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Genetic Reversion via Mitotic Recombination in Ichthyosis with Confetti due to a KRT10 Polyalanine Frameshift Mutation

2016; Elsevier BV; Volume: 136; Issue: 8 Linguagem: Inglês

10.1016/j.jid.2016.04.023

1523-1747

Young Hee Lim, Jingyao Qiu, Corey Saraceni, Barbara Burrall, Keith A. Choate,

Wnt/β-catenin signaling in development and cancer

Ichthyosis with confetti (IWC) is an autosomal dominant disorder of keratinization that is exceedingly rare, with approximately 40 cases reported. Although patients generally demonstrate ichthyosiform erythroderma at birth, the disorder is defined by the hundreds of confetti-like white spots that appear in childhood and grow in size and number over time (Choate and Milstone, 2015Choate K.A. Milstone L.M. Phenotypic expansion in ichthyosis with confetti.JAMA Dermatol. 2015; 151: 15-16Crossref PubMed Scopus (10) Google Scholar, Guerra et al., 2015Guerra L. Diociaiuti A. El Hachem M. Castiglia D. Zambruno G. Ichthyosis with confetti: clinics, molecular genetics and management.Orphanet J Rare Dis. 2015; 10: 115Crossref PubMed Scopus (28) Google Scholar). The histopathology of the red skin surrounding the macules shows perinuclear vacuolization, loss of the granular layer, and parakeratotic hyperkeratosis, whereas the white, revertant macules are histologically normal (Choate and Milstone, 2015Choate K.A. Milstone L.M. Phenotypic expansion in ichthyosis with confetti.JAMA Dermatol. 2015; 151: 15-16Crossref PubMed Scopus (10) Google Scholar). The mutations identified in IWC to date arise de novo and cause frameshift deletions affecting the carboxyl tail of keratin 10 (K10) or keratin 1 (K1), causing type I IWC (IWC-I) and type II IWC (IWC-II), respectively (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). Prior investigation of seven independent IWC-I probands identified distinct mutations causing entry into the same aberrant reading frame invariably replacing the endogenous glycine-rich tail of K10 with a polyarginine sequence (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, Guerra et al., 2015Guerra L. Diociaiuti A. El Hachem M. Castiglia D. Zambruno G. Ichthyosis with confetti: clinics, molecular genetics and management.Orphanet J Rare Dis. 2015; 10: 115Crossref PubMed Scopus (28) Google Scholar). In IWC-II, the K1 glycine tail is maintained, but the final 22 residues of the end domain are replaced with a novel 30 amino acid, nonrepeating sequence (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). Immunohistochemistry of K10 in IWC-I shows mislocalized K10 in aggregates within the nucleolus, with the corresponding decrease in cytosolic intensity; K1 in IWC-II mislocalizes to the nucleus, along with perinuclear collapse of the cytokeratin network (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). In contrast, the white macules in both subtypes not only demonstrate correct cytosolic localization of their respective keratins, but also represent independent copy-neutral loss of heterozygosity events, in which the heterozygous mutant haplotype is lost without aberrations in chromosomal copy number. Via single nucleotide polymorphism (SNP) genotyping, the loss of heterozygosity track was found to span the proximal q arm of chromosome 17 (IWC-I) or chromosome 12 (IWC-II) to the telomere, consistent with genetic reversion via mitotic recombination, a DNA break-induced event that is otherwise rare on a per cell basis (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, O'Keefe et al., 2010O'Keefe C. McDevitt M.A. Maciejewski J.P. Copy neutral loss of heterozygosity: a novel chromosomal lesion in myeloid malignancies.Blood. 2010; 115: 2731-2739Crossref PubMed Scopus (171) Google Scholar). Given that nucleolar K10 is unique to IWC and all patients with IWC-I invariably express polyarginine K10, it was hypothesized that the mislocalization results from gain of the arginine-rich motif, as many RNA-binding proteins utilize their arginine-rich motif to interact with the phosphate backbone of RNA; mutant K10 would similarly associate with and aggregate within the ribosomal RNA-rich environment of the nucleolus (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, Draper, 1999Draper D.E. Themes in RNA-protein recognition.J Mol Biol. 1999; 293: 255-270Crossref PubMed Scopus (342) Google Scholar). However, direct interaction between mutant K10 and ribosome components has not yet been observed, and the role of the polyarginine tail in nucleolar localization and pathobiology of IWC remains unknown. Here, we identify a 28-year-old Caucasian male patient with unaffected parents, who was noted at birth to have mild erythema, widespread scale, and mild palmoplantar keratoderma (IWC100). Before the start of investigation, the Yale Human Investigation Committee approved the study protocol and the patient provided written consent as well as permission to publish his photographs. Other than mild ectropion, no dysmorphic features including nail deformities, nipple hypoplasia, or malformation of the auricle were noted, and no evidence of developmental delay was found (Hendrix et al., 1997Hendrix Jr., J.D. Patterson J.W. Greer K.E. Skin cancer associated with ichthyosis: the MAUIE syndrome.J Am Acad Dermatol. 1997; 37: 1000-1002Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Thicker hyperkeratosis was noted over the extensor surfaces, axillae, and antecubital areas; fine scale covered the entire body including the scalp (Supplementary Figure S1 online). Scales were fine, white, and loosely adherent in some spots, whereas they were larger and plate-like on the dorsal forearms and shins. Confetti-like white spots first appeared in his early twenties, but have remained limited to the forearms (Figure 1a and b) and are less than 5 mm in diameter. Before the development of the white macules, fluorescence in situ hybridization was performed for genetic testing for X-linked ichthyosis, but no steroid sulfatase deletion was detected. Histopathology of the affected skin shared many histological features with IWC-I, including acanthosis, parakeratosis, loss of the granular layer, and perinuclear vacuolization (Figure 1c). In contrast, the white spots showed normal histology (Figure 1d). Given these findings, KRT10 was considered a candidate gene, and direct Sanger sequencing of blood DNA revealed a de novo c.1373G deletion in exon 6, which replaces the normal glycine-serine-rich tail of K10 with a mutant polyalanine motif and extends the end domain by an additional 19 amino acids (Figure 2a and b). Most IWC KRT10 mutations reported thus far lead to a mutant polyarginine tail, and this is the second report of a KRT10 mutation encoding a mutant polyalanine tail (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), though the prior report neither investigated localization of the mutant protein nor mechanism of reversion. To assess mutation pathogenesis, we first performed immunolocalization in skin from this subject, finding overall diminution of suprabasal K10 staining with evidence of filament network collapse and focal aggregates within the nuclei (Figure 2c). These findings were not identified in revertant or normal control skin and are similar to prior observations in polyarginine K10 mutant skin (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). Costaining with the nucleolar marker fibrillarin revealed that K10 aggregates are within the nucleolus. Moreover, calcium-differentiated primary keratinocytes isolated from biopsies of affected skin demonstrated nucleolar aggregates that also costained with fibrillarin (Figure 2c). Immunolocalization of K1, the binding partner of K10, also demonstrated nuclear mislocalization, consistent with prior reports (Supplementary Figure S2 online) (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). To determine the genetic mechanism underlying the revertant mosaicism in this patient, we performed laser capture microdissection of three individual white spots for isolation of DNA and SNP genotyping (Supplementary Materials and Methods and Figure S3 online). Like patients with IWC-I with polyarginine K10, each revertant spot was found to harbor copy-neutral loss of heterozygosity in the proximal q arm of chromosome 17 extending to the telomere, consistent with reversion via mitotic recombination (Figure 2d and Figure S3) (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, O'Keefe et al., 2010O'Keefe C. McDevitt M.A. Maciejewski J.P. Copy neutral loss of heterozygosity: a novel chromosomal lesion in myeloid malignancies.Blood. 2010; 115: 2731-2739Crossref PubMed Scopus (171) Google Scholar). For all three revertant spots, the region of crossover was estimated to fall between SNPs rs6505079 (chr17:28391158, GRCh38/hg38) and rs8078229 (chr17:28500820, GRCh38/hg38), based on binning by 500 SNPs and identifying the point of deviation from a heterozygous b-allele frequency (Figure S4 and Supplementary Materials and Methods online). Polyalanine mutations have been previously reported in the V2 domain of K1 in patients with striate palmoplantar keratoderma or ichthyosis hystrix, Curth-Macklin, though genetic reversion has not been found in either disorder (Richardson et al., 2006Richardson E.S. Lee J.B. Hyde P.H. Richard G. A novel mutation and large size polymorphism affecting the V2 domain of keratin 1 in an African-American family with severe, diffuse palmoplantar keratoderma of the ichthyosis hystrix Curth-Macklin type.J Invest Dermatol. 2006; 126: 79-84Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, Sprecher et al., 2001Sprecher E. Ishida-Yamamoto A. Becker O.M. Marekov L. Miller C.J. Steinert P.M. et al.Evidence for novel functions of the keratin tail emerging from a mutation causing ichthyosis hystrix.J Invest Dermatol. 2001; 116: 511-519Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Whittock et al., 2002Whittock N.V. Smith F.J. Wan H. Mallipeddi R. Griffiths W.A. Dopping-Hepenstal P. et al.Frameshift mutation in the V2 domain of human keratin 1 results in striate palmoplantar keratoderma.J Invest Dermatol. 2002; 118: 838-844Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Polyalanine expansions are also associated with neurological and developmental disorders, including holoprosencephaly, synpolydactyly, and muscular dystrophy (Amiel et al., 2004Amiel J. Trochet D. Clement-Ziza M. Munnich A. Lyonnet S. Polyalanine expansions in human.Hum Mol Genet. 2004; 13: R235-R243Crossref PubMed Scopus (112) Google Scholar, Moumne et al., 2008Moumne L. Dipietromaria A. Batista F. Kocer A. Fellous M. Pailhoux E. et al.Differential aggregation and functional impairment induced by polyalanine expansions in FOXL2, a transcription factor involved in cranio-facial and ovarian development.Hum Mol Genet. 2008; 17: 1010-1019Crossref PubMed Scopus (61) Google Scholar). Many of these polyalanine expansions result in intranuclear aggregation of their respective proteins, with longer tracts demonstrating more aggregates and more severe symptoms (Moumne et al., 2008Moumne L. Dipietromaria A. Batista F. Kocer A. Fellous M. Pailhoux E. et al.Differential aggregation and functional impairment induced by polyalanine expansions in FOXL2, a transcription factor involved in cranio-facial and ovarian development.Hum Mol Genet. 2008; 17: 1010-1019Crossref PubMed Scopus (61) Google Scholar). Mutations in PABPN1 and ARX encoding polyalanine motifs cause oculopharyngeal muscular dystrophy and Partington syndrome, respectively; in each, nuclear aggregates were found to promote cellular stress and death (Calado et al., 2000Calado A. Tome F.M. Brais B. Rouleau G.A. Kuhn U. Wahle E. et al.Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA.Hum Mol Genet. 2000; 9: 2321-2328Crossref PubMed Scopus (199) Google Scholar, Nasrallah et al., 2004Nasrallah I.M. Minarcik J.C. Golden J.A. A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death.J Cell Biol. 2004; 167: 411-416Crossref PubMed Scopus (77) Google Scholar). Polyalanine PABPN1 was further shown to sequester polyA mRNA by binding with high affinity to nascent poly(A) tails, potentially acting as a nuclear RNA trap to prevent proper cytoplasmic transcription of key proteins (Calado et al., 2000Calado A. Tome F.M. Brais B. Rouleau G.A. Kuhn U. Wahle E. et al.Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA.Hum Mol Genet. 2000; 9: 2321-2328Crossref PubMed Scopus (199) Google Scholar). Although the mechanisms of genetic reversion in IWC are yet unknown, IWC is distinguished from other keratinopathies by keratin mislocalization to the nucleus. All prior reports of IWC-I have resulted in replacement of all or a portion of the K10 endogenous tail domain with a polyarginine motif that was thought relevant to reversion via mitotic recombination. Our discovery that IWC due to polyalanine frameshift mutation also leads to nuclear localization and genetic reversion via mitotic recombination suggests that the replacement of the K10 endogenous tail domain may be central not only to disease pathogenesis but also to genetic reversion via mitotic recombination. The authors state no conflict of interest. We would like to thank Jonathan Levinsohn for plotting SNP genotyping data, and Jing Zhou and Rong-Hua Hu for technical assistance. This study was supported by a grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases/NIH (5K08AR056305 and 5R01AR062111) to KAC, and a Clinical Scientist Development Award from the Doris Duke Charitable Foundation to KAC. YHL was supported by the Medical Scientist Training Program at Yale University (NIH NIGMS T32 GM007205) and is a recipient of the Clinical Mentorship Award from the Doris Duke Charitable Foundation. Download .pdf (6.46 MB) Help with pdf files Supplementary Data