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.

DNMT1 downregulation as well as its overexpression distinctly affect mostly overlapping genes implicated in schizophrenia, autism spectrum, epilepsy, and bipolar disorders.

Data on schizophrenia (SZ), epilepsy (EPD) and bipolar disorders (BPD) suggested an association of DNMT1 overexpression whereas certain variants of the gene were predicted to result in its increased expression in autism spectrum disorder (ASD). In addition, loss of DNMT1 in frontal cortex resulted in behavioral abnormalities in mice. Here we investigated the effects of increased as well as lack of DNMT1 expression using Dnmt1tet/tet neurons as a model for abnormal neurogenesis and 10,861 genes showing transcript level dysregulation in datasets from the four disorders. In case of overexpression, 3,211 (∼ 30%) genes were dysregulated, affecting pathways involved in neurogenesis, semaphorin signaling, ephrin receptor activity, etc. A disproportionately higher proportion of dysregulated genes were associated with epilepsy. When transcriptome data of Dnmt1tet/tet neurons treated with doxycycline that downregulated DNMT1 was used, 3,356 genes (∼31%) were dysregulated with a significant proportion involved in pathways similar to those in untreated cells. Both conditions resulted in ∼68% of dysregulated genes wherein a majority showed similar patterns of transcript level changes. Among the genes with transcripts returning to normal levels, ribosome assembly/biogenesis was most significant whereas in absence of DNMT1, a new set of 903 genes became dysregulated and are involved in similar pathways as mentioned above. These findings provide support for overexpression of DNMT1 as well as its downregulation as risk factor for the four disorders and that its levels within a tight range are essential for normal neurodevelopment/mental health.

Genome-scale copy number variant analysis in schizophrenia patients and controls from South India.

Copy number variants (CNVs) are among the main genetic factors identified in schizophrenia (SZ) through genome-scale studies conducted mostly in Caucasian populations. However, to date, there have been no genome-scale CNV reports on patients from India. To address this shortcoming, we generated, for the first time, genome-scale CNV data for 168 SZ patients and 168 controls from South India. In total, 63 different CNVs were identified in 56 patients and 46 controls with a significantly higher proportion of medium-sized deletions (100 kb-1 Mb) after multiple testing (FDR = 2.7E-4) in patients. Of these, 13 CNVs were previously reported; however, when searched against GWAS, transcriptome, exome, and DNA methylation studies, another 17 CNVs with candidate genes were identified. Of the total 30 CNVs, 28 were present in 38 patients and 12 in 27 controls, indicating a significantly higher representation in the former (p = 1.87E-5). Only 4q35.1-q35.2 duplications were significant (p = 0.020) and observed in 11 controls and 2 patients. Among the others that are not significant, a few examples of patient-specific and previously reported CNVs include deletions of 11q14.1 (DLG2), 22q11.21, and 14q21.1 (LRFN5). 16p13.3 deletion (RBFOX1), 3p14.2 duplication (CADPS), and 7p11.2 duplication (CCT6A) were some of the novel CNVs containing candidate genes. However, these observations need to be replicated in a larger sample size. In conclusion, this report constitutes an important foundation for future CNV studies in a relatively unexplored population. In addition, the data indicate that there are advantages in using an integrated approach for better identification of candidate CNVs for SZ and other mental health disorders.

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.

DNMT1: catalytic and non-catalytic roles in different biological processes.

DNMT1 is the main enzyme that uses the information on DNA methylation patterns in the parent strand and methylates the daughter strand in freshly replicated hemimethylated DNA. It is widely known that DNMT1 is a component of the epigenetic machinery mediating gene repression via increased promoter methylation. However, recent data suggest that DNMT1 can also modulate gene expression independent of its catalytic activity and participates in multiple processes including the cell cycle, DNA damage repair and stem cell function. This review summarizes the noncanonical functions of DNMT1, some of which are clearly independent of maintenance methylation. Finally, phenotypic data on altered DNMT1 levels suggesting that maintenance of optimal levels of DNMT1 is vital for normal development and health is presented.

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 15q11.2 CNVs in an Indian population with schizophrenia.

Copy number variants (CNVs) of 15q11.2 yielded conflicting reports on their association with schizophrenia (SZ), indicating the need for replication studies. Because there are no 15q11.2 CNV studies on Indian patients, we began by testing 307 SZ patients and 359 age- and sex-matched controls from South India. Using an improved multiplex ligation probe amplification, six deletions were found in patients and three in controls (p = 0.31), whereas one duplication was found in patients and three in controls (p = 0.63). Analysis of families of two patients and two controls with deletions indicated that the mutations were de novo. In conclusion, there seems to be no significant difference in the frequencies of 15q11.2 CNVs among the controls and patients studied here. Future studies involving a larger number of controls and patients are expected to provide better clarity on the relationship between 15q11.2 CNVs and SZ patients from India.

Improved Multiplex Ligation-dependent Probe Amplification (i-MLPA) for rapid copy number variant (CNV) detection.

BACKGROUND: In Multiplex Ligation-dependent Probe Amplification (MLPA), copy number variants (CNVs) for specific genes are identified after normalization of the amounts of PCR products from ligated reference probes hybridized to genomic regions that are ideally free from normal variation. However, we observed ambiguous calls for two reference probes in an investigation of the human 15q11.2 region by MLPA among 20 controls, due to the presence of single nucleotide polymorphisms (SNPs) in the probe-binding regions. Further in silico analysis revealed that 18 out of 19 reference probes hybridize to regions subject to variation, underlining the requirement for designing new reference probes against variation-free regions. METHODS: An improved MLPA (i-MLPA) method was developed by generating a new set of reference probes to reduce the chances of ambiguous calls and new reagents that reduce hybridization times to 30 min from 16h to obtain MLPA ratio data within 6h. Using i-MLPA, we screened 240 schizophrenia patients for CNVs in 15q11.2 region. CONCLUSION: Three deletions and two duplications were identified among the 240 schizophrenia patients. No variation was observed for the new reference probes. Taken together, i-MLPA procedure helps obtaining non-ambiguous CNV calls within 6h without compromising accuracy.

Characterization of TTAGG telomeric repeats, their interstitial occurrence and constitutively active telomerase in the mealybug Planococcus lilacinus (Homoptera; Coccoidea).

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.