Targeted re-sequencing of linkage region on 2q21 identifies a novel functional variant for hip and knee osteoarthritis.

OBJECTIVE: The aim of the study was to identify genetic variants predisposing to primary hip and knee osteoarthritis (OA) in a sample of Finnish families. METHODS: Genome wide analysis was performed using 15 independent families (279 individuals) originating from Central Finland identified as having multiple individuals with primary hip and/or knee OA. Targeted re-sequencing was performed for three samples from one 33-member, four-generation family contributing most significantly to the LOD score. In addition, exome sequencing was performed in three family members from the same family. RESULTS: Genome wide linkage analysis identified a susceptibility locus on chromosome 2q21 with a multipoint LOD score of 3.91. Targeted re-sequencing and subsequent linkage analysis revealed a susceptibility insertion variant rs11446594. It locates in a predicted strong enhancer element region with maximum LOD score 3.42 under dominant model of inheritance. Insertion creates a recognition sequence for ELF3 and HMGA1 transcription factors. Their DNA-binding affinity is highly increased in the presence of A-allele compared to wild type null allele. CONCLUSION: A potentially novel functional OA susceptibility variant was identified by targeted re-sequencing. This variant locates in a predicted regulatory site and creates a recognition sequence for ELF3 and HMGA1 transcription factors that are predicted to play a significant role in articular cartilage homeostasis.

A GWAS meta-analysis from 5 population-based cohorts implicates ion channel genes in the pathogenesis of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) shows genetic predisposition, however, large-scale, powered gene mapping studies are lacking. We sought to exploit existing genetic (genotype) and epidemiological (questionnaire) data from a series of population-based cohorts for IBS genome-wide association studies (GWAS) and their meta-analysis. METHODS: Based on questionnaire data compatible with Rome III Criteria, we identified a total of 1335 IBS cases and 9768 asymptomatic individuals from 5 independent European genotyped cohorts. Individual GWAS were carried out with sex-adjusted logistic regression under an additive model, followed by meta-analysis using the inverse variance method. Functional annotation of significant results was obtained via a computational pipeline exploiting ontology and interaction networks, and tissue-specific and gene set enrichment analyses. KEY RESULTS: Suggestive GWAS signals (P ≤ 5.0 × 10-6 ) were detected for 7 genomic regions, harboring 64 gene candidates to affect IBS risk via functional or expression changes. Functional annotation of this gene set convincingly (best FDR-corrected P = 3.1 × 10-10 ) highlighted regulation of ion channel activity as the most plausible pathway affecting IBS risk. CONCLUSION & INFERENCES: Our results confirm the feasibility of population-based studies for gene-discovery efforts in IBS, identify risk genes and loci to be prioritized in independent follow-ups, and pinpoint ion channels as important players and potential therapeutic targets warranting further investigation.

A meta-analysis of reflux genome-wide association studies in 6750 Northern Europeans from the general population.

BACKGROUND: Gastroesophageal reflux disease (GERD), the regurgitation of gastric acids often accompanied by heartburn, affects up to 20% of the general population. Genetic predisposition is suspected from twin and family studies but gene-hunting efforts have so far been scarce and no conclusive genome-wide study has been reported. We exploited data available from general population samples, and studied self-reported reflux symptoms in relation to genome-wide single nucleotide polymorphism (SNP) genotypes. METHODS: We performed a GWAS meta-analysis of three independent population-based cohorts from Sweden, Finland, and UK. GERD cases (n=2247) and asymptomatic controls (n=4503) were identified using questionnaire-derived symptom data. Upon stringent quality controls, genotype data for more than 2.5M markers were used for association testing. Bioinformatic characterization of genomic regions associated with GERD included gene-set enrichment analysis (GSEA), in silico prediction of genetic risk effects on gene expression, and computational analysis of drug-induced gene expression signatures using Connectivity Map (cMap). KEY RESULTS: We identified 30 GERD suggestive risk loci (P≤5×10-5 ), with concordant risk effects in all cohorts, and predicted functional effects on gene expression in relevant tissues. GSEA revealed involvement of GERD risk genes in biological processes associated with the regulation of ion channel and cell adhesion. From cMap analysis, omeprazole had significant effects on GERD risk gene expression, while antituberculosis and anti-inflammatory drugs scored highest among the repurposed compounds. CONCLUSIONS: We report a large-scale genetic study of GERD, and highlight genes and pathways that contribute to further our understanding of its pathogenesis and therapeutic opportunities.

GWAS Identifies New Loci for Painful Temporomandibular Disorder: Hispanic Community Health Study/Study of Latinos.

Temporomandibular disorder (TMD) is a musculoskeletal condition characterized by pain and reduced function in the temporomandibular joint and/or associated masticatory musculature. Prevalence in the United States is 5% and twice as high among women as men. We conducted a discovery genome-wide association study (GWAS) of TMD in 10,153 participants (769 cases, 9,384 controls) of the US Hispanic Community Health Study/Study of Latinos (HCHS/SOL). The most promising single-nucleotide polymorphisms (SNPs) were tested in meta-analysis of 4 independent cohorts. One replication cohort was from the United States, and the others were from Germany, Finland, and Brazil, totaling 1,911 TMD cases and 6,903 controls. A locus near the sarcoglycan alpha ( SGCA), rs4794106, was suggestive in the discovery analysis ( P = 2.6 × 106) and replicated (i.e., 1-tailed P = 0.016) in the Brazilian cohort. In the discovery cohort, sex-stratified analysis identified 2 additional genome-wide significant loci in females. One lying upstream of the relaxin/insulin-like family peptide receptor 2 ( RXP2) (chromosome 13, rs60249166, odds ratio [OR] = 0.65, P = 3.6 × 10-8) was replicated among females in the meta-analysis (1-tailed P = 0.052). The other (chromosome 17, rs1531554, OR = 0.68, P = 2.9 × 10-8) was replicated among females (1-tailed P = 0.002), as well as replicated in meta-analysis of both sexes (1-tailed P = 0.021). A novel locus at genome-wide level of significance (rs73460075, OR = 0.56, P = 3.8 × 10-8) in the intron of the dystrophin gene DMD (X chromosome), and a suggestive locus on chromosome 7 (rs73271865, P = 2.9 × 10-7) upstream of the Sp4 Transcription Factor ( SP4) gene were identified in the discovery cohort, but neither of these was replicated. The SGCA gene encodes SGCA, which is involved in the cellular structure of muscle fibers and, along with DMD, forms part of the dystrophin-glycoprotein complex. Functional annotation suggested that several of these variants reside in loci that regulate processes relevant to TMD pathobiologic processes.

Aggrecan core protein of a certain length is protective against hand osteoarthritis.

OBJECTIVE: To study the contribution of aggrecan VNTR (variable number of tandem repeats) polymorphism to clinically differing manifestations of hand osteoarthritis (OA). DESIGN: Five hundred thirty Finnish females representing two academically similar occupations with completely diverse exposure to hand load were included. Radiographs of hands were analysed, the OA findings were graded and the subjects were divided into categories. Aggrecan VNTR alleles were identified by Southern hybridization. Statistical analyses were used to compare joint involvement and pathological findings with the prevalences of the alleles and genotypes. RESULTS: Subjects homozygous for the most common aggrecan VNTR allele, A27 with 27 repeats, had a significantly lower risk of hand OA, with OR 0.46 (95% CI 0.27-0.78) for OA of grade 2 or more. Our results suggest that carrying two copies of the alleles with less than 27 repeats could predispose a subject to a severe hand OA (OR 2.45, 95% CI 1.17-5.12) and carrying two copies of the alleles with more than 27 repeats also increases the risk of the disease (OR 1.73, 95% CI 1.03-2.89). CONCLUSIONS: These findings indicate that allele A27 provides protection from hand OA and that alleles shorter or longer than this may predispose subjects to the disease. Furthermore, they suggest that a certain number of tandem repeats provide for optimal functioning of the aggrecan molecule and that the contribution of genetic factors to the development of hand OA may be even more important than that of environmental factors.

Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF.

BACKGROUND: The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). METHODS: Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. RESULTS: Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). CONCLUSIONS: Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

Haplotype analysis of congenital nephrotic syndrome of the Finnish type in non-Finnish families.

Congenital nephrotic syndrome of the Finnish type (CNF) has an estimated incidence of 1 in 8000 newborns in the genetically isolated population of Finland. Although the disease is most common in Finland, it occurs throughout the world in families without known Finnish origin. In the past, these authors recently localized the CNF gene to the chromosome 19q13.1 region and observed strong linkage disequilibrium to the genetic markers D19S610, D19S608, D19S224, and D19S220 in Finnish families. In these Finnish families, four main CNF haplotype categories have been observed. In the study presented here, haplotype analysis was applied to several non-Finnish CNF families to determine whether the same genetic locus is involved in these families. The results of the haplotype analysis suggest linkage to the 19q13.1 chromosomal region. It was also observed that, in most cases, alleles typically found on CNF chromosomes of Finnish families are also found on CNF chromosomes of non-Finnish families from North America and Europe. In these families, the strongest association was found with marker D19S608. These findings suggest that Finnish and many non-Finnish CNF cases share the same disease locus.

Congenital nephrotic syndrome of the Finnish type is not associated with the Pax-2 gene despite the promising transgenic animal model.

Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disease with an incidence of 1 in 8000 in Finland. CNF is characterized by massive proteinuria and nephrotic syndrome at birth. In a recent report, deregulation of expression of the gene coding for the Pax-2 DNA-binding protein was shown to generate severe kidney abnormalities in transgenic mice resembling the clinical and pathological findings in congenital nephrotic syndrome, making it a candidate gene for CNF. However, in this study, we have unequivocally excluded the Pax-2 gene locus as a causative for congenital nephrotic syndrome of the Finnish type.

Fine mapping and haplotype analysis of the locus for congenital nephrotic syndrome on chromosome 19q13.1.

We have recently localized the gene for congenital nephrotic syndrome of the Finnish type (CNF) to chromosome 19q12-13.1. On the basis of observed recombination events, the gene was localized between markers D19S416/D19S425/D19S213/D19S208/D19S191 and D19S224. Here we have extended the mapping efforts, on the basis of a detailed physical map of the region. By means of three new polymorphic markers--D19S608, D19S609, and D19S610--developed in this study, the critical candidate region could be further restricted. Significant linkage disequilibrium was observed with markers D19S610, D19S608, D19S224, and D19S220, the strongest allelic association being 84% with marker D19S610 at 19q13.1. This suggests that the CNF gene locus lies in close proximity to marker D19S610. Combination of the informative markers revealed four main haplotype categories. Different geographic distribution was observed between these haplotype groups when they were placed on the map of finland according to the birthplaces of grandparents.

Exclusion of eight genes as mutated loci in congenital nephrotic syndrome of the Finnish type.

The congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disease characterized by massive proteinuria already at birth. The gene locus defective in CNF was searched for using polymorphic markers of candidate genes coding for components of the basement membrane (BM). The linkage analyses in 17 Finnish CNF families demonstrated exclusion of obligatory recombination events between the disease and eight genes coding for BM components. The genes coding for the alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 4(IV) chain of type IV collagen, the B1e, B2e and B2t chains of laminin, as well as the BM heparan sulfate proteoglycan core protein were all excluded in this Finnish family material. Since the defect is not in any of the genes coding for major components of BM, the identification of the gene defect will most probably reveal a new gene important for the development and function of the glomerular basement membrane.

Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa.

Dystrophic forms of epidermolysis bullosa (DEB) are associated with mutations in the type VII collagen gene (Col7a1) which encodes the major component of anchoring fibrils. To develop a DEB animal model, type VII collagen deficient mice were generated by targeted homologous recombination. The targeting vector replaced exons 46-69 of Col7a1 with the neomycin-resistance gene, in reverse transcriptional orientation, resulting in elimination of most of the collagenous domain 1. Col7a1 heterozygous (+/-) mice were phenotypically normal. Mating of Col7a1 +/- mice revealed that Col7a1 null (-/-) mice, which were born with extensive cutaneous blistering, died during the first two weeks of life probably due to complications arising from the blistering. Transmission electron microscopy revealed subepidermal blistering below the lamina densa and absence of anchoring fibrils. Immunohistochemical staining with anti-human type VII collagen antibody stained the dermal-epidermal junction in control mice, but did not stain the skin of Col7a1 null mice. Collectively, the DEB mice recapitulate the clinical, genetic, immunohistochemical and ultrastructural characteristics of recessive DEB in humans. These mice provide an animal model to study the pathomechanisms of DEB and serve as a system to test therapeutic approaches, including gene replacement, towards the cure of this devastating skin disease.

Congenital nephrotic syndrome of the Finnish type maps to the long arm of chromosome 19.

Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disease that is characterized by massive proteinuria and nephrotic syndrome at birth. CNF represents a unique, apparently specific dysfunction of the renal basement membranes, and the estimated incidence of CNF in the isolated population of Finland is 1 in 8,000 newborns. The basic defect is unknown, and no specific biochemical defect or chromosomal aberrations have been described. Here we report the assignment of the CNF locus to 19q12-q13.1 on the basis of linkage analyses in 17 Finnish families. Multipoint analyses and observed recombination events place the CNF locus between multiallelic markers D19S416 and D19S224, and the significant linkage disequilibrium observed suggests that the CNF gene lies in the immediate vicinity of the markers D19S224 and D19S220.

