11. Linkage Analysis

Question 1

What is genetic variation? and what effects can it have?

Answer 1

• Genetic variation refers to differences in the DNA sequence between individuals in a population
• Variation can be inherited or due to environmental factors (e.g. drugs, exposure to radiation)

• EFFECTS:

1) Alteration of the amino acid sequence (protein) that is encoded by a gene
2) Changes in gene regulation (where and when a gene is expressed)
3) Physical appearance of an individual (e.g. eye colour, genetic disease risk)
4) Silent or no apparent effect

Question 2

Why is genetic variation important?

Answer 2

1. Genetic variation underlies phenotypic differences among different individuals
2. Genetic variations determine our predisposition to complex diseases and responses to drugs and environmental factors
3. Genetic variation reveals clues of ancestral human migration history

Question 3

What are the different mechanisms of genetic variation?

Answer 3

1. MUTATION/POLYMORPHISM: errors in DNA replication. This may affect single nucleotides or larger portions of DNA
2. GENE FLOW: the movement of genes from one population to another (e.g. migration) is an important source of genetic variation
3. GENETIC RECOMBINATION: shuffling of chromosomal segments between partner (homologous) chromosomes of a pair

Question 4

Give examples of different types of mutation/polymorphism in different types of cells

Answer 4

• Germline mutations: passed on to descendants
• Somatic mutations: not transmitted to descendants
• de novo mutations: new mutation not inherited from either parent ~ can happen when a baby is the womb

Question 5

What is a mutation?

Answer 5

A mutation is a rare change in the DNA sequence that is different to the normal (reference) sequence. The ‘normal’ allele is prevalent in the population and the mutation changes this to a rare ‘abnormal’ variant

Question 6

What is a polymorphism?

Answer 6

A polymorphism is a DNA sequence variant that is common in the population. In this case no single allele is regarded as the ‘normal’ allele. Instead there are two or more equally acceptable alternatives

Question 7

What is the arbitrary cut off point between a mutation and a polymorphism?

Answer 7

Minor allele frequency (MAF) of 1% (i.e. to be classed as a polymorphism, the least common allele must be present in ≥1% of the population)

Question 8

What is genetic recombination?

Answer 8

Happens during prophase I - When there is a line up homologous chromosomes (maternal and paternal chromosomes)

• Crossing over: reciprocal breaking and re-joining of the homologous chromosomes during meiosis
• Results in exchange of chromosome segments and new allele combinations
• Gametes as a results will contain a mixture of maternal and paternal material (recombinant chromosomes)

Question 9

RECAP: What is a genotype, phenotype and an allele?

Answer 9

• The genotype is the genetic makeup of an individual
• The phenotype is the physical expression of the genetic makeup
• Genes are found in alternative versions called alleles - for each characteristic, an organism inherits two alleles, one from each parent; the alleles can be the same or different

Question 10

Recap: What is homozygous, heterozygous and haplotype?

Answer 10

• A homozygous genotype has identical alleles
• A heterozygous genotype has two different alleles
• A haplotype is a group of alleles that are inherited together from a single parent

Question 11

What is a chromosome pair?

Answer 11

• Chromosome pair: homologous chromosomes with genes at the same loci
• The allele at that locus may be the same (homozygous) or different (heterozygous)

Question 12

What are the different classification of genetic disease?

Answer 12

• MENDELIAN / MONOGENIC: disease that is caused by a single gene, with little or no impact from the environment (e.g. PKD)
• NON-MENDELIAN / POLYGENIC: diseases or traits caused by the impact of many different genes, each having only a small individual impact on the final condition (e.g. psoriasis)
• MULTIFACTORIAL: diseases or traits resulting from an interaction between multiple genes and often multiple environmental factors (e.g. heart disease)

Question 13

Genomic variation in disease. How can you identify single variant in linkage analysis?

Answer 13

• Mendelian ~ one gene = one disease
• Complex ~ many genes = one disease
• In linkage analysis, you can use the whole spectrum to identify the single variant (Mendelian) - (graph on slide 12)

Question 14

What is linkage analysis?

Answer 14

• Linkage analysis is a method used to map the location of a disease gene in the genome
• The term ‘linkage’ refers to the assumption of two things being physically linked to each other

Question 15

How can genetic markers be used to identify the location of a disease gene?

Answer 15

Based on their physical proximity

Question 16

What do genetic maps do?

Answer 16

Maps provide a context to orientate yourself and calculate distance between landmarks

Question 17

What do genetic maps do?

