Reduced mitochondrial D-loop methylation levels in sporadic amyotrophic lateral sclerosis

Background: Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms. Results: In the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females. Conclusions: Present data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms

Performance of random forest when SNPs are in linkage disequilibrium

<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) may be correlated due to linkage disequilibrium (LD). Association studies look for both direct and indirect associations with disease loci. In a Random Forest (RF) analysis, correlation between a true risk SNP and SNPs in LD may lead to diminished variable importance for the true risk SNP. One approach to address this problem is to select SNPs in linkage equilibrium (LE) for analysis. Here, we explore alternative methods for dealing with SNPs in LD: change the tree-building algorithm by building each tree in an RF only with SNPs in LE, modify the importance measure (IM), and use haplotypes instead of SNPs to build a RF.</p> <p>Results</p> <p>We evaluated the performance of our alternative methods by simulation of a spectrum of complex genetics models. When a haplotype rather than an individual SNP is the risk factor, we find that the original Random Forest method performed on SNPs provides good performance. When individual, genotyped SNPs are the risk factors, we find that the stronger the genetic effect, the stronger the effect LD has on the performance of the original RF. A revised importance measure used with the original RF is relatively robust to LD among SNPs; this revised importance measure used with the revised RF is sometimes inflated. Overall, we find that the revised importance measure used with the original RF is the best choice when the genetic model and the number of SNPs in LD with risk SNPs are unknown. For the haplotype-based method, under a multiplicative heterogeneity model, we observed a decrease in the performance of RF with increasing LD among the SNPs in the haplotype.</p> <p>Conclusion</p> <p>Our results suggest that by strategically revising the Random Forest method tree-building or importance measure calculation, power can increase when LD exists between SNPs. We conclude that the revised Random Forest method performed on SNPs offers an advantage of not requiring genotype phase, making it a viable tool for use in the context of thousands of SNPs, such as candidate gene studies and follow-up of top candidates from genome wide association studies.</p

Influence of alcohol consumption and alcohol metabolism variants on breast cancer risk among Black women: results from the AMBER consortium

Background: Moderate to heavy alcohol consumption is associated with an increased risk of breast cancer. The etiologic role of genetic variation in genes involved in ethanol metabolism has not been established, with little information available among women of African ancestry. Methods: Our analysis from the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium included 2889 U.S. Black women who were current drinkers at the time of breast cancer diagnosis (N cases = 715) and had available genetic data for four ethanol metabolism genomic regions (ADH, ALDH, CYP2E1, and ALDH2). We used generalized estimating equations to calculate genetic effects, gene* alcohol consumption (≥ 7drinks/week vs. &lt; 7/week) interactions, and joint main plus interaction effects of up to 23,247 variants in ethanol metabolism genomic regions on odds of breast cancer. Results: Among current drinkers, 21% of cases and 14% of controls reported consuming ≥ 7 drinks per week. We identified statistically significant genetic effects for rs79865122-C in CYP2E1 with odds of ER- breast cancer and odds of triple negative breast cancer, as well as a significant joint effect with odds of ER- breast cancer (≥ 7drinks per week OR = 3.92, &lt; 7 drinks per week OR = 0.24, pjoint = 3.74 × 10−6). In addition, there was a statistically significant interaction of rs3858704-A in ALDH2 with consumption of ≥ 7 drinks/week on odds of triple negative breast cancer (≥ 7drinks per week OR = 4.41, &lt; 7 drinks per week OR = 0.57, pint = 8.97 × 10–5). Conclusions: There is a paucity of information on the impact of genetic variation in alcohol metabolism genes on odds of breast cancer among Black women. Our analysis of variants in four genomic regions harboring ethanol metabolism genes in a large consortium of U.S. Black women identified significant associations between rs79865122-C in CYP2E1 and odds of ER- and triple negative breast cancer. Replication of these findings is warranted

Eight common genetic variants associated with serum dheas levels suggest a key role in ageing mechanisms

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant circulating steroid secreted by adrenal glands-yet its function is unknown. Its serum concentration declines significantly with increasing age, which has led to speculation that a relative DHEAS deficiency may contribute to the development of common age-related diseases or diminished longevity. We conducted a meta-analysis of genome-wide association data with 14,846 individuals and identified eight independent common SNPs associated with serum DHEAS concentrations. Genes at or near the identified loci include ZKSCAN5 (rs11761528; p = 3.15×10-36), SULT2A1 (rs2637125; p = 2.61×10-19), ARPC1A (rs740160; p = 1.56×10-16), TRIM4 (rs17277546; p = 4.50×10-11), BMF (rs7181230; p = 5.44×10-11), HHEX (rs2497306; p = 4.64×10-9), BCL2L11 (rs6738028; p = 1.72×10-8), and CYP2C9 (rs2185570; p = 2.29×10-8). These genes are associated with type 2 diabetes, lymphoma, actin filament assembly, drug and xenobiotic metabolism, and zinc finger proteins. Several SNPs were associated with changes in gene expression levels, and the related genes are connected to biological pathways linking DHEAS with ageing. This study provides much needed insight into the function of DHEAS

Rapamycin treatment for amyotrophic lateral sclerosis protocol for a phase II randomized, double-blind, placebo-controlled, multicenter, clinical trial (RAP-ALS trial)

Introduction: Misfolded aggregated proteins and neuroinflammation significantly contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, hence representing therapeutic targets to modify disease expression. Rapamycin inhibits mechanistic target of Rapamycin (mTOR) pathway and enhances autophagy with demonstrated beneficial effects in neurodegeneration in cell line and animal models, improving phenotype in SQSTM1 zebrafish, in Drosophila model of ALS-TDP, and in the TDP43 mouse model, in which it reduced neuronal loss and TDP43 inclusions. Rapamycin also expands regulatory T lymphocytes (Treg) and increased Treg levels are associated with slow progression in ALS patients. Therefore, we planned a randomized clinical trial testing Rapamycin treatment in ALS patients. Methods: RAP-ALS is a phase II randomized, double-blind, placebo-controlled, multicenter (8 ALS centers in Italy), clinical trial. The primary aim is to assess whether Rapamycin administration increases Tregs number in treated patients compared with control arm. Secondary aims include the assessment of safety and tolerability of Rapamycin in patients with ALS; the minimum dosage to have Rapamycin in cerebrospinal fluid; changes in immunological (activation and homing of T, B, NK cell subpopulations) and inflammatory markers, and on mTOR downstream pathway (S6RP phosphorylation); clinical activity (ALS Functional Rating Scale-Revised, survival, forced vital capacity); and quality of life (ALSAQ40 scale). Discussion: Rapamycin potentially targets mechanisms at play in ALS (i.e., autophagy and neuroinflammation), with promising preclinical studies. It is an already approved drug, with known pharmacokinetics, already available and therefore with significant possibility of rapid translation to daily clinics. Findings will provide reliable data for further potential trials. Ethics and dissemination: The study protocol was approved by the Ethics Committee of Azienda Ospedaliero Universitaria of Modena and by the Ethics Committees of participating centers (Eudract n. 2016-002399-28) based on the Helsinki declaration

A Two-Stage Random Forest-Based Pathway Analysis Method

Pathway analysis provides a powerful approach for identifying the joint effect of genes grouped into biologically-based pathways on disease. Pathway analysis is also an attractive approach for a secondary analysis of genome-wide association study (GWAS) data that may still yield new results from these valuable datasets. Most of the current pathway analysis methods focused on testing the cumulative main effects of genes in a pathway. However, for complex diseases, gene-gene interactions are expected to play a critical role in disease etiology. We extended a random forest-based method for pathway analysis by incorporating a two-stage design. We used simulations to verify that the proposed method has the correct type I error rates. We also used simulations to show that the method is more powerful than the original random forest-based pathway approach and the set-based test implemented in PLINK in the presence of gene-gene interactions. Finally, we applied the method to a breast cancer GWAS dataset and a lung cancer GWAS dataset and interesting pathways were identified that have implications for breast and lung cancers

Gallbladder Cancer Predisposition: A Multigenic Approach to DNA-Repair, Apoptotic and Inflammatory Pathway Genes

Gallbladder cancer (GBC) is a multifactorial disease with complex interplay between multiple genetic variants. We performed Classification and Regression Tree Analysis (CART) and Grade of Membership (GoM) analysis to identify combinations of alleles among the DNA repair, inflammatory and apoptotic pathway genetic variants in modifying the risk for GBC. We analyzed 16 polymorphisms in 8 genes involved in DNA repair, apoptotic and inflammatory pathways to find out combinations of genetic variants contributing to GBC risk. The genes included in the study were XRCC1, OGG1, ERCC2, MSH2, CASP8, TLR2, TLR4 and PTGS2. Single locus analysis by logistic regression showed association of MSH2 IVS1+9G>C (rs2303426), ERCC2 Asp312Asn (rs1799793), OGG1 Ser326Cys (rs1052133), OGG1 IVS4-15C>G (rs2072668), CASP8 -652 6N ins/del (rs3834129), PTGS2 -1195G>A (rs689466), PTGS2 -765G>C (rs20417), TLR4 Ex4+936C>T (rs4986791) and TLR2 –196 to –174del polymorphisms with GBC risk. The CART analysis revealed OGG1 Ser326Cys, and OGG1 IVS4-15C>G polymorphisms as the best polymorphic signature for discriminating between cases and controls. In the GoM analysis, the data was categorized into six sets representing risk for GBC with respect to the investigated polymorphisms. Sets I, II and III described low intrinsic risk (controls) characterized by multiple protective alleles while sets IV, V and VI represented high intrinsic risk groups (GBC cases) characterized by the presence of multiple risk alleles. The CART and GoM analyses also showed the importance of PTGS2 -1195G>A polymorphism in susceptibility to GBC risk. In conclusion, the present multigenic approach can be used to define individual risk profiles for gallbladder cancer in North Indian population

Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition