Hemizygosity for a glycine substitution in collagen XVII: unfolding and degradation of the ectodomain.

Defects of collagen XVII, a keratinocyte adhesion protein, are associated with epidermal detachment in junctional epidermolysis bullosa. Although some missense mutations in the collagen XVII gene COL17A1 have been described, the molecular mechanisms leading to disease have remained elusive in these cases. Here we assessed the biologic consequences of a missense mutation by studying the folding and stability of wild-type and mutated recombinant collagen XVII domains. The mutation occurred in a junctional epidermolysis bullosa patient who was compound heterozygous for the novel glycine substitution mutation G633D and the novel nonsense mutation R145X. Collagen XVII mRNA was significantly reduced, indicating nonsense-mediated mRNA degradation and hemizygosity of the patient for the G633D substitution. As glycine residues within the collagen triple helices are important for stable conformation, the thermal stability of the wild-type and mutated eukaryotic recombinant Col15 domain of collagen XVII was assessed. The stability of the mutated fragment was clearly reduced. The midpoint of the helix-to-coil transition, Tm, was 5 degrees C lower than that of wild-type rCol15, indicating abnormal triple-helix folding and susceptibility to proteolysis. Consistently, immunoassays demonstrated reduced amounts of the full-length collagen XVII and absence of the soluble ectodomain in keratinocyte cultures, and lack of the ectodomain from the junctional epidermolysis bullosa skin. These observations show that the glycine substitution G633D in collagen XVII causes abnormal folding and susceptibility to degradation, and thus perturbs the physiologic adhesive functions of collagen XVII in the skin.

Novel homozygous and compound heterozygous COL17A1 mutations associated with junctional epidermolysis bullosa.

Junctional epidermolysis bullosa is a heritable, heterogeneous blistering skin disease with mechanically induced dermal-epidermal separation, mild skin atrophy, nail dystrophy, and alopecia. Four unrelated junctional epidermolysis bullosa families with different phenotypes were investigated here and four novel mutations associated with the disease were identified. Patients 1, 2, and 3 had generalized atrophic benign epidermolysis bullosa, with nonscarring blistering and varying degree of alopecia. Patient 4 had the localisata variant of junctional epidermolysis bullosa, with predominantly acral blistering and normal hair. All patients had mutations in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane protein. Patients 1 and 2 carried homozygous deletions 520delAG and 2965delG, respectively. Patient 3 was compound heterozygous for a missense and a deletion mutation (G539E and 2666delTT), and patient 4 was heterozygous for a known mutation R1226X. The deletions led to premature termination codons and to drastically reduced collagen XVII mRNA and protein levels, consistent with the absence of the collagen in generalized atrophic benign epidermolysis bullosa skin. The missense mutation G539E allowed synthesis of immunoreactive collagen XVII in keratinocytes, but prevented its secretion, thus causing lack of the protein in the skin. The data suggest that different COL17A1 mutations and their combinations can result in a spectrum of biologic and clinical phenotypes of not only generalized atrophic benign epidermolysis bullosa, but also localized junctional epidermolysis bullosa.

Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes.

Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.

The Conradi-Hünermann-Happle syndrome (CDPX2) and emopamil binding protein: novel mutations, and somatic and gonadal mosaicism.

The Conradi-Hünermann-Happle (CHH) syndrome (X-chromosomal dominant chondrodysplasia punctata type II; MIM 302960) is an X-linked dominant disorder that is characterized by ichthyosis, chondrodysplasia punctata, cataracts and short stature. The disease occurs almost exclusively in females and shows increased disease expression in successive generations (anticipation). Recently, causative mutations in the emopamil binding protein (EBP) have been identified. To better appreciate the genetics of this syndrome we analyzed the EBP gene in seven independent families using PCR, conformation-sensitive gel electrophoresis, direct sequencing and restriction enzyme analysis. We found five novel mutations: three nonsense mutations in exon 2 and exon 3 and two frameshift mutations, one deletion in exon 4 and an insertion in exon 5. In two families, known mutations affecting exon 2 were identified. Surprisingly, we failed to detect the mutation in a grandmother exhibiting minor disease symptoms such as sectorial cataract and attribute this to gonadal and somatic mosaicism. Gonadal mosaicism appeared also to be involved in the case of healthy parents having two affected girls, one of whom died due to the disease. We conclude that gonadal mosaicism has to be considered when dealing with seemingly sporadic cases.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.