Genetics 1: Meiosis and Recombination
Explain the different stages of meiosis I
Mitosis
Meiosis
Occurs in all somatic cells to replicate cells
Occurs in germ line cells to produce gametes
2 identical cells are produced
4 non-identical cells are produced (variation)
Daughter cells are diploid (2n = 46)
Daughter cells are haploid (n = 23)
Single one-step process
Two-step process: meiosis I and meiosis II
Mitosis: somatic cell division
Prophase:

The chromosomes condense (become shorter and fatter) and become visible

The centrioles migrate to opposite poles of the cell

Microtubules radiate from each centriole to form the mitotic spindle

The nuclear envelope disintegrates
Metaphase:

The chromosomes become aligned along the equatorial plane of the cell

The centriole of each chromosome attaches to a microtubule of the spindle
Anaphase:

The centromere of each chromosome divides

The spindle contracts and the 2 daughter chromatids separate to opposite poles of the cell
Telophase:

A nuclear envelope develops around each set of daughter chromosomes

Cytokinesis: the cell cytoplasm separates to form 2 new diploid daughter cells
Meiosis I: reduction division (the chromosome number is halved)
Prophase I:

The chromosomes condense and become visible

Synapsis: homologous chromosomes align directly opposite each other to form bivalents

Recombination: crossing over occurs between homologous regions of non-sister chromatids

Chiasmata formation: homologous recombinant chromosomes remain attached where crossing over
has occurred at chiasmata
Metaphase I:

The nuclear membrane disintegrates

The mitotic spindle is formed

The chromosomes become aligned along the equatorial plane of the cell

The centriole of each chromosome attaches to a microtubule of the spindle
Anaphase I:

The spindle contracts and the chromosomes separate to opposite poles of the cell
Telophase I:

Each set of haploid chromosomes has completely separated to opposite poles of the cell

The cell cleaves into 2 new daughter gametes (secondary spermatocytes/oocytes)
Explain what occurs at meiosis II
Meiosis II:

Similar to mitosis

Each chromosome exists as a pair of chromatids

The chromosomes align along the equatorial plane of the cell

The 2 chromatids of each chromosome separate to opposite poles of the cell

2 new haploid daughter gametes are formed (spermatids or ova)
Fertilisation: the fusion of 2 haploid gametes to form a diploid zygote
Meiosis is important in maintaining the diploid number of chromosomes in the offspring since it produces
haploid gametes
Draw a diagram of meiosis showing segregation of two pairs of chromosome
Explain how recombination of chromosomes occurs
Meiosis introduces genetic variation in the offspring:
1. Meiotic recombination:

Each chromatid contains DNA portions from each parental homologous chromosome due to
recombination during prophase I

Each chromosome forms at least one chiasma

Small, medium and large chromosomes have 1, 2 and 3 chiasmata respectively
2. Independent assortment of chromosomes:

Different bivalents separate to gametes independently of each other

Therefore each gamete receives a selection of parental chromosomes
Genetics 2: Polymorphism and Inheritance of Variation
Define a polymorphism
Polymorphism: the occurrence of two or more genetically determined forms (e.g. alleles) in a population
in such frequencies that the rarest of them could not be maintained by mutation alone
Most polymorphisms are silent (i.e. they do not affect a gene) since they are present in intron DNA

Polymorphism: a variant present in a population at a frequency of more than 1%

Mutation: a variant present in a population at a frequency of less than 1%
Define genetic markers

Locus: the site of a gene on a chromosome

Allele: an alternative form of a gene found at the same locus on homologous chromosomes

Marker: a flag in a genome which is always inherited with a given allele
The precise location of the marker within the genome is known

Linkage: the association of any known marker with another marker more often than by chance (i.e. in
more than 50% of cases)

Genetic linkage: 2 alleles are inherited together more often than by chance
Genetic linkage often occurs due to physical linkage

Physical linkage: 2 loci are situated close together on the same chromosome; therefore the alleles at
these loci are inherited together more often than by chance (genetic linkage)

Recombination fraction (θ): a measure of the physical distance separating 2 loci
The recombination fraction indicates the likelihood of a cross-over occurring between the 2 loci
For θ = 0.05: there is on average a 95% chance that the alleles of 2 loci will be inherited together

CentiMorgan (cM): the unit of measurement for genetic linkage (not physical distance)
If 2 loci are 1 cM apart: a cross-over occurs between them on average in 1/100 meioses
1 cM corresponds to a physical distance of ~ 1 Mb (106 bp)

LOD score: a measure of the likelihood of 2 loci being linked
LOD score > 3: confirmation that 2 loci are linked (103:1 in favour)
LOD score < -2: confirmation that 2 loci are not linked (102:1 in favour)
Describe how markers are used to investigate variation
Markers: the marker allele will co-segregate with the disease allele

Microsatellites: short sequence repeats (2-4 bp)

Minisatellites: large sequence repeats (500bp)

SNPs: single base changes within an allele
Microsatellites
Minisatellites
Single nucleotide
polymorphisms (SNPs)
Highly polymorphic
•
•
•
Randomly distributed
•
Easily detectable
•
Stable
Linkage analysis: used to map disease in families
•
•
•
•
Explain how markers are used to follow inheritance
Draw a pedigree
Describe variation in humans
Describe the inheritance of variation
Describe recessive, dominant and X-linked inheritance
Describe co-dominant inheritance
Modes of inheritance:

Autosomal inheritance: determined by an autosomal gene

Sex-linked inheritance: determined by an allosomal gene

Recessive inheritance: 2 alleles are required for phenotypic expression

Dominant inheritance: 1 allele is required for phenotypic expression

Co-dominant inheritance: both alleles are expressed (e.g. blood groups)
Autosomal recessive inheritance
Autosomal dominant inheritance
Usually no family history (asymptomatic carriers)
At least 1 parent is affected
Transmitted by males and females
Horizontal transmission: affected individuals are usually
in a single generation
Vertical transmission: affected individuals are in multiple
generations
Males and females are affected in equal proportions
25% of the offspring are typically affected
50% of the offspring are typically affected
E.g. cystic fibrosis
E.g. osteogenesis imperfecta; achondroplasia
Compound heterozygote: 2 different defective alleles are
present at the same locus
Variable expressivity: clinical features may vary
Consanguinity increases the autosomal recessive risk
Reduced penetrance: abnormal clinical features may be
absent in a heterozygous individual
X-linked recessive
X-linked dominant
Transmitted by female carriers to their sons
Transmitted by males and females
No male-to-male transmission occurs
No male-to-male transmission occurs
Males are almost exclusively affected
Males and females are affected in equal proportions
If mother is a carrier: 50% of sons are typically affected
and 50% of daughters are typically carriers
If father is affected: all daughters are affected
If father is affected: 50% of daughters are carriers;
normal sons
E.g. haemophilia; Duchenne muscular dystrophy
If mother is affected: 50% of sons and daughters are
affected
E.g. hypophosphatemic rickets
Females are usually less severely affected than males
Genetics 3: Aneuploidies and Other Chromosome Aberrations
Describe the normal karyotype, chromosome banding and nomenclature
Karyotype: the chromosome constitution of an individual

Diploid cells (i.e. somatic cells): 46 chromosomes (22 pairs of autosomes and 1 pair of allosomes)

Haploid cells (i.e. gametes): 23 chromosomes
Chromosome banding:

G (giemsa) banding: used to identify individual chromosomes

The banding pattern of each chromosome is specific

Idiogram: shows the banding pattern of each chromosome

Karyogram: shows each chromosome pair in descending order of size
Chromosome nomenclature:

Bands are labelled according to the chromosome number, arm and distance from the centromere

Each chromosome  2 arms: p (short) and q (long)

Each arm  regions

Each region  bands

Numbering is outwards from the centromere

E.g. 15q32: chromosome 15, arm q, region 3, band 2

Karyotype nomenclature:
Male karyotype: 46,XY
Female karyotype: 46,XX
Chromosome morphology:

Metacentric chromosomes: the centromere is located centrally

Acrocentric chromosomes: the centromere is located terminally
Chromosomes 13, 14, 15, 21 and 22 are acrocentric
They often have satellites on their short arms which contain the genes for rRNA

Submetacentric chromosomes: the centromere is located in an intermediate position
Draw a diagram of a balanced translocation and explain why these are generally not deleterious
Common chromosomal abnormalities:
1.
Structural: translocations, deletions, insertions, inversions, rings
2.
Numerical: aneuploidy, polyploidy
3.
Mosaicism: different cell lines (mixoploidy)
Translocations: transfer of genetic material from one chromosome to another
Translocations of somatic cells are associated with cancer

Balanced translocation: the chromosome complement is complete, with no loss or gain of genetic
material due to exchange of genetic material between chromosomes
Usually the chromosome number remains at 46 and all genetic material is present
Therefore the person is generally normal, unless breakage has occurred within a functional gene

Unbalanced translocation: the chromosome complement is incomplete, and contains an incorrect
amount of genetic material

Reciprocal translocation: involves breakage of 2 chromosomes, with subsequent exchange of
segments between them to form 2 new derivative chromosomes

Robertsonian translocation: involves breakage of 2 acrocentric chromosomes at or near their
centromeres, with subsequent fusion of their long arms to form 1 new derivative chromosome
The short arms of each chromosome are lost: this is insignificant since they only contain genes for
rRNA
Draw a diagram showing possible meiotic products from someone with a balanced translocation
All genetic material is present in a balanced translocation parent
At meiosis: the chromosomes segregate and generate significant imbalance
Gametes from a balanced translocation:

50% are unbalanced for a translocation

25% are balanced for a translocation

25% are normal
Offspring from a balanced translocation parent may have an unbalanced set of chromosomes
This may lead to:

Miscarriage

Birth of an infant with multiple abnormalities (mentally and/or physically handicapped)
Describe how 3 different chromosome aberrations lead to Down syndrome
Aneuploidy: the loss or gain of one or more individual chromosomes
Non-disjunction: failure of 2 members of a homologous chromosome pair (bivalent) to separate

Monosomy: loss of a single chromosome  lethal in the case of autosomal monosomy

Trisomy: gain of 1 homologous chromosome
Usually caused by non-disjunction in anaphase I of maternal meiosis I: the pair of homologous
chromosomes fails to separate
(May also be caused by non-disjunction during anaphase II of meiosis II: the pair of sister chromatids
fails to separate)
A disomic gamete is formed: it receives 2 homologous chromosomes
If this disomic gamete is fertilised by a normal gamete, a trisomic zygote results

Tetrasomy: gain of 2 homologous chromosomes
Down syndrome: incidence and clinical features

Incidence: 1 in 650 to 1 in 700

Newborn period: severe hypotonia, sleepy, excess nuchal skin (back of the neck)

Craniofacial features: protruding tongue, small ears, upward sloping palpebral fissures (below the
eyes), bilateral epicanthic folds (inner eyelids), Brushfield spots (on the iris), broad nasal bridge

Limbs: single palmar crease, abnormally short 5th finger, wide gap between first and second toes

Cardiac: atrial and ventricular septal defects

Other: short stature, duodenal atresia (digestive tract)
Down syndrome: natural history

IQ range: 25-75

Relatively advanced social skills

Children are happy and affectionate

Adult height: 150 cm

Severe cardiac anomaly causes early death in 15-20% of cases

Average life expectancy: 50-60 years (omitting cardiac anomaly)

Most adults develop Alzheimer’s disease in later life
Down syndrome: causes
1.
Non-disjunction: trisomy 21 (95% of cases)

Trisomy 21: there is an extra chromosome 21

Caused by non-disjunction of the pair of homologous chromosomes 21 in maternal meiosis I

This results in the formation of 2 disomic gametes (and 2 nullisomic gametes)

If a disomic gamete is fertilised by a normal gamete, the zygote is trisomic for chromosome 21
Causes of non-disjunction:

There is a strong association between advancing maternal age and incidence of Down syndrome

Maternal age may cause predisposition to non-disjunction since there is an ageing effect on the
primary oocyte

There may be an age-related reduction in immunological competence: this allows survival of trisomic
embryos

Radiation

Delayed fertilisation after ovulation

Genetic control of non-disjunction (as in Drosophila)
2.
Translocation: (3% of cases)

Caused by a Robertsonian translocation involving chromosome 21 and another acrocentric
chromosome

A balanced translocation parent carrying the 14q21q translocation can produce a gamete with the
translocation chromosome 21 and a normal chromosome 21

13q21q and 14q21q: 10% risk of Down syndrome in offspring

21q21q: all offspring will have Down syndrome
3.
Mosaicism: post-zygotic non-disjunction during mitosis (2% of cases)

Mosaicism: the presence of 2 or more cell lines with different genetic constitutions, which are derived
from a single zygote

Chromosome mosaicism: caused by non-disjunction in an early embryonic mitotic division

Mosaicism may also be caused by a mutation in a somatic or germ-line cell division

Children are less severely affected than the full syndrome since the embryo shows 33% mosaicism for
trisomy 21
Describe 2 common autosomal and 2 common sex chromosome aneuploidies
Autosomal aneuploidies: Patau syndrome (trisomy 13) and Edwards syndrome (trisomy 18)

Incidence: 1 in 5000 to 1 in 10000

Most infants die within the first few days/weeks of life

Severe learning difficulties in the rare event of long-term survival

Cardiac abnormalities occur in 90% of cases

Craniofacial features: severe bilateral cleft lip and palate; cyclopia (in some cases)

There is a strong association between advancing maternal age and incidence

90% of cases are caused by non-disjunction: the extra chromosome is usually of maternal origin

10% of cases are caused by mosaicism or unbalanced translocations
Sex chromosome aneuploidies:
Turner syndrome: 45,X

Incidence: 1 in 3000 live female births

Features in 2nd trimester: generalised oedema or swelling localised to the neck

Features at birth: either normal or may have intra-uterine oedema with puffy extremities, neck
webbing, low posterior hair-line, short 4th metacarpels, aorta defect (coarctation in 15% of cases)

Normal intelligence

Adult features: short stature and ovarian failure

Short stature: average adult height is 145 cm without growth hormone treatment
Ovarian failure: leads to primary amenorrhea and infertility

Treatment: oestrogen replacement therapy (in adolescents) for development of secondary sexual
characteristics and long-term prevention of osteoporosis

80% of cases are caused by loss of a sex chromosome in paternal meiosis (45,X)

20% of cases are caused by chromosome mosaicism (e.g. 45,X/46,XX)
Klinefelter syndrome: 47,XXY

Incidence: 1 in 1000 live male births

Childhood features: clumsiness, mild learning difficulties (particularly verbal skills)

10-20 point decrease in verbal IQ

Adult features: taller than average, with long lower limbs

30% of adults show moderately severe gynecomastia (breast enlargement)

Infertile, with small soft testes: absence of sperm in the semen

In adults: increased incidence of leg ulcers, osteoporosis and breast carcinoma

Cause: the extra X chromosome arises from the mother or the father (47,XXY)

Some cases are caused by mosaicism (46,XY/47,XXY)

Males with more than 2 X chromosomes show severe learning difficulties and more marked physical
characteristics than Klinefelter men
XXX females: 47,XXX

Incidence: 1 in 1000 of all females

No physical abnormalities

10-20 point decrease in IQ

Cause: the extra X chromosome arises from an error in maternal meiosis I

Normal fertility

Offspring have normal karyotypes

Females with more than 3 X chromosomes show high incidence of learning difficulties
Phenotype deterioration: the severity of learning difficulties is proportional to the number of X
chromosomes present
XYY males: 47,XYY

Incidence: 1 in 1000 newborn males

2-3% of institutionalised males are XYY due to antisocial behaviour or learning difficulties

Most XYY males do not have learning difficulties or antisocial behaviour

Emotional immaturity, impulsive behaviour

Normal fertility

Normal physical appearance and above average stature

10-20 point decrease in IQ

Cause: extra Y chromosome arises from non-disjunction in paternal meiosis II or as a post-zygotic
event (mitotic error)
Describe why sex determination is not solely based on sex chromosome karyotype
Sex determination:

The sex chromosomes determine the sex of an individual

Presence of a Y chromosome  male

The Y chromosome is much smaller than the X chromosome: it carries only a few functional genes

Female gametes: ova carry an X chromosome

Male gametes: sperm carry either an X or a Y chromosome

Until 6 weeks after fertilisation: the embryonic gonads are undifferentiated

Differentiation into a phenotypic male or female occurs 6 weeks after conception:
The embryo develops into a female unless the testis-determining factor is present
This stimulates the undifferentiated gonads to develop into testes

Testis-determining factor: located in the sex-determining region of the Y chromosome (SRY)

XX males: the SRY determining gene is translocated from the Y to the X chromosome
Infertile phenotypic males (some genes on the Y chromosome are required for spermatogenesis)
Karyotype: 46,XX

XY females: the SRY determining gene is lost from the Y chromosome by a mutation or deletion
Infertile phenotypic females
Karyotype: 46,XY
Genetics 4: Prenatal Diagnosis of Genetic Diseases
Describe the indications for prenatal diagnosis
Ideal situation: couples who have a high prior risk of having an abnormal baby should be identified and
counselled before a pregnancy
Less ideal alternative: couples who have a high prior risk of having an abnormal baby should be identified
in early pregnancy to allow them to consider all available prenatal diagnostic options
Common indications of prenatal diagnosis include:

High risk of aneuploidy:
E.g. due to advanced maternal age

Family history of a chromosome, single-gene or structural abnormality

Increased risk predicted on the result of a screening test
E.g. detection of a structural anomaly in the foetus on routine ultrasound screening
Describe the use of non-invasive tests: maternal serum screening and ultrasound
Ultrasound:
Use: prenatal diagnosis of structural abnormalities (e.g. neural tube defects or cardiac anomalies)
Limitations: requires specialised expensive equipment and a skilled and experienced operator
Down syndrome:
Down syndrome foetuses have increased foetal nuchal translucency: increased thickness of the fold of skin
on the back of the neck
Ultrasound is used to measure nuchal pad thickness to screen for Down syndrome
Maternal serum screening:
Use: screening for neural tube defects (NTD) and Down syndrome at around 16 weeks gestation
Neural tube defects: e.g. spina bifida and anencephaly
Detection: by assay of α-fetoprotein (αFP) in the maternal serum
If the foetus has an open NTD: αFP leaks into the maternal serum which leads to an elevated αFP level
Maternal serum αFP screening:

Not 100% sensitive since there is an overlap between the curves for maternal serum αFP levels in
normal and affected pregnancies

Not 100% specific since factors other than an open NTD may cause elevated levels of maternal serum
αFP (e.g. a twin pregnancy)
Down syndrome:
Detection: by analysing the level of particular biochemical markers in the maternal serum
Maternal serum marker
Marker level in affected pregnancies vs. normal levels
α-fetoprotein (αFP)
Reduced level
Unconjugated estriol (µE3)
Reduced level
Pregnancy-associated plasma protein A (PAPP-A)
Reduced level
Human chorionic gonadotropin (free ß-hCG)
Elevated level
Inhibin-A
Elevated level
Down syndrome screening:
Risk of Down syndrome increases with:

Increasing nuchal translucency (detected by ultrasound)

Increasing maternal age

Changes in the levels of biochemical markers found in the maternal serum
Down syndrome can be screened for in pregnancy by accounting for these risk factors:
1.
Maternal age: strong association between maternal age and increasing risk of Down syndrome
2.
Maternal serum markers:
3.
Nuchal translucency: Down syndrome foetuses have increased foetal nuchal translucency
Test
Time (weeks)
Risk factors accounted for
Triple test
14-21
Maternal age, αFP, µE3 and hCG
Quadruple test
14-21
Maternal age, αFP, µE3, hCG and inhibin-A
NT scan
11-13
Nuchal translucency
Combined test
10-13
Nuchal translucency, maternal age, free ß-hCG and PAPP-A
Integrated test
1st trimester
Nuchal translucency, PAPP-A (1st trimester) and αFP, free ß-hCG, µE3 and
inhibin A (2nd trimester)
NICE antenatal care guidelines:
April 2007: 75% detection and less than 3% false positive

Integrated test

Combined test

Serum integrated test
Nasal bones:

Poor prenatal diagnostic tool since there is racial variation in nasal bone characteristics
Confirmation of Down syndrome requires cytogenetic analysis of foetal material obtained by an invasive
procedure (amniocentesis or chorionic villus sampling)
Describe the use of invasive tests: amniocentesis and chorionic villus sampling
Specific prenatal diagnosis of chromosome and single-gene disorders usually requires an invasive test: this
involves obtaining material of foetal origin
Amniocentesis:
Optimal time: 16 weeks gestation
Method:

Equipment: 22G needle with stylet

Aspire 15-20 ml of amniotic fluid via the abdominal wall under ultrasound guidance

Discard the first 2 ml of fluid to remove maternal cells
Cytogenetic analysis:

Centrifuge the sample to yield a pellet of foetal cells and supernatant fluid

Supernatant fluid: used for prenatal diagnosis of neural tube defects by assay of α-fetoprotein

Cell pellet: used for chromosome analysis and biochemical or DNA studies
The cell pellet is cultured in multiple cultures for 14 days to stimulate cell growth
Culture failure rate: 0.5%
Maternal contamination is rare
Mosaicism in amniotic fluid culture:

Mosaicism: it is possible to find 2 or more cell lines with different chromosomal constitutions in
amniotic fluid culture

This is mainly due to culture artefact; therefore mosaicism must be present in more than 2 cultures for
it to be significant
Risks and complications:

Miscarriage risk: 0.5-1%

A late mid-trimester abortion may be necessary if the result is abnormal

*Rh negative women get Anti D within 72 hours

There is a 1.3% risk of liquor leakage

Infection risk: less than 0.1%
Chorionic villus sampling (CVS):
Optimal time: 11-12 weeks
Method:

Transabdominal (or transcervical) aspiration of chorionic villi under ultrasound guidance
Chorionic villi are derived from the outer cell layer of the blastocyst (i.e. the trophoblast)

Maternal decidua must be removed before sample analysis
Cytogenetic analysis:

A provisional result is possible within 24 hours by direct chromosome analysis of chorionic villi

Direct culture of trophoblast cells is possible

Culture of chorionic villi: ideal for chromosome analysis
Chorionic villi are cultured for 14 days
Culture failure rate: 0.2%
Mosaicism: 0.5%
False negative result: 0.03%
Risks/complications:

Miscarriage risk: 0.5-2% (CVS is a technically demanding procedure)

There is a high risk of limb abnormalities if CVS is performed before 9-10 weeks gestation

*Rh negative women get Anti D within 72 hours
Advantage: CVS offers first-trimester prenatal diagnosis and therefore an early termination is possible
*Rh: blood group antigen which is present in 80% of women
Rh sensitisation: in Rh negative women there is an autoimmune response against the foetus
Foetal blood sampling:
Primary use: to obtain a foetal blood sample to test for foetal anaemia
Optimal time: 18 weeks
Method: either of the following:

Transplacental insertion: into the umbilical cord/placenta

Transamniotic insertion: into the intrahepatic vein
Transamniotic insertion is preferred since the loss rate is lower
Amniocentesis and CVS are preferred for obtaining a sample for chromosome analysis
Describe the use of karyotype analysis and FISH for detection of chromosomal abnormalities
Karyotype (chromosome) analysis:
1.
Prepare a metaphase spread of suitable cells by treating the cells to prevent spindle formation
Suitable cells include: chorionic villi and cells from amniotic fluid
The cell sample must be cultured for 10-12 days to obtain a sufficient number of cells for analysis
Metaphase spread: the preparation of chromosomes during the metaphase stage of mitosis in which
they are fully condensed
2.
G banding: provides high-quality chromosome analysis
Chromosomes are treated with trypsin to denature proteins
Chromosomes are stained with Giemsa (DNA-binding dye) which gives each chromosome a
characteristic pattern of light and dark bands
3.
Count the number of chromosomes present in a given number of cells using metaphase spreads
4.
Analyse the banding pattern of each pair of homologous chromosomes in 3-5 metaphase spreads
Each chromosome has a specific banding pattern
Karyogram: shows each pair of homologous chromosomes in descending order of size
Karyotype analysis allows detection of any large rearrangement of DNA without any prior knowledge of
the defect (e.g. aneuploidy, translocation or deletion)
FISH: fluorescent in situ hybridisation

Aim: to detect and localise the presence of specific DNA sequences on chromosomes

Principle: a fluorescent single-stranded DNA probe anneals to a very closely related target sequence
(i.e. one which shows a high degree of sequence similarity)

FISH allows interphase cells to be studied, as well as metaphase cells
Advantage: results are obtained rapidly with interphase FISH

FISH is used widely for clinical diagnosis of several diseases which cause developmental problems
E.g. the common aneuploidy syndromes: trisomy 13, 18 and 21

Chromosome specific fluorescent DNA probes are 99.6% informative
FISH method:

A fluorescent probe is incubated with the patient sample (interphase or metaphase spread) to allow
hybridisation

A fluorescent microscope is used to visualise the region where hybridisation has occurred
Elective late karyotyping: performed at ~32 weeks
Advantages
Disadvantages
No miscarriage risk
May cause iatrogenic prematurity (early birth)
Allows antenatal diagnosis and pregnancy termination
Termination of pregnancy is late
Describe the management options following prenatal diagnosis
A compromise must be made between the risk of the potential condition and the risk of the prenatal
diagnostic procedure
Abortion Act 1967: amended by the Human Fertility and Embryology Act 1990
Pregnancy can be terminated within 24 weeks under one of the following circumstances if it:
1.
Puts the life of the mother at risk
2.
Poses a risk to the mental and physical health of the pregnant woman
3.
Poses a risk to the mental and physical health of the foetus
4.
Shows there is evidence of extreme foetal abnormality i.e. the child would be seriously physically or
mentally handicapped after birth and during life
Management options:

Termination of pregnancy

Continuation of pregnancy:
The multidisciplinary team must: support the parents’ decision, offer continuous ultrasound
monitoring and make detailed plans for the following:

o
Mode of delivery
o
Monitoring in labour
o
Neonatal resuscitation
o
Post-mortem
o
Postnatal care: e.g. surgical, cardiac, neurological
Genetic counselling:
The multidisciplinary team must: calculate the risk of recurrence of a genetic disease, offer
management strategies for future pregnancies and detail implications related to other family
members
Describe the use of PCR for mutation detection with examples



PCR allows one to amplify a specific, small region of the genome from a patient. This DNA can then be analysed
easily for mutations.
Advantages: you start with very little DNA - down to 1 cell. Very fast analysis within 1 day. Can be automated.
Disadvantage: easy to get contamination. Need to know the precise region you are interested in - you can only
amplify about 2 kb at a time.
Detection of a CFTR mutation by PCR




Allele-specific PCR, also known as amplification refractory mutation system or ARMS test can be used to detect any
known point mutation.
Use different primers to recognise the normal and mutant sequences.
Over 1000 different CFTR mutations have been identified so it is extremely difficult to test for all of them.
Generally test for about 5 mutations which make up 80% of all cases.
PCR detection of trisomy 21



Use PCR to amplify microsatellites on chromosome 21.
If there are three alleles, clearly trisomy 21 is present.
If there are two different allele sizes, they would be in a 1:1 ratio in a diploid cell, but a 2:1 ratio in a cell with a
trisomy 21.
Genetics 5: Genetics of Childhood Diseases
Explain the underlying defects for phenylketonuria and other diseases in the same biochemical
pathway
‘One gene-one enzyme concept’:

Metabolic processes are a sequence of linked steps

Each step is controlled by a specific enzyme

Each enzyme is coded for by a gene
Phenylketonuria (PKU): autosomal recessive disorder of amino acid metabolism
Cause:

Deficiency of phenylalanine hydroxylase (PAH)

PAH: catalyses conversion of phenylalanine  tyrosine
Symptoms:

Phenylalanine accumulates: it is converted into phenylpyruvic acid which is excreted in the urine

Tyrosine is deficient: it is necessary for melanin formation (tyrosine  DOPA  melanin)

Reduction in melanin formation: affected children have a fair complexion (often blond hair and blue
eyes)

Untreated children have severe mental retardation and epilepsy (they often have convulsions)
Treatment:

Restrict phenylalanine in the diet
N.B. phenylalanine is an essential amino acid and cannot be entirely removed from the diet

Monitor the blood level of phenylalanine to provide a sufficient dietary quantity of phenylalanine

If PKU is detected in early infancy mental retardation (and epilepsy) can be prevented by restricting
dietary phenylalanine
Diagnosis:

Guthrie test (performed in the 1st week of life): tests for elevated blood phenylalanine levels
Related disorders:

Hypothyroidism: tyrosine  thyroxine

Alkaptonuria: (tyrosine ) homogentisic acid (in urine)  acetoacetic acid ( CO2 + H2O)

Albinism: (tyrosine ) DOPA  melanin
Describe the clinical features and genetic defects in common disorders of carbohydrate metabolism
Galactosaemia: autosomal recessive disorder of monosaccharide metabolism

Cause: deficiency of galactose 1-phosphate uridyl transferase
Galactose 1-phosphate uridyl transferase is necessary for galactose metabolism

Symptoms: vomiting, lethargy, failure to thrive and jaundice in the 2nd week of life

Complications: mental retardation, cataracts and cirrhosis of the liver

Diagnosis: screen for the presence of galactose in the urine

Treatment: early diagnosis and feeding infants with milk substitutes which lack galactose and lactose
prevents complications
Hereditary fructose intolerance: autosomal recessive disorder of monosaccharide metabolism

Cause: deficiency of fructose 1-phosphate aldolase

Dietary fructose is present in honey, fruit and certain vegetables and with glucose in cane sugar

Symptoms: range from minimal to failure to thrive, vomiting, jaundice and convulsions
Symptoms present at different ages depending on when fructose is introduced into the diet

Diagnosis: screen for the presence of fructose in the urine; enzyme assay on an intestinal mucosal or
liver biopsy sample

Treatment: dietary restriction of fructose conveys a good long-term prognosis
Describe the clinical features and genetic defects in common disorders of steroid metabolism
Congenital adrenal hyperplasia (CAH): autosomal recessive disorder of steroid metabolism

Cause: deficiency of 21-hydroxylase (90% of patients), 11ß-hydroxylase or 3ß-dehydrogenase

Symptoms: ambiguous genitalia; lack of cortisol and aldosterone and excess androgen
In newborn females: virilisation of the external genitalia (pseudohermaphroditism) occurs
In adolescent males: the increase in androgen production stimulates growth of the phallus
¼ of infants have the salt-losing form of CAH: circulatory collapse occurs in the 2nd week of life

Complications: gender identity and assignment of gender are problematic; there may be damaging
psychological effects

Treatment: replacement cortisol (and fludrocortisone for the salt-losing form); steroid replacement
must be increased during periods of stress (e.g. surgery) or recurrent illness
Virilised females: may require plastic surgery
Describe the clinical features and genetic defects in common sphingolipidoses
Sphingolipidoses (lipid storage diseases): inability to degrade sphingolipid, resulting in progressive
deposition of lipid/glycolipids primarily in the brain, liver and spleen
Tay-Sachs disease: autosomal recessive sphingolipidosis

Cause: deficiency of the α subunit of ß-hexosaminidase
This leads to accumulation of GM2 ganglioside

Incidence: 1 in 3600 among Ashkenazi Jews

Symptoms:
By 6 months: poor feeding, lethargy and floppiness
6-12 months: lack of developmental milestones (developmental regression)
Progressive symptoms: feeding becomes difficult, deafness, visual impairment and spasticity
Death often occurs in the 3rd year due to respiratory infection

Diagnosis: reduced serum levels of hexosaminidase A
Gaucher disease:

Diagnosis: reduced glucosylceramide ß-glucosidase activity in white blood cells

Symptoms:
Type I (adult onset): febrile episodes; limb/joint/trunk pain; a tendency to pathologic fractures;
enlarged liver and spleen; mild anaemia
Type II (infantile onset): failure to thrive and hepatosplenomegaly (3-6 months); developmental
regression, neurological deterioration, spasticity and fits (by 6 months); death in the 2nd year

Treatment for type I: symptomatic pain relief; removal of enlarged spleen (since it causes a secondary
anaemia due to hypersplenism)
Describe the clinical features and genetic defects in common urea cycle disorders
Urea cycle: occurs in liver cells

5 step metabolic pathway that removes waste nitrogen from the amino groups of amino acids

Ammonia + bicarbonate + 2 ATP  urea

Enzyme deficiencies in the urea cycle lead to protein intolerance due to accumulation of ammonia in
the body

Elevated ammonia levels are toxic to the CNS and may lead to coma or death
Urea cycle disorders: all autosomal recessive, except OTC deficiency (X-linked)

CPSI deficiency

OTC deficiency

Citrullinaemia: ASS deficiency

Argininosuccinic aciduria: ASL deficiency

Hyperargininaemia: ARG deficiency
Explain the classification of congenital defects
Malformation: a primary structural defect of an organ or tissue which results from an inherent abnormality
in development

E.g. congenital heart abnormalities (ventricular or atrial septal defects), cleft lip and neural tube
defects (anencephaly)

Malformations involving a single organ generally show multifactorial inheritance: i.e. the defect is
caused by the interaction of several genes with other factors
Disruption: a secondary structural defect of an organ or tissue which results from external factors
disturbing the normal developmental process

E.g. amputation of digits when an amniotic band/strand becomes entwined around the digits

Extrinsic factors that can disrupt normal development include: ischemia, infection and trauma

A disruption is not genetic by definition; genetic factors may predispose individuals to disruption
Deformation: a defect involving distortion of a structure resulting from abnormal mechanical force

E.g. hip dislocation and mild ‘positional’ talipes (‘club foot’)

Causes may include: lack of amniotic fluid (oligohydramnios) or intra-uterine crowding due to
twinning or a structurally abnormal uterus

Deformations occur late in pregnancy and convey a good prognosis with appropriate treatment since
the underlying organ is otherwise normal in structure
Dysplasia: a defect involving abnormal of organisation of cells into tissue

E.g. skeletal dysplasias: thanatophoric dysplasia is caused by a mutation in FGFR3
This results in short flat bones, a small thorax and a large head

Main cause: single-gene defect

Effects are usually observed in all parts of the body in which the defective tissue is present

There is a high recurrence risk for siblings and/or offspring
Sequence: a pattern of multiple abnormalities which result from a single primary factor

E.g. Potter sequence: chronic leakage of amniotic fluid or defective urinary output causes
oligohydramnios
This causes foetal compression which results in squashed facial features, hip dislocation, talipes and
pulmonary hypoplasia
This usually results in early neonatal death from respiratory failure
Syndrome: a consistent pattern of multiple abnormalities which have a known underlying cause

E.g. Down syndrome

Underlying causes: chromosome abnormalities or single-gene defects
Association: the non-random occurrence of multiple abnormalities which cannot be explained by a
sequence or syndrome

There is a lack of consistency of abnormalities between individuals

The underlying cause is generally unknown: it is not usually genetic

There is a low recurrence risk for siblings and/or offspring

E.g. VATER association: Vertebral, Anal, Tracheo-Esophageal and Renal abnormalities
Explain how non-genetic factors lead to congenital abnormalities
Genetic causes of congenital abnormalities:

Chromosome abnormalities (6%): caused by an abnormality in chromosome number (loss/gain of
chromosome material)

Single-gene defects (7.5%): caused by a point mutation within a single gene

Multifactorial inheritance (20-30%)
Neural tube defects (NTDs): e.g. spina bifida and anencephaly

Most NTDs show multifactorial inheritance

Cause: defective closure of the developing neural tube during the 1st month of embryonic life

Severe symptoms

Recurrence risk: 4-5% in first-degree relatives in areas where NTDs are common

Incidence in the UK is highest in Celtic populations (they may have an adverse susceptibility gene)

No single susceptibility genes have been identified for NTDs

Polymorphisms in the MTHFR (methylene tetrahydrofolate reductase) gene: may be a susceptibility
factor for NTDs

Digenic inheritance: an interaction between PAX1 mutations and Pdgfra mutations causes severe
NTDs in 100% of double mutant embryos in mice
Environmental factors:
Teratogen: an environmental agent which can cause a congenital defect by interfering with normal
embryonic/foetal development

E.g. rubella; thalidomide
Potential teratogenic effects of a drug depend upon: timing of drug administration and dosage
Low-grade teratogens are difficult to detect since:

There is a relatively small number of reported cases

There is a relatively high background incidence of congenital defects

Many pregnant women take medication at some stage during pregnancy
Exhaustive tests are performed before drugs are approved for use by pregnant women

Agents which are teratogenic during pregnancy include: vitamin A, alcohol, lithium, tetracycline,
warfarin and streptomycin
Thalidomide:

Formerly used as a sedative

Associated with severe limb abnormalities (phocomelia) in babies whose mothers had taken
thalidomide during the 1st trimester

Critical period for foetal damage: 20-35 days after conception

40% of thalidomide babies died in early infancy due to severe internal abnormalities of the heart,
kidneys or GI tract
Maternal infections:
Rubella:

Damages 15-25% of infected pregnancies if the mother is infected during the 1st trimester

Symptoms: cardiac defects, cataracts, retinitis, microcephaly

Prevention of congenital rubella infection: widespread immunisation by administration of the MMR
vaccine in early childhood or the rubella vaccine to adolescent women
Cytomegalovirus (CMV) retinitis:

Damages 5% of infected pregnancies

No immunisation is available against CMV

Symptoms: eye defects, deafness, microcephaly
Toxoplasmosis:

Maternal infection during the 1st trimester: 20% risk of foetal infection

Maternal infection during the 2nd and 3rd trimesters: 75% risk of foetal infection

No vaccines are available against toxoplasmosis

Symptoms: eye defects, deafness, microcephaly, hydrocephalus
Genetics 6: Genetic Services within the NHS
Describe the aims, methods and practice of genetic counselling
Clinical genetics consists of a variety of specialties, for example:

Prenatal diagnosis

Cancer genetics

Dysmorphology: recognition of disorders by looking at clinical symptoms

Neurogenetics…
Genetic counselling is a branch of clinical genetics
Genetic counselling: patients or relatives at risk of developing or transmitting a (potentially) hereditary
disorder are advised of:

Consequences of the disorder

Probability of developing or transmitting the disorder

Ways to prevent or ameliorate the disorder
Examples:

A couple who has had a child with a serious disorder must consider the recurrence risk of the disorder
in future children

Individuals with a family history of a serious disorder must consider the likelihood of developing the
disorder themselves or transmitting it to future generations
Common theme in genetic counselling: communication and education which addresses concerns related
to the development and/or transmission of a hereditary disorder
Steps in genetic counselling:
1.
Diagnosis: involves establishing a medical diagnosis, prognosis and possible treatment
2.
Risk assessment: involves looking at the mode of inheritance of the disorder and the risk of developing
and/or transmitting it
3.
Discussion of options: involves discussing the options available for dealing with the risks
4.
Communication
5.
Long-term contact and support
Communication and support: important to allow the individuals concerned to reach their own fully
informed decisions
1. Establishing the diagnosis:
Establishing an accurate diagnosis involves:
1.
Taking a history (family history and medical history)
2.
Performing a full examination
3.
Undertaking appropriate investigations (including chromosome and molecular studies)
2. Calculating and presenting the risk:
This involves:

Quantification: calculation of the numerical value of the risk

Qualification: conveying the full nature of the risk (i.e. the severity and long-term burden of a disorder)

Placing risks in context: this allows the individuals concerned to independently decide the extent of a
risk (i.e. if the risk is high or low)
3. Discussing the options:
The counsellor must ensure that the individuals concerned are provided with all of the information
necessary for them to make an independent informed decision

Ignore the risk: if the risk is low/moderate or if the condition is treatable

Have no (further) children

Adoption

Artificial insemination using donor sperm (AID)

Use of donor ova

Pre-implantation genetic diagnosis

Prenatal diagnosis followed by selective termination of pregnancy
Consanguinity:
Consanguineous relationship: occurs between blood relatives who have at least one common ancestor no
more remote than a great-great grandparent
Consanguinity presents a problem in genetic counselling: there is an increased risk for an autosomal
recessive disorder in future offspring

In the offspring of consanguineous marriages: there is an increased incidence of congenital
abnormalities and late-onset conditions (i.e. conditions that present later in life)

In the offspring of first cousins: the incidence of congenital abnormalities is twice that in the general
population (incidence ~3%)
Interpretation of standard genetics laboratory reports (cytogenetic and molecular genetic)
Parameters governing population genetic screening, current screening programmes and guidelines
for the introduction of such screening programmes
Criteria for a screening programme:
Disease: should have

A high incidence in a target population (i.e. the disease should be sufficiently common)

Potentially serious effects on health that are amenable to prevention or amelioration (treatment)
Test: should be

Accurate and reliable

Highly sensitive
Sensitivity: the proportion of cases that are detected
Measurement: determining the proportion of false negative results

Highly specific:
Specificity: the extent to which the test detects only affected individuals
Measurement: determining the proportion of false positive results
Programme: should

Be offered in a fair and equitable manner and be widely available

Involve voluntary participation

Be morally acceptable to a substantial proportion of the target population to which it is offered

Provide readily-available information and counselling

Be relatively inexpensive
Screening: involves prenatal screening, neonatal screening and population carrier screening
Prenatal screening: (see Genetics 4)

Neural tube defects: screening by assay of maternal serum α-fetoprotein and by ultrasound scans

Trisomy 21: screening by maternal age, biochemical markers in maternal serum and increased nuchal
fold translucency in ultrasound scans
Neonatal screening:

Widespread newborn screening programmes have been introduced for: PKU, galactosaemia and
congenital hypothyroidism
Early treatment of these disorders prevents the development of learning difficulties

Guthrie test: a blood test which is carried out at 7 days to identify ~10 conditions including PKU

Duchenne muscular dystrophy: widespread screening is still not available

Cystic fibrosis: national screening is available; early diagnosis allows early treatment which leads to an
improved prognosis
Population carrier screening:

Involves widespread screening for carriers of autosomal recessive disorders in high-incidence
populations
Disorder
Ethnic group with high incidence
α-Thalassaemia
China and eastern Asia
ß-Thalassaemia
Indian subcontinent and Mediterranean countries
Sickle-cell anaemia
Afro-Caribbeans
Tay-Sachs disease
Ashkenazi Jews
Cystic fibrosis
Western European Caucasians
Summary of genetic services within the NHS:
Genetic services within the NHS cover a large geographical area
There are ~22 genetic service centres within the UK

Regional Genetics Services

Clinical and laboratory services

Teaching/university hospital services
Genetics 7: Population Genetics
Describe allele frequencies in a population based on the Hardy-Weinberg Equilibrium
Hardy-Weinberg principle: the relative frequencies of the different genotypes remain constant from one
generation to the next
E.g. in an ‘ideal’ population: there is an autosomal locus with 2 alleles, A and a, which have frequencies of p
and q respectively (before reading this example think about what frequency actually means)

p+q=1
The frequency of each genotype can be determined by a Punnett square:
Male gametes
Female gametes
A

A
a
(p)
(q)
AA
Aa
2
(p)
(p )
(pq)
a
Aa
aa
(q)
(pq)
(q2)
p2 + 2pq + q2 = 1
For an autosomal recessive disorder:

2pq: carrier frequency

q2: frequency of affected homozygotes
Hardy-Weinberg equilibrium: the maintenance of allele frequencies in a large randomly mating
population in which there is no disturbance by external factors

I.e. dominant traits do not increase at the expense of recessive traits

A population is in Hardy-Weinberg equilibrium for a particular genotype if the relative frequencies of
each genotype remain constant with frequencies of p2, 2pq and q2

Population: a group of individuals present in the same area

Allele frequency: the relative number of alleles at a particular locus within a population

External factors disturb the Hardy-Weinberg equilibrium by changing the allele frequencies
o
Selection for or against a particular genotype
o
Mutation
o
Migration (gene flow)
o
Small population size
o
Non-random mating: proceeds by 2 mechanisms (1) assortative mating and (2) consanguinity
Mutation:

If a particular locus shows a high mutation rate: there is a steady increase in the frequency of mutant
alleles

The introduction of mutant alleles is balanced by the loss of mutant alleles due to reduced
reproductive fitness of affected individuals

For a population in Hardy-Weinberg equilibrium: these two opposing factors have ~ equal effects
Explain founder and migration effects in a population
Migration: gene flow

Migration and subsequent intermarriage introduce new alleles into a population

This leads to a change in the allele frequencies

Gene flow: the slow diffusion of alleles across a racial or geographical boundary
E.g. B blood group allele: originated in Asia and slowly spread west
Small population size: population isolation

Random genetic drift: in a small isolated population a particular allele may be transmitted to a high
proportion of offspring by chance

This leads to a marked change in allele frequency from one generation to the next
Founder effects:

A small group of people create a new isolated population

Some individuals of the group are carriers of a defective allele

The defective allele may become established at high frequency in the population

Several rare autosomal recessive disorders have a high incidence in small populations due to a founder
effect combined with genetic isolation

E.g. Hopi Indians in Arizona: high incidence of albinism
Affected males do not do farming due to susceptibility to bright light; they stay indoors
They have greater opportunity for reproductive activity in the absence of unaffected peers
Therefore the relative frequency of the defective allele increases
Explain consanguinity and assortative mating
Non-random mating: may lead to an increase in the relative frequency of affected homozygotes
Assortative mating:

The tendency for individuals to chose partners who share characteristics (e.g. height, intelligence,
racial origin)

E.g. if there is assortative mating among individuals with autosomal recessive deafness, there will be a
small increase in the relative frequency of affected homozygotes
Consanguinity:

Marriage between blood relatives who have at least one common ancestor no more remote than a
great-great-grandparent

Consanguinity leads to an increase in the relative frequency of affected homozygotes and a decrease
in the relative frequency of heterozygotes
Explain how selection affects disease occurrence; using two examples explain the basis of
heterozygous advantage
Fitness (biological/reproductive): the ability of an individual to survive and produce fertile offspring

Individuals with advantageous characteristics have increased fitness and are selected for

Individuals with deleterious characteristics have reduced fitness and are selected against
Negative selection:

Individuals with deleterious characteristics have reduced reproductive fitness (i.e. they produce less
offspring compared with unaffected control members of the same population)

Negative selection: this leads to a gradual decrease in the frequency of the defective allele and hence
a reduction in the frequency of affected homozygotes
Heterozygote advantage:

Carriers of a recessive disease allele have slightly increased fitness compared with unaffected
homozygotes

Serious autosomal recessive disorders have a high incidence in large populations due to a
heterozygote advantage
E.g. 1: Sickle-cell anaemia:

Affected homozygotes (sickle-cell disease): severely anaemic; persistent ill-health

Heterozygotes (sickle-cell trait): immune to malarial infection because their red blood cells undergo
sickling and are destroyed if they are invaded by the parasite

In areas where malaria is endemic: heterozygotes have increased biological fitness compared with
unaffected homozygotes

Therefore the relative frequency of heterozygotes tends to increase
E.g. 2: Cystic fibrosis:

The disease is lethal before reproductive age and affected males are sterile

Therefore a gruel decline in the incidence of cystic fibrosis would be expected; this is not the case

Speculative advantage for carriers: resistance to a previously endemic gastrointestinal disease
Calculate the carrier frequency for a recessive disease
Estimation of the carrier frequency of an autosomal recessive disease in H-W equilibrium:

Incidence = frequency of affected homozygotes = q2

Carrier frequency ≈ 2×√q2
Calculation of the carrier frequency of an autosomal recessive disease in H-W equilibrium:

Incidence = frequency of affected homozygotes = q2

p+q=1

Carrier frequency = 2pq
Calculate the risk of having an affected child in a family where a relative has a known disease
Genetics 8: DNA Mutations and Genetic Diseases
Understand the different consequences of somatic and germline mutations
Mutation: a heritable change in the structure/amount of genetic material in an organism
Mutations are caused by mutagens or occur spontaneously

Mutagens: chemical/physical agents which greatly increase the mutation rate (e.g. ionising radiation,
certain chemicals)

Spontaneous mutations: occur as a result of errors in DNA replication and repair
Somatic mutations:

Occur in somatic cells

Somatic mutations are not inherited by the next generation

Synonymous/silent mutations: no phenotypic effect (i.e. the mutation does not cause a change in the
encoded polypeptide)

Non-synonymous: there is a phenotypic effect (i.e. the mutation causes a change in the encoded
polypeptide)

Synonymous mutations occur much more frequently compared with non-synonymous mutations

Ageing: may occur due to the accumulation of somatic mutations

Cancer: occurs when a mutation stimulates uncontrollable cell division
Germline mutations:

Occur in germ cells/gametes during gamete formation

Germline mutations can be inherited by the next generation once established in the germline
They are inherited in a Mendelian fashion

Each individual carries a set of mutated genes
Understand the genetics of cystic fibrosis and Duchenne/Becker muscular dystrophy as examples of
autosomal recessive and X-linked recessive disease
Cystic fibrosis:
Genetics: autosomal recessive disease

High incidence in Western European Caucasians: 1 in 2000 to 1 in 3000

Most common autosomal recessive disease in children of Western European origin
This is most likely due to heterozygote advantage (increased heterozygote resistance to enteric
diseases)
Clinical features:

Commonly affected organs: lungs and pancreas

Chronic lung disease: caused by recurrent infection which eventually leads to lung failure and
secondary cardiac failure
Treatment: antibiotics and physiotherapy; heart-lung transplant (for lung and heart failure)

Impaired pancreatic function: reduced enzyme secretion due to blockage of pancreatic ducts
Treatment: oral supplements of pancreatic enzymes

Salty sweat

Sticky viscous secretions of the lungs, pancreas and other secretory organs

Meconium ileus: perinatal gut obstruction caused by thickening of the meconium

Male sterility: caused by congenital bilateral absence of the vas deferens

Average life expectancy: ~30 years
Mapping of the CF gene:
Linkage analysis: allows single-gene disorders to be mapped by studying the co-segregation of genetic
markers with the defective disease allele

The segregation of a disease is studied over several generations in large families with polymorphic
markers from each chromosome

Eventually a polymorphic marker will be identified which co-segregates with the disease allele more
often than would be expected by chance: i.e. the marker and the disease allele are linked
Molecular linkage analysis: lots of nearby markers are used to follow the segregation of a disease
This allows the gene responsible for any genetically-determined phenotype to be found since the marker
allele which segregates with the disease is positioned near the disease allele on the same chromosome
Markers used in molecular linkage analysis include:

Restriction fragment polymorphisms (RFPs)

Single nucleotide polymorphisms (SNPs)
CFTR protein:

CFTR (CF transmembrane conductance regulator) gene: codes for the CFTR protein

Locus of the CFTR gene: chromosome 7q31

The CFTR protein is an ATP Binding Cassette (ABC) transporter

Primary function of the CFTR protein: ATP-dependent chloride channel
Normal mode of action of the CFTR protein:

When CFTR proteins are activated by cAMP: chloride channels open and Cl- ions leave the cell

This stimulates closure of epithelial sodium channels and intracellular Na+ absorption is reduced

This reduces the intracellular NaCl concentration

Normal mucous is produced
Mode of action of the CFTR protein in CF:

The CFTR protein does not function

Therefore Cl- ions remain inside the cell

Na+ ions diffuse into the cell down the electrochemical gradient

The intracellular NaCl concentration increases; therefore water moves into the cell down the water
potential gradient

This results in the secretion of sticky mucous
Mutations in the CFTR gene: result in CF

There are over 1000 mutations in the CFTR gene which cause CF

Mutations in the CFTR gene are predominantly point mutations

Mutations include: missense, frameshift, splice-site, nonsense and deletion (rare)
Missense mutation: a point mutation which codes for a different amino acid
Nonsense mutation: a substitution which results in the generation of a stop codon
Frameshift mutation: a mutation (insertion/deletion of 1 or more bases) which changes the reading
frame of the codon triplets

Mutations in the CFTR gene reduce the functional activity of CFTR proteins

In heterozygotes: 1 copy of the CFTR gene is mutated
Phenotypic effect: there is no disease phenotype since enough CFTR proteins are made for normal cell
function

In homozygotes/compound heterozygotes: both copies of the CFTR gene are mutated
Phenotypic effect: CF symptoms are present since the mutation significantly reduces normal CFTR
protein activity; therefore ion transport is disrupted

o
Homozygote: the mutations on both alleles are identical
o
Compound heterozygote: the mutations on each allele are different
Different classes of CFTR gene mutations have different effects on CFTR gene expression (i.e.
different phenotypic severities)
Phenotypic severity: depends on the functional activity of CFTR proteins

CFTR protein activity:
< 3%: severe CF (characterised by male sterility, lung disease and impaired pancreatic function)
3-8%: mild CF (characterised by male sterility and lung disease)
8-12%: mildest CF (characterised by male sterility)

∆F508: the most common CFTR gene mutation; it is caused by a missense mutation
It prevents the CFTR protein from being transported from the ER to the cytoplasmic membrane
Homozygotes for the ∆F508 mutation have severe CF
Clinical applications and future prospects:

Prenatal diagnosis is offered for CF

No curative treatment is available for CF yet

Symptomatic treatment: supplements of pancreatic enzymes; physiotherapy; antibiotics; heart-lung
transplant

Experimental treatment: drugs which traffic the ∆F508 CFTR protein, activate residual mutated
protein and inhibit Na+ reabsorption; gene therapy
Functional effects of mutations on a protein:
Loss-of-function mutations:
Consequences: either reduced activity of the protein or complete loss of the protein
Haploinsufficiency mutations: loss-of-function mutations in the heterozygous state in which half normal
levels of the protein result in phenotypic effects; result in autosomal dominant disorders
Gain-of-function mutations:
Consequences: either increased levels of gene expression or development of a new function(s) of the
protein (e.g. toxicity)
Gain-of-function mutations: result in dominant disorders
Huntington disease: autosomal dominant disease
Symptoms: late onset, neurodegenerative disease which leads to premature death
The mutation in Huntington disease: trinucleotide expansion

Cause: expansion of a CAG (polyglutamine) repeat sequence (i.e. large CAG repeat sequences)

CAG codon: codes for glutamine

CAG trinucleotide repeat sequence: codes for huntingtin

Huntingtin: expressed in cells of the CNS; unknown function

Expansion of the CAG repeat sequence: results in aggregation of huntingtin in the CNS

Anticipation: expansion of the triplet repeat sequence increases in subsequent generations
Therefore autosomal dominant diseases tend to manifest at an earlier age and/or increase in severity
in subsequent generations

Repeat expansion: causes the age of onset to decrease and penetrance to increase
I.e. the longer the CAG repeat sequence, the earlier the onset of disease and the more likely the onset
of disease
(Other diseases involving repeat expansion include: fragile X, fragile 16 and myotonic dystrophy)
Duchenne muscular dystrophy:
Clinical features: severe progressive myodegenerative disease

At birth: clinically normal

At 3-5 years: slowly progressive muscle weakness- difficulty in running and climbing stairs

At 11 years: most patients have to use a wheelchair due to severe proximal muscle weakness

Average life expectancy: ~18 years

Cause of death: cardio-respiratory failure due to intercostal and diaphragm muscle weakness and due
to respiratory infection

