Association of HLA DRB1-DQB1 Haplotypes with the Risk for Neuromyelitis Optica among South Indians
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.355130
Source of Support: None, Conflict of Interest: None
Keywords: Autoimmune, haplotypes, HLA class II, neuromyelitis optica (NMO), NMO antibodies
Neuromyelitis optica (NMO) is an autoimmune inflammatory demyelinating disease of the central nervous system in which optic nerves and spinal cord are predominantly affected.,,, The NMO shares many radiological and clinical features with multiple sclerosis (MS).,, The presence of aquaporin 4 IgG antibody guides the treatment options for the NMO. This AQP4-IgG autoantibody is highly specific (91%; 85%–99%) and sensitive (73%; 58%–76%) and has less common occurrence in MS. Therefore, combinations of clinical, MRI, and serological characteristics have been proposed to distinguish NMO from MS. The characteristic clinical features of NMO are acute optic neuritis that is bilateral and longitudinally extensive transverse myelitis (LETM). The other symptoms are paroxysmal tonic spasms of the trunks and limbs and bladder dysfunction, nausea, vomiting, hiccups, and acute respiratory decompensation with cervicomedullary extension.,,
The prevalence of NMO varies among various ethnic groups. In India, the prevalence of NMO is 2.7/100,000 according to a recent population-based epidemiological survey. Based on the population epidemiological survey, several studies have shown the strong association of NMO with HLA class II genes.,,, Some alleles have shown to be associated with risk of NMO and some are protective.,,,, The HLA-DRB1*03 was found to be a risk factor for NMO, whereas DRB1*15 was observed to be protective against NMO in Brazilian patients. HLA-DPB1*15:01 allele is the most common allele in Japanese patients. In a recent study, Asgari et al. reported a high frequency of DQB1*04:2 in NMO patients., In another study, DRB1*03 allele was significantly high in NMO patients, whereas a negative association was observed with DRB1*11. HLA-DRB1*03 allele is highly frequent in NMO-IgG positive patients. The HLA DRB1*03 and DRB1*10 was shown to be a risk factor for NMO in Indian patients, but limited studies are available representing Indians for the possible association of HLA DR and DQ alleles with NMO susceptibility, and there are no studies pertaining to the association of HLA DRB1 and DQB1 haplotypes with NMO susceptibility. Hence, the present study was undertaken to explore the possible role of HLA DRB1-DQB1 haplotypes in the susceptibility of NMO in Indian patients.
Study subjects and sampling
We enrolled a total of 158 subjects, which included 38 NMO patients visiting the Neurology department in Nizam's Institute of Medical Sciences (NIMS), Hyderabad, India, and 120 age, gender and ethnicity-matched healthy controls. The diagnosis of NMO was based on revised Wingerchuk's 2006 criteria. The patients with acute myelitis and at least two out of three supportive criteria, which include NMO-IgG positivity, spinal cord MRI lesion extending over three or more vertebral segments, and brain MRI not meeting MS diagnostic criteria, are diagnosed as NMO. Here, 5 ml of blood was collected from each subject in EDTA vacutainer, and 5 ml of blood was collected in plain vacutainer, which was subjected to centrifugation at 3000 rpm for 10 min for the separation of serum. These serum samples were aliquoted and stored at −20°C until analysis. The study was approved by the Institutional Ethics Committee of NIMS, Hyderabad, India. Informed consent was obtained from all the subjects.
Genomic DNA isolation and HLA DR and DQ typing
The genomic DNA was extracted from whole blood using the standard phenol–chloroform extraction protocol. The PCR SSP method using Histo Type DR/DQB (SSP Kit), BAG Health Care GmbH, Germany was used for HLA-DRB1 and DQB1 genotyping.
Estimation of serum NMO-IgG antibodies
Estimation of serum NMO-IgG antibodies was done using human NMO-IgG antibodies kit as per the manufacturer's instructions.
Allele and genotype frequencies were analyzed by simple gene counting method. The allele and genotype distribution and test of associations were performed using an online website called VassarStats for statistical computation (www.faculty.vassar.edu/lowry/VassarStats.html). P values of <0.05 were considered as significant.
