The association between disability progression, relapses, and treatment in early relapse onset MS: an observational, multi-centre, longitudinal cohort study.

The indirect contribution of multiple sclerosis (MS) relapses to disability worsening outcomes, and vice-versa, remains unclear. Disease modifying therapies (DMTs) are potential modulators of this association. Understanding how these endo-phenotypes interact may provide insights into disease pathogenesis and treatment practice in relapse-onset MS (ROMS). Utilising a unique, prospectively collected clinical data from a longitudinal cohort of 279 first demyelinating event cases followed for up to 15 years post-onset, we examined indirect associations between relapses and treatment and the risk of disability worsening, and vice-versa. Indirect association parameters were estimated using joint models for longitudinal and survival data. Early relapses within 2.5 years of MS onset predicted early disability worsening outcomes (HR = 3.45, C.I 2.29-3.61) per relapse, but did not contribute to long-term disability worsening thereinafter (HR = 0.21, C.I 0.15-0.28). Conversely, disability worsening outcomes significantly contributed to relapse risk each year (HR = 2.96, C.I 2.91-3.02), and persisted over time (HR = 3.34, C.I 2.90-3.86), regardless of DMT treatments. The duration of DMTs significantly reduced the hazards of relapses (1st-line DMTs: HR = 0.68, C.I 0.58-0.79; 3rd-line DMTs: HR = 0.37, C.I 0.32-0.44) and disability worsening events (1st-line DMTs: HR = 0.74, C.I 0.69-0.79; 3rd-line DMTs: HR = 0.90, C.I 0.85-0.95), respectively. Results from time-dynamic survival probabilities further revealed individuals having higher risk of future relapses and disability worsening outcomes, respectively. The study provided evidence that in ROMS, relapses accrued within 2.5 years of MS onset are strong indicators of disability worsening outcomes, but late relapses accrued 2.5 years post onset are not overt risk factors for further disability worsening. In contrast, disability worsening outcomes are strong positive predictors of current and subsequent relapse risk. Long-term DMT use and older age strongly influence the individual outcomes and their associations.

Novel plasma and brain proteins that are implicated in multiple sclerosis.

Understanding how variations in the plasma and brain proteome contribute to multiple sclerosis susceptibility can provide important insights to guide drug repurposing and therapeutic development for the disease. However, the role of genetically predicted protein abundance in multiple sclerosis remains largely unknown. Integrating plasma proteomics (n = 3301) and brain proteomics (n = 376 discovery; n = 152 replication) into multiple sclerosis genome-wide association studies (n = 14 802 cases and 26 703 controls), we employed summary-based methods to identify candidate proteins involved in multiple sclerosis susceptibility. Next, we evaluated associations of the corresponding genes with multiple sclerosis at tissue-level using large gene expression quantitative trait data from whole-blood (n = 31 684) and brain (n = 1194) tissue. Further, to assess transcriptional profiles for candidate proteins at cell-level, we examined gene expression patterns in immune cell types (Dataset 1: n = 73 cases and 97 controls; Dataset 2: n = 31 cases and 31 controls) for identified plasma proteins, and in brain cell types (Dataset 1: n = 4 cases and 5 controls; Dataset 2: n = 5 cases and 3 controls) for identified brain proteins. In a longitudinal multiple sclerosis cohort (n = 203 cases followed up to 15 years), we also assessed the corresponding gene-level associations with the outcome of disability worsening. We identified 39 novel proteins associated with multiple sclerosis risk. Based on five identified plasma proteins, four available corresponding gene candidates showed consistent associations with multiple sclerosis risk in whole-blood, and we found TAPBPL upregulation in multiple sclerosis B cells, CD8+ T cells and natural killer cells compared with controls. Among the 34 candidate brain proteins, 18 were replicated in a smaller cohort and 14 of 21 available corresponding gene candidates also showed consistent associations with multiple sclerosis risk in brain tissue. In cell-specific analysis, six identified brain candidates showed consistent differential gene expression in neuron and oligodendrocyte cell clusters. Based on the 39 protein-coding genes, we found 23 genes that were associated with disability worsening in multiple sclerosis cases. The findings present a set of candidate protein biomarkers for multiple sclerosis, reinforced by high concordance in downstream transcriptomics findings at tissue-level. This study also highlights the heterogeneity of cell-specific transcriptional profiles for the identified proteins and that numerous candidates were also implicated in disease progression. Together, these findings can serve as an important anchor for future studies of disease mechanisms and therapeutic development.

Ensemble machine learning identifies genetic loci associated with future worsening of disability in people with multiple sclerosis.

Limited studies have been conducted to identify and validate multiple sclerosis (MS) genetic loci associated with disability progression. We aimed to identify MS genetic loci associated with worsening of disability over time, and to develop and validate ensemble genetic learning model(s) to identify people with MS (PwMS) at risk of future worsening. We examined associations of 208 previously established MS genetic loci with the risk of worsening of disability; we learned ensemble genetic decision rules and validated the predictions in an external dataset. We found 7 genetic loci (rs7731626: HR 0.92, P = 2.4 × 10-5; rs12211604: HR 1.16, P = 3.2 × 10-7; rs55858457: HR 0.93, P = 3.7 × 10-7; rs10271373: HR 0.90, P = 1.1 × 10-7; rs11256593: HR 1.13, P = 5.1 × 10-57; rs12588969: HR = 1.10, P = 2.1 × 10-10; rs1465697: HR 1.09, P = 1.7 × 10-128) associated with risk worsening of disability; most of which were located near or tagged to 13 genomic regions enriched in peptide hormones and steroids biosynthesis pathways by positional and eQTL mapping. The derived ensembles produced a set of genetic decision rules that can be translated to provide additional prognostic values to existing clinical predictions, with the additional benefit of incorporating relevant genetic information into clinical decision making for PwMS. The present study extends our knowledge of MS progression genetics and provides the basis of future studies regarding the functional significance of the identified loci.

Pathogenic genetic variants identified in Australian families with paediatric cataract.

OBJECTIVE: Paediatric (childhood or congenital) cataract is an opacification of the normally clear lens of the eye and has a genetic basis in at least 18% of cases in Australia. This study aimed to replicate clinical gene screening to identify variants likely to be causative of disease in an Australian patient cohort. METHODS AND ANALYSIS: Sixty-three reported isolated cataract genes were screened for rare coding variants in 37 Australian families using genome sequencing. RESULTS: Disease-causing variants were confirmed in eight families with variant classification as 'likely pathogenic'. This included novel variants PITX3 p.(Ter303LeuextTer100), BFSP1 p.(Glu375GlyfsTer2), and GJA8 p.(Pro189Ser), as well as, previously described variants identified in genes GJA3, GJA8, CRYAA, BFSP1, PITX3, COL4A1 and HSF4. Additionally, eight variants of uncertain significance with evidence towards pathogenicity were identified in genes: GJA3, GJA8, LEMD2, PRX, CRYBB1, BFSP2, and MIP. CONCLUSION: These findings expand the genotype-phenotype correlations of both pathogenic and benign variation in cataract-associated genes. They further emphasise the need to develop additional evidence such as functional assays and variant classification criteria specific to paediatric cataract genes to improve interpretation of variants and molecular diagnosis in patients.

Generation and characterisation of four multiple sclerosis iPSC lines from a single family.

Multiple sclerosis (MS) is a complex neuroinflammatory/degenerative disease of the central nervous system (CNS) that results in the formation of demyelinated lesions and axon degeneration. MS aetiology is complex, with genetics estimated to account for ∼48% of MS risk (International Multiple Sclerosis Genetics Consortium, 2019). Despite this, families with a high incidence of MS are rare. We have generated four induced pluripotent stem cell (iPSC) lines from individuals with relapsing-remitting and secondary progressive MS within a single family. The generation of disease-specific iPSC lines from multiple members of a single family will facilitate MS genetic and functional studies.

Developing a clinical-environmental-genotypic prognostic index for relapsing-onset multiple sclerosis and clinically isolated syndrome.

Our inability to reliably predict disease outcomes in multiple sclerosis remains an issue for clinicians and clinical trialists. This study aims to create, from available clinical, genetic and environmental factors; a clinical-environmental-genotypic prognostic index to predict the probability of new relapses and disability worsening. The analyses cohort included prospectively assessed multiple sclerosis cases (N = 253) with 2858 repeated observations measured over 10 years. N = 219 had been diagnosed as relapsing-onset, while N = 34 remained as clinically isolated syndrome by the 10th-year review. Genotype data were available for 199 genetic variants associated with multiple sclerosis risk. Penalized Cox regression models were used to select potential genetic variants and predict risk for relapses and/or worsening of disability. Multivariable Cox regression models with backward elimination were then used to construct clinical-environmental, genetic and clinical-environmental-genotypic prognostic index, respectively. Robust time-course predictions were obtained by Landmarking. To validate our models, Weibull calibration models were used, and the Chi-square statistics, Harrell's C-index and pseudo-R 2 were used to compare models. The predictive performance at diagnosis was evaluated using the Kullback-Leibler and Brier (dynamic) prediction error (reduction) curves. The combined index (clinical-environmental-genotypic) predicted a quadratic time-dynamic disease course in terms of worsening (HR = 2.74, CI: 2.00-3.76; pseudo-R 2=0.64; C-index = 0.76), relapses (HR = 2.16, CI: 1.74-2.68; pseudo-R 2 = 0.91; C-index = 0.85), or both (HR = 3.32, CI: 1.88-5.86; pseudo-R 2 = 0.72; C-index = 0.77). The Kullback-Leibler and Brier curves suggested that for short-term prognosis (≤5 years from diagnosis), the clinical-environmental components of disease were more relevant, whereas the genetic components reduced the prediction errors only in the long-term (≥5 years from diagnosis). The combined components performed slightly better than the individual ones, although their prognostic sensitivities were largely modulated by the clinical-environmental components. We have created a clinical-environmental-genotypic prognostic index using relevant clinical, environmental, and genetic predictors, and obtained robust dynamic predictions for the probability of developing new relapses and worsening of symptoms in multiple sclerosis. Our prognostic index provides reliable information that is relevant for long-term prognostication and may be used as a selection criterion and risk stratification tool for clinical trials. Further work to investigate component interactions is required and to validate the index in independent data sets.

Generation of MNZTASi001-A, a human pluripotent stem cell line from a person with primary progressive multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune and neurodegenerative disease that results in immune cell infiltration of the central nervous system (CNS) and demyelination in young adults. Substantial progress has been made in developing disease modifying therapies for people with relapsing-remitting MS, but options remain limited for people with primary progressive MS (PPMS). PPMS accounts for ∼15% of MS diagnoses. Herein, we generated a human induced pluripotent stem cell line (hiPSC) from a person with clinically definite PPMS. This disease-specific hiPSC line will be useful for studying PPMS in vitro, allowing the generation of immune and CNS cell types.

A 127 kb truncating deletion of PGRMC1 is a novel cause of X-linked isolated paediatric cataract.

Inherited paediatric cataract is a rare Mendelian disease that results in visual impairment or blindness due to a clouding of the eye's crystalline lens. Here we report an Australian family with isolated paediatric cataract, which we had previously mapped to Xq24. Linkage at Xq24-25 (LOD = 2.53) was confirmed, and the region refined with a denser marker map. In addition, two autosomal regions with suggestive evidence of linkage were observed. A segregating 127 kb deletion (chrX:g.118373226_118500408del) in the Xq24-25 linkage region was identified from whole-genome sequencing data. This deletion completely removed a commonly deleted long non-coding RNA gene LOC101928336 and truncated the protein coding progesterone receptor membrane component 1 (PGRMC1) gene following exon 1. A literature search revealed a report of two unrelated males with non-syndromic intellectual disability, as well as congenital cataract, who had contiguous gene deletions that accounted for their intellectual disability but also disrupted the PGRMC1 gene. A morpholino-induced pgrmc1 knockdown in a zebrafish model produced significant cataract formation, supporting a role for PGRMC1 in lens development and cataract formation. We hypothesise that the loss of PGRMC1 causes cataract through disrupted PGRMC1-CYP51A1 protein-protein interactions and altered cholesterol biosynthesis. The cause of paediatric cataract in this family is the truncating deletion of PGRMC1, which we report as a novel cataract gene.

Utilising multi-large omics data to elucidate biological mechanisms within multiple sclerosis genetic susceptibility loci.

BACKGROUND: Genome-wide association studies (GWAS) have succeeded in identifying over 200 susceptibility loci for multiple sclerosis (MS). However, the potential functional variants and the mechanisms by which these loci affect MS risk remain largely unexplained. OBJECTIVES: We used summary data-based Mendelian randomisation to prioritise risk genes and infer potential biological mechanisms for MS risk loci. METHODS: The data used consisted of DNA methylation (n = 1980) QTL (mQTL) and gene expression (n = 31,684) QTL (eQTL) derived from whole blood as well as MS GWAS summary statistics (14,802 cases, 26,703 controls). The findings were further evaluated using data derived from independent brain mQTL (n = 1160) and eQTL (n = 1194). RESULTS: In whole blood, we identified two independent genomic loci (lincRNA: RP11-326C3.13 and TNFSF14) with consistent genome-wide significant pleiotropic associations across different omics layers. In brain tissue, a similar effect for the RP11-326C3.13 locus was observed but not for TNFSF14, indicating a potential tissue-specific effect for the TNFSF14 locus. CONCLUSION: We provide in silico evidence for the putative biological mechanisms by which the identified DNA methylation sites and target genes are functionally relevant to MS development in different tissues. Future research targeting these genes and DNA methylation sites will determine their roles in the pathophysiology of MS.

A multi-ethnic genome-wide association study implicates collagen matrix integrity and cell differentiation pathways in keratoconus.

Keratoconus is characterised by reduced rigidity of the cornea with distortion and focal thinning that causes blurred vision, however, the pathogenetic mechanisms are unknown. It can lead to severe visual morbidity in children and young adults and is a common indication for corneal transplantation worldwide. Here we report the first large scale genome-wide association study of keratoconus including 4,669 cases and 116,547 controls. We have identified significant association with 36 genomic loci that, for the first time, implicate both dysregulation of corneal collagen matrix integrity and cell differentiation pathways as primary disease-causing mechanisms. The results also suggest pleiotropy, with some disease mechanisms shared with other corneal diseases, such as Fuchs endothelial corneal dystrophy. The common variants associated with keratoconus explain 12.5% of the genetic variance, which shows potential for the future development of a diagnostic test to detect susceptibility to disease.

Rapid and efficient cataract gene evaluation in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes.

Cataract is the leading cause of blindness worldwide. Congenital or paediatric cataract can result in permanent visual impairment or blindness even with best attempts at treatment. A significant proportion of paediatric cataract has a genetic cause. Therefore, identifying the genes that lead to cataract formation is essential for understanding the pathological process of inherited paediatric cataract as well as to the development of new therapies. Despite clear progress in genomics technologies, verification of the biological effects of newly identified candidate genes and variants is still challenging. Here, we provide a step-by-step pipeline to evaluate cataract candidate genes in F0 zebrafish using CRISPR-Cas9 ribonucleoprotein complexes (RNP). Detailed descriptions of CRISPR-Cas9 RNP design and formulation, microinjection, optimization of CRISPR-Cas9 RNP reagent dose and delivery route, editing efficacy analysis as well as cataract formation evaluation are included. Following this protocol, any cataract candidates can be readily and efficiently evaluated within 2 weeks using basic laboratory supplies.

Multiple sclerosis risk variants regulate gene expression in innate and adaptive immune cells.

At least 200 single-nucleotide polymorphisms (SNPs) are associated with multiple sclerosis (MS) risk. A key function that could mediate SNP-encoded MS risk is their regulatory effects on gene expression. We performed microarrays using RNA extracted from purified immune cell types from 73 untreated MS cases and 97 healthy controls and then performed Cis expression quantitative trait loci mapping studies using additive linear models. We describe MS risk expression quantitative trait loci associations for 129 distinct genes. By extending these models to include an interaction term between genotype and phenotype, we identify MS risk SNPs with opposing effects on gene expression in cases compared with controls, namely, rs2256814 MYT1 in CD4 cells (q = 0.05) and rs12087340 RF00136 in monocyte cells (q = 0.04). The rs703842 SNP was also associated with a differential effect size on the expression of the METTL21B gene in CD8 cells of MS cases relative to controls (q = 0.03). Our study provides a detailed map of MS risk loci that function by regulating gene expression in cell types relevant to MS.

The voltage-gated calcium channel CaV1.2 promotes adult oligodendrocyte progenitor cell survival in the mouse corpus callosum but not motor cortex.

Throughout life, oligodendrocyte progenitor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes. OPCs express cell surface receptors and channels that allow them to detect and respond to neuronal activity, including voltage-gated calcium channel (VGCC)s. The major L-type VGCC expressed by developmental OPCs, CaV1.2, regulates their differentiation. However, it is unclear whether CaV1.2 similarly influences OPC behavior in the healthy adult central nervous system (CNS). To examine the role of CaV1.2 in adulthood, we conditionally deleted this channel from OPCs by administering tamoxifen to P60 Cacna1c fl/fl (control) and Pdgfrα-CreER:: Cacna1c fl/fl (CaV1.2-deleted) mice. Whole cell patch clamp analysis revealed that CaV1.2 deletion reduced L-type voltage-gated calcium entry into adult OPCs by ~60%, confirming that it remains the major L-type VGCC expressed by OPCs in adulthood. The conditional deletion of CaV1.2 from adult OPCs significantly increased their proliferation but did not affect the number of new oligodendrocytes produced or influence the length or number of internodes they elaborated. Unexpectedly, CaV1.2 deletion resulted in the dramatic loss of OPCs from the corpus callosum, such that 7 days after tamoxifen administration CaV1.2-deleted mice had an OPC density ~42% that of control mice. OPC density recovered within 2 weeks of CaV1.2 deletion, as the lost OPCs were replaced by surviving CaV1.2-deleted OPCs. As OPC density was not affected in the motor cortex or spinal cord, we conclude that calcium entry through CaV1.2 is a critical survival signal for a subpopulation of callosal OPCs but not for all OPCs in the mature CNS.

Association of Genetic Variation With Keratoconus.

Importance: Keratoconus is a condition in which the cornea progressively thins and protrudes in a conical shape, severely affecting refraction and vision. It is a major indication for corneal transplant. To discover new genetic loci associated with keratoconus and better understand the causative mechanism of this disease, we performed a genome-wide association study on patients with keratoconus. Objective: To identify genetic susceptibility regions for keratoconus in the human genome. Design, Setting, and Participants: This study was conducted with data from eye clinics in Australia, the United States, and Northern Ireland. The discovery cohort of individuals with keratoconus and control participants from Australia was genotyped using the Illumina HumanCoreExome single-nucleotide polymorphism array. After quality control and data cleaning, genotypes were imputed against the 1000 Genomes Project reference panel (phase III; version 5), and association analyses were completed using PLINK version 1.90. Single-nucleotide polymorphisms with P < 1.00 × 10-6 were assessed for replication in 3 additional cohorts. Control participants were drawn from the cohorts of the Blue Mountains Eye Study and a previous study of glaucoma. Replication cohorts were from a previous keratoconus genome-wide association study data set from the United States, a cohort of affected and control participants from Australia and Northern Ireland, and a case-control cohort from Victoria, Australia. Data were collected from January 2006 to March 2019. Main Outcomes and Measures: Associations between keratoconus and 6 252 612 genetic variants were estimated using logistic regression after adjusting for ancestry using the first 3 principal components. Results: The discovery cohort included 522 affected individuals and 655 control participants, while the replication cohorts included 818 affected individuals (222 from the United States, 331 from Australia and Northern Ireland, and 265 from Victoria, Australia) and 3858 control participants (2927 from the United States, 229 from Australia and Northern Ireland, and 702 from Victoria, Australia). Two novel loci reached genome-wide significance (defined as P < 5.00 × 10-8), with a P value of 7.46 × 10-9 at rs61876744 in patatin-like phospholipase domain-containing 2 gene (PNPLA2) on chromosome 11 and a P value of 6.35 × 10-12 at rs138380, 2.2 kb upstream of casein kinase I isoform epsilon gene (CSNK1E) on chromosome 22. One additional locus was identified with a P value less than 1.00 × 10-6 in mastermind-like transcriptional coactivator 2 (MAML2) on chromosome 11 (P = 3.91 × 10-7). The novel locus in PNPLA2 reached genome-wide significance in an analysis of all 4 cohorts (P = 2.45 × 10-8). Conclusions and Relevance: In this relatively large keratoconus genome-wide association study, we identified a genome-wide significant locus for keratoconus in the region of PNPLA2 on chromosome 11.

Lipid-related genetic polymorphisms significantly modulate the association between lipids and disability progression in multiple sclerosis.

OBJECTIVE: To investigate whether lipid-related or body mass index (BMI)-related common genetic polymorphisms modulate the associations between serum lipid levels, BMI and disability progression in multiple sclerosis (MS). METHODS: The association between disability progression (annualised Expanded Disability Status Scale (EDSS) change over 5 years, ΔEDSS) and lipid-related or BMI-related genetic polymorphisms was evaluated in a longitudinal cohort (n=184), diagnosed with MS. We constructed a cumulative genetic risk score (CGRS) of associated polymorphisms (p<0.05) and examined the interactions between the CGRS and lipid levels (measured at baseline) in predicting ΔEDSS. All analyses were conducted using linear regression. RESULTS: Five lipid polymorphisms (rs2013208, rs9488822, rs17173637, rs10401969 and rs2277862) and one BMI polymorphism (rs2033529) were nominally associated with ΔEDSS. The constructed lipid CGRS showed a significant, dose-dependent association with ΔEDSS (ptrend=1.4×10-6), such that participants having ≥6 risk alleles progressed 0.38 EDSS points per year faster compared with those having ≤3. This CGRS model explained 16% of the variance in ΔEDSS. We also found significant interactions between the CGRS and lipid levels in modulating ΔEDSS, including high-density lipoprotein (HDL; pinteraction=0.005) and total cholesterol:high-density lipoprotein ratio (TC:HDL; pinteraction=0.030). The combined model (combination of CGRS and the lipid parameter) explained 26% of the disability variance for HDL and 27% for TC:HDL. INTERPRETATION: In this prospective cohort study, both lipid levels and lipid-related polymorphisms individually and jointly were associated with significantly increased disability progression in MS. These results indicate that these polymorphisms and tagged genes might be potential points of intervention to moderate disability progression.

Rediscovering the value of families for psychiatric genetics research.

As it is likely that both common and rare genetic variation are important for complex disease risk, studies that examine the full range of the allelic frequency distribution should be utilized to dissect the genetic influences on mental illness. The rate limiting factor for inferring an association between a variant and a phenotype is inevitably the total number of copies of the minor allele captured in the studied sample. For rare variation, with minor allele frequencies of 0.5% or less, very large samples of unrelated individuals are necessary to unambiguously associate a locus with an illness. Unfortunately, such large samples are often cost prohibitive. However, by using alternative analytic strategies and studying related individuals, particularly those from large multiplex families, it is possible to reduce the required sample size while maintaining statistical power. We contend that using whole genome sequence (WGS) in extended pedigrees provides a cost-effective strategy for psychiatric gene mapping that complements common variant approaches and WGS in unrelated individuals. This was our impetus for forming the "Pedigree-Based Whole Genome Sequencing of Affective and Psychotic Disorders" consortium. In this review, we provide a rationale for the use of WGS with pedigrees in modern psychiatric genetics research. We begin with a focused review of the current literature, followed by a short history of family-based research in psychiatry. Next, we describe several advantages of pedigrees for WGS research, including power estimates, methods for studying the environment, and endophenotypes. We conclude with a brief description of our consortium and its goals.

Genome-wide linkage scan for loci influencing plasma triglyceride levels.

We conducted a genome-wide linkage scan to detect loci that influence the levels of fasting triglycerides in plasma. Fasting triglyceride levels were available at 4 time points (visits), 2 pre- and 2 post-fenofibrate intervention. Multipoint identity-by-descent (MIBD) matrices were derived from genotypes using IBDLD. Variance-component linkage analyses were then conducted using SOLAR (Sequential Oligogenic Linkage Analysis Routines). We found evidence of linkage (logarithm of odds [LOD] ≥3) at 5 chromosomal regions with triglyceride levels in plasma. The highest LOD scores were observed for linkage to the estimated genetic value (additive genetic component) of the log-normalized triglyceride levels in plasma. Our results suggest that a chromosome 10 locus at 37 cM (LODpre = 3.01, LODpost = 3.72) influences fasting triglyceride levels in plasma regardless of the fenofibrate intervention, and that loci in chromosomes 1 at 170 cM and 4 at 24 cM ceases to affect the triglyceride levels when fenofibrate is present, while the regions in chromosomes 6 at 136 to 162 cM and 11 at 39 to 40 cM appear to influence triglyceride levels in response to fenofibrate.

Rare, potentially pathogenic variants in 21 keratoconus candidate genes are not enriched in cases in a large Australian cohort of European descent.

Many genes have been suggested as candidate genes for keratoconus based on their function, their proximity to associated polymorphisms or due to the identification of putative causative variants within the gene. However, very few of these genes have been assessed for rare variation in keratoconus more broadly. In contrast, VSX1 and SOD1 have been widely assessed, however, the vast majority of studies have been small and the findings conflicting. In a cohort of Australians of European descent, consisting of 385 keratoconus cases and 396 controls, we screened 21 keratoconus candidate genes: BANP, CAST, COL4A3, COL4A4, COL5A1, FOXO1, FNDC3B, HGF, IL1A, IL1B, ILRN, IMMP2L, MPDZ, NFIB, RAB3GAP1, RAD51, RXRA, SLC4A11, SOD1, TF and VSX1. The candidate genes were sequenced in these individuals by either whole exome sequencing or targeted gene sequencing. Variants were filtered to identify rare (minor allele frequency <1%), potentially pathogenic variants. A total of 164 such variants were identified across the two groups with no variants fulfilling these criteria in cases in IL1RN, BANP, IL1B, RAD51 or SOD1. The frequency of variants was compared between cases and controls using chi-square or Fishers' Exact tests for each gene with at least one rare potentially pathogenic variant identified in the case cohort. The number of rare potentially pathogenic variants per gene ranged from three (RXRA) to 102 (MPDZ), however for all genes, there was no difference in the frequency between the cases and controls. We conclude that rare potentially pathogenic variation in the 21 candidate genes assessed do not play a major role in keratoconus susceptibility and pathogenesis.

Transcriptome and proteome profiling reveals stress-induced expression signatures of imiquimod-treated Tasmanian devil facial tumor disease (DFTD) cells.

As a topical cancer immunotherapy, the toll-like receptor 7 ligand imiquimod activates tumor regression via stimulation of immune cell infiltration and cytotoxic responses. Imiquimod also exerts direct pro-apoptotic effects on tumor cells in vitro, but a role for these effects in imiquimod-induced tumor regression remains undefined. We previously demonstrated that cell lines derived from devil facial tumor disease (DFTD), a transmissible cancer threatening the survival of the Tasmanian devil (Sarcophilus harrisii), are sensitive to imiquimod-induced apoptosis. In this study, the pro-apoptotic effects of imiquimod in DFTD have been investigated using RNA-sequencing and label-free quantitative proteomics. This analysis revealed that changes to gene and protein expression in imiquimod treated DFTD cells are consistent with the onset of oxidative and endoplasmic reticulum stress responses, and subsequent activation of the unfolded protein response, autophagy, cell cycle arrest and apoptosis. Imiquimod also regulates the expression of oncogenic pathways, providing a direct mechanism by which this drug may increase tumor susceptibility to immune cytotoxicity in vivo. Our study has provided the first global analysis of imiquimod-induced effects in any tumor cell line. These findings have highlighted the potential of cell stress pathways as therapeutic targets in DFTD, and will allow for improved mechanistic use of imiquimod as a therapy in both the Tasmanian devil and human cancers.

Common genetic variation within miR-146a predicts disease onset and relapse in multiple sclerosis.

Despite extensive studies focusing on the changes in expression of microRNAs (miRNAs) in multiple sclerosis (MS) compared to healthy controls, few studies have evaluated the association of genetic variants of miRNAs with MS clinical course. We investigated whether a functional polymorphism in the MS associated miR-146a gene predicted clinical course (hazard of conversion to MS and of relapse, and annualized change in disability), using a longitudinal cohort study of persons with a first demyelinating event followed up to their 5-year review. We found the genotype (GC+CC) of rs2910164 predicted relapse compared with the GG genotype (HR=2.09 (95% CI 1.42, 3.06), p=0.0001), as well as a near-significant (p=0.07) association with MS conversion risk. Moreover, we found a significant additive interaction between rs2910164 and baseline anti-EBNA-1 IgG titers predicting risk of conversion to MS (relative excess risk due to interaction [RERI] 2.39, p=0.00002) and of relapse (RERI 1.20, p=0.006). Supporting these results, similar results were seen for the other EBV-correlated variables: anti-EBNA-2 IgG titers and past history of infectious mononucleosis. There was no association of rs2910164 genotype for disability progression. Our findings provide evidence for miR-146a and EBV infection in modulating MS clinical course.