Targeted Sequence Capture and High-Throughput Sequencing in the Molecular Diagnosis of Ichthyosis and Other Skin Diseases

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Immunohistochemical staining indi- 1The Wellcome Trust Center for Human cause of Olmsted syndrome. Am J Hum cated that MBTPS2 is mainly expressed Genetics, Nuffield Department of Clinical Genet 90:558–64 Medicine, University of Oxford, Oxford, UK; Mevorah B, Goldberg I, Sprecher E et al. (2005) in the upper granular layer in normal 2 Centre for Cutaneous Research, The Blizard Olmsted syndrome: mutilating palmo- skin, as previously shown (Aten et al., Institute, Barts & The London School of plantar keratoderma with periorificial 2010); however, in OS skin, MBTPS2 Medicine and Dentistry, Queen Mary keratotic plaques. J Am Acad Dermatol 53: University of London, London, UK; S266–72 was expressed throughout the epi- 3 dermis (Figure 2c). There was no Department of Dermatology, Jundishapur Naiki M, Mizuno S, Yamada K et al. (2012) University of Medical Sciences, Ahvaz, Iran; MBTPS2 mutation causes BRESEK/BRESHECK 4 apparent difference in MBTPS2 locali- Genetic Department, Kerman University of syndrome. Am J Med Genet A 158A:97–102 zation in the skin of a KFSD patient with 5 Medical Sciences, Kerman, Iran and Darwin Oeffner F, Fischer G, Happle R et al. (2009) IFAP the p.N508S mutation (Aten et al., Building, University College London Genetics syndrome is caused by deficiency in MBTPS2, 2010). It is unclear why this is but it Institute, University College London, London, an intramembrane zinc metalloprotease essen- may be because of differences in UK tial for cholesterol homeostasis and ER stress E-mail: [email protected] response. Am J Hum Genet 84:459–67 processing of the mutants in the two 6These authors contributed equally to this work Olmsted HC (1927) Keratodermia palmaris et diseases. and should be considered joint first author. plantaris congenitalis. Am J Dis Child In summary, we demonstrate a novel 33:757–64 association between an MBTPS2 muta- Sakai J, Nohturfft A, Goldstein JL et al. (1998) tion and an X-linked form of OS. This SUPPLEMENTARY MATERIAL Cleavage of sterol regulatory element- expands the number of disorders linked Supplementary material is linked to the online binding proteins (SREBPs) at site-1 requires version of the paper at http://www.nature.com/jid interaction with SREBP cleavage-activating to MBTPS2 mutations and reveals clin- protein. Evidence from in vivo competition ical heterogeneity associated with dif- studies. J Biol Chem 273:5785–93 REFERENCES ferent MBTPS2 mutations. Tang L, Liang J, Wang W et al. (2011) A novel Written, informed consent was Aten E, Brasz LC, Bornholdt D et al. (2010) mutation in MBTPS2 causes a broad pheno- Keratosis follicularis spinulosa decalvans is obtained from all family members or typic spectrum of ichthyosis follicularis, caused by mutations in MBTPS2. Hum Mut atrichia, and photophobia syndrome in a their legal guardians. This study was 31:1125–33 large Chinese family. J Am Acad Dermatol approved by the South East NHS Bergonse FN, Rabello SM, Barreto RL et al. (2003) 64:716–22 Research Ethics Committee and was Olmsted syndrome: the clinical spectrum of Tang L, Zhang L, Ding H et al. (2012) Olmsted mutilating palmoplantar keratoderma. Pediatr syndrome: a new case complicated with performed according to the Declaration Dermatol 20:323–6 of Helsinki Principles. easily broken hair and treated with oral Cambiaghi S, Tadini G, Barbareschi M et al. retinoid. J Dermatol 39:1–2 (1995) Olmsted syndrome in twins. Arch Yaghoobi R, Omidian M, Sina N et al. (2007) CONFLICT OF INTEREST Dermatol 131:738–9 Olmsted syndrome in an Iranian family: report The authors state no conflict of interest. Ding YG, Wang JY, Qiao JJ et al. (2010) A novel of two new cases. Arch Iran Med 10:246–9 mutation in MBTPS2 causes ichthyosis folli- Ye J, Rawson RB, Komuro R et al. (2000) ER stress ACKNOWLEDGMENTS cularis, alopecia and photophobia (IFAP) induces cleavage of membrane-bound ATF6 We thank Barts and The London Charity for its syndrome in a Chinese family. Br J Dermatol by the same proteases that process SREBPs. financial support (to DPK). 163:886–9 Mol Cell 6:1355–64 1,6 2,6 Larregue M, Callot V, Kanitakis J et al. (2000) Zelenski NG, Rawson RB, Brown MS et al. Alireza Haghighi , Claire A. Scott , Olmsted syndrome: report of two new cases 2 3 (1999) Membrane topology of S2P, a Daniel S. Poon , Reza Yaghoobi , and literature review. J Dermatol 27:557–68 4 protein required for intramembranous Nasrollah Saleh-Gohari , Lin Z, Chen Q, Lee M et al. (2012) Exome cleavage of sterol regulatory element-bind- Vincent Plagnol5 and David P. Kelsell2 sequencing reveals mutations in TRPV3 as a ing proteins. JBiolChem274:21973–80

Targeted Sequence Capture and High-Throughput Sequencing in the Molecular Diagnosis of Ichthyosis and Other Skin Diseases

Journal of Investigative Dermatology (2013) 133, 573–576; doi:10.1038/jid.2012.332; published online 20 September 2012

TO THE EDITOR Mendelian disorders of cornification, integument (Oji et al., 2010). The two The inherited ichthyoses are a large and characterized by scaling and/or hyper- main nonsyndromic ichthyosis groups genetically heterogeneous group of keratosis over the majority of the are autosomal recessive congenital ichthyosis (ARCI), encompassing lamellar ichthyosis (LI), congenital ichthyosi- Abbreviations: ARCI, autosomal recessive congenital ichthyosis; CIE, congenital ichthyosiform erythroderma; HI, harlequin ichthyosis; KPI, keratinopathic ichthyosis; LI, lamellar ichthyosis; NGS, next- form erythroderma (CIE), and harlequin generation sequencing; SPPK, striate palmoplantar keratoderma; SNP, single-nucleotide polymorphism ichthyosis (HI), in which patients are

www.jidonline.org 573 CA Scott et al. Sequence Capture in Molecular Diagnosis of Skin Diseases

usually born as collodion babies; and Table 1. Genes implicated in ichthyosis and other skin diseases on the keratinopathic ichthyoses (KPI), the two custom capture microarray main groups of which are epidermolytic Cytogenetic Disease(s) with ichthyosis (EI) and superficial epider- Gene OMIM location (hg19) skin phenotype molytic ichthyosis (SEI) (Oji et al., 2010). KPI are linked to mutations in Exons captured keratin genes. Mutations in ABCA12 are ABHD5 *604780 3p21.33 CDS associated with HI, the most deva- AP1S1 *603531 7q22.1 MEDNIK stating and frequently lethal form of ALOXE3 *607206 17p13.1 ARCI (CIE) ichthyosis (Akiyama et al., 2005; Kelsell et al., 2005). However, mutations in ALOX12B *603741 17p13.1 ARCI (CIE) multiple genes (Table 1), including CDSN *602593 6p21.33 Hypotrichosis, PSS ABCA12, are linked to LI and CIE, CSTA *184600 3q21.1 AREI which display clinical overlap (Akiya- CYP4F22 *611495 19p13.12 ARCI (LI) ma et al., 2003). DSG1 *125670 18q12.1 SPPK1 As mutations in multiple genes are linked to nonsyndromic and syndromic DSP *125647 6p24.3 DCWHK, LAEB, SFWHS, SPPK2 ichthyosis, undertaking genetic screen- GJB2 *121011 13q12.11 BPS, HID, KID, PPK with deafness ing by PCR and Sanger sequencing in a GJB3 +603324 1p34.3 EKV gene-by-gene approach is labor-inten- GJB4 *605425 1p34.3 EKV sive and expensive. Screening known ichthyosis genes simultaneously in a KRT1 *139350 12q13.13 AEI, EI, EPPK, ICM, NEPPK, SPPK3 patient (or multiple patients) could en- KRT2 *600194 12q13.13 SEI able a swifter identification of gene LOR *152445 1q21.3 LK mutations in patients and facilitate pre- NIPAL4 *609383 5q33.3 ARCI (CIE) natal diagnosis and genetic counseling. Next-generation sequencing (NGS) POMP *613386 13q12.3 KLICK has proven effective in the discovery of SLC27A4 *604194 9q34.11 IPS mutations in novel disease-associated SLURP1 *606119 8q24.3 MDM genes, for example, by exome sequen- SPINK5 *605010 5q32 Atopy, NS cing (Ng et al., 2009) or by targeted STS #308100 Xp22.31 XLI sequence capture of chromosomal re- gions previously linked to a condition TGM1 *190195 14q12 ARCI (CIE, LI) (Blaydon et al., 2011, 2012). For con- TGM5 *603805 15q15.2 APSS ditions associated with mutations in Exons, introns multiple known genes, targeted gene ABCA12 *607800 2q35 ARCI (CIE, LI, HI) capture coupled with NGS has shown promise in the molecular diagnosis of Abbreviations: AEI, annular epidermolytic ichthyosis; APSS, acral peeling skin syndrome; ARCI, autosomal recessive congenital ichthyosis; AREI, autosomal recessive exfoliative ichthyosis; BPS, Bart- disease (Berg et al., 2011; Artuso et al., Pumphrey syndrome; CDS, Chanarin-Dorfman syndrome; CIE, congenital ichthyosiform erythroder- 2012). Here, we designed a custom ma; DCWHK, dilated cardiomyopathy with woolly hair and keratoderma; EI, epidermolytic ichthyosis; NimbleGen microarray for sequence EKV, erythrokeratoderma variabilis; EPPK, epidermolytic palmoplantar keratoderma; HI, harlequin ichthyosis; HID, hystrix-like ichthyosis with deafness; ICM, ichthyosis histrix, Curth-Macklin type; IPS, capture and NGS of the ABCA12 gene ichthyosis prematurity syndrome; KID, keratitis–ichthyosis–deafness syndrome; KLICK, keratosis locus, as well as the exons of 23 other linearis–ichthyosis congenital–keratoderma; LAEB, lethal acantholytic epidermolysis bullosa; LI, genes linked to nonsyndromic and lamellar ichthyosis; LK, loricrin keratoderma; MDM, mal de Meleda; MEDNIK, mental retardation, syndromic forms of ichthyosis, acral enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia; NEPPK, nonepidermo- lytic palmoplantar keratoderma; NS, Netherton syndrome; PPK, palmoplantar keratoderma; PSS, peeling skin syndrome/peeling (APSS) peeling skin syndrome; SEI, superficial epidermolytic ichthyosis; SFWHS, skin fragility–woolly hair skin syndrome (PSS) or striate palmoplan- syndrome; SPPK, striate palmoplantar keratoderma; XLI, X-linked recessive ichthyosis. tar keratoderma (SPPK; Table 1), on pooled DNA consisting of 14 genomic DNA samples (Supplementary Informa- zygous whole-exon 8 deletion was found, and this is likely to be the tion and Supplementary Table S1 online). previously discovered by multiplex second disease-causing mutation in Sam- We discovered 12 sequence var- and oligonucleotide array analysis ple 11. In contrast, a homozygous large iants, which, to our knowledge, have (Thomas et al., 2006) in Sample 11 101 bp deletion (p.I1128EfsX38) within not been reported previously (three was not detected, indicating that cur- exon24ofABCA12 was detected by this deletions leading to frameshifts, seven rently NGS may not be sensitive en- capture method. Known whole-exon missense, one nonsense, and one splice ough to detect all complex or large deletions have been undetectable in site), and four known disease-asso- insertion/deletion mutations. However, other sequence capture investigations ciated mutations (Table 2). The hetero- a predicted intronic splice site mutation (Berg et al., 2011). Other non-microarray

574 Journal of Investigative Dermatology (2013), Volume 133 CA Scott et al. Sequence Capture in Molecular Diagnosis of Skin Diseases

Table 2. Mutations found in this study using custom sequence capture and NGS Gene/NCBI Mutation discovered Mutation Disease Sample ID Gender nucleotide ID Exon in this study type Reference/comment

ARCI (CIE/LI) 1 F ALOX12B 2 c.266T4C (p.I89T)1 MS This study (NM_001139.2) 2F— —— —— 3MCYP4F22 6 c.712G4A MS This study (NM_173483.3) (p.A238T)2 4FABCA12 28 c.4070C4A MS This study (NM_173076.2) (p.S1357X)2 28 c.4139A4G (p.N1380S)2 MS Lefevre et al. (2003) (HGMD no. CM032165) 5M— —— —— 6M— —— —— SPPK 7 F DSG1 4 c.277delG FS This study (NM_001942.2) (p.D93IfsX19)2 APSS 8 F— —— —— ARCI (HI) atypical, 9MABCA12 40 c.5936C4G MS This study chrysalis (NM_173076.2) (p.A1979G)2 46 c.6858delT FS This study (p.F2286LfsX6)2 ARCI (HI) 10 F ABCA12 24 c.3381_3481del FS This study (NM_173076.2) (p.I1128EfsX38)1 11 F ABCA12 — c.2332+2T4G2 SS This study. Known to (NM_173076.2) (intron 17) be heterozygous for a whole-exon 8 deletion (Thomas et al., 2006) 12 F ABCA12 2 c.130C4G (p.R44G)2 MS This study (NM_173076.2) 16 c.2033A4G (p.N678S)2 MS rs147218173 TGM1 2 c.401A4G (p.Y134C)2 MS Farasat et al. (2009), (NM_000359.2) rs147916609 13 F ABCA12 3 c.179G4G (p.R60P)2 MS This study (NM_173076.2) 12 c.1300C4T (p.R434X)2 NS Akiyama et al. (2005), (HGMD no. CM052164) Unaffected mother 14 F ABCA12 12 c.1446A4C (p.E482D)2 MS This study of a HI baby (NM_173076.2) 51 c.7444C4T (p.R2482X)2 NS Akiyama et al. (2007), (HGMD no. CM073950) NIPAL4 1 c.197G4A (p.R66Q)2 MS This study (NM_001099287.1) Abbreviations: APSS, acral peeling skin syndrome; ARCI, autosomal recessive congenital ichthyosis; CIE, congenital ichthyosiform erythroderma; F, female; FS, frameshift; HI, harlequin ichthyosis; LI, lamellar ichthyosis; M, male; MS, missense; NS, nonsense; SPPK, striate palmoplantar keratoderma; SS, splice site. 1Homozygous. 2Heterozygous. based NGS platforms with individual 2011) and 11 (Kelsell et al., 2005) from Before this study, we extracted RNA patient DNA bar coding are now avail- previous studies, and Sample 14 (an from the skin of Sample 14 and, using able, which may show more efficacy in unaffected mother of an HI patient) in the ABCA12 single nucleotide poly- large insertion/deletion detection. whom ABCA12 mutations had not been morphism (SNP) rs10498030 found in ABCA12 mutations were found in all identified using PCR and Sanger se- the genomic DNA of Sample 14 as a HI patients on the microarray, includ- quencing methods described previously marker, confirmed by reverse trans- ing in Samples 10 (Rajpopat et al., (Lefevre et al., 2003). cription–PCR and Sanger sequencing

www.jidonline.org 575 CA Scott et al. Sequence Capture in Molecular Diagnosis of Skin Diseases

that only one allele of ABCA12 was DNA pooling and sequence capture SUPPLEMENTARY MATERIAL expressed. This suggested the presence on a single microarray was feasible in Supplementary material is linked to the online of a regulatory ABCA12 gene mutation. this study because of the small number version of the paper at http://www.nature.com/jid However, NGS and confirmation by a of patient samples. However, other restriction digest revealed the presence technologies are becoming available REFERENCES of p.R2482X in exon 51 in Sample 14. that allow for sequence enrichment for Akiyama M (2006) Harlequin ichthyosis and other PCR and Sanger sequencing with rede- candidate genes without library pre- autosomal recessive congenital ichthyoses: signed exon 51 primers confirmed this paration plus indexing samples both for the underlying genetic defects and pathome- chanisms. J Dermatol Sci 42:83–9 mutation and indicated that biased candidate genes and whole exome Akiyama M, Sakai K, Sato T et al. (2007) Com- amplification of the wild-type allele before NGS. The power of this technol- pound heterozygous ABCA12 mutations in- had occurred during the previous PCR ogy has already had an impact on new cluding a novel nonsense mutation underlie amplification of exon 51 (due to Sam- disease gene discovery and diagnostics, harlequin ichthyosis. Dermatology 215:155–9 ple 14 being heterozygous for SNP and the continuing marked reductions Akiyama M, Sawamura D, Shimizu H (2003) The rs3738884 in the original exon 51 in cost to run these genetic technologies clinical spectrum of nonbullous congenital ichthyosiform erythroderma and lamellar reverse primer sequence), resulting in will allow molecular diagnosis as a ichthyosis. Clin Exp Dermatol 28:235–40 the mutation being missed. standard investigation in patient diag- Akiyama M, Sugiyama-Nakagiri Y, Sakai K et al. Homozygous or compound heterozy- nosis and clinical management. (2005) Mutations in lipid transporter ABCA12 gous ABCA12 deletion or truncation in harlequin ichthyosis and functional recovery by corrective gene transfer. J Clin CONFLICT OF INTEREST mutations usually lead to HI, whereas Invest 115:1777–84 missense mutations cause LI or more The authors state no conflict of interest. Artuso R, Fallerini C, Dosa L et al. (2012) rarely CIE (reviewed in Akiyama, 2006). Advances in Alport syndrome diagnosis using Screening multiple genes simultaneously ACKNOWLEDGMENTS next-generation sequencing. Eur J Hum in patients allows variants in multiple We acknowledge Barts and The London Charity, Genet 20:50–7 Action Medical Research, and ShHIRT: The Berg JS, Evans JP, Leigh MW et al. (2011) Next genes to be identified and thus potential Harlequin Ichthyosis Charity for funding our work. generation massively parallel sequencing of phenotype–genotype correlations to be 1 2 Claire A. Scott , Vincent Plagnol , targeted exomes to identify genetic mutations established. For example, in this study, 1 1 Daniela Nitoiu , Philip J. Bland , in primary ciliary dyskinesia: implications for Sample 12, an adult patient with a milder 1 application to clinical testing. Genet Med Diana C. Blaydon , Catherine M. 13:218–29 HI phenotype, harbored two missense 1 1 Chronnell , Daniel S. Poon , Blaydon DC, Biancheri P, Di WL et al. (2011) changes in ABCA12,aswellasthe 3 4 David Bourn ,La´szlo´ Ga´rdos , Inflammatory skin and bowel disease linked to missense TGM1 mutation p.Y134C, 4 5 Andrea Csa´sza´r , Mariann Tihanyi , ADAM17 deletion. NEnglJMed365:1502–8 which has been previously linked to CIE 6 7 Malcolm Rustin , Nigel P. Burrows , Blaydon DC, Etheridge SL, Risk JM et al. (2012) (Farasat et al., 2009). Potentially, the inter- 8 9 Chris Bennett , John I. Harper , RHBDF2 mutations are associated with action of these three missense changes in 10 11 Bernard Conrad , Ishwar C. Verma , tylosis, a familial esophageal cancer syn- two genes linked to ichthyosis could be 12 12 drome. Am J Hum Genet 90:340–6 Saleem M. Taibjee , Celia Moss , detrimental enough to cause HI, but with 1 1 Farasat S, Wei MH, Herman M et al. (2009) Novel Edel A. O’Toole and David P. Kelsell a less severe phenotype than normal. transglutaminase-1 mutations and genotype- phenotype investigations of 104 patients with A heterozygous DSG1 mutation 1Centre for Cutaneous Research, Blizard autosomal recessive congenital ichthyosis in p.D93IfsX19 was found in SPPK patient Institute, Barts and The London School of the USA. J Med Genet 46:103–11 Sample 7, which indicates that the patient Medicine and Dentistry, Queen Mary Kelsell DP, Norgett EE, Unsworth H et al. (2005) 2 has SPPK1. Disease-causing mutations University of London, London, UK; University Mutations in ABCA12 underlie the severe were not identified in Samples 2, 5, 6, College London Genetics Institute, University congenital skin disease harlequin ichthyosis. College London, London, UK; 3Northern Am J Hum Genet 76:794–803 or 8. Mutations in genes not present on Genetics Service, Institute of Human Genetics, Lefevre C, Audebert S, Jobard F et al. (2003) the microarray, or complex mutations in Central Parkway, Newcastle upon Tyne, UK; Mutations in the transporter ABCA12 are 4 known ichthyosis genes that were not Pediatric Department, Zala County Hospital, associated with lamellar ichthyosis type 2. 5 detected by NGS, could form the genetic Zalaegerszeg, Hungary; Zala Megyei Ko´rha´z, Hum Mol Genet 12:2369–78 Genetikai Laborato´rium, Zalaegerszeg, basis of disease in the other patients. 6 Ng SB, Turner EH, Robertson PD et al. (2009) Hungary; Department of Dermatology, Royal Targeted capture and massively parallel sequen- In summary, our study expands the 7 Free Hospital, London, UK; Dermatology, cing of 12 human exomes. Nature 461:272–6 numbers of mutations associated with Addenbrooke’s Hospital, Cambridge, ARCI and SPPK1 and demonstrates that Cambridgeshire, UK; 8Department of Clinical Oji V, Tadini G, Akiyama M et al. (2010) Revised Genetics, Yorkshire Regional Clinical Genetics nomenclature and classification of inherited sequence capture coupled with NGS can ichthyoses: results of the First Ichthyosis Service, Leeds, UK; 9Department of Paediatric Consensus Conference in Soreze 2009. be used successfully as an accurate and Dermatology, University College London J Am Acad Dermatol 63:607–41 cost-effective tool in the molecular diag- Institute of Child Health and Great Ormond nosis of ichthyosis and other skin dis- Street Hospital, London, UK; 10Division of Rajpopat S, Moss C, Mellerio J et al. (2011) eases. Where no disease-causing muta- Human Genetics and Cytogenetics, Bern Harlequin ichthyosis: a review of clinical and molecular findings in 45 cases. Arch Derma- tion is identified in a targeted sequence University Children’s Hospital, Bern, Switzerland; 11Center of Medical Genetics, tol 147:681–6 capture approach, whole-exome/genome New Delhi, India and 12Birmingham Children’s Thomas AC, Cullup T, Norgett EE et al. (2006) sequencing could be undertaken to iden- Hospital, Birmingham, UK ABCA12 is the major harlequin ichthyosis tify novel loci associated with disease. E-mail: [email protected] gene. J Invest Dermatol 126:2408–13

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