The mean age of NMO patients was 33.05 ± 11.8 years. The incidence of NMO was high in females than in males (M:F = 1:8.5). The NMO-IgG antibodies positive was observed in 76.31% of patients and negative in 23.68% of patients. The normal antinuclear antibody (ANA) levels were seen in 81.57% of cases, and abnormal ANA level was found in 18.42% of patients. The optic neuritis was seen in 50% of patients, and definite NMO was observed in 42% of patients. The MRI findings show that 34.21% of the patients present with the lesion in the cervical spine whereas cervico dorsal lesion was seen in 44.73% of the patients. The maximum number of patients (44%) showed 5–8 vertebrae involvement, whereas 9–16 in 21.05% of the patients and 1–4 vertebrae involvement in 26.31% of the patients. The spincteric dysfunction was present in 36.84% of the patients. Fifty percent of the patients had two attacks, whereas 31.57% of the patients had one attack during the course. The CSF analysis revealed that 52.63% of patients showed normal CSF parameters while pleocytosis was seen in 26.31% of the patients, oligoclonal antibodies were seen in 5.2% of the patients, and elevated protein level was found in 15.78% of the patients. The data were segregated based on NMO-IgG positivity and NMO-IgG negative status; we did not find any significant differences in these clinical characteristics.
Association of HLA DRB1 and DQB1 alleles in patients with NMO
Our analysis showed significant association of HLA-DRB1*10 allele (OR 2.63, 95% CI: 1.18–5.8, P = 0.02) with NMO, whereas DRB1*14 showed protective role against NMO (OR 0.33: 95% CI: 0.11–0.94, P = 0.043) [Table 1]. HLA-DRB1*10 allele also showed significant association in patients with NMO-IgG positive antibody (OR 3.28: 95% CI: 1.42–7.5, P = 0.006) [Table 2]. There was no significant association of HLA-DQB1 with NMO, and NMO-IgG antibodies were observed [Table 3] and [Table 4].
Association of HLA DRB1 - DQB1 haplotypes in patients with NMO
As shown in [Table 5], our extensive haplotype analysis revealed that HLA-DRB1*10-DQB1*05 (OR 2.61, 95% CI: 1.11–6.1, P = 0.03), HLA-DRB1*10-DQB1*06 (OR 2.78, 95% CI: 1.0–8.0, P = 0.05), HLA-DRB1* 15-DQB1*03 (OR 4.5, 95% CI: 1.81–11.5, P = 0.001) were found in higher frequency in cases and strongly associated with the risk of NMO, whereas DRB1*14-DQB1*05 (OR 0.20, 95% CI: 0.06–0.721 P = 0.008) showed a negative association with NMO. Though statistically insignificant, the frequency of HLA DRB1*03-DQB1*05 (15.7%) was high in NMO patients as compared to control (7.5%). The frequency of distribution of HLA DRB1 allele was analyzed with the clinical characteristics of NMO disease. We found a significant association of HLA DRB1*O4 with spasm (OR: 6.7: 95% CI: 1.28–34.8; P = 0.02). Though statistically insignificant, the increased frequency of HLA DRB1*10 in patients with visual symptoms (18.42% vs. 13.16%), presence of spasm (20% vs. 13.04%), sphincter dysfunction (17.86% vs. 14.58), and CSF abnormality (19.44% vs. 12.50%) was observed.
The prevalence of NMO is high in women than in men,, and the median age of onset of the disease is under 30 years. We also found similar findings wherein the ratio of male to female ratio was 1:8.5 and the mean age of onset of the disease was 33 years. In another study, Barhate et al. also found similar results, wherein they reported 1:7.8 and the mean age of onset was 26.5 years. In the present study, the NMO-IgG antibody positivity was observed in 76.31% of patients and negative in 23.68% of patients, similar to Barhate et al., showing 70% IgG positive NMO cases. In another study, NMO-IgG positive was observed in 80.9% of NMO patients, and out of 21 patients, 11 patients having LETM in which 6 (54.5%) were positive for NMO-IgG. The patients with NMO-IgG positivity predicts poor visual outcome, a relapse after longitudinally extensive myelitis and risk of conversion to classical NMO. In a recent study in Korean NMO patients, bilateral simultaneous involvement was more common in AQP4-Ab positive and exhibited more severe visual loss than seronegative patients. In the current study, we did not observe any significant difference in visual symptoms in NMO-IgG positive and NMO-IgG negative patients.
Several case-control and genome-wide association studies have clearly demonstrated that HLA class II genes play a crucial role in the genetic susceptibility of NMO and provide underlying pathogenesis and differential diagnosis in demyelinating diseases like MS. The current study, to the best of our knowledge, is the first comprehensive analysis of HLA-DRB1-DQB1 haplotypes in relation to NMO susceptibility from India. The data demonstrate independent association of HLA DRB1*10 allele with NMO risk whereas DRB1*14 allele showed a protective role. HLA-DRB1*10 allele also showed significant association in patients with NMO-IgG antibody positive. However, in a study, Lekha Pandit et al. found that HLA DRB1*03 and HLA DRB1*10 were associated with the risk in south Indian NMO patients. In the current study, we did not observe any significant association of HLA DRB1*03 with NMO susceptibility, which is in contrast to several case-control studies in different ethnic groups.,, In a study in Muslim Arab Israeli patients, they observed a positive association with the HLA-DRB1*04:04 and HLA-DRB1*10:01 alleles and a strong negative association with the HLA-DRB1*07 and HLA-DQB1*02:02 alleles. In the present study, we found a significant association of HLA DRB1*04 with spasms in NMO patients.
In Caucasian populations, the frequency of HLA DRB1*01 and DRB1*03 allele was found to be high in NMO-IgG positive patients.,, In a study, Asgari et al. demonstrated the presence of high frequency of HLA DQB1*04:02 in NMO patients but did not observe any correlation with HLA DRB1*03. HLADQA1*012 was found in high frequency in NMO-IgG negative patients. High frequency of HLA DRB1*01, DRB1*03, and low frequency of HLA DRB1*15 were observed in Brazilian NMO patients. In a study, Warabi et al. found poor prognosis following interferon-1b treatment in patients with NMO-specific allele DPB1*15:01.
Studies in Japanese NMO patients have indicated that HLADRB1*1602 and HLA-DPB1*0501 were found to be strongly associated with NMO, whereas HLA DRB1*09 showed lower frequency and was protective against NMO., HLA DRB1*1602 was found to be a risk factor in anti-AQP4 antibody-positive patients in Han Chinese with a high relapse rate. In Afro-Carribeans NMO patients, HLA DRB1*03 was present in high frequency whereas HLA DR*1501 was absent. In the present study, HLA DRB1*15 had no effect on the risk of NMO; however, it was associated with the risk of MS (data not shown). This observation is in accordance with several studies.,
In the present study, we found a significant association of DRB1*10-DQB1*05, HLA-DRB1*10-DQB1*06, HLA-DRB1*15-DQB1*03 haplotypes with the risk of NMO, whereas DRB1*14-DQB1*05 showed protective role against NMO. Though HLA DRB1*03 is an established risk factor for NMO in various ethnic groups, we did not observe any significant association with NMO; however, the frequency of HLA DRB1*03-DQB1*05 (15.7%) haplotypes was high as compared to control (7.5%), indicating that in the present study, HLADRB1*03 allele may be the risk factor in combination with HLA DQB1*05. Although many studies have focused on the HLA DRB1, DQB1 alleles, none of them have explored the haplotypes based association that might have contributed to disparities in association. However, studies have shown the association of HLA DRB1-DQB1 haplotypes in the risk of MS in Northern Europe, Mediterranean population., The elevated risk of MS was observed in patients with DRB1*15:01-DQB1*06:02 haplotypes as compared to DRB1*1501 alone. Similarly, DRB1*1501-DQB1*0701 haplotypes were found to be high in MS patients as compared to the control group. In another study, Jones et al. emphasized that the associations of DRB1 and DQB1 alleles may influence the severity of the disease.
In conclusion, we demonstrate that HLA-DRB1*10-DQB1*05, HLA-DRB1*15-DQB1*03 haplotypes are found to have significant risk for the NMO susceptibility in the Indian population. Considering the small sample size, further studies are needed with a large number of patients.
We are thankful to patients for participation in the study.
RK, MAK performed clinical investigations contributed samples and MAK conceptualized and also edited the manuscript. VKK provided research material. SKK, KK conducted experiments and analyzed the data. SKK, VKK wrote and corrected the manuscript.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This work was supported by a grant from the Department of Biotechnology, Government of India No.BT/PR4582/MED/12/547/2012 and Lupin Limited, India.
Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]