Deep Causal Learning: Representation, Discovery and Inference

Causal learning has attracted much attention in recent years because causality reveals the essential relationship between things and indicates how the world progresses. However, there are many problems and bottlenecks in traditional causal learning methods, such as high-dimensional unstructured variables, combinatorial optimization problems, unknown intervention, unobserved confounders, selection bias and estimation bias. Deep causal learning, that is, causal learning based on deep neural networks, brings new insights for addressing these problems. While many deep learning-based causal discovery and causal inference methods have been proposed, there is a lack of reviews exploring the internal mechanism of deep learning to improve causal learning. In this article, we comprehensively review how deep learning can contribute to causal learning by addressing conventional challenges from three aspects: representation, discovery, and inference. We point out that deep causal learning is important for the theoretical extension and application expansion of causal science and is also an indispensable part of general artificial intelligence. We conclude the article with a summary of open issues and potential directions for future work

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

<p>Abstract</p> <p>Background</p> <p><it>Alu </it>methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within <it>Alu </it>elements (<it>Alu</it>) and its methylation status have not well characterized. This information is useful for understanding natural status of <it>Alu </it>in the genome and helpful for developing an optimal assay to quantify <it>Alu </it>hypomethylation.</p> <p>Methods</p> <p>Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A <it>Cac</it>8I COBRA-DHPLC assay was developed to detect methylated-<it>Alu </it>proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0.</p> <p>Results</p> <p>From the results of 427 <it>Alu </it>bisulfite clone sequences, we found that only 27.2% of CpG sites within <it>Alu </it>elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between <it>Alu </it>clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-<it>Alu </it>in a <it>Cac</it>8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of <it>Alu </it>elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.<it>Sss</it>I, respectively. The proportion of methylated-<it>Alu </it>copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%).</p> <p>Conclusions</p> <p>Most <it>Alu </it>CpG sites are deaminated in the genome. 27% of <it>Alu </it>CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. <it>Alu </it>hypomethylation in primary gastric carcinomas could be detected with the <it>Cac</it>8I COBRA-DHPLC assay quantitatively.</p

P16 Methylation as an Early Predictor for Cancer Development From Oral Epithelial Dysplasia: A Double-blind Multicentre Prospective Study

AbstractBackgroundSilencing of P16 through methylation and locus deletion is the most frequent early events in carcinogenesis. The aim of this study is to prospectively determine if early P16 methylation is a predictor for oral cancer development.MethodsPatients (n=181) with mild or moderate oral epithelial dysplasia (OED) were recruited into the double blind multicentre cohort. P16 methylation was analyzed using the MethyLight assay. Progression of OEDs was monitored for a minimum 3year follow-up period.FindingsP16 methylation-informative cases (n=152) were enrolled in the prospective multicenter cohorts with an ultimate compliance of 96.7%. OED-derived squamous cell carcinomas were observed in 21 patients (14.3%) during the follow-up (median, 41.0months). The cancer progression rate from the P16 methylation-positive patients was significantly increased when compared to P16 methylation-negative patients [27.1% vs 8.1%; adjusted odds ratio=4.6; P=0.006]. When the P16 methylation-positive criteria were used as a biomarker for early prediction of cancer development from OEDs, sensitivity and specificity of 62% and 76% were obtained, respectively.InterpretationP16 methylation is unequivocally a marker for determining the malignant potential of OED and there is no need for further research regarding this aspect.FundingNational Basic Research Programs of China (2011CB504201 and 2015CB553902), Beijing Science and Technology Commission (Z090507017709016), and Beijing Municipal Administration of Hospital (XM201303) to Dajun Deng. The funding agencies have no role in the actual experimental design, patient recruitment, data collection, analysis, interpretation, or writing of this manuscript

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7

Background: It is well known that P16INK4A, P14ARF, P15INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16INK4A expression is mediated with CBX7.Results: In this study, we found that the upregulation of P16INK4A, P14ARF, P15INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p &lt; 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16INK4A, P14ARF and P15INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells.Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16INK4A, P14ARF, P15INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters

Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history

<p>Abstract</p> <p>Background</p> <p>A2143G mutation of <it>23S rRNA </it>gene of <it>H. pylori </it>results in clarithromycin (CLR) resistance. To investigate the prevalence of the CLR resistance-related A2143G mutation of the <it>H. pylori</it>-specific <it>23S rRNA </it>gene in Chinese subjects with and without CLR use history, 307 subjects received the treatment with amoxicillin and omeprazole (OA) and 310 subjects received a placebo in 1995, and 153 subjects received a triple therapy with OA and CLR (OAC) in 2000. DNA was extracted from fasting gastric juice at the end of the intervention trial in 2003. <it>H. pylori </it>infection was determined by <it>H. pylori</it>-specific <it>23S rRNA </it>PCR, ELISA, and¹³C-urea breath test assays. Mutations of the <it>23S rRNA </it>gene were detected by RFLP assays.</p> <p>Results</p> <p>The presence of <it>23S rRNA </it>due to <it>H. pylori </it>infection in the OA group remained lower than that in the placebo group 7.3 yrs after OA-therapy [51.1% (157/307) vs. 83.9% (260/310), p = 0.0000]. In the OAC group, the <it>23S rRNA </it>detection rate was 26.8% (41/153) three yrs after OAC-treatment. The A2143G mutation rate among the <it>23S rRNA</it>-positive subjects in the OAC group [31.7% (13/41)] was significantly higher than that in the OA group [10.2% (16/157)] and the placebo group [13.8% (36/260)]. The frequency of the AAGGG → CTTCA (2222–2226) and AACC → GAAG (2081–2084) sequence alterations in the OAC group was also significantly higher than those in the OA group and the placebo group.</p> <p>Conclusion</p> <p>Primary prevalence of the A2143G mutation was 10~14% among Chinese population without history of CLR therapy. Administration of CLR to eliminate <it>H. pylori </it>infection increased the prevalence of the A2143G mutation in Chinese subjects (32%) significantly.</p

A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues

<p>Abstract</p> <p>Background</p> <p><it>p16 </it>Methylation is a potential biomarker for prediction of malignant transformation of epithelial dysplasia. A probe-based, quantitative, methylation-specific PCR (MSP) called MethyLight may become an eligible method for detecting this marker clinically. We studied oral mucosa biopsies with epithelial dysplasia from 78 patients enrolled in a published 4-years' followup cohort, in which cancer risk for patients with <it>p16 </it>methylation-positive dysplasia was significantly higher than those without <it>p16 </it>methylation (by 150-bp MSP and bisulfite sequencing; +133 ~ +283, transcription starting site, +1). The <it>p16 </it>methylation status in samples (<it>N </it>= 102) containing sufficient DNA was analyzed by the 70-bp classic (+238 ~ +307) and 115-bp novel (+157 ~ +272) MethyLight assays, respectively.</p> <p>Results</p> <p><it>p16 </it>Methylation was detectable in 75 samples using the classic MethyLight assay. The methylated-<it>p16 </it>positive rate and proportion of methylated-<it>p16 </it>by the MethyLight in MSP-positive samples were higher than those in MSP-negative samples (positive rate: 37/44 vs. 38/58, <it>P</it>=0.035, two-sided; proportion [median]: 0.78 vs. 0.02, <it>P <</it>0.007). Using the published results of MSP as a golden standard, we found sensitivity, specificity, and accuracy for this MethyLight assay to be 70.5%, 84.5%, and 55.0%, respectively. Because amplicon of the classic MethyLight procedure only partially overlapped with the MSP amplicon, we further designed a 115-bp novel MethyLight assay in which the amplicon on the sense-strand fully overlapped with the MSP amplicon on the antisense-strand. Using the 115-bp MethyLight assay, we observed methylated-<it>p16 </it>in 26 of 44 MSP-positive samples and 2 of 58 MSP-negative ones (<it>P </it>= 0.000). These results were confirmed with clone sequencing. Sensitivity, specificity, and accuracy using the 115-bp MethyLight assay were 59.1%, 98.3%, and 57.4%, respectively. Significant differences in the oral cancer rate were observed during the followup between patients (≥60 years) with and without methylated-<it>p16 </it>as detected by the 115-bp MethyLight assay (6/8 vs. 6/22, P = 0.034, two-sided).</p> <p>Conclusions</p> <p>The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the detection of <it>p16 </it>methylation clinically.</p

Nucleosomes Correlate with In Vivo Progression Pattern of De Novo Methylation of p16 CpG Islands in Human Gastric Carcinogenesis

BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP) assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation "seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC) samples (36/40) was significantly higher than that observed in gastritis (19/45) or normal samples (7/13) (P<0.01). Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5'UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01). In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens. CONCLUSIONS/SIGNIFICANCE: It was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5'UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo

Polycomb CBX7 Directly Controls Trimethylation of Histone H3 at Lysine 9 at the p16 Locus

BACKGROUND: H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter