Dr Vicki Maltby

Background: Pregnancy in women with multiple sclerosis (wwMS) is associated with a reduction of long-term disability progression. The mechanism that drives this effect is unknown, but converging evidence suggests a role for epigenetic mechanisms altering immune and/or central nervous system function. In this study, we aimed to identify whole blood and immune cell-specific DNA methylation patterns associated with parity in relapse-onset MS. Results: We investigated the association between whole blood and immune cell-type-specific genome-wide methylation patterns and parity in 192 women with relapse-onset MS, matched for age and disease severity. The median time from last pregnancy to blood collection was 16.7¿years (range = 1.5¿44.4¿years). We identified 2965 differentially methylated positions in whole blood, 68.5% of which were hypermethylated in parous women; together with two differentially methylated regions on Chromosomes 17 and 19 which mapped to TMC8 and ZNF577, respectively. Our findings validated 22 DMPs and 366 differentially methylated genes from existing literature on epigenetic changes associated with parity in wwMS. Differentially methylated genes in whole blood were enriched in neuronal structure and growth-related pathways. Immune cell-type-specific analysis using cell-type proportion estimates from statistical deconvolution of whole blood revealed further differential methylation in T cells specifically (four in CD4+ and eight in CD8+ T cells). We further identified reduced methylation age acceleration in parous women, demonstrating slower biological aging compared to nulligravida women. Conclusion: Differential methylation at genes related to neural plasticity offers a potential molecular mechanism driving the long-term effect of pregnancy on MS outcomes. Our results point to a potential ¿CNS signature¿ of methylation in peripheral immune cells, as previously described in relation to MS progression, induced by parity. As the first epigenome-wide association study of parity in wwMS reported, validation studies are needed to confirm our findings.

Background: Cladribine is a useful therapeutic option in RRMS with moderate to high disease activity. Its oral formulation and tolerability make it a useful alternative to infusion therapies. Cladribine is known to deplete CD19+ B lymphocytes, but its effect on immunoglobulin subsets is unclear. Objective: To identify whether cladribine therapy in pwMS reduces immunoglobulin subset levels as a surrogate marker of infection risk. Methods: A ¿real-world¿ retrospective analysis of 341 pwMS presenting to a single tertiary centre between March 2017 and July 2021. Differences in immunoglobulin levels between cladribine, other disease-modifying therapies and no active treatment were assessed using a univariate ANOVA. Results: Three hundred and forty-one patients had immunoglobulin levels assessed, with 29 patients treated with cladribine. The mean IgG, IgM and IgA levels on cladribine therapy were 10.44 ± 0.40, 0.99 ± 0.09 and 2.04 ± 0.18 g/L respectively. These were not significantly different from patients not on active treatment. There was a statistically significant reduction in IgG and IgM levels for patients treated with ocrelizumab (9.37 ± 0.19 and 0.68 ± 0.04 g/L) and natalizumab (8.72 ± 0.53 and 0.69 ± 0.12 g/L) compared to patients not on treatment. Conclusion: Cladribine therapy for RRMS was not associated with immunoglobulin subset deficiencies. This is contrasted to ocrelizumab and natalizumab which demonstrate significant reductions in both IgG and IgM levels.

Background: Cognitive impairment is a common symptom of multiple sclerosis (MS). The effect of cognitive impairment in people with MS on employment, quality of life and mental health is known, however, few studies have investigated if cognitive deficits contribute to the economic burden of MS. Objective: To investigate if cognitive impairment correlates with the economic burden of MS. Methods: The client service receipt inventory was used to determine cost to the healthcare system, participant out of pocket cost, community cost and total societal cost. Quality of life was evaluated using the EuroQoL. Participants cognitive performance was assessed with the Audio Recorded Cognitive Screen and the symbol digit modalities test. Spearman's rank correlation coefficient (r) was used to gauge the strength of the correlation between domain scores and cost metrics. Results: Memory, speed of writing and the symbol digit modalities test were all negatively correlated with all aspects of cost of care (r = 0.24¿0.59, P < 0.5). This was found to be independent of other factors, such as EDSS or mental health indices. Conclusion: Cognitive deficits are independently correlated with the economic burden of MS and should be monitored as part of routine care.

Background: The variation in multiple sclerosis (MS) disease severity is incompletely explained by genetics, suggesting genetic and environmental interactions are involved. Moreover, the lack of prognostic biomarkers makes it difficult for clinicians to optimise care. DNA methylation is one epigenetic mechanism by which gene¿environment interactions can be assessed. Here, we aimed to identify DNA methylation patterns associated with mild and severe relapse-onset MS (RMS) and to test the utility of methylation as a predictive biomarker. Methods: We conducted an epigenome-wide association study between 235 females with mild (n = 119) or severe (n = 116) with RMS. Methylation was measured with the Illumina methylationEPIC array and analysed using logistic regression. To generate hypotheses about the functional consequence of differential methylation, we conducted gene set enrichment analysis using ToppGene. We compared the accuracy of three machine learning models in classifying disease severity: (1) clinical data available at baseline (age at onset and first symptoms) built using elastic net (EN) regression, (2) methylation data using EN regression and (3) a weighted methylation risk score of differentially methylated positions (DMPs) from the main analysis using logistic regression. We used a conservative 70:30 test:train split for classification modelling. A false discovery rate threshold of 0.05 was used to assess statistical significance. Results: Females with mild or severe RMS had 1472 DMPs in whole blood (839 hypermethylated, 633 hypomethylated in the severe group). Differential methylation was enriched in genes related to neuronal cellular compartments and processes, and B-cell receptor signalling. Whole-blood methylation levels at 1708 correlated CpG sites classified disease severity more accurately (machine learning model 2, AUC = 0.91) than clinical data (model 1, AUC = 0.74) or the wMRS (model 3, AUC = 0.77). Of the 1708 selected CpGs, 100 overlapped with DMPs from the main analysis at the gene level. These overlapping genes were enriched in neuron projection and dendrite extension, lending support to our finding that neuronal processes, rather than immune processes, are implicated in disease severity. Conclusion: RMS disease severity is associated with whole-blood methylation at genes related to neuronal structure and function. Moreover, correlated whole-blood methylation patterns can assign disease severity in females with RMS more accurately than clinical data available at diagnosis.

Importance: Multiple sclerosis (MS) is usually diagnosed in women during their childbearing years. Currently, no consensus exists on whether pregnancy can delay the first episode of demyelination or clinically isolated syndrome (CIS). Objective: To investigate the association of pregnancy with time to CIS onset. Design, Setting, and Participants: This multicenter cohort study collected reproductive history (duration of each pregnancy, date of delivery, length of breastfeeding) on all participants between September 1, 2016, and June 25, 2019. Adult women being treated at the MS outpatient clinics of 4 tertiary hospitals in 2 countries (Charles University and General University Hospital in Prague, Czech Republic; Royal Melbourne Hospital in Melbourne, Australia; Alfred Hospital in Melbourne, Australia; and John Hunter Hospital in Newcastle, Australia) were recruited to participate in the study. Preexisting data (date of CIS onset, date of birth, sex, date of clinical onset, and Expanded Disability Status Scale result) were collected from MSBase, an international registry of long-term prospectively collected data on patients with MS. Data analyses were performed from June 1, 2019, to February 3, 2020. Exposures: Gravida (defined as any pregnancy, including pregnancy that ended in miscarriage and induced abortion) and parity (defined as childbirth after gestational age of more than 20 weeks, including livebirth and stillbirth) before CIS onset. Main Outcomes and Measures: Time to CIS onset. The following were assessed: (1) whether women with previous pregnancies and childbirths had a delayed onset of CIS compared with those who had never been pregnant and those who had never given birth, and (2) whether a dose response existed, whereby a higher number of gravidity and parity was associated with a later onset of CIS. Results: Of the 2557 women included in the study, the mean (SD) age at CIS onset was 31.5 (9.7) years. Of these women, before CIS onset, 1188 (46%) had at least 1 pregnancy and 1100 (43%) had at least 1 childbirth. The mean (SD) age at first pregnancy was 23.3 (4.5) years and at first childbirth was 23.8 (4.5) years. Women with previous pregnancies and childbirths had a later onset of CIS compared with those who had never been pregnant (HR, 0.68; 95% CI, 0.62-0.75; P <.001), with a median delay of 3.3 (95% CI, 2.5-4.1) years. Women who had given birth also had a later CIS onset compared with women who had never given birth (HR 0.68; 95% CI, 0.61-0.75; P <.001), with a similar median delay of 3.4 (95% CI, 1.6-5.2) years. A higher gravidity and parity number was not associated with delay in CIS onset. Conclusions and Relevance: This study suggests an association between previous pregnancies and childbirths and timing of CIS onset, but having more pregnancies or childbirths did not appear to be associated with a later CIS onset. Further studies are needed to help explain the mechanisms behind the associations between pregnancy and onset of multiple sclerosis 2020 American Medical Association. All rights reserved.

Histone post-translational modifications (PTMs) alter chromatin structure by promoting the interaction of chromatinmodifying complexes with nucleosomes. The majority of chromatin-modifying complexes contain multiple domains that preferentially interact with modified histones, leading to speculation that these domains function in concert to target nucleosomes with distinct combinations of histone PTMs. In Saccharomyces cerevisiae, the NuA3 histone acetyltransferase complex contains three domains, the PHD finger in Yng1, the PWWP domain in Pdp3, and the YEATS domain in Taf14; which in vitro bind to H3K4 methylation, H3K36 methylation, and acetylated and crotonylated H3K9, respectively. While the in vitro binding has been well characterized, the relative in vivo contributions of these histone PTMs in targeting NuA3 is unknown. Here, through genome-wide colocalization and by mutational interrogation, we demonstrate that the PHD finger of Yng1, and the PWWP domain of Pdp3 independently target NuA3 to H3K4 and H3K36 methylated chromatin, respectively. In contrast, we find no evidence to support the YEATS domain of Taf14 functioning in NuA3 recruitment. Collectively our results suggest that the presence of multiple histone PTM binding domains within NuA3, rather than restricting it to nucleosomes containing distinct combinations of histone PTMs, can serve to increase the range of nucleosomes bound by the complex. Interestingly, however, the simple presence of NuA3 is insufficient to ensure acetylation of the associated nucleosomes, suggesting a secondary level of acetylation regulation that does not involve control of HAT-nucleosome interactions.

Background: The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. Methods: Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. Results: UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. Conclusion: These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.

Lymphocytic hypophysitis is an organ-specific autoimmune disease characterised by destruction of pituitary hormone-secreting cells due to attack by self-reactive T lymphocytes. The spectrum of pituitary autoantibodies characterised by indirect immunofluorescence (IF) in these patients has not been substantially defined. The purpose of this study was to determine the spectrum of pituitary autoantibodies in 16 lymphocytic hypophysitis patients. Pituitary sections were prepared from guinea pigs and sera from 16 lymphocytic hypophysitis patients (13 biopsy proven and 3 suspected cases) and 13 healthy controls were evaluated for immunoreactivity to the pituitary tissue by immunofluorescence. A single patient was found to have high titre pituitary autoantibodies against guinea pig pituitary tissue. Immunoreactivity was directed against cells of the intermediate lobe. We present the case report of the patient who is a 24 year old woman that presented with headaches, polyuria and polydipsia. A uniformly enlarged pituitary mass was visible on MRI and a diagnosis of suspected lymphocytic hypophysitis was made. Based on our IF study, we postulate this patient has an autoimmune process directed towards the major cell type in the intermediate lobe, the melanotroph. Pre-adsorption with peptides representing adrenocorticotropic hormone, a-melanocyte stimulating hormone or ß-endorphin did not affect the IF signal suggesting our patient's pituitary autoantibodies may target some other product of Proopiomelanocortin (POMC) processing, such as corticotrophin-like intermediate peptide or ¿-lipoprotein. Alternatively, the autoantibodies may target a peptide completely unrelated to POMC processing. © 2013 Springer Science+Business Media New York.