability in the nucleotide sequence of the diagnosed DNA
molecule, called a H Y B R I D I Z A T I O N P R O B E.
ED
G E N E T I C M A R K E R is any defined locus in the genome that is polymorphic and is located
near a gene, whose mutation can underlie a genetic disease. Although without any direct coding
function, specific alleles of a polymorphic marker might be linked to a mutation in a nearby disease
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gene, thus defining a pathologic haplotype (see also above practical Immunogenetics and Multiple
Allelism). In that case, this specific allele of the linked genetic marker can be used as a surrogate
R

genetic change to identify persons carrying a mutation in the nearby disease gene. This procedure is
called I N D I R E C T D N A – D I A G N O S T I C S. This should be opposed to the D I R E C T
O

D N A – D I A G N O S T I C S, where the very presence or absence of a pathologic mutation in the

gene underlying the genetic disease in question is analysed and evaluated. The purpose of DNA-
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diagnostics is to identify carrier heterozygotes of autosomal or gonosomal recessive diseases, or even

dominant diseases, if incomplete penetrance or adult onset is to be assumed (postnatal diagnostic), or
affected foetuses, being it homozygotes or compound heterozygotes for recessive diseases or
heterozygotes for inborn dominant diseases (prenatal diagnostic).
AU

R E S T R I C T I O N E N D O N U C L E A S E S are bacterial enzymes with endonucleolytic

activity dependent on exact recognition of a specific sequence motif in DNA. When such a typical
recognition sequence is present, a corresponding restriction endonuclease will cut the DNA molecule
N

within it. There are several hundreds of commercially available restriction endonuclease, each
recognizing its typical target sequence (see Table 11.1. for several examples). Variability in DNA
U

sequence thus results in the variability of resulting DNA fragments after cleavage with a restriction
endonuclease. Target sequences for restriction endonucleases thus provide a simple example of
genetic markers, with two possible alleles for each particular site: the site can be either present, and
the restriction endonuclease used cuts within it, or the nucleotide sequence of the target site is
changed, and thus not recognized and not cut. This type of genetic polymorphism is collectivelly
called RESTRICTION FRAGMENT LENGHT POLYMORPHIS
M (R F L P).
To reveal the resulting restriction fragments, DNA, after being cleaved by a restriction endonucease,
is subject to E L E C T R O P H O R E S I S. During this process, DNA fragments, embedded within
specific gels (agarose or polyacrilamid gels) is allewed to travel in electric field; DNA, being a
polyanion due to the regular phosphate risidues making the phospho-sugar backbone, travels towards
the anode (+). The longer the fragment, the slower movement it shows and, consequently, travels the
shorter distance from a start position in a unit of time. Short fragments travel more quickly and the
distance they leave behind is longer.
When macromolecular DNA, such as human genomic DNA, is subject to restriction enzyme
91
cleavage and electrophoresis, millions of different fragments are generated, resulting in a continuum
of fragment lengths from very long to very short, with no possibility to recognise any individual
fragment. This is only made possible by hybridization. Hybridization is not carried out with the
agarose or polyacridamid gel directly, but with a nitrocellulose or nylon membrane exactly
corresponding to the gel with regard to position of all resulting DNA fragments (a sort of gel-
“photocopy” on the membrane). The process of transferring the DNA fragments from the gel to the
membrane is called B L O T T I N G. The DNA is carried from the gel to the membrane by
molecules of sodium hydroxide, at the same time leading to its denaturation. This specific version of
blotting is called S O U T H E R N B L O T T I N G, according to its inventor, Dr. Ed Southern.
The denatured DNA on the membrane is now accessible to hybridization with a probe, usually
corresponding to a gene in question, or to a sequence located close to it, called G E N O M I C
P R O B E if exactly corresponding to a DNA sequence as it exist in chromosomal DNA.
Another variant of a hybridization probe is c D N A. It represents an artificial DNA molecule
corresponding to exons only. It can be synthetized in vitro in a tube out of mRNA, by using a viral

PY
enzyme reverse transcriptase.
The total procedure of restriction fragment length polymorphism is illustrated on the figure 11.1
below.

O
C
Fig. 11.1.
ED
IZ
R
O
TH
AU
N
U

92
Examples
Figure 11.1 below gives a schematic outline of a “molecular” genotype of a hypothetical person,
where M represents a part of the maternal chromosome (i.e. inherited from the mother) and P stands
for the corresponding part of the paternal chromosome. Two essential elements are depicted in both
the chromosomal DNA parts – a gene (let’s assume that mutation within it can result in a genetic
disease) and a nearby polymorphic marker. The polymorphic marker is located 2 centimorgans
behind the gene and it is defined by a probe and by a polymorphism is restriction endonucleases
target sequences. Target sequences for two restriction endonucleases, E1 and E2, are marked by
arrows in both alleles. It is apparent that the two chromosomal parts are identical but a single E2
target sequence, last but one from the right. For that particular restriction cleavage site, only the M
chromosome carries the site, whereas the P chromosome is devoid of it, because the sequence of
nucleotides necessary for recognition and cleavage by the E2 restriction endonuclease carries a subtle
change from the consensus sequence, which does not allow for its recognition. We are thus able to

