PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molmedLink to Publisher's site
 
Mol Med. 1997 February; 3(2): 124–135.
PMCID: PMC2230062

Detection of novel LAMC2 mutations in Herlitz junctional epidermolysis Bullosa.

Abstract

BACKGROUND: Laminin 5, an anchoring filament attachment protein within the lamina lucida of the basement membrane zone involved in the pathogenesis of junctional epidermolysis bullosa (JEB), consists of three polypeptide subunits, the alpha 3, beta 3, and gamma 2 chains which are encoded by the LAMA3, LAMB3, and LAMC2 genes, respectively. To facilitate identification of pathogenetic mutations in LAMC2, a strategy based on direct amplification of genomic DNA by PCR or mRNA by RT-PCR, followed by heteroduplex analysis of the PCR products, was developed. MATERIALS AND METHODS: Primer pairs for amplification of the complete cDNA as well as the 23 individual exons in the genomic DNA, which encode the entire gamma 2 chain of laminin 5, were established. The primers for amplification of exons from genomic DNA were positioned at least 24 bp away from the intron-exon borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of the entire coding sequence of LAMC2 mRNA were used. The amplified sequences were scanned for pathogenetic mutations and sequence variations in JEB patients and unrelated control individuals by heteroduplex analysis by means of conformation sensitive gel electrophoresis (CSGE). RESULTS: Utilizing the strategy developed in this study, we identified pathogenetic mutations in three patients with the Herlitz (lethal) variant of JEB, and eight intragenic normal polymorphisms, which are useful for linkage analysis, in the LAMC2 gene. CONCLUSIONS: The methodology described in this study is capable of detecting single-base substitutions or small insertions and deletions in the LAMC2 gene. Demonstration of mutations in this gene in JEB patients further emphasizes the role of laminin 5 in providing integrity to the cutaneous basement membrane zone.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Burgeson RE, Chiquet M, Deutzmann R, Ekblom P, Engel J, Kleinman H, Martin GR, Meneguzzi G, Paulsson M, Sanes J, et al. A new nomenclature for the laminins. Matrix Biol. 1994 Apr;14(3):209–211. [PubMed]
  • Aumailley M, Krieg T. Laminins: a family of diverse multifunctional molecules of basement membranes. J Invest Dermatol. 1996 Feb;106(2):209–214. [PubMed]
  • Kallunki P, Sainio K, Eddy R, Byers M, Kallunki T, Sariola H, Beck K, Hirvonen H, Shows TB, Tryggvason K. A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment. J Cell Biol. 1992 Nov;119(3):679–693. [PMC free article] [PubMed]
  • Gerecke DR, Wagman DW, Champliaud MF, Burgeson RE. The complete primary structure for a novel laminin chain, the laminin B1k chain. J Biol Chem. 1994 Apr 15;269(15):11073–11080. [PubMed]
  • Ryan MC, Tizard R, VanDevanter DR, Carter WG. Cloning of the LamA3 gene encoding the alpha 3 chain of the adhesive ligand epiligrin. Expression in wound repair. J Biol Chem. 1994 Sep 9;269(36):22779–22787. [PubMed]
  • Vailly J, Verrando P, Champliaud MF, Gerecke D, Wagman DW, Baudoin C, Aberdam D, Burgeson R, Bauer E, Ortonne JP. The 100-kDa chain of nicein/kalinin is a laminin B2 chain variant. Eur J Biochem. 1994 Jan 15;219(1-2):209–218. [PubMed]
  • Vidal F, Baudoin C, Miquel C, Galliano MF, Christiano AM, Uitto J, Ortonne JP, Meneguzzi G. Cloning of the laminin alpha 3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa. Genomics. 1995 Nov 20;30(2):273–280. [PubMed]
  • Pulkkinen L, Gerecke DR, Christiano AM, Wagman DW, Burgeson RE, Uitto J. Cloning of the beta 3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa. Genomics. 1995 Jan 1;25(1):192–198. [PubMed]
  • Airenne T, Haakana H, Sainio K, Kallunki T, Kallunki P, Sariola H, Tryggvason K. Structure of the human laminin gamma 2 chain gene (LAMC2): alternative splicing with different tissue distribution of two transcripts. Genomics. 1996 Feb 15;32(1):54–64. [PubMed]
  • Vailly J, Szepetowski P, Mattei MG, Pedeutour F, Burgeson R, Ortonne JP, Meneguzzi G. The genes for nicein/kalinin 125- and 100-kDa subunits, candidates for junctional epidermolysis bullosa, map to chromosomes 1q32 and 1q25-q31. Genomics. 1994 May 1;21(1):286–288. [PubMed]
  • Uitto J, Pulkkinen L, Christiano AM. Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes. J Invest Dermatol. 1994 Nov;103(5 Suppl):39S–46S. [PubMed]
  • Christiano AM, Uitto J. Molecular diagnosis of inherited skin diseases: the paradigm of dystrophic epidermolysis bullosa. Adv Dermatol. 1996;11:199–214. [PubMed]
  • Christiano AM, Uitto J. Molecular complexity of the cutaneous basement membrane zone. Revelations from the paradigms of epidermolysis bullosa. Exp Dermatol. 1996 Feb;5(1):1–11. [PubMed]
  • Uitto J, Pulkkinen L. Molecular complexity of the cutaneous basement membrane zone. Mol Biol Rep. 1996;23(1):35–46. [PubMed]
  • Ganguly A, Rock MJ, Prockop DJ. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10325–10329. [PubMed]
  • Christiano AM, Anhalt G, Gibbons S, Bauer EA, Uitto J. Premature termination codons in the type VII collagen gene (COL7A1) underlie severe, mutilating recessive dystrophic epidermolysis bullosa. Genomics. 1994 May 1;21(1):160–168. [PubMed]
  • Aberdam D, Galliano MF, Vailly J, Pulkkinen L, Bonifas J, Christiano AM, Tryggvason K, Uitto J, Epstein EH, Jr, Ortonne JP, et al. Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5). Nat Genet. 1994 Mar;6(3):299–304. [PubMed]
  • Baudoin C, Miquel C, Gagnoux-Palacios L, Pulkkinen L, Christiano AM, Uitto J, Tadini G, Ortonne JP, Meneguzzi G. A novel homozygous nonsense mutation in the LAMC2 gene in patients with the Herlitz junctional epidermolysis bullosa. Hum Mol Genet. 1994 Oct;3(10):1909–1910. [PubMed]
  • Pulkkinen L, Christiano AM, Airenne T, Haakana H, Tryggvason K, Uitto J. Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa. Nat Genet. 1994 Mar;6(3):293–297. [PubMed]
  • Vailly J, Pulkkinen L, Christiano AM, Tryggvason K, Uitto J, Ortonne JP, Meneguzzi G. Identification of a homozygous exon-skipping mutation in the LAMC2 gene in a patient with Herlitz's junctional epidermolysis bullosa. J Invest Dermatol. 1995 Mar;104(3):434–437. [PubMed]
  • Verrando P, Blanchet-Bardon C, Pisani A, Thomas L, Cambazard F, Eady RA, Schofield O, Ortonne JP. Monoclonal antibody GB3 defines a widespread defect of several basement membranes and a keratinocyte dysfunction in patients with lethal junctional epidermolysis bullosa. Lab Invest. 1991 Jan;64(1):85–92. [PubMed]
  • Fine JD, Bauer EA, Briggaman RA, Carter DM, Eady RA, Esterly NB, Holbrook KA, Hurwitz S, Johnson L, Lin A, et al. Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. A consensus report by the Subcommittee on Diagnosis and Classification of the National Epidermolysis Bullosa Registry. J Am Acad Dermatol. 1991 Jan;24(1):119–135. [PubMed]
  • McLean WH, Lane EB. Intermediate filaments in disease. Curr Opin Cell Biol. 1995 Feb;7(1):118–125. [PubMed]
  • McLean WH, Pulkkinen L, Smith FJ, Rugg EL, Lane EB, Bullrich F, Burgeson RE, Amano S, Hudson DL, Owaribe K, et al. Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization. Genes Dev. 1996 Jul 15;10(14):1724–1735. [PubMed]
  • Smith FJ, Eady RA, Leigh IM, McMillan JR, Rugg EL, Kelsell DP, Bryant SP, Spurr NK, Geddes JF, Kirtschig G, et al. Plectin deficiency results in muscular dystrophy with epidermolysis bullosa. Nat Genet. 1996 Aug;13(4):450–457. [PubMed]
  • Pulkkinen L, Smith FJ, Shimizu H, Murata S, Yaoita H, Hachisuka H, Nishikawa T, McLean WH, Uitto J. Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy. Hum Mol Genet. 1996 Oct;5(10):1539–1546. [PubMed]
  • Pulkkinen L, McGrath JA, Christiano AM, Uitto J. Detection of sequence variants in the gene encoding the beta 3 chain of laminin 5 (LAMB3). Hum Mutat. 1995;6(1):77–84. [PubMed]
  • Kivirikko S, McGrath JA, Pulkkinen L, Uitto J, Christiano AM. Mutational hotspots in the LAMB3 gene in the lethal (Herlitz) type of junctional epidermolysis bullosa. Hum Mol Genet. 1996 Feb;5(2):231–237. [PubMed]
  • Peral B, Gamble V, San Millán JL, Strong C, Sloane-Stanley J, Moreno F, Harris PC. Splicing mutations of the polycystic kidney disease 1 (PKD1) gene induced by intronic deletion. Hum Mol Genet. 1995 Apr;4(4):569–574. [PubMed]
  • Cooper DN. Human gene mutations affecting RNA processing and translation. Ann Med. 1993 Feb;25(1):11–17. [PubMed]
  • McGrath JA, Pulkkinen L, Christiano AM, Leigh IM, Eady RA, Uitto J. Altered laminin 5 expression due to mutations in the gene encoding the beta 3 chain (LAMB3) in generalized atrophic benign epidermolysis bullosa. J Invest Dermatol. 1995 Apr;104(4):467–474. [PubMed]
  • McGrath JA, Kivirikko S, Ciatti S, Moss C, Christiano AM, Uitto J. A recurrent homozygous nonsense mutation within the LAMA3 gene as a cause of Herlitz junctional epidermolysis bullosa in patients of Pakistani ancestry: evidence for a founder effect. J Invest Dermatol. 1996 Apr;106(4):781–784. [PubMed]
  • McGrath JA, Kivirikko S, Ciatti S, Moss C, Dunnill GS, Eady RA, Rodeck CH, Christiano AM, Uitto J. A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in Herlitz junctional epidermolysis bullosa: prenatal exclusion in a fetus at risk. Genomics. 1995 Sep 1;29(1):282–284. [PubMed]
  • Vailly J, Pulkkinen L, Miquel C, Christiano AM, Gerecke D, Burgeson RE, Uitto J, Ortonne JP, Meneguzzi G. Identification of a homozygous one-basepair deletion in exon 14 of the LAMB3 gene in a patient with Herlitz junctional epidermolysis bullosa and prenatal diagnosis in a family at risk for recurrence. J Invest Dermatol. 1995 Apr;104(4):462–466. [PubMed]

Articles from Molecular Medicine are provided here courtesy of The Feinstein Institute for Medical Research at North Shore LIJ