Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa

Authors

  • Ningning Dang,

    1. Department of Dermatology, St George Hospital, Sydney, Australia;
    2. Department of Dermatology, Jinan Central Hospital, Shandong Province, China;
    3. University of New South Wales, Sydney, NSW, Australia
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  • Dédée F. Murrell

    1. Department of Dermatology, St George Hospital, Sydney, Australia;
    2. University of New South Wales, Sydney, NSW, Australia
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Dedee F Murrell, MA (Cambridge), BMBCh (Oxford), FAAD (USA), MD (UNSW), Chair, Department of Dermatology, St George Hospital, University of NSW, Gray Street, Kogarah, Sydney, NSW 2217, Australia, Tel.: +61 2 9113 2543, Fax: +61 2 9113 2906, e-mail: d.murrell@unsw.edu.au

Abstract

Abstract:  Dystrophic epidermolysis bullosa (DEB) is inherited in both an autosomal dominant DEB and autosomal recessive manner RDEB, both of which result from mutations in the type VII collagen gene (COL7A1). To date, 324 pathogenic mutations have been detected within COL7A1 in different variants of DEB; many mutations are clustered in exon 73 (10.74%) which is close to the 39 amino acid interruption region. Dominant dystrophic epidermolysis bullosa usually involves glycine substitutions within the triple helix of COL7A1 although other missense mutations, deletions or splice-site mutations may underlie some cases. In recessive dystrophic epidermolysis bullosa, the mutations include nonsense, splice site, deletions or insertions, ‘silent’ glycine substitutions within the triple helix and non-glycine missense mutations within the triple helix or non-collagenous NC-2 domain. The nature of mutations in COL7A1 and their positions correlate reasonably logically with the severity of the resulting phenotypes.

Abbreviations:
AFs

anchoring fibrils

COL7A1

the type VII collagen gene

DDEB

dominant dystrophic epidermolysis bullosa

DEB

dystrophic epidermolysis bullosa

LVs

lentiviral vectors

MMP

matrix metalloproteinase

PTC

premature termination codon

RDEB

recessive dystrophic epidermolysis bullosa

RDEB-HS

Hallopeau-Siemens recessive dystrophic epidermolysis bullosa

RDEB-nHS

non-Hallopeau-Siemens recessive dystrophic epidermolysis bullosa

RVs

retroviral vectors

TBDN

transient bullous dermolysis of newborn

Introduction

Dystrophic epidermolysis bullosa (DEB) is an inherited skin fragility disorder in which blistering occurs in the sub-lamina densa zone at the level of anchoring fibrils (AFs) of the dermo-epidermal junction (1). It is inherited in both an autosomal dominant (DDEB) and an autosomal recessive manner (RDEB). The clinical features of DEB have a broad range of severity from isolated nail dystrophy through relatively mild, localized blistering of the extremities to generalized blistering and mutilation, oesophageal blistering and failure to thrive with premature demise (Fig. 1). Both recessive dystrophic epidermolysis bullosa (RDEB) and dominant dystrophic epidermolysis bullosa (DDEB) result from mutations in the type VII collagen gene (COL7A1). The severe Hallopeau-Siemens (HS) RDEB (RDEB-HS) is generally because of premature termination codon (PTC) mutations, resulting from nonsense, frame-shift or splice-site mutations on both COL7A1 alleles (2) which result in either nonsense-mediated decay of the mRNA or truncated polypeptides that are unable to assemble into functional AFs. The milder, non-Hallopeau-?hangover 0>Siemens RDEB (RDEB-nHS) is often caused by compound heterozygous mutations: one PTC mutation and one missense mutation and full-length type VII collagen polypeptides can be synthesized, but they have a different conformation and affect the stabilization of the AF by disulphide bonding or other structural changes. DDEB usually involves glycine substitutions within the triple helix of COL7A1.

Figure 1.

 Clinical presentation of dystrophic epidermolysis bullosa (DEB) patients. Pruriginosa (a) and albopapuloid (b) lesions on the arm and 90% of body surface covered with lesions in dominant dystrophic epidermolysis bullosa (DDEB; severe phenotype). (c) and (d) Mild healed erosions on leg and severely dystrophic toenails in DDEB. (e) Localized atrophic scarring and erosions on the trunk in non-Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (RDEB-nHS). (f) and (g) Widespread blisters, erosions, scars and atrophy and significant nail dystrophy and syndactyly of the feet in RDEB-HS.

Dominant dystrophic epidermolysis bullosa previously included two classical phenotypes, the Cockayne-Touraine type with hypertrophic lesions and the Pasini type with white papular lesions (1,3,4). The pruriginosa form is characterized by severe pruritus and nodular prurigo-like lichenified lesions localized mostly on the lower extremities and extensor forearms (4); lesions are often limited to the pretibial areas (5,6).

Recessive dystrophic epidermolysis bullosa has been classified according to different clinical features into the severe Hallopeau-Siemens type (RDEB-HS) with generalized lesions and scarring of the hands and feet leading to fusion of the digits and severe mucosal involvement; and the milder form, designated RDEB-nHS, which can be localized or generalized and displays very mild or no pseudo-syndactyly and less frequent extra-cutaneous involvement (3). The other recessive forms include the inversa RDEB with blistering in the intertriginous areas, the centripetal RDEB with lesions starting on the extremities and spreading to the trunk; pruriginosa and pretibial types of RDEB also have been described (7,8). Transient bullous dermolysis of newborn (TBDN) usually is a form of DDEB, but less commonly may be transmitted in an autosomal recessive manner with blistering that usually improves markedly during early life or even remits completely (9).

Physiology and structure of COL7A1

Both RDEB and DDEB result from mutations in COL7A1, which is the major component of the AFs that anchor the basal lamina to the dermal collagen fibrils (10,11). The level of expression of COL7A1 in DEB patients shows an inverse correlation with its clinical severity (1). Procollagen VII is a homotrimer composed of three proα1 (VII) chains which are encoded by the 32 kb COL7A1 gene located on chromosome 3p21 (10). The mRNA transcript of approximately 8.9 kb is translated into a proα1 (VII) polypeptide containing 2944 amino acids (12). Each pro α1 (VII) polypeptide chain contains a central triple helical collagenous domain (145 kDa) flanked by both a large amino-terminal non-collagenous (NC-1) domain (145 kDa) and a small carboxyl-terminal non-collagenous (NC-2) domain (30 kDa) (12). COL7A1 has 118 exons. The NC-1 domain is from exon 1 to exon 28 (amino acids 1–1253) and the NC-2 domain is from exon 112 to exon 118 (amino acids 2784–2944) (13). The triple helical domain consists of a repeating Gly-X-Y sequence that is disrupted 19 times by non-collagenous regions, whose largest disruption is 39 amino acid residues in length, known as the ‘hinge’ region. The NC-1 domain consists of submodules with homology to adhesive proteins, including cartilage matrix protein, type III repeats of fibronectin, a von Willebrand factor type A-like motif and a proline- and cysteine-rich domain (C/P) (13; Figs 2, 3 and 4). The triple helical domain contributes to the ultrastructurally recognizable central cross-banded part of the AFs. The NC-1 ‘structural’ domain mediates the attachment of the AFs into the basement membrane above and the islands of collagen IV in the dermis below (14). The NC-2 ‘structural’ domain contains conserved cysteines involved in the formation of disulphide bonds which enable linkage between type VII collagen homotrimers (15). Arg-Gly-Asp (RGD) sequences, four of which are in the NC-1 domain of human type VII collagen have been shown to serve as integrin-mediated attachment sites for cells to adhere to extracellular matrix components such as fibronectin (16).

Figure 2.

 Anchoring fibril assembly and the consequences of the major types mutations on type VII collagen protein synthesis in dystrophic epidermolysis bullosa (DEB). The left side shows the physiology of type VII collagen and the right side shows the pathology. I: proα1 (VII) polypeptides are synthesized in ribosomal complex. II: Three of these chains assemble into a triple helical type VII collagen molecule – homotrimers. At stages III & IV, two homotrimers form antiparallel tail-to-tail dimers with a central carboxy-terminal overlap and with the amino-termini outwards, a portion of the NC-2 domain is removed, and the association of the monomers in stabilized by intermolecular disulphide bonds. Stages V & VI: a large number of dimer molecules assemble into anchoring fibrils and the complete NC-1 domain keeps the adhesive property at both ends. Mature anchoring fibrils are stabilized by transglutaminase cross-links in vivo. Stage VII: premature termination codon mutations (PTC) decrease the amount of the mutated transcripts and result in truncated non-functional polypeptides which are unable to assemble into anchoring fibrils, then causing Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (RDEB-HS). VIII: Missense mutations alter homotrimer formation and/or subsequent stabilization of the dimer molecules by disulphide bonds result in decreased stability and/or alter function of VII collagen known as milder type of non-Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (RDEB-nHS). IX: Glycine substitutions often happen in triple helix region of COL7A1 affecting the correct folding and the secretion of type VII collagen, resulting in dominant dystrophic epidermolysis bullosa (DDEB). (Modified from Jarvikallio and Uitto, Human Mutation 10:338–347, 1997).

Figure 3.

 COL7A1 missense and nonsense mutations in dystrophic epidermolysis bullosa (DEB) patients. The red lettering signifies dominant and the black signifies recessive.

Figure 4.

 COL7A1 deletions, insertions, splice mutations in dystrophic epidermolysis bullosa (DEB) patients.

In the extracellular milieu, the collagen VII homotrimers form tail-to-tail antiparallel dimers, a portion of NC-2 domain is removed and the association of the homotrimers is stabilized by intermolecular disulphide bonds in the overlapping carboxyl-terminal regions (15; Fig. 2). The function of the NC-2 domain is not known and the amino acid sequence contains a Kunitz-type proteinase inhibitor motif (17) which may be a feedback inhibitor secreted by the cleaved propeptide to inhibit the specific C proteinase or have other inhibitory activity towards tissue proteinases. Because the NC-2 domain mediates dimerization of type VII collagen homotrimers before polymerization into AFs, it has been predicted that mutations in NC-2 could affect the processing of collagen VII (18).

Mutation analysis in RDEB

The data summarizing all COL7A1 mutations was first checked in the publicly available section of the Human Genome Medical Database (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=COL7A1) and additional ones were gathered by cross-referencing this against the PubMed Search engine as cloning and sequencing of COL7A1 in 1992 using the search terms ‘collagen VII’ and ‘epidermolysis bullosa’ until submission in August 1, 2007. All languages were included. Around 324 pathogenic mutations have been detected within COL7A1 in different variants of DEB including 43 nonsense, 127 missense, 65 deletion, 28 insertion, 9 insertion-deletion, 51 splice-site and 1 regulatory mutations (Table 1, Figs 3 and 4). Many mutations (20 dominant and 15 recessive) are clustered in exon 73 (10.74%), which suggests that this exon represents a region in which mutations commonly affect the function of AFs (Fig. 5; 19).

Table 1.   COL7A1 mutations published to date in dystrophic epidermolysis bullosa patients
MutationsE/IPro LocConsequencesPhenotypeReferences
  1. E/I, exon/intron; Pro Loc, protein location; SP, signal peptide; VWA, von Willebrand factor A domain; CMP, cartilage matrix protein; Fn3, fibronectin III-like domains; C/P, cysteine & proline rich region; THC, triple helical collagenous domain; AA39, 39 amino acid ‘hinge’ region; KM, kunitz module; RDEB, recessive dystrophic epidermolysis bullosa; RDEB-HS, Hallopeau-Siemens recessive dystrophic epidermolysis bullosa; RDEB-nHS, non-Hallopeau-Siemens recessive dystrophic epidermolysis bullosa; RDEB-I, RDEB inversa; RDEB-Pt, RDEB pretibial; RDEB-Pr, RDEB pruriginosa; DDEB, Dominant dystrophic epidermolysis bullosa; DDEB-CT, DDEB Cockayne-Touraine; DDEB-Pt, DDEB pretibial; DDEB-Pr, DDEB pruriginosa; DDEB-Pa, DDEB pasini; TBDN, transient bullous dermolysis of the newborn; gen, Generalized; loc, Localized; PTC, Premature termination codon mutation; DTC, Delayed termination codon mutation; GS, Glycine substitution mutation; Mis, Missense mutation; Spl, Splice site mutation; Gen, generalized; Loc, localized.

  2. –: Not determined; # may be a polymorphism.

  3. *De novo mutations.

Nonsense
 R20XE1SPPTCRDEBPfender (2003), Prenat Diagn 23, 447
 R109XE3CMPPTCRDEBPfender (2003), Prenat Diagn 23, 447
 R137XE3CMPPTCRDEB-HSSawamura (2005), J Hum Genet 50, 543
 R185XE5CMPPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 Q189XE5CMPPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 R226XE5CMPPTCRDEB-HSChristiano (1996), J Invest Dermatol 106, 679
 R236XE6Fn3PTCRDEB-IHovnanian (1994), Am J Hum Genet 55, 289
 Q251XE6Fn3PTCRDEB-HSChristiano (1996), Mol Med 2, 59
 Q281XE6Fn3PTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 Y311XE7Fn3PTCRDEB-HSChristiano (1995), J Clin Invest 95, 1328
 R525XE12Fn3PTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 R578XE13Fn3PTCRDEB-HSDunnill (1994), Hum Mol Genet 3, 1693
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 Q1211XE27VWAPTCPulkkinen (1999), Matrix Biol 18, 29
 R1340XE33THCPTCRDEB-HSHovnanian (1994), Am J Hum Genet 55, 289
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 R2332XE90THCPTCRDEBPfender (2003), Prenat Diagn 23, 447
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 Q2417XE94THCPTCRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 R2471XE97THCPTCRDEB-nHSChristiano (1996), Am J Hum Genet 58, 671
 R2492XE98THCPTCRDEB-nHS genGardella (2002), J Invest Dermatol 119, 1456
 R2610XE105THCPTCRDEB-nHSHovnanian (1997), Am J Hum Genet 61, 599
 R2685XE109THCPTCRDEB-HSGardella (1999), Hum Mutat 13, 439
 Q2796XE113AcidicPTCRDEBPulkkinen (1999), Matrix Biol 18, 29
 Q2827XE115AcidicPTCRDEB-nHSSato-Matsumura (2002), Arch Dermatol 138, 269
 R2814XE115AcidicPTCRDEB-HSChristiano (1996), Mol Med 2, 59
 E2857XE116AcidicPTCRDEB-nHSShimizu (1996), J Invest Dermatol 106, 119
Missense
 R886PE20Fn3MisRDEB-nHSPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 Y1250SE28C/PMisRDEB-nHSRyoo (2000), J Dermatol Sci 26, 125
 G1338VE34THCGSRDEB-HSDang (2007), J Dermatol Sci 46, 169–176
 G1347WE34THCGSRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 G1347RE34THCGSRDEB-nHSTerracina (1998), J Invest Dermatol 111, 744
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 G1519DE44THCGSTBDN(Recessive)Hammami-Hauasli (1998), J Invest Dermatol 111, 1124
 G1522EE45THCGSDDEB, TBDN (Dominant)Whittock (1999), J Invest Dermatol 113, 673, Fassihi (2005), Br J Dermatol,153, 1058
 G1522RE45THCGSRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 G1557RE48THCGSDDEBChristiano (1996), Am J Hum Genet 58, 671
 G1595RE50THCGSDDEBSato-Matsumura (2002), Arch Dermatol 138, 269
 G1604RE50THCGSRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G1616RE51THCGSRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 G1652RE53THCGSRDEB-nHSCserhalmi-Friedman (1997), Arch Dermatol Res289, 640
 G1655G(4965c-t)E53THCPTC-normalRDEB-nHS locPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 G1664AE54THCGSRDEB-nHS locGardella (2002), J Invest Dermatol 119, 1456
 G1664EE54THCGSRDEBNakamura (2004), J Dermatol Sci 34, 195
 G1673RE54THCGSRDEB-nHSVarki (2007), J Med Genet 44, 181
 R1696CE55THCGSRDEBPfender (2003), Prenat Diagn 23, 447
 P1699LE55THCMisRDEB-PtGardella (2002), J Invest Dermatol 119, 1456
 G1703EE56THCGSRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G1719RE58THCGSRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 G1755D*E59THCGSDDEB-PrPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 R1772WE61THCMisRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G1776RE61THCGSDDEBWhittock (1999), J Invest Dermatol 113, 673
 G1776AE61THCGSDDEBNakamura (2004), J Dermatol Sci 34, 195
 G1782RE61THCGSRDEB-nHS genChristiano (1996), J Invest Dermatol 106, 778
 G1782VE61THCGSRDEBKern (2006), J Invest Dermatol 126, 1006
 G1791EE61THCGSDDEB-PrMellerio (1999), J Invest Dermatol 112, 984
 G1812RE63THCGSRDEB-nHS genMasunaga (2000), J Invest Dermatol 114, 204
 G1814CE63THCGSRDEB-nHSDang (2007), J Dermatol Sci 46, 169–176
 G1815RE63THCGSDDEBSato-Matsumura (2002), Arch Dermatol 138, 269
 Q1924PE69THCPulkkinen (1999), Matrix Biol 18, 29
 P1940PE7039AARDEB-nHSTerracina (1998), J Invest Dermatol 111, 744
 R1957QE7239AAMisRDEB-nHSSawamura (2005), J Hum Genet 50, 543
 G1982WE72THCGSRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 R2002CE73THCPulkkinen (1999), Matrix Biol 18, 29
 G2003RE73THCGSDDEBChristiano (1996), J Invest Dermatol 106, 778
 G2006DE73THCGSDDEBHammami-Hauasli (1998), J Biol Chem 273, 19228
 G2006SE73THCGSDDEBMallipeddi (2003), Br J Dermatol 149, 810
 G2006AE73THCGSDDEBWhittock (1999), J Invest Dermatol 113, 673
 R2008GE73THCMisRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 R2008CE73THCMisRDEB-nHSKon (1998), J Invest Dermatol 111, 534
 G2009RE73THCGSRDEB-nHSWinberg (1997), Hum Mol Genet 6, 1125
 G2012S*E73THCGSDDEBGardella (2002), J Invest Dermatol 119, 1456
 G2012D*E73THCGSDDEBMatsuba (2002), Clin Exp Dermatol 27, 56
 G2015EE73THCGSDDEBHammami-Hauasli (1998), J Biol Chem 273, 19228
 G2025SE73THCGSRDEBPfender (2003), Prenat Diagn 23, 447
 G2025AE73THCGSRDEB-nHSHovnanian (1997), Am J Hum Genet 61, 599
 G2028R*E73THCGSDDEB-PrMurata (2000), Arch Dermatol Res 292, 477
 G2028AE73THCGSDDEBMurata (2000), Arch Dermatol Res 292, 477
 P2029SE73THCMisRDEB-nHSRyoo (2001), J Dermatol Sci 26, 125
 G2031SE73THCGSRDEB-HSNordal (2001), Br J Dermatol 144, 151
 G2034WE73THCGSDDEBRouan (1998), J Invest Dermatol 111, 1210
 G2034RE73THCGSDDEB-CTKon (1997), J Invest Dermatol 109, 684
 G2034EE73THCGSDDEBKern (2006), J Invest Dermatol 126, 1006
 G2037EE73THCGSDDEB-PaJonkman (1999), J Invest Dermatol 112, 815
 G2040SE73THCGSDDEB-CTChristiano (1994), Proc Natl Acda Sci U S A 91, 3549
 G2040DE73THCGSDDEBWhittock (1999), J Invest Dermatol 113, 673
 G2040V*E73THCGSDDEBRouan (1998), J Invest Dermatol 111, 1210
 G2043R*E73THCGSDDEBChristiano (1995), J Invest Dermatol 104, 438
 G2043W*E73THCGSDDEBMecklenbeck (1999), J Invest Dermatol 112, 398
 G2046VE73THCGSDDEBWhittock (1999), J Invest Dermatol 113, 673
 G2049EE73THCGSRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 G2055EE73THCGSDDEBChristiano (1996), Am J Hum Genet 58, 671
 E2059GE73THCMisRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 R2063GE74THCMisRDEB-IHovnanian (1997), Am J Hum Genet 61, 599
 R2063WE74THCMisRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 G2064RE74THCGSDDEBRouan (1998), J Invest Dermatol 111, 1210
 G2064EE74THCGSDDEBSawamura (2005), J Hum Genet 50, 543
 G2067R*E74THCGSDDEBPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 R2069CE74THCMisRDEB-IKahofer (2003), Pediatr Dermatol 20, 243
 G2070EE74THCGSDDEBGardella (2002), J Invest Dermatol 119, 1456
 G2070RE74THCGSDDEBZhang (2003), Clin Exp Dermatol 28, 437
 G2073DE74THCGSRDEB-nHS genDunnill (1996), J Invest Dermatol 107, 171
 G2076D*E75THCGSDDEB-PaKon (1997), J Invest Dermatol 109, 684
 G2079E*E75THCGSDDEBKon (1997), J Invest Dermatol 108, 224
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 G2132DE78THCGSRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G2192SE82THCGSRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G2207RE83THCGSDDEB-CTKon (1998), J Invest Dermatol 111, 534
 G2210VE83THCGSDDEBDang (2007), J Dermatol Sci 46, 169–176
 V2226EE84THCMisRDEB-nHSRyoo (2001), J Dermatol Sci 26, 125
 P2229SE84THCMisRDEB-nHSRyoo (2001), J Dermatol Sci 26, 125
 P2232RE84THCMisRDEB-nHSRyoo (2001), J Dermatol Sci 26, 125
 G2233CE84THCGSRDEB-nHSRyoo (2001), J Dermatol Sci 26, 125
 G2233SE84THCGSRDEB-nHS genSalas-alanis (2000), Int J Dermatol 39, 436
 G2239DE85THCGSDDEB-PrTamai (1998), J Invest Dermatol 111, 1509
 G2242RE85THCGSDDEB-PrLee (1997), J Invest Dermatol 108, 947
 G2242EE85THCGSDDEB-PrTamai (1998), J Invest Dermatol 111, 1509
 G2251EE86THCGSTBDN(Dominant)Hammami-Hauasli (1998), J Invest Dermatol 111, 1124
 G2263VE86THCGSRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G2287RE87THCGSDDEBShimizu (1999), J Invest Dermatol 113, 419
 G2287VE87THCGSDDEBPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 G2316RE89THCGSRDEBShimizu (1999), J Invest Dermatol 113, 419
 G2348R*E91THCGSDDEBCserhalmi-Friedman (1999), Exp Dermatol 8, 143
 G2351RE91THCGSRDEB-nHSChristiano (1996), Am J Hum Genet 58, 682
 G2366CE91THCGSRDEB-nHSSawamura (2005), J H5m Genet 50, 543
 G2366SE92THCGSRDEB-nHSHashimoto (1999), Exp Dermatol 8, 140
 G2366VE92THCGSDDEB-PrChuang (2004), Clin Exp Dermatol 29, 304
 G2369SE93THCGSDDEB-PrMellerio (1999), J Invest Dermatol 112, 984
 G2375SE93THCGSRDEB-HSFassihi (2006), J Dermatol Sci Feb 12
 G2395DE94THCGSRDEBMayama (1999), J Invest Dermatol 112, 568
 G2413EE94THCGSRDEBKern (2006), J Invest Dermatol 126, 1006
 M2415IE94THCMisRDEB-nHS locTiteux (2006), Hum Mutat 27, 291
 V2448V(7344G-A)E95THCPTC-normalRDEB-nHSGardalla (1996), Am J Hum Genet 59, 292
 G2569RE103THCGSRDEB-nHSChristiano (1996), Am J Hum Genet 58, 671
 G2575RE103THCGSRDEB-nHSShimizu (1996), J Invest Dermatol 106, 119
 R2622WE105THCMisRDEB-IGardella (2002), J Invest Dermatol 119, 1456
 G2623CE105THCGSDDEB-PtChristiano (1995), Hum Mol Genet 4, 1579
 G2632RE106THCGSDDEBPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 G2653RE107THCGSRDEB-nHSChristiano (1996), Am J Hum Genet 58, 671
 G2671VE108THCGSRDEB-HSKon (1997), J Invest Dermatol 108, 224
 G2674DE108THCGSRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 G2674RE108THCGSRDEB-nHSChristiano (1996), Am J Hum Genet 58, 671
 G2689RE109THCGSRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 G2695SE109THCGSPulkkinen (1999), Matrix Biol 18, 29
 G2713DE110THCGSDDEBRouan (1998), J Invest Dermatol 111, 1210
 G2713RE110THCGSDDEB-PrMellerio (1999), J Invest Dermatol 112, 984
 G2719AE110THCGSRDEB-HSFassihi (2006), J Dermatol Sci Feb 12
 G2737RE110THCGSRDEBKern (2006), J Invest Dermatol 126, 1006
 G2740AE110THCGSRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 G2749RE111THCGSRDEB-HSChristiano (1996), Am J Hum Genet 58, 671
 G2775SE112THCGSRDEB-nHSKon (1998), J Invest Dermatol 111, 534
 R2791W#E113AcidicMisDDEBWhittock (1999), J Invest Dermatol 113, 673
 M2798KE113AcidicMisRDEB-nHSChristiano (1993), Nature Genet 4, 62
 C2875FE117KMMisRDEB-nHSSawamura (2005), J Hum Genet 50, 543
Deletions
 154delGE2CMPIn frame deletionWhittock (1999), J Invest Dermatol 113, 673
 189delGE2CMPPTCRDEB-HSChristiano (1997), J Invest Dermatol 109, 390
 520delGE4I5CMPPulkkinen (1999), Matrix Biol 18, 29
 520del7I4E5CMPPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 579delCE5CMPPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 642delGTE5CMPPTCRDEB-HSChristiano (1997), Hum Mutat 10, 408
 847-6del5I6E7Fn3PTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 884delGE7Fn3PTCRDEB-HSChristiano (1996), Mol Med 2, 59
 1172delATE9Fn3PTCRDEB-HSChristiano (1994), Genomics 21, 160
 1474del11E11Fn3PTCRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 1474del8E11Fn3PTCRDEB-HSSawamura (2005), J Hum Genet 50, 543
 1898delGE14Fn3PTCRDEB-nHSChristiano (1996), J Invest Dermatol 106, 766
 1930delCE15Fn3PTCRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 2482delCTE19Fn3PTCRDEB-IGardella (2002), J Invest Dermatol 119, 1456
 2638del25E20Fn3PTCRDEB-nHS genHammami-Hauasli (1997), J Invest Dermatol 109, 384
 2785delACE21Fn3PTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 3054delAE23Fn3RDEB-HSFassihi (2006), J Dermatol Sci Feb 12
 3148delCE24VWAPTCRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
 3472delCE26VWAPTCRDEB-HSMasse (2005), Clin Exp Dermatol 30, 289
 3474delAE26VWAPTCRDEB-HSPulkkinen (1999), Matrix Biol 18, 29
 3478delCE26VWAPTCRDEB-nHSDang (2007), J Dermatol Sci 46, 169–176
 3567delGTE27VWAPTCRDEB-HSDang (2007), J Dermatol Sci 46, 169–176
 3839delCE31THCPTCRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 3857delAE31THCPTCRDEB-nHS genMasunaga (2000), J Invest Dermatol 114, 204
 3858delGE31THCPTCRDEB-HSChristiano (1994), Genomics 21, 160
 4095delGE35THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 4172delCE36THCPTCRDEB-nHS genGardella (2002), J Invest Dermatol 119, 1456
 4249delGE38THCPTCRDEB-HSPulkkinen (1999), Matrix Biol 18, 29
 4317delCE39THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 4429delGE41THCPTCRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 4767delAE49THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 4834del8E51THCPTCRDEB-nHS locGardella (2002), J Invest Dermatol 119, 1456
 4918delGE52THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 4919delGE52THCPTCRDEB-HSVarki (2007), J Med Genet 44, 181
 4929delTE52THCPTCRDEB-HSCsikos (2005), Br J Dermatol 152, 879
 5012delAE54THCPTCRDEB-HSKon (1998), J Invest Dermatol 111, 534
 5092delGE55THCPTCRDEB-nHSPulkkinen (1999), Matrix Biol 18, 29
 5504delAE64THCPTCRDEB-nHS genIshiko (2004), Exp Dermatol 13, 229
 5657delAE67THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 5772delGE69THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 5818delCE7039AAPTCRDEB-HSChristiano (1995), J Clin Invest 95, 1328
 5819delCE7039AARDEBPfendner (2003), Prenat Diagn 23, 447
 6041delAGE73THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 6075delCE73THCRDEB-nHSChristiano (1996), J Invest Dermatol 106, 766
 6081delCE73THCPTCRDEB-nHSChristiano (1996), J Invest Dermatol 106, 766
 6081del28E73THCSplDDEBSakuntabhai (1998), Am J Hum Genet 63, 737
 6269delCE75THCRDEBPfender (2003), Prenat Diagn 23, 447
 6751-2delAI85THCIn-frame skippingRDEB-HSPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 6847del27E87THCIn-frame skippingDDEBSakuntabhai (1998), Am J Hum Genet 63, 737
 6849del18E87THCIn-frame deletionDDEBPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 6863del16E87THCPTCDDEBCserhalmi-Friedman (1998), Lab Invest 78, 1483
 7178delTE94THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 7288del29E95THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 7634delGE102THCPTCRDEB-HSVarki (2007), J Med Genet 44, 181
 7729delCE103THCPTCRDEB-HSChristiano (1996), Mol Med 2, 59
 7786delGE104THCPTCRDEB-nHS genMellerio (1997), J Invest Dermatol 109, 246
 8074delGE109THCPTCRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
 8091delGE109THCPTCRDEB-HSKon (1998), J Invest Dermatol 111, 534
 8117delC*E110THCPTCRDEB-nHSPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 8441-15del20E115AcidicIn-frame deletionRDEB-nHSCsikos (2005), Br J Dermatol 152, 879
 8441-14del21E115AcidicPTCRDEB-HSDunnill (1996), J Invest Dermatol 107, 171
 8523del14E115AcidicIn-frame deletionRDEB-nHS locBruckner (1995), J Cell Biol 131, 551
 8697del11E117KMPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 8700delCE117KMRDEBPulkkinen (1999), Matrix Biol 18, 29
 8760delGE117KMDTCRDEB-nHSChristiano (1996), J Invest Dermatol 106, 766
Insertions
 62dupTE1SPPTCRDEBKern (2006), J Invest Dermatol 126, 1006
 111insAE2CMPPTCRDEB-HSPulkkinen (1999), Matrix Biol 18, 29
 114insAE2CMPChristiano (1996), J Invest Dermatol 106, 679
 325insCGE3CMPPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 344insGE3CMPPTCRDEB-HSGardella (2002), Br J Dermatol 147, 450
 434insGCATE4CMPPTCRDEB-HSSato-Matsumura (2003), Acta Derm Venerol 83, 137
 497insAE4CMPPTCRDEB-HSChristiano (1996), J Invest Dermatol 106, 679
 638insAE5CMPPTCRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
 806insTE6Fn3PTCRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
 1344insGCE11Fn3Whittock (1999), J Invest Dermatol 113, 673
 2470insGE19Fn3PTCRDEB-HSChristiano (1994), Genomics 21, 160
 2587insCCE19Fn3PTCRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
 3005dup20E23Fn3PTCRDEB-nHSHorev (2003), Clin Exp Dermatol 28, 80
 3943insGE32THCPTCRDEB-HSUitto (2000), Hum Gene Ther,11, 2267
 3948insTE32THCPTCRDEB-HSSalas-Alanis (1998), Br J Dermatol 138, 852
 4172insCE36THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 4524insTE47THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 4871insCE51THCPTCRDEB-nHS genGardella (2002), J Invest Dermatol 119, 1456
 5031ins9E54THCIn frame insertionRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 5048insGAAAE54THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 5964insCE72THC THCPTCRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 6081insCE73THCPTCRDEB-HSPfender (2003), Prenat Diagn 23, 447
 6522insCE80THCPulkkinen (1999), Matrix Biol 18, 29
 6527insCE80THCPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 6691insCE84THCPTCRDEB-nHS genSalas-alanis (2000), Int J Dermatol 39, 436
 7483insAE99THCRDEB-HSFassihi (2006), J Dermatol Sci, Feb 12
 8245insCE111AcidicRDEBPfender (2003), Prenat Diagn 23, 447
 8505insCE115THCPTCRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
Insertion deletion
 1592del11ins25E12Fn3PTCRDEB-HSHilal (1993), Nat Genet 5, 287
 3088del5ins6E23Fn3PTCRDEB-HSPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 4738del3insAE49THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 5103delCCinsGE56THCPTCRDEB-nHSChristiano (1996), Am J Hum Genet 58, 671
 5944-5945delGGinsTAE72THCPTCRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 7310del6ins25E95THCPTCRDEB-nHSWhittock (1999), J Invest Dermatol 113, 673
 7759-18del5ins4I103THCIn-frame skippingRDEB-nHSPosteraro (2005), Biochem Biophy Res Commun 338, 1391
 8068del17ins2E110THCWhittock (1999), J Invest Dermatol 113, 673
 8069del17insGAE110THCGSDDEBSawamura (2005), J Hum Genet 50, 543
Splice
 267-3C-GI2CMPPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 267-1G-CI2CMPPTCRDEB-PtGardella (2002), J Invest Dermatol 119, 1456
 425A-GI3CMPPTCRDEB-HSGardella (1996), Am J Hum Genet 59, 292
 426+1G>AI3CMPPTCRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 427-3C-GI3CMPSpl errorRDEB-HSKon (1998), J Invest Dermatol 111, 534
 520G-AE4CMPPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 682+1G-AI5CMPPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 682+1G-CI5CMPRDEB-HSDang (2007), J Dermatol Sci 46, 169–176
 847-6G-AI6Fn3PTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 976-3C-AI7Fn3PTCRDEB-nHS genCsikos (2005), Br J Dermatol 152, 879
 1637-1G-AI12Fn3PTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 1781-1G-CI13Fn3PTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 2314+5G>AI17Fn3SplRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 3550+2T>GI26VWAIn-frame deletionRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 3551-3T>GI26VWASplRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 3832-2A>GI30THCSplRDEB-nHSKern (2006), J Invest Dermatol 126, 1006
 4048-1G>AI35THCIn-frame skippingRDEB-HSUitto (2000), Hum Gene Ther 11, 2267
 4118C-TE35THCWhittock (1999), J Invest Dermatol 113, 673
 4119+1G-CI35THCRDEBPfender (2003), Prenat Diagn 23, 447
 4119+1G-TI35THCSpl errorRDEB-HSKon (1998), J Invest Dermatol 111, 534
 4120-1G-CI35THCSplTBDN dominantChristiano (1997), J Invest Dermatol 109, 811
 4783-1G-AI49THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 IVS51+G-AI51THCIn-frame skippingRDEB-HSMasse (2005), Clin Exp Dermatol 30, 289
 4899+1G>AI51THCSplRDEB-HSKern (2006), J Invest Dermatol 126, 1006
 4980-16C-TI53THCGardella (2002), Br J Dermatol 113, 673
 4980+1G-TI53THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 5097G-AE55THCIn-frame deletionRDEB-nHS genWhittock (1999), J Invest Dermatol 113, 673
 5532+1G-AI64THCSpl/PTCRDEB-PrMellerio (1999), J Invest Dermatol 112, 984
 IVS64+4A-GI64THCSplRDEB-nHSHorev (2003), Clin Exp Dermatol 28, 80
 5604+2G>CI66THCSplRDEB-nHSSawamura (2005), J Hum Genet 50, 543
 5771AI69THCSpl errorRDEB-nHSKon (1998), J Invest Dermatol 111, 534
 5772+1GI69THCPulkkinen (1999), Matrix Biol 18, 29
 5820GE7039AAIn-frame skippingRDEB-nHSTerracina (1998), J Invest Dermatol 111, 744
 6180+1GI73THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 6216+5GI74THCSpl errorRDEB-HSKon (1998), J Invest Dermatol 111, 534
 6501G-AE79THCIn-frame deletionRDEB-nHS genChristiano (1996), Mol Med 2, 59
 6501+1G-CI79THCIn-frame deletionRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 6501+1G-TI79THCIn-frame skippingRDEB-HSGardella (2002), J Invest Dermatol 119, 1456
 6573+1G>CI81THCPTCRDEB-HSTamai (1997), Lab Invest 76, 209
 6573+1G-AI81THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 6619-2A-TI82THCIn-frame deletionRDEB-nHSChristiano (1996), J Invest Dermatol 106, 766
 6899AI87THCSplDDEB-PrJiang (2002), Acta Derm Venereol 82, 187
 6900+4AI87THCPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
 6900+4AI87THCSplDDEBDang (2007), J Dermatol Sci 46, 169–176
 7875+1GI105THCRDEBPfender (2003), Prenat Diagn 23, 447
 7930I106THCPTCRDEB-HSHovnanian (1997), Am J Hum Genet 61, 599
 7930I106THCRDEBPfender (2003), Prenat Diagn 23, 447
 8045A-GE108THCExon skipDDEB-PtKon (1998), J Invest Dermatol 111, 534
 8109+2T>AI109THCSplRDEB-nHSSawamura (2005), J Hum Genet 50, 543
 8227-1G-CI110THCDDEBPulkkinen (1999), Matrix Biol 18, 29
 8358+1G>TI112AcidicSplRDEB-nHSSawamura (2005), J Hum Genet 50, 543
 8439A-GE114AcidicPTCRDEB-HSWhittock (1999), J Invest Dermatol 113, 673
Regulatory
 96C-TPromoterPromoterNo transcriptionRDEB-HSGardella (2000), Hum Mutat 16, 275
Calculation
 Nonsense 43     
 Missense 123     
 Deletion 61     
 Insertion 28     
 Indels 9     
 Splice 50     
 Regulatory 1     
 Total 315     
Figure 5.

 A schematic presentation of exons 70–73 of COL7A1. The 39 amino acid hinge region (pink) of the triple helical domain (THD) is encoded by amino acid from 1940 to 1978. Exon 73 is encoded by amino acid residues 1994–2060 (Green) and RGD tripeptide sequence in exon 73 is outlined by bright blue rectangle. The mutations are indicated on the top of sequences (bold are glycine substitutions mutations), 20 dominant and 15 recessive mutations (indicated in red and blue, respectively) are clustered in exon 73 just after the 39 amino acid interruption region. G1812R and G1519D are located within a long uninterrupted stretch of collagen VII triple helix. G2775S and G2749R are located close to C terminal ends of the triple helix, while G1347R is closed to N terminal ends of the triple helix.

In RDEB, mutations include nonsense, splice site, deletions or insertions, ‘silent’ glycine substitutions within the triple helix and non-glycine missense mutations within the triple helix or non-collagenous NC-2 domain (20; Figs 3 and 4). RDEB-HS is generally because of nonsense, frame-shift or splice-site mutations on both alleles leading to PTCs which result in nonsense-mediated decay or truncated polypeptides (2). The truncation of the polypeptides leads to accelerated nonsense-mediated mRNA decay such that the level of the transcripts is significantly reduced, resulting in very few collagen VII monomers being made which are unable to assemble into functional AFs and hence the skin is fragile (2,20,21; Fig. 2). PTC mutations do not cause a clinical phenotype in the heterozygous state, but when in the homozygous state or combined with another PTC mutation they can result in severe RDEB (RDEB-HS). In few cases, RDEB-HS may occur because of combinations of two missense mutations or compound heterozygosity of a missense and a PTC mutation (22).

