ORIGINAL ARTICLE ABCA12 Is the Major Harlequin Ichthyosis Gene Anna C. Thomas1, Tom Cullup2, Elizabeth E. Norgett1, Tara Hill3, Stephanie Barton4, Beverly A. Dale5, Eli Sprecher6, Eamonn Sheridan7, Aileen E. Taylor8, Robert S. Wilroy9, Celia DeLozier10, Nigel Burrows11, Helen Goodyear12, Philip Fleckman5, Karen G. Stephens5, Lakshmi Mehta13, Rosemarie M. Watson14, Robert Graham15, Roni Wolf16, Anne Slavotinek17, Madelena Martin17, David Bourn4, Charles A. Mein2, Edel A. O’Toole1 and David P. Kelsell1 Harlequin ichthyosis (HI) is the most severe form of autosomal-recessive, congenital ichthyosis. Affected infants have markedly impaired barrier function and are more susceptible to infection. Abnormalities in the localization of epidermal lipids as well as abnormal lamellar granule formation are features of HI skin. Previously, we and others have shown that mutations in the ABCA12 gene encoding an adenosine triphosphate-binding cassette (ABC) transporter underlie the skin disease HI. In this study, we have sequenced the ABCA12 gene in an additional 14 patients and show that all contain mutations, with the majority being either nonsense substitution or frameshift mutations. Eleven HI patients had bi-allelic ABCA12 mutations, whereas in the remaining three HI patients in this study, ABCA12 mutations were detected on only one allele by sequencing. In addition, the one patient from the previous study where no sequence mutations were detected was screened for heterozygous deletions. A combination of oligonucleotide arrays, multiplex PCR analysis and single-nucleotide polymorph- ism genotyping revealed a heterozygous intragenic deletion in exon 8. These mutation data establish ABCA12as the major HI gene. Journal of Investigative Dermatology (2006) 126, 2408–2413. doi:10.1038/sj.jid.5700455; published online 10 August 2006 INTRODUCTION 1Centre for Cutaneous Research, Institute of Cell and Molecular Science, Harlequin ichthyosis (HI, OMIM 242500) is the most severe Queen Mary’s School of Medicine and Dentistry, Queen Mary, University of and often lethal form of recessive congenital ichthyosis (Hsu 2 London, London, UK; Genome Centre, Barts and the London, Queen Mary, et al., 1989; Moreau et al., 1999; Sarkar et al., 2000). Infants University of London, London, UK; 3Agilent Technologies, Cheadle Royal Business Park, Cheshire, UK; 4Northern Genetics Service, Institute of Human born with this condition have hard, thick skin covering most of Genetics, International Centre for Life, Central Parkway, Newcastle upon their body. The skin forms large diamond-shaped plates Tyne, UK; 5Departments of Oral Biology, Biochemistry, Medicine/ separated by deep fissures that restrict movement (Hsu et al., Dermatology, and Laboratory Medicine, University of Washington, Seattle, 1989; Dahlstrom et al., 1995). These skin abnormalities also Washington, USA; 6Laboratory of Molecular Dermatology, Department of Dermatology, Rambam Medical Center, Haifa, Israel; 7Yorkshire Regional affect the shape of the eyelids, nose, lips, and ears. Owing to Genetics Service, St James’s University Hospital, Leeds, UK; 8Department of the impaired barrier function of the skin, neonates struggle to Dermatology, Royal Victoria Infirmary, Newcastle upon Tyne, UK; control water loss, regulate temperature, and are more 9Department of Pediatrics, University of Tennessee Health Science Center, Boling Center for Developmental Disabilities, Memphis, Tennessee, USA; susceptible to infection (Moskowitz et al., 2004). In addition, 10Department of Genetic Medicine and Development, University of Geneva the tightened skin can cause breathing difficulties leading to Medical School, Geneva, Switzerland; 11Department of Dermatology, respiratory failure. In the past, an HI-affected neonate would 12 Addenbrookes NHS Trust, Cambridge, UK; Department of Paediatrics, often die within days of birth; however, with better intensive Birmingham and Solihull NHS Trust, Birmingham, UK; 13Division of Medical Genetics, Schneider Children’s Hospital, New York, USA; 14Department of care and possibly the use of oral retinoids, patients now have Paediatric Dermatology, Our Lady’s Hospital for Sick Children, Dublin, an increased chance of survival (Hsu et al., 1989; Moreau Ireland; 15Department of Dermatology, James Paget Hospital, Great et al., 1999; Elias et al., 2000; Sarkar et al., 2000). HI has been 16 Yarmouth, Norfolk, UK; Dermatology Unit, Kaplan Medical Center, reported in both sexes and in a variety of ethnic backgrounds. Rechovot, Israel and 17Department of Pediatrics, University of California, San Francisco, California, USA Our previous study utilized single-nucleotide polymorph- Correspondence: Professor David P. Kelsell, Centre for Cutaneous Research, ism (SNP) array technology to map the HI locus to chromo- Institute of Cell and Molecular Science, Queen Mary, University of London, 4 some 2q35 and subsequently demonstrated that mutations in Newark Street, Whitechapel, London, UK. the ABCA12 gene were a major cause of HI, with 11 out of 12 E-mail: [email protected] patients studied harboring bi-allelic ABCA12 mutations Abbreviations: ABC, adenosine triphosphate-binding cassette; CGH, (Kelsell et al., 2005). Another study has independently comparative genomic hybridization; HI, Harlequin ichthyosis; NBF, nucleotide-binding fold; SNP, single-nucleotide polymorphism associated ABCA12 mutations with HI (Akiyama et al., Received 17 January 2006; revised 27 March 2006; accepted 19 April 2006; 2005). ABCA12 is a member of the adenosine triphosphate- published online 10 August 2006 binding cassette (ABC) superfamily of active transporters. 2408 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology AC Thomas et al. Harlequin Ichthyosis Gene Mutations in ABC genes underlie a variety of other diseases, all 14 patients described in this study. Examples are shown in including cystic fibrosis, Tangier disease, pseudoxanthoma Figure 1 and Table S1 gives the specific details of mutations elasticum, X-linked adrenoleukodystrophy and Dubin–John- found in each patient. Figure 2 shows a summary of HI- son syndrome (Hovnanian, 2005; Uitto, 2005). Interestingly, associated ABCA12 mutations found in this and our previous it has been shown that mutations in the first adenosine study. Two patients were compound heterozygotes and nine triphosphate-binding domain of ABCA12 underlie some were found to have homozygous mutations. In three patients, cases of autosomal-recessive lamellar ichthyosis in families mutations were found on one allele only. The majority of from Africa that showed linkage to chromosome 2q33–34 mutations are nonsense substitutions arising from an inserted (Lamellar Ichthyosis type 2, (LI2), OMIM 601277) (Lefevre premature STOP codon, but four separate single base pair et al., 2003). There is evidence to show that ABCA12 is a deletions and one single base pair insertion was detected, lipid-trafficking molecule involved in lamellar body forma- which are likely to result in frameshift mutations. Evidence for tion and studies have reported abnormalities in the localiza- possible founder mutations was found within different ethnic tion of epidermal lipids as well as abnormal lamellar granule groups. The UK patients of Caucasian origin, 66 and 96, had formation in HI skin (Dale et al., 1990; Akiyama et al., 2005). inherited the same mutation in exon 16 (2025delG). The In addition, the introduction of ABCA12 by gene transfer in mutations 7322delC and W1294* were previously identified HI keratinocytes increased lamellar granule lipid secretion in HI patients of Pakistani origin from our other studies (Akiyama et al., 2005). (Kelsell et al., 2005; Rajpar et al., 2005). In addition, the In this study, we have screened a further 14 unrelated Native American HI patient D938-01 was homozygous for patients affected with HI for disease-associated mutations in the the same ABCA12 mutation (W1744* mutation in exon 34) as ABCA12 gene. In addition to sequence analysis, two different previously identified in an unrelated Native American HI copy number analyses were performed to detect possible patient from a previous study (Kelsell et al., 2005). Although compound heterozygous exon or multiple exon deletions. the vast majority of HI-associated ABCA12 mutations identified to date (this study) (Akiyama et al., 2005; Kelsell RESULTS et al., 2005) (Figure 2) would result in a truncated protein, Sequence analysis of the ABCA12 gene patient 95 was found to contain a homozygous missense Sequence analysis of all 53 coding exons of the ABCA12 substitution (G1179R) in the first transmembrane domain of gene revealed likely disease-associated ABCA12 mutations in ABCA12. This residue is conserved in mouse, rat, and a b Figure 1. Sequencing and HPLC data from HI affected patients. (a) Sequences from three HI-affected patients along with normal controls. (b) Denaturing high-performance liquid chromatography traces of exon 24 showing patient 95 (homozygous G1179R), the mother of patient 95 (heterozygous), and a wild-type control. www.jidonline.org 2409 AC Thomas et al. Harlequin Ichthyosis Gene W199* and Q228* R1297* R2203* Q354* Y377* R714* W1294* W1744* Q2161* D2363N G1179R R1881* Exon 6 8 9 10 16 17 23 24 27 28 33 34 37 42 44 47 49 53 ABCA12 Del G Del A Del CG Del C Del 8 Del 23 Del 28−53 Del T Ins T Figure 2. A schematic of the ABCA12 gene showing the location of HI mutations found. Our previously published mutations are shown in blue, whereas novel mutations are shown in black.