Lethal skeletal dysplasia in mice and humans lacking the golgin GMAP-210.

BACKGROUND: Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease. METHODS: We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation. RESULTS: Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied. CONCLUSIONS: GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations.

IL28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes.

Genetic polymorphisms near IL28B are associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV), two processes that require the appropriate activation of the host immune responses. Intrahepatic inflammation is believed to mirror such activation, but its relationship with IL28B polymorphisms has yet to be fully appreciated. We analyzed the association of IL28B polymorphisms with histological and follow-up features in 2335 chronically HCV-infected Caucasian patients. Assessable phenotypes before any antiviral treatment included necroinflammatory activity (n = 1,098), fibrosis (n = 1,527), fibrosis progression rate (n = 1,312), and hepatocellular carcinoma development (n = 1,915). Associations of alleles with the phenotypes were evaluated by univariate analysis and multivariate logistic regression, accounting for all relevant covariates. The rare G allele at IL28B marker rs8099917-previously shown to be at risk of treatment failure-was associated with lower activity (P = 0.04), lower fibrosis (P = 0.02) with a trend toward lower fibrosis progression rate (P = 0.06). When stratified according to HCV genotype, most significant associations were observed in patients infected with non-1 genotypes (P = 0.003 for activity, P = 0.001 for fibrosis, and P = 0.02 for fibrosis progression rate), where the odds ratio of having necroinflammation or rapid fibrosis progression for patients with IL28B genotypes TG or GG versus TT were 0.48 (95% confidence intervals 0.30-0.78) and 0.56 (0.35-0.92), respectively. IL28B polymorphisms were not predictive of the development of hepatocellular carcinoma. CONCLUSION: In chronic hepatitis C, IL28B variants associated with poor response to interferon therapy may predict slower fibrosis progression, especially in patients infected with non-1 HCV genotypes.

CNVs and genetic medicine (excitement and consequences of a rediscovery).

The extensive variability of individual human genomes contributes to phenotypic variability. Structural genomic variants, and copy number variants (CNVs) in particular, have recently been rediscovered as contributors to the genomic plasticity and evolution and as pathoetiologic elements for both monogenic and complex traits. Herein we review some of the consequences of CNVs in the context of human inherited diseases.

FGF21 signalling pathway and metabolic traits - genetic association analysis.

Fibroblast growth factor 21 (FGF21) is a novel master regulator of metabolic profile. The biological actions of FGF21 are elicited upon its klotho beta (KLB)-facilitated binding to FGF receptor 1 (FGFR1), FGFR2 and FGFR3. We hypothesised that common polymorphisms in the FGF21 signalling pathway may be associated with metabolic risk. At the screening stage, we examined associations between 63 common single-nucleotide polymorphisms (SNPs) in five genes of this pathway (FGF21, KLB, FGFR1, FGFR2, FGFR3) and four metabolic phenotypes (LDL cholesterol - LDL-C, HDL-cholesterol - HDL-C, triglycerides and body mass index) in 629 individuals from Silesian Hypertension Study (SHS). Replication analyses were performed in 5478 unrelated individuals of the Swiss CoLaus cohort (imputed genotypes) and in 3030 directly genotyped individuals of the German Myocardial Infarction Family Study (GerMIFS). Of 54 SNPs that met quality control criteria after genotyping in SHS, 4 (rs4733946 and rs7012413 in FGFR1; rs2071616 in FGFR2 and rs7670903 in KLB) showed suggestive association with LDL-C (P=0.0006, P=0.0013, P=0.0055, P=0.011, respectively) and 1 (rs2608819 in KLB) was associated with body mass index (P=0.011); all with false discovery rate q<0.5. Of these, only one FGFR2 polymorphism (rs2071616) showed replicated association with LDL-C in both CoLaus (P=0.009) and men from GerMIFS (P=0.017). The direction of allelic effect of rs2071616 upon LDL-C was consistent in all examined populations. These data show that common genetic variations in FGFR2 may be associated with LDL-C in subjects of white European ancestry.

Effects of long-term averaging of quantitative blood pressure traits on the detection of genetic associations.

Blood pressure (BP) is a heritable, quantitative trait with intraindividual variability and susceptibility to measurement error. Genetic studies of BP generally use single-visit measurements and thus cannot remove variability occurring over months or years. We leveraged the idea that averaging BP measured across time would improve phenotypic accuracy and thereby increase statistical power to detect genetic associations. We studied systolic BP (SBP), diastolic BP (DBP), mean arterial pressure (MAP), and pulse pressure (PP) averaged over multiple years in 46,629 individuals of European ancestry. We identified 39 trait-variant associations across 19 independent loci (p < 5 × 10(-8)); five associations (in four loci) uniquely identified by our LTA analyses included those of SBP and MAP at 2p23 (rs1275988, near KCNK3), DBP at 2q11.2 (rs7599598, in FER1L5), and PP at 6p21 (rs10948071, near CRIP3) and 7p13 (rs2949837, near IGFBP3). Replication analyses conducted in cohorts with single-visit BP data showed positive replication of associations and a nominal association (p < 0.05). We estimated a 20% gain in statistical power with long-term average (LTA) as compared to single-visit BP association studies. Using LTA analysis, we identified genetic loci influencing BP. LTA might be one way of increasing the power of genetic associations for continuous traits in extant samples for other phenotypes that are measured serially over time.

From SNPs to pathways: integration of functional effect of sequence variations on models of cell signalling pathways

Background: Single nucleotide polymorphisms (SNPs) are the most frequent type of sequence variation between individuals, and represent a promising tool for finding genetic determinants of complex diseases and understanding the differences in drug response. In this regard, it is of particular interest to study the effect of non-synonymous SNPs in the context of biological networks such as cell signalling pathways. UniProt provides curated information about the functional and phenotypic effects of sequence variation, including SNPs, as well as on mutations of protein sequences. However, no strategy has been developed to integrate this information with biological networks, with the ultimate goal of studying the impact of the functional effect of SNPs in the structure and dynamics of biological networks. Results: First, we identified the different challenges posed by the integration of the phenotypic effect of sequence variants and mutations with biological networks. Second, we developed a strategy for the combination of data extracted from public resources, such as UniProt, NCBI dbSNP, Reactome and BioModels. We generated attribute files containing phenotypic and genotypic annotations to the nodes of biological networks, which can be imported into network visualization tools such as Cytoscape. These resources allow the mapping and visualization of mutations and natural variations of human proteins and their phenotypic effect on biological networks (e.g. signalling pathways, protein-protein interaction networks, dynamic models). Finally, an example on the use of the sequence variation data in the dynamics of a network model is presented. Conclusion: In this paper we present a general strategy for the integration of pathway and sequence variation data for visualization, analysis and modelling purposes, including the study of the functional impact of protein sequence variations on the dynamics of signalling pathways. This is of particular interest when the SNP or mutation is known to be associated to disease. We expect that this approach will help in the study of the functional impact of disease-associated SNPs on the behaviour of cell signalling pathways, which ultimately will lead to a better understanding of the mechanisms underlying complex diseases.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation.

BACKGROUND: Toll-like receptors (TLRs) are essential components of the immune response to fungal pathogens. We examined the role of TLR polymorphisms in conferring a risk of invasive aspergillosis among recipients of allogeneic hematopoietic-cell transplants. METHODS: We analyzed 20 single-nucleotide polymorphisms (SNPs) in the toll-like receptor 2 gene (TLR2), the toll-like receptor 3 gene (TLR3), the toll-like receptor 4 gene (TLR4), and the toll-like receptor 9 gene (TLR9) in a cohort of 336 recipients of hematopoietic-cell transplants and their unrelated donors. The risk of invasive aspergillosis was assessed with the use of multivariate Cox regression analysis. The analysis was replicated in a validation study involving 103 case patients and 263 matched controls who received hematopoietic-cell transplants from related and unrelated donors. RESULTS: In the discovery study, two donor TLR4 haplotypes (S3 and S4) increased the risk of invasive aspergillosis (adjusted hazard ratio for S3, 2.20; 95% confidence interval [CI], 1.14 to 4.25; P=0.02; adjusted hazard ratio for S4, 6.16; 95% CI, 1.97 to 19.26; P=0.002). The haplotype S4 was present in carriers of two SNPs in strong linkage disequilibrium (1063 A/G [D299G] and 1363 C/T [T399I]) that influence TLR4 function. In the validation study, donor haplotype S4 also increased the risk of invasive aspergillosis (adjusted odds ratio, 2.49; 95% CI, 1.15 to 5.41; P=0.02); the association was present in unrelated recipients of hematopoietic-cell transplants (odds ratio, 5.00; 95% CI, 1.04 to 24.01; P=0.04) but not in related recipients (odds ratio, 2.29; 95% CI, 0.93 to 5.68; P=0.07). In the discovery study, seropositivity for cytomegalovirus (CMV) in donors or recipients, donor positivity for S4, or both, as compared with negative results for CMV and S4, were associated with an increase in the 3-year probability of invasive aspergillosis (12% vs. 1%, P=0.02) and death that was not related to relapse (35% vs. 22%, P=0.02). CONCLUSIONS: This study suggests an association between the donor TLR4 haplotype S4 and the risk of invasive aspergillosis among recipients of hematopoietic-cell transplants from unrelated donors.

Evaluation of adding a second marker to overcome Staphylococcus aureus spa typing homoplasies.

The utility of sequencing a second highly variable locus in addition to the spa gene (e.g., double-locus sequence typing [DLST]) was investigated to overcome limitations of a Staphylococcus aureus single-locus typing method. Although adding a second locus seemed to increase discriminatory power, it was not sufficient to definitively infer evolutionary relationships within a single multilocus sequence type (ST-5).

A large deletion in the adRP gene PRPF31: evidence that haploinsufficiency is the cause of disease.

PURPOSE: To report a large deletion that encompasses more than 90% of PRPF31 gene and two other neighboring genes in their entirety in an adRP pedigree that appears to show only the typical clinical features of retinitis pigmentosa. METHODS: To identify PRPF31 mutation in a dominant RP family (ADRP2) previously linked to the RP11 locus, the 14 exons of PRPF31 were screened for mutations by direct sequencing. To investigate the possibility of a large deletion, microsatellite markers near PRPF31 gene were analyzed by non-denaturing PAGE. RESULTS: Initial screening of PRPF31 gene in the ADRP2 family did not reveal an obvious mutation. A large deletion was however suspected due to lack of heterozygosity for nearly all PRPF31 intragenic single nucleotide polymorphysm (SNPs). In order to estimate the size of the deletion, SNPs and microsatellite markers spanning and flanking PRPF31 were analyzed in the entire ADRP2 family. Haplotype analysis with the above markers suggested a deletion of approximately 30 kb that included the putative promoter region of a novel gene OSCAR, the entire genomic content of genes NDUFA3, TFPT and more than 90% of PRPF31 gene. Sequence analysis of the region flanking the potential deletion showed a high presence of Alu elements implicating Alu mediated recombination as the mechanism responsible for this event. CONCLUSIONS: This mutation provides evidence that haploinsufficiency rather than aberrant function of mutated proteins is the cause of disease in these adRP patients with mutations in PRPF31 gene.

Androgenetic alopecia: identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology.

The pathogenesis of androgenetic alopecia (AGA, male-pattern baldness) is driven by androgens, and genetic predisposition is the major prerequisite. Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms (SNPs) at eight different genomic loci are associated with AGA development. However, a significant fraction of the overall heritable risk still awaits identification. Furthermore, the understanding of the pathophysiology of AGA is incomplete, and each newly associated locus may provide novel insights into contributing biological pathways. The aim of this study was to identify unknown AGA risk loci by replicating SNPs at the 12 genomic loci that showed suggestive association (5 × 10(-8)<P<10(-5)) with AGA in a recent meta-analysis. We analyzed a replication set comprising 2,759 cases and 2,661 controls of European descent to confirm the association with AGA at these loci. Combined analysis of the replication and the meta-analysis data identified four genome-wide significant risk loci for AGA on chromosomes 2q35, 3q25.1, 5q33.3, and 12p12.1. The strongest association signal was obtained for rs7349332 (P=3.55 × 10(-15)) on chr2q35, which is located intronically in WNT10A. Expression studies in human hair follicle tissue suggest that WNT10A has a functional role in AGA etiology. Thus, our study provides genetic evidence supporting an involvement of WNT signaling in AGA development.

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes.

In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function.

INTRODUCTION: The antiretroviral drug efavirenz (EFV) is extensively metabolized into three primary metabolites: 8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV. There is a wide interindividual variability in EFV plasma exposure, explained to a great extent by cytochrome P450 2B6 (CYP2B6), the main isoenzyme responsible for EFV metabolism and involved in the major metabolic pathway (8-hydroxylation) and to a lesser extent in 7-hydroxylation. When CYP2B6 function is impaired, the relevance of CYP2A6, the main isoenzyme responsible for 7-hydroxylation may increase. We hypothesize that genetic variability in this gene may contribute to the particularly high, unexplained variability in EFV exposure in individuals with limited CYP2B6 function. METHODS: This study characterized CYP2A6 variation (14 alleles) in individuals (N=169) previously characterized for functional variants in CYP2B6 (18 alleles). Plasma concentrations of EFV and its primary metabolites (8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV) were measured in different genetic backgrounds in vivo. RESULTS: The accessory metabolic pathway CYP2A6 has a critical role in limiting drug accumulation in individuals characterized as CYP2B6 slow metabolizers. CONCLUSION: Dual CYP2B6 and CYP2A6 slow metabolism occurs at significant frequency in various human populations, leading to extremely high EFV exposure.

Toll-like receptor 2 (TLR2) polymorphisms are associated with reversal reaction in leprosy.

BACKGROUND: Leprosy is characterized by a spectrum of clinical manifestations that depend on the type of immune response against the pathogen. Patients may undergo immunological changes known as "reactional states" (reversal reaction and erythema nodosum leprosum) that result in major clinical deterioration. The goal of the present study was to assess the effect of Toll-like receptor 2 (TLR2) polymorphisms on susceptibility to and clinical presentation of leprosy. METHODS: Three polymorphisms in TLR2 (597C--&gt;T, 1350T--&gt;C, and a microsatellite marker) were analyzed in 431 Ethiopian patients with leprosy and 187 control subjects. The polymorphism-associated risk of developing leprosy, lepromatous (vs. tuberculoid) leprosy, and leprosy reactions was assessed by multivariate logistic regression models. RESULTS: The microsatellite and the 597C--&gt;T polymorphisms both influenced susceptibility to reversal reaction. Although the 597T allele had a protective effect (odds ratio [OR], 0.34 [95% confidence interval {CI}, 0.17-0.68]; P= .002 under the dominant model), homozygosity for the 280-bp allelic length of the microsatellite strongly increased the risk of reversal reaction (OR, 5.83 [95% CI, 1.98-17.15]; P= .001 under the recessive model). These associations were consistent among 3 different ethnic groups. CONCLUSIONS: These data suggest a significant role for TLR-2 in the occurrence of leprosy reversal reaction and provide new insights into the immunogenetics of the disease.

Genes mirror geography within Europe.

Understanding the genetic structure of human populations is of fundamental interest to medical, forensic and anthropological sciences. Advances in high-throughput genotyping technology have markedly improved our understanding of global patterns of human genetic variation and suggest the potential to use large samples to uncover variation among closely spaced populations. Here we characterize genetic variation in a sample of 3,000 European individuals genotyped at over half a million variable DNA sites in the human genome. Despite low average levels of genetic differentiation among Europeans, we find a close correspondence between genetic and geographic distances; indeed, a geographical map of Europe arises naturally as an efficient two-dimensional summary of genetic variation in Europeans. The results emphasize that when mapping the genetic basis of a disease phenotype, spurious associations can arise if genetic structure is not properly accounted for. In addition, the results are relevant to the prospects of genetic ancestry testing; an individual's DNA can be used to infer their geographic origin with surprising accuracy-often to within a few hundred kilometres.