PathVar: A Customisable NGS Variant Calling Algorithm Implicates Novel Candidate Genes and Pathways in Hemiplegic Migraine.

The exponential growth of next-generation sequencing (NGS) data requires innovative bioinformatics approaches to unravel the genetic underpinnings of diseases. Hemiplegic migraine (HM), a debilitating neurological disorder with a genetic basis, is one such condition that warrants further investigation. Notably, the genetic heterogeneity of HM is underscored by the fact that approximately two-thirds of patients lack pathogenic variants in the known causal ion channel genes. In this context, we have developed PathVar, a novel bioinformatics algorithm that harnesses publicly available tools and software for pathogenic variant discovery in NGS data. PathVar integrates a suite of tools, including HaplotypeCaller from the Genome Analysis Toolkit (GATK) for variant calling, Variant Effect Predictor (VEP) and ANNOVAR for variant annotation, and TAPES for assigning the American College of Medical Genetics and Genomics (ACMG) pathogenicity labels. Applying PathVar to whole exome sequencing data from 184 HM patients, we detected 648 variants that are probably pathogenic in multiple patients. Moreover, we have identified several candidate genes for HM, many of which cluster around the Rho GTPases pathway. Future research can leverage PathVar to generate high quality, candidate pathogenic variants, which may enhance our understanding of HM and other complex diseases.

Exploring the Functional Basis of Epigenetic Aging in Relation to Body Fat Phenotypes in the Norfolk Island Cohort.

DNA methylation is an epigenetic factor that is modifiable and can change over a lifespan. While many studies have identified methylation sites (CpGs) related to aging, the relationship of these to gene function and age-related disease phenotypes remains unclear. This research explores this question by testing for the conjoint association of age-related CpGs with gene expression and the relation of these to body fat phenotypes. The study included blood-based gene transcripts and intragenic CpG methylation data from Illumina 450 K arrays in 74 healthy adults from the Norfolk Island population. First, a series of regression analyses were performed to detect associations between gene transcript level and intragenic CpGs and their conjoint relationship with age. Second, we explored how these age-related expression CpGs (eCpGs) correlated with obesity-related phenotypes, including body fat percentage, body mass index, and waist-to-hip ratio. We identified 35 age-related eCpGs associated with age. Of these, ten eCpGs were associated with at least one body fat phenotype. Collagen Type XI Alpha 2 Chain (COL11A2), Complement C1s (C1s), and four and a half LIM domains 2 (FHL2) genes were among the most significant genes with multiple eCpGs associated with both age and multiple body fat phenotypes. The COL11A2 gene contributes to the correct assembly of the extracellular matrix in maintaining the healthy structural arrangement of various components, with the C1s gene part of complement systems functioning in inflammation. Moreover, FHL2 expression was upregulated under hypermethylation in both blood and adipose tissue with aging. These results suggest new targets for future studies and require further validation to confirm the specific function of these genes on body fat regulation.

Exonic mutations in cell-cell adhesion may contribute to CADASIL-related CSVD pathology.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a condition caused by mutations in NOTCH3 and results in a phenotype characterised by recurrent strokes, vascular dementia and migraines. Whilst a genetic basis for the disease is known, the molecular mechanisms underpinning the pathology of CADASIL are still yet to be determined. Studies conducted at the Genomics Research Centre (GRC) have also identified that only 15-23% of individuals clinically suspected of CADASIL have mutations in NOTCH3. Based on this, whole exome sequencing was used to identify novel genetic variants for CADASIL-like cerebral small-vessel disease (CSVD). Analysis of functionally important variants in 50 individuals was investigated using overrepresentation tests in Gene ontology software to identify biological processes that are potentially affected in this group of patients. Further investigation of the genes in these processes was completed using the TRAPD software to identify if there is an increased number (burden) of mutations that are associated with CADASIL-like pathology. Results from this study identified that cell-cell adhesion genes were positively overrepresented in the PANTHER GO-slim database. TRAPD burden testing identified n = 15 genes that had a higher number of rare (MAF < 0.001) and predicted functionally relevant (SIFT < 0.05, PolyPhen > 0.8) mutations compared to the gnomAD v2.1.1 exome control dataset. Furthermore, these results identified ARVCF, GPR17, PTPRS, and CELSR1 as novel candidate genes in CADASIL-related pathology. This study identified a novel process that may be playing a role in the vascular damage related to CADASIL-related CSVD and implicated n = 15 genes in playing a role in the disease.

DNA Methylation Signatures of Multiple Sclerosis Occur Independently of Known Genetic Risk and Are Primarily Attributed to B Cells and Monocytes.

Epigenetic mechanisms can regulate how DNA is expressed independently of sequence and are known to be associated with various diseases. Among those epigenetic mechanisms, DNA methylation (DNAm) is influenced by genotype and the environment, making it an important molecular interface for studying disease etiology and progression. In this study, we examined the whole blood DNA methylation profiles of a large group of people with (pw) multiple sclerosis (MS) compared to those of controls. We reveal that methylation differences in pwMS occur independently of known genetic risk loci and show that they more strongly differentiate disease (AUC = 0.85, 95% CI 0.82-0.89, p = 1.22 × 10-29) than known genetic risk loci (AUC = 0.72, 95% CI: 0.66-0.76, p = 9.07 × 10-17). We also 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.

Oligofructose-Enriched Inulin Intake, Gut Microbiome Characteristics, and the V̇O2 Peak Response to High-Intensity Interval Training in Healthy Inactive Adults.

BACKGROUND: The gut microbiome has been associated with cardiorespiratory fitness. OBJECTIVES: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Genome wide association study of response to interval and continuous exercise training: the Predict-HIIT study.

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 < 1 × 10-5) 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 < 1 × 10-5). 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 < 1 × 10-4) and the validation study (P < × 10-6), 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 .

A genome-wide methylation study of body fat traits in the Norfolk Island isolate.

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 such as reduced genetic diversity and environmental exposures. METHODS AND RESULTS: 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. 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 × 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 × 10-4) implicated the Cadherin (28 genes, Padj = 6.76 × 10-7) and Wnt signaling pathways (38 genes, Padj = 7.78 × 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.

Expression QTL analysis of glaucoma endophenotypes in the Norfolk Island isolate provides evidence that immune-related genes are associated with optic disc size.

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 < 0.05). The primary finding was for BTN3A2, whereby both cis-SNP and transcript were significantly associated with disc size within a conditional regression model. Specifically, this model included rs853676 (ß = 0.23,P = 0.008) and transcript (ß = 0.23, P = 0.03). We also observed a cis-SNP association between optic disc size and LPCAT2 independent of transcript (P = 0.0004). These genes have specific functions in immune system pathways and suggest a role for an inherited immune component of POAG risk. This study also demonstrates an alternate approach to understanding the functional genetic basis of POAG and ocular health more generally.

Australian driving restrictions: how well do neurologists know them?

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.

A Phenomic Scan of the Norfolk Island Genetic Isolate Identifies a Major Pleiotropic Effect Locus Associated with Metabolic and Renal Disorder Markers.

Multiphenotype genome-wide association studies (GWAS) may reveal pleiotropic genes, which would remain undetected using single phenotype analyses. Analysis of large pedigrees offers the added advantage of more accurately assessing trait heritability, which can help prioritise genetically influenced phenotypes for GWAS analysis. In this study we performed a principal component analysis (PCA), heritability (h2) estimation and pedigree-based GWAS of 37 cardiovascular disease -related phenotypes in 330 related individuals forming a large pedigree from the Norfolk Island genetic isolate. PCA revealed 13 components explaining >75% of the total variance. Nine components yielded statistically significant h2 values ranging from 0.22 to 0.54 (P<0.05). The most heritable component was loaded with 7 phenotypic measures reflecting metabolic and renal dysfunction. A GWAS of this composite phenotype revealed statistically significant associations for 3 adjacent SNPs on chromosome 1p22.2 (P<1x10-8). These SNPs form a 42kb haplotype block and explain 11% of the genetic variance for this renal function phenotype. Replication analysis of the tagging SNP (rs1396315) in an independent US cohort supports the association (P = 0.000011). Blood transcript analysis showed 35 genes were associated with rs1396315 (P<0.05). Gene set enrichment analysis of these genes revealed the most enriched pathway was purine metabolism (P = 0.0015). Overall, our findings provide convincing evidence for a major pleiotropic effect locus on chromosome 1p22.2 influencing risk of renal dysfunction via purine metabolism pathways in the Norfolk Island population. Further studies are now warranted to interrogate the functional relevance of this locus in terms of renal pathology and cardiovascular disease risk.

The NRP1 migraine risk variant shows evidence of association with menstrual migraine.

BACKGROUND: In 2016, a large meta-analysis brought the number of susceptibility loci for migraine to 38. While sub-type analysis for migraine without aura (MO) and migraine with aura (MA) found some loci showed specificity to MO, the study did not test the loci with respect to other subtypes of migraine. This study aimed to test the hypothesis that single nucleotide polymorphisms (SNPs) robustly associated with migraine are individually or collectively associated with menstrual migraine (MM). METHODS: Genotyping of migraine susceptibility SNPs was conducted using the Agena MassARRAY platform on DNA samples from 235 women diagnosed with menstrual migraine as per International Classification for Headache Disorders II (ICHD-II) criteria and 140 controls. Alternative genotyping methods including restriction fragment length polymorphism, pyrosequencing and Sanger sequencing were used for validation. Statistical analysis was performed using PLINK and SPSS. RESULTS: Genotypes of 34 SNPs were obtained and investigated for their potential association with menstrual migraine. Of these SNPs, rs2506142 located near the neuropilin 1 gene (NRP1), was found to be significantly associated with menstrual migraine (p = 0.003). Genomic risk scores were calculated for all 34 SNPs as well as a subset of 7 SNPs that were nearing individual significance. Overall, this analysis suggested these SNPs to be weakly predictive of MM, but of no prognostic or diagnostic value. CONCLUSIONS: Our results suggest that NRP1 may be important in the etiology of MM. It also suggests some genetic commonality between common migraine subtypes (MA and MO) and MM. The identification of associated SNPs may be the starting point to a better understanding of how genetic factors may contribute to the menstrual migraine sub-type.

Genome-wide linkage and association analysis of primary open-angle glaucoma endophenotypes in the Norfolk Island isolate.

PURPOSE: Primary open-angle glaucoma (POAG) refers to a group of heterogeneous diseases involving optic nerve damage. Two well-established risk factors for POAG are elevated intraocular pressure (IOP) and a thinner central corneal thickness (CCT). These endophenotypes exhibit a high degree of heritability across populations. Large-scale genome-wide association studies (GWASs) of outbred populations have robustly implicated several susceptibility gene variants for both IOP and CCT. Despite this progress, a substantial amount of genetic variance remains unexplained. Population-specific variants that might be rare in outbred populations may also influence POAG endophenotypes. The Norfolk Island population is a founder-effect genetic isolate that has been well characterized for POAG endophenotypes. This population is therefore a suitable candidate for mapping new variants that influence these complex traits. METHODS: Three hundred and thirty participants from the Norfolk Island Eye Study (NIES) core pedigree provided DNA. Ocular measurements of CCT and IOP were also taken for analysis. Heritability analyses and genome-wide linkage analyses of short tandem repeats (STRs) were conducted using SOLAR. Pedigree-based GWASs of single-nucleotide polymorphisms (SNPs) were performed using the GenABEL software. RESULTS: CCT was the most heritable endophenotype in this cohort (h2 = 0.77, p = 6×10-6), while IOP showed a heritability of 0.39 (p = 0.008). A genome-wide linkage analysis of these POAG phenotypes identified a maximum logarithm of the odds (LOD) score of 1.9 for CCT on chromosome 20 (p = 0.0016) and 1.3 for IOP on chromosome 15 (p = 0.0072). The GWAS results revealed a study-wise significant association for IOP at rs790357, which is located within DLG2 on chr11q14.1 (p = 1.02×10-7). DLG2 is involved in neuronal signaling and development, and while it has not previously been associated with IOP, it has been associated with myopia. An analysis of 12 known SNPs for IOP showed that rs12419342 in RAPSN on chromosome 11 was nominally associated in Norfolk Island (NI; p = 0.0021). For CCT, an analysis of 26 known SNPs showed rs9938149 in BANP-ZNF469 on chromosome 16 was nominally associated in NI (p = 0.002). CONCLUSIONS: These study results indicate that CCT and IOP exhibit a substantial degree of heritability in the NI pedigree, indicating a genetic component. A genome-wide linkage analysis of POAG endophenotypes did not reveal any major effect loci, but the GWASs did implicate several known loci, as well as a potential new locus in DLG2, suggesting a role for neuronal signaling in development in IOP and perhaps POAG. These results also highlight the need to target rarer variants via whole genome sequencing in this genetic isolate.

Genetic association analysis of miRNA SNPs implicates MIR145 in breast cancer susceptibility.

BACKGROUND: MicroRNAs (miRNAs) are important small non-coding RNA molecules that regulate gene expression in cellular processes related to the pathogenesis of cancer. Genetic variation in miRNA genes could impact their synthesis and cellular effects and single nucleotide polymorphisms (SNPs) are one example of genetic variants studied in relation to breast cancer. Studies aimed at identifying miRNA SNPs (miR-SNPs) associated with breast malignancies could lead towards further understanding of the disease and to develop clinical applications for early diagnosis and treatment. METHODS: We genotyped a panel of 24 miR-SNPs using multiplex PCR and chip-based matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis in two Caucasian breast cancer case control populations (Primary population: 173 cases and 187 controls and secondary population: 679 cases and 301 controls). Association to breast cancer susceptibility was determined using chi-square (X (2) ) and odds ratio (OR) analysis. RESULTS: Statistical analysis showed six miR-SNPs to be non-polymorphic and twelve of our selected miR-SNPs to have no association with breast cancer risk. However, we were able to show association between rs353291 (located in MIR145) and the risk of developing breast cancer in two independent case control cohorts (p = 0.041 and p = 0.023). CONCLUSIONS: Our study is the first to report an association between a miR-SNP in MIR145 and breast cancer risk in individuals of Caucasian background. This finding requires further validation through genotyping of larger cohorts or in individuals of different ethnicities to determine the potential significance of this finding as well as studies aimed to determine functional significance.

Association of microRNA 17-92 cluster host gene (MIR17HG) polymorphisms with breast cancer.

Breast cancer incidence and mortality rates are increasing despite our current knowledge on the disease. Ninety-five percent of breast cancer cases correspond to sporadic forms of the disease and are believed to involve an interaction between environmental and genetic determinants. The microRNA 17-92 cluster host gene (MIR17HG) has been shown to regulate expression of genes involved in breast cancer development and progression. Study of single-nucleotide polymorphisms (SNPs) located in this cluster gene could help provide a further understanding of its role in breast cancer. Therefore, this study investigated six SNPs in the MIR17HG using two independent Australian Caucasian case-control populations (GRC-BC and GU-CCQ BB populations) to determine association to breast cancer susceptibility. Genotyping was undertaken using chip-based matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS). We found significant association between rs4824505 and breast cancer at the allelic level in both study cohorts (GRC-BC p = 0.01 and GU-CCQ BB p = 0.03). Furthermore, haplotypic analysis of results from our combined population determined a significant association between rs4824505/rs7336610 and breast cancer susceptibility (p = 5 × 10(-4)). Our study is the first to show that the A allele of rs4824505 and the AC haplotype of rs4824505/rs7336610 are associated with risk of breast cancer development. However, definitive validation of this finding requires larger cohorts or populations in different ethnical backgrounds. Finally, functional studies of these SNPs could provide a deeper understanding of the role that MIR17HG plays in the pathophysiology of breast cancer.

Effects of dietary folate intake on migraine disability and frequency.

BACKGROUND: Migraine is a highly disabling disease affecting a significant proportion of the Australian population. The methylenetetrahydrofolate reductase (MTHFR) C677T variant has been associated with increased levels of homocysteine and risk of migraine with aura (MA). Folic acid (FA), vitamin B6 , and B12 supplementation has been previously shown to reduce increased levels of homocysteine and decrease migraine symptoms. However, the influence of dietary folate intake on migraine has been unclear. The aim of the current study was to analyze the association of dietary folate intake in the form of dietary folate equivalent, FA, and total food folate (TFF) on migraine frequency, severity, and disability. METHODS: A cohort of 141 adult females of Caucasian descent with MA was genotyped for the MTHFR C677T variant using restriction enzyme digestion. Dietary folate information was collected from all participants and analyzed using the "FoodWorks" 2009 package. Folate consumption was compared with migraine frequency, severity, and disability using linear regression. RESULTS: A significant inverse relation was observed between dietary folate equivalent (R(2) = 0.201, B = -0.002, P = .045, 95% confidence interval [CI] [-0.004, -0.001]) and FA (R(2) = 0.255, B = -0.005, P = .036, 95% CI [-0.009, -0.002]) consumption and migraine frequency. It was also observed that in individuals with the CC genotype for the MTHFR C677T variant, migraine frequency was significantly linked to FA consumption (R(2) = 0.106, B = -0.004, P = .029, 95% CI [-0.007, -0.004]). CONCLUSIONS: The results from this study indicate that folate intake in the form of FA may influence migraine frequency in female MA sufferers.

Protocol for assessing maternal, environmental and epigenetic risk factors for dental caries in children.

BACKGROUND: Expenditure on dental and oral health services in Australia is $3.4 billion AUD annually. This is the sixth highest health cost and accounts for 7 % of total national health expenditure. Approximately 49 % of Australian children aged 6 years have caries experience in their deciduous teeth and this is rising. The aetiology of dental caries involves a complex interplay of individual, behavioural, social, economic, political and environmental conditions, and there is increasing interest in genetic predisposition and epigenetic modification. METHODS: The Oral Health Sub-study; a cross sectional study of a birth cohort began in November 2012 by examining mothers and their children who were six years old by the time of initiation of the study, which is ongoing. Data from detailed questionnaires of families from birth onwards and data on mothers' knowledge, attitudes and practices towards oral health collected at the time of clinical examination are used. Subjects' height, weight and mid-waist circumference are taken and Body Mass Index (BMI) computed, using an electronic Bio-Impedance balance. Dental caries experience is scored using the International Caries Detection and Assessment System (ICDAS). Saliva is collected for physiological measures. Salivary Deoxyribose Nucleic Acid (DNA) is extracted for genetic studies including epigenetics using the SeqCap Epi Enrichment Kit. Targets of interest are being confirmed by pyrosequencing to identify potential epigenetic markers of caries risk. DISCUSSION: This study will examine a wide range of potential determinants for childhood dental caries and evaluate inter-relationships amongst them. The findings will provide an evidence base to plan and implement improved preventive strategies.

Reduced expression of α-synuclein in alcoholic brain: influence of SNCA-Rep1 genotype.

α-Synuclein has recently been implicated in the pathophysiology of alcohol abuse due to its role in dopaminergic neurotransmission. In these studies, genetic variability in the α-synuclein gene influences its expression which may contribute to susceptibility to chronic alcohol abuse. Real-time PCR was used to quantify α-synuclein mRNA expression in autopsy samples of human dorsolateral prefrontal cortex. Because of the association between length of the α-synuclein-repeat 1 microsatellite marker and expression levels of the gene, this marker was genotyped in a Caucasian sample of 126 controls and 117 alcoholics using capillary gel electrophoresis. The allele and genotype frequencies of α-synuclein-repeat 1 marker differed significantly between alcoholics and controls. Alcoholics had greater frequencies of the shortest allele found (267 bp). The shortest allele of the α-synuclein-repeat 1 marker was associated with decreased expression of α-synuclein in prefrontal cortex. Individuals with at least one copy of the 267 bp allele were more likely to exhibit an alcohol abuse phenotype. These results suggest that individuals with the 267 bp allele may be at increased risk of developing alcoholism and that genetic variation at the α-synuclein-repeat 1 locus may influence α-synuclein expression in the prefrontal cortex.

A longitudinal analysis of brain volume changes in myelin oligodendrocyte glycoprotein antibody-associated disease.

BACKGROUND AND PURPOSE: Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is a relapsing demyelinating condition. There are several cross-sectional studies showing evidence of brain atrophy in people with MOGAD (pwMOGAD), but longitudinal brain volumetric assessment is still an unmet need. Current recommendations do not include monitoring with MRI and assume distinct attacks. Evidence of ongoing axon loss will have diagnostic and therapeutic implications. In this study, we assessed brain volume changes in pwMOGAD over a mean follow-up period of 2 years and compared this to changes in people with multiple sclerosis (pwMS). METHODS: This is a retrospective single-center study over a 7-year period from 2014 to 2021. MRI brain scans at the time of diagnosis and follow-up in remission were collected from 14 Caucasian pwMOGAD, confirmed by serum myelin oligodendrocyte glycoprotein immunoglobulin G antibody presence, detected by live cell-based assays. Total brain volume (TBV), white matter (WM), gray matter (GM), and demyelinating lesion volumes were assessed automatically using the Statistical Parametric Mapping and FMRIB automated segmentation tools. MRI brain scans at diagnosis and follow-up on remission were collected from 32-matched pwMS for comparison. Statistical analysis was done using analysis of variance. RESULTS: There is evidence of TBV loss, affecting particularly GM, over an approximately 2-year follow-up period in pwMOGAD (p < .05), comparable to pwMS. WM and lesion volume change over the same period were not statistically significant (p > .1). CONCLUSION: We found evidence of loss of GM and TBV over time  in pwMOGAD, similar to pwMS, although the WM and lesion volumes were unchanged.

HLA-DRB1*15:01 and the MERTK Gene Interact to Selectively Influence the Profile of MERTK-Expressing Monocytes in Both Health and MS.

BACKGROUND AND OBJECTIVES: HLA-DRB1*15:01 (DR15) and MERTK are 2 risk genes for multiple sclerosis (MS). The variant rs7422195 is an expression quantitative trait locus for MERTK in CD14+ monocytes; cells with phagocytic and immunomodulatory potential. We aimed to understand how drivers of disease risk and pathogenesis vary with HLA and MERTK genotype and disease activity. METHODS: We investigated how proportions of monocytes vary with HLA and MERTK genotype and disease activity in MS. CD14+ monocytes were isolated from patients with MS at relapse (n = 40) and 3 months later (n = 23). Healthy controls (HCs) underwent 2 blood collections 3 months apart. Immunophenotypic profiling of monocytes was performed by flow cytometry. Methylation of 35 CpG sites within and near the MERTK gene was assessed in whole blood samples of individuals experiencing their first episode of clinical CNS demyelination (n = 204) and matched HCs (n = 345) using an Illumina EPIC array. RESULTS: DR15-positive patients had lower proportions of CD14+ MERTK+ monocytes than DR15-negative patients, independent of genotype at the MERTK SNP rs7422195. Proportions of CD14+ MERTK+ monocytes were further reduced during relapse in DR15-positive but not DR15-negative patients. Patients homozygous for the major G allele at rs7422195 exhibited higher proportions of CD14+ MERTK+ monocytes at both relapse and remission compared with controls. We observed that increased methylation of the MERTK gene was significantly associated with the presence of DR15. DISCUSSION: DR15 and MERTK genotype independently influence proportions of CD14+ MERTK+ monocytes in MS. We confirmed previous observations that the MERTK risk SNP rs7422195 is associated with altered MERTK expression in monocytes. We identified that expression of MERTK is stratified by disease in people homozygous for the major G allele of rs7422195. The finding that the proportion of CD14+ MERTK+ monocytes is reduced in DR15-positive individuals supports prior data identifying genetic links between these 2 loci in influencing MS risk. DR15 genotype-dependent alterations in methylation of the MERTK gene provides a molecular link between these loci and identifies a potential mechanism by which MERTK expression is influenced by DR15. This links DR15 haplotype to MS susceptibility beyond direct influence on antigen presentation and suggests the need for HLA-based stratification of approaches to MERTK as a therapeutic target.

Association of oestrogen-receptor gene (ESR1) polymorphisms with migraine in the large Norfolk Island pedigree.

BACKGROUND: Oestrogen receptor 1 ( ESR1) is located in region 6q25.1 and encodes a ligand-activated transcription factor composed of several domains important for hormone binding and transcription activation. Progesterone receptor ( PGR) is located in 11q22-23 and mediates the role of progesterone interacting with different transcriptional co-regulators. ESR1 and PGR have previously been implicated in migraine susceptibility. Here, we report the results of an association study of these genes in a migraine pedigree from the genetic isolate of Norfolk Island, a population descended from a small number of Isle of Man "Bounty Mutineer" and Tahitian founders. METHODS: A significant number of molecular markers in the ESR1 (143) and PGR (43) genes were evaluated in a sample of 285 related individuals (135 males; 150 females). A pedigree-based analysis in the GenABEL package was used to analyse the results. RESULTS AND CONCLUSIONS: A total of 10 markers in the ESR1 gene showed association with migraine ( P < 0.05) in the Norfolk Island population. No association was detected with PGR . Three haplotypes in ESR1 were found to be associated with migraine ( P = 0.004, 0.03, 0.005). Future genetic studies in larger populations and expression analysis are required to clarify the role of ESR1 in migraine susceptibility.