Two novel mutations on exon 8 and intron 65 of COL7A1 gene in two Chinese brothers result in recessive dystrophic epidermolysis bullosa

Abstract

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen - the main component of the anchoring fibrils at the dermal-epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5' donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.

Figure 1. Clinical manifestations of the two RDEB patients.

Blisters, blood blisters, scarring formation, and hypopigmentation on the dorsal side of the elbows (A, B), tibialis, knees (C), hands, and feet could be seen in the proband. Nails of fingers and toes were all absent (D). Similar lesions could be seen in the proband’s younger brother (E, F).

Figure 2. Pedigree of the RDEB family.

The segregation of the mutations of c.1006_1007delCA on exon 8 (half black symbols) and c.IVS5568+1delG at the donor splicing site of intron 65 (half gray symbols) on the COL7A1 gene is shown. The arrow refers to the proband.

Figure 3. Histopathologic and ultrastructural features of the RDEB proband’s skin lesion.

Hematoxylin and eosin stain (H&E) of the skin lesion indicated a keratinocytes degeneration and necrosis, subepidermal bulla with infiltration of a few inflammatory cells (A, magnification×40; B×100). Under transmission electron microscope, the split was located at the sub-lamina densa where some red blood cells could be seen (asterisk) (C). The arrow refers to the lamina densa. Magnification×10000.

Figure 4. Result of DNA sequencing of COL7A1 mutation in the affected brothers.

It shows compound heterozygosity of the mutations of c.1006_1007delCA (exon 8) (A) and c.IVS5568+1delG (intron 65) (B).

Figure 5. Schematic diagram of the consequences of the mutations identified in the RDEB pedigree.

The mutation on exon 8 (c.1006_1007delCA), standing at NC-1 of the COL7A1 gene, leads to a premature termination codon (stop) located 48 amino acids downstream in exon 9, resulting in truncation of 2561 amino acids downstream, which include part of the NC-1, all the collagenous triple helix, and the NC-2. The mutation discovered on intron 65 (c.IVS5568+1delG), located at the collagenous triple helix domain of the COL7A1 gene, immediately creates a stop codon, consequently producing a truncated protein lacking part of 1089 C-terminal amino acids downstream. Abbreviations: TH, triple helix; HR, hinge region; NC-1, amino terminus; NC-2, carboxyl terminus.