Mutations of the alpha2(V) chain of type V collagen impair matrix assembly and produce ehlers-danlos syndrome type I

Ehlers-Danlos syndrome (EDS) is a heterogeneous connective tissue disorder that severely impairs the structure and function of the skin, joints, eyes and blood vessels. We have identified mutations of the COL5A2 gene, which encodes the alpha2(V) chain of type V collagen, in two unrelated patients with the severe type I form of EDS. The first proband was heterozygous for a 7 bp deletion that resulted in skipping of exon 27 while the second proband was heterozygous for a single nucleotide substitution that resulted in skipping of exon 28. Cultured dermal fibroblasts from both probands produced about equal amounts of the normal and mutant alpha2(V) mRNAs and protein chains. The dermis from the first proband contained a sparse collagen fibrillar network with great variability in collagen fibril sizes and shapes. The dermal collagens were also abnormally soluble. Bone cells from the first proband also produced about equal amounts of the normal and mutant alpha2(V) mRNAs. However, the collagen fibrillar architecture and collagen solubility of the bone matrix were normal. Our findings show that heterozygous mutations of the COL5A2 gene can produce the EDS type I phenotype. They also suggest that type V collagen plays a more important role in collagen fibrillogenesis of dermis than that of bone.      

Cloning of cDNA for rat pro alpha1(V) collagen mRNA. Expression patterns of type I, type III and type V collagen genes in experimental granulation tissue

A cDNA clone for rat pro alpha1(V) collagen mRNA was constructed using PCR amplification, with primers based on human and hamster COL5A1 gene sequences. The clone pRCVA1 is 560 nucleotides long and it encodes for the carboxy propeptide of type V procollagen. Homology shared with type I collagen sequence was 64%, with type II collagen 65% and with type III collagen 61%. To evaluate the spatial and temporal expression of type V collagen mRNA in wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 5, 8, 14, 21, 30, 59 and 84. Specific cDNA probes to pro alpha1(I), pro alpha1(III) and pro alpha1(V) collagen mRNA were used in slot blot, Northern and in situ hybridization. Type I collagen gene expression was upregulated at the initial stage of wound healing, type III collagen gene expression was constant and from the day 14 onwards type I and III collagen gene expressions were at the same level. Type V collagen gene expression was seen at every time point studied but at a considerably lower level than type I and III collagens. In situ hybridization showed that type V collagen was expressed in two different cell types. In conclusion, type V collagen was expressed in the wound healing model from at least day 5 onwards and it was synthesized by fibroblast-like and rounded cells.     

Erratum: analysis of DNA elements that modulate myosin VIIa expression in humans

Usher syndromeIb (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE-19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE-19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A -2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, -2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B.     

5' flanking sequence of the human immediate early responsive gene ccn1 (cyr61) and mapping of polymorphic CA repeat sequence motifs in the human ccn1 (cyr61) locus.

AIMS: The human ccn1 (hccn; hcyr61) gene has been identified previously at the mRNA and protein level as a 1,25-dihydroxyvitamin D(3) and growth factor regulated gene in human osteoblasts. This study aimed to analyse genomic clones containing the human ccn1 (cyr61) gene and to provide the 5' flanking region. METHODS: Genomic clones were isolated by screening a lambda library and by array filter hybridisations of a genomic library. Sequencing was performed using the dye terminator method. Promoter activity was measured after transient transfection using a beta galactosidase assay. CA repeat motifs were studied by a combined PCR/fragment analysis protocol. RESULTS: The human 5' flanking region of 870 nucleotides contains several stretches with high homology to the mouse promoter as well as CA repeat motifs. This first report on the human 5' flanking sequence of the hccn1 (hcyr61) gene provides important insights into regulation pathways for the expression of this 1,25-dihydroxyvitamin D(3) and growth factor responsive early gene. A genomic clone containing the hccn1 (hcyr61) gene region also yielded a CA sequence located 3' of the ccn1 (cyr61) gene. This CA repeat and one of the CA repeat motifs in the promoter were studied in detail and found to be polymorphic. CONCLUSIONS: The 5' flanking sequence of the hccn1 (hcyr61) gene provides insights into the mechanisms of regulation of this immediate early gene product. The CA repeat polymorphisms within the gene region will be useful in the genetic study of disorders affecting bone metabolism.     

Functional analysis of polymorphic variation within the promoter and 5' untranslated region of the neurofibromatosis type 1 (NF1) gene

The regulatory regions of the neurofibromatosis type 1 (NF1) gene have scarcely been screened either for mutations of potential pathological importance or for functional polymorphisms. To address this question, a 987 bp sequence spanning the promoter and 5' flanking sequence of the human NF1 gene was screened for sequence variants in 570 unrelated NF1 patients and 105 controls. Five novel sequence variants were identified, comprising a 14 bp deletion at -142 within the promoter region, three single nucleotide substitutions in the 5'UTR (C + 247T, C + 261G, G + 462C), and a substitution (C + 514T) at the 5' end of the coding region that served to generate a Stop codon. The latter is likely to be of pathological significance since it is predicted to lead to the synthesis of a truncated protein. The functional significance of three of the other variants (14 bp del, C + 261G, G + 462C) was explored by luciferase reporter gene expression and electrophoretic mobility shift assays. The del14 variant demonstrated allele-specific protein binding without altered reporter gene expression and the G + 462C allele showed slightly decreased reporter gene expression.