Identification of a PV-associated functional variant in the ST18 gene locus

Given the fact that a previous GWAS demonstrated an association between PV and variants in the vicinity of the ST18 gene locus [8], we aimed at characterizing this risk region and therefore performed targeted deep sequencing of the ST18 locus in 16 Jewish PV patients, initially comparing the sequencing results to the 1000 Genomes Project (1000GP) data (http://www.1000genomes.org). Since PV is a complex disease, our primary targets were common single nucleotide polymorphisms (SNPs) that were enriched in the sequenced cohort as compared with controls. We therefore examined all SNPs with non-zero frequencies in the public databases that were not in repetitive regions, totaling 789 SNPs (Fig 1). A case-control association analysis for each SNP, using chi-square and permutation test, led to the identification of a genomic haplotype block strongly associated with the propensity to develop PV (p<0.001) (Fig 1 and S1 Fig). We discovered that this risk haplotype harbors two variants previously found to be most significantly associated with PV in the original GWAS, rs4074067 and rs2304365 [8]. Collectively, the SNPs found within the risk haplotype block had a frequency of 50% in the sequenced cohort and of only 8% in the total 1000GP population.

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Fig 1. Targeted deep sequencing of the ST18 gene locus.

Deep sequencing of the ST18 gene locus in 16 PV patients led to identification of 789 genetic variants (black dots), depicted along the ST18 gene locus (X axis, chr8:53,023,392–53,373,519, GRCh37/hg19 assembly; Y axis, negative log-transformed P-values of association score). A case-control association analysis against the 1000 genome project data revealed a large PV-associated haplotype block (in red) residing within an ST18 intron and harboring rs17315309 (arrows).

https://doi.org/10.1371/journal.pgen.1006008.g001

Within the risk haplotype block, we identified a genetic variant, rs17315309 (Fig 1), which displayed significant association to PV based on the deep sequencing data. We then replicated this association in an independent set of 185 Jewish PV patients compared with 183 population-matched healthy controls (p<0.001; S1 Table).

A number of bioinformatic analyses suggested that this variant may be of functional importance and possibly up-regulate ST18 promoter activity. First, using HMR Conserved Transcription Factor Binding Site database, implemented in the UCSC Genome Browser (https://genome.ucsc.edu/), this variant was found to reside within a p53 transcription factor binding site consensus sequence (Fig 2a). The p53 binding motif consists of two very similar, closely located, half-sites, each 10 bp long [12,13], although it has been shown that one decamer is sufficient for p53 or p63 binding and activity [14–19]. Second, the binding motif harboring rs17315309 is located in an intron of ST18, upstream to the gene coding sequence and inside a 170 bp long DNAse hypersensitivity cluster (chr8:53207581–53207750, ENCODE; http://genome.ucsc.edu/ENCODE/), lending further support to the possibility that this region plays a regulatory role. Of note, p53 is known to recognize consensus binding motifs located proximal to the transcription start site of target genes, either within the promoter region or a gene intron [20]. Third, using the 100 Vertebrae Conservation by PhastCons, implemented in the UCSC Genome Browser (http://compgen.cshl.edu/phast/), we found that both rs17315309 and the p53/p63 binding motif are highly conserved with a maximum conservation score of 1 (range 0 to 1). Similarly, using Biobase Transfac Matrix Database (v7.0), implemented in the UCSC Genome Browser (http://www.gene-regulation.com/pub/databases.html), the 10 bp long binding motif containing rs17315309 was found to be remarkably conserved (computed score: 992; maximal score: 1000), supporting the possibility that it represents a biologically functional binding site. Lastly, as rs17315309 results in a T to C substitution at position 6 of the p53/p63 binding motif, we wished to examine the conservation of this nucleotide in the binding site consensus sequence. Both MotifMap (http://motifmap.ics.uci.edu) and Jasper (http://jaspar.genereg.net) databases indicated that position 6 in the half-binding site consensus sequence of both p53 and p63 is highly conserved and consists usually of a T nucleotide with a minimum to no abundance of a C nucleotide (Fig 2a). Taken collectively, these data suggested that modification of the wild type rs17315309 allele within the p53/p63 binding site may be of biological significance.

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Fig 2. Characterization of a PV-associated functional allele.

(a) rs17315309 resides within a p53/p63 half binding site, upstream to the ST18 coding sequence, and results in the substitution of a highly conserved T nucleotide (broken arrow) within the consensus binding sites of p53 and p63 (MotifMap database; http://motifmap.ics.uci.edu/); (b) NHEKs were co-transfected with a luciferase reporter construct under the regulation of a 282 bp fragment from the putative ST18 promoter, harboring either the wild type or the risk allele of rs17315309, and with control (si-Cont), TP53- (si-p53) or TP63- (si-p63) specific siRNAs. Luciferase activity measurements are provided as arbitrary units (a.u.) relative to the luciferase activity measured in cells transfected with wild type allele and control siRNA. Results represent the mean of three independent experiments ± SE (*p<0.05, **p<0.01 and ***p<0.001 by 2-tailed t test).

https://doi.org/10.1371/journal.pgen.1006008.g002

To examine this possibility, we transfected normal human keratinocytes (NHEKs) with a PGL4.17 luciferase reporter construct under the regulation of a 282 bp fragment spanning the p53/p63 binding motif containing either the wild type (T) or the risk (C) rs17315309 allele. The C allele was found to induce a more than 5-fold increase in luciferase activity (p<0.001) (Fig 2b). Moreover, when the construct was co-transfected with p53- or p63-specific siRNAs (S2a and S2b Fig), this effect was markedly attenuated (Fig 2b), indicating that the PV-associated rs17315309 risk allele increases ST18 promoter activity in a p53-/p63-dependent manner. These results are in line with previous data showing that a single nucleotide change in a canonical p53/p63 binding sequence is enough to affect p53 or p63 binding [21].

ST18 promotes PV IgG-induced keratinocyte secretion of pro-inflammatory mediators

Given these data, the physiological roles of ST18 and the fact that ST18 is markedly overexpressed in the non-lesional epidermis of PV patients [8], we sought to ascertain the consequences of ST18 overexpression on pathophysiological hallmarks of the disease.

We first examined the effect of ST18 on keratinocyte secretion of pro-inflammatory cytokines which are believed to contribute to PV disease phenotype [1,4,11]. Overexpression of ST18 (S2c Fig) in the presence of normal serum or control IgG did not affect the secretion of either TNFα, IL-1α or IL-6 (Fig 3). In contrast, when overexpressed in the presence of PV serum, ST18 was found to drive the secretion of all three cytokines (Fig 3a–3c), indicating that ST18 functions by promoting PV-induced keratinocyte secretion of pro-inflammatory cytokines. This effect was seen early with TNFα and IL-1α but late with IL-6 (Fig 3a–3c). We then repeated the same experiments, comparing the effect of control and PV sera to the effect of control IgG and PV IgG. Overexpression of ST18 was found to increase PV serum-induced and PV IgG-induced secretion of TNFα, IL-1α and IL-6, to the same extent, while not affecting the secretion of these cytokines in the presence of control serum or control IgG (Fig 3d–3f).

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Fig 3. ST18 promotes PV serum- and PV IgG-induced release of pro-inflammatory cytokines.

NHEKs were transfected with an ST18 expression vector (ST18, dark grey) or control empty vector (EV, light grey). (a,b,c) Supernatants were collected 2, 4, 6 and 24 hours post exposure to PV serum or control serum and (a) TNFα, (b) IL-1α and (c) IL-6 secretion was measured as described in Materials and Methods; (d,e,f) Supernatants were collected 6 hours (IL-1α and IL-6) or 24 hours (TNFα) post exposure to PV and control serum or PV and control IgG and (d) TNFα, (e) IL-1α and (f) IL-6 secretion was measured as described in Materials and Methods. Results represent the mean of three independent experiments and are expressed as the relative cytokine secretion in percentage, compared to control (empty vector) ± SE (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 by 2-tailed t test).

https://doi.org/10.1371/journal.pgen.1006008.g003

ST18 augments PV IgG-induced keratinocyte cell-cell disadhesion

As disruption of epidermal cell-cell adhesion is a pathogenic hallmark of PV [1], we investigated ST18 effect on PV serum-induced cell-cell disadhesion. For this purpose, we used the dispase-based dissociation assay. In this system, PV serum destabilizes intercellular bonds, compromising epidermal sheet resilience to mechanical stress [22] (Fig 4a). NHEKs overexpressing ST18 and exposed to PV serum exhibited a more than 2-fold decrease in cell-cell adhesion, as compared to cells exposed to PV serum and transfected with an empty vector (p<0.05) (Fig 4b) or with a vector overexpressing a non-relevant gene (S3 Fig). The deleterious effect of ST18 overexpression on cell-cell adhesion was similar when cells were exposed to pooled PV sera or to PV IgG (Fig 4b). Taken together, these results demonstrate that ST18 may also contribute to PV pathogenesis by potentiating PV IgG-induced acantholysis.

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Fig 4. ST18 promotes PV serum and PV IgG-induced cell-cell disadhesion.

