Investigation of the role of ANKH in ankylosing spondylitis

Objective The ank/ank mouse develops a phenotype similar to ankylosing spondylitis (AS) in humans. ANKH, the human homolog of the mutated gene in the ank/ank mouse, has been implicated in familial autosomal-dominant chondrocalcinosis and autosomal-dominant craniometaphyseal dysplasia. This study was undertaken to investigate the role of ANKH in susceptibility to and clinical manifestations of AS. Methods Sequence variants were identified by genomic sequencing of the 12 ANKH exons and their flanking splice sites in 48 AS patients; variants were then screened in 233 patients and 478 controls. Linkage to the ANKH locus was assessed in 185 affected-sibling-pair families. Results Five single-nucleotide polymorphisms were identified within the coding region and flanking splice sites. No association between either susceptibility to AS or its clinical manifestations and these novel polymorphisms, or between disease susceptibility and 3 known promoter variants, was seen. No linkage between the ANKH locus and AS was observed. Multipoint exclusion mapping rejected the hypothesis of a locus of a magnitude λ≥1.4 (logarithm of odds score <-2) (equivalent to a genetic contribution of >10% to the AS sibling recurrence risk ratio) within this area contributing to AS. Conclusion These findings indicate that ANKH is not significantly involved in susceptibility to or clinical manifestations of AS.

Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5′-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate

Objective Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. Methods ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. Results Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5′-untranslated region (5′-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. Conclusion A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5′-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

A novel ACVR1 mutation in the glycine/serine-rich domain found in the most benign case of a fibrodysplasia ossificans progressiva variant reported to date

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant condition, classically characterised by heterotopic ossification beginning in childhood and congenital great toe malformations; occurring in response to a c.617 G>A ACVR1 mutation in the functionally important glycine/serine-rich domain of exon 6. Here we describe a novel c.587 T>C mutation in the glycine/serine-rich domain of ACVR1, associated with delayed onset of heterotopic ossification and an exceptionally mild clinical course. Absence of great toe malformations, the presence of early ossification of the cervical spine facets joints, plus mild bilateral camptodactyly of the 5th fingers, together with a novel ACVR1 mutation, are consistent with the 'FOP-variant' syndrome. The c.587 T>C mutation replaces a conserved leucine with proline at residue 196. Modelling of the mutant protein reveals a steric clash with the kinase domain that will weaken interactions with FKBP12 and induce exposure of the glycine/serine-rich repeat. The mutant receptor is predicted to be hypersensitive to ligand stimulation rather than being constitutively active, consistent with the mild clinical phenotype. This case extends our understanding of the 'FOP-variant' syndrome.

COL1A1 C-propeptide cleavage site mutation causes high bone mass, bone fragility and jaw lesions: A new cause of gnathodiaphyseal dysplasia?

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant condition characterized by bone fragility, irregular bone mineral density (BMD) and fibro-osseous lesions in the skull and jaw. Mutations in Anoctamin-5 (ANO5) have been identified in some cases. We aimed to identify the causative mutation in a family with features of GDD but no mutation in ANO5, using whole exome capture and massive parallel sequencing (WES). WES of two affected individuals (a mother and son) and the mother's unaffected parents identified a mutation in the C-propeptide cleavage site of COL1A1. Similar mutations have been reported in individuals with osteogenesis imperfecta (OI) and paradoxically increased BMD. C-propeptide cleavage site mutations in COL1A1 may not only cause 'high bone mass OI', but also the clinical features of GDD, specifically irregular sclerotic BMD and fibro-osseous lesions in the skull and jaw. GDD patients negative for ANO5 mutations should be assessed for mutations in type I collagen C-propeptide cleavage sites.

A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.

