Methylome of human skeletal muscle after acute & chronic resistance exercise training, detraining & retraining.

DNA methylation is an important epigenetic modification that can regulate gene expression following environmental encounters without changes to the genetic code. Using Infinium MethylationEPIC BeadChip Arrays (850,000 CpG sites) we analysed for the first time, DNA isolated from untrained human skeletal muscle biopsies (vastus lateralis) at baseline (rest) and immediately following an acute (single) bout of resistance exercise. In the same participants, we also analysed the methylome following a period of muscle growth (hypertrophy) evoked via chronic (repeated bouts-3 sessions/wk) resistance exercise (RE) (training) over 7-weeks, followed by complete exercise cessation for 7-weeks returning muscle back to baseline levels (detraining), and finally followed by a subsequent 7-week period of RE-induced hypertrophy (retraining). These valuable methylome data sets described in the present manuscript and deposited in an open-access repository can now be shared and re-used to enable the identification of epigenetically regulated genes/networks that are modified after acute anabolic stimuli and hypertrophy, and further investigate the phenomenon of epigenetic memory in skeletal muscle.

Evidence of a role for GTP cyclohydrolase-1 in visceral pain.

BACKGROUND: The enzyme guanosine triphosphate-cyclohydrolase-1 (GCH-1) is a rate limiting step in the de novo synthesis of tetrahydrobiopterin (BH4) a co-factor in monoamine synthesis and nitric oxide production. GCH-1 is strongly implicated in chronic pain based on data generated using the selective GCH-1 inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP), and studies which have identified a pain protective GCH-1 haplotype associated with lower BH4 production and reduced pain. METHODS: To investigate the role for GCH-1 in visceral pain we examined the effects of DAHP on pain behaviors elicited by colorectal injection of mustard oil in rats, and the pain protective GCH-1 haplotype in healthy volunteers characterized by esophageal pain sensitivity before and after acid injury, and assessed using depression and anxiety questionnaires. KEY RESULTS: In rodents pretreatment with DAHP produced a substantial dose related inhibition of pain behaviors from 10 to 180 mg/kg i.p. (p < 0.01 to 0.001). In healthy volunteers, no association was seen between the pain protective GCH-1 haplotype and the development of hypersensitivity following injury. However, a substantial increase in baseline pain thresholds was seen between first and second visits (26.6 ± 6.2 mA) in subjects who sensitized to esophageal injury and possessed the pain protective GCH-1 haplotype compared with all other groups (p < 0.05). Furthermore the same subjects who sensitized to acid and possessed the haplotype, also had significantly lower depression scores (p < 0.05). CONCLUSIONS & INFERENCES: The data generated indicate that GCH-1 plays a role in visceral pain processing that requires more detailed investigation.

Array-based DNA methylation profiling in follicular lymphoma.

Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P<0.05) correlation between FL methylation values and reduced gene expression was shown for up to 28% of loci. Methylation changes occurred predominantly in B cells with variability in the amount of non-malignant tissue between samples preventing conclusive correlation with survival. This represents an important caveat in attributing prognostic relevance to methylation and future studies in cancer will optimally require purified tumour populations to address the impact of methylation on clinical outcome.

A genetic study of the ghrelin and growth hormone secretagogue receptor (GHSR) genes and stature.

Growth and nutrition are interrelated and influenced by multiple genetic and environmental factors. We studied whether common variants in ghrelin and ghrelin receptor (GHSR) genes could play a role in stature variation in the general population and in families ascertained for obesity. Selected tagging SNPs in the ghrelin and GHSR genes were genotyped in 263 Caucasian families recruited for childhood obesity (1,275 subjects), and in 287 families from a general population (1,072 subjects). We performed familial testing for associations in the entire population and in a sub-set of the samples selected for a case-control study. In the case-control study for height (cases were selected from the obese cohort with mean ZH = 3.17 +/- 0.15 confidence interval (CI) versus controls with mean ZH 0.14 +/- 0.09), we found an association with a 2 base-pair intronic deletion in the GHSR gene (rs10618418) (p = 0.006, odds ratio (OR) 1.86, 95% CI [1.26;2.74] under additive model), although when adjusting for BMI, the association disappeared (p = 0.06). Individuals carrying no deletion or who were heterozygous were significantly more frequent among the tall obese population (52% vs. 36% in controls, p = 0.007, OR 1.97, 95%CI [1.22;3.18]). However, the association was not maintained after correcting for multiple testing. Familial association testing of the ghrelin and GHSR genes and their interaction testing failed to show that any combination of SNPs had any significant effect. Thus, our results suggest that common variants of the ghrelin and GHSR genes are not major contributors to height variation in a French population.

Yes-associated protein (YAP) functions as a tumor suppressor in breast.

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

A new variation in the promoter region, the -604 C>T, and the Leu72Met polymorphism of the ghrelin gene are associated with protection to insulin resistance.

OBJECTIVE: Previous studies suggested that polymorphisms in the coding region of the preproghrelin were involved in the etiology of obesity and might modulate glucose-induced insulin secretion. We evaluated the association of a new variation, -604C>T, in the promoter region of the ghrelin gene, of Leu72Met (247C>A) and of Gln90Leu (265A>T), all haplotype-tagging single nucleotide polymorphisms (SNPs), with measures of insulin sensitivity in 1420 adult individuals. RESEARCH METHODS: The three SNPs were genotyped using ABI PRISM 7900 HT Sequence Detection System. We used multiple linear regression analysis for quantitative traits and THESIAS software for haplotype analysis. RESULTS: We observed a protective effect exerted by Met72 variant of Leu72Met SNP on insulin resistance parameters; a significant decreasing trend from Leu/Leu to Leu/Met and to Met/Met homozygous subjects in triglycerides, fasting insulin levels and HOMA-IR index (P=0.02, 0.01 and 0.003, respectively), and, consistently, an increase in ghrelin levels (P=0.003) was found. A significant decrease from CC to TC and to TT genotypes in insulin levels and HOMA-IR index was also detected (P=0.00l for both), but only in subjects homozygous for Leu72, where the protective effect of Met72 was not present. The haplotype analysis results supported the data obtained by the evaluation of each single SNP, showing the highest value of insulin levels and HOMA-IR index in the -604(c)247(c) haplotype intermediate value in -604(T)247(C) and lowest value in -604(C)247(A). CONCLUSION: Our observations suggest a protective role of the Met72 variant and of -604 T allele in modulating insulin resistance. These SNPs or an unknown functional variant in linkage disequilibrium could increase ghrelin levels and probably insulin sensitivity.

Somatic mitochondrial DNA mutations in primary and metastatic ovarian cancer.

OBJECTIVE: To date, most mtDNA mutations in cancer have been identified in the control region (D-loop) containing the major promoters. However, almost all studies used one sample per tumor and there is no clear evidence whether metastatic deposits harbor different mtDNA variants. To establish whether different mtDNA variants can be found in the same cancer but at different sites, we analyzed a series of unilateral and bilateral primary epithelial ovarian cancers as well as paired metastatic tumor deposits. METHODS: We sequenced the D-loop region in 52 different tumor samples of 35 ovarian cancer cases, as well as matched normal tissues. Seventeen of those 35 cases had bilateral ovarian cancer, with a sample from each tumor analyzed. RESULTS: Eighty-six polymorphisms (4 new in ovarian cancer) were detected, and 9 different somatic mtDNA mutations were found in 26% (9 of 35) of ovarian cancer cases; all were homoplasmic in nature. Six of the mutations were novel in ovarian cancer. In 24% (4 of 17) of cases with bilateral ovarian tumors, different mtDNA variants were found between paired tumors, suggesting the presence of different clonal populations of cancer cells. Metastatic tumor deposits showed identical mtDNA variants to those found in at least one of the ovarian tumors in cases with bilateral ovarian cancer. CONCLUSION: Our data demonstrate that multiple tumor samples from the same patient may harbor different mtDNA variants.

The EIF2AK3 gene region and type I diabetes in subjects from South India.

Mutations in the EIF2AK3 gene underlie susceptibility to the Wolcott-Rallison syndrome, which is a monogenic disease associated with insulin-deficient neonatal diabetes. Furthermore, suggestive evidence of linkage between type 1 diabetes (T1DM) and the EIF2KA3 chromosomal region has been reported in Scandinavian families. We have investigated the hypothesis that polymorphic variants in and around the EIF2AK3 gene might partially account for susceptibility to T1DM in South Indian subjects. Excess transmission of the common alleles of two polymorphic markers (D2S1786 and 15INDEL, located within the gene) downstream of EIF2AK3, either singly (D2S1786, P = 0.01) and 15INDEL (P = 0.02) or as a combination (P < 0.001), were found in 234 families with a T1DM proband. There was also a clear paternal effect for the 15INDEL marker (P = 0.005) on disease susceptibility. The presence of the common allele of both markers was found in decreased frequency in the subjects with normal glucose tolerance compared to probands with T1DM (both P

Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.

Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.

A search for type 1 diabetes susceptibility genes in families from the United Kingdom.

Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome. A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus (IDDM1) explaining 34% of the familial clustering of the disease (lambda(s)=2.5; refs 3,4). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1/MHC, which was still the only major locus detected, were not excluded at lambda(s)=3 and lod=-2, of which two showed evidence of linkage: chromosome 10p13-p11 (maximum lod score (MLS)=4.7, P=3x10(-6), lambda(s)=1.56) and chromosome 16q22-16q24 (MLS=3.4, P=6.5x10(-5), lambda(s)=1.6). These and other novel regions, including chromosome 14q12-q21 and chromosome 19p13-19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based and haplotype mapping approaches must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage.

Quantification of X-chromosome inactivation patterns in haematological samples using the DNA PCR-based HUMARA assay.

Quantification of X-chromosome inactivation patterns (XCIPs) using PCR amplification of the human androgen receptor (HUMARA) locus is potentially valuable in a range of haematological disorders. Of 236 females screened, 203 (86%) were heterozygous. For quantitative XCIPs it was necessary to limit the number of PCR cycles to 20 to reduce preferential amplification of shorter alleles. The optimized PCR method was compared with Southern blotting results using either PGK, HPRT or M27beta in 51 haematologically normal females and blast cells from 27 patients with acute myeloid leukaemia (AML). Reproducible XCIP results were obtained in all 78 samples using digestion with Hpa II prior to amplification (median difference in duplicate values 3%, range 0-17%) and they correlated well with Southern blotting results, r=0.966. Greater variability was observed in the results using Hha I digestion (median difference 4%, range 0-48%). There were marked inconsistencies in repeated analyses of three AML samples and although the HUMARA-Hha I results correlated well overall with Southern blotting in the remaining 75 samples (r=0.922), in nine samples there were still discrepancies with > or = 20% difference between the two values. These results suggest that PCR analysis of the HUMARA locus in Hpa II-digested DNA is suitable for the quantification of XCIPs in haematological samples but results with Hha I should be treated with caution.