|Ahead of print publication
The clinical and immunological profiles of systemic lupus erythematosus patients from Assam, North-East India
Debashree Talukdar1, Akash Protim Gogoi1, Daisy Doley2, Rebecca R Marak2, Sanjeeb Kakati2, Vandana Pradhan3, Anita H Nadkarni4, Shashi Baruah1
1 Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India
2 Department of Medicine, Assam Medical College and Hospital, Dibrugarh, Assam, India
3 Department of Clinical and Experimental Immunology, National Institute of Immunohaematology, Mumbai, Maharashtra, India
4 Department of Haematogenetics, National Institute of Immunohaematology, Mumbai, Maharashtra, India
Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Sonitpur, Tezpur - 784 028, Assam
Source of Support: None, Conflict of Interest: None
Objective: The objective of this study is to document the clinical and immunological profiles of systemic lupus erythematosus (SLE) patients from the Upper Assam region and to evaluate the relevance of autoantibody combinations on the clinical presentation of SLE.
Materials and Methods: One hundred and forty-five SLE patients were enrolled in the study. Anti-nuclear antibody (ANA) profiles and titers of autoantibodies were determined using the ANA blot and enzyme-linked immunosorbent assay, respectively. The patients were clustered based on autoantibody titers and clinical features among the clusters were compared.
Results: Mucocutaneous (87.59%), hematological (69.65%), renal (58.03%), and musculoskeletal (50.34%) manifestations were the common clinical complications, whereas anti-double stranded deoxyribonucleic acid (dsDNA) antibody was noted in 62.76% of the patients. Patient clustering using autoantibody titers revealed that the cluster with a combination of high titers of anti-dsDNA, anti-nucleosome (Nuc), and anti-ribosomal P (Rib-P) antibody had complex disease presentation with higher frequencies of oral/nasal ulcer, serositis, musculoskeletal, hematological, and central nervous system manifestations.
Conclusion: The most common clinical features of the SLE patients were mucocutaneous, hematological, renal and musculoskeletal manifestations, and anti-dsDNA antibody was the most frequent autoantibody. A combination of high titers of anti-dsDNA, anti-Nuc, and anti-Rib-P antibodies showed association with disease complexity but not the combinations of autoantibodies against extractable nuclear antigens.
Keywords: Assam, autoantibody profile, autoantibody titer, North-east India, systemic lupus erythematosus
|How to cite this URL:|
Talukdar D, Gogoi AP, Doley D, Marak RR, Kakati S, Pradhan V, Nadkarni AH, Baruah S. The clinical and immunological profiles of systemic lupus erythematosus patients from Assam, North-East India. Indian J Rheumatol [Epub ahead of print] [cited 2020 Aug 7]. Available from: http://www.indianjrheumatol.com/preprintarticle.asp?id=289180
| Introduction|| |
Systemic lupus erythematosus (SLE) or lupus is a chronic autoimmune disease, characterized by a spectrum of immunological abnormalities and production of autoantibodies resulting in widespread inflammation causing tissue and organ damage. The prevalence of SLE is 6.5 to 178.0 per 100,000 globally with varying epidemiologic information. The incidence rate and severity of clinical manifestations of SLE are higher among Asians and individuals of African origin than Caucasians. Variations observed in the clinical presentation and disease course of SLE among different populations suggest the importance of genetics in disease pathogenesis.
In India, the reported prevalence of SLE is 3.2 per 100,000. Malar rash, arthritis, renal, and hematological manifestations were reported in higher proportions than discoid lesions, serositis, and neurological manifestations among North and West Indian SLE patients., In contrast, serositis and hematological manifestations were reported to be more frequent in a study conducted in SLE patients from Kolkata, East India in comparison to other manifestations. Arthritis and hematological manifestations were reported to be common in SLE patients from South India.,
Several investigators have documented relation between disease manifestations and serology of SLE. However, reports documenting clinical manifestations and associated autoantibodies of SLE patients from the North-eastern region of India are scarce. This is a first detailed study which documents the clinical and autoantibody profiles of SLE patients from one of the North-Eastern states of India, Assam. Besides, we also clustered the patients based on their autoantibody titers to evaluate the association of different autoantibody combinations with the clinical presentation of the disease.