Genome-wide study of familial juvenile hyperuricaemic (gouty) nephropathy (FJHN) indicates a new locus, FJHN3, linked to chromosome 2p22.1-p21

Familial juvenile hyperuricaemic (gouty) nephropathy (FJHN), is an autosomal dominant disease associated with a reduced fractional excretion of urate, and progressive renal failure. FJHN is genetically heterogeneous and due to mutations of three genes: uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1beta (HNF-1β) on chromosomes 16p12, 1q32.1, and 17q12, respectively. However, UMOD, REN or HNF-1β mutations are found in only ~45% of FJHN probands, indicating the involvement of other genetic loci in ~55% of probands. To identify other FJHN loci, we performed a single nucleotide polymorphism (SNP)-based genome-wide linkage analysis, in six FJHN families in whom UMOD, HNF-1β and REN mutations had been excluded. Parametric linkage analysis using a 'rare dominant' model established linkage in five of the six FJHN families, with a LOD score >+3, at 0% recombination, between FJHN and SNPs at chromosome 2p22.1-p21. Analysis of individual recombinants in two unrelated affected individuals defined a ~5.5 Mbp interval, flanked telomerically by SNP RS372139 and centromerically by RS896986 that contained the locus, designated FJHN3. The interval contains 28 genes, and DNA sequence analysis of the most likely candidate, solute carrier family 8 member 1 (SLC8A1), did not identify any abnormalities in the FJHN3 probands. FJHN3 is likely located within a ~5.5 Mbp interval on chromosome 2p22.1-p21, and identifying the genetic abnormality will help to further elucidate mechanisms predisposing to gout and renal failure.

Investigation of the role of ENPP1 and TNAP genes in chondrocalcinosis

Objectives. Extracellular inorganic pyrophosphate (ePPi) inhibits certain forms of pathological mineralization while promoting others. Three molecules involved in ePPi regulation are important candidates for the development of calcium pyrophosphate dihydrate chondrocalcinosis (CPPD CC). These include ANKH, ectonucleotide pyrophosphatase (ENPP1) and TNAP. We have previously showed that genetic variation in ANKH is a cause of autosomal dominant familial CC and also some sporadic cases of CPPD CC. We now investigate the possible role of ENPP1 and TNAP in CPPD CC. Methods. Exons, untranslated regions (UTR) and exon-intron boundaries of ENPP1 and TNAP were sequenced using ABI Big Dye chemistry on automated sequencers. Sixteen variants were identified (3 in ENPP1 and 13 in TNAP) and were subsequently genotyped in 128 sporadic Caucasian CPPD CC patients and 600 healthy controls using a combination of polymerase chain reaction/restriction fragment-length polymorphism analysis or using Taqman. Allele and genotype frequencies were compared between cases and controls using the χ 2 test. Linkage disequilibrium, haplotype and the single nucleotide polymorphism-specific analyses were also performed. This study had 80% power to detect an odds ratio of 2.2 or more at these loci. Results. No difference was observed in the allele or genotype frequencies between patients and controls at either ENPP1 or TNAP. Conclusions. Polymorphisms of ENPP1 and TNAP are not major determinants of susceptibility to CC in the population studied. Further studies of the aetiology of sporadic CPPD CC are required to determine its causes.

A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G→A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.

A mega-analysis of genome-wide association studies for major depressive disorder

Prior genome-wide association studies (GWAS) of major depressive disorder (MDD) have met with limited success. We sought to increase statistical power to detect disease loci by conducting a GWAS mega-analysis for MDD. In the MDD discovery phase, we analyzed more than 1.2 million autosomal and X chromosome single-nucleotide polymorphisms (SNPs) in 18 759 independent and unrelated subjects of recent European ancestry (9240 MDD cases and 9519 controls). In the MDD replication phase, we evaluated 554 SNPs in independent samples (6783 MDD cases and 50 695 controls). We also conducted a cross-disorder meta-analysis using 819 autosomal SNPs with P<0.0001 for either MDD or the Psychiatric GWAS Consortium bipolar disorder (BIP) mega-analysis (9238 MDD cases/8039 controls and 6998 BIP cases/7775 controls). No SNPs achieved genome-wide significance in the MDD discovery phase, the MDD replication phase or in pre-planned secondary analyses (by sex, recurrent MDD, recurrent early-onset MDD, age of onset, pre-pubertal onset MDD or typical-like MDD from a latent class analyses of the MDD criteria). In the MDD-bipolar cross-disorder analysis, 15 SNPs exceeded genome-wide significance (P<5 x 10(-8)), and all were in a 248 kb interval of high LD on 3p21.1 (chr3:52 425 083-53 822 102, minimum P=5.9 x 10(-9) at rs2535629). Although this is the largest genome-wide analysis of MDD yet conducted, its high prevalence means that the sample is still underpowered to detect genetic effects typical for complex traits. Therefore, we were unable to identify robust and replicable findings. We discuss what this means for genetic research for MDD. The 3p21.1 MDD-BIP finding should be interpreted with caution as the most significant SNP did not replicate in MDD samples, and genotyping in independent samples will be needed to resolve its status.

Genome-wide meta-analysis of common variant differences between men and women

The male-to-female sex ratio at birth is constant across world populations with an average of 1.06 (106 male to 100 female live births) for populations of European descent. The sex ratio is considered to be affected by numerous biological and environmental factors and to have a heritable component. The aim of this study was to investigate the presence of common allele modest effects at autosomal and chromosome X variants that could explain the observed sex ratio at birth. We conducted a large-scale genome-wide association scan (GWAS) meta-analysis across 51 studies, comprising overall 114 863 individuals (61 094 women and 53 769 men) of European ancestry and 2 623 828 common (minor allele frequency >0.05) single-nucleotide polymorphisms (SNPs). Allele frequencies were compared between men and women for directly-typed and imputed variants within each study. Forward-time simulations for unlinked, neutral, autosomal, common loci were performed under the demographic model for European populations with a fixed sex ratio and a random mating scheme to assess the probability of detecting significant allele frequency differences. We do not detect any genome-wide significant (P < 5 x 10(-8)) common SNP differences between men and women in this well-powered meta-analysis. The simulated data provided results entirely consistent with these findings. This large-scale investigation across ~115 000 individuals shows no detectable contribution from common genetic variants to the observed skew in the sex ratio. The absence of sex-specific differences is useful in guiding genetic association study design, for example when using mixed controls for sex-biased traits.

Significant evidence of one or more susceptibility loci for endometriosis with near-Mendelian inheritance on chromosome 7p13-15

BACKGROUND: Endometriosis is a common disease with a heritable component. The collaborative International Endogene Study consists of two data sets (Oxford and Australia) comprising 1176 families with multiple affected. The aim was to investigate whether the apparent concentration of cases in a proportion of families could be explained by one or more rare variants with (near-)Mendelian autosomal inheritance. METHODS AND RESULTS: Linkage analyses (aimed at finding chromosomal regions harbouring disease-predisposing genes) were conducted in families with three or more affected (Oxford: n = 52; Australia: n = 196). In the Oxford data set, a non-parametric linkage score (Kong & Cox (K&C) Log of ODds (LOD)) of 3.52 was observed on chromosome 7p (genome-wide significance P = 0.011). A parametric MOD score (equal to maximum LOD maximized over 357 possible inheritance models) of 3.89 was found at 65.72 cM (D7S510) for a dominant model with reduced penetrance. After including the Australian data set, the non-parametric K&C LOD of the combined data set was 1.46 at 57.3 cM; the parametric analysis found an MOD score of 3.30 at D7S484 (empirical significance: P = 0.035) for a recessive model with high penetrance. Critical recombinant analysis narrowed the probable region of linkage down to overlapping 6.4 Mb and 11 Mb intervals containing 48 and 96 genes, respectively. CONCLUSIONS: This is the first report to suggest that there may be one or more high-penetrance susceptibility loci for endometriosis with (near-)Mendelian inheritance.

Familial isolated hyperparathyroidism is linked to a 1.7 Mb region on chromosome 2p13.3-14

BACKGROUND: Familial isolated hyperparathyroidism (FIHP) is an autosomal dominantly inherited form of primary hyperparathyroidism. Although comprising only about 1% of cases of primary hyperparathyroidism, identification and functional analysis of a causative gene for FIHP is likely to advance our understanding of parathyroid physiology and pathophysiology. METHODS: A genome-wide screen of DNA from seven pedigrees with FIHP was undertaken in order to identify a region of genetic linkage with the disorder. RESULTS: Multipoint linkage analysis identified a region of suggestive linkage (LOD score 2.68) on chromosome 2. Fine mapping with the addition of three other families revealed significant linkage adjacent to D2S2368 (maximum multipoint LOD score 3.43). Recombination events defined a 1.7 Mb region of linkage between D2S2368 and D2S358 in nine pedigrees. Sequencing of the two most likely candidate genes in this region, however, did not identify a gene for FIHP. CONCLUSIONS: We conclude that a causative gene for FIHP lies within this interval on chromosome 2. This is a major step towards eventual precise identification of a gene for FIHP, likely to be a key component in the genetic regulation of calcium homeostasis.

