Background Juvenile idiopathic arthritis (JIA) is considered a complex trait in which the environment interacts with inherited genes to produce a phenotype that shows broad inter-individual variance. A recently completed genome-wide association study (GWAS) identified 24 regions of genetic risk for JIA, for example. However, as is typical for GWAS, most of the regions of genetic risk for JIA (22 of 24) were in non-coding regions of the genome. The studies reported here were undertaken to identify functional elements (other than genes) that might be located within the regions of genetic risk. Methods We used paired end RNA sequencing to identify non-coding RNAs located within 5 kb of the disease-associated SNPs. In addition, we used chromatin immunoprecipitation-sequencing (ChIP-Seq) to identify epigenetic marks associated with enhancer function (H3K4me1 and H3K27ac) in human neutrophils to determine whether there was enrichment of enhancer-associated histone marks in linkage disequilibrium (LD) blocks that encompassed the 22 GWAS SNPs from the non-coding genome. Results In human neutrophils, we identified H3K4me1 and/or H3K27ac marks in 15 of the 22 regions previously as identified as risk loci for JIA. In CD4+ T cells, 18 regions demonstrate H3K4me1 and/or H3K27ac marks. In addition, we identified non-coding RNA transcripts at the rs4705862 and rs6894249 loci in human neutrophils. Conclusion Much of the genetic risk for JIA lies within or adjacent to regions of neutrophil and CD4+ T cell genomes that carry epigenetic marks associated with enhancer function and/or ncRNA transcripts. These findings are consistent with the hypothesis that JIA is fundamentally a disorder of gene regulation that includes both the innate and adaptive immune system. Elucidating the specific roles of these non-coding elements within leukocyte genomes in JIA pathogenesis will be critical to our understanding disease pathogenesis.

Single nucleotide polymorphisms (SNPs) in choline metabolizing genes are associated with disease risk and greater susceptibility to organ dysfunction under conditions of dietary choline restriction. However, the underlying metabolic signatures of these variants are not well characterized and it is unknown whether genotypic differences persist at recommended choline intakes. Thus, we sought to determine if common genetic risk factors alter choline dynamics in pregnant, lactating, and non-pregnant women consuming choline intakes meeting and exceeding current recommendations. Women (n = 75) consumed 480 or 930 mg choline/day (22% as a metabolic tracer, choline-d9) for 10–12 weeks in a controlled feeding study. Genotyping was performed for eight variant SNPs and genetic differences in metabolic flux and partitioning of plasma choline metabolites were evaluated using stable isotope methodology. CHKA rs10791957, CHDH rs9001, CHDH rs12676, PEMT rs4646343, PEMT rs7946, FMO3 rs2266782, SLC44A1 rs7873937, and SLC44A1 rs3199966 altered the use of choline as a methyl donor; CHDH rs9001 and BHMT rs3733890 altered the partitioning of dietary choline between betaine and phosphatidylcholine synthesis via the cytidine diphosphate (CDP)-choline pathway; and CHKA rs10791957, CHDH rs12676, PEMT rs4646343, PEMT rs7946 and SLC44A1 rs7873937 altered the distribution of dietary choline between the CDP-choline and phosphatidylethanolamine N-methyltransferase (PEMT) denovo pathway. Such metabolic differences may contribute to disease pathogenesis and prognosis over the long-term.

Choline and folate are interrelated in 1-carbon metabolism, mostly because of their shared function as methyl donors for homocysteine remethylation. Folate deficiency and mutations of methylenetetrahydrofolate reductase (MTHFR) reduce the availability of a major methyl donor, 5-methyltetrahydrofolate, which in turn may lead to compensatory changes in choline metabolism. This study investigated the hypothesis that reductions in methyl group supply, either due to dietary folate deficiency or Mthfr gene deletion, would modify tissue choline metabolism in a sex-specific manner. Mthfr wild type (+/+) or heterozygous (+/-) knockout mice were randomized to a folate-deficient or control diet for 8 wk during which time deuterium-labeled choline (d9-choline) was consumed in the drinking water (~10 μmol/d). Mthfr heterozygosity did not alter brain choline metabolite concentrations, but it did enhance their labeling in males (P < 0.05) and tended to do so in females (P < 0.10), a finding consistent with greater turnover of dietary choline in brains of +/- mice. Dietary folate deficiency in females yielded 52% higher (P = 0.027) hepatic glycerophosphocholine, which suggests that phosphatidylcholine (PtdCho) degradation was enhanced. Labeling of the hepatic PtdCho in d3 form was also reduced (P < 0.001) in females, which implies that fewer of the dietary choline-derived methyl groups were used for de novo PtdCho biosynthesis under conditions of folate insufficiency. Males responded to folate restriction with a doubling (P < 0.001) of hepatic choline dehydrogenase transcripts, a finding consistent with enhanced conversion of choline to the methyl donor, betaine. Collectively, these data show that several adaptations in choline metabolism transpire as a result of mild perturbations in folate metabolism, presumably to preserve methyl group homeostasis.

Although single nucleotide polymorphisms (SNPs) in folate-mediated pathways predict susceptibility to choline deficiency during severe choline deprivation, it is unknown if effects persist at recommended intakes. Thus, we used stable isotope liquid chromatography-mass spectrometry (LC-MS) methodology to examine the impact of candidate SNPs on choline metabolism in a long-term, randomized, controlled feeding trial among pregnant, lactating, and nonpregnant (NP) women consuming 480 or 930 mg/d choline (22% as choline-d, with d indicating a deuterated trimethyl amine group) and meeting folate-intake recommendations. Variants impairing folate metabolism, methylenetetrahydrofolate reductase (MTHFR) rs1801133, methionine synthase (MTR) rs1805087 [wild-type (WT)], MTR reductase (MTRR) rs1801394, and methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) rs2236225, influenced choline dynamics, frequently through interactions with reproductive state and choline intake, with fewer genotypic alterations observed among pregnant women. Women with these variants partitioned more dietary choline toward phosphatidylcholine (PC) biosynthesis via the cytidine diphosphate (CDP)-choline pathway at the expense of betaine synthesis even when use of betaine as a methyl donor was increased. Choline intakes of 930 mg/d restored partitioning of dietary choline between betaine and CDP-PC among NP (MTHFR rs1801133 and MTR rs1805087 WT) and lactating (MTHFD1 rs2236225) women with risk genotypes. Overall, our findings indicate that loss-of-function variants in folate-metabolizing enzymes strain cellular PC production, possibly via impaired folate-dependent phosphatidylethanolamine-N-methyltransferase (PEMT)-PC synthesis, and suggest that women with these risk genotypes may benefit from choline intakes exceeding current recommendations.-Ganz, A. B., Shields, K., Fomin, V. G., Lopez, Y. S., Mohan, S., Lovesky, J., Chuang, J. C., Ganti, A., Carrier, B., Yan, J., Taeswuan, S., Cohen, V. V., Swersky, C. C., Stover, J. A., Vitiello, G. A., Malysheva, O. V., Mudrak, E., Caudill, M. A. Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.

We characterized the hypolipidemic effects of alpha-lipoic acid (LA, R-form) and examined the associated molecular mechanisms in a high fat fed Zucker rat model. Rats (n = 8) were assigned to a high fat (HF) diet or the HF diet with 0.25% LA (HF-LA) for 30 days and pair fed to remove confounding effects associated with the anorectic properties of LA. Compared with the HF controls, the HF-LA group was protected against diet-induced obesity (102.5±3.1 vs. 121.5±3.6,% change BW) and hypercholesterolemia with a reduction in total-C (−21%), non-HDL-C (−25%), LDL-C (−16%), and total LDL particle number (−46%) and an increase in total HDL particles (∼22%). This cholesterol-lowering response was associated with a reduction in plasma PCSK9 concentration (−70%) and an increase in hepatic LDLr receptor protein abundance (2 fold of HF). Compared with the HF-fed animals, livers of LA-supplemented animals were protected against TG accumulation (−46%), likely through multiple mechanisms including: a suppressed lipogenic response (down-regulation of hepatic acetyl-CoA carboxylase and fatty acid synthase expression); enhanced hepatic fat oxidation (increased carnitine palmitoyltransferase Iα expression); and enhanced VLDL export (increased hepatic diacylglycerol acyltransferase and microsomal triglyceride transfer protein expression and elevated plasma VLDL particle number). Study results also support an enhanced fatty acid uptake (2.8 fold increase in total lipase activity) and oxidation (increased CPT1β protein abundance) in muscle tissue in LA-supplemented animals compared with the HF group. In summary, in the absence of a change in caloric intake, LA was effective in protecting against hypercholesterolemia and hepatic fat accumulation under conditions of strong genetic and dietary predisposition toward obesity and dyslipidemia.