Skip Navigation


Human Molecular Genetics Advance Access originally published online on August 18, 2004
Human Molecular Genetics 2004 13(20):2473-2482; doi:10.1093/hmg/ddh263
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/20/2473    most recent
ddh263v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (37)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lefèvre, C.
Right arrow Articles by Fischer, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lefèvre, C.
Right arrow Articles by Fischer, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, Vol. 13, No. 20 © Oxford University Press 2004; all rights reserved

Mutations in ichthyin a new gene on chromosome 5q33 in a new form of autosomal recessive congenital ichthyosis

Caroline Lefèvre1, Bakar Bouadjar3, Aysen Karaduman4, Florence Jobard1, Safa Saker2, Meral Özguc5, Mark Lathrop1, Jean-François Prud'homme2 and Judith Fischer1,*

1Centre National de Génotypage and 2Généthon, Evry, France, 3Department of Dermatology, CHU Bab-El-Oued, Algiers, Algeria and 4Department of Dermatology and 5DNA and Cell Bank Tübitak, Hacettepe University, Ankara, Turkey

Received June 16, 2004; Accepted August 4, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report the genomic localization by homozygosity mapping and the identification of a gene for a new form of non-syndromic autosomal recessive congenital ichthyosis. The phenotype usually presents as non-bullous congenital ichthyosiform erythroderma with fine whitish scaling on an erythrodermal background; larger brownish scales are present on the buttocks, neck and legs. A few patients presented a more generalized lamellar ichthyosis. Palmoplantar keratoderma was present in all cases, whereas only 60% of the patients were born as collodion babies. Six homozygous mutations including one nonsense and five missense mutations were identified in a new gene, ichthyin, on chromosome 5q33 in 23 patients from 14 consanguineous families from Algeria, Colombia, Syria and Turkey. Ichthyin encodes a protein with several transmembrane domains which belongs to a new family of proteins of unknown function localized in the plasma membrane (PFAM: DUF803), with homologies to both transporters and G-protein coupled receptors. This family includes NIPA1, in which a mutation was recently described in a dominant form of spastic paraplegia (SPG6). We propose that ichthyin and NIPA1 are membrane receptors for ligands (trioxilins A3 and B3) from the hepoxilin pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The clinically and genetically heterogeneous group of autosomal recessive congenital ichthyoses (ARCI) is characterized by generalized desquamation of the skin, usually with erythema (1–3). Most of the patients are born as collodion babies. The disease may progress to two main clinical forms, either lamellar ichthyosis (LI) or non-bullous congenital ichthyosiform erythroderma (NCIE). The estimated incidence is between 1 in 300 000 and 1 in 500 000 for both the forms. LI differs from NCIE in the characteristics of the scales, which are large, adherent, dark and pigmented, and in the absence of skin erythema. In NCIE, the scales are fine and white on an erythematous background, although they are larger and grayish on the limbs (3). Overlapping phenotypes have been described, and may depend on the age of the patient and the region of the body. LI is characterized histologically by orthohyperkeratosis and mild focal parakeratosis. Hyperkeratosis associated with an increase in stratum corneum thickness, a normal or prominent granular layer, and increased mitoses suggest a hyperproliferative epidermal defect in NCIE (3). Prominent dermal blood vessels and an upper lymphocytic infiltrate may explain the erythroderma. The terminal differentiation of the epidermis is disturbed in both the forms, leading to a reduced barrier function and defects in the stratum corneum lipid composition (2–5). LI is considered to be a retention ichthyosis, in comparison to NCIE, which is more a hyperproliferative disease (5,6).

ARCI has been shown to be genetically heterogeneous. Genes have been localized in five forms: LI1 (MIM 242300) on chromosome 14q11; LI2 (MIM 601277) on chromosome 2q33–35; LI3 (MIM 604777) on chromosome 19p12–q12; non-lamellar, non-erythrodermic congenital ichthyosis (NNCI) on chromosome 19p13.2–p13.1 (MIM 604781); and LI5 (MIM 606545), also known as NCIE1 (MIM 242100), on chromosome 17p13 (7–11). Four of these genes have been identified to date: transglutaminase 1 (TGM1) for LI1 (12,13), two lipoxygenases (ALOXE3 and ALOX12B) for LI5/NCIE1 (14), and ABCA12 for LI3 (15). Another gene, CGI-58, or ABHD5, has been shown to underlie a syndromic form of NCIE2 (MIM 242100) called Chanarin-Dorfman syndrome (16). A genome-wide scan of two large consanguineous families with three and four affected children, respectively, allowed us to localize a gene on chromosome 5q33. Twelve additional consanguineous families were then found to have ichthyosis linked to chromosome 5q33. By microsatellite genotyping, we refined the linkage interval to 2.3 Mb on the basis of recombination events. Haplotype analysis reduced the interval to 1 Mb. Twelve candidate genes were excluded by sequencing, prior to identification of mutations in a new gene ichthyin. Ichthyin codes for a protein that we propose to be a receptor for a ligand, probably trioxilin A3, from the hepoxilin pathway with R-chirality.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical and histological features and patient origins
We analyzed 14 families comprising 23 patients (12 females and 11 males) and 50 non-affected family members. Individuals for whom DNA was available are shown in Figure 1. All families are consanguineous from first cousin marriages, except two families in which the parents are second cousins (F1) or second cousins once removed (F14) (Table 1). Most families were from Mediterranean countries (eight from Algeria, four from Turkey and one from Syria), and one was from Colombia.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Pedigrees of families F1–F14. Mutations in ichthyin. Sequences are shown in two affected patients from families F4 and F10, in one parent and in one normal control.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Origin of families and mutations
 
The majority of patients (60%) were born as collodion babies except for the patients from four Algerian families (F2, F4, F9 and F14) and two Turkish families (F6 and F8), who presented a clinical picture resembling NCIE. They presented generalized ichthyosis with erythema, fine whitish scaling on the face and trunk, and larger brownish scaling on the neck, buttocks and legs. However, some of the families showed a more lamellar phenotype. Light microscopy of skin biopsies revealed typical histopathological features in the patients corresponding to their phenotype NCIE or LI. Patients with NCIE phenotype showed hyperkeratosis, thickening of the stratum corneum and moderate acanthosis and parakeratosis. We observed a normal or lightly prominent granular layer and a mild dermal perivascular lymphocytic infiltrate. In patients with lamellar phenotype, there was an important orthohyperkeratosis, moderate acanthosis, a normal or reduced granular layer, and a mild papillomatosis with dilatation of dermal capillaries. All the patients presented palmoplantar keratoderma, often yellowish with fissures, and some had clubbing of nails (Fig. 2).



View larger version (121K):
[in this window]
[in a new window]
  		Figure 2. Upper panel: Left, torso of patient (C1) from family F8. The fine grayish scales are especially prominent around the umbilicus and in the axillary region. Right, shoulders and upper back of patient (C3) from family F1. Fine scales are on an erythrodermic background. Middle panel: Left and right, hands of the same patient (C3) from family F1 showing whitish convex nails and typical palmar keratoderma. Bottom panel: Left, dorsal surface of feet of patient (C1) from family F8 showing plantar keratoderma. Right, lower legs and feet of patient (C3) from family F1 showing fine whitish scales on an erythrodermic background. Center, feet from the brother (C2) of the patient (C3) from family F1.

 
Linkage, linkage disequilibrium and haplotype analysis
We first localized the ichthyin gene on chromosome 5q33.2–q34 by homozygosity mapping in two consanguineous families (F1 and F14) with three and four affected individuals. A region of homozygosity between the markers D5S410 (AFM191xd8) and D5S422 (AFM211yc7) was confirmed with a few additional microsatellite markers, prior to screening of our DNA collection to find more families with ichthyosis linked to this new locus. In total 81 families were analyzed; 64 were consanguineous, and 17 were non-consanguineous, but had more than one affected family member. The six known ichthyosis localizations had been previously excluded in all of these families using a panel of 30 microsatellite markers covering the loci on chromosomes 2q33–35, 3p21, 14q11, 17p13, 19p12–q12 and 19p13.2–p13.1 (7–11). In 12 of these families (F2–F13), linkage of ichthyosis to the new localization was found. We then performed fine mapping with a total of 29 microsatellites (Fig. 3) in a 15.1 Mb interval between markers AFM336xe9 (D5S2077) and AFMb351xf9 (D5S2050). The maximum pairwise LOD score at {Theta}=0.00 for D5S378 was 16.38. A co-segregating region of 1 Mb was homozygous in all patients, defined by recombination events with loss of homozygosity in one patient from family 5 for the centromeric marker AFMa083xb9 (D5S2112), and in a patient from family 14 for the telomeric marker AFMb343xe9 (D5S2049). The results of genotyping are presented in Figure 3. Patients from seven families (F5–F11) shared a common haplotype for 2–5 markers (2–7–4–4–3) inside the interval including the gene; these seven families, which had different geographical origins (Algeria, Colombia and Turkey), were later shown to carry the same missense mutation. Three other families from Algeria (F2–F4) exhibited an identical nonsense mutation, but only two of them shared the same haplotype.



View larger version (64K):
[in this window]
[in a new window]
  		Figure 3. Patients' haplotypes and corresponding mutations. Loss of homozygosity is indicated by gray color. Inside the smallest segregating interval between markers a083xb9 and D5S378, mutations and common alleles are in red. The allele number is indicated as 0 when no genotyping result is available. Paternal and maternal alleles for each affected child are presented.

 
Exclusion of candidate genes and identification of mutations in a novel gene ichthyin
Five genes were sequenced in the initial 2.3 Mb interval between the markers UT2159 and AFMb343xe9 (LOC91937, HAVCR1, HAVCR2, CRSP9 and MGC26988) and all seven known genes in the refined 1 Mb interval (CYFIP2, PRO133, ADAM19, SOX30, FLJ20546, FLJ38273 and ENTH). No mutations were found in the coding regions or the exon–intron boundaries of these 12 genes. Therefore, we started to analyze the human mRNAs and ESTs from the GenBank, between the markers AFMa083xb9 (D5S212) and AFMb343xe9 (D5S2049). One mRNA (AK026158 and AF131815) was of particular interest, as it was highly expressed in epidermal tissues, including skin and keratinocytes. Sequencing of the corresponding gene revealed six different mutations in patients with ARCI.

Structure of the ichthyin gene and its cDNA
The longest mRNA (XM_351632) predicted by automatic analysis, corresponding to the ichthyin gene (LOC348938 DUF803.2), consists of a 3157 bp sequence and codes for a protein of 466 amino acids (XP_351633). This sequence was subsequently removed from the database and a new predicted sequence (XM_371777) of 3077 bp was proposed corresponding to a cDNA (AK026158 and FLJ22505) of 3094 bp, which codes for a protein of 404 amino acids. BLAST analysis between this mRNA and the BAC sequence AC008676 supported the existence of six exons as described in other public databases. We performed 5'-rapid amplification of cDNA ends (RACE) with five different primers and subcloned some of these 5'-RACE products, but we failed to identify any elongation of the 5' end. The sequence was checked by overlapping RT–PCR, and the products were sequenced and compared with the sequences from public databases. BLAST analysis revealed one mouse (NM_172524) and one rat mRNA ortholog (XM_220330) showing homologies of 83 and 84% for the nucleotide sequence, and of 74 and 75% for the protein sequence, respectively. The mouse mRNA of 3299 bp is reported to be full-length, whereas the rat mRNA is only 1272 bp long. Multiple nucleotide alignments (http://prodes.toulouse.inra.fr/multalin/) of orthologs from human, mouse and rat showed also a highly conserved coding sequence with a homology of around 80%.

