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Yohya Shigehara, Shujiro Okuda, Georges Nemer, Adele Chedraoui, Ryota Hayashi, Fadi Bitar, Hiroyuki Nakai, Ossama Abbas, Laetitia Daou, Riichiro Abe, Maria Bou Sleiman, Abdul Ghani Kibbi, Mazen Kurban, Yutaka Shimomura, Mutations in SDR9C7 gene encoding an enzyme for vitamin A metabolism underlie autosomal recessive congenital ichthyosis, Human Molecular Genetics, Volume 25, Issue 20, 15 October 2016, Pages 4484–4493, https://doi.org/10.1093/hmg/ddw277
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Abstract
Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of hereditary skin disorder characterized by an aberrant cornification of the epidermis. ARCI is classified into a total of 11 subtypes (ARCI1-ARCI11) based on their causative genes or loci. Of these, the causative gene for only ARCI7 has not been identified, while it was previously mapped on chromosome 12p11.2-q13.1. In this study, we performed genetic analyses for three Lebanese families with ARCI, and successfully determined the linkage interval to 9.47 Mb region on chromosome 12q13.13-q14.1, which was unexpectedly outside of the ARCI7 locus. Whole-exome sequencing and the subsequent Sanger sequencing led to the identification of missense mutations in short chain dehydrogenase/reductase family 9C, member 7 (SDR9C7) gene on chromosome 12q13.3, i.e. two families shared an identical homozygous mutation c.599T > C (p.Ile200Thr) and one family had another homozygous mutation c.214C > T (p.Arg72Trp). In cultured cells, expression of both the mutant SDR9C7 proteins was markedly reduced as compared to wild-type protein, suggesting that the mutations severely affected a stability of the protein. In normal human skin, the SDR9C7 was abundantly expressed in granular and cornified layers of the epidermis. By contrast, in a patient’s skin, its expression in the cornified layer was significantly decreased. It has previously been reported that SDR9C7 is an enzyme to convert retinal into retinol. Therefore, our study not only adds a new gene responsible for ARCI, but also further suggests a potential role of vitamin A metabolism in terminal differentiation of the epidermis in humans.
Introduction
Autosomal recessive congenital ichthyosis (ARCI) is a genetically heterogeneous disorder of epidermal keratinization of the skin. ARCI is clinically characterized by dryness and scaling of the whole body surface. Pathologically, the conditions are considered to be due to dysfunction of underlying keratinocyte differentiation, which results in the characteristic distinctive scaling skin (1). The clinical conditions vary according to each type of the ichthyosis, while it is often too hard to determine the causative gene based on clinical features. To date, a total of 10 genes responsible for ARCI have been identified, which are TGM1 (ARCI1; MIM 242300) (2,3), ALOX12B (ARCI2; MIM 242100) (4), ALOXE3 (ARCI3; MIM 606545) (4), ABCA12 (ARCI4; MIM 601277/242500) (5,6), CYP4F22 (ARCI5; MIM 604777) (7), NIPAL4 (ARCI6; MIM 612281) (8), LIPN (ARCI8; MIM 613943) (9), CERS3 (ARCI9; MIM 615023) (10), PNPLA1 (ARCI10; MIM 615024) (11), and ST14 (ARCI11; MIM 602400) (12). In addition, ARCI has also previously been mapped on chromosome 12p11.2-q13 (ARCI7; MIM 615022); the causative gene, however, has not yet been reported (13).
In the present study, we performed extensive genetic analyses for three consanguineous Lebanese families with ARCI (Families 1–3). We mapped the susceptible locus of these families to chromosome 12q13.13-q14.1, which was just outside of the ARCI7 locus. Through whole-exome sequencing and Sanger sequencing, we identified missense mutations in short chain dehydrogenase/reductase family 9C, member 7 (SDR9C7) gene on chromosome 12q13.3 in all the three families analyzed. Furthermore, we investigated how the mutations affected expression of SDR9C7 protein both in vitro and in vivo.
Results
Clinical description

Clinical and histological features of patients with ARCI. (A–H) Affected individuals in all the three families have had dry skin with large scales on the whole body surface, hyperkeratosis over elbows and knees, palmoplantar keratoderma and recurrent dermatophytic infections of the skin and onychomycosis. (A,B,F) 10-year-old girl in Family 1. (C,D) 9-year-old girl in Family 2. (E) 19-year-old male in Family 3. (G)(H) 13-year-old girl in Family 1. (I) Histological features of a skin biopsy from the affected individual of Family 2 was compatible for lamellar ichthyosis. Hematoxylin-eosin staining. Scale bar: 100 μm.
Results of initial haplotype analysis and whole-exome sequencing
Using the genomic DNA of the family members as templates, we performed haplotype analysis with microsatellite markers around all the 10 known genes for ARCI. None of the families, however, showed linkage to any of these genes (data not shown). We then forwarded to analyze the ARCI7 locus on chromosome 12 for these families. We initially found that affected individuals in Family 1 finely showed a homozygous haplotype within and around the ARCI7 locus (Supplementary Material, Fig. S1). On the other hand, Families 2 and 3 were excluded from the ARCI7 locus (data not shown). Based on recombination events, the linkage interval of Family 1 was defined to be 45.0 Mb in size, flanked by markers D12S1648 and D12S1660, which almost completely included the ARCI7 locus (Supplementary Material, Fig. S1). Subsequently, we performed whole-exome sequencing using genomic DNA from 1 affected and 1 unaffected (carrier) members of Family 1, and analyzed the data. Within the linkage interval between markers D12S1648 and D12S1660, we identified 363 and 482 single nucleotide variants (SNVs) in the affected and the carrier individuals, respectively. Of these SNVs, we searched for genes with non-synonymous rare variants (minor allele frequency ≤ 0.01) that were homozygous and heterozygous in the affected and the carrier individuals, respectively. As a result, a total of 5 genes, WNT1, SDR9C7, MON2, HELB and GLIPR1L2, were extracted (Table 1). To our surprise, however, all these candidate genes did not residue within the ARCI7 locus. Instead, they were located downstream of the ARCI7 locus (Table 1).
The results of whole-exome sequencing for Family 1: List of non-synonymous rare variants that were homozygous and heterozygous in affected and unaffected (carrier) individuals, respectively
Ch12 position (NC_000012.12) . | 48,979,627 bp . | 56,929,515 bp . | 62,532,629 bp . | 66,310,496 bp . | 75,422,951 bp . |
---|---|---|---|---|---|
Reference allele | T | A | A | A | G |
Alternate allele | A | G | G | C | A |
dbSNP rs# | rs61758378 | rs770729222 | rs143346269 | rs141956990 | rs144813686 |
Gene | WNT1 | SDR9C7 | MON2 | HELB | GLIPR1L2 |
cDNA change | c.264T>A | c.599T>C | c.1592A>G | c.1568A>C | c.632G>A |
Amino acid change | p.Ser88Arg | p.Ile200Thr | p.Gln531Arg | p.Gln523Pro | p.Arg211Gln |
Variant type | missense | missense | missense | missense | Missense |
ExAC-MAF | 0.0030 | 0.000008 | 0.0005 | 0.0178 | 0.0004 |
1000 Genomes-MAF | 0.0028 | N/A | 0.0010 | 0.0088 | 0.0006 |
SIFT score (result) | 0.46 (tolerated) | 0.02 (deleterious) | 0.26 (tolerated) | 0.09 (tolerated) | 0.9 (tolerated) |
PolyPhen-2 score (result) | 0.124 (benign) | 0.701 (possibly damaging) | 0 (benign) | 0.864 (possibly damaging) | 0.004 (benign) |
Ch12 position (NC_000012.12) . | 48,979,627 bp . | 56,929,515 bp . | 62,532,629 bp . | 66,310,496 bp . | 75,422,951 bp . |
---|---|---|---|---|---|
Reference allele | T | A | A | A | G |
Alternate allele | A | G | G | C | A |
dbSNP rs# | rs61758378 | rs770729222 | rs143346269 | rs141956990 | rs144813686 |
Gene | WNT1 | SDR9C7 | MON2 | HELB | GLIPR1L2 |
cDNA change | c.264T>A | c.599T>C | c.1592A>G | c.1568A>C | c.632G>A |
Amino acid change | p.Ser88Arg | p.Ile200Thr | p.Gln531Arg | p.Gln523Pro | p.Arg211Gln |
Variant type | missense | missense | missense | missense | Missense |
ExAC-MAF | 0.0030 | 0.000008 | 0.0005 | 0.0178 | 0.0004 |
1000 Genomes-MAF | 0.0028 | N/A | 0.0010 | 0.0088 | 0.0006 |
SIFT score (result) | 0.46 (tolerated) | 0.02 (deleterious) | 0.26 (tolerated) | 0.09 (tolerated) | 0.9 (tolerated) |
PolyPhen-2 score (result) | 0.124 (benign) | 0.701 (possibly damaging) | 0 (benign) | 0.864 (possibly damaging) | 0.004 (benign) |
The results of whole-exome sequencing for Family 1: List of non-synonymous rare variants that were homozygous and heterozygous in affected and unaffected (carrier) individuals, respectively
Ch12 position (NC_000012.12) . | 48,979,627 bp . | 56,929,515 bp . | 62,532,629 bp . | 66,310,496 bp . | 75,422,951 bp . |
---|---|---|---|---|---|
Reference allele | T | A | A | A | G |
Alternate allele | A | G | G | C | A |
dbSNP rs# | rs61758378 | rs770729222 | rs143346269 | rs141956990 | rs144813686 |
Gene | WNT1 | SDR9C7 | MON2 | HELB | GLIPR1L2 |
cDNA change | c.264T>A | c.599T>C | c.1592A>G | c.1568A>C | c.632G>A |
Amino acid change | p.Ser88Arg | p.Ile200Thr | p.Gln531Arg | p.Gln523Pro | p.Arg211Gln |
Variant type | missense | missense | missense | missense | Missense |
ExAC-MAF | 0.0030 | 0.000008 | 0.0005 | 0.0178 | 0.0004 |
1000 Genomes-MAF | 0.0028 | N/A | 0.0010 | 0.0088 | 0.0006 |
SIFT score (result) | 0.46 (tolerated) | 0.02 (deleterious) | 0.26 (tolerated) | 0.09 (tolerated) | 0.9 (tolerated) |
PolyPhen-2 score (result) | 0.124 (benign) | 0.701 (possibly damaging) | 0 (benign) | 0.864 (possibly damaging) | 0.004 (benign) |
Ch12 position (NC_000012.12) . | 48,979,627 bp . | 56,929,515 bp . | 62,532,629 bp . | 66,310,496 bp . | 75,422,951 bp . |
---|---|---|---|---|---|
Reference allele | T | A | A | A | G |
Alternate allele | A | G | G | C | A |
dbSNP rs# | rs61758378 | rs770729222 | rs143346269 | rs141956990 | rs144813686 |
Gene | WNT1 | SDR9C7 | MON2 | HELB | GLIPR1L2 |
cDNA change | c.264T>A | c.599T>C | c.1592A>G | c.1568A>C | c.632G>A |
Amino acid change | p.Ser88Arg | p.Ile200Thr | p.Gln531Arg | p.Gln523Pro | p.Arg211Gln |
Variant type | missense | missense | missense | missense | Missense |
ExAC-MAF | 0.0030 | 0.000008 | 0.0005 | 0.0178 | 0.0004 |
1000 Genomes-MAF | 0.0028 | N/A | 0.0010 | 0.0088 | 0.0006 |
SIFT score (result) | 0.46 (tolerated) | 0.02 (deleterious) | 0.26 (tolerated) | 0.09 (tolerated) | 0.9 (tolerated) |
PolyPhen-2 score (result) | 0.124 (benign) | 0.701 (possibly damaging) | 0 (benign) | 0.864 (possibly damaging) | 0.004 (benign) |
Results of additional haplotype analysis

Fine mapping of ARCI phenotype on chromosome 12q13.13-q14.1. Pedigrees and Results of haplotype analysis for Family 1 (A), Family 2 (B), and Family 3 (C). Disease-related haplotypes are coloured in red. The linkage interval defined in Families 2 and 3 is indicated by dotted squares (B, C).

Identification of rare variants in the SDR9C7 gene. (A) Schematic representation of the position of the ARCI7 locus and the linkage interval of Families 1–3 determined in this study. Position of the candidate genes and microsatellite markers is shown on the top. The SDR9C7 gene is coloured in red. Note that the linkage interval of Families 2 and 3 are apart from the ARCI7 locus. (B) Identification of a homozygous rare variant c.599T>C (p.Ile200Thr) in the SDR9C7 gene of Family 1. (C) Identification of a homozygous rare variant c.214C>T (p.Arg72Trp) in the SDR9C7 gene of Family 2.
Identification of rare variants in the SDR9C7 gene in all the three families
When we looked back to the results of exome sequencing of Family 1, we found that a rare variant in the SDR9C7 gene was only included in the renewed linkage interval (Fig. 3A, Table 1). This variant was a nucleotide substitution in exon 3 of the SDR9C7 gene (c.599T ≥ C; p.Ile200Thr; rs770729222). We performed Sanger sequencing and confirmed that members of Family 1 definitely carried this variant (Fig. 3B). Subsequently, using the genomic DNA from members of Families 2 and 3, we amplified all exons and exon-intron boundary sequences of the SDR9C7 by polymerase chain reaction (PCR), and directly-sequenced the PCR products. The results demonstrated that Family 2 had a missense variant c.214C > T (p.Arg72Trp; rs530109812) in exon 1 (Fig. 3C), and Family 3 carried c.599T ≥ C (p.Ile200Thr) in exon 3 of the SDR9C7 gene, respectively (data not shown). It is noteworthy that Family 3 had the identical SDR9C7 variant with Family 1, which reflected the fact that Families 1 and 3 were from the same region in Lebanon and they shared an identical haplotype around the SDR9C7 gene (Fig. 2A and C). Screening assays with restriction enzymes revealed that both the variants completely co-segregated with the disease phenotype and were excluded from 300 population-matched unrelated unaffected individuals (600 chromosomes) (Supplementary Material, Fig. S2; data not shown).
Expression of the mutant SDR9C7 proteins in cultured cells

Expression studies of SDR9C7 in cultured cells. (A) Wild-type (Wt), as well as R72W and I200T mutant SDR9C7, without any tags (Tag (-)) were overexpressed in HEK293T cells, and were detected by western blots (WBs) with two distinct anti-SDR9C7 antibodies (clones 10B4 and 4B5). (B) N-terminal Flag-tagged SDR9C7 proteins were overexpressed in HEK293T cells and were analyzed by WBs with anti-Flag and anti-SDR9C7 (clone 10B4) antibodies. WB with anti-β-actin antibody was also performed to show equal loading (A,B). The results clearly demonstrated that expression level of both the mutant proteins was much lower than that of Wt protein.
Expression of the SDR9C7-mRNA in human skin

Results of RT-PCR. Expression of the SDR9C7-mRNA was detected in the skin and normal human keratinocyte (NHK), but not in normal human fibroblast (NHFb). GAPDH was amplified as a positive control.
Localization of the SDR9C7 protein in human skin

Results of indirect-immunofluorescence (IIF) on skin sections. (A–D) We performed double-IIF studies with mouse monoclonal anti-SDR9C7 clone 10B4 (left panels) and rabbit polyclonal anti-loricrin (middle panels) antibodies on paraffin sections of a healthy control individual (A, B) and an affected individual of Family 2 (C, D). The right panels are merged images, and counterstaining with DAPI is shown in blue. SDR9C7 was expressed in granular layer (GL) and cornified layer (CL) of the epidermis in the control skin (A, B). In contrast, SDR9C7 expression in the cornified layer was much lower in the patient’s skin (C, D). Note that a strong red signal in the uppermost cornified layer in the patient’s epidermis (asterisk in panel D) turned out to be a background as it was also detected in stainings without applying primary antibodies (data not shown). There were no obvious differences in expression of loricrin between the control and the affected individuals (middle panels). Scale bars: 100 μm (A), 20 μm (B).
Discussion
In this study, we found three Lebanese families with ARCI without showing linkage to any known causative genes, and identified mutations in the SDR9C7 gene in all the three families analyzed. The SDR9C7 protein, previously known as SDR-O, belongs to the SDR enzyme superfamily (14,15). Although amino acid sequences of SDR9C7 do not show high homology with any other SDR proteins, it has characteristic structural and sequence motifs common for SDR proteins, i.e. the α/β folding motif Rossmann-fold, the glycine rich cofactor binding motif TGxxxGxG and the active centre motif YxxxK (14). It is less likely that the mutations p.Arg72Trp and p.Ile200Thr identified in this study directly affect the latter two sequence motifs. However, based on the crystal structure of an SDR member 17-β-hydroxysteroid dehydrogenase type 1 (17-β-HSD1) (PDB ID 1i5rA), 200Ile in SDR9C7 protein corresponding to 183Ile in α6 of 17-β-HSD1 is predicted to be critical for stabilization of the structural motif via a hydrophobic interaction with the 264Met corresponding to 240Phe in β6 of 17-β-HSD1 (16,17). The substitution from Ile (non-polar) to Thr (polar) at the amino acid residue 200 most likely disrupts the hydrophobic interaction, severely affecting the conformation of the SDR9C7 protein including the Rossmann-fold, which is supported by reduced expression of the p.Ile200Thr-Mut SDR9C7 in vitro (Fig. 4). Concerning the mutation p.Arg72Trp, it was hard to precisely predict the effect on the conformation, because of difficulty in performing a reliable homology modelling. Nevertheless, marked reduction of the p.Arg72Trp-Mut protein both in vitro and in vivo suggested that the mutation would result in disruption of the protein structure (Figs. 4, 6C and D).
SDR proteins are known to catalyze activation/inactivation of prostaglandins, retinoids and several classes of steroid hormones, and as such they catalyze metabolism of various ligands for nuclear receptors (18). Although little is known about the cofactor and the substrate of SDR9C7 protein, it has been reported that SDR9C7 showed a weak activity to convert retinal into retinol in the presence of the cofactor NADH (18). Therefore, SDR9C7 appears to be involved in vitamin A metabolism, while it may also have additional functions. In this study, we have shown that SDR9C7 is abundantly expressed in the granular and the cornified layers of the epidermis where the epidermal keratinocytes are terminally differentiated (Fig. 6A and B, Supplementary Material, Fig. S5). An importance of the vitamin A metabolism in the epidermal differentiation can easily be expected because of the fact that derivatives of vitamin A are widely used for treatment of various cutaneous disorders including ichthyosis. In addition, it is noteworthy that patients with SDR9C7 mutations tend to have recurrent fungal skin infections, suggesting that SDR9C7 is largely involved in barrier function of the epidermis, especially to prevent fungal infections. However, it remains unknown why the patients did not frequently suffer from bacterial, viral or candidal infections. It might be because the deformity of the skin barrier did not affect the innate immunity against these infections including cathelicidins and defensins for instance.
Our data not only provide a novel causative gene for ARCI, but also raise a possibility that SDR9C7 can become a new therapeutic molecule for various cutaneous disorders, even though further analyzes are definitely required to reveal the precise role of SDR9C7 in the epidermis, as well as the functional consequences resulting from the SDR9C7 mutations identified in this study.
Materials and Methods
DNA extraction
Informed consent was obtained from all subjects and approval for this study was provided by the Institutional Review Board of Niigata University and American University of Beirut Medical Center. The study was conducted in adherence to the Declaration of Helsinki Principles. Peripheral blood samples were collected from the family members as well as unrelated healthy control individuals of Lebanese origin, and genomic DNA was extracted from each sample with the QIAamp DNA Blood Midi Kit (Qiagen Inc., Valencia, CA, USA).
Haplotype analysis
Using the genomic DNA from the family members as templates, microsatellite markers close to all the known genes for ARCI (ARCI1-6 and ARCI8-11), as well as those for the ARCI7 locus on chromosome 12, were amplified by PCR. The PCR products were analyzed on 8% polyacrylamide gels, and genotypes were determined by visual inspection.
Whole-exome sequencing
Whole-exome sequencing was performed using genomic DNA from members of Family 1 and the SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacture’s recommendations. Alignment was performed to the hg19 reference genome with Burrows-Wheeler Aligner (BWA) (19), and it was processed with SAMtools (20) and Picard. Genome Analysis Toolkit (GATK) (21) was applied to base quality score recalibration, indel realignment, duplicate removal, and SNVs and indels discovery. Variants were annotated with SnpEff (22) and ANNOVAR (23).
Mutation analysis of SDR9C7 gene
Using the genomic DNA from the family members as templates, all exons including exon-intron boundaries of the SDR9C7 gene were PCR-amplified using gene specific primers (Supplementary Material, Table S2). The amplified PCR products were directly-sequenced in an ABI 3130xl genetic analyzer using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA, USA).
Screening assays for the SDR9C7 variants
To screen for the variant c.599T > C (p.Ile200Thr) in the SDR9C7 gene, intron 2-exon 3 boundary sequences of the SDR9C7 were amplified by PCR using a forward primer (5’-ACAGG GGT GAGAGAAGCATC-3’) and a reverse primer (5’-TGACTCCAG GTTCTCCTTGC-3’). The PCR products were digested by a restriction enzyme DdeI at 37 °C for 3 h, and analyzed on 8.0% polyacrylamide gels. For the variant c.214C > T (p.Arg72Trp), exon 1-intron 1 boundary sequences of the SDR9C7 were PCR-amplified using a mismatch forward primer (5’-GAGGGAT CCCAGAAACTTCAGCGGGATACCTCCGA-3’) and a reverse primer (5’-CATGCAGTGGCTTCCAATCAG-3’). Note that a T > G nucleotide substitution was introduced into the forward primer to generate a PvuI restriction enzyme site only in PCR products from Wt alleles (underlined). The PCR products were digested with PvuI at 37 °C for 3 h, and analyzed on 7.5% polyacrylamide gels.
Generation of expression vectors
Using first-strand cDNA generated from total RNA of normal human skin as a template, cDNA sequences containing the full-length coding region of the SDR9C7 were amplified by PCR using primers listed on Supplementary Material, Table S2. The amplified product was cloned into EcoRI and XhoI sites of the mammalian expression vectors pCXN2.1 (24) and pCMV-Tag2A (Agilent Technologies, Santa Clara, CA). The expression constructs for the p.Arg72Trp and the Ile200Thr Mut-SDR9C7 were generated using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and the Wt-SDR9C7-expression vectors as a template.
SDR9C7-cDNA sequences encoding amino acid residues 1-105, 106-210 and 211-313 were PCR-amplified using the pCXN2.1-Wt-SDR9C7 vector as a template (Supplementary Material, Table S2). The PCR products were subsequently cloned into EcoRI and SalI sites of the pEGFP-C1 vector (Takara Bio Inc., Shiga, Japan) which expresses N-terminal GFP-tagged proteins.
Transient transfection and WBs
HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were plated in 35-mm dishes on the day before transfection. Expression vectors were transfected into the cells using lipofectamine 2000 (Invitrogen). Cells were harvested 24 h after the transfection. The total cell lysate was separated on 4–12% NuPAGE gels (Invitrogen), and WBs were subsequently performed according to a previously described method (25). The primary antibodies used were mouse monoclonal anti-SDR9C7 clone 10B4 (diluted 1:1,000; LifeSpan BioSciences, Inc.), mouse monoclonal anti-SDR9C7 clone 4B5 (diluted 1:500; LifeSpan BioSciences, Inc.), mouse monoclonal anti-Flag (diluted 1:1,000; Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-GFP (diluted 1:2,000; MBL, Nagoya, Japan), and rabbit polyclonal anti-β-actin (diluted 1:3,000; Sigma-Aldrich).
RT-PCR
Total RNA was extracted from a skin sample of a healthy control individual, as well as cultured NHK and NHFb, using the RNeasy Mini Kit (Qiagen). Note that NHK was cultured in a medium containing 1.2 mM CaCl2 in order to induce differentiation. The total RNA was subsequently reverse-transcribed with oligo-dT primers and Superscript III (Invitrogen). Using the first-strand cDNA as a template, cDNA sequences of the SDR9C7 and the GAPDH genes were amplified by PCR using gene-specific primers (Supplementary Material, Table S2). The PCR products were run on 1.0% agarose gels.
IIF studies
IIF studies were performed on paraffin or fresh frozen sections following the methods described previously with minor modifications (25,26). The primary antibodies used were mouse monoclonal anti-SDR9C7 clone 10B4 (diluted 1:100; LifeSpan BioSciences, Inc.), mouse monoclonal anti-SDR9C7 clone 4B5 (diluted 1:150; LifeSpan BioSciences, Inc.), rabbit polyclonal anti-loricrin (diluted 1:200; Novus Biologicals, Littleton, CO, USA), rabbit polyclonal anti-involucrin (diluted 1:500; Abcam, Cambridge, MA, USA), mouse monoclonal anti-filaggrin (clone SPM181; prediluted; GeneTex, Irvine, CA, USA), and rabbit polyclonal anti-keratin 1 (diluted 1:500; BioLegend Inc., San Diego, CA, USA).
Web resources
The URLs for data presented herein are as follows.
Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm.nih.gov/Omim/
GenBank: http://www.ncbi.nlm.nih.gov/Genbank/
PubMed: www.ncbi.nlm.nih.gov/sites/entrez
Ensembl Genome Browser: http://www.ensembl.org/
SIFT: http://sift.jcvi.org/
PolyPhen-2: http://genetics.bwh.harvard.edu/pph2/
Picard: http://broadinstitute.github.io/picard/
dbSNP: http://www.ncbi.nlm.nih.gov/projects/SNP/
RCSB Protein Data Bank (PDB): http://www.rcsb.org/pdb/home/home.do
Supplementary Material
Supplementary Material is available at HMG online.
Acknowledgements
We acknowledge the family members involved in this study. We thank Drs. Satoshi Ishii (Akita University, Japan) and Junichi Miyazaki (Osaka University, Japan) for supplying pCXN2.1 vector.
Conflict of Interest statement. None declared.
Funding
This study was supported in part by a grant from the Naito Foundation, Japan (to Y.S.) and by an MPP and URB grants from the American University of Beirut Medical Center (to M.K.).
References
Author notes
These authors equally contributed to this work.