Analysis of CpG C-to-T mutations in neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a dominant disorder caused by mutations in the NF1 gene; approximately 100 NF1 gene mutations have been published. The CpG C-to-T transition is a frequent mutation mechanism in genetic disorders. To estimate its frequency in NF1, we employed a PCR-restriction digestion method to examine 17 CpGs in 65 patients, and also screened for a CpG nonsense transition (R1947X) that occurs in 1-2% of patients. The analysis revealed disease-related CpG C-to-T transitions (including a nonsense mutation that may be as frequent as R1947X) as well as a benign variant and another mutation at a CpG. Four patients showed CpG mutations in analysis of 18 sites (17 surveyed by restriction digest, plus the R1947X assay), including three C-to-T transitions and one C-to-G transversion. These 18 sites represent one-fifth of the 91 CpGs at which a C-to-T transition would result in a nonsense or nonconservative missense mutation. Thus, it is feasible that the CpG mutation rate at NF1 might be similar to that seen in other disorders with a high mutation rate, and that recurrent NF1 mutations may frequently reside at CpG sites. Hum Mutat 11:411, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of phenotype between patients with Prader-Willi syndrome due to deletion 15q and uniparental disomy 15

Prader-Willi syndrome (PWS) is a complex multiple anomaly syndrome that has been shown to result from deficient expression of paternal chromosome 15(q11-q13). In most cases, it is caused either by deletion of this region in the paternally inherited chromosome 15 or by maternal uniparental disomy (UPD) of chromosome 15. In order to determine whether there are phenotypic differences between patients whose PWS is caused by these two different mechanisms, 54 affected individuals (37 with deletion, 17 with UPD) were personally examined and studied using molecular techniques. The previously recognized increased maternal age in patients with UPD and increased frequency of hypopigmentation in those with deletion were confirmed. Although the frequency and severity of most other manifestations of PWS did not differ significantly between the two groups, those with UPD were less likely to have a “typical” facial appearance. In addition, this group was less likely to show some of the minor manifestations such as skin picking, skill with jigsaw puzzles, and high pain threshold. Females and those with UPD were also older, on average. Possible mechanisms by which these differences could occur and the implications of these differences for diagnosis are described. Am. J. Med. Genet. 68:433–440, 1997. © 1997 Wiley-Liss, Inc.     

Methylenetetrahydrofolate reductase variant and schizophrenia/depression

Patients with methylenetetrahydrofolate reductase (MTHFR) deficiency often show psychiatric manifestations. Since a common variant of the MTHFR gene, T677(Ala), responsible for the thermolabile MTHFR with less than 50% specific MTHFR activity, has been reported, we examined whether the T677 allele is associated with psychiatric disorders in an unrelated Japanese population consisting of 297 schizophrenics, 32 patients with major depression, 40 patients with bipolar disorder, and 419 controls. The genotype homozygous for the T677 allele was significantly frequently observed in schizophrenics with an odds ratio of 1.9 (P = 0.0006), and in patients with major depression with an odds ratio of 2.8 (P = 0.005). Our data suggest associations of the MTHFR gene variant with schizophrenia and depression in the Japanese. Am. J. Med. Genet. 74:526–528, 1997. © 1997 Wiley-Liss, Inc.     

表观遗传与肿瘤代谢研究进展

表观遗传学主要关注DNA甲基化、组蛋白修饰、染色质重塑，以及非编码RNA等超越DNA序列的基因调控机制。表观遗传机制参与了个体发育、细胞命运决定和肿瘤发生等众多生物学过程。其中表观遗传信息以各种染色质修饰和高级结构的形式存储于基因组中，它的建立和维持与细胞代谢紧密相关。肿瘤细胞中存在的代谢改变包括有氧糖酵解、葡萄糖摄取量增加、谷氨酰胺代谢异常活跃、利用非主要供能物质供能等，这些改变满足了肿瘤发生发展过程中旺盛的能量和物质需求，帮助细胞适应缺氧的肿瘤微环境，进而为肿瘤增殖、侵袭、迁移等生物活动提供支持。肿瘤细胞的表观遗传修饰与代谢之间存在复杂的相互关系，一方面肿瘤细胞中的代谢产物作为表观修饰酶的辅因子、修饰供体或拮抗分子影响表观修饰景观；另一方面表观遗传修饰可以直接改变代谢酶和转运蛋白的表达或通过影响信号转导和转录因子的表达调控细胞代谢。本文综述了不同表观遗传学过程与肿瘤细胞代谢之间的相互作用，并展望两者在肿瘤治疗中的潜在应用前景。     

Epigenetic clocks in the pediatric population: when and why they tick?

Recent research efforts have provided compelling evidence of genome-wide DNA methylation alterations in pediatrics. It is currently well established that epigenetic clocks, composed of DNA methylation sites, can estimate the gestational and chronological age of cells and tissues from different ages. Also, extensive research is aimed at their correlation with early life exposure and pediatric diseases. This review aimed to systematically summarize the epigenetic clocks in the pediatric population. Publications were collected from PubMed and Web of Science databases up to Apr 2021. Epigenetic clocks, DNA methylation clocks, epigenetic age acceleration or deceleration, pediatric and the pediatric population were used as search criteria. Here, we first review the currently applicative pediatric epigenetic clocks. We then highlight the interpretation for epigenetic age deviations in the pediatric population and their association with external factors, developmental trajectories, and pediatric diseases. Considering the remaining unknown of pediatric clocks, research strategies into them are also discussed. In all, pediatric epigenetic clocks may act as potent tools to understand development, growth and diseases in early life.     

外周血GP5、APC、GSTP1基因甲基化联合检测与乳腺癌相关性的研究

目的探索外周血GP5、APC、GSTP1基因甲基化联合检测与乳腺癌相关性。方法收集了2020年5月—2020年11月同济大学附属东方医院就诊的172例女性患者,其中有35例乳腺癌患者,90例乳腺良性疾病患者和47例其他组织来源的恶性肿瘤患者。实时荧光定量PCR法测定外周血中GP5、APC、GSTP1的基因甲基化表达水平,并与癌胚抗原（carcinoembryonic antigen, CEA）、糖类抗原153（carbohydrate antigen 153, CA153）、糖类抗原125（carbohydrate antigen 125, CA125）及乳腺钼靶进行比较。通过一致性检验和受试者工作特征（receiver operating characteristic, ROC）曲线分析并比较不同检测方法对乳腺癌的评价指标。分析三种基因联合检测的甲基化阳性率与乳腺癌临床病理特征相关性。结果35例乳腺癌患者和90例乳腺良性疾病患者外周血GP5、APC、GSTP1基因甲基化联合检测与组织病理结果的Kappa一致性为0.770。ROC曲线分析外周血基因甲基化联合检测的曲线下面积（area under the curve, AUC）为0.913(95%CI： 0.849~0.956),其中灵敏度为88.57%,特异度为92.22%,高于乳腺钼靶（AUC=0.765）,高于血清CEA、CA125、CA153联合检测（AUC=0.761）。外周血基因甲基化与3种血清肿瘤指标联合检测的AUC最高为0.949（95%CI： 0.894~0.980)。35例乳腺癌患者外周血GP5、APC、GSTP1基因甲基化联合检测表达阳性率（88.6%）高于3种血清肿瘤标志物联合检测阳性率（28.6%）;术前外周血基因甲基化表达的阳性率与年龄、淋巴结转移、肿瘤分期、肿瘤类型及脉管侵犯均无关（P>0.05）。47例其他组织来源恶性肿瘤与35例乳腺癌患者比较分析结果显示,外周血GP5、APC、GSTP1基因甲基化检测与病理的Kappa一致性为0.849。ROC曲线分析外周血基因甲基化检测的AUC为0.936(95%CI： 0.859~0.978),灵敏度为88.57%,特异度高达95.74%。结论外周血GP5、APC、GSTP1基因甲基化联合检测具有较高的灵敏度和特异度。外周血GP5、APC、GSTP1基因甲基化有望成为诊断乳腺癌的非侵入性生物标志物。     

