Herlitz Junctional Epidermolysis Bullosa: Novel and Recurrent Mutations in the LAMB3 Gene and the Population Carrier Frequency  Aoi Nakano, Ellen Pfendner, Leena Pulkkinen, Jouni Uitto  Journal of Investigative Dermatology  Volume 115, Issue 3, Pages 493-498 (September 2000) DOI: 10.1046/j.1523-1747.2000.00086.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic illustration of the LAMB3 polypeptide with domain organization and locations of all LAMB3 mutations disclosed thus far in the junctional forms of EB. PTC mutations are shown above the molecule, while missense mutations are shown below. Novel mutations disclosed in this study are shown in bold type. PTC mutations associated with non-Herlitz variants are underlined, while the mutations shown in italics are found in both Herlitz and non-Herlitz JEB. Journal of Investigative Dermatology 2000 115, 493-498DOI: (10.1046/j.1523-1747.2000.00086.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Identification and verification of mutations 1365delCA/464insT in the LAMB3 gene of Family 17. (A) Heteroduplex analysis of the PCR product spanning exon 12 of the LAMB3 gene revealed a heteroduplex band in the mother (lane M, arrow), whereas the father (lane F) showed the homoduplex band only. (B) DNA sequencing of the mother's PCR product revealed the heterozygous deletion of CA at nucleotide position 1365 (1365delCA), as shown in the lower panel; the wild-type sequence is shown in the top panel. (C) Verification of the maternal mutation by digestion with Pml I. The mutation created a new restriction enzyme site for Pml I, which cuts the 455 bp PCR product to 277 bp and 178 bp bands in the mutant allele, whereas the 457 bp PCR product remains uncut in the normal allele (the size difference reflects the 2 bp deletion). (D) Heteroduplex analysis of the PCR product spanning exon 6 of the LAMB3 gene revealed one heteroduplex band in the mother (lane M, asterisk), whereas the father (lane F) showed an additional heteroduplex band (arrow). The presence of a common heteroduplex band in both the mother's and the father's DNA (asterisk) could be explained by a T/C polymorphism which was detected in the nucleotide position 384 by sequencing (not shown). (E) DNA sequencing of the father's DNA revealed insertion of T at nucleotide position 464 (464insT), as shown in the lower panel; the wild-type sequence is shown in the top panel. (F) Verification of the paternal mutation by digestion with Mnl I. The mutation abolished the restriction enzyme site for Mnl I, which cuts the 280 bp PCR product in the case of the normal allele to 66 bp, 62 bp, 55 bp, 54 bp, 30 bp, 8 bp, and 5 bp bands, and in the case of the mutant allele to 85 bp, 66 bp, 62 bp, 55 bp, 8 bp, and 5 bp bands (the size difference reflects the 1 bp insertion). MW, molecular weight markers φX174/HaeIII; C, control. Journal of Investigative Dermatology 2000 115, 493-498DOI: (10.1046/j.1523-1747.2000.00086.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Prenatal testing in Family 20. (A) The fetuses (lanes A and B; dizygotic twins) and parents (lanes M and F; mother and father, respectively) are heterozygous for the R635X mutation which creates a new restriction enzyme site for Bgl II. In the case of the mutant allele, this endonuclease cuts the 593 bp PCR product to 430 bp and 163 bp bands (lane PC; positive control), whereas in case of the normal allele the PCR product remains uncut (lane C; negative control). (B) DNA from both fetuses and the father is digested by Hae III, which cuts the Y chromosome PCR product of 300 bp to 216 bp and 84 bp bands, whereas the X chromosome product remains uncut. Thus, both fetuses are males, excluding maternal contamination of DNA, and consequently both fetuses were predicted to be healthy male carriers of the R635X mutation. MW, molecular weight markers φX174/HaeIII. Journal of Investigative Dermatology 2000 115, 493-498DOI: (10.1046/j.1523-1747.2000.00086.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Detection of recurrent mutations in the LAMB3 gene in patients with H-JEB. (A) The R42X mutation creates a new restriction enzyme site for Dde I, which cuts the 498 bp PCR product in the normal allele to 272 bp, 103 bp, 85 bp, and 38 bp, and in the mutant allele to 240 bp, 103 bp, 85 bp, 38 bp, and 32 bp bands. (B) The 957ins77 mutant allele results in a 428 bp band in PCR, whereas the normal allele is detected as a 351 bp band. (C) The Q243X mutation creates a new restriction enzyme site for Bfa I, which cuts the 407 bp PCR product in the normal allele to 323 bp and 84 bp bands, and in the mutant allele to 218 bp, 105 bp, and 84 bp bands. (D) The R569X mutation abolishes the restriction enzyme site for Cac8 I, which cuts the 593 bp PCR product in the normal allele to 232 bp, 219 bp, 44 bp, 38 bp, 37 bp, and 23 bp bands, and in the mutant allele to 270 bp, 219 bp, 44 bp, 37 bp, and 23 bp bands (the smaller bands are not visible). (E) The 565–2A→G mutation creates a new restriction enzyme site for Nla IV, which cuts the 266 bp PCR product in the normal allele to 192 bp and 74 bp bands, and in the mutant allele to 123 bp, 74 bp, and 69 bp bands. (F) The R972X mutation creates a new restriction enzyme site for AIwN I. The normal allele of 324 bp remains uncut in AIwN I digestion, whereas this enzyme cuts the PCR product to 207 bp and 117 bp bands in the mutant allele. (G) The R144X mutation creates a new restriction enzyme site for Hph I. The normal allele of 451 bp remains uncut in Hph I digestion, whereas this enzyme cuts the PCR product to 234 bp and 217 bp bands in the mutant allele in the heterozygous parents (lanes M and F). MW, molecular weight markers φX174/HaeIII; P, patient; M, mother; F, father; C, control. Journal of Investigative Dermatology 2000 115, 493-498DOI: (10.1046/j.1523-1747.2000.00086.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions