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1

Question

Answer

2

Lecture 1

Human Pedigrees.

3

What are consanguineous marB2:C42riages?

Marriages between siblings.

4

Describe are the characteristics of autosomal dominant inheritance.

50% chance of inheritance from heterozygous and recessive parents. Doesn't reflect allele frequencies in population - Mutation may be lethal.

5

Given example of an autosomal dominant disease.

Achondroplasia. Apert Syndrome.

6

What is Achondroplasia?

Dominant mutation. Dwarfism. Most individuals heterozygotic (homozygosity is lethal).

7

Describe the mutation that gives rise to Achondroplasia.

A mutation in the Fibroblast receptor 3 (FGFR3) at base 1138 (GGG=>AGG or sometimes CGG) changing glycine to arginine.

8

What's unusual about Achondroplasia?

All unaffected have the same base mutated.

9

What is Apert syndrome?

Autosomal dominant disorder causing malformations of the skull.

10

What causes Apert syndrome?

Mutation of the FGFR2 at 10q26 during spetmatogenesis. Correlation with Paternal age observed.

11

Why may Apert syndrome be dominant?

May provide a selective advantage at a gametic or genetic level.

12

What is the penetrance of Apert syndrome?

100%

13

What is the significance of Apert syndrome occurring in spermatogenesis?

Only someone of the sperm carry the mutation. This is called gonadal mosaicism.

14

Describe the characteristics of autosomal recessive inheritance.

Appears in progeny of unaffected parents. Males and females affected equally therefore not sex linked. Ratio in a small family may be unclear. Often present in the phenotype of offspring of family members.

15

Given example of autosomal recessive diseases.

Cystic fibrosis.

16

What are the symptoms of cystic fibrosis?

Increased mucus secretion in London pancreas. Salty sweat.

17

What causes cystic fibrosis?

Mutation in the CF transmembranal conductance regulator CFTR (chloride channel). 3 basepair deletion of phenylalanine at DeltaF508.

18

How is cystic fibrosis diagnosed?

Heel prick blood screening.

19

Describe the characteristics of X-linked dominant inheritance.

Affected Males passed on to their daughters but not their sons.

20

Give an example of an X linked dominant disease.

Hypophosphatemia.

21

What are the symptoms of Hypophosphatemia?

Growth impairment and childhood Ricketts.

22

What causes Hypophosphatemia?

Mutation in the PHEX gene at Xp22.

23

Why may heterozygous females no express Hypophosphatemia?

X inactivation (lyonization).

24

How is Hypophosphatemia treated? What is the effectiveness?

Vitamin D and oral phosphate. More effective in heterozygous females than affected males.

25

Describe the characteristics of X linked recessive inheritance.

More frequent in Males are rather than females. Females only inhert if both parents carry it. No male to male transmission. Heterozygote females unaffected unless healthy X is inactivated. Affected females may arise from a new germ line mutation in the father. Affected Males unable to reproduce.

26

Give an example of an X linked recessive disease.

Duchenne Muscular Dystrophy.

27

What are the symptoms of duchenne muscular dystrophy?

Severe progressive muscular atrophy.

28

What causes Duchenne muscular dystrophy?

Frame shift inducing deletions. Affect dystrophin protein.

29

What are the issues of X linked inheritance?

Lyonization makes it difficult to distinguish between dominant and recessive trait. The family pedigrees are usually small. Male lethality.

30

Give an example of male lethality.

Incontinentia Pigmenti.

31

What are the symptoms of Incontinentia Pigmenti?

Females showed abnormalities in pigmentation of skin which disappear by the age of 20 because of cell regeneration. Skewed X inactivation in females.

32

What causes Incontinentia Pigmenti?

Mutation in IKK gamma gene (NEMO) at Xq28.

33

Describe the characteristics of Y linked inheritance.

Male to male inheritance. Being male is Y linked (SRY - Testis determining factor). Male sterility is caused by deletions in sperm promoting genes therefore obviously not heritable.

34

Give an example of Y linked inheritance.

Hairy ears.

35

Describe the characteristics of mitochondrial inheritance.

