Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood.
ABSTRACT: BACKGROUND: Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers. RESULTS: Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMR) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients. CONCLUSIONS: These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.
Project description:Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.
Project description:BACKGROUND: There is a substantial genetic component for birthweight variation, and although there are known associations between fetal genotype and birthweight, the role of common epigenetic variation in influencing the risk for small for gestational age (SGA) is unknown. The two imprinting control regions (ICRs) located on chromosome 11p15.5, involved in the overgrowth disorder Beckwith-Wiedemann syndrome (BWS) and the growth restriction disorder Silver-Russell syndrome (SRS), are prime epigenetic candidates for regulating fetal growth. We investigated whether common variation in copy number in the BWS/SRS 11p15 region or altered methylation levels at IGF2/H19 ICR or KCNQ10T1 ICR was associated with SGA. METHODS: We used a methylation-specific multiplex-ligation-dependent probe amplification assay to analyse copy number variation in the 11p15 region and methylation of IGF2/H19 and KCNQ10T1 ICRs in blood samples from 153 children (including 80 SGA), as well as bisulfite pyrosequencing to measure methylation at IGF2 differentially methylated region (DMR)0 and H19 DMR. RESULTS: No copy number variants were detected in the analyzed cohort. Children born SGA had 2.7% lower methylation at the IGF2 DMR0. No methylation differences were detected at the H19 or KCNQ10T1 DMRs. CONCLUSIONS: We confirm that a small hypomethylation of the IGF2 DMR0 is detected in peripheral blood leucocytes of children born SGA at term. Copy number variation within the 11p15 BWS/SRS region is not an important cause of non-syndromic SGA at term.
Project description:Genome wide DNA methylation profiling of normal kidney (n=36), nephrogenic rest (n=22) and Wilms tumour (n=37) was performed using the Illumina 450k array. Two papers were composed after analysis of this data (1) describes comparative analysis of 22 matched normal kidney-Wilms tumour pairs which identified biomarker differentially methylated regions (DMRs) that could be detected in patient blood; (2) describes comparative analysis of 20 matched trios which identified changes in methylation associated with progression from the precursor lesion towards tumourigenesis. Bisulfte converted DNA from 95 samples of normal kidney, nephrogenic rest and Wilms tumour was hybridised to Illumina HumanMethylation450 bead chips.
Project description:DNA methylation profiling reveals important differentially methylated regions (DMRs) of the genome that are altered during development or that are perturbed by disease. To date, few programs exist for regional analysis of enriched or whole-genome bisulfate conversion sequencing data, even though such data are increasingly common. Here, we describe an open-source, optimized method for determining empirically based DMRs (eDMR) from high-throughput sequence data that is applicable to enriched whole-genome methylation profiling datasets, as well as other globally enriched epigenetic modification data.Here we show that our bimodal distribution model and weighted cost function for optimized regional methylation analysis provides accurate boundaries of regions harboring significant epigenetic modifications. Our algorithm takes the spatial distribution of CpGs into account for the enrichment assay, allowing for optimization of the definition of empirical regions for differential methylation. Combined with the dependent adjustment for regional p-value combination and DMR annotation, we provide a method that may be applied to a variety of datasets for rapid DMR analysis. Our method classifies both the directionality of DMRs and their genome-wide distribution, and we have observed that shows clinical relevance through correct stratification of two Acute Myeloid Leukemia (AML) tumor sub-types.Our weighted optimization algorithm eDMR for calling DMRs extends an established DMR R pipeline (methylKit) and provides a needed resource in epigenomics. Our method enables an accurate and scalable way of finding DMRs in high-throughput methylation sequencing experiments. eDMR is available for download at http://code.google.com/p/edmr/.
Project description:The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.
Project description:BACKGROUND:Silver-Russell syndrome (SRS) is characterized by growth failure and dysmorphic features. Major (epi)genetic causes of SRS are loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy of chromosome 7 (upd(7)mat). However, IGF2, CDKN1C, HMGA2, and PLAG1 mutations infrequently cause SRS. In addition, other imprinting disturbances, pathogenic copy number variations (PCNVs), and monogenic disorders sometimes lead to SRS phenotype. This study aimed to clarify the frequency and clinical features of the patients with gene mutations among etiology-unknown patients with SRS phenotype. RESULTS:Multigene sequencing was performed in 92 out of 336 patients referred to us for genetic testing for SRS. The clinical features of the patients were evaluated based on the Netchine-Harbison clinical scoring system. None of the patients showed 11p15 LOM, upd(7)mat, abnormal methylation levels for six differentially methylated regions (DMRs), namely, PLAGL1:alt-TSS-DMR on chromosome 6, KCNQ1OT1:TSS-DMR on chromosome 11, MEG3/DLK1:IG-DMR on chromosome 14, MEG3:TSS-DMR on chromosome 14, SNURF:TSS-DMR on chromosome 15, and GNAS A/B:TSS-DMR on chromosome 20, PCNVs, or maternal uniparental disomy of chromosome 16. Using next-generation sequencing and Sanger sequencing, we screened four SRS-causative genes and 406 genes related to growth failure and/or skeletal dysplasia. We identified four pathogenic or likely pathogenic variants in responsible genes for SRS (4.3%: IGF2 in two patients, CDKN1C, and PLAG1), and five pathogenic variants in causative genes for known genetic syndromes presenting with growth failure (5.4%: IGF1R abnormality (IGF1R), SHORT syndrome (PIK3R1), Floating-Harbor syndrome (SRCAP), Pitt-Hopkins syndrome (TCF4), and Noonan syndrome (PTPN11)). Functional analysis indicated the pathogenicity of the CDKN1C variant. The variants we detected in CDKN1C and PLAG1 were the second and third variants leading to SRS, respectively. Our patients with CDKN1C and PLAG1 variants showed similar phenotypes to previously reported patients. Furthermore, our data confirmed IGF1R abnormality, SHORT syndrome, and Floating-Harbor syndrome are differential diagnoses of SRS because of the shared phenotypes among these syndromes and SRS. On the other hand, the patients with pathogenic variants in causative genes for Pitt-Hopkins syndrome and Noonan syndrome were atypical of these syndromes and showed partial clinical features of SRS. CONCLUSIONS:We identified nine patients (9.8%) with pathogenic or likely pathogenic variants out of 92 etiology-unknown patients with SRS phenotype. This study expands the molecular spectrum of SRS phenotype.
Project description:BACKGROUND: Aberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors. METHODS: The methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms. RESULTS: Among 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations. CONCLUSIONS: Our analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma.
Project description:We systemically identified tuberculosis (TB)-related DNA methylation biomarkers and further constructed classifiers for TB diagnosis. TB-related DNA methylation datasets were searched through October 3, 2020. Limma and DMRcate were employed to identify differentially methylated probes (DMPs) and regions (DMRs). Machine learning methods were used to construct classifiers. The performance of the classifiers was evaluated in discovery datasets and a prospective independent cohort. Eighty-nine DMPs and 24 DMRs were identified based on 67 TB patients and 45 healthy controls from 4 datasets. Nine and three DMRs were selected by elastic net regression and logistic regression, respectively. Among the selected DMRs, two regions (chr3: 195635643-195636243 and chr6: 29691631-29692475) were differentially methylated in the independent cohort (p = 4.19 × 10<sup>-5</sup> and 0.024, respectively). Among the ten classifiers, the 3-DMR logistic regression classifier exhibited the strongest performance. The sensitivity, specificity, and area under the curve were, respectively, 79.1%, 84.4%, and 0.888 in the discovery datasets and 64.5%, 90.3%, and 0.838 in the independent cohort. The differential diagnostic ability of this classifier was also assessed. Collectively, these data showed that DNA methylation might be a promising TB diagnostic biomarker. The 3-DMR logistic regression classifier is a potential clinical tool for TB diagnosis, and further validation is needed.
Project description:We propose a statistical algorithm MethylPurify that uses regions with bisulfite reads showing discordant methylation levels to infer tumor purity from tumor samples alone. MethylPurify can identify differentially methylated regions (DMRs) from individual tumor methylome samples, without genomic variation information or prior knowledge from other datasets. In simulations with mixed bisulfite reads from cancer and normal cell lines, MethylPurify correctly inferred tumor purity and identified over 96% of the DMRs. From patient data, MethylPurify gave satisfactory DMR calls from tumor methylome samples alone, and revealed potential missed DMRs by tumor to normal comparison due to tumor heterogeneity.
Project description:Differential marking of genes in female and male gametes by DNA methylation is essential to genomic imprinting. In female gametes transcription traversing differentially methylated regions (DMRs) is a common requirement for de novo methylation at DMRs. At the imprinted Gnas cluster oocyte specific transcription of a protein-coding transcript, Nesp, is needed for methylation of two DMRs intragenic to Nesp, namely the Nespas-Gnasxl DMR and the Exon1A DMR, thereby enabling expression of the Gnas transcript and repression of the Gnasxl transcript. On the paternal allele, Nesp is repressed, the germline DMRs are unmethylated, Gnas is repressed and Gnasxl is expressed. Using mutant mouse models, we show that on the paternal allele, ectopic transcription of Nesp traversing the intragenic Exon1A DMR (which regulates Gnas expression) results in de novo methylation of the Exon1A DMR and de-repression of Gnas just as on the maternal allele. However, unlike the maternal allele, methylation on the mutant paternal allele occurs post-fertilisation, i.e. in somatic cells. This, to our knowledge is the first example of transcript/transcription driven DNA methylation of an intragenic CpG island, in somatic tissues, suggesting that transcription driven de novo methylation is not restricted to the germline in the mouse. Additionally, Gnasxl is repressed on a paternal chromosome on which Nesp is ectopically expressed. Thus, a paternally inherited Gnas cluster showing ectopic expression of Nesp is "maternalised" in terms of Gnasxl and Gnas expression. We show that these mice have a phenotype similar to mutants with two expressed doses of Gnas and none of Gnasxl.