Title: KRT5 and KRT14 Mutations in Epidermolysis Bullosa Simplex with Phenotypic Heterogeneity, and Evidence of Semidominant Inheritance in a Multiplex Family
Abstract: Epidermolysis bullosa simplex (EBS), which shows blister formation within the basal layer of the epidermis, is primarily caused by mutations in KRT5 and KRT14 (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, Fine et al., 2014Fine J.D. Bruckner-Tuderman L. Eady R.A. Bauer E.A. Bauer J.W. Has C. et al.Inherited epidermolysis bullosa: Updated recommendations on diagnosis and classification.J Am Acad Dermatol. 2014; 70: 1103-1126Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Most cases result from dominant negative missense mutations in either one of these genes, with an autosomal dominant inheritance. Most mutations are in KRT14 compared with KRT5, reflecting, at least in part, a high frequency of hotspot mutations, such as p.Arg125His and p.Arg125Cys, in KRT14 (Coulombe and Lee, 2012Coulombe P.A. Lee C.H. Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath.J Invest Dermatol. 2012; 132: 763-775Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Only 16 keratin mutations, have been shown to cause the autosomal recessive form of EBS, all of them in KRT14 (Garcia et al., 2011Garcia M. Santiago J.L. Terron A. Hernandez-Martin A. Vicente A. Fortuny C. et al.Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex.Br J Dermatol. 2011; 165: 683-692Crossref PubMed Scopus (21) Google Scholar). In addition to mutations in KRT5 and KRT14, up to approximately 10% of autosomal recessive cases of EBS have been shown to result from mutations in TGM5 encoding transglutaminase 5, whereas relatively few cases, mostly autosomal recessive, have been shown to result from mutations in other genes, including JUP, DSP, PKP1, EXPH5, PLEC, and DST-e (Uitto et al., 2016Uitto J. Bruckner-Tuderman L. Christiano A.M. McGrath J.A. Has C. South A.P. et al.Progress towards treatment and cure of epidermolysis bullosa: Summary of the DEBRA International Research Symposium EB2015.J Invest Dermatol. 2016; 136: 352-358Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Uitto et al., 2016Uitto J. Vahidnezhad H. Youssefian L. Genotypic heterogeneity and the mode of inheritance in epidermolysis bullosa.JAMA Dermatol. 2016; 152: 517-520Crossref PubMed Scopus (8) Google Scholar). As part of an effort to characterize a large cohort of epidermolysis bullosa patients in Iran, a country of close to 80 million inhabitants in which approximately 38% of marriages are consanguineous, patients with clinical presentation and immunoepitope mapping suggestive of EBS (see Supplementary Figure S1 online) were studied by whole-genome homozygosity mapping (see Supplementary Figure S2a and b online), followed by Sanger sequencing (Barzegar et al., 2015Barzegar M. Asadi-Kani Z. Mozafari N. Vahidnezhad H. Kariminejad A. Toossi P. Using immunofluorescence (antigen) mapping in the diagnosis and classification of epidermolysis bullosa: a first report from Iran.Int J Dermatol. 2015; 54: e416-e423Crossref PubMed Scopus (10) Google Scholar) (Table 1). This study was approved by the institutional review board of the Pasteur Institute of Iran, and the patients gave written informed consent to participate in research and to have their images and medical histories published. Eleven families out of 12 were found to harbor mutations in either KRT5 or KRT14 (Figure 1). In seven families, a heterozygous missense mutation, three of them previously unreported, was found to affect a fully conserved amino acid residue (Figure 1a–c and h–k). In four families, there were multiple affected individuals, and one of the parents and/or other affected family members were shown to harbor the same mutation as found in the proband, consistent with autosomal dominant inheritance. In three cases with a heterozygous missense mutation, the parents were clinically normal and had the wild-type KRT14 sequence, suggesting de novo origin of the mutation (families 4, 7, and 11; Table 1). In addition, one of these patients with severe generalized blistering with p.Arg125Cys mutation in KRT14 (family 7) was found to be compound heterozygous with a p.Gly183Glu substitution in KRT5; the latter mutation has been previously suggested to serve as a modifier of the severity of EBS phenotype (Kowalewski et al., 2007Kowalewski C. Hamada T. Wozniak K. Kawano Y. Szczecinska W. Yasumoto S. et al.A novel autosomal partially dominant mutation designated G476D in the keratin 5 gene causing epidermolysis bullosa simplex Weber-Cockayne type: a family study with a genetic twist.Int J Mol Med. 2007; 20: 75-78PubMed Google Scholar). In one family (family 10), a previously unreported 18-base pair de novo in-frame deletion in exon 6 of KRT14 was identified in a patient with generalized severe blistering and extensive palmoplantar keratoderma (see Supplementary Figure S1c). This mutation results in deletion of six amino acids from the keratin 14 polypeptide in the 2B domain critical for keratin filament assembly. Because the association of palmoplantar keratoderma with EBS is rare, this finding, together with previous observations (Livingston et al., 2001Livingston R.J. Sybert V.P. Smith L.T. Dale B.A. Presland R.B. Stephens K. Expression of a truncated keratin 5 may contribute to severe palmar—plantar hyperkeratosis in epidermolysis bullosa simplex patients.J Invest Dermatol. 2001; 116: 970-974Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, Titeux et al., 2006Titeux M. Mazereeuw-Hautier J. Hadj-Rabia S. Prost C. Tonasso L. Fraitag S. et al.Three severe cases of EBS Dowling-Meara caused by missense and frameshift mutations in the keratin 14 gene.J Invest Dermatol. 2006; 126: 773-776Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), suggests that mutations altering the sequence of this domain in keratin 14 can result in palmoplantar keratoderma. Thus, most patients studied here harbored heterozygous mutations, apparently resulting in dominant negative interference of the association of keratin 5 and keratin 14 to form heterodimers during intracellular assembly of intermediate filaments in basal keratinocytes.Table 1Mutation analysis of KRT5 and KRT14 in Iranian families affected by epidermolysis bullosa simplexFamilyGeneExonMutation at DNA LevelInheritance1Epidermolysis bullosa simplex.Homozygous/HeterozygousMutation at Protein LevelPredicted ConsequencePhenotype1Epidermolysis bullosa simplex.PolyPhen2 score2http://genetics.bwh.harvard.edu/pph2/. The values range from 0–1.000, the latter being most damaging.Score of PROVEAN (cutoff = –2.5)3http://provean.jcvi.org/index.php. The values below the cutoff score –2.5 indicate deleterious mutations.Mutation First Reported1KRT51c.530A>GADHeterozygousp.Asn177SerMissenseEBS, localized1.000 (Probably damaging)–4.373 (Deleterious)Liovic et al., 2004Liovic M. Bowden P.E. Marks R. Komel R. A mutation (N177S) in the structurally conserved helix initiation peptide motif of keratin 5 causes a mild EBS phenotype.Exp Dermatol. 2004; 13: 332-334Crossref PubMed Scopus (13) Google Scholar2KRT52c.557T>CADHeterozygousp.Leu189ProMissenseEBS, localized0.939 (Possibly damaging)–6.095 (Deleterious)This study3KRT57c.1283C>AADHeterozygousp.Ala428AspMissenseEBS, localized1.000 (Probably damaging)–5.670 (Deleterious)This study4KRT57c.1424A>GADHeterozygousp.Glu475GlyMissenseEBS, generalized severe0.969 (Probably damaging)–6.367 (Deleterious)Lane et al., 1992Lane E.B. Rugg E.L. Navsaria H. Leigh I.M. Heagerty A.H. Ishida-Yamamoto A. et al.A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.Nature. 1992; 356: 244-246Crossref PubMed Scopus (341) Google Scholar5KRT141c.92delTARHomozygousp.Ile31ThrfsX39FrameshiftEBS, generalized intermediateN/AN/ABatta et al., 2000Batta K. Rugg E.L. Wilson N.J. West N. Goodyear H. Lane E.B. et al.A keratin 14 'knockout' mutation in recessive epidermolysis bullosa simplex resulting in less severe disease.Br J Dermatol. 2000; 143: 621-627Crossref PubMed Google Scholar6KRT141c.200delGARHomozygousp.G67AfsX51FrameshiftEBS, generalized severeN/AN/AThis study7KRT141c.373C>TADHeterozygousp.Arg125CysMissenseEBS, generalized severe0.946 (Possibly damaging)–6.729 (Deleterious)Coulombe et al., 1991Coulombe P.A. Hutton M.E. Letai A. Hebert A. Paller A.S. Fuchs E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (533) Google Scholar8KRT144c.915G>AARHomozygousp.Trp305TerNonsenseEBS, generalized severeN/AN/ACorden et al., 1998Corden L.D. Mellerio J.E. Gratian M.J. Eady R.A. Harper J.I. Lacour M. et al.Homozygous nonsense mutation in helix 2 of K14 causes severe recessive epidermolysis bullosa simplex.Hum Mutat. 1998; 11: 279-285Crossref PubMed Scopus (41) Google Scholar9KRT146c.1130T>CAD/SemidominantHeterozygous/Homozygousp.Ile377ThrMissenseEBS, localized in heterozygous state; EBS, generalized severe in homozygous state0.999 (Probably damaging)–4.646 (Deleterious)Rugg et al., 2007Rugg E.L. Horn H.M. Smith F.J. Wilson N.J. Hill A.J. Magee G.J. et al.Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations.J Invest Dermatol. 2007; 127: 574-580Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar10KRT146c.1241del18ADHeterozygous—In-frame deletionGeneralized EBS with severe palmoplantar hyperkeratosisN/AN/AThis study11KRT147c.1253T>GADHeterozygousp.Leu418ArgMissenseEBS, localized1.000 (Probably damaging)–5.504 (Deleterious)This studyAbbreviations: AD, autosomal dominant; AR, autosomal recessive; N/A, not applicable; PROVEAN, Protein Variation Effect Analyzer.1 Epidermolysis bullosa simplex.2 http://genetics.bwh.harvard.edu/pph2/. The values range from 0–1.000, the latter being most damaging.3 http://provean.jcvi.org/index.php. The values below the cutoff score –2.5 indicate deleterious mutations. Open table in a new tab Abbreviations: AD, autosomal dominant; AR, autosomal recessive; N/A, not applicable; PROVEAN, Protein Variation Effect Analyzer. In addition to these putative autosomal dominant families with EBS, three families with a constellation suggestive of an autosomal recessive inheritance pattern were found to harbor homozygous KRT14 mutations, c.92delT, p.Trp305Ter, and c.200delG, the last mutation being previously unreported to our knowledge. Both parents of the probands were heterozygous carriers of the corresponding mutations (Figure 1). Of particular interest was a large multiplex family (family 9) with affected individuals in five generations (Figure 1h). Three of the family members had generalized spontaneous blistering (see Supplementary Figure S1b), and the rest of the affected individuals had a relatively mild blistering tendency limited to palms and soles when subjected to considerable trauma or heat (see Supplementary Figure S1). Initial attempts to identify candidate genes in this family were performed with whole genome-wide single nucleotide polymorphism-based homozygosity mapping using the Illumina Infinium Exome-24 Bead Chip (240,000 markers) (Illumina, San Diego, CA) which showed conserved homozygosity blocks in all three affected individuals on chromosome band 17q21, corresponding to ITGA3, JUP, and KRT14 as candidate genes (see Supplementary Figure S2a and c). The phenotypes in patients reported with mutations in ITGA3 and JUP are very different from those with KRT14 mutations, the latter patients not having renal and pulmonary involvement (Has et al., 2012Has C. Sparta G. Kiritsi D. Weibel L. Moeller A. Vega-Warner V. et al.Integrin alpha3 mutations with kidney, lung, and skin disease.N Engl J Med. 2012; 366: 1508-1514Crossref PubMed Scopus (170) Google Scholar) or cardiomyopathy and curly hair (Boente et al., 2016Boente M.D. Nanda A. Baselaga P.A. Kelsell D.P. McGrath J.A. South A.P. Cardiomyopathy diagnosed in the eldest child harboring p.S24X mutation in JUP.Br J Dermatol. 2016; ([e-pub ahead of print])http://dx.doi.org/10.1111/bjd.14617Google Scholar); accordingly, sequencing of JUP did not show any pathogenic sequence variants. Because the patients with ITGA3 mutation show early lethality due to renal and pulmonary complications (Has et al., 2012Has C. Sparta G. Kiritsi D. Weibel L. Moeller A. Vega-Warner V. et al.Integrin alpha3 mutations with kidney, lung, and skin disease.N Engl J Med. 2012; 366: 1508-1514Crossref PubMed Scopus (170) Google Scholar), this gene was not sequenced here. Sequencing of KRT14 showed the presence of a c.1130T→C transition that resulted in amino acid substitution p.Ile377Thr (Figure 1h). Segregation analysis of all 12 patients showed that individuals with generalized blistering and with consanguineous parents (first cousin marriages) were homozygous for the C allele (C/C), whereas the remaining nine patients with milder disease were heterozygous for T/C (family 9, Figure 1h). The wild-type sequence T/T was found in a clinically unaffected individual. Thus, this family shows mild localized blistering in heterozygous carriers of the mutation, whereas homozygous mutations result in generalized, more severe disease, consistent with semidominant inheritance. Such a semidominant mode of inheritance was also reported in an EBS family with DST-e mutation (Groves et al., 2010Groves R.W. Liu L. Dopping-Hepenstal P.J. Markus H.S. Lovell P.A. Ozoemena L. 