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Large Intragenic KRT1 Deletion Underlying Atypical Autosomal Dominant Keratinopathic Ichthyosis

2016; Elsevier BV; Volume: 136; Issue: 10 Linguagem: Inglês

10.1016/j.jid.2016.06.016

1523-1747

Takuya Takeichi, Lu Liu, Alya Abdul‐Wahab, James R. McMillan, Kristina L. Stone, Masashi Akiyama, Michael A. Simpson, Maddy Parsons, Jemima E. Mellerio, John A. McGrath,

Hair Growth and Disorders

Mutations in KRT1 (keratin 1) or KRT10 (keratin 10) underlie a spectrum of diseases known as keratinopathic ichthyoses (Hotz et al., 2016Hotz A. Oji V. Bourrat E. Jonca N. Mazereeuw-Hautier J. Betz R.C. et al.Expanding the clinical and genetic spectrum of KRT1, KRT2 and KRT10 mutations in keratinopathic ichthyosis.Acta Derm Venereol. 2016; 96: 473-478Crossref PubMed Scopus (39) Google Scholar, Oji et al., 2010Oji V. Tadini G. Akiyama M. Blanchet Bardon C. Bodemer C. Bourrat E. et al.Revised nomenclature and classification of inherited ichthyoses: results of the First Ichthyosis Consensus Conference in Sorèze 2009.J Am Acad Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Most commonly, heterozygous missense mutations within the head/tail domains result in autosomal dominant epidermolytic ichthyosis (EI), although a spectrum of mutations (some of which may be recessive) underlie a diverse collection of phenotypes that include generalized, annular, superficial (occasionally; mostly keratin 2), and nevoid forms of EI, as well as palmoplantar keratoderma (diffuse, focal, striate), severe variants such as Curth-Macklin ichthyosis, and most recently, ichthyosis with confetti (IWC) (for review on keratin diseases, see Knöbel et al., 2015Knöbel M. O'Toole E.A. Smith F.J. Keratins and skin disease.Cell Tissue Res. 2015; 360: 583-589Crossref PubMed Scopus (53) Google Scholar). The latter usually results from downstream frameshift mutations that result in an arginine- or alanine-rich tail to the keratin leading to nuclear retention of mutant keratin and/or intermediate filament dysfunction (Choate et al., 2010Choate K.A. Lu Y. Zhou J. Choi M. Elias P.M. Farhi A. et al.Mitotic recombination in patients with ichthyosis causes reversion of dominant mutations in KRT10.Science. 2010; 330: 94-97Crossref PubMed Scopus (139) Google Scholar, Choate et al., 2015Choate K.A. Lu Y. Zhou J. Elias P.M. Zaidi S. Paller A.S. et al.Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti.J Clin Invest. 2015; 125: 1703-1707Crossref PubMed Scopus (44) Google Scholar). Almost all pathogenic variants reported, including in EI and IWC, have been point mutations (www.interfil.org/). In this report, we describe an atypical heterozygous KRT1 mutation, a large approximately 2.2 kb intragenic deletion mutation in an autosomal dominant pedigree containing six affected individuals over four generations with heterogeneous clinical features resembling IWC or EI. The proband is a 64-year-old woman. She was born as a collodion baby, but within a few weeks she became erythrodermic with just a few islands of sparing. Palm and sole skin became thicker although the rest of her skin was reported to be fragile. With increasing age, several pale macules developed on her trunk and limbs. She also reported a previous ulcerated basal cell carcinoma on the forehead (surgically excised). Examination of her skin as an adult revealed diffuse erythroderma with hundreds of confetti-like spots on the trunk and extremities in keeping with IWC (Figure 1a), and palmoplantar keratoderma. Nails showed longitudinal ridging but no hair abnormalities were observed. After informed and written consent, skin biopsies were obtained from both the affected and unaffected areas of the abdomen (Figure 1b and c). Transmission electron microscopy from the affected area showed focal keratinocyte aggregation and early cytolysis through the spinous layer as well as perinuclear shells/vacuoles (Figure 1d). In addition, there was an increased number of keratohyalin granules that were coarse and angulated noted from the upper-spinous layer to the granular layer (Supplementary Figure S1 online). Immunofluorescence microscopy on skin samples from affected and unaffected skin (Figure 1e) and unrelated healthy control skin was performed using antikeratin antibodies (Figure 1f). There was a marked reduction in staining intensity for keratin 1 and 10 in the affected epidermis compared with unaffected or control skin. Moreover, no nuclear keratin 1/10 labeling was detected. Unaffected patient skin labeling resembled control skin apart from keratin 2 staining that was increased in the unaffected patient skin compared with both affected patient skin and control. Immunostaining for keratin 5 and 14 showed no major differences between any of the three skin samples. Within the rest of the family (Figure 1g; proband is II-2), her late mother (I-2) and her daughter (III-2) had similarly abnormal skin, whereas the proband's three grandchildren (IV-1, IV-2, and IV-3, now aged 12 years, 9 years, and 6 years, respectively) all had skin changes resembling EI with erythema, erosions, and large islands of sparing on the face, neck, chest, and abdomen (Figure 1h), as well as moderate palmoplantar keratoderma (Figure 1i). Indeed, keratoderma was present in all affected individuals, but none of the youngest generation had clinical signs of IWC. Next, after obtaining ethics approval and informed consent, we extracted genomic DNA from either peripheral blood or saliva samples from the patient, other family members, and controls in compliance with the Helsinki Guidelines. Using four affected members' DNAs (I-2, II-2, III-2, and IV-2), whole-exome sequencing was performed on the Illumina HiSeq 2000 (San Diego, CA), as described elsewhere (Campbell et al., 2014Campbell P. Morton P.E. Takeichi T. Salam A. Roberts N. Proudfoot L.E. et al.Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR.J Invest Dermatol. 2014; 134: 2570-2578Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Then, copy number variation analysis was performed by using four exome data sets. After excluding pathogenic mutations in genes implicated in different forms of ichthyosis, the exome shared variant profile was filtered to look for novel heterozygous mutations. The only shared gene abnormality was a heterozygous deletion in KRT1. We then confirmed the accurate deletion points in intron 1 and intron 5 in KRT1 of genomic DNA by PCR and Sanger sequencing (Figure 2a and b; see Supplementary Methods online for details). The deletion spans 2,209 bp of genomic DNA (chr12:53,071,005-53,073,213; GRCh37/hg19) and at a protein level it removes (in-frame) part of the 1A domain and all of the 1B and 2A domains (amino acids 197–375) (Figure 2c). This deletion is not represented in the dbSNP database, the 1,000 Genomes database, DECIPHER, or in 2,000 unrelated European in-house control exomes. By semiquantitative PCR using genomic DNA, we were also able to demonstrate the reduced presence of the deletion mutation in the proband's pale skin spots compared with her erythematous skin or peripheral blood DNA (Figure 2d; see also Supplementary Methods for details). Apart from generating a truncated keratin 1 peptide, it is also likely that the deletion mutation creates other transcripts that are degraded at RNA level because KRT1 expression was reduced in red (lesional) skin in two subjects tested (Supplementary Figure S2 online). Microsatellite analysis (Supplementary Figure S3 online) provides support for mitotic recombination as the likely corrective mechanism for the IWC phenotype, as has been shown in detail elsewhere (Choate et al., 2010Choate K.A. Lu Y. Zhou J. Choi M. Elias P.M. Farhi A. et al.Mitotic recombination in patients with ichthyosis causes reversion of dominant mutations in KRT10.Science. 2010; 330: 94-97Crossref PubMed Scopus (139) Google Scholar, Choate et al., 2015Choate K.A. Lu Y. Zhou J. Elias P.M. Zaidi S. Paller A.S. et al.Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti.J Clin Invest. 2015; 125: 1703-1707Crossref PubMed Scopus (44) Google Scholar): typically there is copy neutral loss of heterozygosity from a proximal breakpoint to the telomere with mitotic recombination allowing expression of the normal allele. In contrast to the reported cases of IWC, however, the causative pathology differs from that seen in our pedigree. The prototypic molecular pathology of IWC involves frameshift mutations toward the 3′ end of the gene that lead to a polyarginine or polyalanine keratin tail, protein mislocalization to the nucleus, and intermediate filament collapse (Hotz et al., 2016Hotz A. Oji V. Bourrat E. Jonca N. Mazereeuw-Hautier J. Betz R.C. et al.Expanding the clinical and genetic spectrum of KRT1, KRT2 and KRT10 mutations in keratinopathic ichthyosis.Acta Derm Venereol. 2016; 96: 473-478Crossref PubMed Scopus (39) Google Scholar; Lim et al., 2016Lim Y.H. Qiu J. Saraceni C. Burrall B.A. Choate K.A. Genetic reversion via mitotic recombination in ichthyosis with confetti due to a KRT10 polyalanine frameshift mutation.J Invest Dermatol. 2016; 136: 1725-1728Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The much larger upstream in-frame deletion in our case appears to result in clinical features that overlap with IWC although the skin pathology, as well as the extended phenotype in the family, clearly incorporates some of the features of EI. Notably, transmission electron microscopy showed mixed findings with keratin filament aggregation and cytolysis in the spinous layer, as well as prominent keratohyalin granules, both consistent with EI, although some perinuclear shells/vacuoles supported IWC. Thus, although more than 50 mutations in KRT1 have been reported, our pedigree appears to expand genotype-phenotype correlation and underscores the considerable heterogeneity underlying keratinopathic ichthyoses. Our findings also demonstrate that copy number analysis by next-generation sequencing is a useful tool to identify pathogenic changes that elude conventional Sanger sequencing of keratin genes. The authors state no conflict of interest. We acknowledge financial support from the Department of Health via the UK National Institute for Health Research Comprehensive Biomedical Research Centre award to Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. This study was also supported in part by the Great Britain Sasakawa Foundation no. 4314 and Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation (S2404) from the Japan Society for the Promotion of Science. We thank Dr Claire Fuller for additional clinical information. Download .pdf (.81 MB) Help with pdf files Supplementary Data