| Materials and Methods|| |
It was a hospital-based, cross-sectional study conducted at Assam Medical College and Hospital (AMC and H), Dibrugarh and Tezpur University, Tezpur of Assam. Ethical Committee approval was obtained from both the participating institutes. One hundred and forty-five SLE patients fulfilling American College of Rheumatology classification criteria for SLE were enrolled in the study, which was conducted over a period of 2 years (2015–2017). Postmenopausal and pregnant women, smokers, and diabetic patients were excluded from the study. Written consents were obtained from the patients. Predesigned questionnaires were filled up from hospital records and direct patient evaluation by rheumatologists. Disease activity of the patients was assessed using SLE disease activity index (SLEDAI) score. Blood was collected at the time of patient enrollment and serum was stored at −80°C till tested. Routine blood, urinalysis, and other biochemical tests were performed. Chest X-ray, two-dimensional echocardiography, and electrocardiography were performed in the recommended patients. C3 and C4 levels were measured by using nephelometry (Agappe Diagnostics, India).
Antinuclear antibodies (ANAs) were detected by indirect immunofluorescence assay (IFA) using ANA-HEp-2 kit (Aesku. Diagnostics GmbH). A serum dilution of 1:80 was considered as cutoff for this assay. Autoantibody profile of each patient was determined by using AESKUBLOTS ANA-17 Pro (Aesku. Diagnostics GmbH) kit. This ANA blot kit can detect immunoglobulin G antibodies against 17 different antigens like double-stranded deoxyribonucleic acid (dsDNA), nucleosome (Nuc), histone, smith D1 (SmD1), ribosomal P0 (Rib-P0), Sjögren's-syndrome-related antigen A/Ro60 (SSA/Ro60), Ro52/tripartite motif containing protein 21 (Ro52/TRIM21), Sjögren's-syndrome-related antigen B/La (SSB/La), U1small nuclear ribonucleoprotein, centromere protein B, proliferating cell nuclear antigen, anti-mitochondrial antibody M2 antigen, Scl-70, PM/Scl, Jo-1, Mi-2 and Ku in human serum or plasma. The titers of anti-dsDNA, anti-Nuc, anti-SmD1, anti-SSA (mixture of SSA/Ro60 and Ro52) and anti-SSB/La antibodies were determined using enzyme-linked immunosorbent assay (ELISA) kits of Human Diagnostics Worldwide GmbH. Aesku. Diagnostics ELISA kits were used to determine the titer of anti-Rib-P (mixture of P0, P1 and P2) antibody.
Statistical analyses were conducted using XLSTAT-Base solution software (Addinsoft). Spearman correlation analysis was used to investigate the correlation between the parameters. The neutrophil-to-lymphocyte ratio (NLR) of the patient group with renal complications was compared with the group without renal complications by using the Mann–Whitney U-test. The receiver operating characteristic (ROC) curve analysis was performed to determine the optimal value of NLR for predicting renal complications in the SLE patients. K-means clustering was performed by using the titers of the six autoantibodies; anti-dsDNA, anti-Nuc, anti-Rib-P, anti-SmD1, anti-SSA and anti-SSB/La as variables to cluster the patients having similar autoantibody titers together. The clustering analyses were run for four times (K = 2, 3, 4 and 5) and the optimal number of clusters for the patients was determined by using the elbow plot. The values of disease duration and ELISA titers between the clusters were compared using Student's t-test, whereas analysis of variance was used to compare the overall differences. Frequencies of patients having specific medications and clinical manifestations between the clusters were compared using the Fisher's exact test, whereas Chi-square test was used to compare overall differences. The results were considered statistically significant at P < 0.05.
| Results|| |
The study group consisted of 140 female and 5 male SLE patients making the female: male ratio 28:1. Majority of patients belonged to Tibeto-Burman (59.3%) linguistic group followed by Indo-Aryan (33.8%) and Austro-Asiatic (6.9%) groups. The mean age of the patient group was 25.89 ± 8.13 years which also included six pediatric cases. The mean age at disease onset of the patient group was 23.5 ± 8.1 years. The mean SLEDAI score of the patient group was 12.3 ± 6.63. The percent of patients with mild, moderate, and severe disease activity were 25.5%, 54.5%, and 20%, respectively. The patients were mainly prescribed corticosteroids and hydroxychloroquine (HCQ). The percent of patients prescribed corticosteroids and HCQ were 64.14% and 55.17%, respectively.
The major clinical complications of the patients were mucocutaneous manifestations followed by hematological, renal, and musculoskeletal manifestations [Table 1]. Abnormal liver function was also observed as a subclinical complication since in many patients higher than normal levels of bilirubin (12.73%) and the liver enzymes (42.74%), including aminotransferases, serum glutamic oxaloacetic transaminase (31.25%) and serum glutamic pyruvic transaminase (10.34%) and alkaline phosphatase enzyme (21.15%) were reported. Of the SLE patients with abnormal levels of liver enzymes, 8 (13%) patients also had the symptoms of myositis. Leukocytosis was observed in 12.6% of the SLE patients. A positive association of leukocytosis was observed with neutrophil count (r = 0.349, P = 0.0005) which suggested neutrophilia was the cause of leukocytosis in the patients. Interestingly, NLR was found to be higher in patients with renal complications than those without renal complications (Mann–Whitney test; P = 0.0032). The area under the ROC curve (AUC) for NLR was 0.662 (95% confidence interval (CI): 0.559–0.765). The optimal cutoff value of NLR for predicting renal complications in the patients was >3.05 with a sensitivity of 62.26% and a specificity of 67.86%. The levels of the two complement proteins C3 and C4 were observed to be negatively correlated with SLEDAI (r = −0.453, P = <0.0001 and r = −0.438, P = <0.0001). The erythrocyte sedimentation rate (ESR) also showed weak but positive correlation with SLEDAI (r = 0.299, P = 0.001).
All the patients were ANA-IFA positive. Anti-dsDNA antibodies were the most frequent autoantibody observed in ANA blot followed by anti-SSA/Ro60 antibody [Table 2]. Anti-dsDNA (r = 0.221, P = 0.008) and anti-Nuc (r = 0.301, P = 0.0002) antibodies were found to be weakly correlating with SLEDAI. The mean levels of the studied autoantibodies against dsDNA, Nuc, Rib-P, SmD1, SSA, and SSB/La in the patients were 133 ± 83.71 U/ml, 264 ± 142.5 U/ml, 329.4 ± 158.8 U/ml, 312 ± 116.4 U/ml, 308.3 ± 108.4 U/ml, and 278.7 ± 133.9 U/ml, respectively.
Around 71.03% of the patients had two or more than two types of autoantibodies. The patients were clustered into four clusters based on their autoantibody titers by using K-means clustering. No significant difference of disease duration [overall P = 0.471, [Table 3] and number of patients having medications (corticosteroids; overall P = 0.708 and HCQ; overall P = 0.726) was observed between these clusters and overall. Cluster I patients could be characterized by high titers of autoantibodies against extractable nuclear antigens (ENAs); Rib-P, SmD1, and SSA. In cluster II, the titer of autoantibody against the ENA; SSA was high. Cluster III patients had comparatively low titers of all the autoantibodies in comparison to the other clusters. Cluster IV patients had high titers of autoantibodies against dsDNA, Nuc, and Rib-P.
|Table 3: Characteristics of the clusters of SLE patients based on autoantibody titers|
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Interestingly, the first two clusters with high titers of anti-ENA antibodies had no significant difference of clinical manifestations with cluster III. In contrast, cluster IV had relatively complex disease presentation in comparison to the other clusters. The frequencies of central nervous system (CNS) manifestations (vs. CIII, P = 0.057), hematological (CIII, P = 0.052), musculoskeletal (vs. CI, P = 0.059 and CII, P = 0.036), serositis (vs. CII, P = 0.05 and CIII, P = 0.024), and oral/nasal ulcer (vs. CI, P = 0.048; CII, P = 0.022 and CIII, P = 0.015) were higher in this cluster [Table 3].
| Discussion|| |
The present study was conducted to document the clinical and immunological profiles of SLE patients from the Upper Assam region. The patient group had an early disease onset as compared to white and black populations and was similar with the patients from Philippines and Malaysia. Mucocutaneous manifestations were the major clinical feature of the patients. The high frequencies of mucocutaneous and hematological manifestations observed in the patients were within the earlier reported range of 52%–98% and 26%–83.8%, respectively, for Asian cohorts. The frequency of renal complications of the patients was often comparable to neighboring South-East Asian countries, which is higher in comparison to some other parts of India., Interestingly, arthritis cases were observed to be lower than majority of the studies reported from India, and a number of South-East Asian countries., The high frequency of anti-dsDNA antibody observed in the patients was within the range of 43%–65% as seen in other Asians. The high frequency of anti-SSA/Ro60 antibodies observed in patients of Assam is comparable to SLE patients from Hong Kong and Singapore., These observations suggest that clinical and serological profiles of SLE patients of the region have a distinct pattern sharing similarity as well as dissimilarity with patient cohorts from India and neighboring Asian countries. This is not surprising as Tibeto-Burman population was the major linguistic group of our study population which has genetic affinity with neighboring South-East Asian populations. Besides, a high degree of admixture of the ancestral Tibeto-Burman population with other populations of the region, including the Indo-Aryan population native to other parts of India as well as the second major population of our study, favors the relevance of the present study to the local, general population.
Renal involvement is a serious complication of SLE, which is often observed to be a cause of mortality in SLE patients. In consistent with a recent study, we too noted correlation between renal complications and NLR supporting NLR as a marker for lupus nephritis in patients with SLE. The hematological manifestation, leukocytosis of SLE patients has been reported to be caused by granulocytosis. In our patients, neutrophilia was found to be the factor contributing to leukocytosis. We observed negative correlations of the complement proteins C3 and C4 with SLEDAI, while a positive correlation of ESR with SLEDAI as reported in earlier studies.,
Anti-Nuc antibody was observed to show higher correlation with SLEDAI followed by anti-dsDNA antibody. These data are in agreement with previous observation which showed anti-Nuc antibodies as a useful tool in the diagnosis and assessment of disease activity in SLE patients, especially in patients who are negative for anti-dsDNA antibodies.
Co-occurrence of particular sets of autoantibodies was observed in our patient group. Therefore, we tried to identify the subsets of SLE patients characterized by specific autoantibody combinations to evaluate possible association of such combinations with clinical characteristics of the patients. However, unlike earlier studies which used autoantibody profiles as the basis for clustering, we used autoantibody titers as variables for clustering our patients., The major concerns while clustering the patients were disease duration and drug treatment, both of which can influence characteristics of SLE patients. However, our clustering system could be expected to remain unaffected by these factors as no significant difference of the two factors was observed between the clusters.
We observed that the first two clusters of patients had high titers of autoantibodies either to a combination of ENAs or a single ENA, respectively, whereas the third cluster had low titers of all the autoantibodies. However, there was no significant difference in the frequencies of clinical manifestations among these three clusters. In the fourth cluster, we observed high autoantibody titers against both chromatin-related antigens; dsDNA and Nuc and the SLE specific ENA; Rib-P. The patients of this cluster had higher occurrences of musculoskeletal manifestations than cluster I and II, serositis than cluster II and III, oral/nasal ulcer than cluster I, II and III along with CNS and hematological complications than cluster III. The disease complexity of the patients in cluster IV suggests combinatorial effect of high titers of anti-dsDNA, anti-Nuc, and anti-Rib-P antibodies had greater impact on disease complexity of SLE patients than that of anti-ENA antibody combinations alone. Further, previous studies have also reported that high titer of anti-dsDNA or anti-Nuc antibody heightens SLE disease severity.,
The limitation of the study was that it was a hospital-based study with a relatively smaller sample size. Therefore, a study with a larger sample size on the SLE patients of the region will be instructive to validate the findings of the study.
| Conclusion|| |
This study reported that mucocutaneous, hematological, renal, and musculoskeletal were the most common clinical manifestations, whereas anti-dsDNA antibody was the most frequent autoantibody of the SLE patients of the Upper Assam region. NLR was observed as an effective marker to predict renal manifestations of the patients. In addition, our study suggests that SLE patients having a combination of high titers of anti-dsDNA, anti-Nuc, and anti-Rib-P antibodies could be more prone to develop multiple clinical manifestations contributing to the disease complexity of the patients.
| Acknowledgment|| |
We would like to thank all the participants for their cooperation in the study. Thanks to Indrani Kalita, Laboratory Technician, Tezpur University and Shakuntala Das, Senior Research Fellow and Manoj Barman, Laboratory Technician, AMC and H, Dibrugarh for their assistance in data acquisition.
We received the grant DBT-NER/Health/38/2013 from Department of Biotechnology, Government of India and the grant UGC-SAP (DRS II) (No. F.3-1 / 2015/UGC DRS II [SAP II]) from University Grants Commission of India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]