Prognosis: respiratory care and assisted respiration allow survival into late 20s

Becker muscular dystrophy (BMD): milder condition than DMD; age of onset ~11 years; much longer
life expectancy
Muscle histology in DMD-affected individuals:

There is a variation in the size of muscle fibres

Necrosis of muscle fibres occurs followed by phagocytosis of necrotic (dead) fibres

In early stages: active regeneration of muscle occurs to replace/repair lost/damaged fibres

In later stages: the replication capacity of muscle stem cells is exhausted; muscle fibres are replaced by
fat and connective tissue (pseudohypertrophy)
Genetics: X-linked recessive disease

Incidence of DMD: 1 in 3500
DMD gene:

DMD (or dystrophin) gene: codes for dystrophin; largest known human gene

Locus of the DMD gene: Xp21

Predominantly expressed by 3 promoters in skeletal and heart muscle and in the brain
Dystrophin:

Size: 427 kDa

Location: at the cytoplasmic side of the muscle membrane

Structure: dimer composed of 4 domains (actin-binding domain, central and rod domain, cysteine-rich
domain and –COOH terminal domain)

Function: links intracellular actin with extracellular laminin via dystroglycan (DGC)
Mutations in the DMD gene: result in DMD or BMD

Mutation rate: 1 in 10,000; 1/3 of DMD cases are caused by a new mutation

2/3 of mutations: result from deletions of all or part of the DMD gene
Deletions (and duplications) arise due to unequal crossing over during maternal meiosis

1/3 of mutations: result from point mutations
Point mutations arise due to an error in DNA replication during paternal meiosis

Deletions in DMD/BMD: whole exons are removed and the flanking exons are spliced together;
therefore a functional protein may be produced if there is an in-frame fusion of exons
DMD mutations: deletions disturb the translational reading frame; this results in complete absence of
dystrophin or production of non-functional dystrophin
BMD mutations: deletions do not alter the reading frame since there is an in-frame fusion of exons;
there is a reduction in dystrophin production which has residual function
Understand how different mutations are associated with different disease severities in both diseases
Understand the concept of X-inactivation
Male and female cells have the same level of expression of X-linked genes because each female cell has
one active and one inactive copy of the X chromosome
X-inactivation:

Occurs during embryonic development (~1000 cells)

One X-chromosome is inactivated in each cell: this exists as a Barr body

Thereafter the same X chromosome remains inactivated in all subsequent daughter cells

X-inactivation is usually random: i.e. there is an equal chance of either of the two X chromosomes in
the cells of a heterozygous female being inactivated

Skewed inactivation: abnormal X chromosomes are usually preferentially inactivated
In manifesting heterozygotes: X-inactivation is skewed towards the normal X chromosome
Clinical manifestation: carrier females who are manifesting heterozygotes show symptoms of the
disorder
Homozygous females: XmXm

Generally show the same phenotype severity as affected males
Female carriers: XmX

Female carriers are usually asymptomatic since there is either random inactivation or skewing of Xinactivation towards the abnormal X chromosome
E.g. haemophilia A and B; enzyme deficiency diseases (G6PDH deficiency, OTC deficiency)

Manifesting heterozygotes: show the disease phenotype since there is skewing of X-inactivation
towards the normal X chromosome
Female-DMD carriers:

Random inactivation: the normal X chromosome is inactivated in ~50% of cells
Muscle fibres are multinucleate; therefore there is no clinical phenotype as long as 1 nucleus expresses
the normal dystrophin gene

Manifesting heterozygotes: the active X chromosome in most of the cells is an abnormal X
chromosome; therefore clinical manifestation occurs
Understand the importance of genetics for diagnosis and treatment of genetic disease and disease
caused by genetic factors
Genetics 9: The Genetics of Complex Traits
Monogenic inheritance: controlled by a single gene

Single-gene disorders: result from 1 mutated gene

All affected individuals have the mutated gene

The gene product is either defective or absent
Polygenic inheritance: controlled by many genes

Phenotype expression is determined by many genes (polygenes) at different loci

Each gene involved exerts a small additive effect: i.e. the effects of the genes are cumulative

Polygenes generate a normal distribution (e.g. height, body mass, IQ)

Normal distribution: a symmetrical bell-shaped curve distributed evenly about a mean

Polygenic inheritance is the genetic component of multifactorial inheritance
Multifactorial inheritance: controlled by the combined effects of many genes (polygenes) and
environmental factors

Multifactorial disorders: not caused by a single gene

Threshold effect: there is a non-linear relationship between the number of genes and expression of
the disease phenotype
I.e. a threshold exists above which the abnormal phenotype is expressed

Low penetrance: genetically predisposed individuals may not actually be affected by the disorder

Epistasis: genes at different loci interact with each other to amplify the genetic signal

Multiple genes give the potential for a continuously variable phenotype since the phenotype is
determined by the interaction between genes and the environment
Disease susceptibility genes:

Disease susceptibility genes: polymorphic alleles which may function slightly differently

Genes promote susceptibility to a disease; they do not code for a defective protein

Combination of susceptibility genes gives predisposition to a disease
Predisposition is caused by the number of interacting genes, not by a specific combination of genes

Environmental factors trigger the disease (e.g. viral infection, diet)

Disease may develop only on exposure to certain environmental factors (e.g. smoking)
Demonstration of the genetic component of a complex trait:
Population/migration studies:
Migrants moving from a population with low incidence of a particular disease to one with a high incidence:

If the incidence of the disease in the migrant group rises: environmental factors are more significant to
the cause of disease

If low incidence of the disease is maintained in the migrant group: genetic factors are more significant
to the cause of disease
Family studies:
If there is a higher frequency of disease in relatives of an affected individual than in the general population,
this suggests that there is genetic susceptibility to the disease
Families share a common environment; therefore family studies cannot prove genetic susceptibility
λs: the ratio of the risk to siblings of patients to the population incidence

The greater the λs value, the greater the contribution of genetic susceptibility to the disease
Twin studies:
Comparison of the frequency of a disease between monozygotic (identical) and dizygotic (non-identical)
twins:
Concordance: either both members or neither member of a pair of twins is affected

If monozygotic twins have higher concordance rates than dizygotic twins: genetic factors are more
significant to the cause of disease

If monozygotic and dizygotic twins have similar concordance rates: environmental factors are more
significant to the cause of disease
Adoption studies:
Comparison of the frequency of a disease in individuals who remain with their biological parents with
those who are adopted out of their biological family

If frequency of disease is in adopted individuals is similar to their biological parents: genetic factors are
more significant to the cause of disease

If frequency of disease in adopted individuals is similar to their adoptive parents: environmental
factors are more significant to the cause of disease
Association studies: (case-control study)
Comparison of the frequency of a particular polymorphic allele in an affected patient population with that
of a healthy, control population:
If the frequency of the polymorphism is significantly different in the 2 populations: this provides evidence
for a positive or negative association
Candidate gene: a gene which is thought to play a role in the disease
Factors which influence choice of the candidate gene:

Function

Location

Expression
The control population must be carefully selected to control for the affected patient population: this allows
the difference in allele frequency in the 2 populations to be attributed to disease
Control factors include:

Age

Sex

Ethnicity

Environment
E.g. HLA histocompatibility complex on chromosome 6:

There is a strong HLA association between ankylosing spondylitis and the B27 allele

98% of patients with ankylosing spondylitis have the B27 allele vs. 5% of controls

Therefore the following inferences can be drawn:
o
There is a strong association between having the B27 allele and developing ankylosing
spondylitis; however…
o
The B27 allele is not an absolute requirement for developing ankylosing spondylitis
o
The B27 allele does not cause disease on it is own
Autoimmune disease: involves development of an immune response against the ‘self-antigen’

The immune response cannot be resolved since the self-antigens are always present

Therefore there is a continuous immune response (which leads to inflammation)

Many autoimmune diseases are linked to a particular locus

Therefore family history of a disease is not only relevant for that particular disease since another
autoimmune disease may be linked to the same locus as the disease which has a family history