We used a dispase-based dissociation assay to evaluate the effect of ST18 overexpression on cell adhesion. NHEKs transfected with a ST18 expression vector (ST18) or with a control vector (EV) were grown to confluency in the presence of PV serum and control serum or PV IgG and control IgG. (a) Epidermal sheets were released from the tissue plates and subjected to mechanical stress as described in Materials and Methods; (b) the resulting fragments were counted. Results represent the mean of three independent experiments and are expressed as number of fragments ± SE (*p<0.05, **p<0.01, ***p<0.001 by 2-tailed t test).

https://doi.org/10.1371/journal.pgen.1006008.g004

Ethics statement

The study was conducted according to a protocol approved by our institutional review board and the National Committee for Genetic Studies of the Israeli Ministry of Health in accordance with the Declaration of Helsinki Principles (102-2006/TLV-0537-15). All family members provided written informed consent to participate in this study

Patients

All family members provided written informed consent to participate in this study. Blood samples were obtained from all participants according to a protocol approved by our institutional review board and the National Committee for Genetic Studies of the Israeli Ministry of Health in accordance with the Declaration of Helsinki Principles. The diagnosis of PV was posed based upon clinical features, suprabasal separation on histology, positive direct and indirect immunofluorescence microscopy, and/or ELISA detection of anti-Dsg Abs. Genomic DNA was extracted from peripheral blood leukocytes using the 5 Prime ArchivePure DNA Blood kit (5 Prime Inc., Gaithersburg, MD, USA).

PV sera and PV IgG preparation

We used two different mixes of pooled sera from newly diagnosed PV patients (n = 3 and 4) with active disease and an anti-Dsg3 titer above122 relative units/ml, as measured by the anti-desmoglein 3 ELISA (IgG) test kit (Euroimmune AG, Luebeck, Germany). The sera were obtained prior to the initiation of any systemic immunosupressive treatment. PV IgGs were purified as previously described [39] and used at a final concentration of 65 μg/ml.

Targeted deep sequencing of the ST18 locus

DNA enrichment was performed using HaloPlex kit (Agilent Technologies, Santa Clara, CA, USA) and sequencing was conducted on a MiSeq system sequencer (Illumina, San Diego, CA, USA) with 150 bp paired-end reads. A total of 463407 bp were included in the capture design, covering the entire ST18 gene (chr8: 53,023,399–53,373,519, GRCh37/hg19 assembly) as well as 10 kb downstream and 50 kb upstream to the gene and an additional 2 Mb located upstream and downstream to the gene and predicted to harbor putative regulatory regions (https://www.encodeproject.org).

The sequencing data were processed using MiSeq Reporter 2.0.26 and Casava softwares (Illumina, San Diego, CA, USA) and analyzed for quality control using FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were aligned to the Genome Reference Consortium Human Build 37 (GRCh37/hg19) using Burrows-Wheeler Aligner [40] and variant detection was achieved using The Genome Analysis Toolkit [41]. Variants were annotated by ANNOVAR [42] and the frequency of each variant was determined using data from dbSNP138, the 1000 Genome Project and an in-house database. Case-control association test for variants was performed with chi-square, and permutation test, using the Caucasian population from the 1000 Genome Project data (http://www.1000genomes.org) as a control.

rs17315309 genotyping

To screen for the rs17315309 allele, we PCR-amplified a 317 bp fragment, with ReddyMix PCR Master Mix (Thermo scientific, NH, USA) and the following primers 5`- TGCTTGCCGTTTGTAAGATG-3`and 5`-AGCCTGGTTCAAGAGCCTTC-3`. Cycling conditions were as follows: 94°C, 4min; 94°C, 30 sec; 61°C, 30 sec; 72°C 30 sec, for 2 cycles, 94°C, 30 sec; 59°C, 30 sec; 72°C 30 sec, for 2 cycles, 94°C, 30 sec; 57°C, 30 sec; 72°C 30 sec, for 38 cycles, 72°C for 10 min. The T allele is associated with the presence of a recognition site for endonuclease NspI (New England Biolabs, Hitchin, UK). After incubation at 37°C for 16 hours followed by 20 min of inactivation at 65°C, the digested PCR products were electrophoresed in ethidium bromide-stained 3% agarose gels.

