1. Nebojsa Mirkovic, Carles Ferrer-Costa, Marc A. Marti-Renom, Alvaro N.A. Monteiro, Andrej Sali Functional Consequences of the SNPs : BRCA1, Membrane Transporters and More

2. Single Nucleotide Polymorphisms (SNPs) single base pair replacements of appreciable allelic frequency in the population ( >1% ); predominant form of human genetic variation (90%); number predicted to range from one to several million per genome; predicted frequency: 1/1000bp; predicted number per gene: 4-12 on average (limited datasets); classification by genomic location: ncSNPs, cSNPs (synonymous, non-synonymous: 24,000-40,000 per genome).

3. identify gene(s) that underlie numerous genetic disorders and multifactorial traits (eg, pharmacological response); SNPs are probably the biggest class of pathogenic changes in the human genome (coding and regulatory regions); several genetic variants connected to genetic disorders ( E4 allele of APOE with Alzheimer disease, FV Leiden allele with deep-venuos thrombosis, and CCR532 with resistance to HIV infection ); numerous indirect evidence of functional impact; markers of choice in genetic studies. Significance of SNP Analysis

4. Br 32 Br 42 * Br Identification of Sequence Variants in Genes of Interest

5. Project Goal What is the likelihood that a given SNP destroys the function of a protein? Approach design a classification function that can predict functional impact of a particular SNP and relies on a combination of sequence, structure and genetic properties from a well-characterized set of nsSNPs; build ModSNP, a structural database of SNPs, containing protein structure models for all known nsSNPs; apply the classification function to a number of specific examples and to all the SNPs in ModSNP.

6. BRCA1 Project

7. Human BRCA1 Tandem BRCT Repeats 200 aa RING NLS BRCT Globular regions Nonglobular regions BRCA1 BRCT repeats Williams, Green, Glover. Nat.Struct.Biol. 8, 838, 2001

8. Missense Mutations in BRCT Domains by Function transcription activation cancer associated V1665M D1692N G1706A D1733G M1775V P1806A M1652K L1657P E1660G H1686Q R1699Q K1702E Y1703H F1704S L1705P S1715N S1722F F1734L G1738E G1743R A1752P F1761I F1761S M1775E M1775K L1780P I1807S V1833E A1843T M1652T V1653M L1664P T1685A T1685I M1689R D1692Y F1695L V1696L R1699L G1706E W1718C W1718S T1720A W1730S F1734S E1735K V1736A G1738R D1739E D1739G D1739Y V1741G H1746N R1751P R1751Q R1758G L1764P I1766S P1771L T1773S P1776S D1778G D1778H D1778N M1783T A1823T V1833M W1837R W1837G S1841N A1843P T1852S P1856T P1859R C1787S G1788D G1788V G1803A V1804D V1808A V1809A V1809F V1810G Q1811R P1812S N1819S C1697R R1699W A1708E S1715R P1749R M1775R M1652I A1669S no transcription activation unknown not cancer associated unknown

9. Mapping of the Mutations on the Surface of BRCA1 BRCT Domains R1699Q/W G1743R K1702F L1657P D1692N D1773G E1660G

10. Location of Putative BRCT-Protein Interaction Site RMSMVVSGLTPEEFMLVYKFARKHHITLTNLITEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYF WVTQSIKERKMLNEHDFEVRGDVVNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQL CGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNGFHAIGQMCEAPVVTREWVLDSVALYQCQELDTYLI PQIP

11. Mutation Features MutationMutation likelihood Phylo- genetic entropy Accessi bility Residue rigidity Neighor- hood rigidity Percent relative volume change Polarity change Percent relative ASA change Charge change M1652I 10.27B-0.53-1.252 0.1 0 0 00 00 M1652K 0.27B-0.53-1.253 0.2 -2 0 00 10 V1653M 10.00B-1.17-0.8316 0.8 0 0 00 00 L1657P 0.00E-0.65-1.05-32 -1.8 6 00 00 E1660G 0.43E 0.18-0.67-57 -2.6 1-7 00 00 V1665M 10.00B-0.95-0.8716 0.8 0 0 00 00 Y1703H 00.00B-0.35-0.58-21 -1.4 -20 00 10