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). Most previous reports on KRT14 mutations have been limited to families with few affected individuals, and it is therefore unclear if other KRT14 mutations are also semidominant. To our knowledge, our study expands the mutation database by reporting previously undescribed findings on dominant negative missense mutations in KRT5 and KRT14 and adding a previously unreported in-frame six-amino acid deletion in keratin 14. This study further adds one previously unreported frameshift, premature termination codon-causing mutation to the KRT14 database in autosomal recessive EBS. Demonstration of the dose effect of a mutation in one family, consistent with semidominant inheritance, further increases our understanding of the genotype/phenotype correlations in this group of disorders. Regarding the mode of inheritance of EBS, in general the autosomal dominant form is by far more common than the recessive variant, with only 16 distinct mutations of the latter type having been published in KRT14 so far (Garcia et al., 2011Garcia M. Santiago J.L. Terron A. Hernandez-Martin A. Vicente A. Fortuny C. et al.Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex.Br J Dermatol. 2011; 165: 683-692Crossref PubMed Scopus (21) Google Scholar). In our cohort, four out of 11 families, with a total of seven patients with homozygous mutations, were noted, attesting to the effect of consanguinity on heritable diseases in the Iranian population. Another example of the preponderance of homozygous mutations in EBS in the Iranian cohort of consanguineous families is the report of Kindler syndrome with complete homozygosity of mutations in FERMT1 in all families (Youssefian et al., 2015Youssefian L. Vahidnezhad H. Barzegar M. Li Q. Sotoudeh S. Yazdanfar A. et al.The Kindler syndrome: a spectrum of FERMT1 mutations in Iranian families.J Invest Dermatol. 2015; 135: 1447-1450Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Knowledge of family-specific mutations provides information useful for genetic counseling regarding the risk of recurrence in future pregnancies in the same or subsequent generations (Uitto, 2009Uitto J. Progress in heritable skin diseases: translational implications of mutation analysis and prospects of molecular therapies.Acta Derm Venereol. 2009; 89: 228-235Crossref PubMed Scopus (30) Google Scholar). Specific mutations can also allow prognostication at the newborn stage of disease severity. An example of such prognostic utility of the knowledge of mutations in the keratin genes is the recent study indicating that a specific mutation, p.Glu477Lys in KRT5, is strongly associated with mortality in EBS (Sathishkumar et al., 2016Sathishkumar D. Orrin E. Terron-Kwiatkowski A. Browne F. Martinez A.E. Mellerio J.E. et al.The p.Glu477Lys mutation in keratin 5 is strongly associated with mortality in generalized severe epidermolysis bullosa simplex.J Invest Dermatol. 2016; 136: 719-721Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Thus, the expanded mutation database forms the basis for application of precision medicine for management of these patients. The authors state no conflict of interest. The authors thank Mina Tabrizi, Mohsen Siavashi, and Maryam Abiri (Tehran University of Medical Sciences) for assistance in sample collection and processing and Hakon Hakonarson and Cecilia Kim (Children's Hospital of Philadelphia) for assistance in homozygosity mapping. Carol Kelly assisted in manuscript preparation. This work partially fulfills requirements for the PhD thesis of HV. Download .pdf (.51 MB) Help with pdf files Supplementary Figures S1 and S2