Digenic Inheritance in Epidermolysis Bullosa Simplex

Abstract

complementary DNA epidermolysis bullosa EB simplex TO THE EDITOR Epidermolysis bullosa (EB) encompasses a heterogeneous group of hereditary disorders characterized by blistering of the skin upon exposure to mechanical stress. EB simplex (EBS) is the most common form of EB and the first keratin disorder whose genetic basis was elucidated in humans (Uitto et al., 2007Uitto J. Richard G. McGrath J.A. Diseases of epidermal keratins and their linker proteins.Exp Cell Res. 2007; 313: 1995-2009Crossref PubMed Scopus (80) Google Scholar). EBS has been associated with mutations in at least eight distinct genes (Intong and Murrell, 2012Intong L.R. Murrell D.F. Inherited epidermolysis bullosa: new diagnostic criteria and classification.Clin Dermatol. 2012; 30: 70-77Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Although the very rare forms of superficial EBS have been associated with mutations in PKP1 and DSP encoding plakophilin 1 and desmoplakin (Jonkman et al., 2005Jonkman M.F. Pasmooij A.M. Pasmans S.G. et al.Loss of desmoplakin tail causes lethal acantholytic epidermolysis bullosa.Am J Hum Genet. 2005; 77: 653-660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar; McGrath and Mellerio, 2010McGrath J.A. Mellerio J.E. Ectodermal dysplasia-skin fragility syndrome.Dermatol Clin. 2010; 28: 125-129Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the more common type of EBS is usually due to mutations in KRT5 and KRT14 encoding basal cell keratins (Sprecher, 2010Sprecher E. Epidermolysis bullosa simplex.Dermatol Clin. 2010; 28: 23-32Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), although it is occasionally caused by mutations in other genes including PLEC1, ITGA6, ITGB4, and DST (Jonkman et al., 2002Jonkman M.F. Pas H.H. Nijenhuis M. et al.Deletion of a cytoplasmic domain of integrin beta4 causes epidermolysis bullosa simplex.J Invest Dermatol. 2002; 119: 1275-1281Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar; Pfendner et al., 2005aPfendner E. Rouan F. Uitto J. Progress in epidermolysis bullosa: the phenotypic spectrum of plectin mutations.Exp Dermatol. 2005; 14: 241-249Crossref PubMed Scopus (79) Google Scholar; Groves et al., 2010Groves R.W. Liu L. Dopping-Hepenstal P.J. et al.A homozygous nonsense mutation within the dystonin gene coding for the coiled-coil domain of the epithelial isoform of BPAG1 underlies a new subtype of autosomal recessive epidermolysis bullosa simplex.J Invest Dermatol. 2010; 130: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). K5 and K14 assemble into 10-nm keratin intermediate filaments that underlie the formation of the cytoskeleton of epithelial cells (Candi et al., 2005Candi E. Schmidt R. Melino G. The cornified envelope: a model of cell death in the skin.Nat Rev Mol Cell Biol. 2005; 6: 328-340Crossref PubMed Scopus (1267) Google Scholar). Most KRT5 and KRT14 mutations have been shown to disrupt the central α-helical segment of these molecules, thereby leading to reduced resistance to mechanical stress and skin blistering, although other mechanisms may underlie rare phenotypes caused by mutations affecting less well-conserved areas of the keratin molecules (Uttam et al., 1996Uttam J. Hutton E. Coulombe P.A. et al.The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.Proc Natl Acad Sci USA. 1996; 93: 9079-9084Crossref PubMed Scopus (153) Google Scholar; Gu et al., 2003Gu L.H. Kim S.C. Ichiki Y. et al.A usual frameshift and delayed termination codon mutation in keratin 5 causes a novel type of epidermolysis bullosa simplex with migratory circinate erythema.J Invest Dermatol. 2003; 121: 482-485Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar; Sprecher et al., 2003Sprecher E. Yosipovitch G. Bergman R. et al.Epidermolytic hyperkeratosis and epidermolysis bullosa simplex caused by frameshift mutations altering the v2 tail domains of keratin 1 and keratin 5.J Invest Dermatol. 