PolyPhen and SIFT: Tools for predicting functional effects of SNPs

Epi 244

Spring 2009

Sam S. Oh

Frazer et al. Nat Rev Genet, 2009;10:241-251

Human genome variation

• 3.2 billion base pairs (bp)• 99.9% similarity across individuals

– 3.2 million bp dissimilar

• ~11 million SNPs– Coding vs. non-coding (intron and intergenic regions)– Most are synonymous

DNA → RNA → Protein

Example: sickle-cell anemia• A to T SNP of beta-globin gene results in

glutamate (hydrophilic) to valine (hydrophobic) substitution

Example: MTHFR

• Folate metabolism

Finding MTHFR SNPs

Highlight all refSNP numbers (use scroll bar)

and copy

Highlight all refSNP numbers (use scroll bar)

and copy

Note Build number(currently Build 130)

SIFT

• Sorting Intolerant From Tolerant • Predicts tolerability of AA substitution

effects (i.e., non-synonymous SNPs) based on– Sequence homology – Physical properties of amino acids

• Can be applied to naturally occurring nonsynonymous polymorphisms and laboratory-induced missense mutations

Copy all SNP IDs and paste into SIFT. Choose

“Submit Query”

Compare Build numbers

Getting more info for rs2274974

Enter “rs2274974”

Allele info

Protein nameContig name

mRNA name

Position of SNP in mRNA, protein, contig

Flanking sequence, IUPAC code, flanking seq

Build number

Select protein

Scroll down

Note AA1, AA2, and position

Copy FASTA-formatted protein sequence

Paste FASTA-formatted protein sequence

Enter AA substitution[Letter1-position-Letter2]

Substitution occurs at AA 566

Scroll down

Scroll down

Check tolerance of AA substitutions

Tolerance of specified substitution

“Substitution at pos 566 from G to E is predicted to AFFECT PROTEIN FUNCTION with a score of 0.01.

Polymorphism Phenotyping

• Tool for prediction of possible impact of amino acid substitution (i.e., non-synonymous SNPs) on protein structure and function based on:– Amino acid sequence

• What part of the protein did the SNP occur? (E.g., active site, binding site, transmembrane region)

– Multiple alignments with homologous proteins and mammalian orthologues

• How compatible is the substitution based on proteins of comparable sequence?

– 3D structural properties with the substituted amino acid• What is the substitution’s effect on the protein’s physiochemistry?

(E.g., hydrophobicity, electrostatic interactions, ligand binding)

PolyPhen data flow

Four potential predictions

• Probably damaging– It is with high confidence supposed to affect protein

function or structure

• Possibly damaging– It is supposed to affect protein function or structure

• Benign– Most likely lacking any phenotypic effect

• Unknown– Lack of data do not allow PolyPhen to make a

prediction

Copy FASTA-formatted protein sequence

Enter AA position, ancestral AA, and substituted AA

Enter SNP rs#

In dbSNP Build 129, corresponds to protein NP_005948.3

Query vs. SNP Collection

Query SNP Collection

Prediction Probably damaging

Probably damaging

PSIC 2.093 2.172

db SNP Build# N/A 126

References

• NCBI dbSNP– http://www.ncbi.nlm.nih.gov/sites/entrez

• SIFT– http://sift.jcvi.org/

• PolyPhen– http://genetics.bwh.harvard.edu/pph/index.html