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Molecular Analysis of 250 Patients with Autosomal Recessive

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Molecular Analysis of 250 Patients with Autosomal Recessive See related commentary on pg 1319 ORIGINAL ARTICLE Molecular Analysis of 250 Patients with Autosomal Recessive Congenital Ichthyosis: Evidence for Mutation Hotspots in ALOXE3 and Allelic Heterogeneity in ALOX12B Katja-Martina Eckl1, Silvia de Juanes2, Janine Kurtenbach1, Marc Na¨tebus1, Jenny Lugassy3,4,5, Vinzenz Oji6, Heiko Traupe6, Marie-Luise Preil7, Francisco Martı´nez8, Josef Smolle9, Avikam Harel10, Peter Krieg2, Eli Sprecher3,4,5 and Hans C. Hennies1,11 In recent years several new genes for autosomal recessive congenital ichthyosis (ARCI) have been identified. However, little is known about the molecular epidemiology and pathophysiology of this genetically and clinically heterogeneous group of severe disorders of keratinization. ARCI is characterized by intense scaling of the whole integument often associated with erythema. We and others have shown that mutations in ALOX12B and ALOXE3, coding for the lipoxygenases 12R-LOX and eLOX-3 predominantly synthesized in the epidermis, can underlie this rare condition. Here we have surveyed a large group of 250 patients with ARCI for mutations in these two genes. We have identified 11 different previously unreported mutations in ALOX12B and ALOXE3 in 21 ARCI patients from 19 unrelated families and demonstrated that mutations in the two genes are the second most common cause for ARCI in this cohort of patients. Examination of the molecular data revealed allelic heterogeneity for ALOX12B and two mutational hotspots in ALOXE3. Functional analysis of all missense mutations and a splice site mutation demonstrated that complete loss of function of the enzymes underlies the phenotype. Our findings further establish the pivotal role of the 12-lipoxygenase pathway during epidermal differentiation. Journal of Investigative Dermatology (2009) 129, 1421–1428; doi:10.1038/jid.2008.409; published online 8 January 2009 INTRODUCTION substrates containing one or more (Z,Z)-1,4-pentadiene Lipoxygenases (LOX) represent a widely distributed family of moieties (reviewed by Brash, 1999; Ku¨hn and Thiele, non-heme, iron-containing dioxygenases that catalyze the 1999). Within the mammalian LOX family, a distinct subclass regioselective and stereoselective dioxygenation of fatty acid of epidermis-type LOX has been found to be preferentially synthesized in the skin and few other epithelial tissues (Brash et al., 1997, 2007; Boeglin et al., 1998; Kinzig et al., 1999; 1Division of Dermatogenetics, Cologne Center for Genomics, University of 2 Heidt et al., 2000; Krieg et al., 2002). The genes for the Cologne, Cologne, Germany; Division of Genome Modifications and human epidermal LOX, 15-LOX-2, 12R-LOX, and eLOX-3, Carcinogenesis, German Cancer Research Center, Heidelberg, Germany; 3Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of map closely together on human chromosome 17p13.1 (Krieg Technology, Haifa, Israel; 4Center for Translational Genetics, Rappaport et al., 2001). Their differentiation-dependent expression Institute for Research in the Medical Sciences, Technion – Israel Institute of pattern in epithelial tissues suggests a common physiological Technology, Haifa, Israel; 5Laboratory of Molecular Dermatology and Department of Dermatology, Rambam Health Care Campus, Haifa, Israel; role in the regulation of proliferation and differentiation of 6Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany; epithelial cells, especially keratinocytes. The epidermal 7TOMESA Clinic, Bad Salzschlirf, Germany; 8Unidad de Genetica, Hospital 12R-LOX and eLOX-3 differ from all other mammalian LOX 9 Universitario La Fe, Valencia, Spain; Department of Dermatology, Medical in their unique structural and enzymatic features (Boeglin University of Graz, Graz, Austria; 10Pediatric Dermatology Unit, Dana’s Children’s Hospital, Sourasky Medical Center, Tel Aviv, Israel and 11Center et al., 1998; Kinzig et al., 1999; Krieg et al., 1999) as both for Molecular Medicine Cologne, University of Cologne, Cologne, Germany proteins contain an extra domain located at the surface of the Correspondence: Dr Hans C. Hennies, Division of Dermatogenetics, Cologne catalytic subunit. 12R-LOX represents the only mammalian Center for Genomics, University of Cologne, Zu¨lpicher Str. 47, 50674 Ko¨ln, LOX that forms products with R chirality, and, unlike all other Germany. E-mail: [email protected] LOX, eLOX-3 does not exhibit dioxygenase activity but acts Abbreviations: 12R-HPETE, 12R-hydroperoxy-5Z,8Z,10E,14Z- as a hydroperoxide isomerase (Yu et al., 2003). Both enzymes eicosatetraenoic acid; ARCI, autosomal recessive congenital ichthyosis; LOX, lipoxygenase participate in the same pathway and convert arachidonic Received 8 July 2008; revised 3 November 2008; accepted 6 November acid via 12R-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic 2008; published online 8 January 2009 acid (12R-HPETE) to the corresponding hepoxilin-like & 2009 The Society for Investigative Dermatology www.jidonline.org 1421 K-M Eckl et al. Molecular Analysis of 250 Patients with ARCI epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosatrienoic acid. Table 1. Summary of previously unreported mutations Mutations in ALOX12B and ALOXE3, the genes for 12R-LOX in ALOX12B and ALOXE3 including two hotspot and eLOX-3, were found in patients with autosomal recessive mutations identified in patients with ARCI in this study congenital ichthyosis (ARCI; Jobard et al., 2002; Eckl et al., Mutation Protein Exon Frequency1 2005). We and others have shown that those mutations completely eliminate the catalytic activity of the LOX ALOX12B enzymes, suggesting that mutational inactivation of either c.1A4G p.Met1? 1 1 12R-LOX or eLOX-3 is causally linked to the ARCI phenotype c.583T4C p.Phe195Leu 5 1 (Eckl et al., 2005; Yu et al., 2005). Furthermore, 12R-LOX c.942_943insTTTA p.Ala316ProfsX59 8 1 deficiency in the mouse clearly recapitulates the human c.1144A4G p.Lys382Glu 9 1 phenotype (Epp et al., 2007; Moran et al., 2007). c.1153delG p.Val385TyrfsX30 9 1 ARCI, including lamellar ichthyosis, non-bullous conge- nital ichthyosiform erythroderma, and congenital fine scal- c.1272_1273insC p.Lys425GlnfsX24 9 1 ing, forms a clinically and genetically heterogeneous group of c.1625_1626delAA p.Lys542ArgfsX13 12 2 severe keratinization disorders with a prevalence of approxi- c.1654+3A4G splice defect 12 2 mately 1 in 200,000 persons in the European and northern American populations (Traupe, 1989; Williams et al., 2005; ALOXE3 Oji and Traupe, 2006; Schmuth et al., 2007). Affected c.434G4A p.Arg119GlyfsX12 3 2 newborns often present with a so-called collodion mem- 4 2 brane. After loss of this encasement in the first weeks of life, c.700C T p.Arg234X 6 11 patients exhibit a generalized scaling, which varies from c.719delA p.Lys240ArgfsX40 6 1 patient to patient in extent, color, and degree of adherence. c.1031_1039del9 p.Gln344_Ala347delinsPro 8 2 An underlying erythema is often seen, but mostly mild. c.1889C4T3 p.Pro630Leu 14 15 Further features may include alopecia, hypohydrosis, as well 1Number of independent chromosomes with the mutation identified in as marked hyperlinearity and hyperkeratosis of the palms and 250 ARCI patients described here and by Eckl et al., 2005. 2Mutation described previously by Jobard et al., 2002. soles. Although mutations in six genes, TGM1, ALOX12B, 3 ALOXE3, ABCA12, Ichthyin, and CYP4F22, have been found, Mutation described previously by Eckl et al., 2005. more loci must exist, as 30–40% of all ARCI patients do not have mutations in any of these genes. Only transglutaminase 1, 12R-LOX, and eLOX-3, the products of TGM1, ALOX12B, mutation remained undetected as we expect compound and ALOXE3, respectively, have been analyzed functionally heterozygosity for those patients, and may reside in so far, and only the pathophysiology of ARCI caused by regulatory regions or farther in the introns or represent larger deficiency of transglutaminase 1 has been analyzed in more deletions. All mutations were excluded from a panel of 100 detail. matched control persons. Here we present 11 previously unreported inactivating Patient ISA carried two homozygous missense mutations mutations in either ALOX12B or ALOXE3, seen in 19 in ALOXE3, predicted to result in p.Arg145His and independent ARCI cases out of 250 patients analyzed. In p.Leu237Met, respectively. Both mutant enzymes, however, contrast to ALOX12B, showing extended allelic heterogene- were shown to be enzymatically active (Eckl et al., 2005). We ity, we have identified two mutational hotspots in ALOXE3. then tested a double mutant, which also showed normal LOX Using an in vitro assay, we have further demonstrated the activity. As the first mutation, c.434G4A, altered the last ablation of enzyme activity of mutant 12R-LOX and eLOX-3. base of exon 3 and prediction of splice sites (Reese et al., 1997; Nalla and Rogan, 2005) suggested an effect on RNA RESULTS splicing, we used a mini-gene assay to analyze the RNA. We Mutation analysis in ALOX12B and ALOXE3 cloned genomic DNA of the patient spanning intron 1 to Consanguineous families were prescreened for regions of exon 5 of ALOXE3 into a eukaryotic expression vector and homozygosity as described elsewhere (Mizrachi-Koren et al., transfected the construct into HEK 293 cells. Analysis of total 2005; Lugassy et al., 2008). After analyzing TGM1 by direct RNA revealed that the mutation c.434G4A leads to sequencing, mutation analysis was extended by sequencing complete skipping of exon 3 (Figure 1). A second, hetero- all 30 exons and exon/intron boundaries of
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