1. PolyPhen and SIFT: Tools for predicting functional effects of SNPs Epi 244 Spring 2009 Sam S. Oh

2. 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

3. DNA → RNA → Protein

4. Example: sickle-cell anemia A to T SNP of beta-globin gene results in glutamate (hydrophilic) to valine (hydrophobic) substitution

5. Example: MTHFR Folate metabolism

6. Finding MTHFR SNPs

7. Highlight all refSNP numbers (use scroll bar) and copy

8. Note Build number (currently Build 130)

9. 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

10. Copy all SNP IDs and paste into SIFT. Choose “Submit Query” Compare Build numbers

12. Getting more info for rs2274974 Enter “rs2274974”

13. Allele info Protein name Contig 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

14. Copy FASTA-formatted protein sequence

15. Paste FASTA-formatted protein sequence Enter AA substitution [Letter1-position-Letter2]

16. Substitution occurs at AA 566 Scroll down

17. Check tolerance of AA substitutions

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

19. 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)

20. PolyPhen data flow

21. 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

22. Copy FASTA-formatted protein sequence Enter AA position, ancestral AA, and substituted AA

24. Enter SNP rs# In dbSNP Build 129, corresponds to protein NP_005948.3

25. Query vs. SNP Collection QuerySNP Collection PredictionProbably damaging PSIC2.0932.172 db SNP Build#N/A126

26. 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