Non-bullous congentital ichthyosiform erythroderma associated with homozygosity for a novel missense mutation in an ATP binding domain of ABCA12

ARTICLE

ejd.2011.1638

Auteur(s) : Sadia Nawaz¹, Muhammad Tariq¹, Ilyas Ahmad¹, Naveed Altaf Malik¹, Shahid Mahmood Baig¹, Niklas Dahl², Joakim Klar² joakim.klar@igp.uu.se

¹ Human Molecular Genetics Laboratory, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

² Department of Immunology, Genetics and Pathology, Uppsala University, 752 85 Uppsala, Sweden

Reprints: J. Klar

Autosomal recessive congenital ichthyosis (ARCI) is a non-syndromic, heterogeneous group of disorders of keratinization leading to abnormal skin scaling over the whole body. The two main clinical forms of ARCI are non-bullous congenital ichthyosiform erythroderma (NCIE) and lamellar ichthyosis (LI). Patients with LI usually present with large, dark, plate-like cutaneous scales with minimal erythroderma, whereas patients with NCIE have erythroderma with overlying fine white scales. Intermediate forms sharing features of both LI and NCIE exist and either of these two phenotypes may be associated with mutations in several genes that underlie ARCI. To date, ARCI have been associated with mutations in seven genes, namely TGM1 (OMIM 190195), ABCA12 (OMIM 607800), ALOXE3 (OMIM 607206), ALOX12B (OMIM 603741), CYP4F22 (OMIM 611495), NIPAL4 (OMIM 609383) and LIPN (OMIM 613924) [1-7]. The ABCA12 gene on chromosome 2q34 encodes a keratinocyte ATP-binding transporter involved in lipid trafficking [8-11]. Two splice variants of ABCA12 (ABCA12L and ABCA12S) have been described and both isoforms share structural features of all ABCA proteins and contain two ATP-binding domains (nucleotide binding domains, NBD) and two transmembrane (TM) domains. The majority of cases with the rare and severe subtype of ARCI, Harlequin ichthyosis (HI), are homozygous or compound heterozygous for truncating mutations in this gene [9, 11]. On the other hand, missense mutations in ABCA12, with a presumably milder effect on the protein, are associated with LI or NCIE [12, 13].

We present herein the identification of a large consanguineous Pakistani pedigree with five members affected by ARCI. Genetic investigations revealed that the affected individuals are homozygous for a novel ABCA12 mutation.

Materials and Methods

Subjects

A large, six generation consanguineous Pakistani family was investigated, with five members, two affected males and three females segregating autosomal recessive congenital ichthyosis (figure 1). The patients present with NCIE, including small, fine, superficial scales over the entire body and erythroderma. Palmoplantar keratoderma was observed with hyperhidrosis of palms and soles affected by recurrent skin infections. The legs show erythroderma as well as large, brownish scales with some similarities to LI. All affected members suffered from mild ectropion and madarosis (figure 2A-C). Individual VI:1 complained of impaired hearing and bleeding in the external auditory canal due to scaling. Histological analysis of a skin biopsy from individual IV:4 after hematoxylin and eosin (H&E) staining showed hyperkeratosis and a thickening of the epidermis accompanied by infiltration of inflammatory cells in the superficial dermis (figure 2E-F). All participants gave informed consent and the study was approved by the local ethical committee at NIBGE.

Genetic analysis

Genomic DNA was extracted from peripheral blood from five affected family members, healthy siblings and their parents, according to standard procedures. SNP genotyping was performed on DNA samples from four affected family members (V:II, V:5 and VI:I) using the GeneChip Mapping 250K array (Affymetrix) according to the manufacturer's protocol. Homozygosity mapping and sorting of genomic regions were performed as described previously with the dedicated software AutoSNPa [14, 15]. A cut-off of >130 homozygous SNPs was used for the selection of candidate regions. Selected regions were further investigated by genotyping all available family members with microsatellite markers derived from known and novel polymorphic repeats (primer sequences are available upon request). Two point LOD scores were calculated using the MLINK program of LINKAGE computer package [16], assuming an autosomal recessive inheritance, equal male to female recombination rate, full penetrance and a disease allele frequency of 0.00001. Equal allele frequencies of the genotyped markers were used in the calculations. The pedigree was drawn with the Cyrillic software, version 2.1.3 (Cherwell Scientific Publishing Ltd, Oxford, UK) and the haplotype analysis was performed manually. PCR amplification of microsatellite markers in the chromosome 2q33-35 region was performed as described previously [17]. All exons of the ABCA12 gene including the 5^’-UTR and intron-exon boundaries were analyzed for mutations by bi-directional sequencing of genomic DNA from two affected family members. Exon 31 was subsequently sequenced on all available family-members. The sequence reactions were generated using the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Invitrogen, San Diego, USA) according to manufacturer's protocol and separated on an ABI 3700 (Applied Biosystems, Foster City, CA). Primer sequences are available upon request.

Bioinformatic analysis

We used the National Center for Biotechnology Information (NCBI) Entrez Genome Map viewer and Ensemble Human Genome Server database for the localization of microsatellite marker loci. The Genome Database was used for information on microsatellite markers and their primer sequences. The ABCA12 sequence is based on GenBank accession number NM_173076.2. Sequences for human ABCA12 protein and its orthologs (Mus musculus, Gallus gallus, and Danio rerio) were retrieved from HomoloGene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene) and subsequently aligned using ClustalW2 [18]. The degree of evolutionary conservation of amino acid positions was calculated using the ConSurf (http://consurf.tau.ac.il) web server [19].

Results

Autozygosity mapping using Affymetrix GeneChip Mapping 250K Array identified a single homozygous region in affected family members on chromosome 2q. The region spans 8 Mb (758 SNPs) and contains 106 genes, including the ABCA12 gene. Subsequent linkage analysis using microsatellites spanning the ABCA12 gene loci gave a maximum two-point LOD of 2.63, suggesting linkage to the region. Sequencing of ABCA12 revealed a homozygous c.4676G>T transition in all five affected family members and all parents were shown to be heterozygous carriers. The mutation is located in exon 31 and results in a p.G1559V substitution within the first nucleotide binding domain of ABCA12 (figure 3A). The amino acid, p.G1559, is a predicted functional residue and highly conserved (ConSurf conservation score=9.8) and evolutionary conserved between mammals, birds and fish (figure 3B). The transition was excluded on 200 control chromosomes of Pakistani origin and on 200 control chromosomes of Swedish origin.

Discussion

In most types of ichthyosis the pathogenesis is associated with perturbed formation of the intercellular lipid layer or cornified cell envelope, resulting in a defective epidermal barrier [9, 11, 20]. Several mutated genes underlying ichthyosis encode proteins directly involved in the production, transport or assembly of the lipid layer and the cornified envelope. To date, more than 50 ABCA12 gene mutations have been described, of which the majority are truncating loss-of-function ABCA12 mutations associated with HI [21]. We show herein that homozygosity for a novel c.4676G>T transition in the ABCA12 gene, resulting in a p.G1559V substitution, causes NCIE in five members of an extended family. The clinical presentation was similar in all five affected individuals. The mutation is located in the functionally important first ATP binding domain of the protein. Ten distinct ABCA12 mutations have previously been associated with the NCIE sub-phenotype and all patients with this clinical presentation have missense mutations on at least one allele. Mutations in the first ATP binding domain of ABCA12 is generally associated with the slightly more severe form of ARCI, LI, and only one of the previously identified NCIE associated missense mutations is located in this domain [21]. The moderate phenotype in our family may be explained by residual ABCA12 activity and it can be assumed that the p.G1559V mutation does not completely abolish lipid transport function. This would be consistent with previously reported ABCA12 missense variants associated with NCIE.

The ABCA12 gene belongs to the large superfamily of the ATP-binding cassette (ABC) transporter genes, which bind ATP for the transport of various molecules across the cell membrane. All ABCA subfamily members are thought to be lipid transporters and ABCA12 is specifically associated with lipid transport in keratinocytes [22]. Lack of ABCA12 leads to the disruption of lamellar granulae lipid transport, predominantly glucosylceramide, resulting in defective intercellular lipid layers of the stratum corneum [9-11]. Interestingly, ABCA12 gene transfers in vitro have shown that lipid transport, at least in part, may be rescued in ABCA12 deficient keratinocytes, which may have future therapeutic implications [10].

The phenotypic overlap within the ARCI group of disorders makes the identification of novel mutations important in order to better understand the genotype-phenotype correlations. This report, together with future genetic analysis of patients with ichthyosis, will hopefully lead to better delineation of ARCI.

Acknowledgements

Financial support: This work was supported by the The Swedish Institute (to S.N., I.A. and M.T.), the Swedish Research Council VR K2010-66X-10829-17-3 (to N.D.) and Swedish Links Asia VR 348-2008-6069 (to N.D. and S.B.), the Swedish Society for Medical Research (J.K.), Uppsala University and University Hospital. Conflict of interest: none.

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