1. Novel Mutations in the LAMB3 Gene Shared by Two Japanese Unrelated Families with Herlitz Junctional Epidermolysis Bullosa, and Their Application for Prenatal Testing 
Yasuko Takizawa, Hiroshi Shimizu, Leena Pulkkinen, Yoshiki Hiraoka, John A. McGrath, Kaoru Suzumori, Sadakazu Aiso, Jouni Uitto, Takeji Nishikawa 
Journal of Investigative Dermatology 
Volume 110, Issue 2, Pages (February 1998)
DOI: /j x
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Immunopathologic and ultrastructural features of the proband of family 2. (a) Indirect immunofluorescence of the proband’s skin with monoclonal antibody GB3 against laminin 5 shows negative staining. (b) In contrast, labeling with GB3 in normal control skin demonstrates bright linear staining at the dermal-epidermal junction. (c, d) Transmission electron microscopy of the proband’s skin. (c) Blister formation occurs within the lamina lucida and the electron dense lamina densa (arrowheads) is present along the blister floor (scale bar, 1 μm). (d) Higher magnification of the dermal-epidermal junction reveals hypoplastic hemidesmosomes (scale bar, 1 μm). (e) In control skin, the hemidesmosomes are larger, are more electron dense, and have conspicuous inner (arrowheads) and outer (→) hemidesmosomal plaques (scale bar, 0.5 μm).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Haplotype analysis of LAMB3 alleles containing Q166X and W610X mutations in families 1 and 2. The haplotypes segregating with Q166X and W610X mutations are presented on white and gray backgrounds, respectively.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Identification and verification of the maternal nonsense mutation Q166X in family 1. (A) Top, pedigree of the nuclear family: lane 1, the proband with H-JEB; the brother (lane 2), father (lane F), and mother (lane M) were clinically unaffected. Fetal DNA (lane 3) was obtained from chorionic villus samples at the DNA-based prenatal diagnosis at10 wk gestation. Bottom, heteroduplex analysis of the PCR product spanning exon 6 of the LAMB3 gene. When compared with the control (lane C), slower migrating bands were observed with the DNA from all members of the family (lanes F, M, 1, 2, and 3). (B) Direct sequence analysis of the exon 6 in the father (lane F), the brother (lane 2), and the fetus (lane 3) revealed silent polymorphisms only (see text), whereas exon 6 of the proband (→) and the mother (data not shown) revealed a heterozygous C-to-T transition at nucleotide position 496 (Q166X mutation) as compared with the normal sequence shown below. (C) Verification of the mutation by ASO with wild-type and mutant probes that span the site of mutation. Only the proband (lane 1) and the mother (lane M) hybridized to both mutant and wild-type probes, whereas those of the father, the brother, and the fetus hybridized with the wild-type probe only. The pattern of PCR products stained by ethidium bromide (top panel) indicates that comparable amounts of DNA were used for ASO.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Identification and verification of the paternal nonsense mutation W610X in family 1. (A) Top, the pedigree of the family is described in the legend to Fig 3(A); bottom, heteroduplex analysis of the PCR product spanning exon 14 of the LAMB3 gene revealed a heteroduplex band only in the mother, that was subsequently found to be due to a silent polymorphism. (B) Direct sequencing of the exon 14 of the proband revealed a heterozygous G-to-A transition at nucleotide position 1830 (W610X mutation) (→. (C) The presence of the mutation was verified by restriction enzyme digestion. The mutation created a new recognition site for BclI restriction enzyme. Digestion of the proband’s (lane 1), the father’s (lane F), the brother’s (lane 2), and the fetal DNA (lane 3) fragmented the mutant allele to 360 bp and 233 bp bands, whereas the normal allele resisted digestion, indicating heterozygosity for the mutation. The mutation also abolished a restriction endonuclease site for Van 91I restriction enzyme. In the case of the normal allele, the 593 bp fragment was digested to 358 bp and 235 bp fragments, whereas in the case of the mutant allele, the 593 bp fragment resisted digestion. The PCR product from the mother (lane M) was not digested by BclI but was fragmented by Van91I, similar to the normal control (lane C).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions