Identification of a novel mutation in complement receptor 2 in Chinese familial systemic lupus erythematosus

Yuewu Tang1, Yi Luo2

1Department of Nephrology, Three Gorges Hospital, School of Medicine, Chongqing University, Chongqing, China
2Department of Blood Transfusion, Three Gorges Hospital, School of Medicine, Chongqing University, Chongqing, China

Keywords: Complement receptor 2, mutation, sequencing, systemic lupus erythematosus.

Abstract

Objectives: This study aims to analyze the relationship between complement receptor 2 (CR2) gene mutation and the clinical phenotype in Chinese familial systemic lupus erythematosus (SLE).

Patients and methods: A total of one Chinese familial SLE patients (median age: 30.25 years; range, 22 to 49 years) were included between January 2017 and December 2018. The clinical features and diagnoses of familial SLE patients were analyzed using whole-exome sequencing (WES) of genomic deoxyribonucleic acid (DNA) samples. Sanger sequencing was used to verify candidate mutations detected in the examined family.

Results: The mother and her three daughters were diagnosed with SLE. The clinical characteristics showed that the patient and her mother were diagnosed with lupus nephritis. The eldest daughter had decreased renal function and lower serum albumin levels. Immunological index analysis showed that all four patients were positive for anti-SSA and antinuclear antibody (ANA), but that only the second daughter was positive for anti-double-stranded DNA (dsDNA). Complement 3 (C3) was significantly decreased in all patients, while evaluation of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) showed that the second and third daughters had mild active SLE. The mother and eldest daughter were treated with prednisolone combined with cyclophosphamide, while the other two daughters were treated with prednisolone alone. The WES and Sanger sequencing analyses revealed an unreported missense T>C mutation c.2804 in the 15th exon of the CR gene in all four patients.

Conclusion: We identified a novel c.2804 (exon 15) T>C mutation in the CR gene of Chinese familial SLE. This mutation was previously reported, suggesting that the CR gene c.2804 (exon 15) T>C mutation is the probable cause of SLE in this family.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease with a variety of clinical manifestations that can affect various bodily organs and tissues.[1] The incidence of SLE varies from 0.3/100,000 person-years to 23.2/100,000 person-years worldwide, and women are more frequently affected by age and ethnic group.[2] The etiology of SLE has not been well understood; genetic constitution, environmental triggers, and hormone milieu have been reported to contribute to disease development and activity.[3]

The pathogenesis of SLE is complex, involving the innate and adaptive immune systems, autoantibodies, and immune complexes, as well as the complement system.[4] In addition, different genetic and environmental factors are involved in its disease pathogenesis.[5]

Cumulating evidence shows that genetic factors play a central role in SLE. Previous studies have shown that lupus susceptibility is related to genetic factors, and several susceptibility sites have been clearly identified.[6,7] However, current genetic research cannot explain the entirety of SLE heritability, and other associated gene susceptibilities remain largely unknown.[8] Clinical observations indicate that complement activation is a core process in human SLE, with approximately 75% of SLE patients showing low C3 and C4 levels, particularly when renal is involved.[9]

Human complement receptor 2 (CR2) is encoded by a single gene with 20 exons located on chromosome 1q32.2.[10] A previous study demonstrated that CR2 could bind C3 degradation products during complement activation,[10] suggesting that CR2 might play an essential role in immunity.[11] Additional studies showed that polymorphisms of the CR2 gene were associated with osteonecrosis in SLE[12] and that polymorphisms such as rs3813946, rs1048971, and rs17615 were associated with lupus susceptibility in Caucasian and Chinese lupus patients.[13]

Next-generation sequencing technologies have been widely used for sequencing exomes, transcriptomes, and genomes,[14] particularly in the mutant landscape of genetic diseases.[15,16] In this study, we aimed to report the analysis of familial SLE by whole-exome sequencing (WES) and the discovery of a novel c.2804 (exon 15) T>C mutation in the CR2 gene.

