No abstract

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and 11) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105 -155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (~5 2 0 0 nucleotides) and amino acid sequences obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that niceinkalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain 111 that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since niceinkalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.The basement membrane (BM) is a type of extracellular matrix which provides an attachment substrate for overlying cell populations and plays a crucial role in organizing tissue architecture, tissue compartmentalization, differentiation of adjacent cells and filtration of nutrients. Since mechanisms mediating these functions are poorly understood, a considerable amount of research has been devoted to the molecular characterization of BM components and elucidation of their biochemical properties. Some differences exist from one type of BM to another and no less than 14 shared macromolecules have already been found [l].We previously reported the isolation of a novel epidermal BM component, called nicein, which is found in various BM species that we investigated with the monoclonal antibody GB3 [2, 31. This glycosylated protein is composed of three major subunits of 100,125 and 150 kDa, as shown by immunoprecipitation assays performed on keratinocytes in culture [3]. Since nicein is secreted by these cells, we isolated the protein by immunoaffinity chromatography from conditioned

The mRNA's of several integrin subunits are alternatively spliced in the region encoding cytoplasmic domains, that may potentially provide alternative integrin-cytoskeleton interactions and transmembrane signaling pathways. We identified a novel cytoplasmic tail variant of the human E1 subunit by reverse transcriptase polymerase chain reaction. This fourth i~1 variant, named 01D, is specific for skeletal and cardiac muscle. The determined genomic organization of the T-region of the human E1 gene reveals that/$1D is produced by alternative splicing of mRNA. In addition, we show that the expression of /]ID is developmentally regulated during murine myoblast differentiation, suggesting a role for/]ID in myogenesis.

The inherited mechanobullous disorder, junctional epidermolysis bullosa (JEB), is characterized by extensive blistering and erosions of the skin and mucous membranes. The diagnostic hallmarks of JEB include ultrastructural abnormalities in the hemidesmosomes of the cutaneous basement membrane zone, as well as an absence of staining with antibodies against the anchoring filament protein, laminin 5. Therefore, the three genes encoding alpha 3, beta 3 and gamma 2 chains of laminin 5, known as LAMA3, LAMB3 and LAMC2, are candidate genes for JEB. We have previously demonstrated mutations in the LAMB3 and LAMC2 genes in several families with JEB. We initiated mutation analysis from an affected child by PCR amplification of individual LAMA3 exons, followed by heteroduplex analysis. Nucleotide sequencing of heteroduplexes identified a homozygous nonsense mutation within domain I/II of the alpha 3 chain. These findings provide the first evidence that nonsense mutations within the LAMA3 gene are also involved in the pathogenesis of JEB, and indicate that mutations of all three genes of laminin 5 can result in the JEB phenotype.

ObjectiveTo compare the clinical features of patients showing a classical phenotype of facioscapulohumeral muscular dystrophy (FSHD) with genetic and epigenetic characteristics of the FSHD1 and FSHD2 loci D4Z4 and SMCHD1.MethodsThis is a national multicenter cohort study. We measured motor strength, motor function, and disease severity by manual muscle testing sumscore, Brooke and Vignos scores, clinical severity score (CSS), and age-corrected CSS, respectively. We correlated these scores with genetic (D4Z4 repeat size and haplotype; SMCHD1 variant status) and epigenetic (D4Z4 methylation) parameters.ResultsWe included 103 patients: 54 men and 49 women. Among them, we identified 64 patients with FSHD1 and 20 patients with FSHD2. Seven patients had genetic and epigenetic characteristics of FSHD1 and FSHD2, all carrying repeats of 9–10 D4Z4 repeat units (RU) and a pathogenic SMCHD1 variant. In the remaining patients, FSHD was genetically excluded or remained unconfirmed. All clinically affected SMCHD1 mutation carriers had a D4Z4 repeat of 9–16 RU on a disease permissive 4qA haplotype. These patients are significantly more severely affected by all clinical scales when compared to patients with FSHD1 with upper-sized FSHD1 alleles (8–10 RU).ConclusionThe overlap between FSHD1 and FSHD2 patients in the 9–10 D4Z4 RU range suggests that FSHD1 and FSHD2 form a disease continuum. The previously established repeat size threshold for FSHD1 (1–10 RU) and FSHD2 (11–20 RU) needs to be reconsidered.Clinicaltrials.gov identifierNCT01970735.