Passed on from mother to daughter because mitochondria come from the egg cell.

36

Give an example of mitochondrial inheritance along with the gene responsible.

Diabetes mellitus. Deafness. tRNA(Leu)(UUR) gene.

37

What is locus heterogeneity?

When a single disorder is caused by mutations in genes at different chromosomal loci i.e. the same phenotype is elicited by mutations in different genes.

38

What is Complementation?

Mutations in different genes crossed causing heterozygous progeny to show the normal phenotype.

39

What is allelic heterogeneity?

Variation in the intensity of the phenotype between mutant alleles.

40

Give an example of allelic heterogeneity.

Duchenne muscular dystrophy -severe; Becker muscular dystrophy -mild.

41

How can mutations in the same gene elicit a different phenotype?

Mutation of the DNA binding or androgen binding site in the Androgen Receptor gene causes insensitivity (feminisation). Mutation of the CAG repeat the the spinal bulbar muscular atrophy (SBMA) (neurodegeneration).

42

What are PolyQ diseases?

Polyglutamine diseases. (Most triplet diseases).

43

What causes Myotonic dystrophy?

Triplet CTG expansion on 19q.

44

Describe the basis of severity of Myotonic dystrophy.

Severity is proportional to number of repeats. Age of onset decreases with decreasing number of repeats. Number of repeats increases with each generation. Affected individuals inherit 1 allele with normal number and 1 allele with expanded number.

45

What is non-penetrance?

Not all individuals express the phenotype. Usually caused by onset of phenotype later than final age of individual. Causes skipping of generations.

46

What causes variable expression? Give an example.

Epistatic effects or incomplete dominance of alleles. Waardenberg syndrome type 1.

47

Describe the features of genetic analysis in drosophila.

RF measured between genes. Inbred strains with known parental genotypes are analysed. Crosses controlled. Many offspring bred. Chi squared test applied.

48

Describe the features of genetic analysis in humans.

RF measured between markers. Outbred strains with unknown parental genotypes analysed. No mating control. Small pedigreed. Lodscore calculated.

49

What is genetic mapping?

Measuring recombination between disease gene and other polymorphic gene loci.

50

Why id protein polymorphism rare?

Proteins usually conserved to preserve function.

51

Give an example of polymorphic proteins.

Blood group antigens. HLA genes.

52

Describe how DNA polymorphic heterozygotes can be determined.

Electrophoresis of the genes extracted via specific endonucleases. Heterozygotes will form 2 distinct bands. Homozygotes will form a single concentrated band.

53

What is RFLP? What is it used for?

Restriction Fragment Length Polymorphism. Comparison of homologous molecules.

54

How is RFLP carried out?

DNA digested with specific restriction endonucleases. Fragments with mutations at restriction sites will be longer therefore form a new band on the agarose gel.

55

Lecture 2

Linkage Analysis in Humans.

56

What is Linkage?

Tendency of genes on a chromosome to be inherited dogether during meiosis.

57

Why may some genes be more likely to be linked?

Genes that are closer together have a smaller chance of having a crossing over event occuring between them therefore a greater chance of being co-inherited.

58

Name two types of DNA polymorphisms.

Restriction fragment length polymorphisms. Tandem repeat polymorphisms.

59

What is the percentage of informative individuals with restriction fragment length polymorphism? Why?

50%. That's the maximum recombination frequency.

60

What type of tandem repeats are commonly used?

Mini satellites. Micro satellites.

61

What are mini satellites?

Array of tandem repeats 0.5 to 40 KBP. Each repeat 10 to 100 base pairs.

62

How are mini satellites used for polymorphism analysis?

DNA digested by Hinf1. Detected by southern blot analysis of PCR.

63

What are the advantages of using mini satellites?

Most individuals heterozygous (~100%).

64

What are the disadvantages of using mini satellites?

Clustered towards the end of chromosomes therefore can't be used for internal loci.

65

What are micro satellites?

Dinucleotide repeats (GA).Randomly distributed in the genome.

66

How are micro satellites used for polymorphism analysis?