DNA methylation profile in patients with indolent systemic mastocytosis

BackgroundMastocytosis is a clinically heterogeneous, usually acquired disease of the mast cells with a survival time that depends on the onset of the disease and ranges from skin‐limited to systemic disease, including indolent and more aggressive variants. The crucial element in pathogenesis is the presence of oncogenic KIT somatic mutation D816V. Further epigenetic alterations are responsible for regulating the expression of genes. It is essential to identify indicators of disease progression, and the specific clinical picture to establish an appropriate therapeutic strategy.ObjectiveThe aim of this study was to analyze the relation of mastocytosis symptoms and epigenetic changes, and to identify epigenetic predictors of the disease.MethodsGlobal DNA methylation profile analysis was performed in peripheral blood collected from 73 patients with indolent systemic mastocytosis (ISM) and 43 healthy adult volunteers. Levels of 5‐methylcytosine (5‐mC) and 5‐hydroxymethylcytosine (5‐hmC) were determined using an ELISA‐based method, while the methylation of the Alu and LINE‐1 repeats were assayed with the quantitative methylation‐specific PCR technique. A questionnaire interview was conducted among the study participants to collect data on possible epigenetic modifiers. Additionally, the methylation profile was compared between three human mast cell lines: ROSA KIT D816V, ROSA KIT WT, and HMC‐1.1 KIT V560G, in order to assess the association between KIT mutations and methylation profile.ResultsA significantly lower level of DNA hydroxymethylation (5‐hmC) in the blood was found in patients with ISM as compared to the controls (0.022% vs. 0.042%, p = 0.0001). Differences in the markers of global DNA methylation (5‐mC, Alu, LINE‐1) were not statistically significant, although they did indicate generally higher DNA methylation in patients with mastocytosis. The 5‐hmC level was significantly associated with allergy (p = 0.011) in patients with ISM, showing a higher level of 5‐hmC in patients with allergy as compared to patients without allergy. The in vitro study revealed significant differences between the studied cell lines at the level of 5‐mC, Alu, and LINE‐1.ConclusionsThis study confirms that epigenetic changes are involved in mastocytosis, and suggests that allergy may be an important epigenetic modifier of the disease. A possible association between KIT mutations and methylation status observed in human mast cell lines requires further investigation in human studies.Clinical ImplicationsEpigenetic alterations are involved in mastocytosis pathology. The possible role of allergy as an important epigenetic modifier suggests the more impaired function of mast cells in ISM patients without allergy.Capsule summaryDecreased DNA demethylation in the blood DNA of patients with ISM confirms that epigenetic alterations are involved in mastocytosis pathology. We observed a possible role of allergy as an important epigenetic modifier. There is a possible association between KIT mutations and the methylation status observed in human mast cell lines.     

DNA methylation markers have universal prognostic value for anal cancer risk in HIV‐negative and HIV‐positive individuals

Anal cancer has increasing incidence and is preceded by high‐grade anal intraepithelial neoplasia (HGAIN; AIN2–3). Previously, we identified and validated several methylation markers for accurate detection of anal cancer and HGAIN with cancer risk in HIV‐positive (HIV+) men who have sex with men (MSM). This study aimed to evaluate these markers in HIV‐negative risk groups. A cross‐sectional series of 176 tissue samples of anal cancer, AIN3, AIN2, AIN1 and control biopsies obtained in HIV‐negative women and men was tested for six methylation markers (ASCL1, LHX8, SST, WDR17, ZIC1 and ZNF582). Accuracy for detection of AIN3 and cancer (AIN3+) was determined by univariable and multivariable mixed‐effect ordinal logistic regression. Methylation levels of all markers increased with increasing severity of disease (P < 0.0001) and were comparable to results in HIV+ MSM. All markers showed high accuracy for AIN3+ detection [area under the curve (AUC): 0.83–0.86]. The optimal marker panel (ASCL1 and ZIC1; AUC = 0.85 for AIN3+) detected 98% of cancers at 79% specificity. In conclusion, DNA methylation markers show a high diagnostic performance for AIN3+ detection in HIV+ and HIV‐negative risk groups, justifying broad application of methylation analysis for anal cancer prevention programmes.     

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.     

Downregulation of Waf1, C2, C3, and major histocompatibility complex class I loci within an 18-cM region of chromosome 17 in adenovirus-transformed mouse cells

In this study, the expression of the p53 tumor suppressor gene and the p53-regulated Mdm2 and Waf1 genes was evaluated in adenovirus (Ad)-transformed mouse cells. The expected levels of p53 mRNA and protein and Mdm2 mRNA were detected in all transformed cells. However, the level of Waf1 mRNA was markedly reduced in Ad12-transformed cells and in some Ad5-transformed cells. Waf1 expression was not reduced in untransformed mouse cells infected with Ad12 or Ad5. Expression of the class 1 major histocompatibility complex (MHC) locus was downregulated in 13 Ad-transformed cell lines (derived from four different strains of mice) that exhibited reduced expression of Waf1. Waf1 is located in mouse chromosome 17 proximal to the MHC class I locus. To determine whether other chromosome 17 genes were downregulated, the cells were examined for expression of other genetic loci. Of those tested, only the C2 and C3 complement loci were expressed in mouse fibroblasts. Expression of C2 (which is within the MHC) and expression of C3 (which is 15 cM distal to the MHC) were downregulated in those transformed cells in which Waf1 and MHC class I were downregulated. The Ad12- and Ad5-transformed cells that expressed low levels of Waf1, MHC class I, C2, and C3 formed tumors in syngeneic adult mice. These data suggest that the downregulation of multiple genes within the 32 Mb of mouse chromosome 17 that includes the Waf1 locus to the C3 locus occurs in Ad mouse-cell transformation and may contribute to the tumorigenicity of transformed cells. Mol. Carcinog. 18:213–220, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.