Richard Myers

New genomic tools for molecular studies of evolutionary change in threespine sticklebacks

The dramatic radiation of sticklebacks in different post-glacial environments provides a unique opportunity to study the molecular mechanisms that underlie rapid evolutionary change in vertebrates. We have developed a number of genomic and genetic tools to facilitate further study of a wide range of morphological, physiological and behavioral traits in sticklebacks. A large collection of microsatellite markers has previously been developed for use in genome-wide linkage mapping of interesting traits in crosses between different stickleback forms. cDNA libraries have been generated and EST sequencing projects have begun to isolate stickleback homologs of developmental control genes. Large insert BAC libraries have been built to compare chromosome regions of interest from both anadromous and freshwater stickleback populations. Large scale fingerprinting of one of these libraries has been used to assemble overlapping contigs of BAC clones for chromosome walking and positional cloning. Together with recent development of methods to make transgenic sticklebacks, these tools should make it possible to identify the molecular basis of many different evolutionary traits in stickleback, and to begin to answer longstanding questions about the numbers and types of mutations that control the appearance of new morphological, physiological, and behavioral traits during vertebrate evolution.

Genetic Variation in the Proximal Promoter of ABC and SLC Superfamilies: Liver and Kidney Specific Expression and Promoter Activity Predict Variation

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (−250 to +50 bp) and flanking 5′ sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (π) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

Failure to replicate an association of SNPs in the oxidized LDL receptor gene (OLR1) with CAD

BackgroundThe lectin-like oxidized LDL receptor LOX-1 (encoded by OLR1) is believed to play a key role in atherogenesis and some reports suggest an association of OLR1 polymorphisms with myocardial infarction (MI). We tested whether single nucleotide polymorphisms (SNPs) in OLR1 are associated with clinically significant CAD in the Atherosclerotic Disease, VAscular FuNction, & Geneti C Epidemiology (ADVANCE) study.MethodsADVANCE is a population-based case-control study of subjects receiving care within Kaiser Permanente of Northern California including a subset of participants of the Coronary Artery Risk Development in Young Adults (CARDIA) study. We first resequenced the promoter, exonic, and splice site regions of OLR1 and then genotyped four single nucleotide polymorphisms (SNPs), including a non-synonymous SNP (rs11053646, Lys167Asn) as well as an intronic SNP (rs3736232) previously associated with CAD.ResultsIn 1,809 cases with clinical CAD and 1,734 controls, the minor allele of the coding SNP was nominally associated with a lower odds ratio (OR) of CAD across all ethnic groups studied (minimally adjusted OR 0.8, P = 0.007; fully adjusted OR 0.8, P = 0.01). The intronic SNP was nominally associated with an increased risk of CAD (minimally adjusted OR 1.12, p = 0.03; fully adjusted OR 1.13, P = 0.03). However, these associations were not replicated in over 13,200 individuals (including 1,470 cases) in the Atherosclerosis Risk in Communities (ARIC) study.ConclusionOur results do not support the presence of an association between selected common SNPs in OLR1 and the risk of clinical CAD.

Technetium-99m pyrophosphate myocardial uptake in patients with stable angina pectoris.

99m-technetium (Tc) pyrophosphate myocardial scintigrams of 55 patients with stable angina pectoris were compared with those of 13 normal subjects. The mean scintigraphic score, obtained by averaging the blinded interpretations of four readers scoring on an integral scale from 0 to 4, was significantly higher for the patients with angina than for the control subjects (1.36 compared with 0.48, P less than 0.001). Among the patients with angina, those who had a prior myocardial infarction had a higher mean scintigraphic grade than those without a previous infarction (1.73 versus 1.15, P less than 0.005), and the mean grade in both groups was higher than that of control subjects (P less than 0.001). Radionuclide uptake was predominantly diffuse in the patients with angina pectoris (70%), although in those with greater uptake accumulation tended to be localized. Three of the 68 subjects had high levels of radionuclide uptake but no clinical evidence of acute myocardial injury. This study demonstrates that excess myocardial accumulation of 99m-Tc pyrophosphate can occur in patients with stable angina pectoris.

S14-03 From trait to base pairs: Parallel evolution of pelvic reduction in three-spined sticklebacks occurs by repeated deletion of a tissue-specific pelvic enhancer at Pitx1

There are approximately 400 breeds of dog worldwide, displaying significant differences in body size, coat type, leg length, head shape, and more. We have undertaken a genome wide association study (GWAS), involving hundreds of dogs to identify the genes and mutations responsible for this range of variation. A total of 835 dogs from nearly 80 breeds were genotyped using the Affymetrix version 2.0 canine SNP chip. The dataset included 41,678 SNPs spanning all 38 autosomes and the x-chromosome after removing SNPs that had call rates of <90%, were heterozygous in >60% of individuals or could not be successfully reproduced. We then assessed the data for a large number of phenotypes. Among the most interesting traits we have focused on is disproportionate dwarfism or chondrodysplasia, a defining trait for at least nineteen breeds such as the Corgi, Dachshund and Basset Hound. Multi-breed GWAS revealed a single major locus associated with the trait. Homozygosity mapping, second generation sequencing, and cDNA analysis identified a conserved, expressed retrogene that segregates exclusively with the phenotype. In an effort to find the original source of the retrogene, we sequenced a number of dog breeds as well as a set of wolves from around the world. While the retrogenes is not present in any of the wolf populations, we do find the combination of gene and haplotype necessary to create the original retrotransposition in wolves from Europe and the Middle East. This combination was not found in any of the modern dogs breeds suggesting that the original chondrodysplastic mutation predated the formation of breeds and the ancestor of all chondrodysplastic dogs was unlike any of the breeds commonly associated with the phenotype today. Other traits have been similarly examined. To map coat variation we took advantage of both intra and inter breed differences to identify loci for three phenotypes: length, curl, and growth pattern. By comparing data from Dachshunds of differing phenotype to the larger multi-breed analysis we have been able to identify the gene and mutation that controls the facial hair growth patterns seen in breeds like schnauzers. Similarly we have found a keratin that controls the degree of curl, and a growth factor that dictates fur length. Combinations of genotypes at these three genes can account for over 90% of fur phenotypes observed among American Kennel Club (AKC) breeds, demonstrating the value of the canine system for understanding the genetic underpinnings of seemingly complex phenotypes.