Molecular presentation of the JEB patients
A total of 69 patients with clinically suspected recessive JEB were recruited for deep intronic variant analyses. All 69 patients underwent an initial test with WES sequencing (3 caused by LAMA3 variants, 24 caused by LAMB3 variants, 9 caused by LAMC2 variants, 14 caused by ITGB4 variants and 19 caused by COL17A1 variants), but the genetic diagnosis of 9 cases (1 with LAMB3, 2 with ITGB4 and 6 with COL17A1) was inconclusive (Table 1).
The Pat.13 was clinically diagnosed by recessive JEB, but only the LAMB3 c.28+1G>A heterozygous variant was identified by WES (Table 2). The phenotype of Pat.52, Pat.53, Pat.57, Pat.61, Pat.65 and Pat.68 were consistent with localized to intermediate recessive JEB caused by mutations in COL17A1 gene, but only one heterozygous mutation (c.1507del in Pat.52, c.3482_3483del in Pat.53, c.4088del in Pat.57, c.1612del in Pat.61, c.3340_3349del in Pat.65, and c.3281del in Pat.68) was found in each patient (Table 2). The Pat. 45 and Pat.48 were clinically diagnosed by recessive JEB, but only the ITGB4 (c.3931G>T in Pat.45 and c.1805A>T in Pat.48) heterozygous variant was identified by WES.
Clinical presentation of the JEB patients
The Pat.13 presented with erythema, scars, scattered haemorrhagic blisters, depigmentation, dyspigmentation and dystrophic nails (Fig.1A). Pat.52, Pat.53, Pat.57, Pat.61, Pat.65 and Pat.68 presented with alopecia, nail dystrophy, enamel dental problems, oral erosions, disseminated tense blisters on the erythema and skin healing with dyspigmentation (Fig. 2-3). Pat. 45 and Pat.48 presented with scattered blisters and depigmentation. All patients developed skin fragility at an early age and all of them denied family history.
We then applied whole genome sequencing to Pat.13, Pat.52, Pat.53, Pat.57, Pat.61, and Pat.65 to genetically diagnoses them.
Novel c.-38+2T>C promoter mutation in the LAMB3 gene
LAMB3 mutational screening disclosed the novel c.-38+2T>C mutation within the 5′ untranslated region (UTR) in Pat.13, which was approximately 1400bp away from the coding region (Fig.1B). The LAMB3 c.-38+2T>C mutation was absent in the population database gnomAD and predicted to cause aberrant transcript.
Minigene assays showed the c.-38+2T>C-allele induced a larger band than the wildtype in HEK293 cells (Fig.1C). Sanger sequencing of RT-PCR products proved that the c.-38+2T>C induced retention of 120bp intron (Fig. 1C). Wild-type alleles were still present in cells transfected with the mutant 38+2T>C construct. However, we failed to find the wild-type 420bp band, suggesting that the high transfection efficiency resulted in a much higher abundance of aberrant splicing band than normal splicing bands. To verify its effect, the cDNA covering exons 1–6 from the Pat.13′ blood was amplified and analysed, the data showed that the c.-38+2T>C and c.28+1G>A mutations induced the abolishment expression of the LAMB3 (Fig.1D).
The deep intronic mutations identified in COL17A1 gene
We detected three deeply located novel COL17A1 intronic mutations (c.4156+117G>A variant in Pat.52, Pat.57 and Pat.65, c.2039-104G>A variant in Pat.53 and c.1267+237dupC variant in Pat. 61) by WGS (Table 2), all absent or with a minor allele frequency in the population database gnomAD and predicted to cause aberrant transcript.
The COL17A1 c.4156+117G>A deep intronic mutation
The Pat.57 was identified to carry a paternal c.4156+117G>A variant in COL17A1 gene (Fig.2B-C). The variant is located in intron 52. The mutated c.4156+117G>A revealed two aberrant transcripts13, including the 849bp M1 and 562bp M2 transcripts, in addition to the wildtype transcript (952bp) (Fig.2D). Sanger sequencing of subcloned RT-PCR products showed that the 849bp band resulted from skipping 103bp at the 5´splice site of exon 53, resulted in a frameshift transcript, and was the predominant product of the mutated group. The relatively weak 562bp band completely missed the exon 52 and resulted in an in-frameshift transcript (Fig.2D).
Loss of COL17A1 impairs hemidesmosomes structure leading to mucocutaneous fragility
Decreased protein levels of COL17A1 were observed in the skin biopsy of Pat.57 compared with the control levels (Fig.2E upper panel). Transmission electron microscopy (TEM) examination revealed that hemidesmosomes were poorly developed in Pat.57 (Fig.2E lower panel). Consequently, the c.4156+117G>A was classified as pathogenic.
The COL17A1 c.4156+117G>A hotspot deep intronic mutation
The Pat.52 and Pat.65 also carried c.4156+117G>A variant (Fig.3A-D). The c.4156+117G>A variant was observed in 3 out of 5 patients from this study, indicating this alteration may represent a common deep intronic mutation in COL17A1 gene.
The COL17A1 c.2039-104G>A and c.1267+237dupC deep intronic mutations
The Pat.53 carried a COL17A1 c.2039-104G>A variant (Fig.4 A-B). The variant is located in intron 24. The c.2039-104G>A variant revealed a larger aberrant transcript corresponding to an in-frameshift pseudo-exon of 171 bp insertion and a smaller product corresponding to the correctly processed transcript (Fig. 4C).
The c.1267+237dupC COL17A1 variant identified in Pat.61 resulted in skipping of the exon 15 and exon 16 which was 243bp smaller than the wildtype allele in HaCaT cells (Fig.4D-F).
Pat.13 was caused by LAMB3 compound heterozygous mutations and presented with intermediate JEB. Pat.52, Pat.53, Pat.57, and Pat.61 were caused by biallelic COL17A1 PTC mutations, and presented with localized or intermediate JEB.
HEK293 cells utilised did not demonstrate splicing effect in all cases with COL17A1 deep intronic variants (data not shown).