Jig's Genetics

Lecture 1: Meiosis and Recombination 1) Explain the different stages of meiosis I • 22 pairs of autosomes • 1 pair of sex chromosomes; females are XX, males XY • Each pair of homologous chromosomes align together and form a bivalent.

• DNA is exchanged between the homologues (genetic recombination).

• Homologous chromosomes separate (segregate).

• The resulting cells have just one of each chromosome, but there are still two chromatids.

• The number of chromosomes has halved; this is sometimes called the reduction division.

2) Explain what occurs at meiosis II • Effectively the same as mitosis.

• The two chromatids of each chromosome separate to the daughter cells.

• Overall, meiosis starts with diploid cells and results in haploid cells which form the gametes; the eggs and the sperm.

• The X and Y chromosomes form a bivalent in meiosis I so the resulting cells have either an X or a Y chromosome.

3) Draw a diagram of meiosis showing segregation of two pairs of chromosomes

4) Explain how recombination of chromosomes occurs • • •

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Process in which homologous chromosomes are broken (chiasmata/crossovers in meiosis) at the same place and rejoined to give new combinations of alleles Recombination fraction: a measure of distance separating two loci, or more precisely an indication of the likelihood that a cross-over will occur between them If θ = 0.05, this means that on average the synthetic alleles will segregate together 19 times out of 20. If two loci are not linked then θ = 0.5, as on average genes at unlinked loci will segregate together during 50% of meioses

Lecture 2: Polymorphism and Inheritance of Variation 1) Define a polymorphism • A polymorphism is a DNA variation present in a population at a frequency of greater than 1%.

• Most polymorphisms do not affect a gene and are silent- they are in the introns not the exons.

• Mutations are variations of DNA occurring at a frequency of less than 1% in a population.

2) Describe variation in humans • Genetic diversity comes about due to: (a) chiasmata formation (crossing over which allows random exchange of genetic material between homologous chromosomes; (b) independent segregation of homologous chromosomes.

3) Define genetic markers • Markers are “FLAGS” in the genome • We can see them and know where they are and hence follow regions of the genome through meiosis from parent to offspring • They enable “INHERITANCE” to be studied Ideal markers: • Highly Polymorphic - Informative for many individuals • Randomly Distributed - Non clustered at telomeres/centromeres • Easily Assayable - Simple technique, little time • Automation - Too much to do • Stability - Change through generations Types of Markers • Genes • Microsatellites o Short sequence repeats (2-4bp) o Highly polymorphic, random, assayable, unstable • Minisatellites o Large repeat sequences (500bp ) o Highly polymorphic, non-random, assayable?, unstable • Single Nucleotide Polymorphisms (most used) o Single base changes e.g. C to T o Polymorphic (2-3 alleles), random, assayable, stable 4) Describe how markers are used to investigate variation • Linkage: non random association of markers with disease (greater than 50% chance) 5) Explain how markers are used to follow inheritance • Genetic Linkage – association is heritable often because of physical linkage – markers are close together on chromosome • Define model to explain inheritance in pedigree • Test model – LOD score/recombination • Powerful technique – most monogenic disorders – map disease • Model may be wrong > miss linkage/misdefine linkage • Phenotype definition critical (Psychiatric disorders) • •

There are about 30,000 genes in the genome any one of which could be responsible for CF. Researchers took hundreds of genetic markers (microsatellites or RFLPs) scattered through the genome and looked at their inheritance in about 300 families with CF. For each genetic marker they determined what the recombination fraction was and the LOD score for this θ. With about 300 markers spread throughout the genome, you should be able to find neighbouring ones linked to the CF disease.

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You should be able to narrow down the region with the CF gene using more markers and more families to a few cM. You can now look for mutations in all the genes in the region and hopefully identify the gene responsible for CF.

6) Draw a pedigree

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Affected male

Normal male Normal female Mating

Affected female Propositus

Consanguineous mating Parents with son and daughter in order of birth Dizygotic twins

Heterozygous for autosomal gene Carrier X-linked recessive gene Dead

Monozygotic twins Sex unspecified Number of children of sex indicated Generations in roman numerals and within generations by arabic numerals. Eg III4

Abortion or stillbirth or unspecified sex

Female with children by two males

Zygosity uncertain

7) Describe recessive, dominant, and X-linked inheritance Recessive • Need mutations in both copies of the gene to show the phenotype. Heterozygous carriers are not affected. A is the dominant wild type allele, a is the recessive disease allele.

• Pattern of inheritance in a family: - Affects males and females in equal proportions - Usually affects individuals in one generation in a single sibship - 1/4 children affected - Often does not affect previous and subsequent generation because these have non-symptomatic carriers.

- Consanguinity in the parents is frequent • e.g. cystic fibrosis – is caused by a mutation in the CFTR gene this encodes a chloride channel in the lungs, gut and many other tissues. (chromosome 7)- if you have a mutation in one copy, the other copy of the gene produces enough protein to give normal phenotype. However if you have 2 mutant copies of the gene, you make no CFTR protein and have cystic fibrosis Dominant • Just one copy of the mutant gene causes the disease. A is the dominant disease causing allele, a the recessive wild type allele.

• Pattern of inheritance in a family: - Affects males and females in equal proportion - Vertical transmission - Disease present in every generation - 50% of offspring affected - Variable expressivity (variable severity) - Reduced penetrance (some carriers not affected) - New mutations frequently observed E.g. Oestogenesis imperfecta is caused by mutations in the collagen gene. Collagen is made up of a triple chain of collagen protein molecules. If one mutant version of the protein is incorporated then the structure of the collagen triplet is destroyed X-Linked • Females are XX, males are XY, but females and males have the same level of X-linked gene expression • Each cell in a female has one active and one inactive copy of the X chromosome. The genes on the active X are expressed normally so there is the same amount of protein in females as in males with one X and one Y. In the development of the embryo, both X chromosomes in the female are active, however after about the 1000 cell stage one of them is inactivated, therefore if for example the mutant gene is inactivated then the female is unaffected.

• Duchene muscular dystrophy is an example of an X-linked inheritance. Here, the cells, which are not expressing normal DMD, survive with DMD protein made by neighbouring cells.

• Pattern of inheritance in a family: - Affects males almost exclusively - Transmitted from carrier females to their sons - An affected male can have affected grandsons through his daughters who are carriers - No transmission from affected males to sons - Variable expression in heterozygous females 8) Describe co-dominant inheritance • When both alleles are expressed in the heterozygote.

• e.g. blood types: - An O individual is homozygous for the O allele - An AB individual is heterozygous for the A and B alleles

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An A individual is either Heterzygous for A and O alleles; or homozygous for the A allele.

A B individual is either heterozygous for B and O alleles; or homozygous for the B allele.

Lecture 3: Aneuploidy and Other Chromosome Aberrations 1) Describe the normal karyotype, chromosome banding and nomenclature • 46 chromosomes, 22 pairs of autosomes, 1 pair of sex chromosomes (diploid) • Somatic cells are diploid, ova/sperm (gametes) have 23 chromosomes (haploid) • G (Giemsa)-banding used to identify chromosomes • The bands are labelled according to chromosome number; long (q) and short (p) arm; and distance from centromere • Acrocentric chromosomes have satellite short arms containing genes for ribosomal RNA (13, 14, 15, 21, 22)

2) Draw a diagram of a balanced translocation and explain why are these generally not deleterious?

Chromosome aberrations: • Structural – translocations, deletions, insertions, inversions, rings • Numerical – aneuploidy, loss or gain • Mosaicism – different cell lines •

Chromosome aberrations cause:1. 60% of early spontaneous miscarriages 2. 4-5% still births 3. 7.5% of all conceptions, 0.6% of live births •

Balanced meaning that two pieces of chromosome are exchanged. The person in genetically normal because the DNA of both chromosomes is present (translocation of somatic cells is frequently associated with cancer).

3) Draw a diagram showing possible meiotic products from someone with a balanced translocation

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As we can see from above gametes from a balanced translocation may have an unbalanced set of chromosomes.

Reciprocal translocation is where there are breaks in 2 chromosomes, where segments are exchanged to form 2 new derivative chromosomes • Robertsonian translocation is where there are breaks at the centromere of 2 acrocentric chromosomes (ones with satellites), with fusion of the long arms to form 1 new derivative.

4) Describe how 3 different chromosome aberrations lead to Down’s syndrome. Why is this important to know in terms of genetic counselling?

Anueploidy – Numerical abnormalities involving the loss or gain of one or more chromosomes • Monosomy - loss of a single chromosome is almost always lethal • Trisomy - gain of one chromosome can be tolerated • Tetrasomy - gain of two chromosomes can be tolerated • •

95% of Down’s cases are as a result of the trisomy 21 as a result of non-disjunction during maternal meiosis I. Due to: - Probably ageing effect on primary oocyte.

- Age-related reduction in immunological competence allows survival of trisomic embryos.

- Radiation - Delayed fertilisation after ovulation - Genetic control – drosophila

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Translocation is the cause of 3% of Down’s cases. There is a high risk of further Down’s babies.

Due to: - 1/3 of parents are carriers of translocation - 2/3 de novo translocation in child - Robertsonian - breakage of acrocentric chromosomes (13,14,15,21,22) and fusion of their long arms (1:1000 incidence) - No effect in parent - loss of short arm OK - High risk of further Down’s babies - 13q21q and 14q21q - 10% risk of Down’s - 21q21q - all offspring will have Down’s

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Mosaicism is the cause of 2% of Down’s cases. Children are less severely affected.

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Down’s syndrome: 5) Describe 2 common autosomal and 2 common sex chromosome aneuploidies.

• Autosomal: Trisomy 13- Patau’s syndrome, Trisomy 18 - Edward’s syndrome - Both usually result in death in 1st few weeks of life.

- 1:3000-5000 incidence.

- Bilateral cleft lip and palate.

- 90% cardiac abnormalities.

- Mental retardation if longer term survival.

- 10% cases due to translocations/mosaicism.

