| Objective:Epidermolysis bullosa (EB) has been divided into clinically distinct subcategories, including the simplex, junctional and dystrophic variants. Elucidation of BMZ generprotein systems and development of mutation detection strategies have allowed identification of mutations in 10 different BMZ genes which can explain the clinical heterogeneity of EB. These include mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) genes in EBS; mutations in the type VII collagen gene (COL7A1) in the DEB; mutations in the laminin 5 genes (LAMAS, LAMB3 and LAMC2) in JEB; aberrations in the type XVII collagen gene (COL17A1) in generalized atrophic benign EB(GABEB); mutations in the a 6 and b4 integrin genes in a distinct JEB with pyloric atresia.In this study, we want to prove the relationship between the genotype and phenotype within different forms of EB.Methods:Skin biopsies from EB patients were processed for IF mapping and electron microscopy. When staining for antibodies were absent or reduced, genes were screened for mutations.Results:EBS families:Combined missense heterozygous mutations, KRT5-E168D and KRT14-A413T, were observed in the EBS proband and her affected daughter. The KRT5-E168D was a novel mutation. E168D was located in the last amino acid of the H1 region of the KRT5 gene. The H1 region, the end of the 1A and 2B and the L12 linker region are predicted to correspond to major contact sites between oligomers during polymerization of keratins.The amino acid sequences in these important sites are very conserved in most IFs implying that they are important to the filament assembly. With respect to positions, the affected alanine is located in the helix termination motif of the KRT14 gene, another site of pathogenic mutation clusters.However, the nucleotide at position 413 is not always occupied by alanine which compares a segment in the helix termination motif among type I keratins and other types of Ifs.Interestingly, the other nucleotides substituted at position 413 are all polar and large than alanine. This could indicate that the disruption caused by the KRT14-A413T mutation might not be as significant as previously assumed. On its own, it could explain the normal phenotype in the mother and brother of the proband. In R-EBS, the female proband had a severe phenotype including generalized blisters, mucosal involvement and EB naevi. Immunofluorescence mapping and electron microscopy were consistent with a diagnosis of EBS. Staining for Keratin 14 (K14) was negative. The basal layer, however, reacted with monoclonal antibodies to keratins 6 (K6) and 16 (K16). Mutation screening from genomic DNA showed that the proband was homozygous for the truncation mutation Y204X in exon 3 of KRT14, and both unaffected parents were heterozygous for a single KRT14 Y204X mutation. Our proband’s phenotype was very severe as an infant but has improved with age, it may be due to an alternative keratin pairing with K5 in the skin of patients with this condition, to compensate for the loss of K14. It is interesting that K6 and K16 were expressed in the proband’s skin, as these are normally positive in hyperproliferative skin, e.g. in psoriasis and wound healing. An analysis base on the position of the mutation in the keratin structure showed that EBS-DM results from 1A,2B domain, EBS-K and EBS-W are L1-2 domain.DEB families:We report 14 families with different forms of dystrophic epidermolysis bullosa (DEB) with 23 different COL7A1 allelic mutations,9 of which were novel mutations.4 cases of RDEB-HS combined two PTC mutations and 3 cases of RDEB-HS combined a PTC in one allele with a second spice-site or silent glycine substitution mutation in the other allele. Four recessive silent glycine substitution mutations were found, G2775S and G1673R (novel), G1338V (novel) and G2791A. Family 2 with DDEB combined R2791W and novel G2210V and had a Pasini phenotype in most individuals, but two members of the family had severe DDEB pruriginosa. Dominant dystrophic epidermolysis bullosa usually involves glycine substitutions within the triple helix of COL7A1 although other missense mutations, deletions or splice-site mutations may underlie some cases. In RDEB, mutations include nonsense, splice site, deletions or insertions,’silent’glycine substitutions within the triple helix and non-glycine missense mutations within the triple helix or non-collagenous NC-2 domain.JEB families:Two consanguineous families presented with different severities of junctional epidermolysis bullosa (JEB). Family 1 had widespread muco-cutaneous fragility, died within the first 7 months of life. In keeping with Herlitz JEB, skin biopsy labelling was negative for the GB3 antibody against laminin 5. Molecular screening revealed homozygosity for the mutation IVS21-1G>A in the LAMC2 gene; the parents being heterozygous carriers. This splice site mutation is predicted to create a new acceptor splice site 1-bp into exon 22, leading to a frameshift and downstream premature termination codon. This mutation therefore provides an explanation for the negative laminin 5 staining and the clinical phenotype of Herlitz JEB. Family 2 was had two children over 10 years of age with absent nails, localized blisters, pitted dental enamel and corneal erosions. In contrast to Family 1, skin biopsy labelling showed reduced, but not absent, laminin 5 immunoreactivity. A homozygous 39-base pair deletion in LAMC2, that removes the last 16 nucleotides of exon 9 and the first 23 bases of intron 9. was identified; the parents being heterozygous carriers. Three different cryptic splice variants are predicted, two of which are in frame (including skipping of exon 9). The presence of in-frame transcripts would account for the partial laminin 5 immunoreactivity and the non-Herlitz phenotype. Taken together, these cases show that the nature of the pathogenic laminin 5 mutations can account for the clinical diversity of JEB and is useful in counselling of families.Three novel ITGB4 mutations were identified in three families with JEB-PA:two splice-site and one insertion mutation. Two families with lethal phenotypes were due to combinations of PTC and missense mutations (658de1C/R252C and 3903dupC/G273D, respectively). One family had a brother with JEB-PA and a sister with JEB without PA. Both had mild skin involvement with blistering of the feet in the summertime. Both were homozygous for ITGB4 264G-A/3111-1G-A. Two cases in our report had no gastrointestinal symptoms or signs of PA., One was an infant born at 33 weeks gestation, with marked aplasia cutis of the neck, prominent subcutaneous veins, and dysmorphic facies, who died after 1 day. The other is described above. Specifically, most of the JEB-H cases demonstrate PTC mutation in both alleles of one of the laminin 5 gene, while JEB-nH variants are frequently associated with missense mutation in one or both alleles. In lethal JEB-PA, mutations usually consist of premature termination codons (PTC) affecting both ITGB4 alleles, which result in the complete absence ofα6β4 integrin; missense or splice site mutations are more prevalent in non-lethal forms.Conclusions:Usually lethal phenotype combines two PTC mutations, cause mRNA decay or synthesize truncated non-functional polypeptides; non-lethal phenotype results from missense, nonsense, splice site, insertion or deletion mutations. It is not only the nature, but also the position, of mutations reflected in the protein functional domains that affect the phenotype of EB. There’s no continuity between phenotype and genotype, however, different mutations could predict clinical phenotype.Significance:To study the genotype and phenotype correlation of EB can enhance knowledge for keratin, collagen and extracellular matrix, not only to further comprehend the basement membrane in healthy people and patients, but also to find new treatments in the future. This research will help to understand the genetic diversity and complexity of the skin disease, but also help to further understanding of other acquired and genetic diseases... |
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