Homozygous α6 Integrin Mutation in Junctional Epidermolysis Bullosa with Congenital Duodenal Atresia

Junctional epidermolysis bullosa with congenital pyloricor duodenal atresia is a distinct variant within this groupof autosomal recessive blistering skin diseases. In thisstudy we demonstrate, for the first time, a homozygousmutation in the α6 integrin gene (ITGA6) in a family withthree affected individuals. For this purpose, we first de-termined the genomic organization of ITGA6, and placedthe gene on chromosome 2q by high resolution radiationhybrid mapping. Heteroduplex analysis of PCR productscontaining the individual exons of ITGA6, followed by di-rect nucleotide sequencing, revealed that the probandwas homozygous for a G-to-T transversion in the +1position of intron 12. This mutation, 1856+1G →T, affectsan invariant base of the 5 ′ donor splice site predicting ab-errant splicing involving exon 12. The mutation was veri-fied in the proband’s DNA by restriction enzymedigestion which also confirmed that the parents wereheterozygous carriers of this mutation. Altered express-ion of α6 integrin, which forms a heterodimer with the β4subunit at the dermal–epidermal junction, would explainfragility and blistering as a result of minor trauma to theskin.INTRODUCTIONJunctional epidermolysis bullosa (JEB) is a heterogeneous groupof autosomal recessive skin diseases characterized by fragility ofthe dermal–epidermal junction ( 1,2). The ultrastructural hallmarkof JEB is abnormality of the hemidesmosome-anchoring filamentattachment complex at the cutaneous basement membrane zone(BMZ), and tissue separation occurs within the lamina lucida atthe level of anchoring filaments or at the lamina lucida/basalkeratinocyte interphase at the level of hemidesmosomes ( 3). Thehemidesmosomes consist of at least four protein components, theα6β4 integrin, the 230 kDa and the 180 kDa bullous pemphigoidantigens, as well as plectin/HD1, while the anchoring filamentsconsist predominantly, if not exclusively, of laminin 5 (4,5). Thepolypeptide subunits of the hemidesmosomes and anchoringfilaments are encoded by eight distinct genes, each of which couldpotentially serve as a candidate gene for the junctional forms of EB.Previously, a number of mutations in the classic lethal (Herlitz)type of JEB have been demonstrated in the three genes, LAMA3,LAMB3 and LAMC2, encoding the subunit polypeptides oflaminin 5 (6). In non-lethal variants of JEB, specific mutationshave been found, in addition to laminin 5 genes, also in the genesencoding the hemidesmosomal proteins. Specifically, in a variantknown as generalized atrophic benign EB (GABEB), mutationsreside in the gene encoding the 180 kDa bullous pemphigoidantigen/type XVII collagen ( 7,8), while in a form associated withlate-onset muscular dystrophy mutations reside in the plectin/HD1 gene ( 9–12). Furthermore, in one case with JEB associatedwith pyloric atresia, compound heterozygosity for a paternal 1 bpdeletion leading to premature termination codon and a maternalin-frame exon skipping mutation in the β4 integrin gene has beenreported (13). In this study, we describe the first mutation in thegene encoding the α6 integrin subunit, which forms a hetero-dimer with the β4 subunit, in a family with a variant of JEBassociated with congenital duodenal atresia.RESULTSIntron–exon organization and fine mapping of ITGA6The complete genomic organization of the human ITGA6 genewas determined by PCR, using primers derived from the cDNAsequence (GenBank no. X53586; 14). The gene consists of 27exons, one of which (exon 5B) is an additional exon present onlyin certain tissue-specific alternate transcripts (Fig. 1a).A human-specific PCR marker for the ITGA6 gene wasdeveloped for radiation hybrid mapping using the Genebridge4panel. Using the RHMapper program, the resultant data vectorplaced the gene 12.33 cR from framework marker WI-3728 on