Genetic analysis of NCSTN for potential association with hidradenitis suppurativa in familial and nonfamilial patients

Dear Editor, Hidradenitis suppurativa (HS; also known as acne inversa) is an autosomal chronic inflammatory inherited disease.1 Several risk factors are associated with HS, such as smoking, obesity, infection and family history.1 Based on a genome‐wide scan in a four‐generation HS family, recent genetic advances in the understanding of HS have led to the conclusion that genes that encode enzymes within the γ‐secretase family play a key role in HS.2 To find genetic variants that are associated with HS susceptibility, research has focused on sequencing the three essential subunits of γ‐secretase in kindreds of patients with HS.3 To date, 20 mutations in nicastrin (NCSTN) have been reported in African American, Chinese, Japanese, South Welsh and French multiplex kindreds, with NCSTN being the most commonly observed subunit.4 5 6 7 8 Mutations in NCSTN include four deletions (c.210_211delAG, c.487delC, c.1752delG and c.582+1delG) that result in a frame shift. In addition, 10 nonsense mutations that prematurely stop the translation of the mRNA sequence and induce loss of function (p.R117X, p.R434X, p.Y565X, p.S590AfsX3, p.V75I, p.D185N, p.P211R, p.Q216P, p.Q568X and p.Q420X) have been identified, which could potentially disrupt the structure of NCSTN, leading to impaired activity. To further explore the potential genetic association of NCSTN to HS susceptibility, we applied next‐generation sequencing (NGS) technology to interrogate the entire NCSTN gene [15 679 base pairs (GRCh37/hg19)] from DNA samples extracted from the whole blood of 443 subjects with HS enrolled in two large phase III clinical trials, Pioneer I (NCT01468207) and Pioneer II (NCT01468233) (http://www.multivu.com/players/English/7298751-abbvie-phase-3-hidradenitis-suppurativa-trial-results/). Sequencing was initially completed in 95 subjects (57 with family history, 38 with no family history). Select variants within NCSTN that were identified in the 95 patients were further interrogated in all 443 subjects (Table 1). The allelic odds ratio (OR) was computed, as well as other models of genetic association (dominant, recessive, co‐dominant and overdominant) stratified by ethnicity. Control group data were formed using data combined from the 1000 Genomes (Phase I) and Perlgen (HapMap) European (EUR) and African ancestry (AFR) populations, as our participants were either white or black. ORs were reported with the control group as the reference category. Fisher's exact method was used to compute P‐values and confidence intervals using R version 3.1.0 (http://www.R-project.org). Additional models of association were calculated using the SNPassoc package (http://CRAN.Rproject.org/package=SNPassoc).9 10 Model selection utilizing Akaike's information criterion was used to determine the most appropriate genetic model of association. Demographic and clinical characteristics of patients with hidradenitis suppurativa (HS) Data are n (%) unless otherwise indicated. BMI, body mass index. Demographic and clinical characteristics of patients with hidradenitis suppurativa (HS) Data are n (%) unless otherwise indicated. BMI, body mass index. We identified one nonsense mutation, p.R117X (rs387906896), in a white subject with a family history of HS. This mutation has been previously identified in Chinese and African American patients with HS; this is the first study to report this mutation in a white subject.2 4 This nonsense mutation results in an amino acid change from arginine to a premature termination codon at amino acid 117. The premature termination codon at amino acid 117 results in a fragmentation between the intra‐ and extracellular domains, and dysfunction of truncated NCSTN is a foreseeable consequence. One missense mutation was also identified, P.A410V (rs147225198), in NCSTN exon 11. To our knowledge, this mutation has not been previously reported in any patient with HS. P.A410V was present in a white patient with Hurley stage III and without a family history of HS. P.A410V (rs147225198) showed a higher minor allele frequency (MAF) relative to controls from the European ExAC (Exome Aggregation Consortium) population (0·0011 vs. 0·0003, OR 3·5; P = 0·19). The mutation results in a change of amino acid from alanine to valine in the bend region of the extracellular domain, which is thought to be responsible for substrate recruitment by γ‐secretase. The 410 amino acids of NCSTN in the ectodomain function as a receptor that recognizes the downstream proteins.11 The valine to alanine mutation, which adds an R group, may result in additional hydrophobicity and conformational change of the NCSTN ectodomain, which is associated with γ‐secretase protein activity (Fig. S1a, b; see Supporting Information) (http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=4UPC).11 In vitro functional work is needed to confirm this. Three noncoding single nucleotide polymorphisms (SNPs) were also identified that showed a significant OR when compared with the control groups (AFR and EUR, depending on ethnicity). The MAF of rs16831883 was higher in white subjects with HS relative to the control database. In contrast, rs6427515 showed a higher significant change in MAF in the black subjects relative to controls (0·47 vs. 0·33, OR 1·45; P < 0·01). Similarly, rs1004607 also showed a higher MAF in black subjects with HS relative to controls (0·020 vs. 0 in the control group, OR 0·91; P = 0·03) (Tables 2 and 3). Standard genome browser tools such as UCSC Genome Browser, identified that rs16831883 lies in a DNA splice region and rs1004607 lies in a DNase I hypersensitivity cluster. Using the noncoding variant functional prediction tool GWAVA, none of these SNPs predictive scores reached ‘functional’ threshold.12 In addition, a recent publication functionally examined rs6427515 as part of a haplotype with five other linked disequilibrium SNPs; in vitro analysis showed no significant effect on NCSTN function.13 Further in vitro validation work needs to be performed in order to identify if these SNPs have functional significance. Clinical data for the two individuals who carry the two mutations nsSNP, nonsynonymous single nucleotide polymorphism; HS, hidradenitis suppurativa; BMI, body mass index. Clinical data for the two individuals who carry the two mutations nsSNP, nonsynonymous single nucleotide polymorphism; HS, hidradenitis suppurativa; BMI, body mass index. Frequency, allelic odds ratios (OR) and P‐values of carriage of single nucleotide polymorphisms (SNPs) of NCSTN that are associated with hidradenitis suppurativa susceptibility in different ethnic groups The OR is reported as case–control, and calculations are based on Fisher's exact test. All SNPs shown were in Hardy–Weinberg equilibrium with P > 0·60 in controls. MAF, minor allele frequency; CI, confidence interval. Frequency, allelic odds ratios (OR) and P‐values of carriage of single nucleotide polymorphisms (SNPs) of NCSTN that are associated with hidradenitis suppurativa susceptibility in different ethnic groups The OR is reported as case–control, and calculations are based on Fisher's exact test. All SNPs shown were in Hardy–Weinberg equilibrium with P > 0·60 in controls. MAF, minor allele frequency; CI, confidence interval. In summary, this is the largest study to date on potential genetic associations underlying HS. After deep sequencing of NCSTN for single nucleotide variant in subjects with HS, we identified two nonsynonymous mutations, one of which had previously been described in African American and Chinese patients with HS. In addition, several intronic polymorphisms within the NCSTN were identified that were present at a significantly higher frequency in the HS population relative to healthy controls. Whether these SNPs have functional significance is not known. A clear conclusion from this analysis is that the vast majority of subjects enrolled in the HS studies did not display genetic variants in NCSTN. Targeted NGS of NCSTN in all patients may have identified some additional variants in this gene; however, the low number identified in the first 95 patients suggests that additional and as‐yet unknown genes also predispose to the development of this disorder. Clearly, further studies are warranted to identify additional factors that contribute to the aetiology and mechanism of this disease. We thank the subjects for their participation and for providing their DNA for genetic research in the Pioneer I (M11‐313) and Pioneer II (M11‐810) studies. Funding sources: The studies were sponsored by AbbVie. Conflicts of interest: AbbVie contributed to the study design, research and interpretation of data, and writing, reviewing and approving the publication. All authors are AbbVie employees and may hold AbbVie stocks or options. Fig S1. The structure of nicastrin (NCSTN) showing the locations of the variants Ala410Val and Arg117Ter.