Cells cultures

NHEKs were extracted from skin discarded during plastic surgery procedures, after written informed consent had been obtained as previously described [43]. The keratinocytes were seeded on feeder plates containing 3T3-J2 fibroblasts and were grown in medium Green containing 42.5% DMEM (Biological Industries, Beit-Haemek, Israel), 42.5% DMEM/F12 (Biological Industries, Beit-Haemek, Israel), 10% FCII serum, 1% penicillin-streptomycin, 1mM L-glutamine, 1mM sodium pyruvate, 5 μg/mL Insulin, 0.2 mM adenine, 0.5 μg/mL hydrocortisone, 2nM Triiodothyronine, 0.1 nM cholera toxin and 10 ng/mL EGF, and were frozen upon 90% confluence. Cell were then thawed and cultured in KGM media (Lonza, Basel, Switzerland).

For the dispase-based dissociation assay, NHEKs were extracted from foreskin using the same conditions and were thawed and cultured in M154 media (Life Technologies, Carlsbad, CA).

ST18 overexpression

A 8.0 kb-clone containing the ST18 open reading frame in a pCMV6-Entry vector (4.9kb) was purchased from Origene Technologies Company (Rockville, MD, USA). The empty pCMV6-Entry was used as a control. Additionally, a 6.6 kb-clone containing the CNBD2 open reading frame in a pCMV6-Entry vector (4.9kb) was purchased from Origene Technologies Company (Rockville, MD, USA) and used a negative control for protein overexpression. NHEKs were cultured to 80% confluence and subjected to a transient transfection using lipofectamine2000 (Life Technologies, Carlsbad, CA).

p53/p63 binding site luciferase reporter assay

A 282 bp ST18 gene fragment spanning rs17315309 was PCR-amplified using ReddyMix PCR Master Mix (Thermo scientific, NH, USA), primers 5’-AAAATTAGGTACCGCGTTCAAGCACTCTATTACCT-3’ and 5’-AAAAGGACTCGAGGCTTGCCGTTTGTAAGATGA-3’, and DNA extracted from two patients homozygous for rs17315309 wild-type allele T, and for rs17315309 minor allele C, respectively. Cycling conditions were as follows: 94°C, 4 min; 94°C, 30 sec; 61°C, 30 sec; 72°C 30 sec, for 2 cycles, 94°C, 30 sec; 59°C, 30 sec; 72°C 30 sec, for 2 cycles, 94°C, 30 sec; 57°C, 30 sec; 72°C 30 sec, for 38 cycles, 72°C for 10 min. The resulting amplicons were cloned into pGL4.17 vector (Promega, Madison, WI, USA).

NHEKs were co-transfected with the various pGL4.17 vectors and Renilla expression vector and control siRNAs (Life Technologies, Carlsbad, CA) or p53 specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) or p63 specific siRNA (Dharmacon, Inc., Lafayette, CO) with lipofectamine2000 and Opti-MEM medium (Life Technologies, Carlsbad, CA). Efficiency of gene knock down was assessed by qRT-PCR (S2 Fig). Cells were grown in KGM medium (Biological Industries, Beit-Haemek, Israel). Twenty-four hours post transfection, cells were harvested and luciferase expression was evaluated using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and Tecan Infinite M200 device (Tecan Group Ltd, Männedorf, Switzerland)

Cytokine secretion measurement

Supernatant collected from NHEKs was evaluated using Elisa assays specific for IL-1α (Human IL-1 alpha/IL-1F1 DuoSet, R&D systems, Minneapolis, MN, USA), TNF-α (Human TNF-alpha Quantikine HS ELISA, R&D systems, Minneapolis, MN, USA) and IL-6 (Human IL-6 DuoSet, R&D systems, Minneapolis, MN, USA). All ELISA assays were read and quantified using Tecan Infinite M200 device (Tecan Group Ltd, Männedorf, Switzerland). For IL-6, a Human Cytokine Array / Chemokine Array 41-Plex (Eve Technologies Corporation, Calgary, Alberta, Canada) was additionally used using a Millpore MILLIPLEX kit (Merck KGaA, Darmstadt, Germany) read by BioPlex 200 (Bio-Rad, Hercules, CA, USA)

Dispase-based dissociation assay

NHEKs were grown to confluence in triplicates on 6-well plates and exposed to PV or normal serum in a 1:10 dilution or to PV or control IgG antibodies in a final concentration of 65 μg/ml. After 24h the cells were washed twice with PBS, incubated in 2 ml of dispase II (2.4 units/ml, Roche Diagnostics, Basel, Switzerland) at 37°C for 40 minutes and detached from the plate as monolayers. Cell sheets were carefully transferred to 15 ml tube containing 5 ml PBS and subjected to mechanical stress using 5 inversions. The number of fragments was counted by two independent evaluators.

Statistics

All pairwise comparisons were performed using the 2-tailed Student’s t test, unless otherwise indicated. Differences were considered significant if the P value was less than 0.05.