Human non-synonymous SNP: molecular function, evolution and disease Shamil Sunyaev Genetics Division, Brigham & Women’s Hospital Harvard Medical School Harvard-M.I.T. Division of HST

Effect on molecular function Phenotype Natural selection Medical Genetics Structural Biology Biochemistry Evolutionary Genetics

Predicting the effect of mutations in proteins

Why is this useful? n Understanding variation in molecular function and structure n Evolutionary genetics: comparison of polymorphism and divergence rates between different functional categories is a robust way to detect selection

Linkage analysis Rare

Classical association studies ControlDisease Common

Why is this useful? n Rare human developmental disorders / mouse mutagenesis screens: linkage studies are impossible n Genetics of complex disease: SNP prioritization n Genetics of complex disease: Rare variants

Technically, polymorphism should not exist!

Quantitative trait Mendelists Biometricians Forces to maintain variation: Selection Mutation

Common disease / Common variant Trade off (antagonistic pleiotropy) Balancing selection Recent positive selection Reverse in direction of selection Examples APOEAlzheimer’s disease AGTHypertension CYP3AHypertension CAPN10Type 2 diabetes

Individual human genome is a target for deleterious mutations ! ~40% of human Mendelian diseases are due to hypermutable sites Frequency of deleterious variants is directly proportional to mutation rate (q=  /s)

Multiple mostly rare variants Many deleterious alleles in mutation-selection balance Examples Plasma level of HDL-C Plasma level of LDL-C Colorectal adenomas

What about late onset phenotypes?

Harmful mutations n Function: damaging n Evolution: deleterious n Phenotype: detrimental n Advantageous pseudogenization (Zhang et al. 2006) n Gain of function disease mutations n Sickle Cell Anemia

protein multiple alignment profile

PolyPhen

Prediction rate of damaging substitutions possibly probably Disease mutations Divergence 82%57% 9% 3% Polymorphism 27% 15%

10% of PolyPhen false-positives are due to compensatory substitutions

Polyphen Phylogenetic measures PAM * * BLOSUM * * BLOSUM * * BLOSUM * * Site-specific structural/phylogenetic measures * * * Estimate of selection coefficient Williamson et al., PNAS 2005

de novo mutation effect spectrum NO DELETERIOUS POLYMORPHISM LOTS OF DELETERIOUS POLYMORPHISM Effect of new mutation may range from lethal, to neutral, to slightly beneficial

Mutation effect spectrum NO DELETERIOUS POLYMORPHISM LOTS OF DELETERIOUS POLYMORPHISM ?

Neutral mutation model Human ACCTTGCAAAT Chimpanzee ACCTTACAAAT Baboon ACCTTACAAAT Prob(TAC->TGC)  Prob(TGC->TAC) Prob(XY 1 Z->XY 2 Z) 64x3 matrix

Strongly detrimental mutations

Effectively neutral mutations

Mildly deleterious mutations

54 genes, 757 individuals inflammatory response 236 genes, individuals DNA repair and cell cycle pathways 518 genes, individuals

The majority of missense mutations observed at frequency below 1% are deleterious Frequency itself is a reliable predictor of function!

Fitness and selection coefficient Wild typeNew mutation N 1 = 4 N 2 = 3 Fitness 1 N1N1 N2N2 = 1 – s Selection coefficient

Mildly deleterious mutations 54 genes, 757 individuals inflammatory response 236 genes, individuals DNA repair and cell cycle pathways 518 genes, individuals

Fraction of detectable polymorphism

Human effective population size present past Estimation of selection coefficient - simulation

Human effective population size present past -log(s) F singl (s)F MAF>25% (s) Selection coefficient SNP probability to be observed

Classical association studies ControlDisease Common

“Mutation enrichment” association studies ControlDisease Rare

ControlDisease “Mutation enrichment” association studies

Rare missense variants in NPC1L1 gene contributes to variability in cholesterol absorption and plasma levels of low-density lipoproteins (LDLs) Cohen J et al., PNAS 2006 in press Nonsynonymous sequence variants in ABCA1 gene were significantly more common in individuals with low HDL-C ( 95th percentile). Cohen J et al., Science 2004 Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas Fearnhead NS et al., PNAS 2004 “Mutation enrichment” association studies

Cholesterol

Adopted from Brewer et al., 2003

Effect of rare nsSNPs on HDL-C

What about common alleles of smaller effect? n Population of 3500 individuals with known plasma levels of HDL-C n Population includes both genders and three ethnic groups n 839 SNPs genotyped n Independent population of 800 individuals for validation

What about common alleles of smaller effect? n Introduce a linear model (ANCOVA) n Subsequently add SNPs to the linear model n Include SNPs based on the likelihood ratio test n Prioritizing SNPs based on conservation did not help

Effect of common SNPs on HDL-C HDL

And a different population … HDL

Acknowledgements The lab: Gregory Kryukov, Steffen Schmidt, Saurabh Asthana, Victor Spirin, Ivan Adzhubey Bioinformatics:Human genetics: Vasily RamenskyJonathan Cohen