Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes

Abstract

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.

Figure 1

Phenotype of the proband. The clinical phenotype included features of both the GABEB and JEB Herlitz subtypes. A, Proband seen, at the age of 7 mo, with generalized skin blistering but with minimal involvement of the mucous membranes and with normal body height and weight. B, Paronychia-like affection of the fingers, as typically seen in JEB Herlitz.

Figure 2

Pedigree and LAMB3 and COL17A1 mutations. A, Pedigree demonstrating recessive inheritance of the mutations. Areas with horizontal stripes indicate paternal COL17A1 mutations; dotted areas, maternal COL17A1 mutations; blackened areas, maternal LAMB3 mutations. B, Paternal COL17A1 mutation, which was novel—a heterozygous 2669 T→G transversion in exon 37—and was designated “L855X.” Verification of the mutation by AspI digestion demonstrated that the father and the paternal grandmother were heterozygous for this mutation. C, Maternal COL17A1 mutation, which was a heterozygous 3781C→T transition in exon 51 and was designated “R1226X” (McGrath et al. ; Jonkman et al. ; Schumann et al. 1997). The mutation was also found, by TaqI digestion, in the mother's and both the half-sisters' DNA, but it was not found in a control or in the father. D, LAMB3 mutation R635X (Kivirikko et al. 1996), which was verified by both BglII digestion of the PCR amplification products of exon 14 and by dideoxynucleotide sequencing (not shown). In addition to the proband, the mother was heterozygous for this mutation. In B–D, the numbers above the agarose-gel images denote the individuals in the pedigree, and “C” denotes the control.

Figure 3

Northern blotting of collagen XVII and laminin 5 mRNA. A strong signal of the 6-kb collagen XVII mRNA was detected in control keratinocytes (left panel). In contrast, collagen XVII mRNA that was isolated from the JEB patient's keratinocytes (left panel) produced a negative signal, suggesting nonsense-mediated mRNA decay in these cells. The blot was intentionally overexposed to reveal potentially small amounts of mRNA in the patient's cells. When laminin 5 cDNA was used for hybridization, a strong 3.5-kb laminin 5 mRNA band was seen in normal keratinocytes (right panel). The keratinocytes of the patient with JEB produced a clearly weaker band (right panel). GAPDH cDNA was used as a reference probe to correct for minor differences in RNA loading. Densitometric analysis of the laminin 5 signals indicated that the patient's cells contained ∼55% of the laminin 5 mRNA seen in controls. C = control; Pat = patient.

Figure 4

Collagen XVII and laminin 5 protein expression in situ. Indirect immunofluorescence staining of control (A and B) and JEB (C and D) skin biopsy specimens, with antibodies to collagen XVII (A and C) and laminin 5 (B and D). In normal skin, both collagen XVII (A) and laminin 5 (B) antibodies showed a linear fluorescence at the dermal-epidermal junction. In the proband's skin, collagen XVII was absent (C ), and the laminin 5 staining was attenuated (D).