TDP-43 maintains chondrocyte homeostasis and alleviates cartilage degradation in osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most prevalent age-related disorder due to cartilage degradation. Previous studies have identified aberrant chondrocyte homeostasis under extracellular stress as a key pathological mechanism behind cartilage degradation in OA. TDP-43, a DNA/RNA-binding protein has been demonstrated to participate in processing many extracellular stress responses; however, understanding of the role of TDP-43 in OA is limited. This study aims to investigate the role of TDP-43 in chondrocyte homeostasis and cartilage degradation in OA. METHODS: The role of TDP-43 during degradation of cartilage is examined by experimental posttraumatic OA animal models and human cartilage specimens. Cartilage degradation is assessed by histological analysis, qPCR, and Western blot. The molecular mechanisms are investigated in vitro using human primary chondrocytes. RESULTS: TDP-43 decreases significantly in degenerated cartilage. TDP-43 concentration is positively correlated with IL-1ß concentration in synovial fluid derived from OA patients (Pearson r = 0.95, CI (95%) [0.80, 0.99], P < 0.0001). Intra-articular injection of recombinant TDP-43 significantly alleviates cartilage degradation and subchondral bone remodeling in vivo. In vitro mechanistic analyses show that TDP-43 maintains chondrocyte homeostasis under oxidative stress through regulating stress granule dynamics via G3BP1. CONCLUSION: The present study indicates that TDP-43 maintains chondrocyte homeostasis under oxidative stress and alleviates cartilage degeneration in osteoarthritis, identifying TDP-43 as a potential target for the diagnosis and treatment of knee OA.

A Novel IFITM5 Variant Associated with Phenotype of Osteoporosis with Calvarial Doughnut Lesions: A Case Report.

Osteogenesis imperfecta (OI) and other decreased bone density disorders comprise a heterogeneous group of heritable diseases with skeletal fragility. Recently, it was discovered that mutations in SGMS2, encoding sphingomyelin synthetase 2, result in aberrant sphingomyelin metabolism and lead to a novel form of OI termed osteoporosis with calvarial doughnut lesions (OP-CDL) with moderate to severe skeletal fragility and variable cranial hyperostotic lesions. This study describes a Japanese family with the skeletal phenotype of OP-CDL. The affected individuals have moderately severe, childhood-onset skeletal fragility with multiple long-bone fractures, scoliosis and bone deformities. In addition, they exhibit multiple CDLs or calvarial bumps with central radiolucency and peripheral radiopacity. However, SGMS2 sequencing was normal. Instead, whole-exome sequencing identified a novel IFITM5 missense mutation c.143A>G (p.N48S) (classified as a VUS by ACMG). IFITM5 encodes an osteoblast-restricted protein BRIL and a recurrent c.-14C>T mutation in its 5' UTR region results in OI type V, a distinctive subtype of OI associated with hyperplastic callus formation and ossification of the interosseous membranes. The patients described here have a phenotype clearly different from OI type V and with hyperostotic cranial lesions, feature previously unreported in association with IFITM5. Our findings expand the genetic spectrum of OP-CDL, indicate diverse phenotypic consequences of pathogenic IFITM5 variants, and imply an important role for BRIL in cranial skeletogenesis.

A population-based study identifies an association of THBS2 with intervertebral disc degeneration.

OBJECTIVE: To clarify the genetic mechanisms underlying intervertebral disc degeneration (IDD), we examined the associations between single-nucleotide polymorphisms (SNPs) and indicated as coefficient of interaction term (IDD) in a general population in Japan. METHODS: This was a cross-sectional study. In 1,605 participants, C2-3 to L5/S1 in the total spine magnetic resonance imaging (MRI) were evaluated using the Pfirrmann's scoring system. Disc scores of 4 and 5 were defined as IDD. Eight SNPs in eight genes associated with IDD were examined at each disc level, considering the non-genetic risk factors of age, sex, and body mass index (BMI). RESULTS: The highest odds ratio was found for rs9406328 in the THBS2 gene at disc level T12-L1 (OR 1.27, 95%CI 1.05 to 1.53), and this association was strengthened after adjustment for age using logistic regression (OR 1.37, 95%CI 1.12 to 1.67). Among participants aged <50 years and 50-59, the average IDD score in those with 2 risk alleles of rs9406328 was markedly higher than in those with 0 or 1 risk allele, and the difference is much wider than the elderly participants. It indicates the genetic effect of rs9406328 is stronger in the younger age groups. Finally, multiple linear regression analyses of the association between rs9406328 and IDD, adjusted for age, sex, and BMI at each disc level, showed a statistical interaction between age and the number of risk alleles at C7-T1, T3-4 and T4-T5 as well as T12-L1. CONCLUSION: CONCLUSION: The association between rs9406328 in THBS2 and IDD was replicated. The contributions of genetic and environmental factors to IDD differed by disc level.

Response to Lefebvre et al.

Congenital scoliosis (CS) is a common vertebral malformation with incidence of up to 1 of 1000 births worldwide. Recently, TBX6 has been reported as the first disease gene for CS: about 10% of CS patients are compound heterozygotes of rare null mutations and a common haplotype composed by 3 SNPs in TBX6. Lefebvre et al in this journal reported that 2 patients with spondylocostal dysostosis (SCD), a rare skeletal dysplasia affecting spine and ribs also have TBX6 mutations: 1 carried the microdeletion and a rare missense variant, and another 2 rare missense variants. We investigated the pathogenicity of the 3 missense variants in SCD by a luciferase assay. The results were negative for the proposal of Lefebvre et al. We consider these 2 SCD patients are more probably compound heterozygotes of null mutations and a common risk haplotype just as CS patients with TBX6 mutations.

Genome-wide DNA methylation profile implicates potential cartilage regeneration at the late stage of knee osteoarthritis.

OBJECTIVE: The aim of this work was to characterize the genome-wide DNA methylation profile of cartilage from three regions of tibial plateau isolated from patients with primary knee osteoarthritis (OA), providing the first DNA methylation study that reflects OA progression. METHODS: The unique model system was used to section three regions of tibial plateau: the outer lateral tibial plateau (oLT), the inner lateral tibial plateau (iLT) and the inner medial tibial plateau (iMT) regions which represented the early, intermediate and late stages of OA, respectively. Genome-wide DNA methylation profile was examined using Illumina Infinium HumanMethylation450 BeadChip array. Comparisons of the iLT/oLT and iMT/oLT groups were carried out to identify differentially methylated (DM) probes (DMPs) associated with OA progression. DM genes were analyzed to identify the gene ontologies (GO), pathways, upstream regulators and networks. RESULTS: No significant DMPs were identified in iLT/oLT group, while 519 DMPs were identified in iMT/oLT group. Over half of them (68.2%) were hypo-methylated and enriched in enhancers and OpenSea. Upstream regulator analysis identified many microRNAs. DM genes were enriched in transcription factors, especially homeobox genes and in Wnt/ß-catenin signaling pathway. These genes also showed changes in expression when analyzed with expression profiles generated from previous studies. CONCLUSION: Our data suggested the changes in DNA methylation occurred at the late stage of OA. Pathways and networks enriched in identified DM genes highlighted potential etiologic mechanism and implicated the potential cartilage regeneration in the late stage of knee OA.

Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common form of human myelopathy caused by a compression of the spinal cord by ectopic ossification of spinal ligaments. To elucidate the genetic basis for OPLL, we have been studying the ttw (tiptoe walking; previously designated twy) mouse, a naturally occurring mutant which exhibits ossification of the spinal ligaments very similar to human OPLL (refs 3,4). Using a positional candidate-gene approach, we determined the ttw phenotype is caused by a nonsense mutation (glycine 568 to stop) in the Npps gene which encodes nucleotide pyrophosphatase. This enzyme regulates soft-tissue calcification and bone mineralization by producing inorganic pyrophosphate, a major inhibitor of calcification. The accelerated bone formation characteristic of ttw mice is likely to result from dysfunction of NPPS caused by predicted truncation of the gene product, resulting in the loss of more than one-third of the native protein. Our results may lead to novel insights into the mechanism of ectopic ossification and the aetiology of human OPLL.

Domain-specific mutations in TGFB1 result in Camurati-Engelmann disease.

Camurati-Engelmann disease (CED, MIM 131300) is an autosomal dominant, progressive diaphyseal dysplasia characterized by hyperosteosis and sclerosis of the diaphyses of long bones. We recently assigned the CED locus to an interval between D19S422 and D19S606 at chromosome 19q13.1-q13.3, which two other groups confirmed. As the human transforming growth factor-1 gene (TGFB1) is located within this interval, we considered it a candidate gene for CED.

Recommendations for standardization and phenotype definitions in genetic studies of osteoarthritis: the TREAT-OA consortium.

