CCN6 regulates mitochondrial respiratory complex assembly and activity.

Cellular communication network factor 6 (CCN6) mutations are linked with Progressive Pseudo Rheumatoid Dysplasia (PPRD) a debilitating musculoskeletal disorder. The function of CCN6 and the mechanism of PPRD pathogenesis remain unclear. Accordingly, we focused on the functional characterization of CCN6 and CCN6 mutants. Using size exclusion chromatography and native polyacrylamide gel electrophoresis we demonstrated that CCN6 is present as a component of the mitochondrial respiratory complex in human chondrocyte lines. By means of siRNA-mediated transfection and electron microscopy we showed that moderate reduction in CCN6 expression decreases the RER- mitochondria inter-membrane distance. Parallel native PAGE, immunoblotting and Complex I activity assays furthermore revealed increase in both mitochondrial distribution of CCN6 and mitochondrial respiratory complex assembly/activity in CCN6 depleted cells. CCN6 mutants resembling those linked with PPRD, which were generated by CRISPR-Cas9 technology displayed low level of expression of mutant CCN6 protein and inhibited respiratory complex assembly/activity. Electron microscopy and MTT assay of the mutants revealed abnormal mitochondria and poor cell viability. Taken together, our results indicate that CCN6 regulates mitochondrial respiratory complex assembly/activity as part of the mitochondrial respiratory complex by controlling the proximity of RER with the mitochondria, and CCN6 mutations disrupt mitochondrial respiratory complex assembly/activity resulting in mitochondrial defects and poor cell viability.

Progressive Pseudorheumatoid Dysplasia resolved by whole exome sequencing: a novel mutation in WISP3 and review of the literature.

BACKGROUND: Progressive pseudorheumatoid dysplasia (PPRD) is a rare autosomal-recessive, non-inflammatory arthropathy, shown to be caused by mutations in the WNT1-inducible signaling pathway protein 3 (WISP3) gene. Although several hundred cases were reported worldwide, the diagnosis remains challenging. Subsequently, the syndrome is often unrecognized and misdiagnosed (for instance, as Juvenile Idiopathic Arthritis), leading to unnecessary procedures and treatments. The objective of the current study was to identify the molecular basis in a family with PPRD and describe their phenotype and course of illness. PATIENTS AND METHODS: We present here a multiply affected consanguineous family of Iraqi-Jewish descent with PPRD. The proband, a 6.5 years old girl, presented with bilateral symmetric bony enlargements of the 1st interphalangeal joints of the hands, without signs of synovitis. Molecular analysis of the family was pursued using Whole Exome Sequencing (WES) and homozygosity mapping. RESULTS: WES analysis brought to the identification of a novel homozygous missense mutation (c.257G > T, p.C86F) in the WISP3 gene. Following this diagnosis, an additional 53 years old affected family member was found to harbor the mutation. Two other individuals in the family were reported to have had similar involvement however both had died of unrelated causes. CONCLUSION: The reported family underscores the importance of recognition of this unique skeletal dysplasia by clinicians, and especially by pediatric rheumatologists and orthopedic surgeons.

Novel COL2A1 variant (c.619G>A, p.Gly207Arg) manifesting as a phenotype similar to progressive pseudorheumatoid dysplasia and spondyloepiphyseal dysplasia, Stanescu type.

Progressive pseudorheumatoid dysplasia (PPRD) is a rare, autosomal-recessive condition characterized by mild spondyloepiphyseal dysplasia (SED) and severe, progressive, early-onset arthritis due to WISP3 mutations. SED, Stanescu type, is a vaguely delineated autosomal-dominant dysplasia of unknown genetic etiology. Here, we report three individuals from two unrelated families with radiological features similar to PPRD and SED, Stanescu type who share the same novel COL2A1 variant and were matched following discussion at an academic conference. In the first family, we performed whole-exome sequencing on three family members, two of whom have a PPRD-like phenotype, and identified a heterozygous variant (c.619G>A, p.Gly207Arg) in both affected individuals. Independently, targeted sequencing of the COL2A1 gene in an unrelated proband with a similar phenotype identified the same heterozygous variant. We suggest that the p.Gly207Arg variant causes a distinct type II collagenopathy with features of PPRD and SED, Stanescu type.

Ccn6 Is Required for Mitochondrial Integrity and Skeletal Muscle Function in Zebrafish.

Mutations in the CCN6 (WISP3) gene are linked with a debilitating musculoskeletal disorder, termed progressive pseudorheumatoid dysplasia (PPRD). Yet, the functional significance of CCN6 in the musculoskeletal system remains unclear. Using zebrafish as a model organism, we demonstrated that zebrafish Ccn6 is present partly as a component of mitochondrial respiratory complexes in the skeletal muscle of zebrafish. Morpholino-mediated depletion of Ccn6 in the skeletal muscle leads to a significant reduction in mitochondrial respiratory complex assembly and activity, which correlates with loss of muscle mitochondrial abundance. These mitochondrial deficiencies are associated with notable architectural and functional anomalies in the zebrafish muscle. Taken together, our results indicate that Ccn6-mediated regulation of mitochondrial respiratory complex assembly/activity and mitochondrial integrity is important for the maintenance of skeletal muscle structure and function in zebrafish. Furthermore, this study suggests that defects related to mitochondrial respiratory complex assembly/activity and integrity could be an underlying cause of muscle weakness and a failed musculoskeletal system in PPRD.

Progressive pseudorheumatoid dysplasia: a case series report.

rogressive pseudorheumatoid dysplasia (PPRD) is a rare autosomal-recessive, noninflammatory arthropathy. Several cases have been reported worldwide; however, diagnosis remains challenging. Three unrelated children with PPRD were retrospectively studied. All three patients in this study were initially misdiagnosed. The misdiagnoses included juvenile rheumatoid arthritis, myodystrophy and idiopathic short stature. The time from the onset of symptoms to a definitive diagnosis was 3 to 8 years. Clinical signs and radiological phenotypes were analyzed carefully, and they were all consistent with the characteristics of PPRD and noninflammatory polyarticular enlargement. The small joints of both the hands and lower limbs are the most affected. The imaging findings of the patients were flat vertebrae with beak- or bullet-like changes in front of the cone and peripheral metaphysis widening. DNA samples obtained from the family were sequenced to identify the causal gene using whole-exome sequencing (WES). Four Wnt1-inducible signaling pathway protein 3 (WISP3) mutations were verified. c.271delC was not reported previously. The other three mutations, namely, c.136C>T (p. Gln46*), c.667T>G (p. Cys223Gly) and c.589+2T>C, were previously identified. All three patients had a long journey to diagnosis. Early genetic diagnosis can help prevent unnecessary treatments and procedures in patients. Growth hormone is not a good choice for treatment.

Multi-scale mechanical investigation of articular cartilage suffered progressive pseudorheumatoid dysplasia.

BACKGROUND: Progressive pseudorheumatoid dysplasia is a rare skeletal dysplasia mainly caused by abnormal autosomal recessive inheritance. Although the main function of cartilage is mechanical support and the characteristics of this disease is the degradation of AC, previous studies on it had been mainly focused on clinical and genetic aspects and the mechanical behavior of the cartilage affected by PPRD is still ambiguous. In this study, we investigate the mechanics and structure of the cartilage suffered disease at multi-scale, from individual chondrocytes to the bulk-scale tissue. METHODS: Depth-sensing indenter were employed to investigate the mechanics of cartilage; we performed atomic force microscope nanoindentation to investigate the cell mechanics and scanning electron microscopy were used to explore the structure feature and chemical composition. FINDINGS: The elastic modulus of chondrocytes harvested from cartilage suffered from progressive pseudorheumatoid dysplasia is significantly higher than from normal cartilage, same trend were also found in tissue level. Moreover, denser collagen meshwork and matrix calcification were also observed. INTERPRETATION: The elastic modulus of cartilage should closely related to its denser structure and the calcification, and may potentially be an indicator for clinical diagnosis. The stiffening of chondrocytes during PPRD progression should play a rather important role in its pathogenesis.

A Novel Homozygous Frameshift Mutation in CCN6 Causing Progressive Pseudorheumatoid Dysplasia (PPRD) in a Consanguineous Yemeni Family.

Background: Progressive pseudorheumatoid dysplasia (PPRD) inherited in an autosomal recessive fashion, is a disabling disease, characterized by platyspondyly, irregularities of the vertebral bodies, narrowing of the intervertebral discs and intraarticular spaces, widening of the epiphysis-metaphysis, polyarthralgia, multiple joint contractures, and disproportionate short stature. A number of studies have been performed on this deformity in various populations around the globe, including the Arab population. Mutations in CCN6, located on 6q22, are reported to cause this anomaly. Case Presentation: The present study describes the investigation of a consanguineous family of Yemeni origin. Clinical examination of the patient revealed short stature with progressive skeletal abnormalities, stiffness and enlargement of small joints of the hands along with restriction of movements of proximal interphalangeal (PIP) and distal interphalangeal (DIP) joints with weakness and gait disturbance. Sanger sequencing revealed a novel homozygous frameshift deletion mutation (c.746delT; p.Val249Glyfs*10) in CCN6 which may lead to NMD (Nonsense mediated decay). This mutation expands the spectrum of pathogenic variants in CCN6 causing PPRD.