20.02.22 Finding disease related genes using cytogenetics, linkage etc.

Question 1

Ways apparently balanced translocations have found disease genes

Answer 1

1. Sub-mciroscopic imbalance: Investigated with FISH or array CGH.
2. Disruption of gene
3. Gene separated from cis acting regulatory elements (e.g. promoter, rearrangement switching regulatory elements with another gene, changes in chromatin structure)

Question 2

Example of a gene found by sub-mciroscopic imbalance

Answer 2

CHARGE syndrome. Deletion in chromosome 8 in 2 patients, other patients screened for variants in genes within deletion overlap region. Identified mutations in CHD7.

Question 3

Example of a gene found by Disruption of gene

Answer 3

• e.g. Translocation in patient between chromosome 5 and 8 had a SOTOS phenotype (overgrowth, dysmorphism, dev delay). Found breakpoint disrupted NSD1. Tested further patients and found point muations and microdeletions in NSD1.
• RUNX1 most commonly dysregulated in leukemia. Originally found as a t(8;21). Subsequently found in other translocations in other myeloid and lymphoblastic neoplasms t(3;21) or t(12;21)

Question 4

Example of a gene found due to separation from cis acting regulatory elements

Answer 4

e.g. Aniridia (severe hypoplasia of the iris). PAX6 haploinsufficiency at 11p13. Translocations with 3’ breakpoint of PAX6 (regulatory region).

Question 5

Genes found by deletions/duplications

Answer 5

• Microdeletions/duplications may account for 15% of mutations underlying monogenic diseases.
• e.g. patients with deletion of 17p13.3 had Miller-Dieker syndrome (type 1 lissencephaly with facial dysmorphism). Patients with smaller deletions had isolated lissencephaly. Identified LIS1 as gene responsible for MDS.

Question 6

Inversions leading to gene identification

Answer 6

• Breakpoints can directly interfere with gene or have a positional effect.
• Inversion 7q22.1;7q31.1 seen in autistic siblings. Breakpoints mapped to coding sequence of several cytochrome P450 genes.

Question 7

Potential problems with translocation and phenotype

Answer 7

• Link could be a coincidence
• Often many genes involved, may be more than 1 responsible
• Positional effects difficult to identify

Question 8

What is linkage

Answer 8

Tendency for genes and genetic markers to be inherited together as they are located nearby on the same chromosome.

Question 9

What is linkage mapping

Answer 9

• Mapping a disease locus to a chromosomal region.

- Co-inheritance of a genetic trait with a particular allele of a polymorphic DNA marker of known chromosomal location.

Question 10

What does linkage mapping rely on

Answer 10

• As a consequence of random meiotic recombination, the smaller the genetic distance between two loci, the greater the chance that they will be coinherited.
• Requires informative meioses. Determine parental origin.
• Diseases showing mendelian inheritance (i.e. not de novo variants)

Question 11

Steps of linkage analysis

Answer 11

• Identify cohort of patients with disease of interest
• Genotype markers (SNPs or STRs) across whole genome of cohort.
• Identify regions with linkage to disease, based on LOD scores.
• Fine mapping of region with more densely packed markers to look for candidate genes within a region
• Sequence candidate genes to look for pathogenic mutations in affected individuals.

Question 12

What is an LOD score

Answer 12

• LOD= Logarithm of odds
• Log of the ratio of odds that two loci are linked with a specified recombination frequency θ, to the odds that they are unlinked.
• Compares the likelihood of obtaining test data if two loci are linked o the possibility of observing the same data by chance.

Question 13

When is linkage accepted

Answer 13

When LOD score is > 3.

Question 14

When is linkage excluded

Answer 14

When LOD score is

Question 15

What is recombination frequency θ

Answer 15

Frequency with which a single chromosomal crossover will take place between two genes during meiosis.

Question 16

Two types of linkage analysis

Answer 16

1. Parametric

2. Non-parametric

Question 17

What is parametric linkage

Answer 17

• Standard LOD score analysis
• Needs large families with a large number of meioses.
• Need a specific genetic model (inheritance pattern, penetrance, gene frequency)
• Scans 20Mb sections

Question 18

Problems with parametric linkage

Answer 18

• Error prone
• Problems with locus heterogeneity, need to specify genetic model
• Not appropriate for common diseases (not precise model, difficult to diagnose, incomplete penetrance, genetic heterogeneity)

Question 19

What is non-parametric linkage

Answer 19

-Linkage analysis without the need for a genetic model

Question 20

Other types of analysis

Answer 20

• Affected sib-pairs: Random inheritance predicts siblings share 0, 1, 2 alleles with frequency 1/4, 1/2, 1/4. Used to identify diabetes type 1 susceptibility locus upstream of insulin gene.
• Linkage analysis on “affected only”: more stringent disease phenotype with nearly Mendelian inheritance pattern. E.g. APOE gene and late onset Alzheimer’s disease.

Question 21

What is the future of linkage analysis

Answer 21

-Combining with WGS. Provides statistical support that a variant is associated with disease.

Question 22

What is autozygosity mapping

Answer 22

• Used to identify genes associated with autosomal recessive disease (particularly consanguineous families)
• Assumes affected individuals will be homozygous (autozygous) for markers at disease locus
• Does not need parameters and can be informative when testing a small number of individuals.
• Assumes that within a limited population, all affected individuals inherited the same disease-causing allele.

Question 23

What is identity by descent

Answer 23

• Two alleles are both copies of one specific allele that was present in a recent ancestor
• More likely for rarer alleles.

Question 24

What is the coefficient of inbreeding (F)

Answer 24

The probability a child of a consanguineous marriage will be homozygous for a specific gene derived from a common ancestor.

Question 25

What F number indicates parents are first cousins

Answer 25

F=16.

Question 26

Steps of autozygosity mapping

Answer 26

• Identify a cohort with a recessive phenotype. Ideally one family with multiple affected probands
• Genotype a large number of SNPs or microsatellites across the genome
• Identify regions of homozygosity, shared by all affecteds. e.g. IBDfinder.
• Fine map (narrow) candidate region by using more polymorphic markers.
• Sequence genes in candidate region

Question 27

Factors affecting autozygosity mapping

Answer 27

• Number of informative affected and unaffected individuals
• Frequency of allele in population. The rarer the more likely homozygosity represents autozygosity (IBD)
• Degree of relatedness. THe more remotely related, the smaller the proportion of the genome shared from common ancestor, due to recombinations.

Question 28

Problems with autozygosity mapping

Answer 28

• Unexpected allelic heterogeneity
• Identification of IBD, unrelated to disease locus
• Inflation of LOD scores due to underestimation of the extent of inbreeding.

Question 29

What is SAMPLE (shadow autozygosity mapping by linkage exclusion)

Answer 29

-Strategy to identify genetic basis of lethal autosomal recessive disorders by SNP genotyping unaffected individuals in complex consanguineous pedigrees to infer limited chromosomal regions compatible with linkage to a potential disease locus.

Question 30

Use of autozygosity mapping in the future

Answer 30

• Use of NGS has aided gene discovery.

- Can identify regions of interest and also interrogate genes of interest to identify causative mutation.

Question 31

What is positional cloning

Answer 31

• Method of identifying a gene solely on its approximate location
• Used to narrow region of interest after linkage analysis or autozygosity mapping.
• Prior knowledge of gene/protein not required.
• Used to identify DMD and CFTR.

Question 32

What is chromosome walking

Answer 32

-Method used to produce clones covering the candidate region.

1. Starts with a known marker at the end of a candidate region
2. DNA fragment with marker used as a probe to screen a genomic library to identify other clones containing the marker and adjacent sequences.
3. Repeat step with new clones to identify further overlapping clones.

Question 33

Problem with chromosome walking

Answer 33

• Very slow

- Repeat sequences cause issues such as non-specific probe binding

Question 34

What is chromosome jumping

Answer 34

• Restriction digest of DNA to produce fragments that are separated by pulse-field electrophoresis.
• Fragments (80-130kb) in length, containing a known marker near one end, are ligated to form a circle
• Start and marker are now close together. Circle is cut and cloned into another vector, Can be used as another probe for chromosome jumping or walking.
• Genes within cloned genomic regions can be identified by zoo blots or screening cDNA libraries.

Question 35

Other ways to determine the causative gene in a candidate region

Answer 35

• Based on function of protein, homologous genes, expression studies in affected tissues
• Identify variants present in affected individuals and absent in unaffected relatives.