OBJECTIVE: To address the need for standardization of osteoarthritis (OA) phenotypes by examining the effect of heterogeneity among symptomatic (SOA) and radiographic osteoarthritis (ROA) phenotypes. METHODS: Descriptions of OA phenotypes of the 28 studies involved in the TREAT-OA consortium were collected. We investigated whether different OA definitions result in different association results by creating various hip OA definitions in one large population based cohort (the Rotterdam Study I (RSI)) and testing those for association with gender, age and body mass index using one-way ANOVA. For ROA, we standardized the hip-, knee- and hand ROA definitions and calculated prevalence's of ROA before and after standardization in nine cohort studies. This procedure could only be performed in cohort studies and standardization of SOA definitions was not feasible at this moment. RESULTS: In this consortium, all studies with SOA phenotypes (knee, hip and hand) used a different definition and/or assessment of OA status. For knee-, hip- and hand ROA five, four and seven different definitions were used, respectively. Different hip ROA definitions do lead to different association results. For example, we showed in the RSI that hip OA defined as "at least definite joint space narrowing (JSN) and one definite osteophyte" was not associated with gender (P =0.22), but defined as "at least one definite osteophyte" was significantly associated with gender (P=3×10(-9)). Therefore, a standardization process was undertaken for ROA definitions. Before standardization a wide range of ROA prevalence's was observed in the nine cohorts studied. After standardization the range in prevalence of knee- and hip ROA was small. CONCLUSION: Phenotype definitions influence the prevalence of OA and association with clinical variables. ROA phenotypes within the TREAT-OA consortium were standardized to reduce heterogeneity and improve power in future genetics studies.

Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family.

BACKGROUND: Mutations in TRPV4, a gene that encodes a Ca(2+) permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. OBJECTIVES AND METHODS: To examine TRPV4 mutation spectrum and phenotype-genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. RESULTS: TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. CONCLUSION: The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

Muscle oxygenation and fascicle length during passive muscle stretching in ballet-trained subjects.

Muscle stretching transiently decreases muscle-blood flow corresponding to a muscle extension. It may disturb a balance between muscular oxygen demand and oxygen supply to muscles and reduce muscle oxygenation. However, muscle-stretching training may improve blood circulatory condition, resulting in the maintained muscle oxygenation during muscle stretching. The aim of this study was to investigate changes in muscle-blood volume (tHb) and tissue oxygenation index (TOI) during muscle stretching determined by using near-infrared spectroscopy (NIRS) in ballet-trained (BT) and untrained (C) subjects. 11 BT women who regularly perform muscle stretching and 11 C women participated in this study. Fascicle lengths, tHb and TOI in the tibialis anterior muscle were measured during passive plantar flexion from ankle joint angles of 120° (baseline) to 140°, 160°, the maximal comfortable position without pain (CP), and the maximal position (MP). At 160°, the % fascicle-length change from baseline was significantly lower in the BT than the C group, however, for the changes in tHb and TOI the significant interaction effect between the 2 groups was not detected. On the other hand, although the increases in the fascicle length from baseline to CP and MP were greater in BT than C, the tHb and TOI reductions were comparable between groups. We concluded that it appears that BT can extend their muscles without excessive reduction in muscle-blood volume and muscle oxygenation at relatively same but absolutely greater muscle-stretching levels than C. The attenuation in these indices during high-level muscle stretching may be associated with the repetitive muscle stretching of long-term ballet training.

Identification of loss-of-function mutations of SLC35D1 in patients with Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases.

BACKGROUND: Schneckenbecken dysplasia (SBD) is an autosomal recessive lethal skeletal dysplasia that is classified into the severe spondylodysplastic dysplasias (SSDD) group in the international nosology for skeletal dysplasias. The radiological hallmark of SBD is the snail-like configuration of the hypoplastic iliac bone. SLC35D1 (solute carrier-35D1) is a nucleotide-sugar transporter involved in proteoglycan synthesis. Recently, based on human and mouse genetic studies, we showed that loss-of-function mutations of the SLC35D1 gene (SLC35D1) cause SBD. OBJECT: To explore further the range of SLC35D1 mutations in SBD and elucidate whether SLC35D1 mutations cause other skeletal dysplasias that belong to the SSDD group. METHODS AND RESULTS: We searched for SLC35D1 mutations in five families with SBD and 15 patients with other SSDD group diseases, including achodrogenesis type 1A, spondylometaphyseal dysplasia Sedaghatian type and fibrochondrogenesis. We identified four novel mutations, c.319C>T (p.R107X), IVS4+3A>G, a 4959-bp deletion causing the removal of exon 7 (p.R178fsX15), and c.193A>C (p. T65P), in three SBD families. Exon trapping assay showed IVS4+3A>G caused skipping of exon 4 and a frameshift (p.L109fsX18). Yeast complementation assay showed the T65P mutant protein lost the transporter activity of nucleotide sugars. Therefore, all these mutations result in loss of function. No SLC35D1 mutations were identified in all patients with other SSDD group diseases. CONCLUSION: Our findings suggest that SLC35D1 loss-of-function mutations result consistently in SBD and are exclusive to SBD.

Testing the druggable endothelial differentiation gene 2 knee osteoarthritis genetic factor for replication in a wide range of sample collections.

OBJECTIVES: To replicate a previously reported association with osteoarthritis (OA) of the promoter single nucleotide polymorphism (SNP) rs10980705 in the endothelial differentiation gene 2 (EDG2). METHODS: Five collections of samples, four from Europe and one from China, were studied. They included patients with 3 OA phenotypes: 1501 with knee OA, 1497 with hip OA and 376 with generalised OA. A total of 2521 controls were also studied. Allele and genotype frequencies of the rs10980705 SNP were analysed in each individual sample collection and in pooled data. In addition, a meta-analysis to incorporate results from the original Japanese report was performed. RESULTS: The association of the rs10980705 SNP with knee OA was not replicated in any of the five sample collections studied or in their combined analysis (odds ratio (OR) 1.10, 95% CI 0.98 to 1.22; p = 0.10). Meta-analysis of all data, including the original Japanese study, did show association with knee OA (OR 1.15, 95% CI 1.06 to 1.26; p = 0.002) but the effect was accounted for by the Japanese data and was less significant than the original report. No association was found with hip OA or with generalised OA. CONCLUSIONS: The original report of a promising genetic association between a druggable G-protein coupled receptor, EDG2, and knee OA has not been replicated. This lack of replication could be due to a modest effect of the promoter polymorphism that will require even larger studies (the winners curse) although a more pronounced effect in the Asian population vs Europeans cannot be excluded.

Novel KIAA0753 mutations extend the phenotype of skeletal ciliopathies.

The skeletal ciliopathies are a heterogeneous group of disorders with a significant clinical and genetic variability and the main clinical features are thoracic hypoplasia and short tubular bones. To date, 25 genes have been identified in association with skeletal ciliopathies. Mutations in the KIAA0753 gene have recently been associated with Joubert syndrome (JBTS) and orofaciodigital (OFD) syndrome. We report biallelic pathogenic variants in KIAA0753 in four patients with short-rib type skeletal dysplasia. The manifestations in our patients are variable and ranging from fetal lethal to viable and moderate skeletal dysplasia with narrow thorax and abnormal metaphyses. We demonstrate that KIAA0753 is expressed in normal fetal human growth plate and show that the affected fetus, with a compound heterozygous frameshift and a nonsense mutation in KIAA0753, has an abnormal proliferative zone and a broad hypertrophic zone. The importance of KIAA0753 for normal skeletal development is further confirmed by our findings that zebrafish embryos homozygous for a nonsense mutation in kiaa0753 display altered cartilage patterning.

An autosomal dominant posterior polar cataract locus maps to human chromosome 20p12-q12.

We assigned the locus for a previously reported new type of autosomal dominant posterior polar cataract (CPP3) to 20p12-q12 by a genome-wide two-point linkage analysis with microsatellite markers. CPP3 is characterized by progressive, disc-shaped, posterior subcapsular opacity. The disease was seen in 10 members of a Japanese family and transmitted in an autosomal dominant fashion through four generations. We obtained a maximum lod score (Zmax) of 3.61 with a recombination fraction (theta) of 0.00 for markers D20S917, D20S885 and D20S874. Haplotype analysis gave the disease gene localization at a 15.7-cM interval between D20S851 and D20S96 loci on chromosome 20p12-q12. Since the BFSP1 that encodes the lens-specific beaded filament structural protein 1 (filensin) has been mapped around the CPP3 region, we performed sequence analysis on its entire coding region. However, no base substitution or deletion was detected in the CPP3 patients. The mapping of the CPP3 locus to 20p12-q12 not only expands our understanding of the genetic heterogeneity in autosomal dominant posterior polar cataracts but also is a clue for the positional cloning of the disease gene.

Sequence analysis of a total of three megabases of DNA in two regions of chromosome 8p.

Large-scale sequencing of genomic regions and in silico gene trapping together represent a highly efficient and powerful approach for identifying novel genes. We performed megabase-level sequence analyses of two genomic regions on human chromosome 8p (8p11.2 and 8p22-->p21.3), after covering those segments with sequence-ready contigs composed of 74 cosmids, 14 BACs, and three PAC clones. We determined continuous nucleotide sequences of 1,856,753 bases on 8p11.2 and 1,210,381 bases on 8p22-->p21.3 by combining the shotgun and primer-walking methods. In silico gene trapping identified four novel genes in the 8p11.2 region and, in the 8p22-->p21.3 region, six known genes (PRLTS, PCM1, MTAMR7, HCAT2, HFREP-1 and PHP) and three novel genes. The distribution of Alu and LINE1 repetitive elements and the densities of predicted exons were different in each region, and Alu-rich portions contained more exonic sequences than LINE1-rich areas.

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene. Pathological and immunohistochemical similarity to human familial amyloidotic polyneuropathy, type I.

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.

Structure of the gene encoding human colligin-2 (CBP2).

Colligins are collagen-binding proteins localized to the endoplasmic reticulum that belong to the superfamily of serine protease inhibitors and play a role in collagen biosynthesis. Previously, we cloned the human colligin-2 gene (CBP2) and mapped it to chromosome 11q13.15. To further characterize the CBP2 gene, we have determined its genomic structure and the 5'-flanking sequence. The CBP2 gene spanned approximately 11 kb of genomic DNA and consisted of five exons. The promoter sequence of the human gene showed significant homology to that of its murine counterpart, which contained several regulatory sequences including heat-shock and retinoic acid-responsive elements. These findings suggest colligin may function as a collagen-specific molecular chaperon and play a role in the process of retinoic acid-induced differentiation.

Novel mutation in exon 18 of the cartilage oligomeric matrix protein gene causes a severe pseudoachondroplasia.

Pseudoachondroplasia (PSACH) is a common skeletal dysplasia characterized by disproportionate short stature, early-onset osteoarthrosis, and dysplasia of the spine, epiphysis, and metaphysis. Multiple epiphyseal dysplasia (MED) is a similar but less severe disorder characterized by dysplasia of the epiphysis. Both disorders are caused by mutations in the cartilage oligomeric matrix protein (COMP) gene. COMP mutations cluster in a region of the gene that encodes calmodulin-like repeats (CLRs) and correlate closely with disease severity. Typically, mutations in exon 13 that composes the seventh CLR produce severe PSACH phenotypes, whereas mutations found elsewhere in the gene produce mild PSACH or MED phenotypes. We have identified a PSACH patient carrying a novel mutation in exon 18 of COMP that composes the C-terminal globular domain. This mutation produced a severe PSACH phenotype with marked short stature and deformities of the spine and extremities. Our results extend the range of disease-causing mutations within the COMP gene and demonstrate the importance of the additional domain of COMP protein in its in vivo function.

Pseudoachondroplasia with de novo deletion [del(11)(q21q22.2)].

Pseudoachondroplasia (PSACH) is a relatively common osteochondrodysplasia characterized clinically by short-limbed short stature with normal face, and radiographically by platyspondyly and dysplasias of epiphyses and metaphyses of the tubular bones. Recently, mutation of cartilage oligomeric matrix protein has been identified in PSACH. However, clinical variability and genetic heterogeneity have been reported in PSACH, indicating a possible existence of a second PSACH gene. Here, we report on a patient with a typical severe form of PSACH who had a de novo interstitial deletion in the long arm of chromosome 11 [del(11)(q21q22.2)]. The size of the deletion was estimated at 0.8-7.3 Mb using fluorescent in situ hybridization (FISH). This deletion may contain or disrupt a second PSACH locus.

Metaphyseal anadysplasia: evidence of genetic heterogeneity.

We report on two unrelated children, a girl and a boy, with regressive metaphyseal dysplasia. Both children had bow legs and a transient growth decline in early childhood. Metaphyseal modifications of the long bones in the children were most conspicuous at an early age and then subsided by age 2 to 3 years. The father of the boy may have had the same disorder, because he was shorter than his sibs and showed mild modifications of the vertebral end plates with mild narrowing of the interpediculate distance of the lumbar spine. The evolution of the metaphyseal dysplasia in the children closely resembled that of metaphyseal anadysplasia (MAD), which is X-linked recessive in inheritance. By contrast, the occurrence of an isolated, affected girl and possible father-to-son transmission reported here were consistent with autosomal dominant transmission, suggesting heterogeneity of MAD. Molecular studies of the type X collagen gene in the boy did not demonstrate any disease-causing mutation.