Genomics and Health

Question 1

State of the art gene discovery

Answer 1

Whole exome next generation sequencing
Filter polymorphisms to give a list of candidate genes
Confirm by Sanger sequencing

Question 2

Traditional gene discovery

Answer 2

Determine mode of inheritance
Recombination mapping using markers
Haplotype analysis of recombinants
Rapid screening of candidate genes for mutations
Confirm by Sanger sequencing

Question 3

Next generation sequencing

Answer 3

Massively parallel sequencing technology
Entire human genome can be sequenced in a day
Captures broader spectrum of mutations than Sanger sequencing
Unselective and requires no pre-knowledge of mutation
Allows detection of mosaic mutations (acquired post-fertilisation and are present at variable frequency within cells)
Has been used to detect new mutations in colorectal cancer which has a large number of different mutations

Question 4

Next generation sequencing mechanism

Answer 4

Small DNA fragments anchored to solid surface
Each molecule copied in situ by PCR to amplify the template
Reversible terminators with reversible fluorescence molecule and blocking molecule (prevents further extension) added along with polymerase and universal primer
Unincorporated nucleotides washed away
Nucleotide fluorescent molecule detected with laser
Fluorescence and blocking molecule removed
Repeated up to 100 times to generate sequence

Question 5

Exon capture

Answer 5

Chip synthesised with reverse transcription PCR (represents exome). RNA converted to cDNA library
Sequencing sample fragmented and applied to the chip
Target genes bind to their complementary sequences on the chip and unbound sample is washed off
Captured material is eluted and sequenced
Used to sequence coding regions of the genome

Question 6

Filtering criteria

Answer 6

Next generation sequencing detects many polymorphisms, so need to be filtered
Mutation likely to cause change in gene expression/protein structure (nonsense, frameshifts)
Mutation not commonly found in SNP databases or control genome sequences
Alleles in affected individuals correspond to mode of inheritance
Same gene mutated in affected, unrelated individuals
No unaffected individuals with putative genotype

Question 7

Cantu syndrome (de novo mutation)

Answer 7

Dominant lethal inheritance
Congenital hypertrichosis (excess hair)
Distinctive facial features
Abnormal skeleton development
Mutation in ABCC9 gene (ATP-dependent K+ transporter) identified by Sanger sequencing
Arg to Gln at position 1154

Question 8

De novo mutations

Answer 8

Sequence affected child and unaffected parent
Average of 74 de novo point mutations in every genome
Sperm more susceptible (80% paternal) and mutations increase with age

Question 9

Gene panels for diagnosis

Answer 9

Exon capture/amplification for a subset of genes

All possible alleles can be identified in one test (PCR requires alleles to be known first)

Question 10

Whole exome sequencing (WES)

Answer 10

Not routine as still costly
Only tests exome, mutations in regulatory regions may be important
WES made positive diagnosis in 25% of cases

Question 11

SNP analysis

Answer 11

Single nucleotide polymorphism (single base pair that commonly varies in humans). Variants are alleles
Refers to one strand (may be homozygous or heterozygous)
Around 40 million SNPs
One allele inherited from each parent

Question 12

Haplotypes

Answer 12

Arrangement of SNPs on a chromosome used as markers

SNPs within a block can stay associated for many generations

Question 13

Genome wide association

Answer 13

SNP arrays used to genotype individuals with and without a trait
Higher SNP allele incidence in individuals with the trait (association)
Candidate genes identified

Question 14

Pharmacogenomics

Answer 14

GWA studies identify SNPs associated with sensitivity/side effects.
Validation by replication difficult. Small sample sizes as adverse reactions are rare