Protective environmental factors for neuromyelitis optica

Abstract

Objective:

To determine whether early environmental factors, such as cesarean delivery, breastfeeding, and exposure to smoking or herpes viruses, are associated with neuromyelitis optica (NMO) risk in children.

Methods:

This is a case-control study of pediatric NMO, multiple sclerosis (MS), and healthy subjects. Early-life exposures were obtained by standardized questionnaire. Epstein-Barr virus, cytomegalovirus, and herpes simplex virus 1 antibody responses were determined by ELISA. Multivariate logistic regression models were used to adjust for age at sampling, sex, race, and ethnicity.

Results:

Early-life exposures were obtained from 36 pediatric subjects with NMO, 491 with MS, and 224 healthy controls. Daycare (odds ratio [OR] 0.33, 95% confidence interval [CI] 0.14, 0.78; p < 0.01) and breastfeeding (OR 0.42, 95% CI 0.18, 0.99; p = 0.05) were associated with lower odds of having NMO compared with healthy subjects. Cesarean delivery tended to be associated with 2-fold-higher odds of NMO compared with having MS/clinically isolated syndrome (OR 1.98, 95% CI 0.88, 4.59; p = 0.12) or with being healthy (OR 1.95, 95% CI 0.81, 4.71; p = 0.14). Sera and DNA were available for 31 subjects with NMO, 189 with MS, and 94 healthy controls. Epstein-Barr virus, herpes simplex virus 1, cytomegalovirus exposure, and being HLA-DRB1*15 positive were not associated with odds of having NMO compared with healthy subjects.

Conclusions:

Exposure to other young children may be an early protective factor against the development of NMO, as previously reported for MS, consistent with the hypothesis that infections contribute to disease risk modification. Unlike MS, pediatric NMO does not appear to be associated with exposures to common herpes viruses.

Neuromyelitis optica (NMO) is characterized by optic neuritis, brainstem deficits, and transverse myelitis and is associated with aquaporin-4 (AQP4) antibodies.¹ Adult studies suggest that genetic susceptibility to NMO is likely present, but most compatible with a complex disorder with an important component of nongenetic factors.^2,3 Environmental risk factors for NMO are thus likely critical to disease onset, but these are largely unknown. In children, cases may follow a viral prodrome whereas in adults molecular mimicry involving T-cell responses against a Clostridium species may be important for disease initiation.^4,5 In Japanese adults, exposure to the organisms Helicobacter pylori or Chlamydophila pneumonia may be associated with AQP4-immunoglobulin G (IgG)-positive NMO.⁶

In contrast with NMO, a number of environmental risk factors have been consistently reported for multiple sclerosis (MS) in adults and children. Epstein-Barr virus (EBV) antibody response is associated with increased risk of MS.^7–10 Early exposure to cytomegalovirus (CMV) may be protective, while HLA-DRB1*15:01 status modifies the association of herpes simplex virus 1 (HSV-1) antibodies with MS.^10,11 Early-life events such as breastfeeding and exposure to other young children may affect MS risk as well.^12–15 The effect of these factors on NMO risk is not known.

The study of environmental factors in pediatric patients offers many advantages. Less bias in the recollection of early childhood events may occur and data are collected during the time of primary exposure to many factors including herpes virus infections. We sought to determine whether early-life events influence the odds of having NMO in children.

METHODS

Standard protocol approvals, registrations, and patient consents.

The project was approved by the local institutional review boards at all participating Pediatric MS Centers (University of California, San Francisco; SUNY at Stony Brook; SUNY at Buffalo; University of Alabama, Birmingham; Boston Children's Hospital; Partners Pediatric MS Center; Mayo Clinic; Children's Hospital Loma Linda; Texas Children's Hospital). When required by the local institutional review board, informed consent and assent were obtained from all parents and patients.

Patients.

For environmental questionnaire data, subjects with NMO, MS, and clinically isolated syndrome (CIS) with high risk of MS were identified through the Pediatric MS Network from May 2011 to June 2013. NMO cases were reviewed by a panel of 4 pediatric demyelinating disease specialists to confirm diagnosis. For serum and DNA studies, MS cases were identified from the aforementioned centers and NMO cases were identified from Stony Brook, University of California, San Francisco, and from the Accelerated Cure Project/Guthy Jackson Foundation. Subjects met published criteria for pediatric NMO and pediatric MS and had disease onset before age 18 years.^16–18 All patients with NMO had their sera tested for AQP4 antibody status (Mayo Clinic) as part of their care. Healthy controls were identified at well visits to general pediatric clinics of the same aforementioned institutions from which cases were enrolled and were part of an ongoing case-control study led by the Pediatric MS Network (R01NS071463, principal investigator Waubant). Healthy controls had no history of autoimmune disease. Race and ethnicity were self-reported according to current NIH guidelines.

