positional cloning of human disease genes: a reversal of scientific priorities D Botstein, et al Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32: JM Rommens, …, LC Tsui, FS Collins Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: FS Collins Positional cloning: let's not call it reverse anymore. Nat Genet 1: 3-6. functional cloning disease phenotype hypothesize function chromosomal position clone the gene Prior To 1989 positional cloning disease phenotype hypothesize function chromosomal position clone the gene AFTER 1989

Botstein’s insight on human genetics gene marker phenotype cause-and-effect statistical correlation positionally close consider a mutagenesis and screening experiment that we might do in animals genetic variations occur naturally and humans screen themselves for diseases cause-and-effect is strongest when disease has Mendelian inheritance pattern MARKER is any sequence that is variable and does not have to be in the gene Q: why should there be a statistical correlation between the marker and gene?

genetic recombination is required to localize Mendelian disease genes in a family based positional cloning study w/o genetic recombination linkage across chromosome would be complete approximately one recombination per chromosome per meiosis human mutation rate is known to be per site per generation there is no benefit to genotyping more markers than genetic recombinations size of family analyzed determines number of genetic recombinations additional experiments needed to identify gene once localized to Mb region

Online Mendelian Inheritance in Man currently lists 2993 phenotypes whose molecular basis is known the success of what came to be known as positional cloning was a tribute to an admission of ignorance; we did not know enough human biology to guess the likely gene for a disease so we focused instead on determining where the gene was on the chromosome; for the overwhelming majority of cases, the answer turned out to be a completely unknown gene that no scientist had hypothesized

interesting traits tend to be less genetic and more environmental interesting to scientists interesting to the public genetics is important environment is important Mendelian diseases psychiatric disorders complex diseases human behaviour

making positional cloning work on diseases that the public cares about despite the successes of positional cloning with Mendelian diseases analogous procedures for complex diseases FAILED spectacularly failure was attributed to inability to get large enough families to compensate for weaker cause-and-effect in diseases that are not entirely genetic sample sizes need not be a limitation if we do not restrict the studies to families and instead use affected individuals from the population human population originated from “family” of 15,000 individuals that survived a near death experience 70,000 years ago (numbers controversial) therefore, human variation occurs in haplotype blocks whereby polymorphisms are statistically correlated on length scales of a few kb’s novel mutations complicate the situation but to a first approximation the way to find complex disease genes is to increase the number of makers

SNPs, haplotypes, and tag SNPs (a) Chromosomal region with (three) sites of variation indicated. (b) A haplotype is a particular combination of SNP alleles along the chromosome. Here we show all 4 observed haplotypes in a surveyed population for a 6000 bp region with 20 SNPs. (c) Genotyping only 3 tag SNPs out of the available 20 is sufficient to distinguish between all observed haplotypes. RA Gibbs, et al The International HapMap Project. Nature 426:

genome wide association study (or GWAS) to find complex disease genes in population based positional cloning simplifying assumptions were made about the nature of disease variants CDCV hypothesis: a few common allelic variants account for most of the genetic variance in disease susceptibility Reich DE, Lander ES On the allelic spectrum of human disease. Trends Genet 17: CDRV hypothesis: a large number of rare allelic variants account for the genetic variance in disease susceptibility Terwilliger JD, Weiss KM Linkage disequilibrium mapping of complex disease: fantasy or reality? Curr Opin Biotechnol 9: one common variant has more public health impact than many rare ones GWAS is easy GWAS is hard

genetic loci found and heritability explained for several complex traits Manolio, et al Nature 461:

feasibility of detecting genetic variants by risk allele frequency and strength of genetic effect

pharmacogenomics: genetic variation in response to treatment adverse drug reactions are a major cause of hospitalization and death; for USA 2.2 million serious cases and 100,000 deaths a year human metabolism to detoxify drugs either makes them more water soluble for excretion in urine or more fat soluble for excretion in stool variability associated with cytochrome P450 enzyme detoxification is 1000-fold; hence one person’s food is another person’s poison BiDil is a patented (FDA in 2005) combination of two generic drugs specifically indicated for African Americans with congestive heart failure Race in a Bottle (Scientific American August 2007)

19q13.13 (IL28B gene) region containing genome wide determinant of response to hepC treatment adapted from D Ge, et al Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461:

percent SVR by genotype and C-allele frequencies by population SVR (sustained virological response) refers to absence of detectable virus at end of follow up evaluation, indicating successful response to treatment individuals homozygous for C-allele respond better to treatment (regardless of population) and C-allele is most often found in East Asian populations

an example of a flawed GWAS study 1 July 2010: this paper claimed to have identified genetic factors for longevity; it was published with much fanfare at Sciencexpress and got extensive media coverage; but within days the result was challenged by sharper eyes

and a lesson on the power of blogs which humbled that much-hyped paper in just a matter of days 7 July 2010: data of Sebastiani et al (right) are unusual in that all of the highest-ranked SNPs stand out by themselves and are not flanked by a column of highly-ranked SNPs as in other studies like the Wellcome Trust Case Control Consortium (left)