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A missense variant in NCF1 is associated with susceptibility to multiple autoimmune diseases

Abstract

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a strong genetic component characterized by autoantibody production and a type I interferon signature1. Here we report a missense variant (g.74779296G>A; p.Arg90His) in NCF1, encoding the p47phox subunit of the phagocyte NADPH oxidase (NOX2), as the putative underlying causal variant that drives a strong SLE-associated signal detected by the Immunochip in the GTF2IRD1GTF2I region at 7q11.23 with a complex genomic structure. We show that the p.Arg90His substitution, which is reported to cause reduced reactive oxygen species (ROS) production2, predisposes to SLE (odds ratio (OR) = 3.47 in Asians (Pmeta = 3.1 × 10−104), OR = 2.61 in European Americans, OR = 2.02 in African Americans) and other autoimmune diseases, including primary Sjögren's syndrome (OR = 2.45 in Chinese, OR = 2.35 in European Americans) and rheumatoid arthritis (OR = 1.65 in Koreans). Additionally, decreased and increased copy numbers of NCF1 predispose to and protect against SLE, respectively. Our data highlight the pathogenic role of reduced NOX2-derived ROS levels in autoimmune diseases.

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Figure 1: The GTF2IRD1GTF2INCF1 region at 7q11.23.
Figure 2: Highly homologous sequence among NCF1, NCF1B and NCF1C.
Figure 3: Determination of the ΔGT/GTGT ratio.

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Acknowledgements

We thank all subjects for their participation in this study. We thank E. Magdangal and Y. Shi for help with DNA preparation and organization. We also thank A. Lusis for valuable discussion and comments. This work was supported by US National Institutes of Health grants R01AR043814 (B.P.T.), R21AR065626 (B.P.T.), R01AR056360 (P.M.G.), R01AR063124 (P.M.G.), U19AI082714 (P.M.G.), R01AR043274 (K.L.S.), R01DE015223 (K.L.S.), R01DE018209 (K.L.S.), R01AR050782 (K.L.S.), R01AR065953 (C.J.L. and K.L.S.), P50AR0608040 (K.L.S. and C.J.L.), U19AI082714 (K.L.S. and C.J.L.) and P60AR062755 (D.L.K. and G.S.G.), the Lupus Foundation of America (B.P.T.), the Alliance for Lupus Research (B.P.T.), the Sjögren's Syndrome Foundation (K.L.S. and C.J.L.), Korea Healthcare Technology R&D Project of the Ministry for Health and Welfare in the Republic of Korea grants HI13C2124 (S.-C.B.) and HI15C3182 (K.K.), National Basic Research Program of China (973 program) grant 2014CB541902 (N.S.), Key Research Program of Bureau of Frontier Sciences and Education Chinese Academy of Sciences grant QYZDJ-SSW-SMC006 (N.S.), Key Research Program of the Chinese Academy of Sciences grant KJZD-EW-L01-3 (N.S.), State Key Laboratory of Oncogenes and Related Genes grant 91-14-05 (N.S.), National Natural Science Foundation of China grant 31630021 (N.S.), Strategic Priority Research Program of the Chinese Academy of Sciences grant XDA12020107 (N.S.). Clinical and Translational Science Institute (CTSI) grants UL1RR033176 (UCLA), UL1TR000124 (UCLA) and UL1TR001450 (MUSC), and funds from the Spaulding-Paolozzi Autoimmunity Center of Excellence (MUSC), the Richard M. Silver, MD, Endowment for Inflammation Research (B.P.T.) and the SmartState® Center of Economic Excellence in Inflammation and Fibrosis Research (B.P.T.). The funders had no role in study design, data collection, analysis and interpretation, writing of the report, or decision to submit the paper for publication.

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Authors

Contributions

J.Z., B.P.T. and N.S. led the study. J.Z., Y.D. and B.P.T. wrote the manuscript. J.Z., J.M., Y.D. and R.Q. performed the experiments. J.Z., J.M., Y.D., J.A.K. and K.K. analyzed the data and performed statistical analysis. S.-Y.B., H.-S.L., Q.-Z.L., E.K.W., M.L., J.G., Z.L., W.T., A.R., C.J.L., K.L.S., B.H.H., J.M.G., D.L.K., G.S.G., S.-C.B. and P.M.G. contributed primarily to sample collection and/or genotyping. All authors reviewed the final manuscript.

Corresponding authors

Correspondence to Jian Zhao, Nan Shen or Betty P Tsao.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 No association between rs117026326 genotypes and transcript levels of NCF1, GTF2I and GTF2IRD1.

(a) Neighboring genes of rs117026326. Genes located within ±300 kb of rs117026326 include GTF2IRD1, GTF2I, LOC101926943, NCF1, GTF2IRD2, STAG3L2, PMS2P5 and GATSL2. Of them, NCF1, which encodes the p47phox subunit of the NOX2 complex, is the most likely SLE-related gene. GTF2I encodes general transcription factor TFII-I; GTF2IRD1 and GTF2IRD2 encode structurally similar and potentially functionally overlapping TFII-I-like transcription factors; LOC101926943 is an uncharacterized long noncoding RNA; STAG3L2 and PMS2P5 are both pseudogenes; and GATSL2 encodes an arginine sensor for the mTORC1 pathway. (b) Association between rs117026326 genotypes and transcript levels of NCF1, GTF2I and GTF2IRD1 in peripheral blood mononuclear cells (PBMCs) from patients with SLE and controls. Data were compared by Spearman correlation or Mann–Whitney test (two-tailed). Center lines and error bars represent means ± s.e.m.

Supplementary Figure 2 PCR-amplification of NCF1-specific sequence.

(a) NCF1-specific PCR primer binding sites. To exclude the influence of NCF1B and NCF1C and obtain correct genotypes of NCF1 variants, we amplified NCF1-specific sequence by PCR. Two NCF1-specific loci were selected as PCR primer binding sites. One locus, targeted by PCR primers P1-R, P2-L and P2*-L as shown below in b, is a GTGT sequence at the beginning of exon 2 of NCF1 (chr7:74,777,267–74,777,270), which is different from the GT deletion (ΔGT) in NCF1B and NCF1C. Another locus, targeted by PCR primer P3-L, is a T allele in intron 6 of NCF1 (chr7:74,783,147), which is different from the G in NCF1B and NCF1C. (b) PCR amplification of NCF1 for sequencing and SNP genotyping. The entire 15.5-kb region of NCF1 was amplified by three PCR reactions (PCR products P1, P2 and P3) for Sanger sequencing. To genotype NCF1 variants, we performed nested PCR and TaqMan assays, in which P2 (a larger PCR product containing p.Arg90His, p.Ser99Gly, intronic-1 and intronic-2) or P2* (a smaller PCR product containing p.Arg90His and p.Ser99Gly only) was obtained using NCF1-specific primer and then used as DNA template for TaqMan SNP genotyping assays.

Supplementary Figure 3 Significant association of p.Arg90His risk genotypes with early age of disease onset in Korean and European-American patients with SLE.

Data were compared by Spearman correlation or Mann–Whitney test (two-tailed). Center lines and error bars represent means ± s.e.m.

Supplementary Figure 4 Evolutionary conservation and computational prediction for functional impact of p.Arg90His and p.Ser99Gly.

(a) Alignments of multiple vertebrate species at p.Arg90His and p.Ser99Gly. Arg90 is an evolutionarily conserved amino acid. This figure was adapted from the UCSC Genome Browser. (b) Assessment of the functional impact of p.Arg90His and p.Ser99Gly. The substitution of Arg90 with a histidine residue encoded by the SLE risk allele was predicted to be deleterious by softwares, including SIFT (Sorting Intolerant From Tolerant; http://sift.bii.a-star.edu.sg/), PolyPhen-2 (Polymorphism Phenotyping v2; http://genetics.bwh.harvard.edu/pph2/), PANTHER (Protein ANalysis THrough Evolutionary Relationships; http://www.pantherdb.org/), MutationTaster (http://www.mutationtaster.org/), MutationAssessor (http://mutationassessor.org/) and FATHMM (Functional Analysis through Hidden Markov Models; http://fathmm.biocompute.org.uk/).

Supplementary Figure 5 No association between p.Arg90His and ROS levels in neutrophils from healthy controls.

Intracellular ROS levels were determined using fluorescent dye DCFH-DA and measured using flow cytometry. Data were compared by Spearman correlation or Mann–Whitney test (two-tailed). Center lines and error bars represent means ± s.e.m.

Supplementary Figure 6 NCF1 variants in the 1000 Genomes Project.

(a) The 1000 Genomes Project inaccessible region at 7q11.23. The ‘pilot’ and ‘strict’ level of stringency in the 1000 Genomes Project are shown as gray and black bars on the top, respectively. NCF1, NCF1B and NCF1C are located in regions that do not meet the ‘strict’ level of stringency in 1000 Genomes Project phases 1 and 3. This figure was adapted from the UCSC Genome Browser. (b) NCF1 variants included in the 1000 Genomes Project. NCF1 variants with MAF >0.5% in at least one ancestral group (n = 8 in phase 1; n = 34 in phase 3) are shown in this table. p.Arg90HisR90H (rs201802880) is not included in either phase 1 or 3. p.Ser99Gly (rs17295741) is included in phase 3, which however shows deviation from Hardy–Weinberg equilibrium (HWE). In addition, deviations from HWE are observed at the four other common NCF1 SNPs (rs368231459, rs587770703, rs62475426 and rs2528941) included in phase 3, which indicates that 1000 Genomes Project data in the NCF1 region are unreliable.

Supplementary Figure 7 Plots of the principal-component analysis (PCA).

(a) PCA of Chinese, European-American (EurAm) and African-American (AfrAm) samples genotyped by Immunochip (IC) along with reference samples from the 1000 Genomes Project. (bd) PCA of Chinese, European-American and African-American subjects genotyped by IC. (e,f) PCA of Korean SLE cases, RA cases and healthy controls in the replication stage.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–11 (PDF 1952 kb)

Supplementary Data

Raw TaqMan data for the R90H variant in NCF1. (PDF 16195 kb)

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Zhao, J., Ma, J., Deng, Y. et al. A missense variant in NCF1 is associated with susceptibility to multiple autoimmune diseases. Nat Genet 49, 433–437 (2017). https://doi.org/10.1038/ng.3782

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