genome variation

Question 1

changes at the DNA level

Answer 1

• substitutions
• deletions
• duplications
• insertions
• inversion
• translocations
• complex changes

Question 2

changes at the RNA level

Answer 2

• RNA processing/initiation
  
  • promoter/initiation site
  • affects amount of RNA
• splicing
  
  • abolition of splice site
  • activation of intronic splice site producing a pseudoexon
• large deletions
  
  • e.g. part of promoter

Question 3

changes at protein level

Answer 3

• silent/synonymous changes
  
  • same amino acid
• substitution, deletion, insertion
• missense (different amino acid)
• nonsense (stop codon)
• duplication, insertion, translocation
• complex rearrangements

Question 4

importance of genomic variation

Answer 4

• cause of disease
• risk of developing disease
  
  • especially with multiple variants
• response to drugs

Question 5

1000 genomes project

Answer 5

• DNA of 1000 healthy adults sequenced
• 14 populations
• ancestry from all continents
• low coverage whole genome, and targeted deep exome sequence data
• x4 coverage

Question 6

discovery aims of HGP

Answer 6

• single nucleotide variants with frequency >1%
• single nucleotide variants with frequency 0.1-0.5% in gene regions
• structural variants
  
  • CNVs, insertions/deletions, inversions
• estimated frequencies of variant alleles

Question 7

genetic variant databases

Answer 7

• gene specific
  
  • p53 gene
• disease specific
  
  • cystic fibrosis
• large scale
  
  • dbSNP

Question 8

dbSNP

Answer 8

• database of short sequence variations
• all organisms
• single nucelotide substitutions, insertions/deletions
• any variations >50 nt in separate dbVAR database

Question 9

dbSNP information

Answer 9

• submissions (public and private)
• validation
  
  • evidence used
  • refSNP ID number for each submission if more than one
• ancestral allele
  
  • by comparison between human and chimp
• links to other relevant resources
• molecular and functional consequences
  
  • computed or submitted
• global MAF
• association studies from GWAS

Question 10

MAF

Answer 10

• minor allele frequency
• default global population frequency
• based on 2500 individuals worldwide from phase 3 of 1000 GP

Question 11

whole exome sequencing

Answer 11

• all exons of protein coding genes sequenced (1% of genome)
• useful for identifying novel rare disease variants
• results analysed using large databases of known variants
  
  • ExAC

Question 12

ExAC

Answer 12

• exome aggregation consortium database
• exome sequences of >60,000 unrelated individuals
  
  • from many disease-specific and population genetics studies
• adult-onset diseases
• no homozygous variants causing childhood-onset mendelian diseases
• gives allele frequency
• 2014

Question 13

ClinVar

Answer 13

• combines information about genomic variation and relates to human health
• germline and somatic variants
• clinical significance from submitters
  
  • from all submitted records for same variant
  • indicates consensus or conflict
• interpretation for >200,000 variants

Question 14

OMIM

Answer 14

• online mendelian inheritance in man
• catalogue of human genes and mendelian genetic disorders
• 6000 phenotypes with known molecular basis
• 4000 genes with phenotype-causing mutations

Question 15

humsavar

Answer 15

• human polymorphisms and disease mutations
• single amino acid variants only (missense)
• 75,000 in total
  
  • disease variants
  • polymorphisms
  • unclassified variants
• links to other databases
  
  • uniprot, dbSNP, OMIM

Question 16

COSMIC

Answer 16

• catalogue of somatic mutations in cancer
• >4 mill coding mutations reported
• combined genome-wide results from >28,000 tumours
• displays distribution of types of variants for each gene
• tissue specific variant information

Question 17

in silico prediction

Answer 17

• distinguish between disease-causing and neutral variants
  
  • expensive to do wet lab work
• interpret large datasets of rare variants
  
  • prioritisation and characterisation
• requires knowledge of amino acid sequence and effects of variants
  
  • mutability

Question 18

amino acid mutability

Answer 18

• from 1000 GP
• arginine most mutable
  
  • 4/6 codons encoding arg have CpG
• leucine low mutability
  
  • no codons with CpG dinucleotides
• CpG mutates at higher rate than other dinucleotides
• more complex amino acids have lowest mutabilities
  
  • trp, phe

Question 19

in silico prediction programs

Answer 19

• >30 available for amino acid substitutions
• for missense:
  
  • sequence based methods
    
    • e.g. SIFT
  • sequence and structure based combinations
    
    • e.g. PolyPhen-2

Question 20

SIFT

Answer 20

• sorting intolerant from tolerant
• align homologous sequences to query
• identify conserved residues unlikely to tolerate substitutions
• PSI-BLAST to create MSA of similar sequences
• calculate probability of any of the 20 amino acids at each position
• calculate output score
  
  • probability for each of the 19 possible substitutions at each position in the aligned target protein
  • 0.05 or less indicates deleterious

Question 21

PolyPhen-2

Answer 21

• polymorphism phenotyping
• structures and annotations of residues improve prediction accuracy
• also qualitative assessment
  
  • benign, possibly damaging, probably damaging
• compare WT allele properties to mutant
• evaluate sequence conservation by aligning homologous sequences
• predicts effect of single substitution or large number (batch mode)
• also database search of already computed predictions

Question 22

HumDiv

Answer 22

• default PolyPhen-2 classifier
• 3000 damaging alleles causing human mendelian disease (uniprot)
• 6000 differences between human proteins and close mammalian homologs assumed as non-damaging

Question 23

HumVar

Answer 23

• another classifier for PolyPhen-2
• 13000 human disease-causing mutation (uniprot)
• 9000 human non-synonymous amino acid substitutions with no annotated disease involvement (non-damaging)
• more appropriate for mendelian disease associated substitutions
  
  • drastic effect

Question 24

SAAPs

Answer 24

• single amino acid polymorphism data analysis
• SAAPdap (predictor)
• SAAPpred (predictor)
• analyses liekly structural effect of amino acid substitution
• considers residue conservation
• requires experimental structure

Question 25

in silico prediction

focus on amino acid susbstitutions

Answer 25

• many changes are very subtle
  
  • difficult to identify in wet lab work
• easier to study protein than non-coding regions of DNA
• improve understanding of how mutation affects protein