Patients and Methods

This retrospective study was conducted at Three Gorges Hospital, Department of Nephrology between January 2017 and December 2018. A total of one Chinese familial SLE patients (median age: 30.25 years; range, 22 to 49 years) were enrolled in this study.

WES and deoxyribonucleic acid (DNA) sequence analysis

Genomic DNA was extracted from the peripheral blood of the patients using the QIAamp Blood DNA Mini Kit (QIAGEN S.p.A, Milano, Italy) according to the manufacturer's instructions. The whole-exome library was constructed using the IDT xGen® Exome Research Panel v1.0 (IDT, Iowa, USA) and sequenced on an Illumina Nova Seq 6000 series sequencer (Illumina, USA), with a sequencing coverage of the target sequence that was ≥99%. For mutation annotation and pathogenicity prediction, the raw data were cleaned and compared with the National Center for Biotechnology Information's Human Reference Genome (HG19) using the BWA software package. Synonymous changes and single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) ≥5% were removed (http://www.ncbi.nlm.nih.gov/projects/SNP). Non-synonymous changes were filtered using the SIFT software (http://sift.jcvi.org). Analysis of the function of mutated genes, their relationship to disease, and the interpretation of sequencing variations were based on guidelines from the American College of Medical Genetics and Genomics and the As Association for Molecular Pathology.

Mutation confirmed by Sanger sequencing

Sanger sequencing was used to verify suspected pathogenic mutations. The polymerase chain reaction (PCR) products were sequenced using ABI 3730XL and analyzed using the DNASTAR software.

Results

The clinical characteristics of the familial SLE

The baseline characteristics of the four family members, including the patient’s mother (Ia, mother), IIa (eldest daughter), IIb (middle daughter), and IIc (youngest daughter), are listed in Table 1 and Figure 1a. The proband patient (IIa, eldest daughter) was a 26-year-old woman who visited our department and reported a history of SLE. The mother and three daughters were all diagnosed with SLE according to the European League Against Rheumatism (EULAR) SLE 2012 criteria.[17] The patient and her mother had lupus nephritis. Patient’s mother and she had mild hypertension. The proteinuria in the mother was nephritic proteinuria, while the proband patient showed nephrotic proteinuria. The patients all showed a mild decrease in estimated glomerular filtration rate (eGFR) and low serum albumin levels. Our laboratory findings showed that all four patients were positive for the anti-SSA and anti-nuclear (ANA) antibodies, but the titers of ANA varied among the four patients. All patients showed hypocomplementemia, with titers of the ANA and anti-SSA antibodies (+++). One of the patient’s sisters showed anti-double-stranded DNA (dsDNA) positivity, while the other three patients were negative for this phenotype. The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was calculated based on symptoms. Renal biopsy showed that the proband patient had lupus nephritis IV according to the 2003 International Society of Nephrology (ISN)/ Renal Pathology Society (RPS) Classification of lupus nephritis (LN) (Figure 2).[18]

Table 1. Baseline information of the familial SLE

Figure 1. Genomic analysis of the familial SLE patients. (a) A detailed family history of SLE patients evaluated in this study. (b) A novel c.2804 T>C mutation of the CR2 gene in the mother and the three daughters evaluated in this study. SLE: Systemic lupus erythematosus.

Figure 2. Patient renal pathology results of proband patient. (a) A total of 16 glomeruli were acquired, and two glomeruli were global sclerotic (PAS staining, x40). (b) Focal segmentations of mesangial cells and mild mesangial matrix hyperplasia, and a cellular fibroid crescent formation (H&E staining, x400). (c) A small amount of granular eosinophilic protein deposition was observed in the mesangial area of the kidney (PAS staining, x400). (d) An electron-dense deposition was observed in the mesangial and subendothelial area of the kidneys of familial SLE patients. SLE: Systemic lupus erythematosus.

Mutational analysis

Using WES, there were about 41,130 variants in common among the mother and her three daughters, two gene mutations have been identified, ITGB3 with c.1960 (exon12) G>A mutation (reference sequence GenBank accession no. NM_000212). However, the ITGB3 mutation is inconsistent with the clinical manifestations of the patients. Another mutation is CR2 (reference sequence GenBank accession no. NM_001006658.2). This is a currently unreported nonsense mutation, characterized by a 2804 T>C nucleotide change (Figure 1b). Thus, the peptide isoleucine at site 935 was replaced by threonine (I935T) in the CR2 protein (Table 2). The PROVEAN test showed that the p. I935T mutation was deleterious (-2.75), and PolyPhen2 analysis predicted that the p. I935T mutation was most likely damaging, with scores of 0.63 and 0.516 in the HumDiv and HumVar models, respectively. ProtParam computed the instability index (II) of the CR2 protein to be 77.98, which classifies the protein as unstable, although the p. I935T substitution did not change the aliphatic or instability index (II). Mutation tasker software hazard prediction showed a score of 0.998, indicating that it is predicted to be disease-causing. The Mendelian Clinically Applicable Pathogenicity (M-CAP) Score and Sorting Intolerant from Tolerant (SIFT) prediction also showed a genetic damaging effect. Based on the above prediction software, the mutation c.2804 (exon 15) T>C in CR2 is likely pathogenic. We also searched the ClinVar database and found that a total of 74 variations were reported for CR2. However, most of them were likely benign or not clinically significant, with only a few classified as pathogenic (Table 3). Therefore, our analysis has added a new pathogenic variation to CR2 that is correlated with symptoms of familial SLE.

Table 2. CR2 mutation of the familial SLE

Table 3. Previous reports of CR2 gene mutation and association with disease

Discussion

Systemic lupus erythematosus has a strong genetic predisposition in human patients and animal models.[18] The genetic basis of SLE has been established using genome-wide association studies (GWAS),[19] which have indicated that the genes responsible for regulating immune regulation pathways are mainly involved in SLE.[20,21] Recent studies have shown that the CR2 gene encodes complement receptor (CR)1 and CR2. CR2 is a 145 kDa glycoprotein that plays a crucial role in regulating normal immunity. For instance, it activates B cells by combining with B cell receptors to target the follicular dendritic cell antigen.[22] A previous report showed that CR2 was expressed in B cells[23] and that changes in CR2 expression had different effects on disease presentation in autoimmune animal models.[24] Moreover, CR2 knockout mice had a severe defect in their humoral response to T-dependent antigens.[25]

Both CR1 and CR2 have been reported to play a role in the development of SLE. Abnormal CR1 and CR2 function may be associated with the production of autoantibodies by altering the action of other SLE susceptibility genes.[26] In CR1/CR2 deficient mice, low levels of IgG3 rheumatoid factor (RF) and total serum IgG3 were observed, while significantly higher levels of IgA were observed in the glomeruli.[27]

Currently, SLE, like most common human illnesses, is recognized as a polygene disease with complex genetic susceptibility. A large number of gene polymorphisms have been associated with SLE susceptibility;[28,29] however, some researchers have hypothesized that SLE may be a mono-gene disease, as a complete deficiency in C1, C2, and C4 can cause SLE,[30] although other genes have also been implicated in single-gene lupus, which provides a valuable insight into the course of the very complex disease.[31] Familial SLE was first reported in 1961,[32] with more cases reported over time.[33] Wang et al.[34] analyzed the clinicopathological characteristics of familial SLE patients and found that compared to sporadic subjects, familial subjects had a higher prevalence of fever, rash, and arthritis, but lower platelet counts. However, another study reported no significant differences in the clinical and serological manifestations between familial and non-familial SLE.[35] Compared to the non-familial cohort, the familial SLE group had poorer SLEDAI scores at the beginning of diagnosis.[36] In our case study, we found a slight difference in serological manifestations and SLEDAI scores among the four SLE patients. In addition, only one patient developed lupus nephritis. Therefore, the link between CR2 and different phenotypes requires further study.

With its rapid development, second-generation sequencing technology has been applied to study several biological problems. It can quickly sequence entire genomes and be scaled up to target genomic regions for deep sequencing.[37] Frangou et al.[38] used next-generation ribonucleic acid (RNA) sequencing technology to investigate the pathogenesis of SLE by comparative transcriptome analysis of the spleen and kidneys of lupus-prone and healthy mice. Sandling et al.[39] identified coding single-nucleotide variations (SNVs) in novel candidate risk sites for adolescent SLE by targeting next-generation sequencing. Next-generation sequencing can also identify micro RNA (miRNA)-based biomarker panels for lupus nephritis.[40] As a result, next-generation sequencing technologies with high throughput and accuracy provide a powerful tool for identifying genomic mutations in autoimmune diseases.[41]

In conclusion, our study identified a novel CR2 gene mutation by next-generation sequencing in close family members with familial SLE. These findings provide evidence that CR2 contributes to the pathogenesis of SLE and this new disease-associated mutation site may provide new clues to the underlying molecular mechanisms of SLE.

Citation: Tang Y, Luo Y. Identification of a novel mutation in complement receptor 2 in Chinese familial systemic lupus erythematosus. Arch Rheumatol 2022;37(4):566-573.

Ethics Committee Approval

The study protocol was approved by the Institute of Three Gorges Hospital Ethics Committee (no. 2017-002-01). The study was conducted in accordance with the principles of the Declaration of Helsinki.

Author Contributions

Collected the data and drafted the paper: Y.W.T.; Conceived the idea and revised the paper: Y.L.

Conflict of Interest

The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Financial Disclosure

The authors received no financial support for the research and/or authorship of this article.

Acknowledgments

We would like to thank the participants involved in the study.

References

  1. Tsokos GC. Autoimmunity and organ damage in systemic lupus erythematosus. Nat Immunol 2020;21:605-14. DOI: 10.1038/s41590-020-0677-6
  2. Rees F, Doherty M, Grainge MJ, Lanyon P, Zhang W. The worldwide incidence and prevalence of systemic lupus erythematosus: A systematic review of epidemiological studies. Rheumatology (Oxford) 2017;56:1945-61. DOI: 10.1093/rheumatology/kex260
  3. Tsokos GC, Lo MS, Costa Reis P, Sullivan KE. New insights into the immunopathogenesis of systemic lupus erythematosus. Nat Rev Rheumatol 2016;12:716-30. DOI: 10.1038/nrrheum.2016.186
  4. Fava A, Petri M. Systemic lupus erythematosus: Diagnosis and clinical management. J Autoimmun 2019;96:1-13. DOI: 10.1016/j.jaut.2018.11.001
  5. Ceccarelli F, Perricone C, Borgiani P, Ciccacci C, Rufini S, Cipriano E, et al. Genetic factors in systemic lupus erythematosus: Contribution to disease phenotype. J Immunol Res 2015;2015:745647. DOI: 10.1155/2015/745647
  6. Mohan C, Putterman C. Genetics and pathogenesis of systemic lupus erythematosus and lupus nephritis. Nat Rev Nephrol 2015;11:329-41. DOI: 10.1038/nrneph.2015.33
  7. Bentham J, Morris DL, Graham DSC, Pinder CL, Tombleson P, Behrens TW, et al. Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus. Nat Genet 2015;47:1457-64. DOI: 10.1038/ng.3434
  8. So HC, Gui AH, Cherny SS, Sham PC. Evaluating the heritability explained by known susceptibility variants: A survey of ten complex diseases. Genet Epidemiol 2011;35:310-7. DOI: 10.1002/gepi.20579
  9. Bao L, Cunningham PN, Quigg RJ. Complement in lupus nephritis: New perspectives. Kidney Dis (Basel) 2015;1:91-9. DOI: 10.1159/000431278
  10. van den Elsen JM, Isenman DE. A crystal structure of the complex between human complement receptor 2 and its ligand C3d. Science 2011;332:608-11. DOI: 10.1126/science.1201954
  11. Holers VM, Kulik L. Complement receptor 2, natural antibodies and innate immunity: Inter-relationships in B cell selection and activation. Mol Immunol 2007;44:64-72. DOI: 10.1016/j.molimm.2006.07.003
  12. Kim TH, Bae SC, Lee SH, Kim SY, Baek SH. Association of complement receptor 2 gene polymorphisms with susceptibility to osteonecrosis of the femoral head in systemic lupus erythematosus. Biomed Res Int 2016;2016:9208035. DOI: 10.1155/2016/9208035
  13. Douglas KB, Windels DC, Zhao J, Gadeliya AV, Wu H, Kaufman KM, et al. Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing. Genes Immun 2009;10:457-69. DOI: 10.1038/gene.2009.27
  14. Le Gallo M, Lozy F, Bell DW. Next-generation sequencing. Adv Exp Med Biol 2017;943:119-48. DOI: 10.1007/978-3-319-43139-0_5
  15. Boycott KM, Vanstone MR, Bulman DE, MacKenzie AE. Rare-disease genetics in the era of next-generation sequencing: Discovery to translation. Nat Rev Genet 2013;14:681-91. DOI: 10.1038/nrg3555
  16. Mardis ER. The impact of next-generation sequencing technology on genetics. Trends Genet 2008;24:133-41. DOI: 10.1016/j.tig.2007.12.007
  17. Petri M, Orbai AM, Alarcón GS, Gordon C, Merrill JT, Fortin PR, et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012;64:2677-86. DOI: 10.1002/art.34473
  18. Kiriakidou M, Ching CL. Systemic lupus erythematosus. Ann Intern Med 2020;172:ITC81-ITC96. DOI: 10.7326/AITC202006020
  19. Deng Y, Tsao BP. Genetic susceptibility to systemic lupus erythematosus in the genomic era. Nat Rev Rheumatol 2010;6:683-92. DOI: 10.1038/nrrheum.2010.176
  20. Rahman AH, Eisenberg RA. The role of toll-like receptors in systemic lupus erythematosus. Springer Semin Immunopathol 2006;28:131-43. DOI: 10.1007/s00281-006-0034-3
  21. Rönnblom L, Alm GV, Eloranta ML. The type I interferon system in the development of lupus. Semin Immunol 2011;23:113-21. DOI: 10.1016/j.smim.2011.01.009
  22. Holers VM. Complement receptors and the shaping of the natural antibody repertoire. Springer Semin Immunopathol 2005;26:405-23. DOI: 10.1007/s00281-004-0186-y
  23. Wu H, Boackle SA, Hanvivadhanakul P, Ulgiati D, Grossman JM, Lee Y, et al. Association of a common complement receptor 2 haplotype with increased risk of systemic lupus erythematosus. Proc Natl Acad Sci U S A 2007;104:3961-6. DOI: 10.1073/pnas.0609101104
