Analysis of parent of origin specific DNA methylation at SNRPN and PW71 in tissues: implication for prenatal diagnosis.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct developmental disorders caused by absence of paternal or maternal contributions of the chromosome region 15q11-q13, resulting from deletions, uniparental disomy (UPD), or rare imprinting mutations. Molecular cytogenetic diagnosis is currently performed using a combination of fluorescence in situ hybridisation (FISH), DNA polymorphism analysis, and DNA methylation analysis. Only methylation analysis will detect all three categories of PWS abnormalities, but its reliability in tissues other than peripheral blood has not been examined extensively. Therefore, we examined the methylation status at the CpG island of the small nuclear ribonucleoprotein associated polypeptide N (SNRPN) gene and at the PW71 locus using normal and abnormal lymphoblast (LB) cell lines (n = 48), amniotic fluid (AF) cell cultures (n = 25), cultured chorionic villus samples (CVS, n = 17), and fetal tissues (n = 18) by Southern blot analysis with methylation sensitive enzymes. Of these samples, 20 LB cell lines, three AF cultures, one CVS, and 15 fetal tissues had been previously diagnosed as having deletions or UPD by other molecular methods. Methylation status at SNRPN showed consistent results when compared with FISH or DNA polymorphism analysis using all cell types tested. However, the methylation pattern for PW71 was inconsistent when compared with other tests and should therefore not be used on tissues other than peripheral blood. We conclude that SNRPN, but not PW71, methylation analysis may be useful for diagnosis of PWS/AS on LB cell lines, cultured amniotic fluid, or chorionic villus samples and will allow, for the first time, prenatal diagnosis for families known to carry imprinting centre defects.     

Alu elements support independent origin of prosimian, platyrrhine, and catarrhine Mhc-DRB genes

The primate major histocompatibility complex (Mhc) genes fall into two classes and each of the classes into several families. Of the class II families, the DRB family has a long and complex evolutionary history marked by gene turnover, rearrangement, and molecular convergence. Because the history is not easily decipherable from sequences alone, Alu element insertions were used as cladistic markers to support the surmised phylogenetic relationships among the DRB genes. Intron 1 segments of 24 DRB genes from five platyrrhine species and five DRB genes from three prosimian species were amplified by PCR and cloned, and the amplification products were sequenced or PCR-typed for Alu repeats. Three Alu elements were identified in the platyrrhine and four in the prosimian DRB genes. One of the platyrrhine elements (Alu50J) is also found in the Catarrhini, whereas the other two (Alu62Sc, Alu63Sc) are restricted to the New World monkeys. Similarly, the four prosimian elements are found only in this taxon. This distribution of Alu elements is consistent with the phylogeny of the DRB genes as determined from their intron 1 sequences in an earlier and the present study. It contradicts the exon 2-based phylogeny and thus corroborates the conclusion that the evolution of DRB exon 2 sequences is, to some extent, shaped by molecular convergence. Taken together, the data indicate that each of the assemblages of DRB genes in prosimians, platyrrhines, and catarrhines is derived from a separate ancestral gene.     

Large-scale methylation analysis of human genomic DNA reveals tissue-specific differences between the methylation profiles of genes and pseudogenes

Cytosine in CpG dinucleotides is frequently found to be methylated in the DNA of higher eukaryotes and differential methylation has been proposed to be a key element in the organization of gene expression in man. To address this question systematically, we used bisulfite genomic sequencing to study the methylation patterns of three X-linked genes and one autosomal pseudogene in two adult individuals and across nine different tissues. Two of the genes, SLC6A8 and MSSK1, are tissue-specifically expressed. CDM is expressed ubiquitously. The pseudogene, psi SLC6A8, is exclusively expressed in the testis. The promoter regions of the SLC6A8, MSSK1 and CDM genes were found to be essentially unmethylated in all tissues, regardless of their relative expression level. In contrast, the pseudogene psi SLC6A8 shows high methylation of the CpG islands in all somatic tissues but complete demethylation in testis. Methylation profiles in different tissues are similar in shape but not identical. The data for the two investigated individuals suggest that methylation profiles of individual genes are tissue specific. Taken together, our findings support a model in which the bodies of the genes are predominantly methylated and thus insulated from the interaction with DNA-binding proteins. Only unmethylated promoter regions are accessible for binding and interaction. Based on this model we propose to use DNA methylation studies in conjunction with large-scale sequencing approaches as a tool for the prediction of cis-acting genomic regions, for the identification of cryptic and potentially active CpG islands and for the preliminary distinction of genes and pseudogenes.     

DNA variability of human genes

We have investigated the level of DNA-based variation (both SNPs and haplotypes) for several thousand human genes. In addition, we have characterized how this variation is distributed in a number of biologically and clinically important ways. First, we have determined how SNPs are distributed within human genes: where they occur relative to various functional regions; levels of variability of human SNPs; pattern of the molecular sequence of SNPs; and how these compare with the corresponding sequence of a chimpanzee. Second, we have determined how these aspects of SNP distribution vary among four human population samples. All genes were sequenced on DNA obtained from 82 unrelated individuals: 20 African-Americans, 20 East Asians, 21 European-Americans, 18 Hispanic-Latinos and three Native Americans. In particular, we looked at patterns of SNP and haplotype sharing among the four larger population samples. Third, we have determined the patterns of linkage disequilibrium among SNPs, which also determines the haplotype variability of each gene. These characteristics also vary substantially among populations. A deeper understanding of these aspects of human genetic variation will be of vital importance when trying to identify the genetic contribution to complex phenotypes such as aging.     

Prediction of methylation CpGs and their methylation degrees in human DNA sequences

DNA methylation plays a key role in the regulation of gene expression. The most common type of DNA modification consists of the methylation of cytosine in the CpG dinucleotide. The detections of DNA methylation have been determined mostly by experimental methods, which were time-consuming and expensive, difficult to meet the requirements of modern large-scale sequencing technology. Accordingly, it is necessary to develop automatic, reliable prediction methods for DNA methylation. In this study, the trinucleotide composition, a 64-dimensional feature vector of the occurrence frequency of 64 trinucleotides in the DNA sequence, was utilized to model SVM for the prediction of CpG methylation degrees in humans. The model was evaluated by jackknife validation and the correlation coefficient (R) and root-mean-square error (RMSE) were 0.8223 and 0.2042, respectively. The proposed method was also used to predict methylation sites, the model was evaluated by jackknife validation and the Matthews correlation coefficient (MCC) and accuracy (ACC) were 0.6263 and 0.8133, respectively. The good results indicated that the proposed method was a useful tool for the investigation of DNA methylation prediction research.     

Molecular detection of B-cell neoplasms by specific DNA methylation biomarkers

A novel, easy to perform PCR-based method employing specific DNA methylation biomarkers to detect B-cell neoplasms in a variety of B-cell lines and B lymphoblastic leukemia (B-ALL) patient specimens has been developed. This method detects as few as 5 B-ALL cells, or 1 B-ALL cell in 1,000,000 normal background blood cells using a single marker, DLC-1 gene CpG island (CGI) methylation. By adding two additional markers PCDHGA12 and RPIB9, over 80% of B-ALL cases were detected in patients’ bone marrow and/or peripheral blood specimens. We have traced clinical B-ALL cases up to 10 years retrospectively and the DLC-1 methylation is correlated with patient clinical status. Thus, this epigenetic-based molecular method demonstrates its potential use in the diagnosis of B-cell neoplasia, in addition to traditional approach such as clinical features, morphology, immunophenotype, and genetic analysis.     

Analysis of IMGSAC autism susceptibility loci: evidence for sex limited and parent of origin specific effects

Background and methods: Autism is a severe neurodevelopmental disorder, which has a complex genetic predisposition. The ratio of males to females affected by autism is approximately 4:1, suggesting that sex specific factors are involved in its development. We reported previously the results of a genomewide screen for autism susceptibility loci in 83 affected sibling pairs (ASP), and follow up analysis in 152 ASP. Here, we report analysis of an expanded sample of 219 ASP, using sex and parent of origin linkage modelling at loci on chromosomes 2, 7, 9, 15, and 16.

Results: The results suggest that linkage to chromosomes 7q and 16p is contributed largely by the male–male ASP (MLS = 2.55 v 0.12, and MLS = 2.48 v 0.00, for the 145 male–male and 74 male–female/female–female ASP on chromosomes 7 and 16 respectively). Conversely linkage to chromosome 15q appears to be attributable to the male–female/female–female ASP (MLS = 2.62 v 0.00, for non-male and male–male ASP respectively). On chromosomes 2 and 9, all ASP contribute to linkage. These data, supported by permutation, suggest a possible sex limited effect of susceptibility loci on chromosomes 7, 15, and 16. Parent of origin linkage modelling indicates two distinct regions of paternal and maternal identity by descent sharing on chromosome 7 (paternal MLS = 1.46 at ~112 cM, and maternal MLS = 1.83 at ~135 cM; corresponding maternal and paternal MLS = 0.53 and 0.28 respectively), and maternal specific sharing on chromosome 9 (maternal MLS = 1.99 at ~30 cM; paternal MLS = 0.02).

Conclusion: These data support the possibility of two discrete loci underlying linkage of autism to chromosome 7, and implicate possible parent of origin specific effects in the aetiology of autism.

     

Analysis of DNA methylation in human BK virus

Human BK virus may cause nephropathy due to viral replication in patients who have undergone renal transplantation. However, the mechanism regulating replication of BKV is still not clear. Previous studies have suggested that epigenetic modifications may play a crucial role in virus replication. In this study, the DNA methylation profiles of five CpG sites located within the promoter/enhancer regions and nine CpG sites located within the early and late coding regions of the replicating BKV genome were investigated. BKV genomic DNA from mature virions and from the early and late phases of replicating BKV were examined for DNA methylation by bisulfite sequencing that covered 14 CpG sites. Our results showed that none of the examined BKV DNA from the various different stages of replication was methylated. This is the first report to analyze the methylation of BKV genomic DNA during viral replication. The results seem to indicate that methylation is not involved in regulation of BKV replication.     

Mixed origin of the current Tunisian population from the analysis of Alu and Alu/STR compound systems

During successive historical periods, Tunisia has been a crossroads of multiple civilizations and their corresponding key population movements. The aim of this study was to provide genetic information relating to the mixed origin of the Tunisian population, and to analyze its genetic relationship with other North African and Mediterranean populations. A set of 16 Alu and 3 Alu/STR compound systems has been analyzed in 268 autochthonous Tunisians from the north-center and the south of the country. Our two sampled populations showed no significant differentiation from one another in any of the three Alu/STR compound systems, whereas the analysis of the 16 Alu markers revealed a significant genetic differentiation between them. A sub-Saharan component shown by the three Alu/STR combinations is more noticeable in our north-center sample than in that of the south. The presence of two Alu/STR combinations specific to North African ancestral populations also suggests that the ancient Berber component is relatively more substantial in the north and center regions than in the south. Our Tunisian samples cluster together with other Berber samples from Morocco and Algeria, underpinning the genetic similarity among North Africans regardless of their current linguistic status (Berber or Arabic).     

人类基因百科全书——ENCODE

DNA元件百科全书计划是美国人类基因组研究院在2003年9月启动的研究项目,旨在解析人类基因组中的所有功能性元件.该项目包含试点和技术推广运用两个阶段,试点阶段选取约1％的人类基因组进行研究,以此来比较所使用研究方法的准确性和可靠性,然后推广运用到全基因组研究.第二阶段从2007年9月开始迅速开展起来,在147种不同细胞类型中开展实验,产生了1640个数据集,这一阶段的研究成果于2012年9月以30篇学术论文的形式发表于国际主流SCI期刊杂志上.ENCODE项目是继人类基因组工程之后的又一重大项目;研究结果表明80.4％的人类基因组都具有生物学活性;其关注的不仅仅是功能性的基因,而是包含了人类基因组序列中的所有功能元件,包括基因、转录本和转录调控区,连同DNA甲基化模式、组蛋白修饰和染色质状态等.这项计划的主要目标是为科学界和医学界提供关于人类基因组重要功能元件的高质量和全方位的注解,从而全面理解人类基因组的功能.本文对ENCODE项目的相关信息进行综述,旨在使人们能认识和应用ENCODE项目的研究成果更好的为疾病预防和人类健康服务.     

Alterations of DNA methylation and clinicopathological diversity of human cancers

Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication.     

DNA methylation profiles of human active and inactive X chromosomes

X-chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. To characterize epigenetic changes that accompany this process, we measured DNA methylation levels in 45,X patients carrying a single active X chromosome (X(a)), and in normal females, who carry one X(a) and one inactive X (X(i)). Methylated DNA was immunoprecipitated and hybridized to high-density oligonucleotide arrays covering the X chromosome, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands (CGIs). While the majority of CGIs show increased methylation levels on the X(i), XCI actually results in significant reductions in methylation at 7% of CGIs. Both intra- and inter-genic CGIs undergo epigenetic modification, with the biggest increase in methylation occurring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI generally have low levels of promoter methylation, while genes that show inter-individual variation in silencing show intermediate increases in methylation. Thus, promoter methylation and susceptibility to XCI are correlated. We also observed a global correlation between CGI methylation and the evolutionary age of X-chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We used our epigenetic map to predict 26 novel genes escaping XCI, and searched for parent-of-origin-specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides a detailed analysis of the epigenetic profile of active and inactive X chromosomes.     

Interindividual variability of electromyographic patterns and pedal force profiles in trained cyclists

The aim of this study was to determine whether high inter-individual variability of the electromyographic (EMG) patterns during pedaling is accompanied by variability in the pedal force application patterns. Eleven male experienced cyclists were tested at two submaximal power outputs (150 and 250 W). Pedal force components (effective and total forces) and index of mechanical effectiveness were measured continuously using instrumented pedals and were synchronized with surface electromyography signals measured in ten lower limb muscles. The intersubject variability of EMG and mechanical patterns was assessed using standard deviation, mean deviation, variance ratio and coefficient of cross-correlation ( with lag time = 0). The results demonstrated a high intersubject variability of EMG patterns at both exercise intensities for biarticular muscles as a whole (and especially for Gastrocnemius lateralis and Rectus femoris) and for one monoarticular muscle (Tibialis anterior). However, this heterogeneity of EMG patterns is not accompanied by a so high intersubject variability in pedal force application patterns. A very low variability in the three mechanical profiles (effective force, total force and index of mechanical effectiveness) was obtained in the propulsive downstroke phase, although a greater variability in these mechanical patterns was found during upstroke and around the top dead center, and at 250 W when compared to 150 W. Overall, these results provide additional evidence for redundancy in the neuromuscular system.