Compound Heterozygosity for Novel Splice Site Mutations in the BPAG2/COL17A1 Gene Underlies Generalized Atrophic Benign Epidermolysis Bullosa Leena Pulkkinen,

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Compound Heterozygosity for Novel Splice Site Mutations in the BPAG2/COL17A1 Gene Underlies Generalized Atrophic Benign Epidermolysis Bullosa Leena Pulkkinen, Lin Lin, Jouni Uitto Journal of Investigative Dermatology Volume 113, Issue 6, Pages 1114-1118 (December 1999) DOI: 10.1046/j.1523-1747.1999.00793.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Diagnostic features of the patient. (A) Extended pedigree of the family of the proband (arrow) with generalized atrophic benign epidermolysis bullosa. (B) Clinical findings in the proband: nonscarring diffuse alopecia present over the scalp. (C) Absent expression of BP180 in the skin. Sections of the patient’s skin (upper and middle panels) or normal neonatal human foreskin (lower panel), were analyzed by IDIF microscopy using anti-BP180 MoAb HD18 (upper and lower panels) or antilaminin-5 MoAb K140 (middle panel). Scale bar: 20 μm. Journal of Investigative Dermatology 1999 113, 1114-1118DOI: (10.1046/j.1523-1747.1999.00793.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Mutation detection in the family with GABEB. (A) Sequence analysis of the proband’s PCR product spanning exon 45 and flanking intronic sequences. A heterozygous G-to-C tranversion was noted in the 3′ acceptor splice site consensus sequence affecting the last nucleotide of intron 44 in the patient’s DNA (left panel), whereas the control sequence has G in the same position of both alleles (right panel). (B) This mutation, designated as 3053–1G→C, abolished the restriction enzyme site for PstI, which was used for verification of the mutation in family members. The mutant allele corresponds to the 272 bp fragment, whereas the normal allele is digested by PstI to 192 and 80 bp fragments. The father (II-3) and the proband (III-3) are heterozygotes for this mutation. (C) Sequencing of the proband’s PCR product spanning exon 51 and flanking intronic sequences revealed a heterozygous G-to-C transversion affecting 5′ donor splice site consensus sequence (left panel), whereas the control sequence harbored G at the same position of both alleles (right panel). (D) The presence of this mutation, designated as 3871+1G→C, was verified in the proband and other family members by ASO. In addition to the proband (III-3) and her mother (II-4), the two maternal aunts (II-5 and II-6), maternal grandmother (I-4), and two daughters of an aunt (III-6 and III-7) were carriers of this mutation. Journal of Investigative Dermatology 1999 113, 1114-1118DOI: (10.1046/j.1523-1747.1999.00793.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Consequences of the splice site mutations at the mRNA level. (A) RT-PCR of the proband’s mRNA (P in lane 1) revealed two bands when the region corresponding to the nucleotides 2796–3488 of BPAG2 and spanning the mutation 3053–1G→C was amplified. Amplification of the control mRNA (C in lane 2) resulted in a 693 bp band only. RT-PCR of the region corresponding to nucleotides 3621–4362 of BPAG2 and spanning the mutation 3871+1G→C revealed multiple bands in the proband’s mRNA (P in lane 3), whereas amplification of the control mRNA (C in lane 4) resulted in a 742 bp band only. (B) Analysis of the aberrant transcript caused by the mutation 3053–1G→C. Sequencing of the subcloned PCR product corresponding to the band of slower mobility (see lane 1 in A) revealed a 40 bp partial retention of intron 44 sequences. This resulted in frameshift and premature termination codon within exon 45, as schematically shown in the lower panel. (C) Schematic presentation of the aberrant transcripts caused by the mutation 3871 + 1G→C. Upper panel: The exon-intron organization of the sequence spanning the region from exon 50 to exon 52 of BPAG2 gene. Normal splicing is illustrated above the gene, and aberrant splicing events involving transcripts 1, 2, and 3, based on sequence analysis of transcripts of different sizes, are shown below the gene. Lower panel: Schematic presentation of the aberrant transcripts 1, 2, and 3. Journal of Investigative Dermatology 1999 113, 1114-1118DOI: (10.1046/j.1523-1747.1999.00793.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Absent expression of BP180 by patient’s keratinocytes. Neonatal human foreskin keratinocytes (lanes 1, 2, and 3) or first passage primary cultures of patient’s keratinocytes (lanes 4, 5, and 6) were incubated overnight with 50 μCi 35S-labeled methionine/cysteine mixture per ml and then analyzed by immunoprecipitation using nonimmune rabbit sera (lanes 1 and 4), polyclonal antibody 594 directed against the NC16a domain of BP180 (lanes 2 and 5), or polyclonal antibody 136 directed against the C-terminal domain of BP180 (lanes 3 and 6). Molecular weight marker positions (in kDa) are indicated on the right. Journal of Investigative Dermatology 1999 113, 1114-1118DOI: (10.1046/j.1523-1747.1999.00793.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions