Genetic variations in the Dravidian population of South West coast of India: Implications in designing case-control studies.

BACKGROUND & OBJECTIVES: Indian data have been largely missing from genome-wide databases that provide information on genetic variations in different populations. This hinders association studies for complex disorders in India. This study was aimed to determine whether the complex genetic structure and endogamy among Indians could potentially influence the design of case-control studies for autoimmune disorders in the south Indian population. METHODS: A total of 12 single nucleotide variations (SNVs) related to genes associated with autoimmune disorders were genotyped in 370 healthy individuals belonging to six different caste groups in southern India. Allele frequencies were estimated; genetic divergence and phylogenetic relationship within the various caste groups and other HapMap populations were ascertained. RESULTS: Allele frequencies for all genotyped SNVs did not vary significantly among the different groups studied. Wright's FSTwas 0.001 per cent among study population and 0.38 per cent when compared with Gujarati in Houston (GIH) population on HapMap data. The analysis of molecular variance results showed a 97 per cent variation attributable to differences within the study population and <1 per cent variation due to differences between castes. Phylogenetic analysis showed a separation of Dravidian population from other HapMap populations and particularly from GIH population. INTERPRETATION & CONCLUSIONS: Despite the complex genetic origins of the Indian population, our study indicated a low level of genetic differentiation among Dravidian language-speaking people of south India. Case-control studies of association among Dravidians of south India may not require stratification based on language and caste.

HLA associations in South Asian multiple sclerosis.

BACKGROUND: Previous efforts to identify Human Leukocyte Antigen (HLA) gene associations with multiple sclerosis (MS) in the South Asian population have been underpowered. AIM: To identify the primary HLA class II alleles associated with MS in Indians. METHODS: We typed HLA-DRB1, -DQA1 and -DQB1 in 419 patients and 451 unrelated controls by polymerase chain reaction using sequence specific oligonucleotide probes (PCR-SSOP). RESULTS: At the gene level DRB1 showed significant evidence of association (p=0.0000012), DQA1 showed only marginal evidence of association (p=0.04) and there was no evidence for association at DQB1 (p=0.26). At the DRB1 locus association is confirmed with the *15:01 (p=0.00002) and the *03 (p=0.00005) alleles. CONCLUSION: Our study confirms that the risk effects attributable to the HLA- DRB1*15:01and DRB1*03 alleles seen in Europeans are also seen in Indians. The absence of any evidence of association with DQB1 alleles reflects the lower linkage disequilibrium between DQB1 alleles and DRB1 risk alleles present in this population, and illustrates the potential value of fine mapping signals of association in different ethnic groups.

Mycophenolate mofetil in the treatment of multiple sclerosis: a preliminary report.

BACKGROUND: Mycophenolate mofetil (MMF) is an affordable and tolerable drug reported to be beneficial in the treatment of multiple sclerosis (MS). AIM: To determine efficacy of MMF as first line disease modifying drug (DMD) in 40 patients with MS seen in our demyelinating disease registry. MATERIALS AND METHODS: The annualized relapse rate (ARR) for 1 year prior to starting MMF therapy and 1 year post treatment was calculated. Pre- and post-treatment expanded disability status scores (EDSS), age at onset of treatment, disease duration, and type of MS were recorded. Wilcoxon rank sum test was used for comparison of ARRs and EDSS before and after treatment. RESULTS: Forty patients included 27 females and 13 males. Mean duration of MMF therapy was 24 months (range 14-33 months). Pre-treatment mean ARR of 0.95 was significantly different from post treatment mean ARR of 0.11 (P=0.0001). Pre-treatment mean EDSS 3.80 (inter quartile range [IQR] 3.5-4.5) was significantly different from post-treatment mean EDSS 2.66 (IQR 1.5-3.0, P=0.0001). No adverse effects were reported that required stopping of medication. Five patients discontinued treatment 6-11 months after starting therapy, two of whom relapsed subsequently. CONCLUSION: Our preliminary results support the use of MMF, a cheap and well-tolerated drug, as first line disease modifying drug in MS. Long-term results in a larger patient cohort is required for validating our preliminary conclusions.

Association of vitamin D and multiple sclerosis in India.

BACKGROUND: Vitamin D deficiency is widely prevalent in India. The association between vitamin D status and multiple sclerosis (MS) has not been previously studied in Indians. OBJECTIVE: The objective of this paper is to determine whether vitamin D status is associated with MS in India. METHODS: In this study 110 MS patients and 108 matched controls were included. Serum 25-hydroxyvitamin D (25(OH)D) was measured in 63 patients in relapse, 77 patients in remission and all controls. Quantity of sun exposure in childhood and body mass index (BMI) were calculated. Patients and controls were genotyped for HLA-DRB1*1501. RESULTS: Patients had significantly lower 25(OH)D levels than matched controls (p = 0.003), and patients in relapse had a significantly lower vitamin D level as compared to those in remission (p = 0.001). Vitamin D deficiency (< 50 nmol/l) was seen in a higher proportion of cases (71.8%) than controls (53.7%) (p = 0.01). Higher quartiles of vitamin D (> 58 nmol/l) showed an inverse relationship with MS (OR = 0.28, CI = 0.11-0.68, p= 0.005). This effect persisted after adjusting for sun exposure. CONCLUSION: The results of our study indicated that serum 25(OH)D shows an inverse relationship with MS in the Indian population. Reverse causality cannot be excluded.

Comparison of live and fixed cell-based assay performance: implications for the diagnosis of MOGAD in a low-middle income country.

Background: Though considered optimal, live cell-based assay (LCBA) is often unavailable for the diagnosis of myelin oligodendrocyte glycoprotein antibody-associated disorders (MOGAD) in resource-poor regions. This study was undertaken to determine the agreement between LCBA and the widely available fixed cell-based assay (FCBA), for recommending testing guidelines within our region. Method: All consecutive patients in our registry with a MOGAD phenotype were tested. The results from a commercially available FCBA (Euroimmun, Germany) were compared with a validated "in-house" LCBA. Clinical and MRI data were available for correlation. Results: Among the 257 patient samples tested, 118 (45.9%) were positive by FCBA titre ≥1: 10 and or LCBA titres ≥1: 160 titre and 139 samples were negative. There was robust agreement between the two assays (agreement 98.8%, Cohen's kappa 0.98 [95% CI- 0.95-1.00], Spearman correlation 0.97 (p < 0.0001). Among five discordant samples, four had clinical and or MRI data which supported an alternate diagnosis. There was a modest correlation between assay titres, particularly for samples with titres ≥ 1:100 in FCBA (Spearman's Rho 0.26, p 0.005). Thirty samples were positive by FCBA at < 1:100 titre and included 1:80 (20),1:40(7) and 1:10 (3) titres. Among them, 80% had clear positive titres when tested by LCBA. Conclusion: The FCBA tested with serum dilutions of 1:10 was highly predictive of MOGAD in our study and compared well with our "in-house" LCBA. The current recommendations for testing at higher dilutions need to be re-examined in light of our findings. The results of our study should ideally be replicated in a larger dataset but at the same time provide some guidance for the accurate diagnosis of MOGAD in resource-poor settings.

Helicobacter pylori infection may influence prevalence and disease course in myelin oligodendrocyte glycoprotein antibody associated disorder (MOGAD) similar to MS but not AQP4-IgG associated NMOSD.

Background: Helicobacter pylori (Hp) persists after colonizing the gut in childhood, and potentially regulates host immune system through this process. Earlier studies have shown that Hp infection in childhood, may protect against MS in later life. Such an association was not seen with AQP4-IgG positive NMOSD, while the association with MOGAD is unclear. Objective: To evaluate frequency of Hp IgG among patients with MOGAD, MS, NMOSD and matched controls and its effect on disease course. To ascertain whether childhood socio economic factors were linked to prevalence of Hp infection. Methods: In all, 99 patients diagnosed to have MOGAD, 99 AQP4 IgG+ NMOSD, 254MS and 243 matched controls were included. Patient demographics, diagnosis, age at disease onset, duration and the last recorded expanded disability status scale (EDSS) were obtained from our records. Socioeconomic and educational status was queried using a previously validated questionnaire. Serum HpIgG was detected using ELISA kits (Vircell, Spain). Result: Frequency of Hp IgG was significantly lower among MOGAD (28.3% vs 44%, p-0.007) and MS (21.2% vs 44%, p-0.0001) but not AQP4-IgG+ NMOSD patients (42.4% vs 44%, p-0.78) when compared to controls. Frequency of Hp IgG in MOGAD & MS patients combined (MOGAD-MS) was significantly lower than those with NMOSD (23.2% vs 42.4%, p- 0.0001). Seropositive patients with MOGAD- MS were older (p-0.001. OR -1.04, 95% CI- 1.01- 1.06) and had longer disease duration (p- 0.04, OR- 1.04, 95% CI- 1.002- 1.08) at time of testing. Educational status was lower among parents/caregivers of this study cohort (p- 0.001, OR -2.34, 95% CI- 1.48-3.69) who were Hp IgG+. Conclusions: In developing countries Hp infection may be a significant environmental factor related to autoimmune demyelinating CNS disease. Our preliminary data suggests that Hp may exert a differential influence - a largely protective role for MS-MOGAD but not NMOSD and may influence disease onset and course. This differential response maybe related to immuno-pathological similarities between MOGAD and MS in contrast to NMOSD. Our study further underscores the role of Hp as a surrogate marker for poor gut hygiene in childhood and its association with later onset of autoimmune diseases.

Evaluating the Role of HLA DRB1 Alleles and Oligoclonal Bands in Influencing Clinical Course of Multiple Sclerosis - A Study from the Mangalore Demyelinating Disease Registry.

BACKGROUND: The possible interaction between genetic and immunological factors in influencing clinical course of multiple sclerosis (MS) has not been studied previously in Indian population. AIM: In this study we evaluated the association of HLA alleles and OCB in affecting clinical course and disability of MS. METHODS: Clinical and demographic features of 145 MS patients who had CSF oligoclonal bands (OCB) tested by isoelectric focussing technique were analyzed, disability status estimated, and HLA DRB1 alleles were genotyped. RESULTS: OCBs were positive in 53.8% (78/145) of all MS cases. Patients with CSF OCB had more frequent relapses and an association with HLA DRB1*15. Early disease onset and a high annualized relapse rate was associated with HLA DRB1*03 allele. A relapsing remitting course for MS was seen with HLA DRB1*03 & 15 while a progressive disease was associated with DRB1*01. Presence of both OCB and HLA DRB1*13 was significantly associated with disability in this cohort. CONCLUSION: The results of our study suggest that an interaction between immunological and genetic factors may influence disease onset, course, and disability in MS.

Role of Viral Infections in Multiple Sclerosis Pathogenesis among Indian Population.

BACKGROUND: The role of viral infections in multiple sclerosis (MS) pathogenesis is unclear. OBJECTIVE: Certain neurotropic viruses previously linked with MS among white population were studied including Epstein-Barr virus, human herpesvirus-6 (HHV-6) and MS-associated retrovirus (MSRV). MATERIAL AND METHODS: Sixty-two MS patients (37 had a recent clinical relapse) and 65 controls with other neurological disorders were included. Blood and cerebrospinal fluid (CSF) samples were obtained and processed with the primary objective of determining whether there was intrathecal multiplication of viruses under study (EBV, HHV6 A and B and human endogenous retrovirus) or a breach in blood-brain barrier associated with viral presence in both peripheral blood and CSF. RESULTS: Evidence of breach in blood-brain barrier was seen in 86.5% of patients as evidenced by abnormal CSF/serum albumin index and or MRI. EBV nuclear antigen (EBNA1 IgG) was seen in 89% of MS patients and 58% controls (P = <0.001). However, HHV6 IgG was similar in both groups (85% versus 81%; P = 0.45). In affinity immunoblotting reaction intrathecal IgG synthesis against EBNA1 antigen was demonstrable in 26% (16/62) of patients and none against HHV6. A subset of patients showed significant elevation in mean copy number of plasma EBV DNA during relapse and there was a trend for the same among patients harboring HHV-6B. No evidence of isolated intrathecal viral presence or multiplication was seen. CONCLUSIONS: The results of our study suggest that viruses studied namely EBV and HHV6 have a role in triggering relapses through a peripheral mechanism, rather than a direct role through intrathecal multiplication.

Coexistence of Autoantibodies and Other Autoimmune Diseases with Multiple Sclerosis and Related Disorders - Experience from the Mangalore Demyelinating Disease Registry (MANDDIR).

