positional cloning of human diseasegenes: a reversal of scientific priorities

D Botstein, et al. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32: 314-331.

JM Rommens, …, LC Tsui, FS Collins. 1989. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065.

FS Collins. 1992. Positional cloning: let's not call it reverse anymore. Nat Genet 1: 3-6.

functional cloning

disease phenotype

hypothesize function

chromosomal position

clonethe gene

Prior To 1989

positional cloning

disease phenotype

hypothesize function

chromosomal position

clonethe gene

AFTER 1989

Botstein’s insight on human genetics

gene

marker

phenotypecause-and-effect

statistical correlation

posi

tiona

lly c

lose

consider a mutagenesis and screening experiment that we might do in animalsgenetic variations occur naturally and humans screen themselves for diseases

cause-and-effect is strongest when disease has Mendelian inheritance patternMARKER 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 tolocalize Mendelian disease genes in afamily based positional cloning study

w/o genetic recombination linkage across chromosome would be completeapproximately one recombination per chromosome per meiosis human mutation rate is known to be 10-8 per site per generationthere is no benefit to genotyping more markers than genetic recombinationssize of family analyzed determines number of genetic recombinationsadditional experiments needed to identify gene once localized to Mb region

Online Mendelian Inheritance inMan 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

OMIM Statistics for 31 March 2011 Autosomal X-Linked Y-Linked Mitochondrial Total

* gene of known sequence 12,605 620 48 35 13,308+ gene of known sequence and phenotype 314 18 0 2 334# phenotype of known molecular basis 2,725 236 4 28 2,993% phenotype of unknown molecular basis 1,632 134 5 0 1,771phenotype suspected to be Mendelian 1,831 130 2 0 1,963

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. 2003. The International HapMap Project. Nature 426: 789-796.

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 susceptibilityReich DE, Lander ES. 2001. On the allelic spectrum of human disease. Trends Genet 17: 502-510

CDRV hypothesis: a large number of rare allelic variants account for the genetic variance in disease susceptibilityTerwilliger JD, Weiss KM. 1998. Linkage disequilibrium mapping of complex disease: fantasy or reality? Curr Opin Biotechnol 9: 578-594

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. 2009. Nature 461: 747-753

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. 2009. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461: 399-401

percent SVR by genotype andC-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 blogswhich humbled that much-hyped paper in just a matter of days

http://www.wired.com/wiredscience/2010/07/Serious-flaws-revealed-in-longevity-genes-study

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)