12. YES charge change + buriedness YES NO <30A 3 ≥60A 3 <90A 3 ≥90A 3 rigid (<-0.7) non-rigid ( ≥ -0.7) exposed buried residue rigidity volume change functional site -- - - - 0 or 1 class phylogenetic entropy polarity change 0 <0 - NO non 0 ≥0≥0 YES + - “Decision” Tree for Predicting Functional Impact of Genetic Variants NO 2 class <60A 3 ≥30A 3 neighborhood rigidity buriedness residue rigidity volume change charge change polarity change phylogenetic entropy other information (helix breaker, turn breaker) other information (helix breaker, turn breaker) + mutation likelihood volume change - residue rigidity volume change polarity change phylogenetic entropy other information (helix breaker, turn breaker) + mutation likelihood functional site buriedness START neighborhood rigidity neighborhood rigidity charge change

13. Rationalization of Functionally Characterized Mutants M1652I ++ Likely replacement, ‘I’ allowed in multiple sequence alignment. L1657P ?- Predicted binding site, large volume change in rigid neighborhood, unlikely replacement at completely preserved position. C1697R -- Two class polarity change, unlikely replacement at completely preserved position. S1715N ?- Not explained. Likely replacement. Prediction of Function of Uncharacterized Mutants M1652T - Large volume change in rigid neighborhood. V1653M + Likely replacement. G1738R - Very large volume change at flexible position, unlikely replacement at completely preserved position. T1773S + Likely replacement, predicted binding site.

14. SNP Web Server

15. Membrane Transporter Project

16. Chang, Roth. Science 293, 1793, 2001 Eco-msbA dimer, 1jsq Domain Organization and Structure of E.Coli ABC Transporter MsbA 1582 crystalized not crystalized A BC A Walker motif A (375-383) B Walker motif B (501-505) C ABC transporter signature (481-489) transmembranenucleotide binding transmembrane intracellular extracellular

17. PMT Database a database of in-site detected and/or verified polymorphisms in various mammalian membrane transporters with ample population genetics data (allelic frequencies, ethnic distribution); 59 proteins divided in two groups (24 and 25 proteins respectively) depending on the SNP-detection approach; 10 ABC transporters, 5 with SNPs reported so far (BSEP, MDR1, MDR3, MRP1, MRP2).

18. http://guitar.rockefeller.edu/modbase Pieper et al., Nucl. Acids Res. 2002.

19. ModPipe Results for Transmembrane Proteins from the PMT Database 49 proteins submitted; 11 proteins could not be modeled; 15 proteins have bad fold assessment; 8 proteins have good fold assessment, but bad model score; 15 proteins have good fold assessment and good model score. ProteinBSEPMDR1MRP2MDR3MRP1 Coverage6/98/133/182/70/5 Coverage of ABC Transporters

20. Modeled regions of MDR1 Predicted Domain Organization and Structure Model of Human ABC Transporter MDR1 11280 modeled not modeled repeat 1repeat 2 intracellular, extracellular ATP-binding loop transmembrane helix ATP-binding cassette 1jj7A (383-625)1g291 (1034-1274) 38 3 62 5 1034 1274

21. MDR1 (ABC-B1) Numb. of models Seq. Id. [%] E-valueCoverage (aa) Mapped SNPs ABC1643-18-94 -63~2403 ABC2124-62~2405 SNPASA Secondary Structure HydrophilicityVolume Blosum score Energy S400NHalfCoil- 0.770+23+ 1- 3.01 L662RHalfCoil- 3.690+24- 2- 4.42 R669CExposedHelix+ 2.710- 62- 3- 14.11 P1051AHalfStrand- 0.560- 23- 1- 3.28 W1108RHalfCoil- 4.440- 15- 3+ 2.80 S1141TExposedCoil+ 0.400+20+ 1- 3.72 V1251IHalfStrand+ 0.800+ 19+ 3- 1.24 T1256KHalfCoil- 1.700+ 42- 1- 4.87 Sequence and structure features of SNPs

22. Significance a comprehensive structural study of the functional impact of mutations focused on a particular protein family; a large and informative mutation set used; rationalization of the characterized mutation set used to develop a predictive tool; possible contribution to genetic studies (eg, candidate gene approach) and medical practice (with other methods).