Isolated by screening genomic library with CA oligonucleotides. Analysed by PCR across array using a single fluorescence labelled primer.

67

What are the advantages of using micro satellites?

Some have high heterozygosity.

68

And what are the disadvantages of using micro satellites?

Cause stutter bands on PCR which show up as artefacts. These are byproducts of that are two base pairs shorter.

69

How are the disadvantages of using micro satellites overcome?

By using triplets or tetramer repeats.

70

What is the recombination factor?

Percentage of the recombinant progeny out of all progeny. Maximum is 50%.

71

Why is the maximum recombination factor 50%?

The chance that a daughter cell will have one of the alleles is 50% since there are only two alleles. Single and double cross overs between loci give an average of 50%.

72

What is the recombination factor used for?

Calculating distance between loci in the centi Morgans (cM).

73

What does one centi Morgan correspond to?

1% of recombination.

74

What is the total map length of the male genome? Explain why that is.

2450 centi Morgans. 49 chiasmata form per male meiosis event. 50% max recombinance. 49x50=2450.

75

Derive the map length of 1cM in base pairs.

3x10^9 / 2563 (average map length for both sexes) = ~1Mbp

76

What are the 3 requirements for genetic mapping (pedigree analysis)?

Family showing disease segregation over 3 generations. Polymorphic DNA markers giving a high probability of heterozygosity. At least one parent must be heterozygous at disease locus and at marker locus (AaBb).

77

What is linkage phase?

Co-inheritance of alleles in the same gamete. Opposite of recombination.

78

How does linkage phase arise?

When crossing over does not affect allele combination.

79

How is the chance of observing a specific combination of alleles calculated for unlinked loci?

Max RF to the power of the number of offspring (0.5^n).

80

How is the chance of observing a specific combination of alleles calculated for linked loci?

(1-RF)^non recombinant offspring x RF^recombinant offspring

81

How can odds ratio be applied to probabilities of combinations with linked and unlinked loci?

By calculating the ratio of chance of a specific combination of linked and unlinked loci.

82

How is lodscore calculated?

Z = Log10 of odds ratio; Z = log10{[(1-?)^s x ?^r)](0.5^n)}

83

How is the position of the disease locus determined from the lodscore?

Determined by the ? (RF) at which Z is the highest.

84

How does lodscore different if phase is known?

Z usually greater if phase known.

85

When may lodscore not be enough for accurate analysis?

If Z is doesn't show significance either for or against linkage.

86

What are the dsignificance borders of lodscore?

Z shows significance for linkage if above 3; significance against linkage if below -2.

87

What is the chance for linkage at lodscore of 3? Why?

1000:1. log10(1000) = 3.

88

What is the chance against linkage at lodscore of -2? Why?

100:1. log10(100) = 2/-2.

89

How can an insignificant lodscore be analysed further?

Using two-point linkage analysis.

90

How is two-point linkage analysis carried out?

By calculating the linkage between a disease gene and 2 or more polymorphic markers. Knowing that and the position of the markers, the gene can be mapped using RF as relative distance from locus.

91

What are the limitations of linkage analysis?

Requires a genetic model for the disease locus. Errors in data entry due to excess recombination. Not useful if locus heterogeneity exists. Limited resolution to 1cM (1Mb).

92

What was the goal of the Human Genome Project?

To create a complete genetic map based on polymorphic loci.

93

How was the HGP carried out?

40 families used for DNA samples including 8 three-generation pedigree falimies (total of 188 meioses). Skeletal map produced using 544 PCR markers (6.7cM apart) to establish genetic location of markers. Framework map produced using 1123 PCR markers (4.9cM apart) to fine-tune clocation.

94

Lecture 3

Genetic Maps and Genotyping.

95

What were the limitations of the Human Genome Project?

Low resolution - 4.9cM between markers.

96

Describe the advancements made on the Human Genome Project.

Icelandic DeCode Genetics provided a more comprehensive map. 146 families with 2-7 children (1257 meioses). 5136 STR loci used. 0.5-1.8cM between loci. Crossing-over 0.1-3cM apart.

97

What are STRs?

Short Tandem Repeats.

98

Why is the order of markers on physical maps different?

Chromosomal Inversions.

99

What are the problems faced by chromosomal mapping?

Female maps 1.65x longer because the recombination frequency in females is greater by a ratio of 1.65. Expansion of map towards the end of chromosomes.

100

What may be the evolutionary ramifications of the difference in recombination rate?

If recombination drives evolution, females would contribute to it more than males.

101

What is a haplotype?

Combination of alleles at linked loci which can be broken by recombination.

102

What are haploypes used for?

Mapping genes with locus heterogeneity.

103

How does linkage equilibrium occur?

reached if haplotypes are present at the expected frequency in the population.

104

How does linkage disequilibrium occur?

When there is a skew in the observed vs. expected frequency of haplotypes.

105

How do new haplotypes arise?

Via mutation of existing haplotypes. Recombination may also give rise to new haplotypes but only after initial mutation.

106

What factors affect establishment of linkage equilibrium?

Recombinance Factor. Number of meiosis. Number of generations. Distance between loci.

107

What is LD used for? How?

Genetic mapping - loci that are close together show LD.

108

Describe the expected inheritance pattern of 2 loci in LD.

Inherited together. LD indicated inheritance from a common ancestor.

109

How is LD calculated?

D = P(AB) - (P(A) x P(B)); P(AB) - Observed, P(A) and P(B) - Expected.

110

What is D'? What does it represent?

Maximum value of D. D'=0 - No LD.; D'=-1 or 1 - Complete LD.

111

What factors influence LD?

Recombination and gene conversion decrease LD; Recurrent mutations increase LD; Genetic drift increases LD; Migration increases LD; Natural selection increases LD.

112

Describe the general pattern of recombination.

Occurs at preferential sites - hotspots. 40% of chr12 hotspots contain CNNCNNTNNCCNC motif.

113

How can hotspots be visualised?

Haplotype maps are used to visualise LD breakdown sites - hotspots.

114

Describe the process of SNP genotyping.

Red/Green pigmented antibodies attached to complimentary nucleotides of a particular SNP are used. Heterozygous SNPs show up yellow (R+G); Homozygous SNPs show up red or green.

115

What was the goal of the HapMap Project?

Creating a haplotype map of the human genome.

116

What can the HapMap project be used for?

Studying patterns in human variation. Locating alleles associated with health.

117

How can disease loci be located using HapMap?

Compare haplotypes of diseased individuals to determine responsible linked loci. Compare recombinants to determine which of the linked loci is disease-causing. If the other linked locus shows a distinct phenotype - may be used for preventive diagnosis (e.g.: locus for diabetes linked with purple hair).

118

What is coupling phase?

Dominant alleles of linked loci are all on 1 chromosome. Recessive alleles on the homologue (Cis).

119

What is repulsion phase?

Each homologous chromosome has one dominant and one recessive allele from the two loci (Trans).

120

What is PRDM9 gene?

Gene coding for a transcription factor PR domain zinc finger protein 9.

121

What are the functions of PRDM9?

N-terminal KRAB (Kruppel-associated box). Central PR/SET domain (histone methyl transferase). C-terminal zinc finger domain (Allows binding of proteins to DNA).

122

What is the importance of PRDM9?

Activation of hotspots in meiosis by the zinc finger domain (absence causes arrest at pachytene stage of prophase therefore sterility).

123

Why does the hotspot activation vary?

Different alleles of PRDM9 which have variant zinc finger domains, cause different patterns of hotspot activation.

124

What is synteny?

Co-inheritance of loci on the same chromosome.

125

Describe an example of syntenic genomes.

Human and mouse genomes are of a similar size (conservation). Coding sequences almost completely syntenic. Different chromosome morphology: Mouse - acrocentric; Human - mostly submetacentric. Non-coding sequences not well conserved.

126

What can synteny analysis be used for?

Prediction of gene locations. Prediction of gene order. Identification of non-human models for disease based on syntenicity of the desired region.

127

Give an example of a non-human model that has been found using synteny analysis.

Waardenberg Syndrome Type 1.