Nonsyndromic orofacial clefts in Chile: LINE-1 methylation and MTHFR variants

Aim: To evaluate the risk of nonsyndromic orofacial clefts (NSOFCs) associated with LINE-1 methylation, as a marker of global DNA methylation, and the effect of MTHFR functional variants on this variable. Patients & methods: LINE-1 methylation was evaluated by bisulfite modification coupled to DNA pyrosequencing in 95 NSOFC cases and 95 controls. In these subjects, MTHFR genotypes for variants c.C677T (rs1801133) and c.A1298C (rs1801131) were obtained. Results: Middle levels (second tertile) of LINE-1 methylation increase the risk of NSOFCs. In addition, LINE-1 methylation depends on c.A1298C genotypes in controls but not in cases. Conclusion: A nonlinear association between global DNA methylation and NSOFCs was detected in this Chilean population, which appears to be influenced by MTHFR functional variants.

Methylation hotspots evidenced by deep sequencing in patients with facioscapulohumeral dystrophy and mosaicism

ObjectiveTo investigate the distribution of cytosine-guanine dinucleotide (CpG) sites with a variable level of DNA methylation of the D4Z4 macrosatellite element in patients with facioscapulohumeral dystrophy (FSHD).MethodsBy adapting bisulfite modification to deep sequencing, we performed a comprehensive analysis of D4Z4 methylation across D4Z4 repeats and adjacent 4qA sequence in DNA from patients with FSHD1, FSHD2, or mosaicism and controls.ResultsUsing hierarchical clustering, we identified clusters with different levels of methylation and separated, thereby the different groups of samples (controls, FSHD1, and FSHD2) based on their respective level of methylation. We further show that deep sequencing–based methylation analysis discriminates mosaic cases for which methylation changes have never been evaluated previously.ConclusionsAltogether, our approach offers a new high throughput tool for estimation of the D4Z4 methylation level in the different subcategories of patients having FSHD. This methodology allows for a comprehensive and discriminative analysis of different regions along the macrosatellite repeat and identification of focal regions or CpG sites differentially methylated in patients with FSHD1 and FSHD2 but also complex cases such as those presenting mosaicism.

Fanconi Anemia Pathway Suppression of R Loops Is a Mechanism of Maintenance of Genomic Stability

Background The FA pathway involvement in homologous recombination and translesion synthesis repair requires binding to DNA by central proteins of the pathway, but interaction with RNA and modulation of genotoxic intermediates such as R loops has not been explored. In this study we explore the incidence of R loops in FA mutant cells. Methods DRIP: duplex RNA hybrid immunoprecipitation-genomic DNA was extracted, sonicated, and subjected to immunoprecipitation using S9.6 DNA:RNA hybrid antibody. Resulting immunoprecipitates were run on agarose gel and stained using ethidium bromide. Bisulfite modification/DNA Sequencing: genomic DNA was extracted and a portion was denatured. After bisulfite modification, coding sequence in GAPDH was amplified by PCR, and the resulting products sequenced. Similarly prepared genomic DNA was prepared, exomic sequencing was performed. Bioinformatic analysis was undertaken on each sample. Immunofluorescence microscopy: cells were cultured in chamber slides, treated or not with mitomycin C, fixed, and stained. Cell culture: mutant and complemented versions of each Results Using anti-RNA:DNA hybrid antibodies, we used a series of assays to test for the presence of R loops in FA mutant cells. Agarose gel electrophoresis demonstrated increased pulldown of RNA:DNA hybrids in FA-D2 and FA-A mutant cells but not FA-J. Similar results were seen in qPCR data designed to detect R loops in multiple sites in G-C rich areas of beta-actin and GAPDH. Immunofluorescence revealed increased signal using the S9.6 antibody in a DNA damage inducible manner in mutant FA-D2 and FA-A cells but not FA-J mutant cells. BRCA2 mutant cells also demonstrated increases in R loops. Exome sequencing revealed that R loops were globally present in far greater amounts in FA-D2 mutant cells. Conclusions RNA:DNA hybrids as represented by R loops are present in greater amounts in FA-D2, FA-A, and BRCA2/FA-D1 mutant cells but not in FA-J mutant cells. Suppression of R loops represents a non-canonical function for the FA pathway in maintenance of genomic stability. Translational potential statement The net cause of genomic instability in FA may be formation of DNA:RNA hybrids, which could be prevented by means of small molecules. Understanding the biology can lead to enhanced treatment of bone marrow failure as well as of FA-associated tumors. Disclosures No relevant conflicts of interest to declare.