BACKGROUND: Co-occurrence of other autoimmune disorders (AID) and autoantibodies in patients with autoimmune demyelinating CNS disorders have not been studied previously in patients of Indian origin. OBJECTIVE: To determine the frequency of concomitant autoimmune disorders, anti-nuclear antibody (ANA) and antithyroid antibody (ATAb) and to evaluate the impact on clinical course of disease. MATERIALS AND METHODS: A total of 111 patients with MS and 152 patients with non-MS demyelinating disorders were included. Demographics, clinical course and disability were recorded. History of other autoimmune disorders (AIDs) in patients and first degree relatives was noted. Serum ANA and ATAb were tested. RESULTS: Concomitant AIDs were seen in 21% of MS and 19% of non-MS patients. Autoimmune thyroid disease was most frequent and seen in 10.8% of MS and 6.6% of non-MS disorders. Frequency of ATAb was significantly higher among MS group (MS 25.5% vs non-MS 13.2% P = 0.04) but that of ANA was similar between the 2 groups (MS 19.8% vs non-MS 26.9% P = 0.17). A positive family history of autoimmune disorders was noted in 20% of MS and 15.1% of non-MS disorders. Clinical course was unaffected by presence of concomitant AID and autoantibodies. CONCLUSION: Cooccurrence of autoantibodies and AID are seen in a significant number of patients with MS and non-MS disorders and influences clinical management.

Overcoming the challenges in diagnosis of AQP4-IgG positive neuromyelitis optica spectrum disorders in resource poor settings using an indigenized and cost effective cell based assay.

BACKGROUND: Diagnosis of neuromyelitis optica spectrum disorders (NMOSD) in India is hindered by limited access to cost effective and sensitive assays for detection of aquaporin-4 antibody (AQP4-IgG) in India. OBJECTIVE: To develop a cost effective, sensitive, cell based assay (CBA) for detection of AQP4-IgG and to evaluate the serological status in patients with NMOSD diagnosed by 2015 diagnostic criteria. METHOD: Stably transfected Chinese hamster ovary (CHO) cell line expressing aquaporin M23 isomer was established. A fixed CBA was developed and validated in 381 samples including clinically definite NMOSD (n = 87), high risk NMOSD (n = 51), other demyelinating disorders (n = 92), other neurological disorders (n = 51) and healthy volunteers (n = 100). We tested the same samples again using a commercially available CBA and compared the results. All assays were performed by 2 independent investigators blinded to clinical and serological status. RESULTS: Our "in house"(Mangalore) assay showed sensitivity of 81.6% (95% CI 71.86-89.11%) for clinically definite NMOSD and 29.41% (95% CI 17.50-43.8%) for high risk NMOSD. Specificity was 100% for both groups. Both assays showed similar results for 67/ 87 (77.01%) patients with definite NMOSD while 4 samples tested positive by our assay alone (Cohen's kappa coefficient [K] - 0.86). Among the high risk group 14/51 (27.5%) samples showed similar results, one patient additionally was positive by the Mangalore assay (K - 0.95).

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4.

OBJECTIVE: To understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin. METHODS: In this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set. RESULTS: Microbiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation. CONCLUSION: Our study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.

CD6 gene polymorphism rs17824933 is associated with multiple sclerosis in Indian population.

BACKGROUND: Multiple sclerosis (MS) prevalence has increased worldwide. The known genetic association for MS in the west has not been studied in detail in nonwhite populations and particularly Indians. OBJECTIVE: The objective of this study was to evaluate some known genetic variations outside the major histocompatibility complex (MHC) region associated with MS in patients of Indian origin. MATERIALS AND METHODS: We investigated 10 gene-associated single nucleotide polymorphisms (SNP's) outside the MHC region in 300 patients and 720 unrelated controls. Genotyping was performed on an ABI7500 real-time polymerase chain reaction genotyping platform using predesigned TaqMan SNP genotyping assays. RESULTS: CD6 gene associated SNP (rs17824933) showed significant association with MS (P = 4.2 × 10-5, odds ratio [OR] = 2.24, confidence interval (CI) = 1.51-3.33). A modest association was also noted for TMEM39A rs1132200 (P = 0.023, OR = 1.41, CI = 1.05-1.91) and IL2RA rs2104286 (P = 0.04, OR = 1.3, CI = 1.006-1.67). In the remaining SNPs, the allele frequencies were overexpressed in patients when compared to healthy controls. CONCLUSION: Our data illustrate the similarity in risk association between Indian and European populations for MS.

Serological markers associated with neuromyelitis optica spectrum disorders in South India.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSDs) represent 20% of all demyelinating disorders in South India. No studies have determined the seroprevalence to both antibodies against aquaporin-4* and antimyelin oligodendrocyte glycoprotein antibody (anti-MOG+) in this population. OBJECTIVE: To identify and characterize seropositive patients for anti-aquaporin-4 antibody (anti-AQP4+) and anti-MOG+ in South India. MATERIALS AND METHODS: We included 125 consecutive patients (15 children) who were serologically characterized using live transfected cells to human M23-AQP4 or full-length MOG. RESULTS: Among a total of 125 patients, 30.4% of patients were anti-AQP4+, 20% were anti-MOG+, and 49.6% were seronegative. No patient was positive for both. Anti-MOG+ patients represented 28.7% (25/87) of seronegative NMOSD. In comparison to anti-AQP4+ patients, anti-MOG+ patients were commonly male, had less frequent attacks and milder disability on expanded disability status score scale. Seronegative patients were also predominantly male, 36% (9/25) had monophasic longitudinally extensive transverse myelitis and disability was comparable with anti-AQP4+ patients. Lumbar cord involvement was common in anti-MOG+ and seronegatives, whereas anti-AQP4+ patients had more cervical lesions. CONCLUSION: Anti-AQP4+/anti-MOG + patients accounted for nearly half of the patients suspected of having NMOSD in South India, indicating that antibody testing may be useful on the management of subgroups with different prognosis.

Relapsing optic neuritis and isolated transverse myelitis are the predominant clinical phenotypes for patients with antibodies to myelin oligodendrocyte glycoprotein in India.

BACKGROUND: Clinical phenotypes of patients with antibodies to myelin oligodendrocyte glycoprotein (anti-MOG+) are unknown in India. OBJECTIVES: Retrospectively to characterise anti-MOG+ patients with inflammatory central nervous system disorders in India. METHOD: A total of 87 patients with non-multiple sclerosis demyelinating disorders (excluding acute disseminated encephalomyelitis) who were seronegative for anti-aquaporin 4 antibody were retrospectively analysed using a cell-based assay for anti-MOG+ status. RESULTS: Twenty-five patients were anti-MOG+ in this cohort. They represented 28.7% (25/87) of patients who tested negative for anti-AQP4+. Sixty-four per cent (16/25) of anti-MOG+ patients were men and had a relapsing course. Patients with recurrent optic neuritis and those with a single attack of acute longitudinally extensive transverse myelitis were the most common phenotypes. CONCLUSION: Relapsing optic neuritis was the most common phenotype, contrasting with a lower risk of relapses in transverse myelitis.

Environmental factors related to multiple sclerosis in Indian population.

BACKGROUND: Multiple sclerosis (MS) is less prevalent among Indians when compared to white populations. Genetic susceptibility remaining the same it is possible that environmental associations may have a role in determining disease prevalence. AIMS: To determine whether childhood infections, vaccination status, past infection with Helicobacter pylori (H.pylori), diet, socioeconomic and educational status were associated with MS. MATERIAL AND METHODS: 139 patients and 278 matched control subjects were selected. A validated environmental exposure questionnaire was administered. Estimation of serum H.pylori IgG antibody was done by ELISA. Patients and controls were genotyped for HLA-DRB1*15:01. RESULTS: In our cohort a significant association was seen with measles (p < 0.007), vegetarian diet (p < 0.001, higher educational status (p < 0.0001) and urban living (p < 0.0001). An inverse relationship was seen with H.Pylori infection and MS (p < 0.001). Measles infection (OR 6.479, CI 1.21-34.668, p < 0.029) and high educational status (OR 3.088, CI 1.212-7.872, p < 0.018) were significant risk factors associated with MS. H.pylori infection was inversely related to MS (OR 0. 319, CI 0.144- 0.706, p < 0.005). CONCLUSIONS: Environmental influences may be important in determining MS prevalence.