Sequence variations in the collagen IX and XI genes are associated with degenerative lumbar spinal stenosis.

BACKGROUND: Degenerative lumbar spinal stenosis (LSS) is usually caused by disc herniation or degeneration. Several genetic factors have been implicated in disc disease. Tryptophan alleles in COL9A2 and COL9A3 have been shown to be associated with lumbar disc disease in the Finnish population, and polymorphisms in the vitamin D receptor gene (VDR) (FokI and TaqI), the matrix metalloproteinase-3 gene (MMP-3) and an aggrecan gene (AGC1) VNTR have been reported to be associated with disc degeneration. In addition, an IVS6-4 a>t polymorphism in COL11A2 has been found in connection with stenosis caused by ossification of the posterior longitudinal ligament in the Japanese population. OBJECTIVE: To study the role of genetic factors in LSS. METHODS: 29 Finnish probands were analysed for mutations in the genes coding for intervertebral disc matrix proteins, COL1A1, COL1A2, COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, and AGC1. VDR and MMP-3 polymorphisms were also analysed. Sequence variations were tested in 56 Finnish controls. RESULTS: Several disease associated alleles were identified. A splice site mutation in COL9A2 leading to a premature translation termination codon and the generation of a truncated protein was identified in one proband, another had the Trp2 allele, and four others the Trp3 allele. The frequency of the COL11A2 IVS6(-4) t allele was 93.1% in the probands and 72.3% in controls (p = 0.0016). The differences in genotype frequencies for this site were less significant (p = 0.0043). CONCLUSIONS: Genetic factors have an important role in the pathogenesis of LSS.

Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients.

BACKGROUND: Congenital nephrotic syndrome (NPHS1) is a rare disease inherited as an autosomally recessive trait. The NPHS1 gene mutated in NPHS1 children has recently been identified. The gene codes for nephrin, a cell-surface protein of podocytes. Two mutations, named Fin-major and Fin-minor, have been found in over 90% of the Finnish patients. In this study, we correlated the NPHS1 gene mutations to the clinical features and renal findings in 46 Finnish NPHS1 children. METHODS: Clinical data were collected from patient files, and kidney histology and electron microscopy samples were re-evaluated. The expression of nephrin was studied using immunohistochemistry, Western blotting, and in situ hybridization. RESULTS: Nephrotic syndrome was detected in most patients within days after birth regardless of the genotype detected. No difference could be found in neonatal, renal, cardiac, or neurological features in patients with different mutations. Nephrin was not expressed in kidneys with Fin-major or Fin-minor mutations, while another slit diaphragm-associated protein, ZO-1, stained normally. In electron microscopy, podocyte fusion and podocyte filtration slits of various sizes were detected. The slit diaphragms, however, were missing. In contrast to this, a nephrotic infant with Fin-major/R743C genotype expressed nephrin in kidney had normal slit diaphragms and responded to therapy with an angiotensin-converting enzyme inhibitor and indomethacin. CONCLUSIONS: The most common NPHS1 gene mutations, Fin-major and Fin-minor, both lead to an absence of nephrin and podocyte slit diaphragms, as well as a clinically severe form of NPHS1, the Finnish type of congenital nephrotic syndrome.

Improved prenatal diagnosis of the congenital nephrotic syndrome of the Finnish type based on DNA analysis.

Haplotype analysis and alpha-fetoprotein quantitation comprise a prenatal diagnosis of congenital nephrosis. Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disease characterized by massive proteinuria and nephrotic syndrome from birth. Prenatal diagnosis of CNF has previously been based on the quantitation of alpha-fetoprotein (AFP) in the amniotic fluid and maternal serum, but an increased AFP is not specific for the disease. We have recently localized the CNF gene to the chromosome 19q13.1 region and observed a strong linkage disequilibrium to the genetic markers D19S610, D19S608, D19S224 and D19S220 in this chromosomal area. Four main CNF-haplotypes have been observed in Finnish kindreds. In the present study, linkage and haplotype analyses have been applied to prenatal diagnosis of six families with a history of CNF. The results diminish the risk of false positive diagnosis and abortions of healthy fetuses in families at risk.

Structure of the gene for congenital nephrotic syndrome of the finnish type (NPHS1) and characterization of mutations.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin.

Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier.