DNMT1 Y495C mutation interferes with maintenance methylation of imprinting control regions.

Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) is a rare autosomal dominant neurological disorder due to missense mutations in DNA methyltransferase 1 (DNMT1). To investigate the nature of the dominant effect, we compared methylomes of transgenic R1wtDnmt1 and R1Dnmt1Y495C mouse embryonic stem cells (mESCs) overexpressing WT and the mutant mouse proteins respectively, with the R1 (wild-type) cells. In case of R1Dnmt1Y495C, 15 out of the 20 imprinting control regions were hypomethylated with transcript level dysregulation of multiple imprinted genes in ESCs and neurons. Non-imprinted regions, minor satellites, major satellites, LINE1 and IAP repeats were unaffected. These data mirror the specific imprinting defects associated with transient removal of DNMT1 in mESCs, deletion of the maternal-effect DNMT1o variant in preimplantation mouse embryos, and in part, reprogramming to naïve human iPSCs. This is the first DNMT1 mutation demonstrated to specifically affect Imprinting Control Regions (ICRs), and reinforces the differences in maintenance methylation of ICRs over non-imprinted regions. Consistent with nervous system abnormalities in the HSAN1E disorder and involvement of imprinted genes in normal development and neurogenesis, R1Dnmt1Y495C cells showed dysregulated pluripotency and neuron marker genes, and yielded more slender, shorter, and extensively branched neurons. We speculate that R1Dnmt1Y495C cells produce predominantly dimers containing mutant proteins, leading to a gradual and specific loss of ICR methylation during early human development.

Generation of a transgenic mouse embryonic stem cell line overexpressing DNMT1.

DNMT1 overexpression is reported in disorders like schizophrenia, bipolar, epilepsy and multiple cancer types. Here, we used non-homologous recombination to generate R1Dnmt1WT-1, a mouse embryonic stem cell (ESC) line carrying a Dnmt1 cDNA transgene to achieve about two-fold overexpression. This ESC line showed increased transcript levels of Sox2 pluripotency marker. R1Dnmt1WT-1 embryoid bodies showed increased levels of Lefty1 (endoderm), Tbxt and Acta2 (mesoderm), and Pax6 (ectoderm) transcripts. This new line showed normal karyotype and microsatellite profiles making it useful in studying carcinogenesis and abnormal neurogenesis due to DNMT1 overexpression.

Comparative Somatic Variant Analysis of a Rare Case with Concurrent Oral Leukoplakia and Oral Submucosal Fibrosis.

Oral leukoplakia (OL) and oral submucosal fibrosis (OSMF) are precancerous conditions with common etiologies but with different risks for oral cancer (OC) progression. In rare cases, both conditions occur in the same patient and provide an opportunity for understanding the common and distinctive variants upon exposure of genetically identical normal cells to the same carcinogen(s). We performed exome sequencing of a patient with OL (hyperplasia, but no dysplasia) and OSMF (grade II) in the opposite cheeks using blood DNA as the reference genome. The overall somatic variant burden was higher in OSMF than OL, but opposite in the case of copy number alterations. OL-specific variants were enriched in genes associated with DNA repair, cell division/cell cycle checkpoint pathways, whereas in OSMF, extracellular matrix-receptor interaction was mainly affected. The proportions of variants in cancer driver genes and cancer driver mutations were similar in both cases indicating no difference in the potential risk associated with the two conditions at the stages sampled. Future studies on rare cases similar to the one described in this report will help in understanding the molecular basis of differences associated with OL and OSMF and shared processes accompanying OC progression.

Genome-wide methylation analysis of post-mortem cerebellum samples supports the role of peroxisomes in autism spectrum disorder.

Aim: We tested the hypothesis that a subset of patients with autism spectrum disorder (ASD) contains candidate genes with high DNA methylation differences (effective values) that potentially affect one of the two alleles. Materials & methods: Genome-wide DNA methylation comparisons were made on cerebellum samples from 30 patients and 45 controls. Results: 12 genes with high effective values, including GSDMD, MMACHC, SLC6A5 and NKX6-2, implicated in ASD and other neuropsychiatric disorders were identified. Monoallelic promoter methylation and downregulation were observed for SERHL (serine hydrolase-like) and CAT (catalase) genes associated with peroxisome function. Conclusion: These data are consistent with the hypothesis implicating impaired peroxisome function/biogenesis for ASD. A similar approach holds promise for identifying rare epimutations in ASD and other complex disorders.

Genetic susceptibility of autism spectrum disorder (ASD) is well recognized but found only in one among 30­50% of identical twins. Epigenetic modifications such as DNA methylation underlie differences in gene function without alteration in the sequence. In this study, we identified large DNA methylation changes in nine gene promoters in cerebellum tissues of patients with ASD. Several were already implicated in ASD and other neuropsychiatric disorders. Promoters of two genes (SERHL and CAT) involved in peroxisome function showed increased methylation and low-level expression. The two abnormalities identified support the hypothesis associating ASD with defects in peroxisomes that are involved in detoxification of reactive oxygen species.