• Sex chromosomal: • Kleinfelter’s syndrome (47, XXY) - 1 in 1000 male live births.

- Clumsiness, mild verbal learning disability (verbal IQ reduced by 10-20 points).

- Taller than average (long lower limbs).

- 30% - moderately severe gynaecomastia.

- All infertile.

- Increased risk of leg ulcers, osteoporosis and breast carcinoma in adult life.

- ‘X’ chromosome from either male or female.

• Turner’s syndrome (45, X) - 1 in 5000 - 10,000 live female births - Generalised oedema and swelling in neck region can be detected in 2nd trimester.

- Can look normal at birth or have puffy extremities and intra-uterine oedema.

- Low posterior hairline, short 4th metacarpals, widely spaced nipples, aorta defect in 15% of cases.

- Normal intelligence.

- short stature - 145 cm without GH treatment - ovarian failure - primary amenorrhoea and infertility - treatment -oestrogen replacement for secondary sexual characteristics and prevention of osteoporosis - 80% due to loss of X or Y chromosome in paternal meiosis - ring chromosome, 1 arm deletion, mosaicism, isochromosome 6) Describe why sex determination is not solely based on sex chromosome karyotype.

• It is possible to have both XX males and XY females.

• XX males result from the translocation of the SRY male determining gene from the Y to the X chromosome. Phenotypically males, testes develop, but sterile because some genes on the Y chromosome are needed for spermatogenesis.

• XY females result from deletion of the SRY gene leading to females who are infertile.

Lecture 4: Prenatal diagnosis of genetic disease Indications for prenatal testing: • High Risk of Aneuploidy o High risk on Down Syndrome screening o Previous aneuploid fetus o Maternal request eg.Age • Known Genetic Disorder o Achondroplasia o Cystic Fibrosis o Haemoglobinopathies o X Linked disorder o Parental Balanced Translocation

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• Structural Anomaly detected in Fetus on Routine Ultrasound Screening 1) Describe the use of non-invasive tests – maternal serum screening and ultrasound.

Maternal serum screening • Looking for abnormal protein levels in mother’s serum.

• Carry out test at 16 weeks.

• Maternal serum α-fetoprotein (MSAFP), low for Down’s and high when the foetus has a neural tube defect such as open spina bifida.

• MSAFP alone identifies about 40% of Down’s syndrome pregnancies.

• The Triple test also measures unconjugated estriol and human chorionic gonadotrophin and together these identify about 70% of cases with a false positive rate of 5%.

• Follow up with ultrasound and invasive tests for DNA or chromosome analysis. NO DNA TEST FOR SPINA BIFIDA ONLY DOWN’S. • Advanced maternal age is associated with increased risk of having a child with Down’s syndrome. Often offer an amniocentesis from about 37 years onward.

Ultrasound • Looking for abnormal structure of the fetus.

• Can be used not only for obstetric indications, such as placental localisation and the diagnosis of multiple pregnancies, but also for the prenatal diagnosis of structural abnormalities which are not associated with known chromosomal, biochemical or molecular defects. Conveys no risk to the mother or foetus but it does require specialised equipment and a skilled and experienced operator.

• For Down’s syndrome, detailed ultrasound scan of the nuchal fold can identify about 70% of cases.

• Open spina bifida and anencephaly can be recognized with ultrasound.

• Maternal serum testing and ultrasound cannot detect most genetic diseases - need to assay the foetal DNA. 2) Describe the use of invasive tests – amniocentesis, chorionic villus sampling/fetoscopy.

Amniocentesis- 0.5%-1% risk of miscarriage.

• Involves the aspiration of 10-20ml of amniotic fluid through the abdominal wall under ultrasound guidance.

• This is usually performed around the 16th week of gestation.

• The sample is spun down to yield a pellet of cells and supernatant fluid.

• The fluid can be used in the prenatal diagnosis of neural tube defects by assay of α-fetoprotein.

• The cells can be cultured and used for chromosome analysis.

Chorionic villus sampling- 2%-3% risk of miscarriage.

• Enables prenatal diagnosis to be undertaken during the 1st trimester.

• Usually carried out at 10-12 weeks gestation under ultrasound guidance by either transcervical or transabdominal aspiration of chorionic villi.

• These are foetal in origin being derived from the outer cell layer of the blastocyst, i.e. the trophoblast.

• Chromosome analysis can be undertaken on chorionic villi either directly, looking at metaphase spreads from actively dividing cells, or following culture.

• Direct chromosome analysis of chorionic villi usually allows a provisional result to be given within 24 hours.

Fetoscopy • Involves visualisation of the foetus by means of an endoscope.

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Undertaken during the 2nd trimester to try to detect the presence of subtle structural abnormalities pointing to a serious underlying diagnosis.

• Has also been used to obtain tissue samples from the foetus which can be analysed as a means of achieving the prenatal diagnosis of several rare disorders.

3) Describe the use of karyotype analysis • Collect cells and prepare spreads of metaphase chromosomes.

• Uses G-banding.