Genomewide significant linkage to migrainous headache on chromosome 5q21

Familial typical migraine is a common, complex disorder that shows strong familial aggregation. Using latent-class analysis (LCA), we identified subgroups of people with migraine/severe headache in a community sample of 12,245 Australian twins (60% female), drawn from two cohorts of individuals aged 23-90 years who completed an interview based on International Headache Society criteria. We report results from genomewide linkage analyses involving 756 twin families containing a total of 790 independent sib pairs (130 affected concordant, 324 discordant, and 336 unaffected concordant for LCA-derived migraine). Quantitative-trait linkage analysis produced evidence of significant linkage on chromosome 5q21 and suggestive linkage on chromosomes 8, 10, and 13. In addition, we replicated previously reported typical-migraine susceptibility loci on chromosomes 6p12.2-p21.1 and 1q21-q23, the latter being within 3 cM of the rare autosomal dominant familial hemiplegic migraine gene (ATP1A2), a finding which potentially implicates ATP1A2 in familial typical migraine for the first time. Linkage analyses of individual migraine symptoms for our six most interesting chromosomes provide tantalizing hints of the phenotypic and genetic complexity of migraine. Specifically, the chromosome 1 locus is most associated with phonophobia; the chromosome 5 peak is predominantly associated with pulsating headache; the chromosome 6 locus is associated with activity-prohibiting headache and photophobia; the chromosome 8 locus is associated with nausea/vomiting and moderate/severe headache; the chromosome 10 peak is most associated with phonophobia and photophobia; and the chromosome 13 peak is completely due to association with photophobia. These results will prove to be invaluable in the design and analysis of future linkage and linkage disequilibrium studies of migraine.

Dominant negative ATM mutations in breast cancer families

BACKGROUND: The ATM gene encoding a putative protein kinase is mutated in ataxia-telangiectasia (A-T), an autosomal recessive disorder with a predisposition for cancer. Studies of A-T families suggest that female heterozygotes have an increased risk of breast cancer compared with noncarriers. However, neither linkage analyses nor mutation studies have provided supporting evidence for a role of ATM in breast cancer predisposition. Nevertheless, two recurrent ATM mutations, T7271G and IVS10-6T-->G, reportedly increase the risk of breast cancer. We examined these two ATM mutations in a population-based, case-control series of breast cancer families and multiple-case breast cancer families. METHODS: Five hundred twenty-five or 262 case patients with breast cancer and 381 or 68 control subjects, respectively, were genotyped for the T7271G and IVS10-6T-->G ATM mutations, as were index patients from 76 non-BRCA1/2 multiple-case breast cancer families. Linkage and penetrance were analyzed. ATM protein expression and kinase activity were analyzed in lymphoblastoid cell lines from mutation carriers. All statistical tests were two-sided. RESULTS: In case and control subjects unselected for family history of breast cancer, one case patient had the T7271G mutation, and none had the IVS10-6T-->G mutation. In three multiple-case families, one of these two mutations segregated with breast cancer. The estimated average penetrance of the mutations was 60% (95% confidence interval [CI] = 32% to 90%) to age 70 years, equivalent to a 15.7-fold (95% CI = 6.4-fold to 38.0-fold) increased relative risk compared with that of the general population. Expression and activity analyses of ATM in heterozygous cell lines indicated that both mutations are dominant negative. CONCLUSION: At least two ATM mutations are associated with a sufficiently high risk of breast cancer to be found in multiple-case breast cancer families. Full mutation analysis of the ATM gene in such families could help clarify the role of ATM in breast cancer susceptibility.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.