Identification of a deep intronic mutation in the COL6A2 gene by a novel custom oligonucleotide CGH array designed to explore allelic and genetic heterogeneity in collagen VI-related myopathies

BACKGROUND: Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. METHODS: We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. RESULTS: A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. CONCLUSIONS: Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes

ADAMTS‐4_v1 Is a Splice Variant of ADAMTS‐4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain

Objective. We have previously reported a mRNA variant of ADAMTS4 (ADAMTS4_v1) in human synovial cell co-cultures obtained from osteoarthritic patients. This RNA message has only been found in OA synovium and if translated would result in a protein identical to ADAMTS4 except that the C-terminal spacer domain would be different. In this study, we set out to determine if ADAMTS4_v1 is translated into a protein, expressed in vivo and acts as a functional aggrecanase. Methods. Polyclonal antibodies were raised against unique C-terminus sequences of ADAMTS4_v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG-tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4_v1 and the resultant plasmid was used to transfect HEK293 cells. ADAMTS4_v1 produced by these cells was purified via the FLAG epitope and the ability of this recombinant protein to cleave aggrecan, biglycan and decorin was investigated. Results. An antibody specific to ADAMTS4_v1 was found to bind to the synovial membrane surface on cryosections and the protein was detected in synovial cell lysates from human OA patient synovium. The recombinant ADAMTS4_v1 demonstrated enzyme activity towards the target substrate in a commercial aggrecanase-1 ELISA assay and was also found to cleave aggrecan at the pathologically important Glu373↓374Ala aggrecanase site. Conclusion. ADAMTS4_v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase and cleaves other proteoglycan substrates. This splice variant may be a major contributor to superficial zone loss of aggrecan in OA cartilage

Hyaline Cartilage Tissue Is Formed through the Co-culture of Passaged Human Chondrocytes and Primary Bovine Chondrocytes

To circumvent the problem of sufficient number of cells for cartilage engineering, we previously developed a two stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage one and compare it to bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data shows that the extracellular matrix accumulation was evident after 2 weeks of co-culture which was one week behind the bP0 control culture. By 3-4 weeks the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulphate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells and to start stage two is just after 3 weeks