Recessive dystrophic epidermolysis bullosa-HS is generally because of nonsense, frame-shift or splice-site (23–26) mutations on both alleles leading to PTCs, which result in nonsense-mediated decay or truncated polypeptides (2). The PTCs enhance mRNA decapping which leads to accelerated nonsense-mediated mRNA decay such that the level of the transcripts is significantly reduced, resulting in very few collagen VII monomers being made which are unable to assemble into functional AFs and hence the skin is fragile (2,20,21; Fig. 2). PTC mutations do not cause a clinical phenotype in the heterozygous state, but in the homozygous state or combined with another PTC mutation they can result in severe RDEB (RDEB-HS). The milder phenotypes of RDEB-nHS are frequently caused by PTCs, small deletions, substitutions of glycine residues in the collagenous domain, splice-site mutations within NC-2 (23–26), delayed termination codons (27), in-frame exon skipping (20,27) or missense substitution mutations involving an amino acid other than glycine (20,28,29), the majority involving arginine residues which result either in the loss of an ionic charge or in the introduction of a bulky chain at external positions of the triple helix (28; Table 1; Fig. 3). Full-length type VII collagen polypeptides can be synthesized (30). These mutations affect a critical amino acid and alter the conformation of the protein, which may still be able to assemble into a small number of AFs, but is likely to be unstable when they laterally aggregate (Fig. 2). About 17 cysteine residues are precisely conserved between the human and the mouse and hamster COL7A1, one cysteine within the triple helical domain (amino acid 2634) has been proposed to form intermolecular disulphide bonds by pairing with the first or second cysteine residue (amino acids 2802 and 2804) within the NC-2 domain of another type VII collagen molecule. About 8 cysteine residues within the NC-2 domain have been suggested to participate in the formation of disulphide bonds which stabilize the anti-parallel association of the two type VII collagen molecules during the extracellular assembly of AFs (12,31). This is consistent with positive but attenuated immunohistochemical staining and reduced numbers of AFs on Electron microscopy (EM) in dominant and recessive DEB in which these cysteine residues are affected (1,30). Missense mutations can sometimes influence the splicing process (32,33). TBDN may also be caused by COL7A1 mutations (34,35) and it is not known why these result in transient disease. Often, on immunofluorescence microscopy, there is stippling of collagen VII in the basal cells, suggesting retention of collagen VII (36,37).

One study showed that patients with RDEB only develop squamous cell carcinoma in the presence of the NC1 domain of type VII collagen. The NC1/laminin 332 interaction induce integrin-mediated signals which is essential for the survival of transformed keratinocytes in the anchorage-independent state and NC2 domains of procollagen VII are not required for α6β4 integrin-mediated survival signalling (38).

Mutation analysis in DDEB

Dominant dystrophic epidermolysis bullosa usually involves glycine substitutions within the triple helix of COL7A1 although other missense mutations, deletions or splice-site mutations may underlie some cases (20,39–44; Fig. 2). These mutations affect critical amino acids in the structure of the triple helix, and hence disrupting them may affect the overall stability of the AFs. Assuming equal expression of wild-type and mutant alleles, seven-eighths of the trimeric molecules contain at least one mutant pro α1 chain and only one-eighth consists solely of normal polypeptides (19).

More than 100 missense mutations that result in a Gly-Xaa substitution have been described in the collagenous domain of COL7A1; half of these mutations have a ‘dominant-negative’ effect causing DDEB (51/102), spanning from amino acids 1522–2791 (Fig. 6). A common region for mutations affects the amino acid residues 2003–2079 (exon 73–75) as part of a 35-triplet stretch of Gly-X-Y which is flanked by non-collagenous sequences of 39 and 6 amino acid residues, just downstream from the 39 amino acide hinge region (Figs 5 and 6) Thirty- five triplet segments are evolutionarily highly conserved in the human, mouse and hamster (17,31). Glycine substitution in this segment may lead to a greater destabilization than in long uninterrupted collagenous segments or close to the N- or C-terminal ends (19).

Figure 6.

 Glycine substitutions (GS) in dystrophic epidermolysis bullosa (DEB). These are all in the NC-2 domain of COL7A1 and the ones above represent dominant dystrophic epidermolysis bullosa (DDEB), whereas the ones below represent recessive dystrophic epidermolysis bullosa (RDEB).

Indeed, toenail dystrophy may be the only clinical manifestation of DDEB in some families. Recent reports have shown some glycine substitutions could lead only to nail dystrophy but no skin blistering when combined with a normal allele, such as G1815R, G1595R and G2251E and G2287R (45,46). As explained below, many glycine to arginine substitutions in COL7A1 do not result in a phenotypic change according to the literature, but not all may have had their toenails well scrutinized. G2689R mutations led to an interesting phenotype, mucosal involvement, nail dystrophy and no skin blisters (25; Table 1).

In the variant of DEB known as TBDN in which there is stippling of collagen VII on Immunofluorescence (IF) antigen mapping without linear staining with antibodies to collagen VII (47), only four distinct mutations have been found in COL7A1, all within the collagenous domain. The first mutation was the splice mutation 4120-1G>C (IVS35-1G>C, at intron 35) (42). The second and third mutations are both glycine substitution mutations, a novel recessive G1519D (in exon 44) and a dominant G2251E (in exon 86) mutation. The G1519D substitution is clinically silent when combined with a normal allele and G2251E merely caused nail dystrophy in heterozygotes, but no skin blistering (9). The fourth mutation, G1522E lies in exon 45 and was identified before in a sporadic case of EB pruriginosa (48). G1522E and G2251E are the first amino acids encoded by exon 45 and exon 86, respectively; G1519D is close to G1522E and the last third amino acid encoded by exon 44 plus IVS35G>C, all of four mutations are close to splice-sites or in splice-sites. However, Hammami-Hauasli did analysis with G1522E using the delilaTM software package and did not predict any splice-site alterations and IVS35G>C is predicted to create a ‘leaky’ site, i.e. normal splicing and in-frame skipping of exon 36 (9). The exact reason why TBDN phenotype results from these mutations is not known, but it is proposed that they alter sites that are important for release of the polypeptide from the rough endoplasmic reticulum (48).

‘Silent’ glycine substitutions in recessive inheritance pattern

Not all glycine substitutions mutations within COL7A1 gene are dominant. Glycine substitutions may be inherited recessively; these substitutions usually exhibit no clinical phenotype in the heterozygous state but when combined with another COL7A1 mutation (nonsense, splice-site, insertion or deletion mutation), the clinical consequences are either mild, moderate or severe RDEB (9). About 32 of such clinically ‘silent’ glycine mutations in COL7A1 are summarized in Table 1. It is not clear why some glycine substitutions are dominant whilst others have no clinical phenotype in the heterozygous state. It may be the position of these mutations within the collagenous domain of COL7A1 and the resulting degree of abnormal folding which influences the consequences at the phenotypic level. Most glycine substitutions that do not result in a clinical phenotype are located close to either the N or C terminal ends of the triple helix or in the middle of the long uninterrupted segments of Gly-X-Y repeats (19). For example, G1812R in exon 63 has no clinical phenotype when combined with normal COL7A1 alleles; however, when combined with a PTC mutation on the other allele (3857delA), it results in RDEB-nHS (49). G1519D did not interfere with folding and secretion of procollagen VII demonstrated by IF staining and immunoblot analysis; this mutation causes pathological consequences only in combination with another COL7A1 gene defect (19). Gly-1519 and 1812 are both located within a long uninterrupted stretch of collagen VII triple helix (Fig. 5). G2775S (5) and G2749R (22) are located close to C terminal ends of the triple helix, while G1347R (50) is close to the N terminal ends of the triple helix. More than half of the silent glycine substitutions are glycine to arginine substitutions acting in a recessive manner which have recently been reported in RDEB-nHS. Modelling studies indicate that glycine to arginine substitutions probably only lead to minor localized disruption of the triple helix structure (51).

De novo mutations

Patients with relatively mild DEB and no family history are frequently diagnosed as de novo or sporadic cases of dominant DEB, although a mild case of recessive DEB cannot be excluded on the basis of clinical and ultrastructural examination (52,53).

The true de novo cases usually develop a dominant mutation in COL7A1; their offspring has a 50% risk of inheriting the mutation. There may also be de novo cases of RDEB (20,40). However, if one parent is a germline mosaic, the risk then depends on the percentage of mutated germline cells. If neither parent is a germline mosaic, then the risk of having another child with de novo DDEB is the same as in the general population (54). In terms of genetic counselling, however, it is practically impossible to exclude that a parent of a child who appears to have a de novo mutation is not a germline mosaic, and therefore it is important to stress to such parents that there is a small but negligible (<1%) chance that they may have a second child with DEB.

Indeed, exon 73 has previously been shown to harbour a large number of glycine substitutions, including de novo mutations. De novo glycine substitutions resulting in DDEB are rare but the following mutations have been reported: G1775D, G2067R (40), G2012S (8), G2012D (55), G2028R (56), G2040V (54), G2043R, G2043W (57), G2076D (58), G2079E (52) and G2348R (59). The de novo mutations usually result from glycine substitutions and are seen in DDEB, but Posteraro et al. detected the 8117delC frameshift mutation in a patient affected with the less severe RDEB-nHS variant as a de novo mutation (40).

Recurrent mutations

Although most COL7A1 mutations have been specific to the individual families, with no ‘hotspot’ mutations, haplotype analysis showed that some recurrent mutations have been found in similar allelic backgrounds in different patients (60). In Italy, six recurrent mutations were found including 497insA, 4738G-A, 7344G-A, 425A-C, G1664A and 8441-14del21 (8). The mutation 2470insC has only been found in Mexico (61). R578X, 7786delG and R2814X mutations are specifically limited to British patients and 5818delC, 6573+1G-C, E2857X are frequent in Japanese (62). A high recurrence of 425A-G was found in central European patients (63).

Position of mutations and the phenotype

The position of a mutation along the type VII collagen molecule together with the genetic background and external factors of the affected individual may influence the phenotype of DEB. R2063G and R2063W mutations which occur at the same arginine 2063 residue were associated with inverse RDEB and RDEB-HS, respectively, suggesting that the nature of the amino acid change for a given position within the COL7A1 may influence the phenotype (28). The position of the PTC within COL7A1 in Japanese RDEB patients correlates with the clinical severity, the combination of PTC located upstream (5818delC and 6573 + 1G-C) within the collagenous domain had complete pseudo-syndactyly whereas when the PTCs were located downstream (6573 + 1G-C and E2857X) and the patients had partial pseudo-syndactyly (64). Similar trends were found in our series of Australasian patients (65). However, not all PTC mutations occurring downstream in the gene result in less severe phenotypes. A recurrent nonsense mutation, R2814X was found in British patients and resulted in generalized severe blistering and scarring. R2814X occuring in exon 114/115 close to the 3′ end of the gene and might be expected to only result in a mildly truncated protein. However, nonsense mutations can result in low steady-state mRNA levels and also have an effect on RNA splicing as this occurs in the last base of exon 114 (66).

It has been shown that missense mutations in the C-terminal region of the triple helix affect the early stages of AF assembly by altering the formation of procollagen VII anti-parallel dimers (67). The position of glycine substitutions is important in determining whether a dominant, recessive or a silent mutation occurs. Most of the mutations clustered around exon 73 within the triple helix of COL7A1 are dominant. Mutations in other regions of the triple helix may cause less dominant negative interference and only result in clinical disease when combined with additional inherited or acquired predisposing factors. Similarly, the position of glycine substitution mutations may affect the susceptibility of type VII collagen to secondary proteolysis with the potential for differences in breakdown products triggering an inflammatory cascade in the patients’ skin (7).

Furthermore, recent studies in siblings with RDEB harbouring the same COL7A1 mutations but very different severities of pseudosyndactyly have shown that those with more activity in matrix metalloproteinase (MMP) genes (by polymorphism analysis) have more severe and complete pseudosyndactyly than those with less MMP activity (68).

Gene therapy and future overview of RDEB

For gene therapy approaches to RDEB, the use of retroviral vectors (RVs), lentiviral vectors (LVs) and non-viral vectors based on the ΦC31 bacteriophage integrase system have been developed (69–71). RV’s efficiently transfer the 9-kb COL7A1 cDNA into keratinocytes of RDEB and achieve correction of the genetic defect (70). Chen et al. (69) delivered full-length type VII collagen using LVs transducted into RDEB keratinocytes and fibroblasts resulting in persistent synthesis and secretion of type VII collagen. Comparable results were obtained when ΦC31 integrase-based gene transfer the COL7A1 cDNA into cells resulting in correction including type VII collagen protein expression, AF formation and dermal-epidermal cohesion (71).

Alternative approaches, in particular, cell therapy involving the injection of allogeneic normal fibroblasts to replace collagen VII are underway as pilot studies and seems very promising (72). Drugs that allow ‘read-through’ of PTC/null mutations may also be beneficial for patients with those types of mutations. Drugs that reduce metalloproteinase activity specifically may assist some patients with overactive MMP isoforms as these patients have been shown to have a worse phenotype than those siblings with the same COL7A1 mutations but normal MMP1 activity (68).

In summary, screening the COL7A1 gene is useful in understanding the different clinical variants of DEB and essential to prenatal diagnosis. In this study, a review of known mutations of COL7A1 and the genotypes and phenotypes of the patients with DEB were undertaken. DDEB usually involves glycine substitutions within the triple helix of COL7A1 although other missense mutations, deletions or splice-site mutations may underlie some cases. The RDEB mutations include nonsense mutations, splice site mutations, deletions or insertions, ‘silent’ glycine substitutions within the triple helix and non-glycine missense mutations within the triple helix domain (Fig. 5). The nature of mutations in COL7A1 and their positions correlate reasonably logical with the severity of the resulting phenotypes of DEB.

Acknowledgements

We thank Jinan Central Hospital, Shandong Province, China and an American-Chinese Grant for supporting Dr Dang’s postdoctoral work with DFM; DebRA Australia and NSW for grants to DFM for partial salary support for NND and Premier Dermatology, Sydney for ongoing research support.

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