PY
distinguish the M chromosome from the P chromosome after cleavage of the chromosomal DNA
with the E2 restriction endonuclease, electrophoretic separation of the resulting fragments, their
transfer to a nitrocellulose or nylon membrane (Southern blotting) and hybridization with the probe.
In that case, the P chromosome would yield a longer fragment than the M chromosome because of

O
the absence of the polymorphic restriction site - often called (–) allele - while the M chromosome
would yield a shorter fragment because of an additional cleavage at the variable restriction site -

C
often called (+) allele. Assuming that there is a difference in the sequence of the gene as well (i.e. the
maternal or the paternal allele of the gene, either of them but not both, would carry a mutation), we
ED
might be able, by analysing other family members both phenotypically and with regard to the (+) and
(–) alleles of the linked polymorphic marker, and relating the results with each other, to decipher a
linkage between the mutated gene allele and a particular RFLP-marker (i.e. to construct both
haplotypes, e.g. mutated maternal gene allele linked to (+) polymorphic allele yielding a shorter
IZ

fragment and wt paternal gene allele linked to (–) polymorphic allele yielding longer fragment, or
vice versa) and use the polymorphism as a surrogate to infer whether the mutation has been passed to
R

the progeny (indirect DNA-diagnostics). Its accuracy is 98%. Gametic crossing over would change
the haplotypes (now the mutated maternal gene allele would become linked to the (–) polymorphic
O

allele resulting in longer fragment and the wt paternal gene allele to the (+) polymorphic allele
yielding the shorter fragment), making the predictions based on indirect DNA-diagnostics wrong.
The probability that this happens is 2%, based on the genetic distance between the gene and the
TH

probe. In contrast, the same procedure performed with the E1 restriction endonuclease would be not
able to discriminate between M and P chromosomes, as both are identical with regard to the presence
or absence of restriction cleavage sites in the region covered by the probe (in fact, not only here but
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throughout the entire locus). Hybridization would this reveal a single fragment, in other words, the
individual is homozygous for this polymorphism and thus noninformative.
N
U

93
Fig. 11.2

PY
O
C
ED
IZ
(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

Using DNA-diagnostics for genotype determination in autosomal recessive diseases:

R

Fig. 11.3.
O

The affected son is a homozygote both for the mutation (he is affected)
and for the RFLP-polymorphism, having on both homologous
TH

chromosomes only the allele 1 (1/1). Both his parents are heterozygotes,
both for the disease allele (therefore they are healthy but have a sick son)
and for the RFLP-polymorphism (1/2). What genotype and phenotype has
AU

the unborn fetus? Solution: It is apparent that the RPLP allele 1 is linked
to the disease-causing mutation. As the fetus is homozygous for the RFLP
allele 2 (2/2), he/she will be healthy and not even a carrier of the mutation.
N

Fig. 11.4.
The parents are heterozygous carriers of the disease-causing mutation
U

(hence they have an affected son), but only the mother is also a
heterozygote for the nearby RFLP-polymorphism (1/2), whereas the father
is homozygous for the RFLP-allele 1 (1/1). The affected son is a
homozygote for the disease-causing mutation (hence affected) and
heterozygote for the nearby RFLP-polymorphism (1/2). Because he must
have inherited both the chromosomes with the mutation, it is apparent that
the mother’s RFLP allele 2 is linked to the mutation, father is in this case
noninformative. What genotype and phenotype has the unborn fetus? Solution: As the fetus has
inherited the RFLP allele 1 from her mother, he/she will be phenotypically normal. As the father
is noninformative for the RFLP polymorphism analysed, we are not able to decide, whether the
he passed a mutated or wild type allele, both being linked to the RFLP allele 1. Therefore, there
is a 50% probability that the child will be a heterozygous carrier for the disease-causing
mutation.

94
Fig. 11.5.
Only partial DNA-diagnostic conclusion can be drawn in this family. With
regard to the disease-causing mutation, the parents are of course
heterozygous carriers and the son is the affected homozygote. The father is
informative, as he is also a heterozygote for the analysed RFLP-
polymorphism and based on the son’s RFLP genotype, we can conclude
that the RFLP allele 2 is linked with the disease-causing mutation, and this
allele has been passed to both the affected son and the new foetus. The
mother, on the other hand, is no informative, as she is homozygote for the
RFLP allele 1. Consequently, both the disease-causing mutation alleles
and the wild type allele at the disease locus are linked with the allele 1 at the nearby RFLP locus and
we are not able to distinguish the disease-causing mutation allele from the wild type allele
inheritance based on this RFLP-polymorphism. The foetus has thus 50% chance of being a
heterozygote carrier for the disease-causing mutation and 50% chance of being affected.

PY
Fig.11.6.

O
The entire family is noninformative, as all members are homozygotes for the
analysed RFLP-polymorphism. With regard to the disease-causing mutation,

C
the parents are again by necessity heterozygous carriers and the son is the
affected homozygote, nevertheless, as both the wild type and the mutated
alleles in both parents are linked with the same RFLP allele (the allele 1), we
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are not able to decide, whether the foetus has inherited the wild type or the
mutant allele from either parent.
IZ

Fig. 11.7.
R

The family is again noninformative. In this case, both parents are apparently
heterozygotes both for the disease-causing mutation (their son is affected)
O

and for the analysed RFLP-polymorphism. Contrary to the situation in Fig.

11.3., however, the affected son, being homozygote for the disease-causing
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mutation, is heterozygote for the RFLP-polymorphism as well. This means

that the disease-causing mutation is linked with the RFLP allele 1 in one
parent and with the RFLP allele 2 in the other parent. Consequently, it is
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again impossible to forecast, whether the foetus has inherited the wild type
or the mutant allele from either parent.

To accomplish DNA-diagnosis in the pedigrees 11.5 – 11.7., we would have to resort to

N

another nearby RFLP-polymorphism, usually by using both a different restriction

endonuclease and a different hybridization probe.
U

Fig. 11.8.
Also in this family is the DNA-diagnosis impossible. The only way how to
reconstruct the haplotypes (i.e. how to decide which allele of the analysed
RFLP-polymorphism is linked with the disease-causing mutation and with
the wild type allele at the disease locus, respectively) is to relate the pattern
of inheritance of the disease and the RFLP-polymorphism; this becomes
impossible if the DNA from the deceased affected son is no more available.
In this case, we have to resort to genetic prognosis (i.e. 25% risk of the
second child being affected and 50% risk of being a carrier).

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

95
Variable number of tandem repeats (VNTR = minisatellites)

The RFLP systems above are as a matter of principle biallelic; at any RFLP locus, only two alleles
are possible, + allele (i.e. the presence of a variable restriction cleavage site) or – allele (its absence).
It follows that the informativeness of such polymorphic markers would be limited (only double
heterozygotes, i.e. at the same time for a trait-underlying allele and an RFLP-allele, are informative –
see the examples above). The probability of any individual being a heterozygote is directly
proportional to the total number of alleles in the population. Polymorphic markers with higher overall
number of alleles would this be more informative than biallelic RFLP-markers. One example of such
a more informative polymorphic marker is provided by the variable numer of tandem repeats
(minisatellites). They represents one class of repetitive DNA elements; the length of a basic sequence
ranges usually from 10–60 base pairs, and this is typically repeated 5-50 times, with high variability
in the number of repetitions. Minisatellites occur at more than 1,000 locations in the human genome,
their distribution is, nevertheless, rather uneven. Their use in DNA-diagnostics thus crucially

PY
depends on whether there is a minisatellite located in the vicinity of a gene of interest.
The overall methodology is rather similar to the RFLP marker detection, involving again DNA
isolation, its restriction enzyme cleavage, electrophoresis of the myriad resulting DNA fragments,
Southern blotting including denaturation and hybridization of the nitrocellulose or nylon membrane

O
with a convenient labelled probe. Contrary to the RFLP markers, however, there is no variability in
the presence or absence of the respective restriction cleavage sites, only enzymes with constant

C
restriction cleavage sites are taken into the analysis. Instead, the DNA fragment between the constant
DNA cleavage sites is very variable in length due to a variation in a number of repeat units. Taking a
ED
sequence of the repeat unit as a hybridization probe, we can thus reveal different VNTR alleles
differing in the actual number of repeats according to the length of the fragment visualized after
hybridization, with more repeats units resulting in longer fragments (Fig. 11.9.). These VNTR
systems are much more polymorphic than the RFLP systems, with more different alleles in the
IZ

population, resulting in much larger proportion of heterozygotes and thereby in a much larger
informativeness.
R

Fig. 11.9.
O
TH
AU
N
U

96
TASKS
11.1. Restriction endonucleases. Find in the sequence bellow target sequences for any two restriction
endonucleases from the Table 11.1. and outline the exact way of DNA cleavage. Count the length of the
resulting fragments, both in the double-stranded DNA and after denaturation.

5'
GTTAACGTGCGCAACGATCCCAAGTTAACTGTGCAGTTAGGATCCCCTCACTCAAG
CAATTGCACGCGTTGCTAGGGTTCAATTGACACGTCAATCCTAGGGGAGTGAGTTC
3'
3'
CTTAGGTCGACACCCCAGGCCTTTACACTTTATGCGAATTCCCGATCTGGGTATGTT
GAATCCAGCTGTGGGGTCCGGAAATGTGAAATACGCTTAAGGGCTAGACCCATACAA
5'

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Tab. 11.1. Selected restriction endonucleases and their target sequences.
Target sequence

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Name of the restriction
Original microorganism 5‘→3‘
enzyme
3‘→5‘

C
G↓G A T C C
Bacillus amyloliquefaciens H Bam HI
C C T A G↑G
ED G↓A A T T C
Escherichia coli RY13 EcoRI
C T T A A↑G
Py G C G C↓Pu
Haemophilus aegyptus Hae II
Pu↑C G C G Py
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G G ↓C C
Haemophilus aegyptus HaeIII
C C ↑G G
R

G C G↓C
Haemophilus haemolyticus HhaI
C↑G C G
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G T Py↓Pu A C
Haemophilus influenzae Rd HindII
C A Pu↑Py T G
TH

G T T ↓A A C
Haemophilus parainfluenzae HpaI
C A A ↑T T G
C↓C G G
Haemophilus parainfluenzae HpaII
G G C↑C
AU

G↓T C G A C
Streptomyces albus G SalI
C A G C T↑G
N
U

97
11.2. Genomic probes and cDNA probes. Figure 11.9. bellow gives an outline of a hypothetical
gene involving three exons and two introns. The figure gives also its restriction map, i.e. relative
positions of restriction endonucleases TaqI a EcoRI. Notice that all the restriction target sites are
nonpolymorphic (i.e. always present) but a single TaqI target sequence in the second intron of the
gene that is variable (i.e. may but need not be present – marked by *). Also indicated are hybridization
probes used for analysis – the genomic probe derived from the second intron and the cDNA probe.
a) Specify the number and the respective lenghts of all the possible DNA fragments generated by the
restriction endonuclease TaqI.

PY
b) After the TaqI restriction cleavage, electrophoresis and Southern blotting, hybridization with the
labelled cDNA probe has been carried out. Specify the number and the respective lenghts of all the
DNA fragments revealed.

O
C
ED
c) Is the cDNA probe suitable to reveal the TaqI restriction polymorphismu in the second intron?
IZ

d) Is the genomic probe derived from the sequence of the second intron suitable for this purpose?
Indicate the lenghts of all the possible fragments revealed.
R
O
TH

Fig. 11.10.
AU
N
U

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

98
11.3. RFLP polymorphisms
Based on the pedigrees of families presenting with an autosomal recessive disease and by virtue of the
performed DNA analysis, decide on the genotypes of II/2 and II/3, separately for the pedigrees 11.11.
and 11.12. Let’s suppose thata the RFLP marker used in DNA analysis is in complete linkage with the
mutated gene.

Fig. 11.11.

PY
O
Fig. 11.12.

C
ED
IZ
R

Based on the pedigrees of families presenting with an gonosomal recessive disease and by virtue of the
O

performed DNA analysis, decide on the genotypes of II/3, separately for the pedigrees 11.13. and 11.14.
Let’s suppose thata the RFLP marker used in DNA analysis is in complete linkage with the mutated
TH

gene.
Fig. 11.13.
AU
N
U

Fig. 11.14.

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)
99
11.4. VNTR polymorphisms in indirect DNA-diagnostics. The pedigree bellow (Fig. 11.15.) shows
a family segregating autosomal dominant polycystic kidney disease (ADPKD), an adult-onset renal
disease presenting with progressive growth of numerous cysts in kidneys leading to increasing
compression of fuctional kidney tissue, finally resulting in kidney failure (called end stage renal
disease – ESRD) necessitating dialysis care or kidney transplantation. The father (I/1) reached this
stage and is after a kidney transplantation, whereas his son (II/1) has been diagnosed by
ultrasonography and is without overt clinical manifestation yet. No kindey impairement could be
found in his daughter (II/2). The mother (I/2) is just pregnant with dizygotic twins.
a) Calculate the genetic prognosis for the fetuses II/3 and II/4, based just on pedigree analysis.

PY
O
b) Calculate the genetic risk for the fetuses II/3 and II/4, based just on the DNA analysis performed

C
with a VNTR polymorphism located 5 cM from the mutated gene.

Fig. 11.15.
ED
IZ
R
O
TH
AU
N
U

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

100
11.5. The pedigree bellow on the Fig. 11.16. shows another family with autosomal dominant
polycystic kidney disease (ADPKD). The father (I/1) is currently on dialysis, all his three childrens
(II/2, II/3 and II/4) have been already diagnosed with the disease based on ultrasonography. Two of
them (the son II/2 and the daughter II/4) already married and wish to know about the risk for their
children.

a) Calculate the genetic prognosis for the fetuses III/1 and III/2, based just on pedigree analysis.

PY
b) Calculate the genetic risk for the fetuses II/3 and II/4, based just on the DNA analysis performed
with a VNTR polymorphism located 5 cM from the mutated gene.

O
C
ED
IZ
R

Fig. 11.16.
O
TH
AU
N
U

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

101
PRACTICAL 12
DNA DIAGNOSTICS II

KEYWORDS
Polymerase chain reaction (PCR), microsatellites, marker and family informativeness, direct and
indirect DNA-diagnostics, genetic anticipation

All the methodology of the Southern blotting and hybridization with labelled probes serves to
visualize individual DNA fragments that are located within or reasonably close to the gene of interest
and are convenient for use in DNA diagnostics, out of millions of other fragments issued from any
restriction cleavage of genomic DNA. This methodology became obsolete with the advent of
polymerase chain reaction that allows for direct amplification of a DNA fragment of interest. This

PY
might involve a variable restriction cleavage site, allowing for direct analysis of its presence or
absence after appropriate DNA fragment amplification, restriction cleavage and electrophoresis; DNA
is stained by specific fluorescent dyes like ethidiumbromide, allowing for visual inspection of DNA
fragments after PCR, restriction cleavage and electrophoresisi by naked eye and very easy scoring of

O
(+) and (–) alleles. Besides, additional DNA markers not easily amenable to analysis by restriction
cleavage can be used, notable microsatellites.

C
Targeted amplification of desired DNA fragments by polymerase chain reaction
ED
The polymerase chain reaction (PCR – Fig. 12.1.) is a method of amplification of a desired stretch of
DNA. A special type of DNA-polymerase is used to synthetize DNA molecules in a tube. This Taq-
polymerase has been isolated from bacteria living in hot springs (Thermus aquaticus) and represents a
IZ

remarkably stable enzyme that is not destroyed even by termeratures close to 100 °C and its reaction
optimum lies higher as for other DNA-polymerases (at 72 °C). As any DNA-polymerase, it needs
R

primers to start the DNA synthesis; primers in PCR are synthetic single stranded DNA
oligonucleotides of ~ 35 nucleotides in length that are oriented with their 3’-termini towards each
O

other. In addition to merely initiating the DNA synthesis, the primers also specify the DNA fragment
to be synthetized; only DNA stretch between both primers can be repeatedly synthetized.
TH

The reaction mixture consists of original genomic DNA, called template DNA (e.g. isolated from
leukocytes of an affected patient and his/her relatives), a pair of primers coresponding to borders of a
DNA stretch to be amplified, the Taq-polymerase and a convenient buffer solution. The polymerase
chain reaction consists of three steps featuring different temperatures:
AU

1. Denaturation = heating the reaction mixture to 94°C for one minute; this leads to breakdown of
hydrogen bonds between complementary baise pairs resulting in single stranded DNA out of the
original double stranded molecule.
N

2. Primer annealing = cooling the reaction mixture to temperatures between 55°C and 65°C, according
to the length and a particular sequence of primers; primers hybridize to their complementary
U

sequences in the genomic DNA. As primers are in a dramatic molar excess to the concentration of the
genomic DNA, probability of reassociation reaction (see above practical DNA diagnostics I) is
negligeable.
3. Primer extension = increasing the reaction temperature to 72°C, i.e. to the reaction optimum of the
Taq-polymerase. This allows for the Taq-polymerase to synthetize new DNA strands, initiating at the
respective 3’-ends of the primers and extending towards the positions of the respective second primer.
These three cycles are repeated on average 30 times; a specific piece of equipment called thermal
cycler, is needed for this. In the completion of every cycle, the newly synthetized DNA molecules can
serve as a template DNA for a next cycle of amplification, leading to exponential increase of total
amount of amplified DNA fragment. After 30 cycles, the concentration thus increases more 106 times.

102
Fig.12.1. Polymerase Chain Reaction

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O
C
ED
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R
O
TH

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

Short tandem repeats (microsatellites)

AU

One of the great advances made possible by the introduction of PCR into experimental and clinical
genetics is the exploitation of new classes of polymorphic markers, called microsatellites, short
N

tandem repeats (STR) or simple sequence repeats (SSRs). Quite apparent from the name,
microsattelites can be regarded as a smaller brother of minisattelites (see above practical DNA
U

diagnostics I). Their basic sequence is only several bps long (typically 2 – 5 bps), the range of
repetitions is similar (typically 5 – 50 repeats) and also highly variable. Their great advantage over
minisatellites is, however, that they are much more copious in the human genome and their
distribution is quite regular, it is thus possible to find a microsatellite in the vicinity of any gene.
There are thousands of microsatellites of the same type (e.g. CA(n)) scattered throughout the human
genome. Any individual microsatellite can thus be only defined by a unique sequence flanking it.
Using this sequence information for primer synthesis, we can thus amplify just the microsattelite of
interest out of thousends other microsattelites. Analogically to the minisattelites, the variablitily in the
number of repeats is directly reflected by the length of the PCR product – the more repeats, the longer
fragment is amplified. Because, however, they are much shorter than minisatellites, the amplification
products can be directly visualized and measured by electrophoresis, obviating any blotting and
hybridization. The microsattelite flanking sequences thus define any microsattelite locus, and the
particular number of repeats (i.e. the length of the PCR product after the amplification with these
unique flanking sequences as primers) thus define particular microsattelite alleles. Microsattelits are
especially valuable in indirect DNA diagnostics (Fig. 12.2).
103
Fig.12.2. Microsattelites in indirect DNA diagnostics. The microsattelite allele
n=22 is linked with a mutation in a gene underlying a hypothetical autosomal dominant
disease.

PCR primer 1 variable repeat Constant region 2

---- G TGC A ACC G TG TC C AGCG C A CCG TGC AC CG T AGC A A TG ----

---- C AC G T TGGC AC AGG TC GC G T n GGC ACG TGGC A TCG T T AC ----

Constant region 1 PCR primer 2

PCR products analysed by gel electrophoresis

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n = 22

O
n = 20 gel

C
n = 18 electrophoresis

C D A B E F
ED
A B
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O

C D E F
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AU
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104
TASKS
12.1. DNA-diagnostics of haemophilia carriers via an intragenic insertion polymorphism
Haemophilia, for the most part, is completely sex-linked gonosomal recessive disease due to an
inherited defect in the blood coagulation system, resulting in easy bruising, inadequate clotting of
traumatic injury or, in the case of severe hemophilia, a spontaneous hemorrhage (see above also
practical Sex and Heredity). Haemophilia is genetically heterogenous disease; two linked X-
chomosome located genes each can independently result in the disease if mutated (see also practical
Genealogy). The Haemophilia A the most common type, present in about 1 in 5,000–10,000 male
births; the gene codes for the blood coagulation factor VIII. Haemophilia B occurs in around 1 in
about 30,000 male births and is caused by a loss-of-function mutation in the nearby located bood
coagulation factor IX gene. There is another genetically distinct form – heamophilia C – a rare
autosomal semodominant form due to a mutation in the coagulation factor XI gene. The correct

PY
clinical diagnosis is a prerequisite of any subsequent DNA diagnosis and usually relies on a
biochemical assessment of either blood coagulation factor in plasma. It has been discovered that the
first intron of the factor IX gene underlying the haemophilia B could be of a variable length, due to a
presence or absence of a particular sequence within it (Fig. 12.3.). This insertion polymorphism can

O
be analysed by PCR-mediated amplification of the respective sequence, resulting in fragments of
different leghts depending on the presence or absence of this particular sequence.

C
Fig.12.3. ED
IZ
R
O
TH
AU
N
U

a) Are the sisters II3, II4, and II5 carriers?

b) How could the results of a prenatal DNA diagnostics performed in the III1 – fetus be interpreted?
Consider two vartiants, either you are not certain about the sex, or you know that the fetus is going to
be a boy.

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

105
12.2. Application of PCR for analysing RFLP-polymorphisms
The RFLP-polymorphisms consist in a variability in sequence motifs that can be recognized and cut
by a restriction endonuclease, with two possible alleles: (+) if the recognition sequence is present and
a corresponding restriction endonuclease can cut within it, or (–) if the restriction endonuclease target
sequence has diverged from the consensus and consequently fails to be recognized and cut. Some
restriction target sequences are always present and therefore of little use as genetic markers (called
constant sites), while others may but need not be present (called variable sites) and therefore of use as
polymorphic markers. For any variable restriction cleavage site, theree genotypes are possible –
individuals can be either homozygotes (+)/(+) or (–)/(–), or heterozygotes (+)/(–). To determine the
respective genotype, a complicated methodology involving the Southern blotting and hybridization
had to be used (see above also practical DNA Diagnostics I). Using PCR, a simpler and more direct
approach to genotyping has become possible.

PY
Von Hippel Lindau syndrome is a rare autosomal dominant adult onset disease with high penetrance
and frequency approaching 1 : 30 000 live births, characterized by the development of retinal and
central nervous system (CNS) hemangioblastomas (benign blood vessel tumors), fluid-filled cysts in
kidneys, pancreas, and genital tract, as well as by an increased risk of developing a type of kidney

O
cancer called clear cell renal cell carcinoma, a type of pancreatic cancer called a pancreatic
neuroendocrine tumor, and pheochromocytoma, a tumour of adrenal glands. The VHL gene is located

C
at 3p25-26 and codes for classical tumour suppressor gene that follows a double-hit scenario. Its main
function is to regulate cellular response to hypoxia, with biallelic mutations or loss of heterozygosity
leading to complex dysregulation of expression of many genes, like vascular endothelial growth
ED
factor, platelet-derived growth factor B, erythropoietin and genes involved in glucose uptake and
metabolism.
An A/G single nucleotide polymorphism (SNP) have been discovered in the first exon at nucleotide
IZ

19, with the G-allele creating target sequence for the restriction endonuclease HaeIII (GGCC - +
allele), while the A at this position constitutes the (–) alele :
R

A
5'- ...TTACAAC GCCTACGG... - 3'
O

G
TH

Importantly, this variable HaeIII restriction site (*) is flanked by two nonpolymorphic constant HaeIII
restriction sites (Fig. 12.4.). We can easily determine the genotype at the variable restriction site by
PCR-amplifying the stretch of DNA comprising all the three HaeIII restriction sites and performing
the restriction cleavage.
AU
N
U

106
a) Based on the locus scheme bellow, specify the (+) and (–) allele and all the three possible
genotypes.

Fig. 12.4.

PY
O
C
ED
IZ

b) In a VHL family bellow, this HaeII polymorphism has been analysed (Fig. 12.5.). Based on the
R

results, can you specify the diagnosis for the IIIrd generation?
O

Fig. 12.5.
TH
AU
N
U

107
c) During the analysis, an archival tissue sample of I/2 could be found and its subsequent analysis
revealed that she was homozygous for the (+) allele of the HaeIII polymorphism. Has this finding any
implications for the DNA diagnosis of the family?

d) Causal mutation in the VHL gene could be identified in this family. It consists in the missense
mutation in exon 3, codon 238 CGG (Arg)→TGG (Trp). This single nucleotide substitution entails a
loss of the MspI (CCGG) restriction target sequence, which can be diagnosed after PCR amplification
of a corresponding DNA segent followed by MspI restriction cleavage (Fig. 12.6a.). This constitutes
the direct DNA diagnostics, as we can directly reveal the presence or absence of the causal DNA
mutation rather then relying on a linked polymorphism. Interpret the results obtained for our family

PY
previously diagnosed via an indirect DNA diagnostics (Fig. 12.6b.).

O
Fig. 12. 6.a

C
ED
IZ
R
O

Fig. 12.6.b
TH
AU
N
U

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

108
12.3. Microsatellites
Microsatellites, also called short tandem repeats (STR) or simple sequence repeats (SSRs) are DNA
polymorphisms made up by short sequence motifs repeated 5 – 50 times, with the actual number of
repeats being highly variable. After PCR amplification with primers corresponding to unique flanking
sequences that are different for each individual microsatellite, the actual number of repetitions
directly translates into the length of the corresponding PCR product. The large variability in repeat
numbers results in high heterozygosity rate and therefore high informativeness (see above). In
addition to DNA diagnostics, they were of paramount use in gene mapping studies preceding
comprehensive human genome characterization, and they continue to be used in a number of other
issues, like mapping of human disease genes and modifyier genes, loss of heterozygosity studies of
tumour suppressor genes (see optional practicum Selected Issues in Cancer Genetics) etc.
Microsatellites are listed in several on-line databases, like GenBank or Ensemble.
Familiar adenomatous polyposis (FAP) is an adult onset autosomal dominant cancer predisposition

PY
syndrome characterized by develoment of numerous benign gut polyps, some of which inevitably
progress into colorectal carcinoma. The predisposing gene APC (Adenomatous Polyposis Coli) is
located in 5q21 and codes for an important tumour suppressor, involved in signal transduction as a
negative regulator of an oncoprotein β-catenin as well as in proper chromosome separation during

O
mitosis as a part of the kinetochore complex. Cellular deficiency in APC thus results in both
activation of oncogenic signalling and propensity to nondisjunction.

C
a) In a family presenting with FAP, a (CA)n microsatellite locus D5S346 located 30 -70 Kb behind
ED
the APC gene has been analysed by PCR amplification and following electrophoresis (Fig. 12.7.).
Based on the results bellow the pedigree, find out who in the III. generation inherited the mutation.
IZ

Fig. 12.7.
R
O
TH
AU
N
U

b) Consider possibilities to confirm or disprove the mutation in III4 a III5.

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

109
12.4. Analysis of trinukleotid expansions
A special type of microsatellite-like repeats has been characterized recently as causally related to a
group of neuronal and neurodegenerative diseases. These involve Fragile X-syndrome, Friedreich’s
ataxia, Myotonic dystrophy, as well as a group of progressive neurodegenerative diseases including
Huntington disease (see also practical Genetic Prognosis), Kennedy disease (SBMA = spinal and
bulbar muscular athrophy), Spinocereberal ataxia (SCA – multiple subtypes), Machado-Joseph
disease (also called Spinocereberal ataxia type 3) and Dentatorubral-pallidoluysian atrophy. Each of
these diseases underlie a microsatellite, which can be either in the promotor region of the gene (CGG
- Fragile X-syndrome), intron (GAA - Friedreich’s ataxia), 3’-untranslated region (CTG - Myotonic
dystrophy) or coding exons (CAG – progressive neurodegenenrative diseases) and the disease
pathophysiology involves a progressive instability of the microsatellite-like repeat in question,
resulting in its expansion (i.e. a progressive increase in repeat numbers from one generation to the
next). The actual number of repeats has a predictive value as to the penetrance and expressivity of the

PY
disease, i.e. the greater expansion extent, the higher probabilty of disease onset at younger age and of
its quicker progression – this phenomenon is called genetic anticipation. Analysis of the length of the
microsatellite in question bears thus a direct clinical message and is subject to DNA-diagnostic
analysis.

O
In a family presenting with the Huntington disease, DNA diagnostics has been untertaken to assess
the extend of GAG repeat expansion (Fig. 12.7.).

C
a) Specify the CAG repeat number in each family member.
ED
b) Draw a conclusion about a risk of Huntington disease manifestation among the IIIrd generation.
Who can be expected to develop the disease (Huntington disease is an adult onset disease)?
IZ
R

d) Is there any principal difference in the interpretation of the microsatellite analysis performed with
O

the Familial Adenomatous Polyposis (12.3.) and the Huntington disease (12.4.)?
TH

Fig.12.8.
AU
N
U

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)
110
OPTIONAL PRACTICAL 1
GENETIC PROGNOSIS AND DNA DIAGNOSTICS
O1.1. 20 years ago, a family asked for advice at a department of medical genetics. At that time, the
family included both healthy parents, a healthy daughter and a son affected with phenylketonuria, an
autosomal recessive metabolic disease (see above practical Genetic Prognosis); importantly, the
mother had been pregnant anew at the time of the consultation request. The parents were not related
with each other.
a) Draw a pedigree of the family and indicate possible genotypes of each family member.

PY
O
C
ED
b) If you were the medical geneticist asked, what advice would you give to the family, e.g. regarding
the genetic prognosis, possibilities of available differential diagnostic procedures, prevention, etc.?
IZ
R

O1.2. The family sought a genetic consultation again. The previous pregnancy gave a healthy son.
The impetus for the new genetic consultation is that the daughter is engaged and intends to start her
O

own family. Her partner has no siblings and his parents are healthy, with no evidence of
phenylketonuria in past generations. The population frequency of phenylketonuria is estimated to be
TH

1:10 000.
a) What is the genetic prognosis for a child born to the engaged couple?
AU
N

b) What would be the genetic prognosis for children of each son, the affected one and the healthy
U

one?

c) What conclusion would you come to, if you were the consulting medical geneticist, as to the
genetic prognosis, possibilities od available differential diagnostic procedures, prevention, etc.?

111
O1.3. To determine, what is the genotype of healthy children in the family are, indirect DNA
diagnostics has been performed. The DNA diagnostics has been conceived as classical RFLP analysis,
after total DNA cleavage with the restriction endonuclease Bgl II and hybridization with an intragenic
radioactively labelled probe derived as a fragment of the human phenylalanine hydroxylase (hPAH)
gene (Fig. V1/1).

a) Is the family informative?

b) What is the probability that the daughter is a heterozygous carrier?

PY
O
c) What is the probability that the healthy son is a heterozygous carrier?

C
ED
d) Can we extend this approach to assess the genotype of the daughter’s fiancé?
IZ
R
O

Fig. O1/1
TH
AU
N
U

112
O1.4. In parallel, direct DNA diagnostics has been approached. An apparent drawback of the direct
DNA diagnostics in phenylketonuria is the existence of allelic heterogeneity, i.e. many mutations
coexist and independently segregate in the population. The direct DNA diagnostic procedure has been
established for the 10 most prevalent mutations. In our population, the most frequent mutation is
R408W in the 12th exon of the hPAH gene, which accounts for 40-50 % of patients with
phenylketonuria. This mutation results in the appearance of the target sequence for the restriction
endonuclease Sty I, which can be evidenced after PCR amplification of the DNA fragment
surrounding the mutation site and subsequent Sty I restriction cleavage (Fig. V1/2):

Fig. O1/2

PY
O
C
ED
Applying this analysis to the family under consultation gave the following result (Fig. V1/3):
IZ

Fig. O1/3
R
O
TH
AU
N
U

a) Interpret the results of the direct DNA diagnostics. What is the genotype of each family member?

b) Based on the results, what is the genetic prognosis of the incipient child of the engaged couple?

c) Would the results of the direct DNA diagnosis performed justify prenatal DNA diagnostics of the
foetus?

113
d) Make recommendation in order to assess the child’s prognosis more precisely. What legal and
ethical concerns are to be considered?

O1.5. Apparently, there is an as yet unidentified mutation in the hPAH gene segregating in the
family together with the usual R408W missense mutation – the unaffected father is by necessity a
heterozygote carrier for this mutation and the affected son is compound heterozygote, having
inherited this unidentified mutation from his father and the R408W missense mutation from the
mother; the same missense mutation is by chance present also in the genotype of the healthy fiancé.
Neither of the other 9 most frequent mutations underlying phenylketonuria in the middle-European
population could be detected by PCR amplification and subsequent restriction cleavage followed by
electrophoresis in any member of this family. A method of choice in such cases is SSCP (Single
Strand Conformation Polymorphism) analysis. It relies on complex folding of denatured single-
stranded DNA molecules that can be markedly influenced by a single nucleotide change and in turn
dramatically change electrophoretic mobility of such folded single-strands, whereas consequences of

PY
such point mutations for electrophoretic mobility of double-stranded DNA fragments are usually
negligible. The SSCP analysis involves usually PCR amplification of all or some exons, denaturation
of the amplification products and their electrophoresis. If there is some deviation from a usual

O
electrophoretic mobility observed, this is indicative of the presence of a mutation, which is verified by
a direct sequencing of the exons under suspect.

C
In the family under consultation, all exons of the hPAH gene have been gradually inspected by SSCP
analysis, examples of electrophoresis results are in Fig. 1/4.
ED
IZ
R
O
TH
AU
N
U

114
Fig. O1/4

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O
C
ED
IZ
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O
TH
AU
N

a) Based on the results, reconstruct genotypes of all family members (use the symbols R408W and
U

6mut for the mutant alleles and + for the wt-allele).

b) Formulate a definitive conclusion of the genetic consultation, including methodology of DNA

diagnostics applicable for the foetus.

(Křenová D., Otová B: How to practice biological and medical genetics, Karolinum Praha 2009)

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