Mutation analysis of the ichthyin gene
Sequencing of the six exons and of the exon–intron boundaries of the ichthyin gene revealed six different homozygous mutations in the 14 consanguineous families (Table 1). One was a nonsense mutation and five were missense mutations: 247C->T (R83X) and 239G->T (G80V) in exon 2; 341C->A (A114N) in exon 4; 437C->T (S146F) and 523C->G (H175N) in exon 5; and 703G->A (G235R) in exon 6. The mutations were all situated in parts of the gene that are highly conserved between mice, rats and humans. None of these sequence variations was found in 100 normal chromosomes from a Mediterranean control population.

Expression analysis
We analyzed tissue expression by ichthyin-specific RT–PCR with primer pairs RT_4 (Table 2) for a 1111 bp fragment using the RAPID-SCANTM gene expression panel and RNAs from cultured keratinocytes, fibroblasts, placenta and lymphocytes (data not shown). The ichthyin transcript was found to be expressed in most tissues tested; it was highly expressed in brain, lung, stomach, skin and leucocytes, and was present at a lower level in the other tissues except liver, thyroid and fetal brain, in which no expression was detectable. Strong expression of ichthyin transcripts was observed in cultured keratinocytes from normal skin biopsies; expression was weaker in cultured fibroblasts from the same skin biopsies, in placenta and in lymphocytes.


View this table:
[in this window]
[in a new window]
  		Table 2. Primer sequences for ichthyin and paralogs: exon amplification and RT–PCR
 
Sequence analysis of the ichthyin protein and identification of conserved residues
The sequence of 404 amino acids corresponds to a protein with a calculated molecular weight of 44 kDa. This sequence was submitted to the topology prediction programs for sequence signals, transmembrane (TM) domains and protein orientation available through the ExPASy server (www.expasy.org), to the protein analyzing tools from the Biology WorkBench (http://biowb.sdsc.edu/) and to the homology searches through protein databases (http://www.ncbi.nlm.nih.gov). The protein is predicted to localize in the plasma membrane, but does not possess a signal sequence. The number of predicted TM domains was eight using the PSORTII program (http://psort.nibb.ac.jp/form2.html) and nine using the SMART program (http://smart.embl-heidelberg.de/); each of these domains consists of 22 amino acids. This structure defines a new family of proteins of unknown function (PFAM: DUF803). By the Dense Alignment Surface (DAS) method (http://www.sbc.su.se/~miklos/DAS/) (17), only seven TM domains were identified; one TM region (amino acids 162–168) was considered too small, as it consisted of only seven amino acids in humans and only three amino acids in the mouse ortholog. It was identified as a secondary hydrophilic TM helix using SOSUI analysis (http://sosui.proteome.bio.tuat.ac.jp/) owing to the absence of amphophilic side chains at the helix ends, which is one of the two arguments used (together with hydrophobicity) to define a TM domain (18). The BPROMPT (http://www.jenner.ac.uk/BPROMPT/) program also predicted seven TM domains for ichthyin. Analysis using the ProtFun2.1 server (http://www.cbs.dtu.dk/services/ProtFun/) classifies ichthyin in functional and gene ontology categories under transport and binding activities.

A new family of proteins with several TM domains
BLAST analysis identified five human paralogs of ichthyin (listed later), of which the first three are the most closely related to ichthyin, with up to 57% homology. Four of these genes have the same basic structure and have 5–8 exons: (1) NIPA1 (Non-Imprinted in Prader–Willi/Angelman syndrome 1) or spastic paraplegia (SPG6) on chromosome 15q11.2; (2) NIPA2 (DUF803.0) also on chromosome 15q11.2; (3) LOC152519 (DUF803.3) on chromosome 4p12; (4) FLJ13955 (DUF803.1) on chromosome 8q22.2; and (5) dJ462023.2 (DUF803.4) on chromosome 1p36.12–p35.1. The FLJ13955 and dJ462023.2 genes are predicted to have 12 exons. Some of these sequences were annotated by automatic analysis pipelines such as GeneWise, Genscan and Gnomon, and could be either too long or incomplete, with the exception of NIPA1 and 2, which have been more extensively analyzed (19,20). Two transcripts were found for NIPA1, one of 2.2 kb and a larger one of 7.5 kb with a long 3'-untranslated region (UTR) which is present at low levels in most tissues, but at higher levels in neuronal tissues (19).

Fifty-two homologues were found in eukaryotes including Caenorhabditis elegans, Drosophila, mice and plants (IPR008521; http://ebi.ac.uk/interpro/). Weaker BLASTP homologies were also found with a hypothetical transport protein in Pseudomonas denitrificans (ORF6; SW:YCB6_PSEDE), and with a C. elegans chemoreceptor with seven TM domains of the sri family (WP:CE33629) (21).

Multiple amino acid alignment confirms both the high homology and the conservation of the positions of the TM regions of the six proteins of the family.

The phylogenetic tree (Tree Top, http://www.genebee.msu.su/services/phtree_reduced.html) of the multiple sequence alignments (CLUSTALW) of the six human DUF803 proteins and 347 human G-protein coupled receptor (GPCR) protein sequences from the GPCR database (GPCRDB, May 2003 release; http://www.gpcr.org/7tm/) identified DUF803 proteins as a branch between glutamate and frizzled/taste2 receptors (22).

The mutations identified in ichthyin include a nonsense mutation in the second exon, which would lead to the synthesis of a truncated protein. Four of the five missense mutations were situated inside one of the predicted TM domains, in the second (aa80), the third (aa146), the fourth (aa175) and the sixth (aa235). The fifth mutation (aa114) is located between the second and third TM domains.

Genotyping on chromosomes 15q11.2 and 8q22.2, and mutation analysis of NIPA2 and FLJ13955 in families in which other known loci had been excluded
Two of the ichthyin paralogs, NIPA2 and FLJ13955, have been described to be strongly expressed in keratinocytes (SAGE). In order to test linkage, the localizations containing these paralogs in a form of ARCI were genotyped in 65 consanguineous ichthyosis families (226 individuals including 103 affected family members). Potential linkage to chromosome 15q11.2 or 8q22.2 was suggested in 12 families, but when the two candidate genes were sequenced for the exons and the exon–intron boundaries, no mutations were found.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The identification of mutations in a new gene with several TM domains, in patients with ARCI is a landmark in the analysis of this group of disorders, for which both low-incidence and wide clinical and genetic heterogeneity have made it difficult to develop a classification scheme using clinical (2,3,6), biochemical (5,6,23) and ultrastructural criteria (24). This supports a metabolic origin of ARCI, which had already been suspected after two genes from the lipoxygenase family (ALOX12B and ALOXE3) were found to be mutated in this disorder (14). This finding was extended by the identification of ALOXE3 as a hydroperoxide isomerase (25). Two other genes which are mutated in ichthyoses, CGI-58 or abhydrolase 5 (ABHD5) (12,13,16) and the transporter ABCA12 (15), could belong to the hepoxilin metabolic pathway, in which membrane arachidonic acid is transformed to hepoxilin, trioxilin and derivatives of R-chirality (26,27). The phenotype of this ARCI caused by mutations in ichthyin is similar to that previously described for other ARCIs, in which the majority of patients present a NCIE phenotype. Not all ARCI patients are born as collodion babies; this was the case in nine of our patients, from four Algerian and two Turkish families.

As was the case for the five genes previously identified in ARCI and in Chanarin-Dorfman syndrome (12–16), homozygosity mapping turned out to be a powerful method for localization of the causative genes. The main difficulty in the present study was the identification of the gene, which necessitated the sequencing of all the genes present in the refined interval, and a search for new genes, which were suspected only by the presence of cDNAs.

A precise characterization of the structure of this novel gene by RT–PCR and 5'- and 3'-RACE was necessary. Moreover, we expected a classical protein of the large GPCRs family with seven TM domains.

The nine TM domains proposed for ichthyin by SMART, which, according to PFAM, define a new family of proteins, which has been designated as DUF803. The nine TM domains are not always found in other proteins of this family, or in the homologs in other species or by using other topology TM prediction programs. For example, only eight TM domains are predicted by SMART in the ichthyin homologue in mouse, with 406 amino acids, and a signal sequence of 30 amino acids. Other topology programs such as DAS or BPROMPT predict only seven TM domains for ichthyin as is the case for NIPA1 and NIPA2, and eight TM domains, instead of nine, for the three other proteins of the family. The eighth supplementary predicted TM domain is localized in the N-terminal portion of the protein, which normally contains the signal sequence. The difficulty in differentiating between a signal sequence and a TM domain (28,29), as well as the discrepancies in the number of TM domains found between topology prediction programs, are well known (30). With its 7–9 TM domains, ichthyin is clearly membrane-associated, but it is not yet possible to determine whether it is a GPCR (GPCRs have seven TM domains, and 10% of them have signal sequences), or whether it is localized in the plasma membrane or in one of the intracellular membrane systems. However, no sequences targeting internal membranes were detected. Even for the extensively studied proteins like presenilin 1, which was assumed to possess 10 hydrophobic regions, recent immunofluorescent studies with specific antibodies to the C- and N-tails led the authors to propose a new model with only seven TM domains (31).

The DUF803 family includes a group of membrane proteins of unknown function, recently revealed by the identification of a heterozygous mutations in NIPA1 (T45R) in two families (20), in one of the numerous types of autosomal dominant spastic paraplegia. Owing to the presence of several TM domains, this protein has been proposed to be a transporter or a receptor for an unknown ligand (19,20). A transporter of the ABC family, ABCA12, has already been found to be mutated in one form of ARCI (15). By analogy with the predicted function of ichthyin, NIPA1, which is a homolog, is proposed to be a receptor for the ligand from the hepoxilin pathway (trioxilin B3). The heterozygous mutation found in NIPA1 could correspond to a dominant negative mutation of this receptor, as observed for several GPCR proteins.

Two receptors for each ligand of the leukotriene pathway, and for LXA4 of the lipoxin pathway, which are parallel to the hepoxilin pathway, have been identified to date: LTB4R and LTB4R2 for LTB4; CYSLTR1 and CYSLTR2 for LTC4/LTD4/LTE4; and FPRL1 and 2 for LXA4 (32,33). NIPA2 is the gene with the closest homology to ichthyin. No mutations have been found in this gene in SPG6, at least in the families studied to date (20). We sequenced the paralogs of ichthyin, in the families with ichthyosis which are not linked to the seven loci identified to date, but found no mutations in NIPA2 (DUF803.0) and FLJ13955 (DUF803.1).

A stable hepoxilin analog, PBT-3, was found to selectively inhibit the binding of antagonists of the thromboxane {alpha} receptor (34), but there is no homology between the thromboxane {alpha} receptor and ichthyin.

The suspicion that ichthyin is a membrane receptor of the hepoxilin pathway offers strong opportunities to provide simple treatments to patients. This type of protein is easily accessible and it should be possible to synthesize both agonists and antagonists with high specificity and sensitivity in the ligand–receptor interactions. It should be noted that these applications could also be useful in the treatment of dry skin, which is far more frequent than ARCI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects and samples
Four dermatologists (B. Bouadjar, M.-J. Rueda, A. Karaduman and M. Nourallah) recorded the clinical data and pedigree information of the families. Blood samples were collected from each participating family member after obtaining written informed consent. DNA extraction from peripheral blood leukocytes and establishment of cell lines were performed at the DNA bank of Généthon using standard procedures.

Genetic analysis
Genotyping was carried out using 400 highly polymorphic microsatellite markers from the ABI panel (Linkage Mapping Set2, LMS2, Applied Biosystems) and a MegaBase capillary sequencer for the genome-wide scan. ABI 377 sequencers were used for fine mapping with publicly available microsatellites. Haplotypes were constructed assuming the most parsimonious linkage phase. Linkage programs were used on the basis of the assumption of autosomal recessive inheritance, full penetrance and a disease frequency of 1 in 500 000 in the general population. Pairwise LOD scores were calculated with the MLINK program of the LINKAGE 5.1 package (37) incorporating consanguineous loops into the pedigree files.

Mutation screening
Mutation analysis was performed in affected patients and in both parents in the 14 families, and in supplementary non-affected sibs in cases of missing parents. We designed intronic oligonucleotide primers flanking the exons for amplification and sequencing of the ichthyin gene (Table 2) using the Primer3 program (http://www-genome.wi.mit.edu/genome_ software/other/primer3.html) (38). Sets of PCR conditions were used as indicated in Table 2. The touch-down PCR reaction was performed in a 45 µl volume containing 50 ng of genomic DNA (in 5 µl) with Hot MasterTM Taq DNA polymerase (Eppendorf): initial denaturation step at 95°C for 5 min, 6 cycles of amplification consisting of 40 s at 94°C, 30 s 68°C, and a 30 s elongation step at 72°C, followed by 30 cycles of 40 s at 94°C, 30 s at optimal annealing temperature, 30 s at 72°C, and a 5 min terminal elongation step. Aliquots of 1–2 µl of purified PCR products were added to 0.5 µl of sense or antisense primer (20 µM) and 2 µl of BigDye terminator mix (Applied Biosystems) in a 15 µl volume. The linear amplification consisted of an initial 5 min denaturation step at 96°C, 25 cycles of 10 s of denaturation at 96°C and a 4 min annealing–extension step at 56–60°C. The reaction products were purified and sequenced on an Applied Biosystems Sequencer 3700. The forward or reverse strands from all patients and controls were sequenced for the entire coding region and the exon–intron boundaries. The sequences were analyzed with the Phred Phrap program on Unix.

Lymphoblastoid, keratinocyte and fibroblast cell cultures, and RNA extraction
Lymphoblastoid cell lines were established using standard procedures. Total RNA from lymphocytes was extracted with the RNA-PLUS (Quantum-Appligene) kit following the manufacturer's instructions. Human keratinocytes and fibroblasts were obtained from the skin removed during routine plastic surgery of a normal individual. The skin sample was processed for primary keratinocyte culture, and the cells were grown according to the standard procedure described by Invitrogen Life Technologies, using products from the same company in serum-free keratinocyte medium supplemented with bovine pituitary extract (25 µg/ml) and recombinant epidermal growth factor (0.1 ng/ml). For primary fibroblast culture we used Dulbecco's Modified Eagle's Medium with 10% fetal calf serum and 2% L-glutamine. Cultures were grown for two passages and were harvested when they reached 90% confluence. Total RNA was isolated using the QIAamp RNA Mini Protocol for isolation of total RNA from cultured cells (QIAGEN) following the manufacturer's instructions. The mRNA was isolated following the Oligotex direct mRNA protocol as provided by the manufacturer (QIAGEN).

5'-RACE and cloning of the amplification products
5'-RACE was performed with Marathon-Ready cDNA from melanoma, placenta and stomach (Clontech) using the Advantage 2 Polymerase Mix (Clontech) following the manufacturer's instructions. The PCR was run with AP1 primer and one of the five gene-specific antisense primers which we designed from the cDNA sequence, starting with an initial denaturation step at 94°C for 1 min, 30 cycles at 94°C for 30 s and 68°C for 4 min. To visualize the PCR products, they were loaded on a 2% agarose gel.

RT–PCR and Rapid-ScanTM gene expression panel
RT–PCR was performed using the RT–PCR kit (Life Technologies) with oligo-dT primers to generate the first strand of cDNA. Amplification of cDNA from keratinocytes, fibroblasts, placenta and lymphocytes was performed with four primer pairs (Table 2) covering the entire coding region, the 3'- and 5'-UTR regions.

A gene expression panel including 24 human tissues was tested following the manufacturer's instructions (Rapid-Scan, OriGene Technologies) using primer pair RT_4 which is specific for the ichthyin transcript. Expression in keratinocytes, fibroblasts, placenta and lymphocytes was also tested.

Accession numbers
Ichthyin.
LocusID LOC348938; mRNA XM_37177 (GI:42657191); AK026158 (GI:10438919); protein XP_351633 (37549911).

Ichthyin orthologs.
Mouse: 9530066K23Rik, LocusID 214112; mouse mRNA NM_172524 (GI:27369725) identical to AK035561 (GI:26330753); mouse protein: NP_766112 (27369726); identical to BAC29107 (GI:26330754). Rat: LocusID LOC303070; mRNA XM_220330.2 (GI:34870677); XM_220330.1 (GI:27669704, record removed); rat XP_220330.2 (GI:34870678); XP_220330.1 (GI:27669704, record removed).

Paralogs.
Chromosome 15q11.2 NIPA2 (non-imprinted in Prader–Willi/Angelman syndrome 2), LocusID 81614; mRNA NM_030922 or BC011775 (GI:33989168). Chromosome 8q22.2 FLJ13955. LocusID 79815; NM_024759 (GI:13376096) identical to AK024017 (GI:10436266); protein NP_079035 (GI:13376097) identical to BAB14779 (GI:10436267)

Online Mendelian inheritance in man (http://www.ncbi.nlm. nih.gov/Omim).
Abhydrolase domain containing 5 (ABHD5), previously CGI58, Comparative Gene Identification 58 [CGI58; MIM 604780]; arachidonate lipoxygenase 3 [ALOXE3; MIM 607206]; arachidonate 12-lipoxygenase, R type [ALOX12B; MIM 603741]; ATP-binding cassette, subfamily A, member 12 [ABCA12; MIM 607800]; Chanarin-Dorfman syndrome [CDS; MIM 275630]; cysteinyl leukotriene receptor 1 [CYSLTR1; MIM 300201]; cysteinyl leukotriene receptor 2 [CYSLTR2; MIM 605666]; ichthyosis non-lamellar and non-erythrodermic congenital [NNCI; MIM 604781]; lamellar ichthyosis [LI; MIM 242300]; lamellar ichthyosis 1 [LI1; MIM 604777]; lamellar ichthyosis 2 [LI2; MIM 601277]; lamellar ichthyosis 3 [LI3; MIM 604777]; lamellar ichthyosis 5 [LI5; MIM 606545]; leukotriene B4 receptor [LTB4R; MIM 601531]; leukotriene B4 receptor 2 [LTB4R2; MIM 605773]; non-bullous ichthyosiform erythroderma [NCIE1, MIM 242100]; non-bullous ichthyosiform erythroderma [NCIE2, MIM 604780]; non-imprinted gene in Prader–Willi/Angelman syndrome chromosome region 1 [NIPA1; MIM 6008145]; non-imprinted gene in Prader–Willi/Angelman syndrome chromosome region 2 [NIPA2; MIM 6008146]; Presenilin 1 [PSEN1; MIM 104311]; transglutaminase 1 [TGM1; MIM 190195]; Spastic paraplegia 6, autosomal dominant [SPG6; MIM 600363].


   ACKNOWLEDGEMENTS
 
We wish to thank the members of the families for their participation in this study and Dr Rueda and Dr Nourallah who identified two of these families. We are grateful to Patrick Danoy for help in cloning techniques, and to Susan Cure for help in writing this manuscript. We would like to acknowledge the technical support of the Généthon DNA bank. This study was supported by the Centre National de Génotypage (CNG), the GIS-Institut des Maladies Rares, the Association Française contre les Myopathies (AFM) and Généthon.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +33 160878357; Fax: +33 160878383; Email: fischer{at}cng.fr


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Williams, M.L. and Elias, P.M. (1985) Heterogeneity in autosomal recessive ichthyosis. Clinical and biochemical differentiation of lamellar ichthyosis and nonbullous congenital ichthyosiform erythroderma. Arch. Dermatol., 121, 477–488.[Abstract/Free Full Text]

  2. Traupe, H. (1989) The Ichthyoses: A Guide to Clinical Diagnosis, Genetic Counseling, and Therapy. Springer, Heidelberg, Germany.

  3. Griffiths, W.A.D., Judge, M.R. and Leigh, I.M. (1998) Disorders of keratinization. In Champion, R.H., Burton, J.L., Burns, D.A. and Breathnach, S.M. (eds), Rook/Wilkinson/Ebling: Textbook of Dematology. Blackwell Science, Oxford, UK, pp. 1483–1530.

  4. Lavrijsen, A.P., Bouwstra, J.A., Gooris, G.S., Weerheim, A., Bodde, H.E. and Ponec, M. (1995) Reduced skin barrier function parallels abnormal stratum corneum lipid organization in patients with lamellar ichthyosis. J. Invest. Dermatol., 105, 619–624.[CrossRef][Web of Science][Medline]

  5. Melnick, B. (1989) Epidermal lipids and the biochemistry of keratinization. In Traupe, H. (ed.), The Ichthyoses: A Guide to Clinical Diagnosis, Genetic Counseling, and Therapy. Springer, Heidelberg, Germany, pp. 14–42.

  6. Hazell, M. and Marks, R. (1985) Clinical, histologic, and cell kinetic discriminants between lamellar ichthyosis and nonbullous congenital ichthyosiform erythroderma. Arch. Dermatol., 121, 489–493.[Abstract/Free Full Text]

  7. Russell, L.J., DiGiovanna, J.J., Hashem, N., Compton, J.G. and Bale, S.J. (1994) Linkage of autosomal recessive lamellar ichthyosis to chromosome 14q. Am. J. Hum. Genet., 55, 1146–1152.[Web of Science][Medline]

  8. Parmentier, L., Lakhdar, H., Blanchet-Bardon, C., Marchand, S., Dubertret, L. and Weissenbach, J. (1996) Mapping of a second locus for lamellar ichthyosis to chromosome 2q33–35. [Erratum (1996) Hum. Mol. Genet., 5, 862–863.] Hum. Mol. Genet., 5, 555–559.[Abstract/Free Full Text]

  9. Fischer, J., Faure, A., Bouadjar, B., Blanchet-Bardon, C., Karaduman, A., Thomas, I., Emre, S., Cure, S., Özgüc, M., Weissenbach, J. et al. (2000) Two new loci for autosomal recessive ichthyosis on chromosomes 3p21 and 19p12–q12 and evidence for further genetic heterogeneity. Am. J. Hum. Genet., 66, 904–913.[CrossRef][Web of Science][Medline]

  10. Krebsova, A., Kuster, W., Lestringant, G.G., Schulze, B., Hinz, B., Frossard, P.M., Reis, A. and Hennies, H.C. (2001) Identification, by homozygosity mapping, of a novel locus for autosomal recessive congenital ichthyosis on chromosome 17p, and evidence for further genetic heterogeneity. Am. J. Hum. Genet., 69, 216–222.[CrossRef][Medline]

  11. Virolainen, E., Wessman, M., Hovatta, I., Niemi, K.M., Ignatius, J., Kere, J., Peltonen, L. and Palotie, A. (2000) Assignment of a novel locus for autosomal recessive congenital ichthyosis to chromosome 19p13.1–p13.2. Am. J. Hum. Genet., 66, 1132–1137.[CrossRef][Web of Science][Medline]

  12. Huber, M., Rettler, I., Bernasconi, K., Frenk, E., Lavrijsen, S.P., Ponec, M., Bon, A., Lautenschlager, S., Schorderet, D.F. and Hohl, D. (1995) Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science, 267, 525–528.[Abstract/Free Full Text]

  13. Russell, L.J., DiGiovanna, J.J., Rogers, G.R., Steinert, P.M., Hashem, N., Compton, J.G. and Bale, S.J. (1995) Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis. Nat. Genet., 9, 279–283.[CrossRef][Web of Science][Medline]

  14. Jobard, F., Lefèvre, C., Karaduman, A., Blanchet-Bardon, C., Emre, S., Weissenbach, J., Özgüc, M., Lathrop, M., Prud'homme, J.F. and Fischer, J. (2002) Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1. Hum. Mol. Genet., 11, 107–113.[Abstract/Free Full Text]

  15. Lefevre, C., Audebert, S., Jobard, F., Bouadjar, B., Lakhdar, H., Boughdene-Stambouli, O., Blanchet-Bardon, C., Heilig, R., Foglio, M., Weissenbach J. et al. (2003) Mutations in the transporter ABCA12 are associated with lamellar ichthyosis type 2. Hum. Mol. Genet., 12, 2369–2378.[Abstract/Free Full Text]

  16. Lefevre, C., Jobard, F., Caux, F., Bouadjar, B., Karaduman, A., Heilig, R., Lakhdar, H., Wollenberg, A., Verret, J.L., Weissenbach J. et al. (2001) Mutations in CGI–58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin–Dorfman syndrome. Am. J. Hum. Genet., 69, 1002–1012.

  17. Cserzo, M., Wallin, E., Simon, I., von Heijne, G. and Elofsson, A. (1997) Prediction of transmembrane alpha-helices in procariotic membrane proteins: the Dense Alignment Surface method. Protein Eng., 10, 673–676.[Abstract/Free Full Text]

  18. Hirokawa, T., Boon-Chieng, S. and Mitaku, S. (1998) SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics, 14, 378–379.[Abstract/Free Full Text]

  19. Chai, J.H., Locke, D.P., Greally, J.M., Knoll, J.H., Ohta, T., Dunai, J., Yavor, A., Eichler, E.E. and Nicholls, R.D. (2003) Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader–Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons. Am. J. Hum. Genet., 73, 898–925.[CrossRef][Web of Science][Medline]

  20. Rainier, S., Chai, J.H., Tokarz, D., Nicholls, R.D. and Fink, J.K. (2003) NIPA1 gene mutations cause autosomal dominant hereditary spastic paraplegia (SPG6). Am. J. Hum. Genet., 73, 967–971.[CrossRef][Web of Science][Medline]

  21. Crouzet, J., Levy-Schil, S., Cameron, B., Cauchois, L., Rigault, S., Rouyez, M.C., Blanche, F., Debussche, L., Thibaut, D. (1991) Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase. J. Bacteriol., 173, 6074–6087.[Abstract/Free Full Text]

  22. Fredriksson, R., Lagerstrom, M.C., Lundin, L.G. and Schioth, H.B. (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol., 63, 1256–1272.[Abstract/Free Full Text]

  23. Bergers, M., Traupe, H., Dunnwald, S.C., Mier, P.D., van Dooren-Greebe, R., Steijlen, P. and Happle, R. (1990) Enzymatic distinction between two subgroups of autosomal recessive lamellar ichthyosis. J. Invest. Dermatol., 94, 407–412.[CrossRef][Web of Science][Medline]

  24. Ghadially, R., Williams, M.L., Hou, S.Y. and Elias, P.M. (1992) Membrane structural abnormalities in the stratum corneum of the autosomal recessive ichthyoses. J. Invest. Dermatol., 99, 755–763.[CrossRef][Web of Science][Medline]

  25. Yu, Z., Schneider, C., Boeglin, W.E., Marnett, L.J. and Brash, A.R. (2003) The lipoxygenase gene ALOXE3 implicated in skin differentiation encodes a hydroperoxide isomerase. Proc. Natl Acad. Sci. USA, 100, 9162–9167.[Abstract/Free Full Text]

  26. Pace-Asciak, C.R. (1994) Hepoxilins: a review on their cellular actions. Biochim. Biophys. Acta., 1215, 1–8.[Medline]

  27. Pace-Asciak, C.R., Reynaud, D., Demin, P. and Nigam, S. (1999) The hepoxilins. A review. Adv. Exp. Med. Biol., 447, 123–132.[Web of Science][Medline]

  28. Chen, C.P., Kernytsky, A. and Rost, B. (2002) Transmembrane helix predictions revisited. Protein Sci., 11, 2774–2791.[CrossRef][Web of Science][Medline]

  29. Lao, D.M., Okuno, T. and Shimizu, T. (2002) Evaluating transmembrane topology prediction methods for the effect of signal peptide in topology prediction. In Silico Biol., 2, 485–494.[Medline]

  30. Wallin, E. and von Heijne, G. (1995) Properties of N-terminal tails in G-protein coupled receptors: a statistical study. Prot. Eng., 8, 693–698.[Abstract/Free Full Text]

  31. Kim, J. and Schekman, R. (2004) The ins and outs of presenilin 1 membrane topology. Proc. Natl Acad. Sci. USA, 101, 905–906.[Free Full Text]

  32. Brink, C., Dahlen, S.E., Drazen, J., Evans, J.F., Hay, D.W., Nicosia, S., Serhan, C.N., Shimizu, T. and Yokomizo, T. (2003) International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors. Pharmacol. Rev., 55, 195–227.[Abstract/Free Full Text]

  33. Funk, C.D. (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 294, 1871–1875.[Abstract/Free Full Text]

  34. Qiao, N., Reynaud, D., Demin, P., Halushka, P.V. and Pace-Asciak, C.R. (2003) The thromboxane receptor antagonist PBT-3, a hepoxilin stable analog, selectively antagonizes the TPalpha isoform in transfected COS-7 cells. J. Pharmacol. Exp. Ther., 307, 1142–1147.[Abstract/Free Full Text]

  35. Drews, J. (2000) Drug discovery: a historical perspective. Science, 287, 1960–1964.[Abstract/Free Full Text]

  36. Lathrop, G.M. and Lalouel, J.-M. (1984) Easy calculations of lod scores and genetic risks on small computers. Am. J. Hum. Genet., 36, 460–465.[Web of Science][Medline]

  37. Rozen, S. and Skaletsky, H.J. (2000) Primer3 on the WWW for general users and for biologist programmers. In Krawetz, S. and Misener, S. (eds), Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp. 365–386.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Med. Genet.Home page
S Farasat, M-H Wei, M Herman, D J Liewehr, S M Steinberg, S J Bale, P Fleckman, and J R Toro
Novel transglutaminase-1 mutations and genotype-phenotype investigations of 104 patients with autosomal recessive congenital ichthyosis in the USA
J. Med. Genet., February 1, 2009; 46(2): 103 - 111.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
P. M. Elias, M. L. Williams, W. M. Holleran, Y. J. Jiang, and M. Schmuth
Thematic review series: Skin Lipids. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism
J. Lipid Res., April 1, 2008; 49(4): 697 - 714.
[Abstract] [Full Text] [PDF]

Home page
 Arch DermatolHome page
M. Harting, N. Brunetti-Pierri, C. S. Chan, J. Kirby, M. K. Dishop, G. Richard, F. Scaglia, A. C. Yan, and M. L. Levy
Self-Healing Collodion Membrane and Mild Nonbullous Congenital Ichthyosiform Erythroderma Due to 2 Novel Mutations in the ALOX12B Gene
Arch Dermatol, March 1, 2008; 144(3): 351 - 356.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. M. Gauthier, Y. Chawengsub, D. H. Goldman, R. E. Conrow, S. Anjaiah, J. R. Falck, and W. B. Campbell
11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1467 - H1472.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
J Dahlqvist, J Klar, I Hausser, I Anton-Lamprecht, M H. Pigg, T Gedde-Dahl Jr, A Ganemo, A Vahlquist, and N Dahl
Congenital ichthyosis: mutations in ichthyin are associated with specific structural abnormalities in the granular layer of epidermis
J. Med. Genet., October 1, 2007; 44(10): 615 - 620.
[Abstract] [Full Text] [PDF]

Home page
 J Child NeurolHome page
R. Bien-Willner, N. Sambuughin, H. Holley, J. Bodensteiner, and K. Sivakumar
Childhood-Onset Spastic Paraplegia With NIPA1 Gene Mutation
J Child Neurol, November 1, 2006; 21(11): 974 - 977.
[Abstract] [PDF]

Home page
 Hum Mol GenetHome page
V. Oji, J. M. Hautier, B. Ahvazi, I. Hausser, K. Aufenvenne, T. Walker, N. Seller, P. M. Steijlen, W. Kuster, A. Hovnanian, et al.
Bathing suit ichthyosis is caused by transglutaminase-1 deficiency: evidence for a temperature-sensitive phenotype
Hum. Mol. Genet., November 1, 2006; 15(21): 3083 - 3097.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
C. Lefevre, B. Bouadjar, V. Ferrand, G. Tadini, A. Megarbane, M. Lathrop, J.-F. Prud'homme, and J. Fischer
Mutations in a new cytochrome P450 gene in lamellar ichthyosis type 3
Hum. Mol. Genet., March 1, 2006; 15(5): 767 - 776.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/20/2473    most recent
ddh263v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (37)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lefèvre, C.
Right arrow Articles by Fischer, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lefèvre, C.
Right arrow Articles by Fischer, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?