Somatic Variants and Exon-Level Copy Number Changes in Five Hyperplastic Oral Leukoplakias.

Oral leukoplakia (OL), an oral potentially malignant disorder, begins with a hyperplastic/hyperkeratotic stage at which no genome-scale somatic single nucleotide variant profiles have been described so far. We performed exome sequencing of five cases at this stage with no evidence of dysplasia to identify genetic alterations (exon-level copy number alterations, indels, and single nucleotide variants), their association with transcript levels, and relationship with oral cancer susceptibility. Pathway enrichment analysis of genes associated with tobacco chewing and age-related mutation signatures, transcripts with variants predicted to be functionally damaging and those with significantly altered levels all indicated the involvement of focal adhesion, ECM-receptor interactions, regulation of cytoskeleton, and DNA repair. Two novel mutations identified in FAT1 tumor suppressor gene were associated with decreased transcript levels. In addition, 16 expressed cancer driver genes contained functionally damaging variants. Many of the affected genes were also reported in dysplastic OL lesions. The presence of variants in cancer driver genes and those shared with oral dysplasias possibly provides a basis for further progression and increased susceptibility to oral cancer.

Dysregulation of schizophrenia-associated genes and genome-wide hypomethylation in neurons overexpressing DNMT1.

Aim: To study the effects of DNMT1 overexpression on transcript levels of genes dysregulated in schizophrenia and on genome-wide methylation patterns. Materials & methods: Transcriptome and DNA methylome comparisons were made between R1 (wild-type) and Dnmt1tet/tet mouse embryonic stem cells and neurons overexpressing DNMT1. Genes dysregulated in both Dnmt1tet/tet cells and schizophrenia patients were studied further. Results & conclusions: About 50% of dysregulated genes in patients also showed altered transcript levels in Tet/Tet neurons in a DNA methylation-independent manner. These neurons unexpectedly showed genome-wide hypomethylation, increased transcript levels of Tet1 and Apobec 1-3 genes and increased activity and copy number of LINE-1 elements. The observed similarities between Tet/Tet neurons and schizophrenia brain samples reinforce DNMT1 overexpression as a risk factor.

Lay abstract DNMT1 controls cytosine methylation, which is often associated with reduced gene expression. Increased levels of DNMT1 is a risk factor for schizophrenia but information on the affected genes is limited. In this study, ∼50% of genes with altered levels of messenger RNAs in schizophrenia patients were also altered in neurons with increased DNMT1. Surprisingly, the neurons with higher DNMT1 levels showed genome-wide decrease in methylation. These findings uncover a new type of gene dysregulation that is independent of DNMT1's catalytic activity.

Analysis of transcript levels of a few schizophrenia candidate genes in neurons from a transgenic mouse embryonic stem cell model overexpressing DNMT1.

Overexpression of DNA Methyltransferase I (DNMT1) is considered as one of the etiological factors for schizophrenia (SZ). However, information on genes subjected to dysregulation because of DNMT1 overexpression is limited. To test whether a larger group of SZ-associated genes are affected, we selected 15 genes reported to be dysregulated in patients (Gad1, Reln, Ank3, Cacna1c, Dkk3, As3mt, Ppp1r11, Smad5, Syn1, Wnt1, Pdgfra, Gsk3b, Cxcl12, Tcf4 and Fez1). Transcript levels of these genes were compared between neurons derived from Dnmt1tet/tet (Tet/Tet) mouse embryonic stem cells (ESCs) that overexpress DNMT1 with R1 (wild-type) neurons. Transcript levels of thirteen genes were significantly altered in Tet/Tet neurons of which, the dysregulation patterns of 11 were similar to patients. Transcript levels of eight out of these eleven were also significantly altered in Tet/Tet ESCs, but the dysregulation patterns of only five were similar to neurons. Comparative analyses among ESCs, embryoid bodies and neurons divided the 15 genes into four distinct groups with a majority showing developmental stage-specific patterns of dysregulation. Reduced Representational Bisulfite Sequencing data from neurons did not show any altered promoter DNA methylation for the dysregulated genes. Doxycycline treatment of Tet/Tet ESCs that eliminated DNMT1, reversed the direction of dysregulation of only four genes (Gad1, Dkk3, As3mt and Syn1). These results suggest that 1. Increased DNMT1 affected the levels of a majority of the transcripts studied, 2. Dysregulation appears to be independent of promoter methylation, 3. Effects of increased DNMT1 levels were reversible for only a subset of the genes studied, and 4. Increased DNMT1 levels may affect transcript levels of multiple schizophrenia-associated genes.