Background Immunohematology reference laboratories provide red blood cell (RBC), platelet (PLT), and neutrophil typing to resolve complex cases, using serology and commercial DNA tests that define clinically important antigens. Broad‐range exome sequencing panels that include blood group targets provide accurate blood group antigen predictions beyond those defined by serology and commercial typing systems and identify rare and novel variants. The aim of this study was to design and assess a panel for targeted exome sequencing of RBC, PLT, and neutrophil antigen–associated genes to provide a comprehensive profile in a single test, excluding unrelated gene targets. Study Design and Methods An overlapping probe panel was designed for the coding regions of 64 genes and loci involved in gene expression. Sequencing was performed on 34 RBC and 17 PLT/neutrophil reference samples. Variant call outputs were analyzed using software to predict star allele diplotypes. Results were compared with serology and previous sequence genotyping data. Results Average coverage exceeded 250×, with more than 94% of targets at Q30 quality or greater. Increased coverage revealed a variant in the Scianna system that was previously undetected. The software correctly predicted allele diplotypes for 99.5% of RBC blood groups tested and 100% of PLT and HNA antigens excepting HNA‐2. Optimal throughput was 12 to 14 samples per run. Conclusion This single‐test system demonstrates high coverage and quality, allowing for the detection of previously overlooked variants and increased sample throughput. This system has the potential to integrate genomic testing across laboratories within hematologic reference settings.

Background Sarcopenia is the age‐related loss of muscle mass, strength, and function. Epigenetic processes such as DNA methylation, which integrate both genetic and environmental exposures, have been suggested to contribute to the development of sarcopenia. This study aimed to determine whether differences in the muscle methylome are associated with sarcopenia and its component measures: grip strength, appendicular lean mass index (ALMi), and gait speed. Methods Using the Infinium Human MethylationEPIC BeadChip, we measured DNA methylation in vastus lateralis muscle biopsies of 83 male participants (12 with sarcopenia) with a mean (standard deviation) age of 75.7 (3.6) years from the Hertfordshire Sarcopenia Study (HSS) and Hertfordshire Sarcopenia Study extension (HSSe) and examined associations with sarcopenia and its components. Pathway, histone mark, and transcription factor enrichment of the differentially methylated CpGs (dmCpGs) were determined, and sodium bisulfite pyrosequencing was used to validate the sarcopenia‐associated dmCpGs. Human primary myoblasts (n = 6) isolated from vastus lateralis muscle biopsies from male individuals from HSSe were treated with the EZH2 inhibitor GSK343 to assess how perturbations in epigenetic processes may impact myoblast differentiation and fusion, measured by PAX7 and MYHC immunocytochemistry, and mitochondrial bioenergetics determined using the Seahorse XF96. Results Sarcopenia was associated with differential methylation at 176 dmCpGs (false discovery rate ≤ 0.05) and 141 differentially methylated regions (Stouffer ≤ 0.05). The sarcopenia‐associated dmCpGs were enriched in genes associated with myotube fusion (P = 1.40E‐03), oxidative phosphorylation (P = 2.78E‐02), and voltage‐gated calcium channels (P = 1.59E‐04). ALMi was associated with 71 dmCpGs, grip strength with 49 dmCpGs, and gait speed with 23 dmCpGs (false discovery rate ≤ 0.05). There was significant overlap between the dmCpGs associated with sarcopenia and ALMi (P = 3.4E‐35), sarcopenia and gait speed (P = 4.78E‐03), and sarcopenia and grip strength (P = 7.55E‐06). There was also an over‐representation of the sarcopenia, ALMi, grip strength, and gait speed‐associated dmCpGs with sites of H3K27 trimethylation (all P ≤ 0.05) and amongst EZH2 target genes (all P ≤ 0.05). Furthermore, treatment of human primary myoblasts with the EZH2 inhibitor GSK343 inhibitor led to an increase in PAX7 expression (P ≤ 0.05), decreased myotube fusion (P = 0.043), and an increase in ATP production (P = 0.008), with alterations in the DNA methylation of genes involved in oxidative phosphorylation and myogenesis. Conclusions These findings show that differences in the muscle methylome are associated with sarcopenia and individual measures of muscle mass, strength, and function in older individuals. This suggests that changes in the epigenetic regulation of genes may contribute to impaired muscle function in later life.

Impairment of endothelial function with aging is accompanied by reduced nitric oxide (NO) production. T-type Ca3.1 channels augment nitric oxide and co-localize with eNOS. Therefore, the hypothesis was that T-type channels contribute to the endothelial dysfunction of aging. Endothelial function was determined in mesenteric arteries (perfusion) and aortae (isometric contraction) of young and old wild-type (WT), Ca3.1, and Ca3.2 knockout mice. NO production was measured by fluorescence imaging in mesenteric arteries. With age, endothelium-dependent subsequent dilatation (following depolarization with KCl) of mesenteric arteries was diminished in the arteries of WT mice, unchanged in Ca3.2 preparations but increased in those of Ca3.1 mice. NO synthase inhibition abolished the subsequent dilatation in mesenteric arteries and acetylcholine-induced relaxations in aortae. NO levels were significantly reduced in mesenteric arteries of old compared to young WT mice. In Ca3.1 and Ca3.2 preparations, NO levels increased significantly with age. Relaxations to acetylcholine were significantly smaller in the aortae of old compared to young WT mice, while such responses were comparable in preparations of young and old Ca3.1 and Ca3.2 mice. The expression of Ca3.1 was significantly reduced in aortae from aged compared to young WT mice. The level of phosphorylated eNOS was significantly increased in aortae from aged Ca3.1 mice. In conclusion, T-type calcium channel-deficient mice develop less age-dependent endothelial dysfunction. Changes in NO levels are involved in this phenomenon in WT and Ca3.1 mice. These findings suggest that T-type channels play an important role in age-induced endothelial dysfunction.

Non-communicable diseases (NCD) such as type-2 diabetes and CVD are now highly prevalent in both developed and developing countries. Evidence from both human and animal studies shows that early-life nutrition is an important determinant of NCD risk in later life. The mechanism by which the early-life environment influences future disease risk has been suggested to include the altered epigenetic regulation of gene expression. Epigenetic processes regulate the accessibility of genes to the cellular proteins that control gene transcription, determining where and when a gene is switched on and its level of activity. Epigenetic processes not only play a central role in regulating gene expression but also allow an organism to adapt to the environment. In this review, we will focus on how both maternal and paternal nutrition can alter the epigenome and the evidence that these changes are causally involved in determining future disease risk.

Epigenetics links perinatal influences with later obesity. We identifed differentially methylated CpG (dmCpG) loci measured at 17 years associated with concurrent adiposity measures and examined whether these were associated with hsCRP, adipokines, and early life environmental factors. Genome-wide DNA methylation from 1192 Raine Study participants at 17 years, identified 29 dmCpGs (Bonferroni corrected p < 1.06E-07) associated with body mass index (BMI), 10 with waist circumference (WC) and 9 with subcutaneous fat thickness. DmCpGs within Ras Association (RalGDS/AF-6), Pleckstrin Homology Domains 1 (RAPH1), Musashi RNA-Binding Protein 2 (MSI2), and solute carrier family 25 member 10 (SLC25A10) are associated with both BMI and WC. Validation by pyrosequencing confirmed these associations and showed that MSI2 , SLC25A10 , and RAPH1 methylation was positively associated with serum leptin. These were also associated with the early environment; MSI2 methylation (β = 0.81, p = 0.0004) was associated with pregnancy maternal smoking, SLC25A10 (CpG2 β = 0.12, p = 0.002) with pre-and early pregnancy BMI, and RAPH1 (β = −1.49, p = 0.036) with gestational weight gain. Adjusting for perinatal factors, methylation of the dmCpGs within MSI2, RAPH1, and SLC25A10 independently predicted BMI, accounting for 24% of variance. MSI2 methylation was additionally associated with BMI over time (17 years old β = 0.026, p = 0.0025; 20 years old β = 0.027, p = 0.0029) and between generations (mother β = 0.044, p = 7.5e-04). Overall findings suggest that DNA methylation in MSI2, RAPH1, and SLC25A10 in blood may be robust markers, mediating through early life factors.

Recent studies have shown that a large proportion of patients classified as essential thrombocythemia (ET) actually have early primary prefibrotic myelofibrosis (prePMF), which implies an inferior prognosis as compared to patients being diagnosed with so-called genuine or true ET. According to the World Health Organization (WHO) 2008 classification, bone marrow histology is a major component in the distinction between these disease entities. However, the differential diagnosis between them may be challenging and several studies have not been able to distinguish between them. Most lately, it has been argued that simple blood tests, including the leukocyte count and plasma lactate dehydrogenase (LDH) may be useful tools to separate genuine ET from prePMF, the latter disease entity more often being featured by anemia, leukocytosis and elevated LDH. Whole blood gene expression profiling was performed in 17 and 9 patients diagnosed with ET and PMF, respectively. Using elevated LDH obtained at the time of diagnosis as a marker of prePMF, a 7-gene signature was identified which correctly predicted the prePMF group with a sensitivity of 100% and a specificity of 89%. The 7 genes included MPO, CEACAM8, CRISP3, MS4A3, CEACAM6, HEMGN, and MMP8, which are genes known to be involved in inflammation, cell adhesion, differentiation and proliferation. Evaluation of bone marrow biopsies and the 7-gene signature showed a concordance rate of 71%, 79%, 62%, and 38%. Our 7-gene signature may be a useful tool to differentiate between genuine ET and prePMF but needs to be validated in a larger cohort of “ET” patients.

Folate, a cofactor for the supply of one-carbon groups, is required by epigenetic processes to regulate cell lineage determination during development. The intake of folic acid (FA), the synthetic form of folate, has increased significantly over the past decade, but the effects of high periconceptional FA intake on cell lineage determination in the early embryo remains unknown. Here, we investigated the effect of maternal high FA (HFA) intake on blastocyst development and expression of key regulatory genes. C57BL/6 adult female mice were fed either Control diet (1 mg FA) for 4 weeks before conception and during the preimplantation period (Con-Con); Control diet for 4 weeks preconception, followed by HFA (5 mg FA) diet during preimplantation (Con-HFA); or HFA diet for 4 weeks preconception and during preimplantation (HFA-HFA). At E3.5, blastocyst cell number, protein, and mRNA expression were measured. In HFA-HFA blastocysts, trophectoderm cell numbers and expression of CDX2, Oct-4, and Nanog were reduced compared with Con-Con blastocysts; Con-HFA blastocysts showed lower CDX2 and Oct-4 expression than Con-Con blastocysts. These findings suggest periconceptional HFA intake induces changes in key regulators of embryo morphogenesis with potential implications for subsequent development.

Folic acid (FA) intake has been associated with increased breast cancer risk in some studies. Although underlying mechanisms are unknown, epigenetic modifications that persistently alter transcription have been suggested. We tested the hypothesis that high FA (HFA) intake alters the adult mammary transcriptome in a manner consistent with increased potential for carcinogenesis, detectable beyond the period of intake. C57BL/6 mice were fed control FA (CFA) (1 mg/kg diet) or HFA (5 mg/kg diet) diets for 4 weeks, followed by AIN93M maintenance diet for 4 weeks. Plasma 5-methyltetrahydrofolate, p-aminobenzoylglutamate and unmetabolised FA concentrations were greater (1.62, 1.56, 5.80-fold, respectively) in HFA compared to CFA mice. RNA sequencing of the mammary transcriptome (~20 million reads) showed 222 transcripts (191 upregulated) differentially expressed between groups. Gene Set Enrichment showed upregulated genes significantly enriched in Epithelial Mesenchymal Transition, Myogenesis and Apical Junction and downregulated genes in E2F targets, MYC targets and G2M checkpoint. Cancer was the most altered Disease and Disorder pathway, with Metastasis, Mammary Tumour and Growth of Tumour the most upregulated pathways. ChIP-seq enrichment analysis showed that targets of histone methyltransferase EZH2 were enriched in HFA mice. This study demonstrates HFA intake during adulthood induces mammary transcriptome changes, consistent with greater tumorigenic potential.