A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T --> C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-beta1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-beta1-mediated induction of cartilage matrix genes through direct interaction with TGF-beta1 and inhibition of TGF-beta1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-beta1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-beta signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-beta.     

Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome

Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in fibrillin-1 (encoded by FBN1 in humans and Fbn1 in mice), a matrix component of extracellular microfibrils. A distinct subgroup of individuals with Marfan syndrome have distal airspace enlargement, historically described as emphysema, which frequently results in spontaneous lung rupture (pneumothorax; refs. 1-3). To investigate the pathogenesis of genetically imposed emphysema, we analyzed the lung phenotype of mice deficient in fibrillin-1, an accepted model of Marfan syndrome. Lung abnormalities are evident in the immediate postnatal period and manifest as a developmental impairment of distal alveolar septation. Aged mice deficient in fibrillin-1 develop destructive emphysema consistent with the view that early developmental perturbations can predispose to late-onset, seemingly acquired phenotypes. We show that mice deficient in fibrillin-1 have marked dysregulation of transforming growth factor-beta (TGF-beta) activation and signaling, resulting in apoptosis in the developing lung. Perinatal antagonism of TGF-beta attenuates apoptosis and rescues alveolar septation in vivo. These data indicate that matrix sequestration of cytokines is crucial to their regulated activation and signaling and that perturbation of this function can contribute to the pathogenesis of disease.     

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans

There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.     

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.     

Rare MTNR1B variants impairing melatonin receptor 1B function contribute to type 2 diabetes

Genome-wide association studies have revealed that common noncoding variants in MTNR1B (encoding melatonin receptor 1B, also known as MT(2)) increase type 2 diabetes (T2D) risk(1,2). Although the strongest association signal was highly significant (P < 1 × 10(-20)), its contribution to T2D risk was modest (odds ratio (OR) of ～1.10-1.15)(1-3). We performed large-scale exon resequencing in 7,632 Europeans, including 2,186 individuals with T2D, and identified 40 nonsynonymous variants, including 36 very rare variants (minor allele frequency (MAF) <0.1%), associated with T2D (OR = 3.31, 95% confidence interval (CI) = 1.78-6.18; P = 1.64 × 10(-4)). A four-tiered functional investigation of all 40 mutants revealed that 14 were non-functional and rare (MAF < 1%), and 4 were very rare with complete loss of melatonin binding and signaling capabilities. Among the very rare variants, the partial- or total-loss-of-function variants but not the neutral ones contributed to T2D (OR = 5.67, CI = 2.17-14.82; P = 4.09 × 10(-4)). Genotyping the four complete loss-of-function variants in 11,854 additional individuals revealed their association with T2D risk (8,153 individuals with T2D and 10,100 controls; OR = 3.88, CI = 1.49-10.07; P = 5.37 × 10(-3)). This study establishes a firm functional link between MTNR1B and T2D risk.     

Heterozygous TGFBR2 mutations in Marfan syndrome

Marfan syndrome is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton and cardiovascular systems associated with defects in the gene encoding fibrillin (FBN1) at 15q21.1 (ref. 1). A second type of the disorder (Marfan syndrome type 2; OMIM 154705) is associated with a second locus, MFS2, at 3p25-p24.2 in a large French family (family MS1). Identification of a 3p24.1 chromosomal breakpoint disrupting the gene encoding TGF-beta receptor 2 (TGFBR2) in a Japanese individual with Marfan syndrome led us to consider TGFBR2 as the gene underlying association with Marfan syndrome at the MSF2 locus. The mutation 1524G-->A in TGFBR2 (causing the synonymous amino acid substitution Q508Q) resulted in abnormal splicing and segregated with MFS2 in family MS1. We identified three other missense mutations in four unrelated probands, which led to loss of function of TGF-beta signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2, a putative tumor-suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders.     

Genome-wide association study and mouse model identify interaction between RET and EDNRB pathways in Hirschsprung disease

Genetic studies of Hirschsprung disease, a common congenital malformation, have identified eight genes with mutations that can be associated with this condition. Mutations at individual loci are, however, neither necessary nor sufficient to cause clinical disease. We conducted a genome-wide association study in 43 Mennonite family trios using 2,083 microsatellites and single-nucleotide polymorphisms and a new multipoint linkage disequilibrium method that searches for association arising from common ancestry. We identified susceptibility loci at 10q11, 13q22 and 16q23; the gene at 13q22 is EDNRB, encoding a G protein-coupled receptor (GPCR) and the gene at 10q11 is RET, encoding a receptor tyrosine kinase (RTK). Statistically significant joint transmission of RET and EDNRB alleles in affected individuals and non-complementation of aganglionosis in mouse intercrosses between Ret null and the Ednrb hypomorphic piebald allele are suggestive of epistasis between EDNRB and RET. Thus, genetic interaction between mutations in RET and EDNRB is an underlying mechanism for this complex disorder.     

Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease

We performed a meta-analysis of 14 genome-wide association studies of coronary artery disease (CAD) comprising 22,233 individuals with CAD (cases) and 64,762 controls of European descent followed by genotyping of top association signals in 56,682 additional individuals. This analysis identified 13 loci newly associated with CAD at P < 5 × 10?? and confirmed the association of 10 of 12 previously reported CAD loci. The 13 new loci showed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6% to 17% increase in the risk of CAD per allele. Notably, only three of the new loci showed significant association with traditional CAD risk factors and the majority lie in gene regions not previously implicated in the pathogenesis of CAD. Finally, five of the new CAD risk loci appear to have pleiotropic effects, showing strong association with various other human diseases or traits.     

Integrated detection and population-genetic analysis of SNPs and copy number variation

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.     

A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease

Susceptibility to Crohn's disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn's disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohn's disease-associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohn's disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.     

Identification of two new loci at IL23R and RAB32 that influence susceptibility to leprosy

We performed a genome-wide association study with 706 individuals with leprosy and 5,581 control individuals and replicated the top 24 SNPs in three independent replication samples, including a total of 3,301 individuals with leprosy and 5,299 control individuals from China. Two loci not previously associated with the disease were identified with genome-wide significance: rs2275606 (combined P = 3.94 × 10(-14), OR = 1.30) on 6q24.3 and rs3762318 (combined P = 3.27 × 10(-11), OR = 0.69) on 1p31.3. These associations implicate IL23R and RAB32 as new susceptibility genes for leprosy. Furthermore, we identified evidence of interaction between the NOD2 and RIPK2 loci, which is consistent with the biological association of the proteins encoded by these genes (NOD2-RIPK2 complex) in activating the NF-κB pathway as a part of the host defense response to infection. Our findings have expanded the biological functions of IL23R by uncovering its involvement in infectious disease susceptibility and suggest a potential involvement of autophagocytosis in leprosy pathogenesis. The IL23R association supports previous observations of the marked overlap of susceptibility genes for leprosy and Crohn's disease, implying common pathogenesis mechanisms.     

Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem of unknown etiology that varies in prevalence among ancestry groups. To identify genetic variants contributing to differences in hepatic fat content, we carried out a genome-wide association scan of nonsynonymous sequence variations (n = 9,229) in a population comprising Hispanic, African American and European American individuals. An allele in PNPLA3 (rs738409[G], encoding I148M) was strongly associated with increased hepatic fat levels (P = 5.9 x 10(-10)) and with hepatic inflammation (P = 3.7 x 10(-4)). The allele was most common in Hispanics, the group most susceptible to NAFLD; hepatic fat content was more than twofold higher in PNPLA3 rs738409[G] homozygotes than in noncarriers. Resequencing revealed another allele of PNPLA3 (rs6006460[T], encoding S453I) that was associated with lower hepatic fat content in African Americans, the group at lowest risk of NAFLD. Thus, variation in PNPLA3 contributes to ancestry-related and inter-individual differences in hepatic fat content and susceptibility to NAFLD.     

Genome-wide association study in individuals of South Asian ancestry identifies six new type 2 diabetes susceptibility loci

We carried out a genome-wide association study of type-2 diabetes (T2D) in individuals of South Asian ancestry. Our discovery set included 5,561 individuals with T2D (cases) and 14,458 controls drawn from studies in London, Pakistan and Singapore. We identified 20 independent SNPs associated with T2D at P < 10(-4) for testing in a replication sample of 13,170 cases and 25,398 controls, also all of South Asian ancestry. In the combined analysis, we identified common genetic variants at six loci (GRB14, ST6GAL1, VPS26A, HMG20A, AP3S2 and HNF4A) newly associated with T2D (P = 4.1 × 10(-8) to P = 1.9 × 10(-11)). SNPs at GRB14 were also associated with insulin sensitivity (P = 5.0 × 10(-4)), and SNPs at ST6GAL1 and HNF4A were also associated with pancreatic beta-cell function (P = 0.02 and P = 0.001, respectively). Our findings provide additional insight into mechanisms underlying T2D and show the potential for new discovery from genetic association studies in South Asians, a population with increased susceptibility to T2D.     

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.     

DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis

Hypophosphatemia is a genetically heterogeneous disease. Here, we mapped an autosomal recessive form (designated ARHP) to chromosome 4q21 and identified homozygous mutations in DMP1 (dentin matrix protein 1), which encodes a non-collagenous bone matrix protein expressed in osteoblasts and osteocytes. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels and suggesting that DMP1 may regulate FGF23 expression.     

TACI is mutant in common variable immunodeficiency and IgA deficiency

The tumor necrosis factor receptor family member TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in B cells. We found that 4 of 19 unrelated individuals with common variable immunodeficiency (CVID) and 1 of 16 individuals with IgA deficiency (IgAD) had a missense mutation in one allele of TNFRSF13B (encoding TACI). One of the four individuals with CVID had a single nucleotide insertion in the other TNFRSF13B allele. None of these mutations were present in 50 healthy subjects. TNFRSF13B mutations cosegregated with the phenotype of CVID or IgAD in family members of four index individuals that we studied. B cells from individuals with TACI mutations expressed TACI but did not produce IgG and IgA in response to the TACI ligand APRIL, probably reflecting impaired isotype switching. These results suggest that TACI mutations can result in CVID and IgAD.     

Mutations in the region encoding the von Willebrand factor A domain of matrilin-3 are associated with multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4-13). These dominant forms of MED (EDM1-3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1-3 genes provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24-p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.     

Mapping of a gene predisposing to early-onset Alzheimer's disease to chromosome 14q24.3.

Genetic linkage studies with chromosome 21 DNA markers and mutation analysis of the beta-amyloid protein precursor gene located in 21q21.3 have indicated that early-onset Alzheimer's disease (EOAD) is a heterogeneous disorder for which at least one other chromosomal locus exists. We examined two extended histopathologically confirmed EOAD pedigrees, AD/A and AD/B, with highly informative short tandem repeat (STR) polymorphisms and found complete linkage of the disease to a (CA)n dinucleotide repeat polymorphism at locus D14S43 in 14q24.3 (Zmax = 13.25 at theta = 0.0). Using additional chromosome 14 STR polymorphisms we were able to delineate the region containing the EOAD gene to an area of, at most, 8.9 centiMorgans between D14S42 and D14S53, flanking D14S43 on both sides.