  24. Del Nagro CJ, Kolla RV, Rickert RC. A critical role for complement C3d and the B cell coreceptor (CD19/ CD21) complex in the initiation of inflammatory arthritis. J Immunol 2005;175:5379-89. DOI: 10.4049/jimmunol.175.8.5379
  25. Ahearn JM, Fischer MB, Croix D, Goerg S, Ma M, Xia J, et al. Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity 1996;4:251-62. DOI: 10.1016/S1074-7613(00)80433-1
  26. Wu X, Jiang N, Deppong C, Singh J, Dolecki G, Mao D, et al. A role for the Cr2 gene in modifying autoantibody production in systemic lupus erythematosus. J Immunol 2002;169:1587-92. DOI: 10.4049/jimmunol.169.3.1587
  27. Boackle SA, Culhane KK, Brown JM, Haas M, Bao L, Quigg RJ, et al. CR1/CR2 deficiency alters IgG3 autoantibody production and IgA glomerular deposition in the MRL/lpr model of SLE. Autoimmunity 2004;37:111-23. DOI: 10.1080/08916930410001685063
  28. Tsang-A-Sjoe MW, Nagelkerke SQ, Bultink IE, Geissler J, Tanck MW, Tacke CE, et al. Fc-gamma receptor polymorphisms differentially influence susceptibility to systemic lupus erythematosus and lupus nephritis. Rheumatology (Oxford) 2016;55:939-48. DOI: 10.1093/rheumatology/kev433
  29. Radanova M, Vasilev V, Dimitrov T, Deliyska B, Ikonomov V, Ivanova D. Association of rs172378 C1q gene cluster polymorphism with lupus nephritis in Bulgarian patients. Lupus 2015;24:280-9. DOI: 10.1177/0961203314555173
  30. Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies and systemic lupus erythematosus. Autoimmunity 2007;40:560-6. DOI: 10.1080/08916930701510673
  31. Alperin JM, Ortiz-Fernández L, Sawalha AH. Monogenic lupus: A developing paradigm of disease. Front Immunol 2018;9:2496. DOI: 10.3389/fimmu.2018.02496
  32. Brunjes S, Zike K, Julian R. Familial systemic lupus erythematosus: A review of the literature, with a report of ten additional cases in four families. Am J Med 1961;30:529-36. DOI: 10.1016/0002-9343(61)90077-8
  33. Michel M, Johanet C, Meyer O, Francès C, Wittke F, Michel C, et al. Familial lupus erythematosus. Clinical and immunologic features of 125 multiplex families. Medicine (Baltimore) 2001;80:153-8. DOI: 10.1097/00005792-200105000-00001
  34. Wang Z, Tang Z, Zhang HT, Hu WX, Liu ZH, Li LS. Clinicopathological characteristics of familial SLE patients with lupus nephritis. Lupus 2009;18:243-8. DOI: 10.1177/0961203308097447
  35. Abutiban F, Mokkaddam K, Ameen E, Moussa M, Alsaeid K. Comparison between familial and sporadic systemic lupus erythematosus in Kuwaiti patients. Lupus 2009;18:86-91. DOI: 10.1177/0961203308094228
  36. Abdwani R, Hira M, Al-Nabhani D, Al-Zakwani I. Juvenile systemic lupus erythematosus in the Sultanate of Oman: Clinical and immunological comparison between familial and non-familial cases. Lupus 2011;20:315-9. DOI: 10.1177/0961203310383299
  37. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005;437:376-80. DOI: 10.1038/nature03959
  38. Frangou E, Grigorioy M, Banos A, Bertsias G, Dermitzakis E, Boumpas DT. PS2:38 Comparative tissue transcriptome analysis by next-generation sequencing reveals novel pathways that characterise genetic susceptibility and developmental biology in systemic lupus erythematosus (sle). LUPUS 2018;5(Suppl 1):A51. DOI: 10.1136/lupus-2018-abstract.86
  39. Sandling J, Hultin Rosenberg L, Farias FHG, Alexsson A, Leonard D, Murén E, et al. S4D:5 Targeted nextgeneration sequencing suggests novel risk loci in juvenile onset systemic lupus erythematosus. LUPUS 2018;5(Suppl 1):A15. DOI: 10.1136/lupus-2018-abstract.24
  40. Su YJ, Lin IC, Wang L, Lu CH, Huang YL, Kuo HC. Next generation sequencing identifies miRNA-based biomarker panel for lupus nephritis. Oncotarget 2018;9:27911-9. DOI: 10.18632/oncotarget.25575
  41. Ma Y, Shi N, Li M, Chen F, Niu H. Applications of next-generation sequencing in systemic autoimmune diseases. Genomics Proteomics Bioinformatics 2015;13:242-9. DOI: 10.1016/j.gpb.2015.09.004