Figure 4.

Genetic analysis. A: Pedigree of the patients with plectin deficiency. The parent generation is labeled I. The children are labeled II. Children II-1 and II-2 are compound heterozygous for the paternal and the maternal mutation. The older, clinically unaffected sister (#) is heterozygous for the paternal mutation. B: Identification of mutations 1287ins3 and Q1518X. Sequencing of a region of exon 9 carrying a heteroduplex identified by CSGE revealed a 3-bp insertion GCT at 1287 (top). Another heteroduplex formation was observed at the beginning of exon 31. Sequencing showed a heterozygous C-to-T change leading to a stop codon designated Q1518X (bottom). C: Verification of mutations by restriction enzyme digestion. I: The paternal mutation Q1518X creates a new BfaI site that leads to the digestion of the normal allele of 510 bp to two fragments of 325 and 185 bp. These fragments were found in the father (F), patient II-1 (C), his brother II-2 (C1), and the unaffected sister II-3 (C2). II: The maternal mutation 1287ins3 leads to the extension of a PvuII fragment of 57 bp for 3 bp creating a 60-bp fragment. This extension was seen in the mother (M), patient II-1 (C), and patient II-2 (C1). D: RNase protection analysis of plectin transcripts in fibroblasts of patient II-1 and a control. Autoradiography of RNase protected bands specific for plectin’s exon E1 (108 nucleotides), E1c (148 nucleotides), and E32 (257 nucleotides) revealed that no apparent reduction in E1c transcript levels in patient’s fibroblasts (left) and abundant expression of E32 and E1 mRNA was present (right). A GAPDH-specific probe protecting 53 nucleotides of GAPDH RNA was used as a control for RNA quantification.