• Can detect any large (>5Mb) rearrangement of DNA without any prior knowledge of the defect e.g. Down’s, any other aneuploidy, translocation or deletion.

• Need to culture samples from an amniocentesis or chorionic villus sample for 10-12 days to obtain enough cells in metaphase.

4) Describe the use of FISH detection of chromosomal abnormalities with examples.

• Based on the unique ability of a portion of single stranded DNA, i.e. a probe, to anneal with its complementary target sequence wherever it is located on a metaphase spread.

• Unlike most other methods used for chromosome analysis, FISH (Fluorescent in situ hybridisation) can also be used to study chromosomes in cells which are resting between cell division in interphase.

• The DNA probe is conjugated with modified nucleotides which, after hybridisation with the patient’s sample, allows the region where hybridisation has occurred to be visualised under UV light.

• Has the major advantage that the results can be obtained quickly.

• Both Angelman’s syndrome and Prader-Will syndrome can be detected using this technique.

5) 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.

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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.

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If there are three alleles, clearly trisomy 21 is present.

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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.

Lecture 5: Genetics of childhood diseases 1) Explain the underlying defects for phenylketonuria and other diseases in the same biochemical pathway.

• Children with PKU, if untreated, are severely mentally retarded and often have convulsions.

• In PKU, the particular enzyme necessary for the conversion of phenylalanine to tyrosine, phenylalanine hydroxylase (PAH), is deficient. In other words, there is a ‘genetic block’ in the metabolic pathway.

• PKU was the first genetic disorder in humans shown to be caused by a specific enzyme deficiency. As a result of the enzyme defect, phenylalanine accumulates and is converted to phenylpyruvic acid and other metabolites which are excreted in the urine.

The enzyme block leads to a deficiency of tyrosine with a consequent reduction in melanin formation.

• Affected children therefore often have blond hair and blue eyes. An obvious method of treating children with PKU would be to replace the missing enzyme but this cannot be done simply by any conventional means of treatment.

• Removal of phenylalanine from the diet has proved to be an effective treatment.

• If PKU is detected early enough in infancy, mental retardation can be prevented by giving a special diet containing a restricted amount of phenylalanine (e.g. avoid sweetener aspartame) • Phenylalanine is an essential amino acid and therefore cannot be entirely removed from the diet.

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2) Describe the molecular basis of 2 disorders of carbohydrate metabolism.

• Galactosaemia • Autosomal recessive disorder due to deficiency of the enzyme galactose-1-phosphate uridyl transferase, which is necessary for the metabolism of the dietary sugar galactose.

• Newborn infants with galactosaemia present with vomiting, lethargy, failure to thrive and jaundice in the second week of life. If untreated, they go on to develop complications which include mental retardation, cataracts and cirrhosis of the liver.

• Galactosaemia can be screened for by the presence of reducing substances in the urine with specific testing for galactose. The complications of galactosaemia can be prevented by early diagnosis and feeding of affected infants with milk substitutes which do not contain galactose or lactose which is broken down to galactose.

• Early diagnosis and treatment are essential if the severe complications are to be prevented.

• Hereditary fructose intolerance • Autosomal recessive disorder due to deficiency of the enzyme fructose-1-phosphate adolase.

• Dietary fructose is present in honey, fruit and certain vegetables and in combination with glucose in cane sugar.

• Individuals with hereditary fructose intolerance present at different ages, depending on when fructose in introduced into the diet.

• Symptoms can be minimal but can be as sever as those seen in galactosaemia which include failure to thrive, vomiting, jaundice and convulsions.

• Diagnosis is confirmed by the presence of fructose in the urine and enzyme assay on an intestinal mucosal or a liver biopsy sample.

• Dietary restriction of fructose is associated with a good long term prognosis.

3) Explain the underlying defects causing disorders of steroid metabolism.

• Congenital adrenal hyperplasia (adrenogenital syndrome) • The diagnosis of CAH should be considered in any newborn female infant presenting with virilization of the external genitalia as this is the commonest cause of ambiguous genitalia in female newborns.

• 21-hydroxylase deficiency accounts for over 90% of cases.

• About ¼ of affected infants will have the salt-losing form, presenting in the 2nd week of life with circulatory collapse.

• Less commonly CAH is due to deficiency of the enzymes 11β-hydroxylase or 3β-dehydrogenase and very rarely occurs as a result of deficiencies of enzymes 17α-hydroxylase and 17,20-lyase.

• Virilization of the external genitalia is caused by an accumulation of the adrenocortical steroids proximal to the enzyme block in the steroid biosynthetic pathway, which may have testosterone-like activity. The possibility of CAH should not be forgotten in male infants presenting with circulatory collapse in the 1st few weeks of life.

• Affected infants, in addition to requiring urgent correct assignment of gender, are treated with replacement cortisol, along with fludrocortisone if they have the salt-losing form.

• Virilized females may require plastic surgery in due course.

• Steroid replacement is lifelong and needs to be increased during intercurrent illness or times of stress, e.g. surgery.

4) Describe the clinical characteristics in common sphingolipidoses.

