The immune cell transcriptome is modulated by vitamin D3 supplementation in people with a first demyelinating event participating in a randomized placebo-controlled trial.

Vitamin D deficiency is a risk factor for developing multiple sclerosis. The PrevANZ trial was conducted to determine if vitamin D3 supplementation can prevent recurrent disease activity in people with a first demyelinating event. As a sub-study of this trial, we investigated the effect of supplementation on peripheral immune cell gene expression. Participants were randomized to 1000, 5000 or 10,000 international units daily of vitamin D3 or placebo. Peripheral blood was collected at baseline and 12 weeks and sent for ribonucleic acid sequencing. Datasets from 55 participants were included. Gene expression was modulated by high dose supplementation. Antigen presentation and viral response pathways were upregulated. Oxidative phosphorylation and immune signaling pathways, including tumor necrosis factor-alpha and interleukin-17 signaling, were downregulated. Overall, vitamin D3 supplementation for 12 weeks modulated the peripheral immune cell transcriptome with induction of anti-inflammatory gene expression profiles. Our results support a dose-dependent effect of vitamin D3 supplementation on immune gene expression.

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.

Improvement of the thalamocortical white matter network in people with stable treated relapsing-remitting multiple sclerosis over time.

Advanced imaging techniques (tractography) enable the mapping of white matter (WM) pathways and the understanding of brain connectivity patterns. We combined tractography with a network-based approach to examine WM microstructure on a network level in people with relapsing-remitting multiple sclerosis (pw-RRMS) and healthy controls (HCs) over 2 years. Seventy-six pw-RRMS matched with 43 HCs underwent clinical assessments and 3T MRI scans at baseline (BL) and 2-year follow-up (2-YFU). Probabilistic tractography was performed, accounting for the effect of lesions, producing connectomes of 25 million streamlines. Network differences in fibre density across pw-RRMS and HCs at BL and 2-YFU were quantified using network-based statistics (NBS). Longitudinal network differences in fibre density were quantified using NBS in pw-RRMS, and were tested for correlations with disability, cognition and fatigue scores. Widespread network reductions in fibre density were found in pw-RRMS compared with HCs at BL in cortical regions, with more reductions detected at 2-YFU. Pw-RRMS had reduced fibre density at BL in the thalamocortical network compared to 2-YFU. This effect appeared after correction for age, was robust across different thresholds, and did not correlate with lesion volume or disease duration. Pw-RRMS demonstrated a robust and long-distance improvement in the thalamocortical WM network, regardless of age, disease burden, duration or therapy, suggesting a potential locus of neuroplasticity in MS. This network's role over the disease's lifespan and its potential implications in prognosis and treatment warrants further investigation.

Not all roads lead to the immune system: the genetic basis of multiple sclerosis severity.

Multiple sclerosis is a leading cause of neurological disability in adults. Heterogeneity in multiple sclerosis clinical presentation has posed a major challenge for identifying genetic variants associated with disease outcomes. To overcome this challenge, we used prospectively ascertained clinical outcomes data from the largest international multiple sclerosis registry, MSBase. We assembled a cohort of deeply phenotyped individuals of European ancestry with relapse-onset multiple sclerosis. We used unbiased genome-wide association study and machine learning approaches to assess the genetic contribution to longitudinally defined multiple sclerosis severity phenotypes in 1813 individuals. Our primary analyses did not identify any genetic variants of moderate to large effect sizes that met genome-wide significance thresholds. The strongest signal was associated with rs7289446 (ß = -0.4882, P = 2.73 × 10-7), intronic to SEZ6L on chromosome 22. However, we demonstrate that clinical outcomes in relapse-onset multiple sclerosis are associated with multiple genetic loci of small effect sizes. Using a machine learning approach incorporating over 62 000 variants together with clinical and demographic variables available at multiple sclerosis disease onset, we could predict severity with an area under the receiver operator curve of 0.84 (95% CI 0.79-0.88). Our machine learning algorithm achieved positive predictive value for outcome assignation of 80% and negative predictive value of 88%. This outperformed our machine learning algorithm that contained clinical and demographic variables alone (area under the receiver operator curve 0.54, 95% CI 0.48-0.60). Secondary, sex-stratified analyses identified two genetic loci that met genome-wide significance thresholds. One in females (rs10967273; ßfemale = 0.8289, P = 3.52 × 10-8), the other in males (rs698805; ßmale = -1.5395, P = 4.35 × 10-8), providing some evidence for sex dimorphism in multiple sclerosis severity. Tissue enrichment and pathway analyses identified an overrepresentation of genes expressed in CNS compartments generally, and specifically in the cerebellum (P = 0.023). These involved mitochondrial function, synaptic plasticity, oligodendroglial biology, cellular senescence, calcium and G-protein receptor signalling pathways. We further identified six variants with strong evidence for regulating clinical outcomes, the strongest signal again intronic to SEZ6L (adjusted hazard ratio 0.72, P = 4.85 × 10-4). Here we report a milestone in our progress towards understanding the clinical heterogeneity of multiple sclerosis outcomes, implicating functionally distinct mechanisms to multiple sclerosis risk. Importantly, we demonstrate that machine learning using common single nucleotide variant clusters, together with clinical variables readily available at diagnosis can improve prognostic capabilities at diagnosis, and with further validation has the potential to translate to meaningful clinical practice change.

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.

A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis.

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.

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.

Ongoing increase in incidence and prevalence of multiple sclerosis in Newcastle, Australia: A 50-year study.

BACKGROUND: Since 1959, multiple sclerosis (MS) prevalence has been estimated for the east coast Australian city of Newcastle. Previous surveys, conducted in 1988 and 2003, have described an increase in the prevalence and incidence of MS. OBJECTIVES: In this study, we evaluated whether these trends continue and provide 50 years of MS epidemiological follow-up for this southern hemisphere city. METHODS: Expressed per 100,000 people, prevalence of MS in Newcastle was calculated for those with a confirmed diagnosis of MS on 9 August 2011 and incidence based on the number of cases with MS diagnosis made during the preceding decade. Data were age-standardised to the total Australian population. Statistical comparisons were undertaken using Poisson regression analysis. RESULTS: In 2011, the estimate of MS prevalence was 124.2, with female-to-male ratio reaching 3.1, a 53% increase in female predominance since 1996. MS incidence increased to 6.7, with a significantly higher proportion of new female cases since the previous survey. CONCLUSION: Prevalence of MS in Newcastle has risen linearly and is contributed to by a substantial increase in new cases over the preceding decade. Female predominance of MS cases continues to increase with a new diagnosis three times more likely in women.

Anxiety Levels Are Independently Associated With Cognitive Performance in an Australian Multiple Sclerosis Patient Cohort.

Neurological and psychological symptoms in multiple sclerosis can affect cognitive function. The objective of this study was to explore the relationship between psychological measures and cognitive performance in a patient cohort. In 322 multiple sclerosis patients, psychological symptoms were measured using the Depression Anxiety and Stress Scale, and cognitive function was evaluated using Audio Recorded Cognitive Screen. Multifactor linear regression analysis, accounting for all clinical covariates, found that anxiety was the only psychological measure to remain a significant predictor of cognitive performance (p<0.001), particularly memory function (p<0.001). Further prospective studies are required to determine whether treatment of anxiety improves cognitive impairment.

The effect of 1 mg folic acid supplementation on clinical outcomes in female migraine with aura patients.

BACKGROUND: Migraine is a common neurovascular condition that may be linked to hyperhomocysteinemia. We have previously provided evidence that reduction of homocysteine with a vitamin supplementation can reduce the occurrence of migraine in women. The current study examined the occurrence of migraine in response to vitamin supplementation with a lower dose of folic acid. METHODS: This was a 6 month randomised, double blinded placebo controlled trial of daily vitamin supplementation containing 1 mg of folic acid, 25 mg of Vitamin B6 and Vitamin B12, on reduction of homocysteine and the occurrence of migraine in 300 female patients diagnosed with migraine with aura. RESULTS: Vitamin supplementation with 1 mg of folic acid, did not significantly decrease homocysteine levels (P = 0.2). The treatment group did not show a significant decrease in the percentage of participants with high migraine disability, severity or frequency at the end of the 6 month intervention (P > 0.1). CONCLUSION: 1 mg of folic acid in combination with vitamin B6 and B12 is less effective in reducing migraine associated symptoms compared to the previously tested dosage of 2 mg folic acid in combination with 25 mg of vitamin B6 and 400 µg of vitamin B12.

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.