rare Mendelian diseases versus common multi-factorial diseases

e.g., cystic fibrosis is one of the most common life-shortening childhood-onset inherited diseases in the United States, affecting 1 in 3900 births; one of every 31 individuals is a carrier of the recessive disease allele

e.g., in the United States, the lifetime risk for developing cancer is slightly less than 1 in 2 for men and slightly more than 1 in 3 for women; although there are subclasses of cancer like early onset breast cancer that obey Mendelian rules, they make up a negligible fraction of the overall disease

Mendelian diseases Complex diseasespeople affected young children older adultshereditability run in families some to nonegenes involved one major gene 10's to 100'senvironment less important very importantcases mapped 2284 (in OMIM) small handful

1980: DNA markers are the key to identifying Mendelian disease genes

Botstein D, White RL, Skolnick M, Davis RW. 1980. Am J Hum Genet 32: 314-331

the fact we cannot experiment on people does not preclude us from doing genetics; all we need are more DNA markers to differentiate individuals; the markers themselves need not cause the disease; they need only be sufficiently close to the gene that does; markers can take many forms including RFLPs, microsatellites, SNPs, etc.

(a) disease cosegregates with marker A; (b) disease cosegregates with marker B prior to a recombination event, and marker C after

power to localize genes is reliant on having sufficient number of recombinations

(a) (b)

1989: successful cloning of CFTR gene responsible for cystic fibrosis

Rommens JM, …, Tsui LC, Collins FS.1989. Science 245: 1059-1065

the F508 mutation is one of over 1500 eventually found in this gene but it is still the most important; this was the first disease gene identified by its chromosomal position instead of by its hypothesized function; Huntington’s was mapped before CF but the unexpected nature of that mutation (triplet repeat) took longer to solve

Mutation Prevalence (%)DF508 79G551D 2.17R117H 0.70

621+1 (G>T) 0.50G542X 0.50

N1303K 0.351717-1 (G>T) 0.28

R1162X 0.14R553X 0.14

3849+10KB (G>T) 0.07R334W 0.07W1282X 0.07

Online Mendelian Inheritance in Man currently lists 2284 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 April 6, 2008 Autosomal X-Linked Y-Linked Mitochondrial Total

* gene of known sequence 11,577 538 48 37 12,200+ gene of known sequence and phenotype 358 30 0 0 388# phenotype of known molecular basis 2,065 191 2 26 2,284% phenotype of unknown molecular basis 1,471 129 5 0 1,605phenotype suspected to be Mendelian 1,954 140 2 0 2,096

1990-6: birth and death of sib pair analysis for linkage based studies of

common multi-factorial diseases

Risch N, Merikangas K. 1996. The future of genetic studies of complex human diseases. Science 273: 1516-1517

linkage analysis had been successfully used to find genes for Mendelian diseases; in 1990, Risch popularized a method (sib pairs) to find genes for complex multi-factorial diseases; that method failed and they wanted to propose a different method that would be more powerful

association studies were to be performed on functional polymorphisms for as many candidate genes as technically feasible, the entire genome if need be, regardless of how impractical that was; at least the number of patients would no longer be a limiting factor

past, present, and (near) future genetic studies of human diseases

the biggest change from before is we are now doing these studies on the general population instead of rare families

we must study common diseases in the general population because rare mutations that cause Mendelian subcategories of disease are not typically responsible for those diseases in the general population

the problem however is that without families we lose statistical power and must compensate by gathering more data

Past Present Futuresamples used rare families populations populationsmethodology linkage (pedigrees) linkage disequilibrium direct associationDNA markers microsatellites HapMap SNPs 1000 genomes

applicable to Mendelian diseasescommon diseases w/common variants

common diseases w/moderate variants

causality link definitely yes probably not probably not

population bottleneck, subsequent recombination, linkage disequilibrium

although the parameters and details of the human population bottleneck are still not settled, the order of magnitude estimates are that our species collapsed to 15,000 individuals 70,000 years ago; assuming few new mutations the only thing that would have happened since that time is recombination, and we can model any particular individual’s genome as a mosaic of segments from these 15,000 ancestral genomes

mutation is the red ; chromosomal stretches derived from common ancestor are yellow; the new stretches due to recombination are blue

we need not test all the functional polymorphisms, just enough markers

to be within linkage disequilibrium

linkage disequilibrium (LD) is the non-random association of two alleles on adjacent loci; there are many reasons why this might happen, but for the HapMap, the assumption is that human variation is intrinsically limited because of the recent population bottleneck

common-disease-common-variant versus common-disease-rare-variant

CDCV hypothesis: a few common allelic variants account for most of the genetic variance in disease susceptibility

Reich 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 susceptibility

Terwilliger JD, Weiss KM. 1998. Linkage disequilibrium mapping of complex disease: fantasy or reality? Curr Opin Biotechnol 9: 578-594

for complex reasons having to do with human population history, linkage disequilibrium would only work in diseases where the CDCV hypothesis is valid; the best justification for the HapMap was that one common variant has more public health impact than many rare variants, so it makes sense to find these first

multiple rare alleles contribute to low plasma HDL cholesterol levels

for 128 individuals with low plasma HDL-C, 21 (16%) had variants not present in the high HDL-C group; conversely, only 3 (2%) of individuals with high plasma HDL-C had variants not present in the low HDL-C group (P < 0.0001); Cohen JC, …, Hobbs HH. 2004. Science 305: 869-872

3 non-synonymous variants in 3 out of 128 outlier samples

15 non-synonymous variants in 21 out of 128 outlier samples

HDL-C levels

candidate genes ABCA1, APOA1, LCAT

International HapMap Consortiumhttp://www.hapmap.org/thehapmap.html.en

International HapMap Consortium. 2005. Nature 437: 1299-1320

phase I genotyped common SNPs of frequency greater than 0.05 in every 5-kb interval for 269 individuals from 3 populations in Africa (YRI), Europe (CEU), and Asia (CHB+JPT)

solid line represents ENCODE region data, dashed line represents neutral model with constant population size and random mating and no ascertainment biases

7 tag SNPs capture all the common variation in a locus on chromosome 2

left plot shows the 7 haplotypes and their respective counts, with colored circles indicating SNP positions where a haplotype has the less common allele; groups of SNPs captured by a single tag SNP (r2 0.8) using a pairwise tagging algorithm have the same color; right plot shows the SNPs mapped to a genealogical tree relating the seven haplotypes for this region

Wellcome Trust Case Control genome wide association studies

Wellcome Trust Case Control Consortium. 2007. Nature 447: 661-678

genotype 500,000 SNPs from HapMap in a British population of 2,000 affected individuals for each of 7 major diseases, with another 3,000 shared individuals for control

of the 14 variants for which there was a strong prior evidence of association to the studied diseases all but two (APOE and INS) were reproduced by this analysis

genome wide scan in seven diseases; y-axis represents statistical significance using -log10 of a p-value

the chromosomes are shown in alternating colors; significant SNPs with p-value <110-5 are in green

a doubling in relative risk for a disease is not as bad as it sounds

Disease Year Sample IdentifiedIncreased

relative riskMacular degeneration

2005 1,700 1 new gene 400% to 600%

Inflammatory bowel disease

2006 4,500 1 new gene 120%

Prostate cancer 2007 17,5002 variants

(just 1 new)123%

Obesity 2007 38,700 1 new gene 67%

Type 2 diabetes 2007 32,5009 variants

(just 3 new)80%

Heart disease 2007 41,600 1 variants 25% to 40%

Couzin J, Kaiser J. 2007. Science 316: 820-822

with the notable exception of macular degeneration there is only a doubling in relative risk; the 120% increase in relative risk for inflammatory bowel disease only bumps the absolute risk from 0.5% to 1.1%

but gene therapy remains elusive 19 years after the cystic fibrosis gene

Jesse Gelsinger (June 18, 1981 to September 17, 1999) was the first person identified as having died in a clinical trial for gene therapy. He was only 18 years old. Gelsinger suffered from ornithine transcarbamylase OTC deficiency, a disease of the liver whose victims are unable to metabolize ammonia, a byproduct of protein breakdown.

Gelsinger was injected with adenoviruses containing the corrected gene in the hope that it would manufacture the much needed enzyme. He died four days later, having suffered a massive immune response, triggered by the viral vector used to transport the gene into his cells. This led to multiple organ failure and brain death.

Food and Drug Administration investigators concluded that scientists involved in the trial, including lead researcher Dr. James M. Wilson (University of Pennsylvania), broke several rules of conduct: (a) Inclusion of Gelsinger as a substitute for another volunteer who had dropped out, despite his having high ammonia levels that should have led to his exclusion from the trial, (2) Failure by the university to report that 2 other patients had experienced serious side effects from the therapy, (3) Failure to mention the deaths of monkeys given a similar treatment, as should be been done for the informed consent.

The university paid the parents an undisclosed amount.