Environmental exposures.

For all subjects seen by the Pediatric MS Network, parents completed standardized questionnaires regarding pregnancy exposures and complications, delivery method, and early childhood exposures. Data extracted from these questionnaires are prospectively entered into a Web-based data capture system, as part of the Pediatric Multiple Sclerosis and Other Demyelinating Diseases database. The data include history of birth by cesarean delivery, breastfeeding and length of breastfeeding (months), exposure to active or passive cigarette smoking for 6 months or more after birth, attendance at daycare, number of siblings and order of birth, type of insurance (private vs other), and level of education of the parents (the latter 2 as surrogates of socioeconomic status).

Herpes virus assays.

Sera were collected from subjects at the time of enrollment. Batched EBV viral capsid antigen (VCA), Epstein Barr nuclear antigen-1 (EBNA1), CMV, and HSV-1 IgG antibody responses were measured blindly at the Oklahoma Medical Research Foundation. Results for normalized ELISAs (Wampole Laboratories, Princeton, NJ) for EBV-VCA, CMV, and HSV-1 are presented as international standardized ratios.¹⁹ EBNA1 ELISA (BioDesign, Carmel, NY) results are reported as relative concentration as previously described.^20,21 Positive anti-EBNA1 responses were ≥4 SDs above a panel of controls known to be EBV seronegative.

Genotyping.

Because the effect of viral exposures on MS risk may depend on HLA-DRB1 status, we measured this risk factor in subjects tested for viral responses.¹⁹ DNA samples of MS, NMO, and control patients were typed in a blinded manner by a gene-specific Taqman assay for single nucleotide polymorphisms associated with HLA-DRB1*1501/1503 alleles, as previously published.¹⁹

Statistical methods.

Statistical analyses were performed using Stata 12.0 statistical software (StataCorp, College Station, TX) and SAS/STAT software (version 9.3; SAS Institute, Inc., Cary, NC). Median [interquartile range] or n (%) was used to describe patient characteristics across the different diagnostic groups. Interbirth interval was defined as the difference in ages between the patient and the nearest younger sibling. Patients with no younger siblings were assigned the highest ranking for interbirth interval. Infant-years exposure was defined as the total amount of time before age 6 that the patient was exposed to a younger sibling or twin younger than 2 years of age. Comparisons were made using χ² test for binary variables and Kruskal-Wallis test for numeric variables. For each risk factor of interest, analysis was performed using multivariable logistic regression models adjusting for sex, race, and ethnicity for environmental questionnaire data and age at blood draw, sex, race, and ethnicity for sera measured variables. A 2-sided p value <0.05 was considered significant.

RESULTS

Demographic features and early environmental exposures.

Early environmental exposure questionnaires were completed by the parents of 36 subjects with NMO, 491 with MS, and 224 healthy controls (table 1). Pediatric subjects with NMO were younger at onset than subjects with MS/CIS (p < 0.01). There were also racial differences across the diagnostic groups (p < 0.01) with more African Americans in the NMO group. NMO cases had 58% frequency of positive results for AQP4-IgG. While AQP4-IgG-positive and -negative NMO cases were combined for the primary analyses, we did observe some demographic differences, with AQP4-IgG-positive individuals more likely to be female and of nonwhite descent than AQP4-IgG-negative subjects with NMO (table e-1 on the Neurology® Web site at Neurology.org).

Table 1.

Subject characteristics

In multivariable analyses adjusted for sex, race, and ethnicity (tables 2 and 3), exposure to daycare was associated with substantially reduced odds of having NMO compared with having MS/CIS (odds ratio [OR] 0.41, 95% confidence interval [CI] 0.17, 0.97; p = 0.04) or with being a healthy control (OR 0.33, 95% CI 0.14, 0.78; p < 0.01). Breastfeeding was also associated with lower odds of having NMO vs being a healthy control (OR 0.42, 95% CI 0.18, 0.99; p = 0.05). Delivery by cesarean section tended to be associated with 2-fold-higher odds of NMO compared with having MS/CIS (OR 1.98, 95% CI 0.88, 4.59; p = 0.12) or with being healthy (OR 1.95, 95% CI 0.81, 4.71; p = 0.14).

Table 2.

Multivariable analyses of early environmental risk factors associated with the odds of pediatric NMO vs MS/CIS, adjusted for sex, race, and ethnicity

Table 3.

Multivariable analyses of early environmental risk associated with the odds of pediatric NMO vs healthy controls, adjusted for sex, race, and ethnicity

Maternal education level was associated with risk of NMO (p = 0.03). College-level education or higher, attained by the mother, reduced the odds of NMO by 84% as opposed to not completing high school (OR 0.16, 95% CI 0.03, 0.79) compared with healthy controls. Other markers of socioeconomic status, insurance type and father's education, as well as exposure to cigarette smoking were not associated with having NMO.

Herpes virus exposures.

For the evaluation of viral susceptibility factors, DNA and serum were available for 31 pediatric NMO cases, 186 pediatric MS/CIS cases, and 94 healthy controls (table e-2). Differences across groups included proportions of patients of nonwhite descent (48.3% for NMO vs 27.2% and 25.3% for MS and healthy subjects, respectively). Sex, age at blood draw, race, and ethnicity were adjusted for in the multivariable analyses.

Compared to patients with MS (table 4), EBV antibody responses were associated with lower odds of NMO (VCA OR 0.14, 95% CI 0.042, 0.49; p = 0.002; EBNA1 OR 0.13, 95% CI 0.038, 0.46; p = 0.002). HSV-1 exposure was also associated with lower odds of having NMO compared to subjects with MS (OR 0.32, 95% CI 0.11, 0.94; p = 0.038). There were no differences for viral risk factors between patients with NMO and healthy controls (table 5).

Table 4.

Multivariable analyses of the odds of having pediatric NMO vs MS/CIS, adjusted for age at blood draw, sex, race, and ethnicity

Table 5.

Multivariable analyses of the odds of having pediatric NMO vs healthy controls, adjusted for age at blood draw, sex, race, and ethnicity

HLA-DRB1*15.

Multivariable analyses demonstrated that having the HLA-DRB1*1501 or 1503 allele tended to be associated with lower odds of NMO (OR 0.45, 95% CI 0.17, 1.15; p = 0.096) compared to patients with MS/CIS but not compared to healthy controls (OR 1.33, 95% CI 0.48, 3.73; p = 0.58).

DISCUSSION

In this study of early environmental risk factors of NMO, we have demonstrated that breastfeeding and daycare exposure may be protective. The immune benefits of breastfeeding are well recognized. For autoimmune diseases, the benefit may be related to transfer of maternal antigens and immune cells, resulting in improved immunity or tolerance.²² Protective effects have been demonstrated for atopic allergies, type 1 diabetes, Crohn disease, MS, and rheumatoid arthritis, although some of these findings remain controversial.^13,22–30 Early exposure to other children has been consistently associated with decreased MS risk, implicating that viral infections early in life are protective.^15,31 Daycare provides one of the largest exposures to pathogens for young children. Our findings suggest that attending daycare also likely protects against the development of NMO. We did not find that exposure to younger or older siblings increased the odds of NMO, but the subjects we evaluated had few siblings and the overall number of included patients with NMO was small, such that an association cannot be excluded.

In MS, while early viral exposures may be protective, EBV infection and, in particular, late EBV infection, resulting in mononucleosis, have been strongly associated with MS risk.^7,32 We found no association of EBV exposure as measured by standard VCA or EBNA1 ELISA and NMO compared with healthy subjects. This was despite the older age of blood draw in the subjects with NMO. These data suggest that EBV infection likely does not increase risk of NMO.

Whereas passive smoking has been implicated in increasing risk of some autoimmune diseases including MS, we did not find evidence of an association of early exposure to cigarette smoke with odds of NMO, although our sample size of subjects with NMO was limited.^33,34 We found delivery by cesarean section tended to be associated with a 2-fold-greater odds of NMO, compared with either healthy controls or subjects with MS. Even though the observation did not reach statistical significance, the magnitude of the point estimate was the same for analyses with both control groups and warrants further investigation in a larger study. The significance of a nonvaginal birth and autoimmune disease has been previously postulated to relate to early differences in infant gut flora.^35,36

Similar to recent findings in adults, we observed demographic differences in AQP4-IgG-positive and -negative subjects with NMO.^37,38 Subjects testing positive were predominantly female and nonwhite whereas the sex ratio in AQP4-IgG-negative subjects with NMO neared 1 and the majority were of white ancestry. Because of limited number of NMO cases, we were not able to further stratify our multivariable analyses by AQP4-IgG status, but this would be of interest in future, larger studies of NMO risk factors.

The strengths of our study include the careful case ascertainment and the use of 2 types of controls coming from the same centers. Nevertheless, we acknowledge some limitations of this study. The number of pediatric subjects with NMO was small. The variables most vulnerable to sample-size limitations in our study may be insurance, smoking, and cesarean delivery, because these show plausible patterns isolated to the NMO group, that if present in a larger sample size may have been significant. Recall bias is of concern in case-control studies, but this may be less of a limitation in the pediatric setting where the early environmental risk factor data are ascertained from parents and at time points closer to exposure than in adult studies. Recall bias is also likely to be similar and less influential in comparison of 2 disease states—MS and NMO. Furthermore, the older age and longer disease duration at serum collection for the NMO group means that viral exposures for the NMO group at time of onset may have been different compared with time of serum testing, although analyses were adjusted for both. However, the older age could not explain a lower frequency of seropositivity for viruses studied.

Pathologic and biological evidence now demonstrate that NMO and MS are separate entities. Our study suggests that risk factors for both disorders may also differ. Larger prospective case-control studies will further identify genetic and environmental risk factors specific to NMO. These may in turn help define more in-depth disease processes at play.

GLOSSARY

AQP4

aquaporin-4

CI

confidence interval

CIS

clinically isolated syndrome

CMV

cytomegalovirus

EBNA1

Epstein Barr nuclear antigen-1

EBV

Epstein-Barr virus

HSV-1

herpes simplex virus 1

IgG

immunoglobulin G

MS

multiple sclerosis

NMO

neuromyelitis optica

OR

odds ratio

VCA

viral capsid antigen

AUTHOR CONTRIBUTIONS

Dr. Graves: design of study, data collection, analysis and interpretation of data, drafting and revising the manuscript. Siri Grandhe: data collection and analysis, revising manuscript. Kelley Weinfurtner: data collection and analysis, revising manuscript. Drs. Krupp and Belman: provided samples, interpretation of data, revising the manuscript. Dr. Ness: provided samples, interpretation of data, revising the manuscript. Dr. Gorman: provided samples, interpretation of data, revising the manuscript. Dr. Weinstock-Guttman: data collection and revising the manuscript. Dr. Patterson and Rodriguez: provided samples, interpretation of data, revising the manuscript. Dr. Chitnis: provided samples, interpretation of data, revising the manuscript. Dr. Aaen: provided samples, interpretation of data, revising the manuscript. Dr. Lotze: provided samples, interpretation of data, revising the manuscript. Dr. Mowry: data collection and analysis, revising the manuscript. Dr. Rose: data collection, revising the manuscript. Timothy Simmons: analysis and interpretation of data, revising the manuscript. Dr. Casper: design of study, data collection, analysis and interpretation of data, revising the manuscript. Dr. James: serotyping, interpretation of data, revising the manuscript. Dr. Waubant: design of study, interpretation of data, revising the manuscript.

STUDY FUNDING

This work was supported by the National MS Society (HC 0165) and NIH (R01NS071463). In addition, work in Dr. James' laboratory was supported by NIH grants U19 AI082714 and U01 AI101934. Dr. Graves' NIH support was through the BIRCWH K12 program (5K12HD052163).

DISCLOSURE

J. Graves was supported by the Foundation for Consortium of Multiple Sclerosis Centers and the NIH Bridging Interdisciplinary Research Careers in Women's Health programs (5K12HD052163) during this work. She has been a one-time advisory board participant for EMD Serono. S. Grandhe and K. Weinfurtner report no disclosures relevant to the manuscript. L. Krupp is supported by the National MS Society, NIH, Robert and Lisa Lourie Foundation, Department of Defense. She has received honoraria, consulting payments, grant support, or royalties from Biogen, MedImmune, Novartis, Teva Neuroscience, Sanofi-Aventis, and EMD Serono. A. Belman reports no disclosures relevant to the manuscript. T. Chitnis has served as a consultant for Biogen Idec, Teva Neurosciences, Novartis, Sanofi-Aventis, and has received grant support from NIH, National MS Society, Guthy-Jackson Charitable Foundation, CMSC and Merck-Serono, and Novartis. J. Ness reports no disclosures relevant to the manuscript. B. Weinstock-Guttman received honoraria for serving in advisory boards and educational programs from Teva Pharmaceuticals, Biogen Idec, Novartis, Acorda EMD Serono, Novartis, Genzyme, and Sanofi. She also received support for research activities from the NIH, National Multiple Sclerosis Society, National Science Foundation, Department of Defense, EMD Serono, Biogen Idec, Teva Neuroscience, Novartis, Acorda, Genzyme, and the Jog for the Jake Foundation. M. Gorman, M. Patterson, M. Rodriguez, T. Lotze, and G. Aaen report no disclosures relevant to the manuscript. E. Mowry is supported by the National MS Society and the NIH. She receives free medication for a randomized control trial from Teva Neuroscience. J. Rose has research funding from Teva Neuroscience and Biogen. He is a member of the Medical Advisory Board for the DECIDE trial, which is funded by Biogen and AbbVie. T. Simmons reports no disclosures relevant to the manuscript. T. Casper has been supported by the National MS Society and the NIH (R01NS071463). He is an ad hoc consultant for Biovest International, Inc. J. James reports no disclosures relevant to the manuscript. E. Waubant is funded by the National MS Society, the NIH, and the Race to Erase MS. She has received honorarium from Teva Neuroscience and Genzyme for 2 educational lectures, and is an ad hoc consultant for Actelion, Sanofi-Aventis, and Roche. She is on an advisory board for a clinical trial of Novartis. Go to Neurology.org for full disclosures.