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Hemiplegic migraine (HM) is a rare subtype of migraine with aura. Given that causal missense mutations in the voltage-gated calcium channel α1A subunit gene CACNA1A have been identified in a subset of HM patients, we investigated whether HM patients without a mutation have an increased burden of such variants in the “CACNA1x gene family”. Whole exome sequencing data of an Australian cohort of unrelated HM patients (n = 184), along with public data from gnomAD, as controls, was used to assess the burden of missense variants in CACNA1x genes. We performed both a variant and a subject burden test. We found a significant burden for the number of variants in CACNA1E (p = 1.3 × 10−4), CACNA1H (p −16) and CACNA1I (p −16). There was also a significant burden of subjects with missense variants in CACNA1E (p = 6.2 × 10−3), CACNA1H (p −16) and CACNA1I (p −16). Both the number of variants and number of subjects were replicated for CACNA1H (p = 3.5 × 10−8; p = 0.012) and CACNA1I (p = 0.019, p = 0.044), respectively, in a Dutch clinical HM cohort (n = 32), albeit that CACNA1I did not remain significant after multiple testing correction. Our data suggest that HM, in the absence of a single causal mutation, is a complex trait, in which an increased burden of missense variants in CACNA1H and CACNA1I may contribute to the risk of disease.

IntroductionMultiple Sclerosis (MS) has a complex pathophysiology that involves genetic and environmental factors. DNA methylation (DNAm) is one epigenetic mechanism that can reversibly modulate gene expression. Cell specific DNAm changes have been associated with MS, and some MS therapies such as dimethyl fumarate can influence DNAm. Interferon Beta (IFNβ), was one of the first disease modifying therapies in multiple sclerosis (MS). However, how IFNβ reduces disease burden in MS is not fully understood and little is known about the precise effect of IFNβ treatment on methylation.MethodsThe objective of this study was to determine the changes in DNAm associated with INFβ use, using methylation arrays and statistical deconvolutions on two separate datasets (total ntreated = 64, nuntreated = 285).ResultsWe show that IFNβ treatment in people with MS modifies the methylation profile of interferon response genes in a strong, targeted, and reproducible manner. Using these identified methylation differences, we constructed a methylation treatment score (MTS) that is an accurate discriminator between untreated and treated patients (Area under the curve = 0.83). This MTS is time-sensitive and in consistent with previously identified IFNβ treatment therapeutic lag. This suggests that methylation changes are required for treatment efficacy. Overrepresentation analysis found that IFNβ treatment recruits the endogenous anti-viral molecular machinery. Finally, statistical deconvolution revealed that dendritic cells and regulatory CD4+ T cells were most affected by IFNβ induced methylation changes.DiscussionIn conclusion, our study shows that IFNβ treatment is a potent and targeted epigenetic modifier in multiple sclerosis.

Multiple sclerosis is a complex autoimmune disease that causes neuronal demyelination and debilitating physical and cognitive symptoms. Epigenetic factors can mediate genetic and environmental effects on disease risk. Here we profiled blood-based DNA methylation in 583 MS cases and 643 healthy controls representing 3 independent study groups. An epigenome-wide association study was performed, incorporating statistical deconvolution to identify immune cell-specific signals, which were validated in purified cell subsets. First, we elucidated the mediation effects of methylation at the HLA risk locus. Second, we identified methylation differences in MS that occur independently of known genetic risk loci and show that these differences more strongly differentiate disease than HLA genotype and the polygenic risk score. Finally, we show that methylation differences in MS occur predominantly in B cells and monocytes and indicate the involvement of cell-specific biological pathways. Overall, this study comprehensively characterizes the immune cell specific epigenetic architecture of MS.

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 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.

A percentage of the population suffers prolonged and persistent post-concussion symptoms (PCS) following average head injuries or develops severe neurological dysfunction following minor head trauma. Genetic variants that may contribute to individual response to head trauma have been investigated in some studies, but to date none have explored the use of machine learning (ML) methods with genomic data to specifically explore outcomes of head trauma. Whole exome sequencing (WES) was completed for three groups of individuals (N = 60): (a) 16 individuals with severe neurological responses to minor head trauma, (b) 26 individuals with persistent PCS and (c) 18 individuals with normal recovery from concussion or mTBI. Gradient boosted tree algorithms were applied to the data using XGBoost. By using variants with CADD scores above 15 in the training set (randomly sampled 70%), we identified signatures that accurately distinguish to accurately distinguish the test groups with an average area under the curve (AUC) of 0.8 (SE = 0.019). Metrics including positive and negative prediction values, as well as kappa were all within acceptable range to support the prediction accuracy. This study illustrates how ML methods in combination with WES data have the potential to predict severe or prolonged responses to head trauma from healthy recovery. KEY MESSAGES: Linear association analysis has been inconclusive in concussion genetics. Non-linear methods as boosted trees can offer better insights in small samples. Strong discrimination trends can be achieved from exome data of cases and controls.

Objective This study is the first to assess if the National Disability Insurance Scheme (NDIS) package allocated to people with multiple sclerosis (pwMS) is correlated with the disability level measured by standardised neurological assessment. Methods We aimed to recruit 10 pwMS per expanded disability status score (EDSS) step, including EDSS 0 (no disability) up to 9 (bedridden), and requested information about their NDIS application. Value of their packages was compared with mobility, cognition and psychological impact. Results Out of 186 pwMS, only 49% of all patients had an NDIS package approved. The mean values of the annual allowance were AU$30 318 for patients with mild disability, AU$38 361 for moderate disability and AU$115 113 for severe disability. There was a striking variability in packages approved, but restricted mobility seems to be the driving factor. Rejection rates were

Abstract Monogenic forms of Alzheimer’s disease (AD) have been identified through mutations in genes such as APP, PSEN1, and PSEN2, whilst other genetic markers such as the APOE ε carrier allele status have been shown to increase the likelihood of having the disease. Mutations in these genes are not limited to AD, as APP mutations can also cause an amyloid form of cerebral small vessel disease (CSVD) known as cerebral amyloid angiopathy, whilst PSEN1 and PSEN2 are involved in NOTCH3 signalling, a process known to be dysregulated in the monogenic CSVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The overlap between AD genes and causes of CSVD led to the hypothesis that mutations in other genes within the PANTHER AD–presenilin pathway may be novel causes of CSVD in a cohort of clinically suspicious CADASIL patients without a pathogenic NOTCH3 mutation. To investigate this, whole exome sequencing was performed on 50 suspected CADASIL patients with no NOTCH3 mutations, and a targeted gene analysis was completed on the PANTHER. ERN1 was identified as a novel candidate CSVD gene following predicted pathogenic gene mutation analysis. Rare variant burden testing failed to identify an association with any gene; however, it did show a nominally significant link with ERN1 and TRPC3. This study provides evidence to support a genetic overlap between CSVD and Alzheimer’s disease.

Background and Aims Natural variation in body fat is explained by both genetic and environmental effects. Epigenetic mechanisms such as DNA methylation can mediate these effects causing changes in gene expression leading to onset of obesity. Studies of genetic isolates have the potential to provide new epigenetic insights with advantages in reduced genetic diversity and environmental exposures. Methods This was an exploratory study of genome-wide DNA methylation in relation to body fat traits in 47 healthy adults from the genetic isolate of Norfolk Island. Quantitative body fat traits (body fat percentage, body mass index, hip circumference, waist circumference, waist-hip-ratio and weight) were carefully measured. DNA methylation data was obtained from peripheral blood using Illumina 450K arrays. Multi-trait analysis was performed using Principal Component Analysis (PCA). CpG by trait association testing was performed using stepwise linear regressions. Results Two components were identified that explained approximately 89% of the phenotypic variance. In total, 5 differential methylated positions (DMPs) were identified at genome-wide significance (P ≤ 2.4 x 10-7), which mapped to GOT2-CDH8, LYSMD3, HIBADH, ADGRD1 and EBF4 genes. Gene set enrichment analysis of 848 genes containing suggestive DMPs (P ≤ 1.0 x 10-4) implicated the Cadherin (28 genes, Padj = 6.76 x 10-7) and Wnt signaling pathways (38 genes, Padj = 7.78 x 10-6). Conclusion This study provides new insights into the epigenetically influenced genes and pathways underlying body fat variation in a healthy cohort and provides targets for consideration in future studies of obesity risk.

Unrecovered War Casualties – Army (UWC-A) is part of the Australian Defence Force (ADF) and investigates unaccounted for Australian soldiers from previous conflicts. In the Asia-Pacific region of W...

To evaluate the performance of novel spiral MRSI and tissue segmentation pipeline of the brain, to investigate neurometabolic changes in normal-appearing white matter (NAWM) and white matter lesions (WML) of stable relapsing remitting multiple sclerosis (RRMS) compared to healthy controls (HCs).Spiral 3D MRSI using LASER-GOIA-W [16,4] was undertaken on 16 RRMS patients and 9 HCs, to acquire MRSI data from a large volume of interest (VOI) 320 cmCompared to HCs, RRMS demonstrated a statistically significant reduction in all mean brain tissues and increase in CSF volume. Within VOI, WM decreased (-10%) and CSF increased (41%) in RRMS compared to HCs (p 0.001). MRSI revealed that total creatine (tCr) ratios of N-acetylaspartate and glutamate+glutamine in WML were significantly lower than NAWM-MS (-9%, -8%) and HCs (-14%, -10%), respectively. Myo-inositol/tCr in WML was significantly higher than NAWM-MS (14%) and HCs (10%). SVM of MRSI yielded accuracy, sensitivity and specificity of 86%, 95%, and 70%, respectively for HCs vs WML, which were higher than HC vs NAWM and WML vs NAWM models.This study demonstrates the benefit of MRSI in evaluating MS neurometabolic changes in NAWM. SVM of MRSI data in the MS brain may be suited for clinical monitoring and progression of MS patients. Longitudinal MRSI studies are warranted.

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.

The aetiology and pathophysiology of complex diseases are driven by the interaction between genetic and environmental factors. The variability in risk and outcomes in these diseases are incompletely explained by genetics or environmental risk factors individually. Therefore, researchers are now exploring the epigenome, a biological interface at which genetics and the environment can interact. There is a growing body of evidence supporting the role of epigenetic mechanisms in complex disease pathophysiology. Epigenome-wide association studies (EWASes) investigate the association between a phenotype and epigenetic variants, most commonly DNA methylation. The decreasing cost of measuring epigenome-wide methylation and the increasing accessibility of bioinformatic pipelines have contributed to the rise in EWASes published in recent years. Here, we review the current literature on these EWASes and provide further recommendations and strategies for successfully conducting them. We have constrained our review to studies using methylation data as this is the most studied epigenetic mechanism; microarray-based data as whole-genome bisulphite sequencing remains prohibitively expensive for most laboratories; and blood-based studies due to the non-invasiveness of peripheral blood collection and availability of archived DNA, as well as the accessibility of publicly available blood-cell-based methylation data. Further, we address multiple novel areas of EWAS analysis that have not been covered in previous reviews: (1) longitudinal study designs, (2) the chip analysis methylation pipeline (ChAMP), (3) differentially methylated region (DMR) identification paradigms, (4) methylation quantitative trait loci (methQTL) analysis, (5) methylation age analysis and (6) identifying cell-specific differential methylation from mixed cell data using statistical deconvolution.