2003; 120: 623-626Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). EBS caused by mutations in KRT5 or KRT14 is usually inherited as an autosomal dominant trait. Occasionally, especially in populations characterized by an elevated coefficient of inbreeding, autosomal recessive inheritance is common (Abu Sa’d et al., 2006Abu Sa’d J. Indelman M. Pfendner E. et al.Molecular epidemiology of hereditary epidermolysis bullosa in a Middle Eastern population.J Invest Dermatol. 2006; 126: 777-781Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the present report, we describe a hitherto reported form of recessive inheritance in EBS. A 2-year-old boy of Jewish Ashkenazi origin was referred to our outpatient clinic because of widespread congenital skin blistering (EBS—other generalized). His parents noticed marked exacerbation during the summer months. His mother as well as maternal grandfather reported a similar but significantly milder phenotype. These two individuals demonstrated blisters, mostly during the summer period and exclusively located on the soles. The grandparents of the proband confirmed that his mother was only mildly affected from early childhood. The father of the proband was totally asymptomatic. On examination, tensed bullae were observed over the dorsal surface of the feet, the legs (Figure 1a), and along the trunk. No milia, mucosal involvement, or nail dystrophy were observed. Examination of the mother and maternal grandfather of the proband failed on several occasions to reveal active signs of disease. A skin biopsy showed subepidermal blister formation, whereas immunohistochemical staining demonstrated keratin staining at the base and roof of the blister (not shown). The family provided written and informed consent according to a protocol approved by the National Committee for Genetic Studies of the Israeli Ministry of Health in accordance with the Declaration of Helsinki guidelines. DNA was extracted from peripheral blood leukocytes using the 5 Prime ArchivePure DNA Blood Kit (5 Prime, Gaithersburg, MD). In an attempt to explain the striking phenotypic discrepancy between the proband and other affected members of his family, we screened all family members for mutations in KRT5 and KRT14. Genomic DNA was PCR-amplified using primer pairs spanning the entire coding sequence, as well as intron–exon boundaries of KRT5 and KRT14, as previously described (Whittock et al., 2000Whittock N.V. Eady R.A. McGrath J.A. Genomic organization and amplification of the human epidermal type II keratin genes K1 and K5.Biochem Biophys Res Commun. 2000; 274: 149-152Crossref PubMed Scopus (19) Google Scholar; Ciubotaru et al., 2003Ciubotaru D. Bergman R. Baty D. et al.Epidermolysis bullosa simplex in Israel: clinical and genetic features.Arch Dermatol. 2003; 139: 498-505Crossref PubMed Scopus (58) Google Scholar). The proband and his asymptomatic father were found to carry a heterozygous G → A transition at position 1163 of the KRT14 complementary DNA (cDNA). This mutation results in the substitution of a histidine for an arginine at amino acid position 388 (p.R388H, Figure 1b). This mutation has previously been associated with recessive inheritance of EBS in a compound heterozygous state (Ciubotaru et al., 2003Ciubotaru D. Bergman R. Baty D. et al.Epidermolysis bullosa simplex in Israel: clinical and genetic features.Arch Dermatol. 2003; 139: 498-505Crossref PubMed Scopus (58) Google Scholar). In addition, the proband was found to carry a heterozygous T → C transition at position 548 of the KRT5 cDNA. This mutation results in the substitution of threonine for isoleucine, p.I183T (Figure 1b), affecting a highly conserved amino-acid residue (Figure 1d, Conseq=9, range 1–9; http://conseq.tau.ac.il/) of predicted functional importance (PolyPhen-2=1, range 1–0, http://genetics.bwh.harvard.edu/pph2/index.shtml; SIFT=0, range 1–0, http://sift.jcvi.org/). The mildly affected mother and maternal grandfather of the proband also carried the same mutation in a heterozygous state (Figure 1b). Using PCR–restriction fragment length polymorphism assays, we confirmed segregation of the mutations with the disease phenotype in the family (Figure 1c) and excluded p.I183T from a panel of 200 population-matched control individuals (400 alleles). The two missense mutations identified in this study, p.I183T and p.R388H, are both located in highly conserved regions within the 1A domain of K5 and 2B domain of K14, respectively. As the two molecules form under physiological conditions a heterodimer (Coulombe et al., 2009Coulombe P.A. Kerns M.L. Fuchs E. Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility.J Clin Invest. 2009; 119: 1784-1793Crossref PubMed Scopus (153) Google Scholar), the presence of mutations in both K5 and K14 is likely to aggravate the consequences of each separate mutation. p.R388H has been reported previously in three cases of EBS. In the first study, it was shown that p.R388H causes EBS-Weber Cockayne in a compound heterozygous state with another nonsense mutation in KRT14 whereas it was phenotypically silent in the father of the affected children (Ciubotaru et al., 2003Ciubotaru D. Bergman R. Baty D. et al.Epidermolysis bullosa simplex in Israel: clinical and genetic features.Arch Dermatol. 2003; 139: 498-505Crossref PubMed Scopus (58) Google Scholar). Abu Sa’d et al., 2006Abu Sa’d J. Indelman M. Pfendner E. et al.Molecular epidemiology of hereditary epidermolysis bullosa in a Middle Eastern population.J Invest Dermatol. 2006; 126: 777-781Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar identified two patients with EBS-Weber Cockayne carrying p.R388H in a heterozygous state with no other mutations in KRT5 or KRT14, suggesting partial penetrance and/or the effect of modifying factors (Ciubotaru et al., 2003Ciubotaru D. Bergman R. Baty D. et al.Epidermolysis bullosa simplex in Israel: clinical and genetic features.Arch Dermatol. 2003; 139: 498-505Crossref PubMed Scopus (58) Google Scholar; Abu Sa’d et al., 2006Abu Sa’d J. Indelman M. Pfendner E. et al.Molecular epidemiology of hereditary epidermolysis bullosa in a Middle Eastern population.J Invest Dermatol. 2006; 126: 777-781Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Several heterozygous mutations have previously been shown to affect codon 183 of KRT5. The mutations p.I183F (Pfendner et al., 2005bPfendner E.G. Sadowski S.G. Uitto J. Epidermolysis bullosa simplex: recurrent and de novo mutations in the KRT5 and KRT14 genes, phenotype/genotype correlations, and implications for genetic counseling and prenatal diagnosis.J Invest Dermatol. 2005; 125: 239-243Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) or p.I183M (Chamcheu et al., 2010Chamcheu J.C. Virtanen M. Navsaria H. et al.Epidermolysis bullosa simplex due to KRT5 mutations: mutation-related differences in cellular fragility and the protective effects of trimethylamine N-oxide in cultured primary keratinocytes.Br J Dermatol. 2010; 162: 980-989Crossref PubMed Scopus (19) Google Scholar) results in a severe form of EBS, whereas p.I183V (Glasz-Bona et al., 2009Glasz-Bona A. Medvecz M. Sajo R. et al.Easy method for keratin 14 gene amplification to exclude pseudogene sequences: new keratin 5 and 14 mutations in epidermolysis bullosa simplex.J Invest Dermatol. 2009; 129: 229-231Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) or p.I183T (this study) results in a milder phenotype. The severity of the phenotype is probably influenced by the size of the substituted amino acid, as phenylalanine and methionine have a larger side chain compared with valine and threonine. Clearly, digenic inheritance together with other modifying traits (Titeux et al., 2008Titeux M. Pendaries V. Tonasso L. et al.A frequent functional SNP in the MMP1 promoter is associated with higher disease severity in recessive dystrophic epidermolysis bullosa.Hum Mutat. 2008; 29: 267-276Crossref PubMed Scopus (82) Google Scholar) may explain in part the highly variable phenotype typical of EB in general, and of EBS more specifically (Sprecher, 2010Sprecher E. Epidermolysis bullosa simplex.Dermatol Clin. 2010; 28: 23-32Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The first example of digenic inheritance of a human phenotype was reported in a case of retinitis pigmentosa (Kajiwara et al., 1994Kajiwara K. Berson E.L. Dryja T.P. Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci.Science. 1994; 264: 1604-1608Crossref PubMed Scopus (608) Google Scholar). Since then, similar cases have been described in many monogenic disorders including nonsyndromic deafness, Bardet–Biedl syndrome, and Waardenburg syndrome type II (reviewed in Badano and Katsanis, 2002Badano J.L. Katsanis N. Beyond Mendel: an evolving view of human genetic disease transmission.Nat Rev Genet. 2002; 3: 779-789Crossref PubMed Scopus (288) Google Scholar). Digenic inheritance has been reported in a severe case of junctional EB, in which two mutations in the COL17A gene and one mutation in the LAMB3 gene were detected and found to be associated with abnormal expression of two functionally related proteins, collagen XVII and laminin 332 (Floeth and Bruckner-Tuderman, 1999Floeth M. Bruckner-Tuderman L. Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes.Am J Hum Genet. 1999; 65: 1530-1537Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). It is of interest that in this case heterozygous carriers of one COL17A1 mutation and of one LAMB3 mutation were asymptomatic (Floeth and Bruckner-Tuderman, 1999Floeth M. Bruckner-Tuderman L. Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes.Am J Hum Genet. 1999; 65: 1530-1537Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A patient with EBS and his affected mother were also found to carry mutations in two distinct EB-associated genes, KRT5 (p.E168D) and KRT14 (p.A413T) (Murrell et al., 2011Murrell D.F. Trisnowati N. Miyakis S. et al.The yin and the yang of keratin amino acid substitutions and epidermolysis bullosa simplex.J Invest Dermatol. 2011; 131: 1787-1790Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The fact that the p.E168D in KRT5 mutation reported in this study affects a highly conserved residue, involved in another EBS-causing mutation (p.E168K; Muller et al., 2006Muller F.B. Kuster W. Wodecki K. et al.Novel and recurrent mutations in keratin KRT5 and KRT14 genes in epidermolysis bullosa simplex: implications for disease phenotype and keratin filament assembly.Hum Mutat. 2006; 27: 719-720Crossref PubMed Scopus (48) Google Scholar), supports a potential pathogenic role for this mutation in causing a disease phenotype. In contrast, p.A413T is most likely a polymorphism of no consequence, as this sequence alteration was found in a substantial number of healthy controls in a heterozygous state (5% of a control population); a healthy individual was found to carry p.A413T in a homozygous state; bioinformatic analysis predicts lack of effect of the p.A413T change on K14 structure; and in contrast with p. A413P, p.A413T did not have any deleterious effect on keratin filament formation as assayed in vitro (Natsuga et al., 2011Natsuga K. Nishie W. Smith B.J. et al.Consequences of two different amino-acid substitutions at the same codon in KRT14 indicate definitive roles of structural distortion in epidermolysis bullosa simplex pathogenesis.J Invest Dermatol. 2011; 131: 1869-1876Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In addition, looking more carefully at the data, Murrell et al., 2011Murrell D.F. Trisnowati N. Miyakis S. et al.The yin and the yang of keratin amino acid substitutions and epidermolysis bullosa simplex.J Invest Dermatol. 2011; 131: 1787-1790Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar even suggested that p.A413T may have a protective effect, which in turn may explain the fact that this polymorphism has been maintained through evolution. Thus, these data suggest that digenic inheritance was not directly involved in causing/modifying the clinical phenotype in the above case. In conclusion, we provide evidence implicating digenic inheritance in determining a clinical phenotype in EBS, suggesting that full sequencing of all EBS-associated genes may improve the quality of genetic counseling in this clinically heterogeneous disease. We acknowledge the participation of all family members in this study.