Gene-environment interactions: Epstein-Barr virus infection and risk of pediatric-onset multiple sclerosis.

BACKGROUND AND OBJECTIVE: Prior Epstein-Barr virus (EBV) infection is associated with an increased risk of pediatric-onset multiple sclerosis (POMS) and adult-onset multiple sclerosis (MS). It has been challenging to elucidate the biological mechanisms underlying this association. We examined the interactions between candidate human leukocyte antigen (HLA) and non-HLA variants and childhood EBV infection as it may provide mechanistic insights into EBV-associated MS. METHODS: Cases and controls were enrolled in the Environmental and Genetic Risk Factors for Pediatric MS study of the US Network of Pediatric MS Centers. Participants were categorized as seropositive and seronegative for EBV-viral capsid antigen (VCA). The association between prior EBV infection and having POMS was estimated with logistic regression. Interactions between EBV serostatus, major HLA MS risk factors, and non-HLA POMS risk variants associated with response to EBV infection were also evaluated with logistic regression. Models were adjusted for sex, age, genetic ancestry, and the mother's education. Additive interactions were calculated using relative risk due to interaction (RERI) and attributable proportions (APs). RESULTS: A total of 473 POMS cases and 702 controls contributed to the analyses. Anti-VCA seropositivity was significantly higher in POMS cases compared to controls (94.6% vs 60.7%, p < 0.001). There was evidence for additive interaction between childhood EBV infection and the presence of the HLA-DRB1*15 allele (RERI = 10.25, 95% confidence interval (CI) = 3.78 to 16.72; AP = 0.61, 95% CI = 0.47 to 0.75). There was evidence for multiplicative interaction (p < 0.05) between childhood EBV infection and the presence of DRB1*15 alleles (odds ratio (OR) = 3.43, 95% CI = 1.06 to 11.07). Among the pediatric MS variants also associated with EBV infection, we detected evidence for additive interaction (p = 0.02) between prior EBV infection and the presence of the GG genotype in risk variant (rs2255214) within CD86 (AP = 0.30, 95% CI = 0.03 to 0.58). CONCLUSION: We report evidence for interactions between childhood EBV infection and DRB1*15 and the GG genotype of CD86 POMS risk variant. Our results suggest an important role of antigen-presenting cells (APCs) in EBV-associated POMS risk.

High serum neurofilament levels are observed close to disease activity events in pediatric-onset MS and MOG antibody-associated diseases.

BACKGROUND: Serum neurofilament light chain (sNfL) is an emerging multiple sclerosis (MS) biomarker which measures neuro-axonal damage. However, understanding its temporal association with disease activity in pediatric-onset MS (POMS) and Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) remains limited. OBJECTIVE: To investigate the association of sNfL levels and time from disease activity in children with MS and MOGAD. METHODS: POMS and MOGAD cases with onset before 18 years of age were enrolled at the University of California San Francisco (UCSF) Regional Pediatric MS Center. Frequency-matched healthy subjects were recruited from general pediatric clinics. Serum samples were tested for MOG-IgG at Mayo Clinic using a live cell-based fluorescent activated cell sorting assay. sNfL levels were measured using single-molecule array (Simoa) technology measured in pg/mL. Data on demographics, clinical features, MRI, CSF, and treatment data were collected by chart review. RESULTS: We included 201 healthy controls healthy controls, 142 POMS, and 20 confirmed MOGAD cases with available sNfL levels. The median (IQR) age at the time of sampling was 15.6 (3.9), 15.5 (3.1), and 8.8 (4.1) years for controls, POMS, and MOGAD, respectively. Median sNfL levels (pg/ml) were higher in POMS (19.6) and MOGAD (32.7) cases compared to healthy controls (3.9) (p<0.001). sNfL levels ≥100 pg/ml were only detected within four months of a clinical event or MRI activity in both POMS and MOGAD cases. In addition, sNfL levels were higher in POMS patients with new/enlarged T2 and gadolinium-enhanced lesions than those without MRI activity within four months of sampling in POMS cases. CONCLUSION: High sNfL levels were observed close to clinical or MRI events in POMS and MOGAD. Our findings support sNfL as a biomarker of disease activity in pediatric demyelinating disorders.

Gene-environment interactions increase the risk of paediatric-onset multiple sclerosis associated with household chemical exposures.

BACKGROUND: We previously reported an association between household chemical exposures and an increased risk of paediatric-onset multiple sclerosis. METHODS: Using a case-control paediatric multiple sclerosis study, gene-environment interaction between exposure to household chemicals and genotypes for risk of paediatric-onset multiple sclerosis was estimated.Genetic risk factors of interest included the two major HLA multiple sclerosis risk factors, the presence of DRB1*15 and the absence of A*02, and multiple sclerosis risk variants within the metabolic pathways of common household toxic chemicals, including IL-6 (rs2069852), BCL-2 (rs2187163) and NFKB1 (rs7665090). RESULTS: 490 paediatric-onset multiple sclerosis cases and 716 controls were included in the analyses. Exposures to insect repellent for ticks or mosquitos (OR 1.47, 95% CI 1.06 to 2.04, p=0.019), weed control products (OR 2.15, 95% CI 1.51 to 3.07, p<0.001) and plant/tree insect or disease control products (OR 3.25, 95% CI 1.92 to 5.49, p<0.001) were associated with increased odds of paediatric-onset multiple sclerosis. There was significant additive interaction between exposure to weed control products and NFKB1 SNP GG (attributable proportions (AP) 0.48, 95% CI 0.10 to 0.87), and exposure to plant or disease control products and absence of HLA-A*02 (AP 0.56; 95% CI 0.03 to 1.08). There was a multiplicative interaction between exposure to weed control products and NFKB1 SNP GG genotype (OR 2.30, 95% CI 1.00 to 5.30) but not for other exposures and risk variants. No interactions were found with IL-6 and BCL-2 SNP GG genotypes. CONCLUSIONS: The presence of gene-environment interactions with household toxins supports their possible causal role in paediatric-onset multiple sclerosis.

Ermin deficiency leads to compromised myelin, inflammatory milieu, and susceptibility to demyelinating insult.

Ermin is an actin-binding protein found almost exclusively in the central nervous system (CNS) as a component of myelin sheaths. Although Ermin has been predicted to play a role in the formation and stability of myelin sheaths, this has not been directly examined in vivo. Here, we show that Ermin is essential for myelin sheath integrity and normal saltatory conduction. Loss of Ermin in mice caused de-compacted and fragmented myelin sheaths and led to slower conduction along with progressive neurological deficits. RNA sequencing of the corpus callosum, the largest white matter structure in the CNS, pointed to inflammatory activation in aged Ermin-deficient mice, which was corroborated by increased levels of microgliosis and astrogliosis. The inflammatory milieu and myelin abnormalities were further associated with increased susceptibility to immune-mediated demyelination insult in Ermin knockout mice. Supporting a possible role of Ermin deficiency in inflammatory white matter disorders, a rare inactivating mutation in the ERMN gene was identified in multiple sclerosis patients. Our findings demonstrate a critical role for Ermin in maintaining myelin integrity. Given its near-exclusive expression in myelinating oligodendrocytes, Ermin deficiency represents a compelling "inside-out" model of inflammatory dysmyelination and may offer a new paradigm for the development of myelin stability-targeted therapies.

Gene-environment interactions increase the risk of pediatric-onset multiple sclerosis associated with ozone pollution.

BACKGROUND: We previously reported a relationship between air pollutants and increased risk of pediatric-onset multiple sclerosis (POMS). Ozone is an air pollutant that may play a role in multiple sclerosis (MS) pathoetiology. CD86 is the only non-HLA gene associated with POMS for which expression on antigen-presenting cells (APCs) is changed in response to ozone exposure. OBJECTIVES: To examine the association between county-level ozone and POMS, and the interactions between ozone pollution, CD86, and HLA-DRB1*15, the strongest genetic variant associated with POMS. METHODS: Cases and controls were enrolled in the Environmental and Genetic Risk Factors for Pediatric MS study of the US Network of Pediatric MS Centers. County-level-modeled ozone data were acquired from the CDC's Environmental Tracking Network. Participants were assigned ozone values based on county of residence. Values were categorized into tertiles based on healthy controls. The association between ozone tertiles and having MS was assessed by logistic regression. Interactions between tertiles of ozone level and the GG genotype of the rs928264 (G/A) single nucleotide polymorphism (SNP) within CD86, and the presence of DRB1*15:01 (DRB1*15) on odds of POMS were evaluated. Models were adjusted for age, sex, genetic ancestry, and mother's education. Additive interaction was estimated using relative excess risk due to interaction (RERI) and attributable proportions (APs) of disease were calculated. RESULTS: A total of 334 POMS cases and 565 controls contributed to the analyses. County-level ozone was associated with increased odds of POMS (odds ratio 2.47, 95% confidence interval (CI): 1.69-3.59 and 1.95, 95% CI: 1.32-2.88 for the upper two tertiles, respectively, compared with the lowest tertile). There was a significant additive interaction between high ozone tertiles and presence of DRB1*15, with a RERI of 2.21 (95% CI: 0.83-3.59) and an AP of 0.56 (95% CI: 0.33-0.79). Additive interaction between high ozone tertiles and the CD86 GG genotype was present, with a RERI of 1.60 (95% CI: 0.14-3.06) and an AP of 0.37 (95% CI: 0.001-0.75) compared to the lowest ozone tertile. AP results indicated that approximately half of the POMS risk in subjects can be attributed to the possible interaction between higher county-level ozone carrying either DRB1*15 or the CD86 GG genotype. CONCLUSIONS: In addition to the association between high county-level ozone and POMS, we report evidence for additive interactions between higher county-level ozone and DRB1*15 and the CD86 GG genotype. Identifying gene-environment interactions may provide mechanistic insight of biological processes at play in MS susceptibility. Our work suggests a possible role of APCs for county-level ozone-induced POMS risk.

High titers of myelin oligodendrocyte glycoprotein antibody are only observed close to clinical events in pediatrics.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG)-IgG is increasingly detected in children with CNS demyelinating diseases. Due to the clinical overlap in children with CNS demyelination with and without MOG-IgG positivity, identifying distinct characteristics would help early diagnosis. OBJECTIVE: To compare the specific features that may help differentiate MOG-IgG positive from negative children with CNS demyelinating diseases. To compare characteristics of patients with high and low MOG-IgG titers. METHODS: Children with CNS demyelinating disorders with onset before 18 years of age who were tested for MOG-IgG at the University of California San Francisco were included. This retrospective study collected the following by chart review: demographic, clinical, MRI, CSF, and treatment data. Serum was tested for MOG-IgG at Mayo Clinic by live cell-based fluorescent activated cell sorting assay with titer ≥1:20 confirming positivity. RESULTS: We assessed 65 Mog-IgG positive and 65 MOG-IgG negative patients. Median (IQR) age of onset was 7.6 (6.6) years for MOG-IgG positive and 13.8 (5.8) years for MOG-IgG negative (p<0.001). The female to male ratio was approximately 1:1 for the MOG-IgG positive group and 3:1 for the negative group (p=0.042). The most common initial diagnosis was demyelinating disease not otherwise specified (52.3%) in the positive group, compared to relapsing-remitting multiple sclerosis (41.5%) in the negative group (p<0.01). Optic nerve involvement (52.3%) was the most common clinical localization at onset for the MOG-IgG positive group, while brainstem/cerebellar (49.2%) localization predominated in the MOG-IgG negative group. The positive group also presented more often with a severe event at disease onset than the negative group (81.5% vs 60.3%; p< 0.002). MOG-IgG positive children had a lower frequency of oligoclonal bands (15.8% vs 57.4%; p<0.001). The frequency of baseline brain and spinal cord MRI abnormalities were similar in both groups; however, MOG-IgG positive patients more often had T2 hyperintense lesions in the optic nerves (26/43 vs 10/41; p<0.001). Disease-modifying medications were used in 64.6% of MOG-IgG positive patients versus 80% of negative children. Of the 32 positive patients with follow-up titers, seven reverted to negative while two who tested negative initially converted to positive. Positive titers greater than 1:160 were only observed within four months of a clinical event (disease onset or relapse). Patients with high and low MOG-IgG titers were comparable in demographic and clinical characteristics. CONCLUSION: Despite some clinical overlap, we report notable demographic, MRI and CSF differences between MOG-IgG positive and negative children with CNS demyelinating disorders at disease onset. High MOG-IgG titers were only observed close to a clinical event.

Novel mutation in HTRA1 in a family with diffuse white matter lesions and inflammatory features.

OBJECTIVE: To investigate the possible involvement of germline mutations in a neurologic condition involving diffuse white matter lesions. METHODS: The patients were 3 siblings born to healthy parents. We performed homozygosity mapping, whole-exome sequencing, site-directed mutagenesis, and immunoblotting. RESULTS: All 3 patients showed clinical manifestations of ataxia, behavioral and mood changes, premature hair loss, memory loss, and lower back pain. In addition, they presented with inflammatory-like features and recurrent rhinitis. MRI showed abnormal diffuse demyelination lesions in the brain and myelitis in the spinal cord. We identified an insertion in high-temperature requirement A (HTRA1), which showed complete segregation in the pedigree. Functional analysis showed the mutation to affect stability and secretion of truncated protein. CONCLUSIONS: The patients' clinical manifestations are consistent with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL; OMIM #600142), which is known to be caused by HTRA1 mutations. Because some aspects of the clinical presentation deviate from those reported for CARASIL, our study expands the spectrum of clinical consequences of loss-of-function mutations in HTRA1.

Unbiased Profiling of Isogenic Huntington Disease hPSC-Derived CNS and Peripheral Cells Reveals Strong Cell-Type Specificity of CAG Length Effects.

In Huntington disease (HD), the analysis of tissue-specific CAG repeat length effects has been challenging, given the difficulty in obtaining relevant patient tissues with a broad range of CAG repeat lengths. We used genome editing to generate an allelic panel of isogenic HD (IsoHD) human embryonic stem cell (hESC) lines carrying varying CAG repeat lengths in the first exon of HTT. Functional analyses in differentiated neural cells revealed CAG repeat length-related abnormalities in mitochondrial respiration and oxidative stress and enhanced susceptibility to DNA damage. To explore tissue-specific effects in HD, we differentiated the IsoHD panel into neural progenitor cells, neurons, hepatocytes, and muscle cells. Transcriptomic and proteomic analyses of the resultant cell types identified CAG repeat length-dependent and cell-type-specific molecular phenotypes. We anticipate that the IsoHD panel and transcriptomic and proteomic data will serve as a versatile, open-access platform to dissect the molecular factors contributing to HD pathogenesis.

Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells.

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.

Acute course of deferoxamine promoted neuronal differentiation of neural progenitor cells through suppression of Wnt/ß-catenin pathway: a novel efficient protocol for neuronal differentiation.

Neural progenitor cells (NPCs) are feasible therapeutically model cells in regenerative medicine. However, a number of obstacles oppose their applications including insufficiency in differentiation protocols. These complications should be overwhelmed to obtain a significant clinical application. Deferoxamine (DFO), as a small molecule with a clinically high-affinity to chelate intracellular Iron, has been granted orphan drug status for treatment of traumatic spinal cord injury, while its neuroprotective function is not well understood. The aim of the present study is evaluating whether DFO could modulate neuronal differentiation process of NPCs. A varies concentrations of DFO were used to promote neuronal differentiation of mouse and human NPCs with different serum condition as an extracellular source of Iron. Several neural markers were assessed by RT-qPCR and Western analysis. Meanwhile ß-catenin content was evaluated as key member of Wnt pathway. The maximal neuronal differentiation rate was observed when treating cells were treated with acute dosage of DFO (100 µM) for 6h in serum free condition. This treatment produced a significant increase in expression of neuronal markers and resulted in dramatically decrease in expression of glial markers. The protein content of ß-catenin was also decreased by this treatment. Despite of chronic concentration of DFO, which reduced the size of EBs apparently due to G1/S arrest of cell cycle as known features of DFO. Application of acute courses of DFO increased neuronal differentiation rate of NPCs in serum free conditions. We concluded that suppression of Wnt/ß-catenin pathway was induced through chelating of intracellular Iron due to DFO treatment. These findings help to understand therapeutic benefit of DFO as a neuroprotective agent.

Livin, a novel marker in lymphoma type distinction.

