11- Linkage Analysis

Question 1

define linkage analysis

Answer 1

a method used to map the location of a disease gene in the genome - make a major assumption that genetic markers in close proximity to disease gene will be co-inherited

Question 2

define genetic variation

Answer 2

differences in the DNA sequence between individuals in a population - inherited or due to environmental factors

Question 3

define genetic linkage

Answer 3

the tendency of genes located on the same chromosome to be inherited together during meiosis

the closer two genes are on a chromosome, the higher the probability that they will be inherited as a unit

Question 4

name the three mechanisms of genetic variation

Answer 4

mutation/ polymorphism, homologous recombination, gene flow

mutation = error in DNA replication concerning rare variants - germline, somatic, de novo

polymorphism = error in DNA replication; a common allele that is a variant of the ‘normal’ allele

homologous recombination = exchanging of genetic material between homologous chromosomes during meiosis

gene flow = movement of genes from one population to another

Question 5

difference between mutation and polymorphism

Answer 5

mutation and polymorphism are errors in DNA replication – mutations concern rare variants producing an abnormal allele whereas polymorphism indicates a common allele that is a variant of the ‘normal’ allele

an MAF score of above 1% indicates polymorphism, whilst below indicates a mutation

Question 6

define homologous recombination

Answer 6

exchanging of genetic material between homologous chromosomes during meiosis

Question 7

describe homologous recombination

Answer 7

occurs during meiosis between two homologous chromosomes – maternal and paternal chromosomes with genes for the same traits – during prophase I

the chromosomes line up, cross over to form a chiasmata, and exchange genetic info, creating allele combinations and recombinant chromosomes = allele shuffling

Question 8

name the three classifications of genetic disease

Answer 8

Mendelian (monogenic), non-Mendelian (polygenic) and multifactorial

monogenic diseases have higher penetrance than polygenic

Question 9

describe Mendelian disease

Answer 9

monogenic Mendelian diseases are rare and caused by one gene

Question 10

describe non-Mendelian disease

Answer 10

polygenic non-Mendelian diseases caused by the cumulative effect of many disease-causing genes and are more common

Question 11

describe multifactorial disease

Answer 11

occur from multiple genes and environmental factors interacting (e.g. cardiovascular diseases)

Question 12

describe genetic and physical linkage mapping

Answer 12

genetic maps provide information about blocks/regions of a chromosome – approximate distance separating any two loci = generated by examining how often chromosomal segments are observed together

physical maps provide more precise information on physical distance = the exact location

Question 13

define co-segregation in genetic linkage

Answer 13

tendency of genetic markers and a trait or disease gene to be inherited together in family pedigrees

two loci close together on the same chromosome are more likely to be inherited together during meiosis = leads to co-segregation

Question 14

what are the principles of genetic linkage?

Answer 14

1. cross overs and recombination
   - cross overs are more likely to occur between loci separated by some distance on a chromosome
   - two loci must be close together to be considered linked, more likely to be inherited together
2. haplotypes
   - define multiple alleles at linked loci = physical grouping of genetic variants that tend to be inherited together
   - chromosomal segments with specific allelic combinations can be tracked through pedigrees and populations

Question 15

name the two types of genetic marker routinely used in linkage studies

Answer 15

microsatellite/ STR markers and SNP markers

STRs = repetitive elements in the genome with variable numbers of repeat units, polymorphic

SNP = single nucleotide polymorphism/ single base change at a specific position, biallelic

Question 16

compare STRs and SNPs as genetic markers - properties

Answer 16

STRs = highly polymorphic but less common. high heterozygosity, widely spaced apart

• highly polymorphic - multiple variations in repeat units within a population
• high heterozygosity = individuals often have two different alleles at any given STR locus
• variability from indels of repeat units during DNA replication
• widely spaced apart on genome
• labour intensive to assign genotypes

SNPs = biallelic, lower polymorphism and heterozygosity, more densely spaced
- biallelic = only two possible alleles
- lower polymorphism = arises from variations in SN nucleotide change
- lower heterozygosity = only two possible alleles
- arise form point mutations = transitions or transversions, less prone to rapid changes
- more densely spaced throughout genome allows for finer mapping
- automatically assigned genotypes

STRs favoured for higher polymorphism and heterozygosity, for regions with lower recombination rates

SNPs favoured for high genome coverage - are more densely distributed, allow for finer mapping

Question 17

describe microsatellite genotyping

Answer 17

• PCR based amplification = amplifies STRs with differently fluorescently-based primers positioned on either side of the repetitive element
• amplified products run through gel electrophoresis - fewer repeat units mean smaller fragments and move faster up gel, appear as bands
• fragment sizes can be used to track different alleles from parent to offspring

uses: forensic investigations, paternity tests, identifies disease genes in linkage analysis

Question 18

compare microsatellite and SNP coverage

Answer 18

microsatellites - some areas can be poorly covered, disease gene may be in uncovered areas

SNP = covers genome well, few gaps, individual SNPs are less informative as they’re biallelic and less polymorphic, may lead to more ambiguity when building haplotypes

Question 19

define a haplotype

Answer 19

a group of alleles inherited together from a single parent

Question 20

describe how to build a haplotype

Answer 20

trach shared chromosomal segments between family members to determine which genomic regions are co-inherited with the disease gene

genotype for several genetic markers between affected and unaffected parents and offspring are collected - used to see what alleles the offspring have inherited, what alleles they all have in common

more than one affected sibling/family member can help identify a recombination event and define a critical disease interval – the smallest region of the genome where the disease gene is likely to be located

the disease gene must be in a chromosomal segment inherited by all affected family members

linkage software uses haplotype analysis to assess recombination and define the critical linkage interval

Question 21

describe statistical analysis of linkage

Answer 21

linkage analysis = mapping the location of a Mendelian disease gene in the genome by assuming genetic markers are in close proximity with the disease gene/ relies on co-segregation

probability of linkage can be assessed using an LOD score – logarithm of the odds score – = represents the probability of two loci being linked versus it being by chance
- implies that the recombination fraction of any observed co-segregation is due to the disease gene being in that region
- higher LOD score = higher chance of linkage

> 3 is genome wide significance and evidence of linkage; below -2 is against linkage. in between is suggestive

Question 22

describe the two forms of analysis for linkage software

Answer 22

parametric analysis = has specific parameters to input - pedigree, data, map and model files

non-parametric analysis = no specific parameters - just input pedigree, data and map files

Question 23

name the 3 stages of variant validation by exome sequencing of linkage

Answer 23

1. variant profile
2. filtering with zygosity, excluding known/common variants, assessing linkage, predicting functionality and prior biological knowledge of gene = all have certain parameters
3. identifying the causal variants