• An inability to degrade sphingolipid, resulting in the progressive deposition of lipid or glycolipid primarily in the brain, liver and spleen.

• CNS involvement results in progressive mental deterioration, often with fits, usually resulting in death in childhood.

• Tay-Sachs disease • Affects 1 in 3600 persons of Ashkenazi-Jewish ancestry. Infants usually present at 6 months with poor feeding, lethargy and floppiness. Loss of developmental milestones or developmental regression will usually become apparent in the 2nd half of the 1st year. Feeding becomes increasingly difficult and the infant progressively deteriorates with deafness, visual impairment and spasticity which progresses to rigidity. Death usually occurs by 3 years of age as a result of respiratory infection. Less severe juvenile, adult and chronic forms are also reported.

• Biochemical confirmation of Tay-Sachs disease is by demonstration of reduced hexosaminidase A levels in serum, white blood cells or cultured fibroblasts. Reduced hexosaminidase A activity is due to deficiency of the α subunit of the enzyme βhexosaminidase which leads to the accumulation of the sphingolipid, GM2 ganglioside.

• Gaucher’s disease • Commonest sphingolipidoses and occurs with increased frequency among persons of Ashkenazi-Jewish ancestry. Two main types based on the age of onset: • Type I (Adult type) • Most common form of Gaucher’s disease. Present with febrile episodes and limb, joint or trunk pain and a tendency to pathological fractures. Clinical examination usually reveals an enlarged spleen and liver. Affected persons often show mild anaemia.

• Type II (Infantile) • CNS is a major feature. Present usually between 3-6 mths of age with failure to thrive and hepatosplenomegaly. By 6mths they begin to show developmental regression and neurological deterioration with spasticity and fits leading on to recurrent pulmonary infection and death in the 2nd year.

• Diagnosis is confirmed by reduced activity of the enzyme glucosylceramide β-glucosidase in white blood cells or cultured fibroblasts. Treatment involves symptomatic relief of pain. Often necessary to remove the large spleen because it causes secondary anaemia due to premature sequestering of red blood cells (hypersplenism).

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6) Explain the classification of congenital defects.

• Malformation – primary structural defect of an organ e.g. atrial septal defects, cleft lip. Usually involves a single organ showing multifactorial inheritance.

• Disruption – secondary abnormal structure of an organ or tissue e.g. amniotic band causing digital amputation. Caused by ischaemia, infection, trauma. Not genetic, but genetic factors can predispose.

• Deformation – abnormal mechanical force distorting a structure e.g. club foot, hip dislocation. Occur late in pregnancy and convey good prognosis since organ is normal in structure.

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Dysplasia – abnormal organisation of cells into tissue e.g. thanatophoric dysplasia, ectodermal dysplasia. Caused by single gene defects, high recurrence risk for siblings/offspring.

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Sequence – multiple abnormalities initiated by primary factor e.g. leakage of amniotic fluid leads to Potter sequence. Could have genetic component as initial factor.

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Syndrome – consistent pattern of abnormalities for specific underlying cause e.g. Down’s syndrome. Caused by single gene defects or chromosome abnormalities.

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Association – non-random occurrence of abnormalities not explained by syndrome e.g. VATER association. Not usually genetic.

Classification is not mutually exclusive e.g. a primary malformation of kidneys can lead to same sequence of events as Potters syndrome – risk estimates therefore a problem.

7) Explain how non-genetic factors lead to congenital abnormalities.

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Teratogen – agent which interferes with normal embryonic or foetal development e.g. rubella virus and thalidomide .

Thalidomide – used as a sedative. Limb abnormalities (phocomelia) occurred when used during foetal period 20-35 days postconception. 40% of babies died in early infancy of internal abnormalities affecting heart, kidney, and GI tract.

Vitamin A, alcohol, lithium, tetracycline, warfarin, streptomycin are all teratogenic during pregnancy in humans.

Maternal infections Rubella – 15-25% babies damaged if mother gets infection during first trimester. Widespread immunization programmes in childhood or adulthood. Microcephaly, eye, heart defects.

CMV – 5% of pregnancies affected. No immunization available. Eye defects, deafness, and microcephaly Toxoplasmosis – 20% (1st trimester) -75% (2nd and 3rd trimesters) risk to foetus. No vaccines. Microcephaly, eye defects, deafness, hydrocephalus.

Lecture 6: Population genetics 1) Describe allele frequencies in a population based on the Hardy-Weinberg Equilibrium (H-WE).

• A gene locus has 2 alleles A and a . These alleles have frequencies of p and q. Therefore p + q = 1 (100%) • •

Allele frequencies and genotype frequencies are constant from generation to generation in a population that is:1. large and randomly mating 2. with no selection, mutation or migration Most autosomal loci in most populations follow HWL (diseases and polymorphisms)

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2) Calculate the carrier frequency for a recessive disease.

• Cystic fibrosis affects 1 in 2000 children born in populations of North European origin.

• What is the carrier frequency for this disease?

• We know that p+q = 1, carrier frequency = 2pq, • q2 is frequency of recessive disease • 1/2000 = q2, therefore q = 0.022 • p = 1-q, therefore p = 1 - 0.22 = 0.978 • 2pq = 2 x 0.978 x 0.022 = 0.043 or 1/23 3) Calculate the risk of having an affected child in a family where a relative has a known disease.

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Female patient has 2/3 chance of being a carrier of either allele from her brother.

Her cousin has 1/8 chance of inheriting allele. However, he also has the population chance of being a carrier.

(2/3 x 1/4 x 1/4) Mo Fa ch

+ (2/3 x pop chance x Mo Fa [1/10,000]

1/4 ) ch

= 0.041 + 0.0033 = 1/23 chance of affected child 5) Explain the effect of mutation, selection and consanguinity on H-WE. • When the allele frequencies in a population predict the genotype frequencies in that population in accordance with the expectations of H-WL, that population is said to be in H-WE except when there is :1. selection 2. migration 3. population isolation 4. founder effects 5. assortative mating 6. consanguinity 7. mutation Mutation • If a particular locus shows a high mutation rate (or a generation is exposed to excess of mutagens e.g. radiation), it will lead to an increase in proportion of mutant alleles in the population.

• This is balanced by reduced fitness of affected individuals.

• A population in H-WE is assumed to have equal opposing factors.

Selection • Decreased reproductive fitness. Deleterious genotype resulting in reduced ability to reproduce. Leads to gradual reduction in frequency of the mutant gene and disturbs HWE. • Increasing reproductive fitness. Carriers of recessive disease alleles show a slight increase in biological fitness compared to unaffected homozygotes (heterozygous advantage).

Consanguinity • Marriage between blood relatives (at least one common ancestor no more remote than a great-great-grandparent, i.e. they share more than 1/32 genes).

• Recessive traits are more frequent in the progeny of consanguineous mating.

6) Explain how founder effects and migration affect H-WE. Founder effects • Occurs when a small group of people, which include carriers of a genetic disorder, create a new population, so that either the carrier status is disproportionate, e.g. rare disorders which occur at high frequency.

• Hopi Indians in Arizona have high incidence of albinism. Thus males did not do outdoor farming because of susceptibility to bright light - providing them with reproductive activity in absence of unaffected peers.

Migration • Alleles introduced into the population by migration and subsequent intermarriage will change allele frequencies.

• Slow diffusion of alleles across a racial or geographic boundary is known as gene flow.

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Examples include B blood group which originated in Asia and DF508 CF mutation/a1-antityrpsin deficiency with highest incidence in Scandinavia.

7) Explain how population isolation and assortative mating effect on H-WE. Population isolation • Refers to populations or subgroups which are (more or less) separated reproductively by geography or culture.

• One mutant allele might be transmitted to a high proportion of offspring, thus disturbing H-WE • .

Assortative mating • Tendency for humans to choose partners who share characteristics such as height, intelligence and racial origin.

• e.g. deaf children go to deaf schools and may marry deaf partners (as they need to communicate) - leads to a small increase in affected homozygotes.

• e.g. red hair might negative assortative mating • .

8) Using two examples explain the basis of heterozygous advantage.

Sickle cell anaemia • Affected homozygotes have severe anaemia.

• Heterozygotes are immune to malaria because if RBCs are infected they undergo sickling and are rapidly destroyed.

• Where malaria is endemic, carriers of sickle cell trait are at an advantage over unaffected homozygotes.

• There will be a tendency for frequency of heterozygotes to increase - affects H-WE. Cystic fibrosis • Since the disease is either lethal before reproductive age and males are sterile you might expect the disease to die out. This is not the case. We do not know what the advantage is for CF carriers, but perhaps it was resistance to a previously endemic diarrhoeal disease.

Lecture 7: DNA Mutations and Genetic Disease 1) Describe the difference consequences of somatic and germline mutations.

• Mutations are continuously occurring in DNA due to radiation, chemical damage, and mistakes in replication. If a mutation occurs in the germline it can be inherited • Mutations which occur in the DNA of somatic cells are not inherited by the next generation. Some such mutations will kill the cell in which they occur or have no effect. Ageing may be largely due to the accumulation of mutations in somatic cells. Cancer occurs when a mutation allows a cell to divide more frequently or start to mutate DNA. 2) Describe how nonsense, missense, and frameshift mutations can cause cystic fibrosis.

• CF is the most common lethal autosomal recessive diease in Caucasians, affecting about 1/2500 children.

• The CF transmembrane regulator gene encodes a chloride protein channel located in the membrane of the secretory cells. The loss of one copy of the gene allows enough channel protein to be made for normal function of the cells, therefore no disease.

However, if both copies of the gene are mutated, then ion transport is disrupted leading to thick mucus and symptoms of CF • The CFTR gene is very large, spans 200 kb and has 27 exons. Nearly 1000 different mutations have been found in the CF gene.

Nearly all nonsense, missense or frameshift mutations. (nonsense= sequence change to include a stop codon; missense= codes for wrong amino acid; frameshift= addition or deletion of one or more bases which puts code out of frame) The most common (70%) is the missense mutation deltaF508 (Δ for deletion and F for phenylalanine). The mutant protein is trapped in the Golgi apparatus and not transported to the surface of the cells.

