The Mediating Role of Kynurenine Pathway Metabolites on the Relationship Between Inflammation and Muscle Mass in Oldest-Old Men.

Tryptophan (TRP) metabolites along the kynurenine (KYN) pathway (KP) have been found to influence muscle. Proinflammatory cytokines are known to stimulate the degradation of TRP down the KP. Given that both inflammation and KP metabolites have been connected with loss of muscle, we assessed the potential mediating role of KP metabolites on inflammation and muscle mass in older men. Five hundred and five men (85.0 ±â€…4.2 years) from the Osteoporotic Fractures in Men cohort study with measured D3-creatine dilution (D3Cr) muscle mass, KP metabolites, and inflammation markers (C-reactive protein [CRP], alpha-1-acid glycoprotein [AGP] and a subsample [n = 305] with interleukin [IL-6, IL-1ß, IL-17A] and tumor necrosis factor-α [TNF-α]) were included in the analysis. KP metabolites and inflammatory markers were measured using liquid chromatography-tandem mass spectrometry and immunoassays, respectively. 23%-92% of the inverse relationship between inflammatory markers and D3Cr muscle mass was mediated by KP metabolites (indirect effect p < .05). 3-hydroxyanthranilic acid (3-HAA), quinolinic acid (QA), TRP, xanthurenic acid (XA), KYN/TRP, 3-hydroxykynurenine (3-HK)/3-HAA, QA/3-HAA, and nicotinamide (NAM)/QA mediated the AGP relationship. 3-HAA, QA, KYN/TRP, 3-HK/XA, HKr ratio, 3-HK/3-HAA, QA/3-HAA, and NAM/QA mediated the CRP. KYN/TRP, 3-HK/XA, and NAM/QA explained the relationship for IL-6 and 3-HK/XA and QA/3-HAA for TNF-α. No mediation effect was observed for the other cytokines (indirect effect p > .05). KP metabolites, particularly higher ratios of KYN/TRP, 3-HK/XA, 3-HK/3-HAA, QA/3-HAA, and a lower ratio of NAM/QA, mediated the relationship between inflammation and low muscle mass. Our preliminary cross-sectional data suggest that interventions to alter D3Cr muscle mass may focus on KP metabolites rather than inflammation per se.

Evidence for inefficient contraction and abnormal mitochondrial activity in sarcopenia using magnetic resonance spectroscopy.

BACKGROUND: Mitochondrial dysfunction has been implicated in sarcopenia. 31 P magnetic resonance spectroscopy (MRS) enables non-invasive measurement of adenosine triphosphate (ATP) synthesis rates to probe mitochondrial function. Here, we assessed muscle energetics in older sarcopenic and non-sarcopenic men and compared with muscle biopsy-derived markers of mitochondrial function. METHODS: Twenty Chinese men with sarcopenia (SARC, age = 73.1 ± 4.1 years) and 19 healthy aged and sex-matched controls (CON, age = 70.3 ± 4.2 years) underwent assessment of strength, physical performance, and magnetic resonance imaging. Concentrations of phosphocreatine (PCr), ATP and inorganic phosphate (Pi) as well as muscle pH were measured at rest and during an interleaved rest-exercise protocol to probe muscle mitochondrial function. Results were compared to biopsy-derived mitochondrial complex activity and expression to understand underlying metabolic perturbations. RESULTS: Despite matched muscle contractile power (strength/cross-sectional area), the ATP contractile cost was higher in SARC compared with CON (low-intensity exercise: 1.06 ± 0.59 vs. 0.57 ± 0.22, moderate: 0.93 ± 0.43 vs. 0.58 ± 0.68, high: 0.70 ± 0.57 vs. 0.43 ± 0.51 mmol L-1  min-1  bar-1  cm-2 , P = 0.003, <0.0001 and <0.0001, respectively). Post-exercise mitochondrial oxidative synthesis rates (a marker of mitochondrial function) tended to be longer in SARC but did not reach significance (17.3 ± 6.4 vs. 14.6 ± 6.5 mmol L-1  min-1 , P = 0.2). However, relative increases in end-exercise ADP in SARC (31.8 ± 9.9 vs. 24.0 ± 7.3 mmol L-1 , P = 0.008) may have been a compensatory mechanism. Mitochondrial complex activity was found to be associated with exercise-induced drops in PCr [citrate synthetase activity (CS), Spearman correlation rho = -0.42, P = 0.03] and end-exercise ADP (complex III, rho = -0.52, P = 0.01; CS rho = -0.45, P = 0.02; SDH rho = -0.45, P = 0.03), with CS also being strongly associated with the PCr recovery rate following low intensity exercise (rho = -0.47, P = 0.02), and the cost of contraction at high intensity (rho = -0.54, P = 0.02). Interestingly, at high intensity, the fractional contribution of oxidative phosphorylation to exercise was correlated with activity in complex II (rho = 0.5, P = 0.03), CS (rho = 0.47, P = 0.02) and SDH (rho = 0.46, P = 0.03), linking increased mitochondrial complex activity with increased ability to generate energy through oxidative pathways. CONCLUSIONS: This study used 31 P MRS to assess ATP utilization and resynthesis in sarcopenic muscle and demonstrated abnormal increases in the energy cost during exercise and perturbed mitochondrial energetics in recovery. Associations between mitochondrial complex activity and the fractional contribution to energy requirement during exercise indicate increased ability to generate energy oxidatively in those with better mitochondrial complex activity.

Association of amino acid metabolites with osteoporosis, a metabolomic approach: Bushehr elderly health program.

INTRODUCTION AND OBJECTIVES: Amino acids are the most frequently reported metabolites associated with low bone mineral density (BMD) in metabolomics studies. We aimed to evaluate the association between amino acid metabolic profile and bone indices in the elderly population. METHODS: 400 individuals were randomly selected from 2384 elderly men and women over 60 years participating in the second stage of the Bushehr elderly health (BEH) program, a population-based prospective cohort study that is being conducted in Bushehr, a southern province of Iran. Frozen plasma samples were used to measure 29 amino acid and derivatives metabolites using the UPLC-MS/MS-based targeted metabolomics platform. We conducted Elastic net regression analysis to detect the metabolites associated with BMD of different sites and lumbar spine trabecular bone score, and also to examine the ability of the measured metabolites to differentiate osteoporosis. RESULTS: We adjusted the analysis for possible confounders (age, BMI, diabetes, smoking, physical activity, vitamin D level, and sex). Valine, leucine, isoleucine, and alanine in women and tryptophan in men were the most important amino acids inversely associated with osteoporosis (OR range from 0.77 to 0.89). Sarcosine, followed by tyrosine, asparagine, alpha aminobutyric acid, and ADMA in women and glutamine in men and when both women and men were considered together were the most discriminating amino acids detected in individuals with osteoporosis (OR range from 1.15 to 1.31). CONCLUSION: We found several amino acid metabolites associated with possible bone status in elderly individuals. Further studies are required to evaluate the utility of these metabolites as clinical biomarkers for osteoporosis prediction and their effect on bone health as dietary supplements.

A Randomized Controlled Clinical Trial in Healthy Older Adults to Determine Efficacy of Glycine and N-Acetylcysteine Supplementation on Glutathione Redox Status and Oxidative Damage.

Glycine and cysteine are non-essential amino acids that are required to generate glutathione, an intracellular tripeptide that neutralizes reactive oxygen species and prevents tissue damage. During aging glutathione demand is thought to increase, but whether additional dietary intake of glycine and cysteine contributes towards the generation of glutathione in healthy older adults is not well understood. We investigated supplementation with glycine and n-acetylcysteine (GlyNAC) at three different daily doses for 2 weeks (low dose: 2.4 g, medium dose: 4.8 g, or high dose: 7.2 g/day, 1:1 ratio) in a randomized, controlled clinical trial in 114 healthy volunteers. Despite representing a cohort of healthy older adults (age mean = 65 years), we found significantly higher baseline levels of markers of oxidative stress, including that of malondialdehyde (MDA, 0.158 vs. 0.136 µmol/L, p < 0.0001), total cysteine (Cysteine-T, 314.8 vs. 276 µM, p < 0.0001), oxidized glutathione (GSSG, 174.5 vs. 132.3 µmol/L, p < 0.0001), and a lower ratio of reduced to oxidized glutathione (GSH-F:GSSG) (11.78 vs. 15.26, p = 0.0018) compared to a young reference group (age mean = 31.7 years, n = 20). GlyNAC supplementation was safe and well tolerated by the subjects, but did not increase levels of GSH-F:GSSG (end of study, placebo = 12.49 vs. 7.2 g = 12.65, p-value = 0.739) or that of total glutathione (GSH-T) (end of study, placebo = 903.5 vs. 7.2 g = 959.6 mg/L, p-value = 0.278), the primary endpoint of the study. Post-hoc analyses revealed that a subset of subjects characterized by high oxidative stress (above the median for MDA) and low baseline GSH-T status (below the median), who received the medium and high doses of GlyNAC, presented increased glutathione generation (end of study, placebo = 819.7 vs. 4.8g/7.2 g = 905.4 mg/L, p-value = 0.016). In summary GlyNAC supplementation is safe, well tolerated, and may increase glutathione levels in older adults with high glutathione demand. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05041179, NCT05041179.

An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.

Epigenome-wide association study of sarcopenia: findings from the Hertfordshire Sarcopenia Study (HSS).

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.

Mitochondrial oxidative capacity and NAD+ biosynthesis are reduced in human sarcopenia across ethnicities.

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.

Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors.

Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro-adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis.

Vitamin B12 deficiency and impaired expression of amnionless during aging.

BACKGROUND: Physical frailty and loss of mobility in elderly individuals lead to reduced independence, quality of life, and increased mortality. Vitamin B12 deficiency has been linked to several age-related chronic diseases, including in the musculo-skeletal system, where vitamin B12 deficiency is generally believed to be linked to poor nutritional intake. In the present study, we asked whether aging and frailty associate with altered vitamin B12 homeostasis in humans and investigated the underlying molecular mechanisms using preclinical models. METHODS: We analysed a subset of the Singapore Longitudinal Aging Study and stratified 238 participants based on age and Fried frailty criteria. Levels of methyl-malonic acid (MMA), a marker for vitamin B12 deficiency, and amnionless, the vitamin B12 co-receptor that anchors the vitamin B12 transport complex to the membrane of epithelial cells, were measured in plasma. In addition, vitamin B12 levels and the molecular mechanisms of vitamin B12 uptake and excretion were analysed in ileum, kidney, liver, and blood using a rat model of natural aging where nutritional intake is fully controlled. RESULTS: We demonstrate that aging and frailty are associated with a higher prevalence of functional vitamin B12 deficiency that can be detected by increased levels of MMA in blood (ρ = 0.25; P = 0.00013). The decline in circulating vitamin B12 levels is recapitulated in a rat model of natural aging where food composition and intake are stable. At the molecular level, these perturbations involve altered expression of amnionless in the ileum and kidney. Interestingly, we demonstrate that amnionless can be detected in serum where its levels increase during aging in both rodents and human (P = 3.3e-07 and 9.2e-07, respectively). Blood amnionless levels negatively correlate with vitamin B12 in rats (r2  = 0.305; P = 0.0042) and positively correlate with the vitamin B12 deficiency marker MMA in humans (ρ = 0.22; P = 0.00068). CONCLUSIONS: Our results demonstrate that aging and frailty cause intrinsic vitamin B12 deficiencies, which can occur independently of nutritional intake. Mechanistically, vitamin B12 deficiency involves the physio-pathological decline of both the intestinal uptake and the renal reabsorption system for vitamin B12. Finally, amnionless is a novel biomarker which can detect perturbed vitamin B12 bioavailability during aging and physical frailty.

Plasma Proteomic Profiles of Cerebrospinal Fluid-Defined Alzheimer's Disease Pathology in Older Adults.

BACKGROUND: Cerebrospinal fluid (CSF) biomarkers of the beta-amyloid and microtubule associated protein tau metabolism have proven the capacity to improve classification of subjects developing Alzheimer's disease (AD). The blood plasma proteome was characterized to further elaborate upon the mechanisms involved and identify proteins that may improve classification of older adults developing an AD dementia. OBJECTIVE: Identify and describe plasma protein expressions that best classify subjects with CSF-defined presence of AD pathology and cerebral amyloidosis. METHODS: We performed a cross-sectional analysis of samples collected from community-dwelling elderly with (n = 72) or without (n = 48) cognitive impairment. CSF Aß1-42, tau, and phosphorylated tau (P-tau181) were measured using ELISA, and mass spectrometry quantified the plasma proteomes. Presence of AD pathology was defined as CSF P-tau181/Aß1-42 > 0.0779, and presence of amyloidosis was defined as CSF Aß1-42 < 724 pg/mL. RESULTS: Two hundred and forty-eight plasma proteins were quantified. Plasma proteins did not improve classification of the AD CSF biomarker profile in the whole sample. When the analysis was separately performed in the cognitively impaired individuals, the diagnosis accuracy of AD CSF profile was 88.9% with 19 plasma proteins included. Within the full cohort, there were 16 plasma proteins that improved diagnostic accuracy of cerebral amyloidosis to 92.4%. CONCLUSION: Plasma proteins improved classification accuracy of AD pathology in cognitively-impaired older adults and appeared representative of amyloid pathology. If confirmed, those candidates could serve as valuable blood biomarkers of the preclinical stages of AD or risk of developing AD.

One-carbon metabolism, cognitive impairment and CSF measures of Alzheimer pathology: homocysteine and beyond.

BACKGROUND: Hyperhomocysteinemia is a risk factor for cognitive decline and dementia, including Alzheimer disease (AD). Homocysteine (Hcy) is a sulfur-containing amino acid and metabolite of the methionine pathway. The interrelated methionine, purine, and thymidylate cycles constitute the one-carbon metabolism that plays a critical role in the synthesis of DNA, neurotransmitters, phospholipids, and myelin. In this study, we tested the hypothesis that one-carbon metabolites beyond Hcy are relevant to cognitive function and cerebrospinal fluid (CSF) measures of AD pathology in older adults. METHODS: Cross-sectional analysis was performed on matched CSF and plasma collected from 120 older community-dwelling adults with (n = 72) or without (n = 48) cognitive impairment. Liquid chromatography-mass spectrometry was performed to quantify one-carbon metabolites and their cofactors. Least absolute shrinkage and selection operator (LASSO) regression was initially applied to clinical and biomarker measures that generate the highest diagnostic accuracy of a priori-defined cognitive impairment (Clinical Dementia Rating-based) and AD pathology (i.e., CSF tau phosphorylated at threonine 181 [p-tau181]/ß-Amyloid 1-42 peptide chain [Aß1-42] >0.0779) to establish a reference benchmark. Two other LASSO-determined models were generated that included the one-carbon metabolites in CSF and then plasma. Correlations of CSF and plasma one-carbon metabolites with CSF amyloid and tau were explored. LASSO-determined models were stratified by apolipoprotein E (APOE) ε4 carrier status. RESULTS: The diagnostic accuracy of cognitive impairment for the reference model was 80.8% and included age, years of education, Aß1-42, tau, and p-tau181. A model including CSF cystathionine, methionine, S-adenosyl-L-homocysteine (SAH), S-adenosylmethionine (SAM), serine, cysteine, and 5-methyltetrahydrofolate (5-MTHF) improved the diagnostic accuracy to 87.4%. A second model derived from plasma included cystathionine, glycine, methionine, SAH, SAM, serine, cysteine, and Hcy and reached a diagnostic accuracy of 87.5%. CSF SAH and 5-MTHF were associated with CSF tau and p-tau181. Plasma one-carbon metabolites were able to diagnose subjects with a positive CSF profile of AD pathology in APOE ε4 carriers. CONCLUSIONS: We observed significant improvements in the prediction of cognitive impairment by adding one-carbon metabolites. This is partially explained by associations with CSF tau and p-tau181, suggesting a role for one-carbon metabolism in the aggregation of tau and neuronal injury. These metabolites may be particularly critical in APOE ε4 carriers.

A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish.

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish.

Extensive natural variation for cellular hydrogen peroxide release is genetically controlled.

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H(2)O(2) release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H(2)O(2) release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10-8) and 54 suggestive associations (p<1.00×10-5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H(2)O(2) release was observed in Down Syndrome (DS) individuals (p<2.88×10-12). Taken together, our results show strong evidence of genetic control of H(2)O(2) in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.

Tandem repeat sequence variation as causative cis-eQTLs for protein-coding gene expression variation: the case of CSTB.

Association studies have revealed expression quantitative trait loci (eQTLs) for a large number of genes. However, the causative variants that regulate gene expression levels are generally unknown. We hypothesized that copy-number variation of sequence repeats contribute to the expression variation of some genes. Our laboratory has previously identified that the rare expansion of a repeat c.-174CGGGGCGGGGCG in the promoter region of the CSTB gene causes a silencing of the gene, resulting in progressive myoclonus epilepsy. Here, we genotyped the repeat length and quantified CSTB expression by quantitative real-time polymerase chain reaction in 173 lymphoblastoid cell lines (LCLs) and fibroblast samples from the GenCord collection. The majority of alleles contain either two or three copies of this repeat. Independent analysis revealed that the c.-174CGGGGCGGGGCG repeat length is strongly associated with CSTB expression (P = 3.14 × 10(-11)) in LCLs only. Examination of both genotyped and imputed single-nucleotide polymorphisms (SNPs) within 2 Mb of CSTB revealed that the dodecamer repeat represents the strongest cis-eQTL for CSTB in LCLs. We conclude that the common two or three copy variation is likely the causative cis-eQTL for CSTB expression variation. More broadly, we propose that polymorphic tandem repeats may represent the causative variation of a fraction of cis-eQTLs in the genome.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence.

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Methylation profiling in individuals with uniparental disomy identifies novel differentially methylated regions on chromosome 15.

The maternal and paternal genomes possess distinct epigenetic marks that distinguish them at imprinted loci. In order to identify imprinted loci, we used a novel method, taking advantage of the fact that uniparental disomy (UPD) provides a system that allows the two parental chromosomes to be studied independently. We profiled the paternal and maternal methylation on chromosome 15 using immunoprecipitation of methylated DNA and hybridization to tiling oligonucleotide arrays. Comparison of six individuals with maternal versus paternal UPD15 revealed 12 differentially methylated regions (DMRs). Putative DMRs were validated by bisulfite sequencing, confirming the presence of parent-of-origin-specific methylation marks. We detected DMRs associated with known imprinted genes within the Prader-Willi/Angelman syndrome region, such as SNRPN and MAGEL2, validating this as a method of detecting imprinted loci. Of the 12 DMRs identified, eight were novel, some of which are associated with genes not previously thought to be imprinted. These include a site within intron 2 of IGF1R at 15q26.3, a gene that plays a fundamental role in growth, and an intergenic site upstream of GABRG3 that lies within a previously defined candidate region conferring an increased maternal risk of psychosis. These data provide a map of parent-of-origin-specific epigenetic modifications on chromosome 15, identifying DNA elements that may play a functional role in the imprinting process. Application of this methodology to other chromosomes for which UPD has been reported will allow the systematic identification of imprinted sites throughout the genome.

Regulation of fibrinogen production by microRNAs.

Elevated levels of fibrinogen are associated with increased risk of cardiovascular disease, whereas low fibrinogen can lead to a bleeding disorder. We investigated whether microRNAs (miRNAs), known to act as post-transcriptional regulators of gene expression, regulate fibrinogen production. Using transfection of a library of 470 annotated human miRNA precursor molecules in HuH7 hepatoma cells and quantitative measurements of fibrinogen production, we identified 23 miRNAs with down-regulating (up to 64% decrease) and 4 with up-regulating effects (up to 129% increase) on fibrinogen production. Among the down-regulating miRNAs, we investigated the mechanism of action of 3 hsa-miR-29 family members and hsa-miR-409-3p. Overexpression of hsa-miR-29 members led to decreased steady-state levels of all fibrinogen gene (FGA, FGB, and FGG) transcripts in HuH7 cells. Luciferase reporter gene assays demonstrated that this was independent of miRNA-fibrinogen 3'-untranslated region interactions. In contrast, overexpression of hsa-miR-409-3p specifically lowered fibrinogen Bß mRNA levels, and this effect was dependent on a target site in the fibrinogen Bß mRNA 3'-untranslated region. This study adds to the known mechanisms that control fibrinogen production, points toward a potential cause of variable circulating fibrinogen levels, and demonstrates that a screening approach can identify miRNAs that regulate clinically important proteins.

Elevated levels of MIC-1/GDF15 in the cerebrospinal fluid of patients are associated with glioblastoma and worse outcome.

For patients with brain tumors identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebrospinal fluid (CSF), would greatly facilitate patient management. MIC-1/GDF15 (growth differentiation factor 15) is a secreted protein of the TGF-beta superfamily and emerged as a candidate marker exhibiting increasing mRNA expression during malignant progression of glioma. Determination of MIC-1/GDF15 protein levels by ELISA in the CSF of a cohort of 94 patients with intracranial tumors including gliomas, meningioma and metastasis revealed significantly increased concentrations in glioblastoma patients (median, 229 pg/ml) when compared with control cohort of patients treated for non-neoplastic diseases (median below limit of detection of 156 pg/ml, p < 0.0001, Mann-Whitney test). However, plasma MIC-1/GDF15 levels were not elevated in the matching plasma samples from these patients. Most interestingly, patients with glioblastoma and increased CSF MIC-1/GDF15 had a shorter survival (p = 0.007, log-rank test). In conclusion, MIC-1/GDF15 protein measured in the CSF may have diagnostic and prognostic value in patients with intracranial tumors.

Modulation of angiogenic and inflammatory response in glioblastoma by hypoxia.

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy.Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.

Infrequent promoter methylation of the MGMT gene in liver metastases from uveal melanoma.

Uveal melanoma is associated with a high mortality rate once metastases occur, with over >90% of metastatic patients dying within less than 1 year from metastases to the liver. The intraarterial hepatic (iah) administration of the alkylating agent fotemustine holds some promise with response rates of 36% and median survival of 15 months. Here, we investigated whether the DNA-repair-protein MGMT may be involved in the variability of response to fotemustine and temozolomide in uveal melanoma. Epigenetic inactivation of MGMT has been demonstrated to be a predictive marker for benefit from alkylating agent therapy in glioblastoma. We found a methylated MGMT promoter in 6% of liver metastases from 34 uveal melanoma patients. The mean MGMT activity measured in liver metastases with negligible liver tissue content was significantly lower than in liver tissue (146 versus 523 fmol/mg protein, p = 0.002). Expression of the MGMT protein was detectable in 50% of 88 metastases by immunohistochemistry on a tissue microarray. Expression was heterogeneous, and in accordance with MGMT activity data, usually lower than in the surrounding liver. Differential MGMT activity/expression between metastasis and liver tissue and more efficient depletion of MGMT with higher doses of alkylating agent therapy using iah delivery may provide the pharmacologic window for the higher response rate. However, these results do not support MGMT methylation status or protein expression as predictive markers for treatment outcome to iah chemotherapy with alkylating agents.