Answer 17

Look at information in blocks or regions (similar to zones on a tube map)

Question 18

What is the unit used in genetic mapping?

Answer 18

Megabase

- 1 megabase = 1 million base pairs

Question 19

What are the principles of genetic linkage?

Answer 19

• Genetic linkage is the tendency for alleles at neighbouring loci to segregate together at meiosis
• Therefore to be linked, two loci must lie very close together
• A haplotype defines multiple alleles at linked loci. • Haplotypes mark chromosomal segments which can be tracked through pedigrees and populations
• Cross-overs are more likely to occur between loci separated by some distance than those close together

Question 20

How are genetic markers used for linkage mapping?

Answer 20

• Uses an observed locus (genetic marker) to draw inferences about an unobserved locus (disease gene)
• If a marker is linked to a disease locus, the same marker alleles will be inherited by two affected relatives more often than expected by chance
• If the marker and the disease locus are unlinked, the affected relatives in a family are less likely to inherit the same marker alleles

Question 21

What are different types of genetic marker?

Answer 21

• Microsatellite markers

* Single nucleotide polymorphisms

Question 22

Expand on microsatellites

Answer 22

• Less common now. Highly polymorphic short tandem repeats of 2 to 6 bp
• Microsatellites may differ in length between chromosomes (heterozygous)
• Are relatively widely spaced apart
• 400 (200) microsatellite markers
• Average spacing 9 cM (20 cM)
• PCR-based system
• Fluorescently-labelled primers
• Manual assignment of genotypes
• Labour intensive
• Whole genome scan 2-3 months

Question 23

Expand on single nucleotide polymorphisms

Answer 23

• Now the genetic marker of choice. Biallelic – a SNP will be one of two possible bases
• Less heterozygous than microsatellites, but spaced much closer together
• More informative
• Most common type of variation
• ~6,000 SNPs
• Spaced throughout the genome
• Microarray-based system
• Genotypes assigned automatically
• Highly automated
• Data returned within 1-2 months

Question 24

What is microsatellite genotyping used for?

Answer 24

• DNA fingerprinting from very small amounts of material
• Standard test uses 13 core loci making the likelihood of a chance match 1 in three trillion
• Paternity testing
• Linkage analysis for disease gene identification

Question 25

What does SNP genotyping microarrays do?

Answer 25

• Provides genome-wide coverage of SNP markers • SNPs are proxy markers; NOT the causal disease variants • Can amplify thousands of markers in a single experiment • Alleles are identified by relative fluorescence - homozygous for allele 1 = green signal - homozygous for allele 2 = red signal - heterozygous (1/2) = yellow signal

Question 26

What is SNP genotyping microarrays typically used for?

Answer 26

• Linkage analysis in families (affected vs unaffected relatives) - homozygosity mapping (autosomal recessive) and mapping of Mendelian traits • GWAS in populations (unrelated cases vs matched controls) - non-Mendelian disorders and multifactorial traits

Question 27

How can the probability of linkage be assessed?

Answer 27

* The probability of linkage can be assessed using a LOD score * LOD = logarithm of the odds score * Assesses the probability of obtaining the test data if the two loci are linked, to the likelihood of observing the same data purely by chance * i.e. calculates a likelihood ratio of observed vs. expected (no linkage, θ=0.5) * Recombination fraction is the proportion of recombinant births (i.e. R / NR+R) * The higher the LOD score, the higher the likelihood of linkage • When talking about linkage, you can never have recombinant fractions of higher than 0.5 as that is 50% recombinant frequency and at this point you are having independent assortment - As there is a 50% chance you are going to get maternal or paternal allele anyway

Question 28

What is the statistical analysis of linkage?

Answer 28

* LOD scores can be calculated across the whole genome using genotype data for many genetic markers in multiple members of a family * LOD scores are additive – different families linked to the same disease locus will increase the overall score * A LOD score equal to or more than 3 is considered evidence for linkage * A LOD score equal to or less than -2 is considered evidence against linkage * Between -2 and 3 is considered suggested evidence

Question 29

EXAMPLE: Adams-Oliver syndrome

Answer 29

• Characterized by presence of terminal transverse limb defects (TTLD) and scalp aplasia cutis congenita (ACC) • Associated features: - Neurological anomalies - Cardiac malformations - Vascular defects (e.g. cutis marmorata telangiectatica congenita, dilated veins)

Question 30

Explain locus refinement and gene identification

Answer 30

• Refinement of minimal linkage interval using microsatellite markers across the region - What do proteins made from this gene do? - reason for disease?