F. M. Pope

A single base mutation in COL5A2 causes Ehlers-Danlos syndrome type II.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders. Recently mutations have been found in the genes for type V collagen in a small number of people with the most common forms of EDS, types I and II. Here we characterise a COL5A2 mutation in an EDS II family. Cultured dermal fibroblasts obtained from an affected subject synthesised abnormal type V collagen. Haplotype analysis excluded COL5A1 but was concordant with COL5A2 as the disease locus. The entire open reading frame of the COL5A2 cDNA was directly sequenced and a single base mutation detected. It substituted a glycine residue within the triple helical domain (G934R) of alpha2(V) collagen, typical of the dominant negative changes in other collagens, which cause various other inherited connective tissue disorders. All three affected family members possessed the single base change, which was absent in 50 normal chromosomes.

Type III collagen mutations in Ehlers Danlos Syndrome type IV and other related disorders

F.M.POPK, A.C.NICHOLLS, P.NARCISI, A.TEMPLE, Y.CHIA, P.FRYER, A.DE PAEPE,* W.P. DE GROOTE,t J.R.McEWAN,: D.A.COMPSTON,§ H.OORTHUYS,^ J.DAVIES** AND D.L.DINWOODIEft MRC Clinical Research Centre, Watford Road, Harrow; * Dienst Medische Genettka, Ghent, Belgium; f .4cademisch Ziekenhuis der Vrije Universileit, Amsterdam, The Netherlands: X Royal Postgraduate Medical School, Hammersmith Hospital; § University Hospital of Wales, Cardiff; ^ Academisch Medisch Centrum, Amsterdam, The Netherlands; ** Imogen Bassett Hospital, New York State, U.S.A. and ff Department of Genetics, Western General Hospital, Edinburgh

Genetic linkage to the type VII collagen gene (COL7A1) in 26 families with generalised recessive dystrophic epidermolysis bullosa and anchoring fibril abnormalities.

To strengthen the evidence for genetic linkage to COL7A1, we have studied 26 generalised recessive dystrophic epidermolysis bullosa (EB) families of British, Italian, Irish, and South African origin. We chose two linkage markers, a COL7A1 PvuII intragenic polymorphism and a highly informative anonymous microsatellite marker, D3S1100, which maps close to the COL7A1 locus at 3p21.1-3. Diagnosis was established by family history, clinical examination, immunofluorescence, and ultrastructural studies. The PvuII marker was informative in 16 families with a maximum lod score (Zmax) of 3.51 at recombination fraction (theta) = 0. The D3S1100 microsatellite was informative in 24 out of 25 families with Zmax = 6.8 at theta = 0.05 (Z = 4.94 at theta = 0) and no obligatory recombination events. These data strongly suggest that COL7A1 mutations cause EB in these families and, combined with previous studies, indicate locus homogeneity. The importance of anchoring fibrils for dermal-epidermal adhesion is further underlined. D3S1100 may later prove useful in prenatal diagnosis of this disease, if used in combination with other markers.

A single base mutation in the gene for type III collagen (COL3A1) converts glycine 847 to glutamic acid in a family with Ehlers-Danlos syndrome type IV. An unaffected family member is mosaic for the mutation

SummaryEhlers-Danlos syndrome type IV, an inherited connective tissue disease, is usually caused by mutations in the gene for type III collagen. Here, we describe a glycine to glutamic acid substitution in a patient with this syndrome. Previous studies had shown that fibroblasts from the patient, his mother and brother secreted a reduced amount of type III collagen and also produced an overmodified form of the protein that was preferentially retained intracellularly. Peptide mapping experiments indicated that the mutation was located within cyanogen bromide peptide 9. This was supported by chemical cleavage analysis and sequencing of cDNA encoding this region. Allele-specific oligonucleotide hybridisation of genomic DNA confirmed that a G to A mutation converted Gly 847 to Glu. The mutation was present in two other affected family members and also in a third, who was clinically unaffected. Further analysis of this unaffected individual revealed reduced mutant:normal ratios in DNA obtained from both blood and hair samples, showing that she was mosaic for the mutation.

Characterisation of a glycine to valine substitution at amino acid position 910 of the triple helical region of type III collagen in a patient with Ehlers-Danlos syndrome type IV.

We have studied a patient with Ehlers-Danlos syndrome type IV. Protein mapping studies of her type III collagen had indicated that cyanogen bromide fragment 9 contained the site of the mutation. Here we describe the mapping of this region for a single base mutation using a chemical modification and cleavage technique. Sequence analysis of cDNA showed a G to T mutation resulting in the substitution of glycine 910 by valine. This was confirmed by allele specific oligonucleotide hybridisation to the probands genomic DNA.

A 27-bp deletion from one allele of the type III collagen gene (COL3A1) in a large family with Ehlers-Danlos syndrome type IV

SummaryA large family with Ehlers-Danlos syndrome type IV (EDS IV) has previously been described. Unlike most cases of EDS IV, fibroblasts from affected members secreted near normal amounts of type III collagen. We have localised the mutation in this family to the CB5 peptide of type III collagen, by using both protein and cDNA mapping techniques. Sequence analysis of cDNA revealed a 27-bp deletion within exon 37, a deletion that removed nine amino acids and maintained the Gly-X-Y repeat of the collagen helix. Further sequencing of genomic DNA confirmed its location, and amplification of DNA from family members showed that it was absent in unaffected individuals but present in all the affected individuals tested. This deletion is flanked by two short direct repeats of CTCC; it may have arisen by slipped mispairing, and has subsequently been transmitted to all affected family members.

LINKAGE OF A POLYMORPHIC MARKER FOR THE TYPE-III COLLAGEN GENE (COL3A1) TO ATYPICAL AUTOSOMAL DOMINANT EHLERS-DANLOS SYNDROME TYPE-IV IN A LARGE BELGIAN PEDIGREE

SummaryWe have examined a large family in which eleven members have a form of autosomal dominant Ehlers-Danlos syndrome type IV. Analysis of fibroblast cultures from affected individuals showed a partial deficiency of type III collagen production. The protein produced was, however, normal in all aspects examined. Using a restriction site polymorphism associated with the structural gene for human type III collagen (COL3A1), we have found tight linkage between the low frequency polymorphic allele and the clinical expression of the disease (lod=3.86 at ϑ=0), identifying the type III collagen gene as the disease locus.

Genetic linkage to the collagen α1 (V) gene (COL5A1) in two British Ehlers–Danlos syndrome families with variable type I and II phenotypes

To investigate the role of COL5A1 as a candidate gene for Ehlers–Danlos syndrome (EDS), we have carried out linkage studies in two large British families with EDS type I/II and type II, respectively. Fourteen living, affected individuals were identified by family history, clinical examination and ultrastructural analysis. A polymorphic intragenic simple sequence repeat at the COL5A1 locus showed linkage to EDS without recombination to give a combined lod score of 5.7. We have previously reported linkage to COL5A1 in an EDS type I/II family which brings the total loci score to 9.8 at zero recombination. Taken together, these data implicate COL5A1 as an important cause of EDS and confirm that types I and II are allelic.

Detection and characterisation of an overmodified type III collagen by analysis of non-cutaneous connective tissues in a patient with Ehlers-Danlos syndrome IV.

The clinical and biochemical observations in a patient with a mild form of Ehlers-Danlos syndrome (EDS) type IV are described. The patients skin fibroblasts produced markedly diminished amounts of type III collagen. SDS-polyacrylamide gel electrophoresis of collagens produced by cells obtained from other, non-cutaneous tissues showed two forms of collagen alpha 1(III) chains, a normal and a slow migrating, mutant form. Further analysis confirmed that the type III collagen molecules containing mutant alpha chains which were overmodified had a lower thermal stability and were poorly secreted into the extracellular medium. The protein defect was mapped by in situ cyanogen bromide digestion and was located in alpha 1(III) CB9, the C-terminal peptide of the collagen triple helix. This study shows that non-cutaneous connective tissues can be a useful source for the study of type III collagen defects in patients with EDS type IV.

Heterogeneity of pseudoxanthoma elasticum: delineation of a new form?

Sixty‐four patients with pseudoxanthoma elasticum (PXE) were investigated in a nationwide study within South Africa and Zimbabwe. Thirty‐nine individuals formed a distinct clinical subgroup. Thèse persons were found exclusively among people of Afrikaner descent, whose origins are mainly derived from Dutch and French‐Huguenot stock. This disorder was inherited as an autosomal recessive trait and presented mild to moderate cutaneous and cardiovascular manifestations. However, after the third decade of life severe visual impairment developed and culminated in blindness in 8 people by the age of 50. The cause of the visual defect was progressive extension of angioid streaks into the macula with neovascularization and haemor‐rhage. Laser therapy may have prevented further bleeding in 4 instances. The severity of ocular involvcment contrasted with the mildness of the skin changes, and in this respect the condition seems to differ from previously delineated autosomal recessive forms of PXE.