Despite advances in immunohistochemical and molecular diagnostics, there are persistent difficulties in differentiating between several subtypes of non-Hodgkin lymphoma (NHL) and classic Hodgkin lymphoma (CHL). Considering high level of livin expression in hematologic malignancies, we aimed to examine the utility of livin expression ratio, as an ancillary biomarker, in distinguishing CHL from NHL in ambiguous cases. We evaluated livin expression in 38 CHL, 23 NHL, and 39 nonneoplastic lymph nodes in paraffin-embedded blocks. Tissue microarray-based semiquantitative immunoflourecent staining was applied for protein expression. Criterion standard of diagnosis was based on selection of only definite cases and not the cases suspected by hemathopathologists. A significant difference was found in the livin/GAPDH mean ratio (M.R) of expression between NHL and CHL cases. A receiver operating characteristic curve analysis confirmed 0.6370 to be the best diagnostic cut-off value for the livin/GAPDH expression M.R in diffuse large B-cell lymphoma (DLBCL) (area under the curve = 0.944); it yielded 92% sensitivity, 94% specificity, likelihood ratios positive 17.5, and likelihood ratios negative 0.07 for diagnosing DLBCL from CHL. Mean ratio of livin/Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression seems to be a valuable index in differentiating DLBCL from CHL. We suggested an optimal cut-off point for livin/GAPDH expression M.R with a high sensitivity and specificity. Thus, in diagnostically difficult cases of DLBCL and CHL, focus on livin as marker may provide useful corroborative information.

The effect of preoperative melatonin on nuclear erythroid 2-related factor 2 activation in patients undergoing coronary artery bypass grafting surgery.

During and after coronary artery bypass grafting (CABG), oxidative stress occurs. Finding an effective way to improve antioxidant response is important in CABG surgery. It has been shown that patients with coronary heart disease have a low Melatonin production rate. The present study aimed to investigate the effects of Melatoninon nuclear erythroid 2-related factor 2(Nrf2) activity in patients undergoing CABG surgery. Thirty volunteers undergoing CABG were randomized to receive 10 mg oral Melatonin (Melatonin group, n = 15) or placebo (placebo group, n = 15) before sleeping for 1 month before surgery. The activated Nrf2 was measured twice by DNA-based ELISA method in the nuclear extract of peripheral blood mononuclear cells of patients before aortic clumps and 45 minutes after CABG operation. Melatonin administration was associated with a significant increase in both plasma levels of Melatonin and Nrf2 concentration in Melatonin group compared to placebo group, respectively (15.2 ± 4.6 pmol/L, 0.28 ± 0.01 versus 1.1 ± 0.59 pmol/L, 0.20 ± 0.07, P < 0.05). The findings of the present study provide preliminary data suggesting that Melatonin may play a significant role in the potentiation of the antioxidant defense and attenuate cellular damages resulting from CABG surgery via theNrf2 pathway.

Apoptosis inhibition or inflammation: the role of NAIP protein expression in Hodgkin and non-Hodgkin lymphomas compared to non-neoplastic lymph node.

BACKGROUND: Inhibitors of Apoptosis (IAP) family play a critical role in apoptosis and inflammatory response. Neuronal Apoptosis Inhibitory Protein (NAIP), as a member of both IAPs and NLR families (NOD-Like Receptor), is a unique IAP harboring NOD (Nucleotide Oligomerization Domain) and LLR (Leucine Rich Repeat) motifs. Considering these motifs in NAIP, it has been suggested that the main function of NAIP is distinct from other members of IAPs. As a member of NLR, NAIP mediates the assembly of 'Inflammasome' for inflammatory caspase activation. Pathologic expression of NAIP has been reported not only in some infectious and inflammatory diseases but also in some malignancies. However, there is no report to elucidate NAIP expression in lymphomatic malignancies. METHODS: In this study, we examined NAIP protein expression in 101 Formalin-Fixed Paraffin-Embedded blocks including samples from 39 Hodgkin Lymphoma and 23 Non Hodgkin Lymphoma cases in comparison with 39 control samples (30 normal and 9 Reactive Lymphoid Hyperplasia (RLH) lymph nodes) using semi-quantitative immuno-flourecent Staining. RESULTS: NAIP expression was not statistically different in lymphoma samples neither in HL nor in NHL cases comparing to normal samples. However, we evaluated NAIP expression in normal and RLH lymph nodes. Surprisingly, we have found a statistically significant-difference between the NAIP expression in RLH (M.R of NAIP/GAPDH expression = 0.6365 ± 0.017) and normal lymph node samples (M.R of NAIP/GAPDH expression = 0.5882 ± 0.047) (P < 0.01). CONCLUSIONS: These findings show that the regulation of apoptosis could not be the main function of NAIP in the cell, so the pathologic expression of NAIP is not involved in lymphoma. But, we concluded that the over expression of NAIP has more effective role in the inflammatory response. Also, this study clarifies the NAIP expression level in lymphoma which is required for IAPs profiling in order to be used in potential translational applications of IAPs.