Genomics and Health Flashcards
What are the traditional routes to gene discovery vs state of the art routes?
Traditional:
= determine mode of inheritance
= recombination mapping using markers
= haplotype analysis of recombinants
= rapid screening of candidate genes for mutations
= confirm by Sanger sequencing
State of the Art:
= whole exome next generation sequencing
= lots of polymorphisms
= filter polymorphisms to give list of candidate genes
= confirm by sanger sequencing
What is next generation sequencing?
EXTRA READING
= includes variety of methods
= use massively parallel processing to sequence millions of DNA fragments in single run (simultaneous)
= Library preparation
= Sequencing
= Base calling
= Data processing
= Variant calling
= Data Analysis
advantages
= speed, cost-effectiveness, high-throughput, accuracy
What is exon capture?
EXTRA READING
= sequencing specific regions of genome such as exons
= selectively isolates and enriches DNA fragments of interest from a sample (= better depth and accuracy)
= DNA fragmentation
= Adapter ligation
= Hybridisation
= Capture
= Amplification
= Sequencing
What is the filtering criteria for disease?
= mutation likely to cause a change in gene expression or protein structure
(e.g. nonsense, strong mis-sense, splice site changes, frameshifts)
= mutation not commonly found in SNP databases or control genome sequences
= alleles in affected individuals correspond to mode of inheritance
(e.g. two mutant alleles for recessive condition)
= same gene mutated in affected, unrelated individuals
= no unaffected individuals with putative disease-causing genotype
e.g. Direct identification of the UVSS-A causal gene by exome sequencing of subjects Kps3 and XP24KO
= UV sensitivity = skin discolouration
What is an example of Identification of de novo mutations?
= Cantú Syndrome
= dominant (lethal) inheritance
= congenital hypertrichosis (excess hair)
= distinctive facial features
= osteochondrodysplasia
= cardiac defects
= Harakalova et al = whole exome sequencing of an affected child + unaffected parents
= 5 candidate genes Sanger sequenced
= mutation confirmed in ABCC9 gene (ATP-dependent K+ transport)
= Arg to Gln = at position 1154
= mutations in ABCC9 confirmed in 13 out of 15 Cantu patients (absent from 5000 control exomes)
What are de novo mutations?
‘New’ mutations
= sequence affected child and unaffected parents
= average of 74 de novo point mutations (in whole genome)
= apply the filtering criteria
= 80% paternal, 2 additional de novo mutations for each year of paternal age
(sperm more susceptible to mutation)
= often discovered through whole exome sequencing
What do sequencing panels of genes do?
e.g. exon capture or exon amplification for subset of genes
= all possible alleles can be identified in one test
= e.g. 450 gene panel for eye disorders - 8 tests
What is whole exome sequencing used for?
Not routine in diagnosis - when all else fails
Often simultaneous gene identification (research) + diagnosis
All mutations can be identified
(ethical questions surrounding incidental findings)
Only 85-95% coverage of protein-coding regions
(technical limitations)
Produces huge amounts of data
What is an example of a clinical whole exome sequencing (WES) trial study?
WES of 250 people with undiagnosed disorders
(@ Baylor College of Medicine)
= most neurological disorders
= positive diagnosis in 25% of cases
= 33 autosomal dominant
= 16 autosomal recessive
= 9 X-lined
BUT exome only
= mutations in regulatory regions may be important
30 patients had unrelated genetic variants
How is DNA sequencing used in Medicine?
Department of health
= 100,000 genomes project
= completed in 2018
= actionable findings for 20-25% rare disease patients
= approx. 50% of cancer cases contain potential for therapy / clinical trial
Uses
= cancer (cancer vs normal genome)
= rare inherited conditions (identify mutations)
= infectious diseases (pathogen genomes and patient genomes)
= new routes for diagnosis
= personalised medicine (targeted treatments)
BUT homozygous loss of function often appears to have no effect
What is SNP analysis?
SNP
= single nucleotide polymorphism
= single base pair that commonly (>1%) varies in human population
(as opposed to rare mutation)
= generally refer to one strand
= variants are alleles
= genotype may be homozygous or heterozygous
= about 40 million SNPs
Analysis
= focuses on approx. 1% single nucleotide differences between individuals
= can be very high throughput
= useful in association studies, genetic ancestry and diagnosis of chromosomal disorders
What are Haplotypes?
= arrangement of SNPs on a chromosome
SNPs within a block can stay associated for many generations
4 - 6 alternative haplotypes for each block
approx. 20 SNPs per block
humans are haplotype mosaics
EXTRA READING
= sets of closely linked genetic variants that tend to be inherited together
= blocks of DNA
= haplotype = combination of SNPs found in particular block of DNA
= can be used to track inheritance of specific genetic variants + identify regions of genome associated with certain traits
How is Genome-Wide Association used?
SNPs array used to genotype individuals with and without the trait
Higher incidence of SNP allele in individuals with the trait vs to those without the trait
= ASSOCIATION
Significance depends on degree of association and number of individuals tested
= very low P value used for significance to avoid false positives
Nearby (linked) candidate genes identified and tested
EXTRA READING
= examines relationship between variations in genome and traits or diseases
= studies involve:
selection of participants
DNA sequencing
Statistical anlysis
Follow-up studies
What is pharmacogenomics?
Pharmacogenetics on a genome-wide scale
= attempty to identify SNPs associated with sensitivity / side effects
Difficult to get large sample sizes
= adverse reactions are rare
Validation by replication difficult
Obvious phenotypic effects of adverse reactions or overdose
EXTRA READING
= study how genetics affects response to drugs
e.g. warfarin response - CYP2C9 and VKORC1
What are some significant genome-wide associations for drug response?