10 research outputs found
Abnormal hypermethylation at imprinting control regions in patients with S-adenosylhomocysteine hydrolase (AHCY) deficiency
S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status
Promoter Methylation Status of ASC/TMS1/PYCARD is Associated with Decreased Overall Survival and TNM Status in Patients with Early Stage Non-small Cell Lung Cancer (NSCLC).
BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, with 5-year overall survival less than 15%. Therefore, it is essential to find biomarkers for early detection and prognosis. Aberrant DNA methylation is a common feature of human cancers and its utility is already recognized in cancer management. The aim of this study was to explore the diagnostic and prognostic value of the promoter methylation status of the ASC/TMS1/PYCARD and MyD88 genes, key adaptor molecules in the activation of the innate immune response and apoptosis pathways.
METHODS: A total of 50 non-small cell lung cancer (NSCLC) patients were enrolled in the study. Methylation of bisulphite converted DNA was quantified by pyrosequencing in fresh frozen malignant tissues and adjacent non-malignant tissues. Associations between methylation and lung function, tumor grade and overall survival were evaluated using receiver-operating characteristics (ROC) analysis and statistical tests of hypothesis.
RESULTS: Methylation level of tested genes is generally low but significantly decreased in tumor tissues (ASC/TMS1/PYCARD, P<0.0001; MyD88, P<0.0002), which correlates with increased protein expression. Three CpG sites were identified as promising diagnostic marker candidates; CpG11 (-63 position) in ASC/TMS1/PYCARD and CpG1 (-253 position) and 2 (-265 position) in MyD88. The association study showed that the methylation status of the ASC/TMS1 CpG4 site (-34 position) in malignant and non-malignant tissues is associated with the overall survival (P=0.019) and the methylation status of CpG8 site (-92 position) is associated with TNM-stage (P=0.011).
CONCLUSIONS: The methylation status of the ASC/TMS1/PYCARD and MyD88 promoters are promising prognostic biomarker candidates. However, presented results should be considered as a preliminary and should be confirmed on the larger number of the samples
HRM analysis confirms pyrosequencing data of patients A, B and F.
<p>High resolution melting (HRM) analysis gives the mean methylation status of the complete PCR amplicons of three imprinted gene DMRs. PCR primers for the <i>MEST</i>, <i>SNRPN</i> and <i>H19</i> DMRs used for pyrosequencing also worked for HRM analysis to confirm pyrosequencing results. DNA of patients A, B and F were analysed together with controls. The mean values ± standard deviation did not differ by more than 10% methylation between pyrosequencing and HRM analysis for any of the analysed DMRs. The bars show the mean methylation values, the error bars the standard deviation of two independent measurements.</p
Methylation levels determined by bisulfite pyrosequencing analysis.
<p>Methylation levels determined by bisulfite pyrosequencing analysis.</p
Genome wide methylation levels in three patients with AHCY deficiency and controls.
<p>Patients A and B showed genome wide methylation levels more than five times as high as those of controls, patient F displayed a genome wide methylation level similar to that of controls. The bars display the ratio of methylation levels of the three patients normalised to that of three control samples in three independent measurements. Variation between the three control samples was included via error propagation.</p
Methylation levels determined by high resolution melting analysis.
<p>Methylation levels determined by high resolution melting analysis.</p
List of analysed patients with AHCY deficiency.
<p>List of analysed patients with AHCY deficiency.</p
Methylation values of single CpG sites analysed in the <i>MEST</i> bisulfite pyrosequencing assay.
<p>DNA methylation [%] determined by pyrosequencing at five consecutive CpG sites in the <i>MEST</i> DMR in the seven patients and controls<b>.</b> The CpG sites within the pyrosequencing assay are ordered according to their indicated position in the genome (Ensembl version 76).</p
Three patients with AHCY deficiency show abnormal hypermethylation in some imprinted gene DMRs.
<p>Bisulfite pyrosequencing results of six imprinted gene DMRs as well as LINE1 and Alu repetitive elements in seven patients and controls. Only patients A, D and F showed abnormal hypermethylation of some, but not all imprinted gene DMRs. No methylation errors were observed for the repetitive elements. The bars show the mean methylation values, the error bars display the standard deviation of two to four independent measurements. Moderately abnormal methylation values are indicated by a single asterisk (*), highly abnormal methylation values by a double asterisk (**).</p