Screening of methylation genes in age-related cataract

AIM： To analyze and screen the methylation status of whole-genome in age-related cataract samples. METHODS： Anterior lens capsule samples were collected from age-related cortical cataract patients over 50 years of age with LOCS III score of nuclear color ≥4 along with control subjects. DNAs were extracted and subjected to methylation microarray for the identification of methylated genes employing the high-throughput sequencing approach. RESULTS： Compared with the control group, 843 sites were found methylated, including 802 hypermethylation sites with 542 corresponding genes, 41 demethylation sites with 29 corresponding sites. COL4 A1, GJA3, SIPA1 L3 were confirmed by mass spectrometry, the results were consistent with high-throughput sequencing. CONCLUSION： DNA methylation microarrays is an efficient way for screening the aberrantly methylated genes. In this study, we are able to screen a few age-related cataract genes such as COL4 A1, GJA3, and SIPA1 L3 for their aberrant methylation patterns in cataract patients however further work is warranted to understand the significance of these findings.

Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis.Based on the hyperbranched rolling circle amplification（HRCA） and microarray techniques,a new method for qualitative detection of methylation was developed.In the present study,padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes.Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction.Utilizing the method of 3-dimensional polyacrylamide gel-based microarray,the HRCA product will be immobilized on the slide to form a DNA microarray,which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.To control the false positive signals,DNA ligase and temperature of ligation/digestion are optimized.Methylation status of four CpG sites located in P15,Ecadherin,hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR.Our research proves that this method is simple and inexpensive,and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.

Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes

With defined culture protocol, human embryonic stem cells （hESCs） are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for car- diac disease therapies. In this study, we successfully generated a highly pure population of human cardio- myocytes （hCMs） （〉95% cTnT＋） from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA meth- ylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene func- tions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription fac- tors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understandingof how the epigenetic machinery coordinates to regulate gene expression in different cell types.