3) Describe the spectrum of mutations causing Duchenne muscular dystrophy.

• Presents between 3 and 5 years with slowly progressive muscle weakness. Most have to use a wheel chair by the age of 11 and death on average by 18 years.

• It is X-linked recessive inheritance affecting 1 in 3,500 males, female carriers usually show no phenotype. Gene isolated in 1987 at Xp21.

• The DMD gene encodes a 427KDa protein called dystrophin, it is encoded by the largest known human gene covering 2.3 mbases of genomic DNA • As the male rarely reaches reproductive age, new mutations must match the copies lost in affected boy. The mutation rate is a third of the incidence of affected males, giving a mutation rate of about 1/10000 – one of the highest known mutation rates known in the human. 1/3 of mutations are stop, frameshift, splicing and promoter mutations. 2/3 of mutations are due to deletions of all or part of the gene. The large size of the gene and the high rate of mutations account for the high mutation frequency.

• Milder Becker muscular dystrophy (1 in 20,000 males) is caused by mutations in the same gene - deletion of only part of the protein, allowing the production of a partially functional protein.

4) Understand the concepts of X-inactivation • Female cells are XX, male cells are XY • BUT male and female cells have the same level of expression of X-linked genes, • BECAUSE each cell in a female has one active and one inactive copy of the X chromosome.

• During embryonic development, up to about 1000 cells, both X chromosomes in a female are expressed equally. At this point, one X-chromosome in each cell of a female is inactivated at random.

• During subsequent cell divisions, the same X chromosome remains inactive.

• All FEMALES have patches of cells with one of their X chromosomes inactivated at random.

• DMD carriers have inherited one DMD-X chromosome and one normal X chromosome. Random inactivation will result in about 50% of cells in which the normal X chromosome is inactivated.

• Muscle fibres are multinucleate; so as long as one nucleus is expressing the DMD protein, no clinical phenotype.

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• • • • •

If X-inactivation is skewed towards the normal X-chromosome clinical manifestation of varying degree may occur in female DMDcarriers.

Carriers in other X-linked Diseases: Generally no disease phenotype Haemophilia A and B: serum proteins, plenty made for the body by the normal cells.

Enzyme deficiencies: as long as enough cells express the enzyme, the phenotype is normal (e.g. G6PDH deficiency, OTC Deficiency).

5) Understand the importance of genetics for diagnosis and treatment of genetic disease and disease caused by genetic factors • Determine the molecular basis inherited diseases • Design drugs for treatment • Genetic testing • Prenatal testing • Gene therapy – insertion of genes in persons cells to treat disease

Lecture 8: Common disorders and multifactorial inheritance 1) Explain polygenic inheritance and the normal distribution.

Polygenic disease • Expression of a phenotype determined by many genes at different loci.

• Each gene involved exerts a small additive effect.

• Threshold exists above which the abnormal phenotype is expressed.

• No one gene is dominant or recessive.

• Heritability - proportion of total phenotypic variance caused by additive genetic variance.

Normal distribution •Some human characteristics show a continuous distribution across the population, resembling a normal (Gaussian) distribution e.g. blood pressure, height, intelligence.

2) Explain multifactorial inheritance – the liability/threshold model.

• Common diseases result from a complex interaction of the effects of multiple different genes, or what is known as polygenic inheritance, with environmental factors and influences, due to what is known as multifactorial inheritance.

• According to the liability/threshold model, all the factors which influence the development of a multifactorial disorder, whether genetic or environmental, can be considered as a single entity known as liability.

• The liabilities of all individuals in a population form a continuous variable, which has a normal distribution in both the general population and in relatives of affected individuals.

• Liability cannot be measured, but forms a continuous variable that has a normal distribution.

• There is a threshold which determines whether abnormal phenotype is expressed.

• However, the curves for these relatives will be shifted to the right, with the extent to which they are shifted being directly related to the closeness of their relationship to the affected index case.

• To account for a discontinuous phenotype with an underlying continuous distribution, it is proposed that a threshold exists above which the abnormal phenotype is expressed. In the general population the proportion beyond the threshold is the population incidence and among relatives the proportion beyond the threshold is the familial incidence.

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•

Greatest risk amongst close relatives of index case e.g spina bifida – 4% risk in 1st degree relative 1% risk in 2nd degree relative • Incidence greatest for relatives of severely affected patients e.g. cleft palate 6% risk in 1st degree relative if bilateral 2% risk in 1st degree relative if unilateral 3) Understand the concept of genetic susceptibility to common disease.

• Inheritance of a single gene mutation can be the main determinant of developing a disease, but it can be modified by environment (severity). Disease may only develop upon exposure to specific factors e.g. smoking. Disease may only develop upon exposure to specific factors e.g. smoking • Gene polymorphism can reduce disease e.g. ALDH1 isoenzyme –flushing reaction to alcohol – reduced alcohol-related liver disease.

• Response to drug treatment – isoniazid inactivation status in treatment of tuberculosis – slow inactivators have risk of side effects of drug • Predisposition to disease 4) Describe the approaches to demonstrate a complex genetic trait.

Population and immigration studies • Is the incidence of a disease different in different populations?

• Study a low incidence immigrant group moving to new group with high incidence.

- If incidence rises then more likely to be environmental.

- Maintenance of low incidence would suggest genetic factors are important.

Family studies • Higher frequency of disease in family relatives than in general population.

- However, families often share a common environment, so may not be genetic.

- Spouses (different genetic background) can serve as controls for environment - look at % of affected relatives of specific relationship e.g. 1o / 2o risks Twin studies • If disease is genetic then identical twins will be similarly affected, whereas non-identical twins will differ.

• To avoid environmental influences study identical twins who were raised apart (rare).

• If disease due to environment then identical and non-identical twins will be similarly affected.

Adoption studies • If frequency of disease in adopted individuals is similar to those with biological parents, then genetic factors important.

• If frequency of disease in adopted individuals is similar to that of adoptive parents then environment more important.

Biochemical studies • Alzheimer’s disease - amyloid deposits in neuronal plaques in the brain consist of APP (amyloid precursor protein).

• Hypertension – dietary salt a contributory factor – lower incidence of hypertension in low-salt users.

Polymorphism associations • • •

Depression (unipolar) – shared HLA alleles in sib-pairs Alzheimer's disease – E2 allele of ApoE confers decreased risk, whereas E4 allele most significant risk factor Schizophrenia – serotonin in brain autopsy, suggesting type 2a receptor involved

5) Identifying genes which cause multifactorial disorders.

Disease associations • Undertaken by comparing the incidence of particular polymorphism in affected patients with the incidence in a carefully matched control group – age, ethnicity, sex, and environment matched.

• If the incidences in the two groups differ significantly, this provides evidence for a positive or negative association.

Linkage analysis • Has proved extremely valuable in mapping single gene disorders by studying the cosegregation of polymorphisms with the disease until a linked marker has been identified. However this method is difficult because: 1. complex maths required 2. variable age of onset 3. informative meioses very small 4. genetic and environmental heterogeneity 6) Explain the genetic susceptibility to diabetes mellitus.

• Two main forms: Type 1 being the rarer juvenile onset insulin-dependent form (IDDM) showing a high incidence of potentially serious renal, retinal and vascular complications. Type 2 is the maturity onset insulin-dependent form and is relatively benign.

• Type 1 has been more closely researched due to its severity and associated complications. Observations point to a multifactorial aetiology with both environmental and genetic contributions. Known environmental factors include diet viral exposure in early childhood and certain drugs. The disease process involves irreversible destruction of insulin-producing islet β-cells in the pancreas by the body’s own immune system, probably as a result of an interaction between infection and an abnormal genetically programmed immune response.

IDDM1 • There are strong associations with the HLA region on chromosome 6p21. 95% of affected individuals have DR3 and/or DR4 alleles compared to 50% in general population. HLA contribution to IDDM susceptibility is determined by the 57th amino acid residue

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where aspartic acid conveys protection, in contrast to other alleles which increase susceptibility. As this was the first susceptibility locus determined for type 1 diabetes it was labelled IDDM1.

• Concordance rate - in monozygotic twins is 50% • Sibling recurrence risk - 6% greater than population IDDM2 • The next locus to be identified was that of the insulin gene on chromosome 11p15 where it was shown that variation in the number of tandem repeats of a 14bp sequence upstream to the gene influences disease susceptibility. Long repeats convey protection by increasing expression of the insulin gene in the foetal thymus gland thereby reducing the likelihood that insulin producing β-cells will be viewed as foreign by the mature immune system.

Lecture 9: Genetic services within the NHS 1) • • • • • • 2)

Describe the aims, methods and practice of genetic counselling What is wrong? (diagnosis) What will happen? (prognosis) Can it be treated? (therapy) Why has it happened? (aetiology) Will it happen again? (recurrence risk) Can it be prevented? (prenatal diagnosis) Parameters governing population genetic screening, current screening programs and guidelines for the introduction of such screening programmes

Disease • high incidence in target population • serious effect on health • treatable or preventable

Test • non-invasive and easily carried out • accurate and reliable • inexpensive

Programme • widespread and equitable availability • voluntary participation • acceptable to the target population • full information and counselling provided

Screening • Sensitivity – proportion of cases that are detected – measure false negatives • Specificity – the extent to which the test detects only affected individuals – measure false positives Prenatal • Neural tube defects o maternal serum aFP / USS • Trisomy 21 / other chromosome abnormalities o maternal age o biochemical markers in maternal serum (aFP, oestriol, hCG) o increased nuchal fold on USS Neonatal • Guthrie card o phenylketonuria o galactosaemia o hypothyroidism • Duchenne muscular dystrophy • Cystic fibrosis Population carrier screening – Thalassaemia; Sickle cell disease; Tay-Sachs disease; Cystic fibrosis

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