M Lathrop

SLCO1B1 variants and statin-induced myopathy--a genomewide study.

BACKGROUNDnLowering low-density lipoprotein cholesterol with statin therapy results in substantial reductions in cardiovascular events, and larger reductions in cholesterol may produce larger benefits. In rare cases, myopathy occurs in association with statin therapy, especially when the statins are administered at higher doses and with certain other medications.nnnMETHODSnWe carried out a genomewide association study using approximately 300,000 markers (and additional fine-mapping) in 85 subjects with definite or incipient myopathy and 90 controls, all of whom were taking 80 mg of simvastatin daily as part of a trial involving 12,000 participants. Replication was tested in a trial of 40 mg of simvastatin daily involving 20,000 participants.nnnRESULTSnThe genomewide scan yielded a single strong association of myopathy with the rs4363657 single-nucleotide polymorphism (SNP) located within SLCO1B1 on chromosome 12 (P=4x10(-9)). SLCO1B1 encodes the organic anion-transporting polypeptide OATP1B1, which has been shown to regulate the hepatic uptake of statins. The noncoding rs4363657 SNP was in nearly complete linkage disequilibrium with the nonsynonymous rs4149056 SNP (r(2)=0.97), which has been linked to statin metabolism. The prevalence of the rs4149056 C allele in the population was 15%. The odds ratio for myopathy was 4.5 (95% confidence interval [CI], 2.6 to 7.7) per copy of the C allele, and 16.9 (95% CI, 4.7 to 61.1) in CC as compared with TT homozygotes. More than 60% of these myopathy cases could be attributed to the C variant. The association of rs4149056 with myopathy was replicated in the trial of 40 mg of simvastatin daily, which also showed an association between rs4149056 and the cholesterol-lowering effects of simvastatin. No SNPs in any other region were clearly associated with myopathy.nnnCONCLUSIONSnWe have identified common variants in SLCO1B1 that are strongly associated with an increased risk of statin-induced myopathy. Genotyping these variants may help to achieve the benefits of statin therapy more safely and effectively. (Current Controlled Trials number, ISRCTN74348595.)

Large-scale test of hypothesised associations between the angiotensin-converting-enzyme insertion/deletion polymorphism and myocardial infarction in about 5000 cases and 6000 controls

BACKGROUNDnThe original report of a possible association between myocardial infarction and the insertion/deletion (I/D) polymorphism of the gene for the angiotensin-1-converting enzyme (ACE) indicated a risk ratio for myocardial infarction with the DD genotype of 1.34 (95% CI 1.05-1.70), and the association was claimed to be particularly strong in a retrospectively defined low-risk subgroup (3.2 [95% CI 1.7-5.9). Subsequent investigations reached varying conclusions, but all were small, and much larger studies were needed.nnnMETHODSn4629 myocardial infarction cases and 5934 controls were compared. Cases were UK men aged 30-54 years and women aged 30-64 years recruited on presentation to hospital with confirmed myocardial infarction. Controls were aged 30-64 years with no history of cardiovascular disease, but were siblings or children of myocardial infarction survivors, or spouses of such relatives. All risk-ratio calculations allow for this relatedness of some of the controls. An updated meta-analysis of previous studies was also conducted.nnnFINDINGSnThe ACE DD genotype was found in 1359 (29.4%) of the myocardial infarction cases and in 1637 (27.6%) of the controls (risk ratio 1.10 [95% CI 1.00-1.21]). The association between myocardial infarction and the DD genotype did not seem to be stronger in the subgroup defined as low risk by previously used criteria (234 [28%] of 836 cases and 911 [28%] of 3253 controls: risk ratio 1.04 [95% CI 0.87-1.24]), or in any other subgroup. Nor was the ACE I/D genotype predictive of subsequent survival.nnnINTERPRETATIONnThis study involved many more cases than any previously reported study of this question, but did not confirm the existence of any substantial association. In an updated meta-analysis of these results with those of previously published studies, the risk ratio for myocardial infarction with the DD genotype seems to lie in the range 1.0 to about 1.1. Although an increase in risk of up to about 10-15% cannot be ruled out, substantially more extreme risks can be. Moreover, there are not especially strong associations in the subgroups previously selected for emphasis. These findings illustrate the need for some studies of candidate genes to involve much larger populations than is customary, without undue emphasis on retrospectively defined subgroups.

Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation.

We have characterized 21 mutations in the type VII collagen gene (COL7A1) encoding the anchoring fibrils, 18 of which were not previously reported, in patients from 15 unrelated families with recessive dystrophic epidermolysis bullosa (RDEB). COL7A1 mutations in both alleles were identified by screening the 118 exons of COL7A1 and flanking intron regions. Fourteen mutations created premature termination codons (PTCs) and consisted of nonsense mutations, small insertions, deletions, and splice-site mutations. A further seven mutations predicted glycine or arginine substitutions in the collagenous domain of the molecule. Two mutations were found in more than one family reported in this study, and six of the seven missense mutations showed clustering within exons 72-74 next to the hinge region of the protein. Patients who were homozygous or compound heterozygotes for mutations leading to PTCs displayed both absence or drastic reduction of COL7A1 transcripts and undetectable type VII collagen protein in skin. In contrast, missense mutations were associated with clearly detectable COL7A1 transcripts and with normal or reduced expression of type VII collagen protein at the dermo/epidermal junction. Our results provide evidence for at least two distinct molecular mechanisms underlying defective anchoring fibril formation in RDEB: one involving PTCs leading to mRNA instability and absence of protein synthesis, the other implicating missense mutations resulting in the synthesis of type VII collagen polypeptide with decreased stability and/or altered function. Genotype-phenotype correlations suggested that the nature and location of these mutations are important determinants of the disease phenotype and showed evidence for interfamilial phenotypic variability.

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Refinement of the Chromosome 5p Locus for Familial Calcium Pyrophosphate Dihydrate Deposition Disease

Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.

Deletions within COL7A1 Exons Distant from Consensus Splice Sites Alter Splicing and Produce Shortened Polypeptides in Dominant Dystrophic Epidermolysis Bullosa

We describe two familial cases of dominant dystrophic epidermolysis bullosa (DDEB) that are heterozygous for deletions in COL7A1 that alter splicing, despite intact consensus splice-site sequences. One patient shows a 28-bp genomic deletion (6081del28) in exon 73 associated with the activation of a cryptic donor splice site within this exon; the combination of both defects restores the phase and replaces the last 11 Gly-X-Y repeats of exon 73 by a noncollagenous sequence, Glu-Ser-Leu. The second patient demonstrates a 27-bp deletion in exon 87 (6847del27), causing in-frame skipping of this exon; consensus splice sites, putative branch sites, and introns flanking exons 73 and 87 showed a normal sequence. Keratinocytes from the probands synthesized normal and shortened type VII collagen polypeptides and showed intracellular accumulation of type VII procollagen molecules. This first report of genomic deletions in COL7A1 in DDEB suggests a role for exonic sequences in the control of splicing of COL7A1 pre-mRNA and provides evidence that shortened type VII collagen polypeptides can alter, in a dominant manner, anchoring-fibril formation and can cause DDEB of differing severity.

A haplotype of the human CXCR1 gene protective against rapid disease progression in HIV-1+ patients

Chemokines and their receptors are key factors in the onset and progression of AIDS. Among them, accumulating evidence strongly indicates the involvement of IL-8 and its receptors, CXCR1 and CXCR2, in AIDS-related conditions. Through extensive investigation of genetic variations of the human CXCR1–CXCR2 locus, we identified a haplotype of the CXCR1 gene (CXCR1-Ha) carrying two nonsynonymous single nucleotide polymorphisms, CXCR1_300 (Met to Arg) in the N terminus extracellular domain and CXCR1_142 (Arg to Cys) in the C terminus intracellular domain. Transfection experiments with CXCR1 cDNAs corresponding to the CXCR1-Ha and the alternative CXCR1-HA haplotype showed reduced expression of CD4 and CXCR4 in CXCR1-Ha cells in human osteosarcoma cells as well as in Jurkat and CEM human T lymphocytes. Furthermore, the efficiency of X4-tropic HIV-1NL4–3 infection was significantly lower in CXCR1-Ha cells than in CXCR1-HA cells. The results were further confirmed by a series of experiments using six HIV-1 clinical isolates from AIDS patients. A genetic association study was performed by using an HIV-1+ patient cohort consisting of two subpopulations of AIDS with extreme phenotypes of rapid and slow progression of the disease. The frequency of the CXCR1-Ha allele is markedly less frequent in patients with rapid disease onset than those with slow progression (P = 0.0003). These results provide strong evidence of a protective role of the CXCR1-Ha allele on disease progression in AIDS, probably acting through modulation of CD4 and CXCR4 expression.

Localization of tub and uncoupling proteins (Ucp) 2 and 3 to a region of rat Chromosome 1 linked to glucose intolerance and adiposity in the Goto-Kakizaki (GK) Type 2 diabetic rat

Uncoupling proteins (UCP) 2 and UCP3 are closely related to themitochondrial membrane protein UCP1, which can dissociate res-piration from ATP synthesis, resulting in the generation of heat(Fleury et al. 1997; Boss et al. 1997; Vidal-Puig et al. 1997).Because this property may allow for disposal of excess energy,uncoupling proteins are regarded as potential candidate genes forobesity and Type 2 diabetes. Another candidate gene for obesity,tub, was identified as an autosomal recessive mutation causingobesity in mouse (Noben-Trauth et al. 1996; Kleyn et al. 1996).Tub, Ucp2, and Ucp3 have been mapped to a region of mouseChromosome (Chr) 7 that shares synteny homology with humanChr 11p15-11q13 (Chung et al. 1996; Noben-Trauth et al. 1996;Kleyn et al. 1996; Fleury et al. 1997; Solanes et al. 1997). Ucp3has been shown in humans and mice to reside within 75–150 kbof Ucp2, co-localizing in P1 and BAC genomic clones (Solanes etal. 1997). Ucp2 and 3 are located at quantitative trait loci (QTL)for obesity in three different mouse models (Seldin et al. 1994;Warden et al. 1995; Taylor and Phillips 1996) and show linkagewith resting metabolic rate in humans (Bouchard et al. 1997). RatcDNAs for Ucp2 (Matsuda et al. 1997; GenBank AB006613) andUcp3 (GenBank U92069) have been cloned, but these genes havenot yet been mapped in rat. Tub has not yet been cloned or mappedin rat.A segment of rat Chr 1 that shares synteny homology withmouse Chr 7 holds areas of interest for several diseases. Includedare regions linked to glucose intolerance and adiposity in the Goto-Kakizaki (GK) nonobese Type 2 diabetic rat (Gauguier et al. 1996;Galli et al. 1996), hypertension in several rat strains (spontane-ously hypertensive, stroke-prone spontaneously hypertensive, re-combinant inbred strains, and Dahl salt-sensitive hypertensive; re-viewed in Jacob et al. 1996), hypertension and renal failure in thefawn-hooded rat (Brown et al. 1996), pancreatic islet morphologyin obese WKY13M F2 Lepr

Refined Genetic Mapping of the Darier Locus to a <1-cM Region of Chromosome 12q24.1, and Construction of a Complete, High-Resolution P1 Artificial Chromosome/Bacterial Artificial Chromosome Contig of the Critical Region

Darier disease (DD) (MIM 124200) is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and by abnormal keratinization. We present linkage analysis showing, in four families, key recombination events that refine the location of the DD locus on chromosome 12q23-24.1 to a region of <1 cM. We have constructed a YAC/P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC)-based physical map that encompasses this refined DD region. The map consists of 35 YAC, 69 PAC, 16 BAC, and 2 cosmid clones that were ordered by mapping 54 anonymous sequence-tagged sites. The critical region is estimated to be 2.4 Mb in size, with an average marker resolution of 37.5 kb. The refinement of the critical interval excludes the ALDH2, RPL6, PTPN11, and OAS genes, as well as seven expressed sequence tags (ESTs) previously mapped in the DD region. The three known genes (ATP2A2, PPP1CC, and SCA2) and the 10 ESTs mapped within the critical region are not obvious candidates for the DD gene. Therefore, this detailed integrated physical, genetic, and partial transcript map provides an important resource for the isolation of the DD gene and, possibly, other disease genes.

SNP Haplotypes in the Angiotensin I-Converting Enzyme (ACE) Gene: Analysis of Nigerian Family Data Using Gamete Competition Models: Gamete Competition for Nigerian ACE SNPs

Gamete competition models were used to explore the relationships between 13 ACE gene polymorphisms and plasma ACE concentration in a set of Nigerian families. Several markers in the 5′ and 3′ regions of the gene were significantly associated with ACE concentration (P < 10‐4). Multi‐locus genotypes comprising different combinations of markers from the 5′ UTR and the 3′ region of the gene were also analysed; in addition to G2350A, in the 3′ region, two markers from the 5′ UTR (A‐5466C and A‐240T) were found to be associated with ACE concentration. These results are consistent with reports that have suggested the presence of at least two ACE‐linked QTLs, and demonstrate the utility of gamete competition models in the exploratory investigation of the relationship between a quantitative trait and multiple variants in a small genomic region.