Epigenetics may play a central, yet unexplored, role in the profound changes that the maternal immune system undergoes during pregnancy and could be involved in the pregnancy-induced modulation of several autoimmune diseases. We investigated changes in the methylome in isolated circulating CD4

Chronic kidney disease (CKD) is a persistent impairment of kidney function. Genome-wide association studies (GWAS) have revealed multiple genetic loci associated with CKD susceptibility but the complete genetic basis is not yet clear. Since CKD shares risk factors with cardiovascular diseases and diabetes, there may be pleiotropic loci at play but may go undetected when using single phenotype GWAS. Here, we used multi-phenotype GWAS in the Norfolk Island isolate (n = 380) to identify new loci associated with CKD. We performed a principal components analysis on different combinations of 29 quantitative traits to extract principal components (PCs) representative of multiple correlated phenotypes. GWAS of a PC derived from glomerular filtration rate, serum creatinine, and serum urea identified a suggestive peak (pmin = 1.67 × 10–7) that mapped to KCNIP4. Inclusion of other secondary CKD measurements with these three kidney function traits identified the KCNIP4 locus with GWAS significance (pmin = 1.59 × 10–9). Finally, we identified a group of two SNPs with increased minor allele frequencies as potential functional variants. With the use of genetic isolate and the PCA-based multi-phenotype GWAS approach, we have revealed a potential pleotropic effect locus for CKD. Further studies are required to assess functional relevance of this locus.

The gut microbiome has been associated with cardiorespiratory fitness.To assess the effects of oligofructose (FOS)-enriched inulin supplementation on the gut microbiome and the peak oxygen uptake (V̇O2peak) response to high-intensity interval training (HIIT).The study was a randomized controlled trial. Forty sedentary and apparently healthy adults [n = 31 women; aged 31.8 ± 9.8 y, BMI (in kg⋅m-2) 25.9 ± 4.3] were randomly allocated to 1) 6 wk of supervised HIIT (4 × 4-min bouts at 85-95% peak heart rate, interspersed with 3 min of active recovery, 3·wk-1) + 12 g·d-1 of FOS-enriched inulin (HIIT-I) or 2) 6 wk of supervised HIIT (3·wk-1, 4 × 4-min bouts) + 12 g·d-1 of maltodextrin/placebo (HIIT-P). Each participant completed an incremental treadmill test to assess V̇O2peak and ventilatory thresholds (VTs), provided a stool and blood sample, and completed a 24-h diet recall questionnaire and FFQ before and after the intervention. Gut microbiome analyses were performed using metagenomic sequencing. Fecal short-chain fatty acids were measured by mass spectrometry.There were no differences in the mean change in V̇O2peak response between groups (P = 0.58). HIIT-I had a greater improvement in VTs than HIIT-P [VT1 (lactate accumulation): mean difference + 4.3% and VT2 (lactate threshold): +4.2%, P 0.05]. HIIT-I had a greater increase in the abundance of Bifidobacterium taxa [false discovery rate (FDR)0.05] and several metabolic processes related to exercise capacity (FDR 0.05). Exploratory analysis of merged data found participants with a greater response to HIIT (V̇O2peak ≥3.5 mL⋅kg-1⋅min-1) had a 2.2-fold greater mean abundance of gellan degradation pathways (FDR 0.05) and a greater, but not significant, abundance of Bifidobacterium uniformis species (P 0.00023, FDR = 0.08).FOS-enriched inulin supplementation did not potentiate HIIT-induced improvements in V̇O2peak but led to gut microbiome changes possibly associated with greater ventilatory threshold improvements in healthy inactive adults. Gellan degradation pathways and B. uniformis spp. were associated with greater V̇O2peak responses to HIIT.

Estimates of mutation rates for various regions of the mitochondrial genome (mtGenome) vary widely, depending on whether they are inferred using a phylogenetic approach or obtained directly from pedigrees. Traditionally, only the control region, or small portions of the coding region have been targeted for analysis due to the cost and effort required to produce whole mtGenome Sanger profiles. Here, we report one of the first pedigree derived mutation rates for the entire human mtGenome. The entire mtGenome from 225 individuals originating from Norfolk Island was analysed to estimate the population mutation rate and compared against published mutation rates. These individuals were from 45 maternal lineages spanning 345 generational events. Mutation rates for various portions of the mtGenome were calculated. Nine mutations (including two transitions and seven cases of heteroplasmy) were observed, resulting in a rate of 0.063 mutations/site/million years (95% confidence interval: 0.033 – 0.118). These mutation rates are approximately 17 times higher than estimates derived from phylogenetic analysis with heteroplasmy detected in 13 samples (n=225, 5.8% individuals). Providing one of the first pedigree derived estimates for the entire mtGenome, this study provides a better understanding of mtGenome evolution and has relevance to many research fields, including medicine, anthropology and forensics.

The theory that multiple sclerosis is related to venous pressure has been discredited due to previous operator dependent diagnostic criteria and premature attempts at treatment. (1) An elevation in venous pressure may only be a component of the compliance changes found in MS. (2) The neck veins may only supply a component of the venous pressure elevation found intracranially. Although a more targeted approach towards neck angioplasty (both towards disease subtype and those with more favorable stenoses) may be beneficial, we would advocate caution. We encourage others to give the venous pressure theory a second chance and to replicate our work.

BACKGROUND In a previous study, multiple sclerosis (MS) was found to be associated with an increase in intracranial arterial pulsation volume and a reduction in venous sinus compliance, affecting pulsation dampening. There was a suggestion that the reduction in compliance of the sagittal sinus in MS was caused by an increase in venous pressure, secondary to transverse sinus stenosis. Some differences were noted depending on the gender of the patients, however, the original study was relatively underpowered for further sub-classification. The purpose of the current study is to enroll a larger number of patients to allow sub-classification on gender and disease type to further evaluate the markers of possible venous pressure alteration. METHODS 103 patients with MS were prospectively recruited from an MS clinic and compared to 50 matched non-MS patients. Using 3DT1 post contrast images, the sagittal sinus cross-sectional area was measured. The narrowest portion of the transverse sinuses was located and the cross sectional areas and wetted circumferences were measured to calculate the minimum hydraulic and effective diameters. The jugular bulb heights were measured. Voxel wise brain morphometry was performed to evaluate atrophy. Statistical analysis was performed using non-parametric methods and was assessed using α≤0.05. RESULTS Compared to controls, the MS patients' sagittal sinuses were 23% larger in cross-section (p

In this article, we introduce the variant call format–diagnostic annotation and reporting tool (VCF-DART), a customized analysis pipeline tool for the rapid annotation of variants from exome or genome sequencing and the generation of reports for genetic diagnostics. VCF-DART uses custom gene lists to categorize variants into specific analysis tiers and to subcategorize them on the basis of standard parameters to facilitate the rapid interrogation of potentially pathogenic variants by human operators. The tool uses publicly available databases to identify a range of data to assist with variant classification and curation processes and includes robust logging of parameters and database versions to allow comparison of analyses performed at different times. VCF-DART—an online analysis pipeline for next-generation sequencing data—is a platform agnostic tool that leverages the use of publicly available databases to improve a laboratory's calling ability and reduce analysis times long-term. It also runs highly efficiently and scales from desk and laptop machines to servers. Overall, VCF-DART provides a simple, customizable, and entirely open-source method to identify genetic variants that may be of clinical importance in a variety of genetically important conditions.

Purpose Germline mutations within melanoma susceptibility genes are present only in minority of melanoma patients and it is expected that additional genes will be discovered with next generation sequence technology and whole-exome sequencing (WES). Materials and Methods Herein we performed WES on a cohort of 96 unrelated Polish patients with melanoma diagnosed under the age of 40 years who all screened negative for the presence of CDKN2A variants. A replication study using a set of 1,200 melanoma patient DNA samples and similarly large series of healthy controls was undertaken. Results We selected 21 potentially deleterious variants in 20 genes (VRK1, MYCT1, DNAH14, CASC3, MS4A12, PRC1, WWOX, CARD6, EXO5, CASC3, CASP8AP2, STK33, SAMD11, CNDP2, CPNE1, EFCAB6, CABLES1, LEKR1, NUDT17, and RRP15), which were identified by WES and confirmed by Sanger sequencing for an association study. Evaluation of the allele distribution among carriers and their relatives in available family trios revealed that these variants were unlikely to account for many familial cases of melanoma. Replication study revealed no statistically significant differences between cases and controls. Conclusion Although most of the changes seemed to be neutral we could not exclude an association between variants in VRK1, CREB3L3, EXO5, and STK33 with melanoma risk.

Post-traumatic stress disorder (PTSD) is triggered by experiencing terrifying event(s) for which there is currently no objective test for a definitive diagnosis. We report a pilot study where two-dimensional (2D) neuro magnetic resonance spectroscopy (MRS), collected at 3 T in a clinical scanner with a 64-channel head coil, identifies neuro deregulation in the PTSD cohort. The control subjects (n = 10) were compared with PTSD participants with minimal co-morbidities (n = 10). The 2D MRS identified statistically significant increases in the total spectral region containing both free substrate fucose and fucosylated glycans of 31% (P = 0.0013), two of multiple fucosylated glycans (Fuc IV and VI) were elevated by 48% (P = 0.002), and 41% (P = 0.02), respectively, imidazole was increased by 12% (P = 0.002), and lipid saturation was increased by 12.5% (P = 0.009). This is the first evidence of fucosylated glycans, reported in animals to be involved in learning and memory, to be affected in humans with PTSD.

OBJECTIVES This study explored the association of children's salivary characteristics, past caries experience, birth weight, and reported maternal prenatal vitamin and mineral supplementation with the dental untreated decay of the child. METHODS This cross-sectional study, a sub-study of Griffith University Environments for Healthy Living birth cohort study, was conducted on 174 mother-child dyads. Mother's prenatal usage of vitamin and mineral supplements; child's birthweight; salivary pH, buffering capacity, and levels of salivary MS and LB were explored as risk indicators. Dental caries experience was assessed using International Caries Detection and Assessment System criteria. Path analysis was conducted to evaluate the association of risk indicators with children's current and past dental caries experience. RESULTS Children's past caries experience (β = 0.332, p = 0.018), and salivary MS counts (β = 0.215, p = 0.032) were positively associated with untreated decay at time of examination. With a trend towards significance, children whose mothers had reported taking iron supplements during pregnancy experienced lower levels of past caries (β = -0.137, p = 0.068) and untreated dental caries (β = -0.046, p = 0.051). CONCLUSIONS This study confirms that a child's levels of untreated decay is positively associated with their past caries, and that it correlates with current levels of salivary MS. Children of mothers who reported to have taken iron supplements during pregnancy experienced less caries throughout their lives. These observations confirm the importance to offspring of monitoring maternal health throughout pregnancy and of early monitoring of children's oral health in preventing future dental disease.

Background Low cardiorespiratory fitness (V̇O2peak) is highly associated with chronic disease and mortality from all causes. Whilst exercise training is recommended in health guidelines to improve V̇O2peak, there is considerable inter-individual variability in the V̇O2peak response to the same dose of exercise. Understanding how genetic factors contribute to V̇O2peak training response may improve personalisation of exercise programs. The aim of this study was to identify genetic variants that are associated with the magnitude of V̇O2peak response following exercise training. Methods Participant change in objectively measured V̇O2peak from 18 different interventions was obtained from a multi-centre study (Predict-HIIT). A genome-wide association study was completed (n = 507), and a polygenic predictor score (PPS) was developed using alleles from single nucleotide polymorphisms (SNPs) significantly associated (P –5) with the magnitude of V̇O2peak response. Findings were tested in an independent validation study (n = 39) and compared to previous research. Results No variants at the genome-wide significance level were found after adjusting for key covariates (baseline V̇O2peak, individual study, principal components which were significantly associated with the trait). A Quantile–Quantile plot indicates there was minor inflation in the study. Twelve novel loci showed a trend of association with V̇O2peak response that reached suggestive significance (P –5). The strongest association was found near the membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2) gene (rs6959961, P = 2.61 × 10–7). A PPS created from the 12 lead SNPs was unable to predict V̇O2peak response in a tenfold cross validation, or in an independent (n = 39) validation study (P > 0.1). Significant correlations were found for beta coefficients of variants in the Predict-HIIT (P –4) and the validation study (P –6), indicating that general effects of the loci exist, and that with a higher statistical power, more significant genetic associations may become apparent. Conclusions Ongoing research and validation of current and previous findings is needed to determine if genetics does play a large role in V̇O2peak response variance, and whether genomic predictors for V̇O2peak response trainability can inform evidence-based clinical practice. Trial registration Australian New Zealand Clinical Trials Registry (ANZCTR), Trial Id: ACTRN12618000501246, Date Registered: 06/04/2018, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=374601&isReview=true.

Epigenetics may play a central, yet unexplored, role in the profound changes that the maternal immune system undergoes during pregnancy and could be involved in the pregnancy-induced modulation of several autoimmune diseases. We investigated changes in the methylome in isolated circulating CD4(+) T-cells in non-pregnant and pregnant women, during the 1(st) and 2(nd) trimester, using the Illumina Infinium Human Methylation 450K array, and explored how these changes were related to autoimmune diseases that are known to be affected during pregnancy. Pregnancy was associated with several hundreds of methylation differences, particularly during the 2(nd) trimester. A network-based modular approach identified several genes, e.g., CD28, FYN, VAV1 and pathways related to T-cell signalling and activation, highlighting T-cell regulation as a central component of the observed methylation alterations. The identified pregnancy module was significantly enriched for disease-associated methylation changes related to multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. A negative correlation between pregnancy-associated methylation changes and disease-associated changes was found for multiple sclerosis and rheumatoid arthritis, diseases that are known to improve during pregnancy whereas a positive correlation was found for systemic lupus erythematosus, a disease that instead worsens during pregnancy. Thus, the directionality of the observed changes is in line with the previously observed effect of pregnancy on disease activity. Our systems medicine approach supports the importance of the methylome in immune regulation of T-cells during pregnancy. Our findings highlight the relevance of using pregnancy as a model for understanding and identifying disease-related mechanisms involved in the modulation of autoimmune diseases. CC BY 4.0Contact Sandra Hellberg Email iconsandra.hellberg@liu.sePublished online: 04 Oct 2021Copyright © 2022 Informa UK Limited This work was supported by the Swedish Foundation for Strategic Research (SB16-0011), Swedish Research Council (K2013-61X-22310-01-4, 2015-030807, 2018-02776) and Lions research grant (Liu-2012-01948).

The pathology of progressive multiple sclerosis (MS) is poorly understood. We have previously assessed DNA methylation in the CD4+ T cells of relapsing–remitting (RR) MS patients compared to healthy controls and identified differentially methylated regions (DMRs) in HLA-DRB1 and RNF39. This study aimed to investigate the DNA methylation profiles of the CD4+ T cells of progressive MS patients. DNA methylation was measured in two separate case/control cohorts using the Illumina 450K/EPIC arrays and data was analysed with the Chip Analysis Methylation Pipeline (ChAMP). Single nucleotide polymorphisms (SNPs) were assessed using the Illumina Human OmniExpress24 arrays and analysed using PLINK. Expression was assessed using the Illumina HT12 array and analysed in R using a combination of Limma and Illuminaio. We identified three DMRs at HTR2A, SLC17A9 and HDAC4 that were consistent across both cohorts. The DMR at HTR2A is located within the bounds of a haplotype block; however, the DMR remained significant after accounting for SNPs in the region. No expression changes were detected in any DMRs. HTR2A is differentially methylated in progressive MS independent of genotype. This differential methylation is not evident in RRMS, making it a potential biomarker of progressive disease.

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.To investigate if cognitive impairment correlates with the economic burden of MS.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.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.Cognitive deficits are independently correlated with the economic burden of MS and should be monitored as part of routine care.

The MinION is a portable DNA sequencer that allows real time sequencing at low capital cost investment. We assessed accuracy and cost-effectivess of the MinION for genetic diagnostic testing of known SCN1A mutations that cause Dravet Syndrome (DS). DNA samples (n = 7) from DS patients previously shown to carry SCN1A mutations via Ion Torrent and Sanger sequencing were sequenced using the MinION. SCN1A amplicons for 8 exons were sequenced using the MinION with 1D chemistry on an R9.4 flow cell. All known missense mutations were detected in all samples showing 100 % concordance with results from other methods. However, the MinION failed to detect the insertions/deletions (INDELs) present in these patients. Nevertheless, these results indicate that MinION is a cost-effective platform for use as an initial screening step in the detection of nucleotide substitution mutations in in SCN1A, especially in under-resourced laboratories or hospitals. Further improvements are required to reliably detect INDELS in this gene.

Background Cladribine tablets (marketed as Mavenclad) are a new oral therapy, which has recently been listed on the pharmaceutical benefits scheme in Australia for the treatment of relapsing multiple sclerosis (MS). The current dosing schedule is for 2 courses given a year apart, which has been shown to be effective for treatment of MS for up to 4 years in 75% of patients (based on annualized relapse rate). However, the reinitiation of therapy after year 4 has not been studied. Objective This study aims to evaluate the safety and efficacy of cladribine tablets over a 6-year period, according to no evidence of disease activity 3. Methods This will be a multicenter, 6-year, phase IV, low interventional, observational study that incorporates clinical, hematological, biochemical, epigenetic, radiological and cognitive biomarkers of disease. Participants considered for treatment with cladribine as part of their routine clinical care will be consented to take part in the study. They will be monitored at regular intervals during the initial course of medication administration in years 1 and 2. After year 3, patients will have the option of redosing, if clinically indicated, or to switch to another disease-modifying therapy. Throughout the duration of the study, we will assess blood-based biomarkers including lymphocyte subsets, serum neurofilament light chain, DNA methylation, and RNA analysis as well as magnetic resonance imaging findings (brain volume and/or lesion load) and cognitive performance. Results This study has been approved by the Hunter New England Local Health District Human Research Ethics Committee. Recruitment began in March of 2019 and was completed by June 2021. Conclusions This will be the first long-term efficacy trial of cladribine, which offers reinitiation of therapy in the 3rd year, based on disease activity, after the initial 2 courses. We expect that this study will indicate whether any of the assessed biomarkers can be used to predict treatment efficacy or the need for future reinitiation of cladribine in people with MS. Trial Registration This study is registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12619000257167) with Universal Trial Number (U1111-1228-2165). International Registered Report Identifier (IRRID) DERR1-10.2196/24969

BACKGROUND Cladribine tablets (marketed as Mavenclad) are a new oral therapy, which has recently been listed on the pharmaceutical benefits scheme in Australia for the treatment of relapsing multiple sclerosis (MS). The current dosing schedule is for 2 courses given a year apart, which has been shown to be effective for treatment of MS for up to 4 years in 75% of patients (based on annualized relapse rate). However, the reinitiation of therapy after year 4 has not been studied. OBJECTIVE This study aims to evaluate the safety and efficacy of cladribine tablets over a 6-year period, according to no evidence of disease activity 3. METHODS This will be a multicenter, 6-year, phase IV, low interventional, observational study that incorporates clinical, hematological, biochemical, epigenetic, radiological and cognitive biomarkers of disease. Participants considered for treatment with cladribine as part of their routine clinical care will be consented to take part in the study. They will be monitored at regular intervals during the initial course of medication administration in years 1 and 2. After year 3, patients will have the option of redosing, if clinically indicated, or to switch to another disease-modifying therapy. Throughout the duration of the study, we will assess blood-based biomarkers including lymphocyte subsets, serum neurofilament light chain, DNA methylation, and RNA analysis as well as magnetic resonance imaging findings (brain volume and/or lesion load) and cognitive performance. RESULTS This study has been approved by the Hunter New England Local Health District Human Research Ethics Committee. Recruitment began in March of 2019 and was completed by June 2021. CONCLUSIONS This will be the first long-term efficacy trial of cladribine, which offers reinitiation of therapy in the 3rd year, based on disease activity, after the initial 2 courses. We expect that this study will indicate whether any of the assessed biomarkers can be used to predict treatment efficacy or the need for future reinitiation of cladribine in people with MS. CLINICALTRIAL This study is registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12619000257167) with Universal Trial Number (U1111-1228-2165). INTERNATIONAL REGISTERED REPORT DERR1-10.2196/24969

Multiple sclerosis (MS) is associated with a breakdown in the intracranial pulse wave dampening or windkessel effect. This is manifest by an increase in the arterial stroke volume and a decrease in the dampening afforded by both the CSF displaced into the spinal canal and the blood displaced by the venous sinus pulsation. There is evidence that the reduction in compliance of the sagittal and straight sinuses in MS is caused by an increase in venous pressure despite the jugular bulb pressures being normal. This implies MS patients have a venous outflow stenosis somewhere between the torcular and jugular bulbs. The purpose of the current study is to define the site, significance and cause of these stenoses.50 patients with MS were prospectively recruited from an MS clinic and compared to 50 matched control patients. Using 3DT1 post contrast images, a survey of the venous sinuses was performed looking for the narrowest portion of the sinuses in each of 4 segments from the sagittal sinus to jugular bulbs. The cross sectional areas and wetted circumferences of the venous sinuses were measured at each site to calculate the minimum hydraulic and effective diameters. The BMI, optic nerve sheath diameters and pituitary heights were measured. Statistical analysis was performed using non-parametric methods and was assessed using α≤0.05.Compared to controls, the MS patients' sagittal sinuses were 24% larger in cross-section (p=0.0001) with an 18% larger wetted circumference (p=0.0001). The MS patients' transverse sinuses had an average effective stenosis of 38% by area (p0.0001) with 8/50 patients having a high grade stenosis of65% by area and 16/50 a low grade stenosis of between 40-65% by area compared to 1/50 low grade stenoses in this segment in the controls. The commonest cause of the stenosis was a giant arachnoid granulation. The optic nerve sheaths were larger in MS than controls (p=0.0006). Comparing MS patients with transverse sinus stenosis to those without, the pituitary height was 16% smaller and BMI 25% larger (p=0.02 and 0.003 respectively) CONCLUSION: In patients with MS, the reduction in venous sinus compliance is associated with venous outflow stenoses in the transverse sinuses which increases the upstream venous pressure and dilates the sagittal sinuses. This finding suggests a continuum exists between MS and idiopathic intracranial hypertension.

Mitochondria supply intracellular energy requirements during exercise. Specific mitochondrial haplogroups and mitochondrial genetic variants have been associated with athletic performance, and exercise responses. However, these associations were discovered using underpowered, candidate gene approaches, and consequently have not been replicated. Here, we used whole-mitochondrial genome sequencing, in conjunction with high-throughput genotyping arrays, to discover novel genetic variants associated with exercise responses in the Gene SMART (Skeletal Muscle Adaptive Response to Training) cohort (n = 62 completed). We performed a Principal Component Analysis of cohort aerobic fitness measures to build composite traits and test for variants associated with exercise outcomes. None of the mitochondrial genetic variants but eight nuclear encoded variants in seven separate genes were found to be associated with exercise responses (FDR rs11061368: DIABLO, rs113400963: FAM185A, rs6062129 and rs6121949: MTG2, rs7231304: AFG3L2, rs2041840: NDUFAF7, rs7085433: TIMM23, rs1063271: SPTLC2). Additionally, we outline potential mechanisms by which these variants may be contributing to exercise phenotypes. Our data suggest novel nuclear-encoded SNPs and mitochondrial pathways associated with exercise response phenotypes. Future studies should focus on validating these variants across different cohorts and ethnicities.

Mitochondria supply intracellular energy requirements during exercise. Specific mitochondrial haplogroups and mitochondrial genetic variants have been associated with athletic performance, and exercise responses. However, these associations were discovered using underpowered, candidate gene approaches, and consequently have not been replicated. Here, we used whole-mitochondrial genome sequencing, in conjunction with high-throughput genotyping arrays, to discover novel genetic variants associated with exercise responses in the Gene SMART (Skeletal Muscle Adaptive Response to Training) cohort (n=62 completed). We performed a Principal Component Analysis of cohort aerobic fitness measures to build composite traits and test for variants associated with exercise outcomes. None of the mitochondrial genetic variants but nine nuclear encoded variants in eight separate genes were found to be associated with exercise responses (FDR(rs11061368: DIABLO, rs113400963: FAM185A, rs6062129 and rs6121949: MTG2, rs7231304: AFG3L2, rs2041840: NDUFAF7, rs7085433: TIMM23, rs1063271: SPTLC2, rs2275273: ALDH18A1). Additionally, we outline potential mechanisms by which these variants may be contributing to exercise phenotypes. Our data suggest novel nuclear-encoded SNPs and mitochondrial pathways associated with exercise response phenotypes. Future studies should focus on validating these variants across different cohorts and ethnicities.AUTHOR SUMMARYPrevious exercise genetic studies contain many flaws that impede the growth in knowledge surrounding change in exercise outcomes. In particular, exercise studies looking at mtDNA variants have looked at very small portions of the mitochondrial genome. Mitochondria are the ‘power house’ of the cell and therefore understanding the mitochondrial genetics behind adaptations to training can help us fill knowledge gaps in current research. Here, we utilised a new mitochondrial genetic sequencing technique to examine all mitochondrial and mitochondrial related genetic variations. We have shown that there were no mitochondrial specific variants that influenced exercise training however there were 9 related variants that were significantly associated with exercise phenotypes. Additionally, we have shown that building composite traits increased the significance of our association testing and lead to novel findings. We will be able to understand why response to training is so varied and increase the effectiveness of exercise training on a host of metabolic disorders.

Driving regulations are complex, particularly for neurological conditions, but accurate application of restrictions is important. This study was designed to investigate knowledge of the Austroads guidelines in medical students, neurological trainees and consultant neurologists using a questionnaire addressing both private and commercial licence restrictions related to five common neurological conditions, namely transient ischaemic attack (TIA), vasovagal episode, unwitnessed blackout, first epileptic seizure and benign paroxysmal positional vertigo. In total, 118 of the 120 returned responses could be analysed. Overall, 50% of all responses were correct. Respondents performed better for private than commercial licences, and consultants performed better than trainees and students. The highest proportion of correct answers was seen for vasovagal attack, and the lowest for TIA. In summary, knowledge of driving restrictions was relatively poor, and regular consultation of the guidelines is recommended. A larger study is warranted and increased education at both medical school and postgraduate levels should be considered.

Background Cognitive impairment is common in multiple sclerosis (MS) but not adequately monitored by Expanded Disability Status Scale assessment. The Audio Recorded Cognitive Screen (ARCS) and Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) are easy-to-use tools to assess cognitive function in clinical practice. Objective To compare the sensitivity of ARCS to BICAMS and their relative predictive value for employment status. Methods MS patients and healthy controls were assessed using the ARCS and the BICAMS consecutively. Receiver Operating Characteristic (ROC) curve analyses were used to compare the two tests. A step-wise, logistic regression analysis was used to identify the cognitive test(s) that best predicted employment status and quality of life. Results : Total ARCS, memory and attention domain scores were moderately correlated with all BICAMS tests (r = 0.3–0.5; P ≤ 0.05). Total ARCS predicts cognitive impairment with good sensitivity and specificity relative to the BICAMS tests (AUC = 0.8; P = 0.00045). Total ARCS detects higher levels of impairment than BICAMS in MS patients (44% versus 21%). The memory domain of the ARCS and the BVMT-R were the best predictors of employment status (OR = 1.12 and 1.14, P Conclusion BICAMS and ARCS have comparable sensitivity for cognitive impairment in MS. Memory assessment from either tests is the best predictor of employment status; however, the BICAMS is a better predictor of work productivity

Primary open-angle glaucoma (POAG) is influenced by both genetic and environmental factors. Despite significant progress in identifying genetic variants associated with POAG, there remains a substantial amount of unexplained heritability. Study design features that may enhance knowledge of the genetic architecture include focusing on multiple quantitative traits related to ocular disorders (i.e. endophenotypes), targeting genetic variants that directly influence gene expression (i.e. cis-eQTLs) and utilising genetically isolated populations to reduce genetic and environmental noise and thus enhance association signals. In this study we performed heritability and blood-based eQTL association analysis of five key POAG endophenotypes in 330 individuals from the Norfolk Island (NI) isolate. Results showed evidence of heritability for all five traits, with H2 estimates ranging from 0.35 for intraocular pressure (IOP) to 0.82 for central corneal thickness (CCT) (P

Background Although many genetic variants have been associated with multiple sclerosis (MS) risk, they do not explain all the disease risk and there remains uncertainty as to how these variants contribute to disease. DNA methylation is an epigenetic mechanism that can influence gene expression and has the potential to mediate the effects of environmental factors on MS. In a previous study, we found a differentially methylation region (DMR) at MHC HLA-DRB1 that was associated within relapsing-remitting MS (RRMS) patients in CD4+ T cells. This study aimed to confirm this earlier finding in an independent RRMS cohort of treatment-naïve female patients. Methods Total genomic DNA was extracted from CD4+ T cells of 28 female RRMS and 22 age-matched healthy controls subjects. DNA was bisulfite-converted and hybridised to Illumina 450K arrays. Beta values for all CpGs were analysed using the DMPFinder function in the MINFI program, and a follow-up prioritisation process was applied to identify the most robust MS-associated DMRs. Results This study confirmed our previous findings of a hypomethylated DMR at HLA-DRB1 and a hypermethylated DMR at HLA-DRB5 in this RRMS patient cohort. In addition, we identified a large independent DMR at MHC, whereby 11 CpGs in RNF39 were hypermethylated in MS cases compared to controls (max. ∆beta = 0.19, P = 2.1 × 10−4). We did not find evidence that SNP genotype was influencing the DMR in this cohort. A smaller MHC DMR was also identified at HCG4B, and two non-MHC DMRs at PM20D1 on chr1 and ERICH1 on chr8 were also identified. Conclusions The findings from this study confirm our previous results of a DMR at HLA-DRB1 and also suggest hypermethylation in an independent MHC locus, RNF39, is associated with MS. Taken together, our results highlight the importance of epigenetic factors at the MHC locus in MS independent of treatment, age and sex. Prospective studies are now required to discern whether methylation at MHC is involved in influencing risk of disease onset or whether the disease itself has altered the methylation profile. Electronic supplementary material The online version of this article (doi:10.1186/s13148-017-0371-1) contains supplementary material, which is available to authorized users.

Epigenetic regulation of various genomic functions, including gene expression, provide mechanisms whereby an organism can dynamically respond to changes in its environment and modify gene expression accordingly. One epigenetic mechanism implicated in human aging and age-related disorders is DNA methylation. Isolated populations such as Norfolk Island (NI) should be advantageous for the identification of epigenetic factors related to aging due to reduced genetic and environmental variation. Here we conducted a methylome-wide association study of age using whole blood DNA in 24 healthy female individuals from the NI genetic isolate (aged 24-47 years). We analysed 450K methylation array data using a machine learning approach (GLMnet) to identify age-associated CpGs. We identified 497 CpG sites, mapping to 422 genes, associated with age, with 11 sites previously associated with age. The strongest associations identified were for a single CpG site in MYOF and an extended region within the promoter of DDO. These hits were validated in curated public data from 2316 blood samples (MARMAL-AID). This study is the first to report robust age associations for MYOF and DDO, both of which have plausible functional roles in aging. This study also illustrates the value of genetic isolates to reveal new associations with epigenome-level data.

Background Migraine is a common neurological disorder which affects a large proportion of the population. The Norfolk Island population is a genetically isolated population and is an ideal discovery cohort for genetic variants involved in complex disease susceptibility given the reduced genetic and environmental heterogeneity. Given that the majority of proteins responsible for mitochondrial function are nuclear encoded, this study aimed to investigate the role of Nuclear Encoded Mitochondrial Protein (NEMP) genes in relation to migraine susceptibility. Methods A gene‐centric association analysis of NEMP genes was undertaken in the most related individuals (n = 315) within the genetically isolated Norfolk Island population. The discovery phase included genes with three or more SNP associations (P

The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices.