Deep Bisulfite Sequencing of Aberrantly Methylated Loci in a Patient with Multiple Methylation Defects

Abstract

NLRP7 is a maternal effect gene as maternal mutations in this gene cause recurrent hydatidiform moles, spontaneous abortions and stillbirths, whereas live births are very rare. We have studied a patient with multiple anomalies born to a mother with a heterozygous NLRP7 mutation. By array-based CpG methylation analysis of blood DNA from the patient, his parents and 18 normal controls on Illumina Infinium HumanMethylation27 BeadChips we found that the patient had methylation changes (delta ß ≥ 0.3) at many imprinted loci as well as at 87 CpGs associated with 85 genes of unknown imprinting status. Using a pseudoproband (permutation) approach, we found methylation changes at only 7-24 CpGs (mean 15; standard deviation 4.84) in the controls. Thus, the number of abberantly methylated CpGs in the patient is more than 14 standard deviations higher. In order to identify novel imprinted genes among the 85 conspicuous genes in the patient, we selected 19 (mainly hypomethylated) genes for deep bisulfite amplicon sequencing on the ROCHE/454 Genome Sequencer in the patient and at least two additional controls. These controls had not been included in the array analysis and were heterozygous for a single nucleotide polymorphism at the test locus, so that allele-specific DNA methylation patterns could be determined. Apart from FAM50B, which we proved to be imprinted in blood, we did not observe allele-specific DNA methylation at the other 18 loci. We conclude that the patient does not only have methylation defects at imprinted loci but (at least in blood) also an excess of methylation changes at apparently non-imprinted loci.

In vitro maturation of oocytes is not associated with altered deoxyribonucleic acid methylation patterns in children from in vitro fertilization or intracytoplasmic sperm injection

“…Next-generation sequencing of bisulfite-treated DNA was used to determine the methylation patterns of several hundred to several thousand individual alleles of a target gene per sample in a single experiment (31). To generate amplicon libraries for the RAD51C promoter, bisulfite-treated blood and placenta DNA samples, respectively, were PCR amplified using forward primer CTTGCTTCCTGGCACGAG-ATGGTGTATAA GTGTGAAAATTTATAAGA and reverse primer CAGGAAA CAGCTATGAC-CCTCTAAAAATTCCTCAACAATCTAAA.…”

On the origin of sperm epigenetic heterogeneity

“…The patterns shown by several of the clones sequenced (Flanagan et al 2006, Kobayashi et al 2007, Marques et al 2008, Hammoud et al 2010, Marques et al 2010, Wu et al 2010, Minor et al 2011, Sato et al 2011, Ankolkar et al 2012, Montjean et al 2013) resemble at a small scale the binomial (sequences fully methylated vs fully unmethylated) distribution of sequencing patterns obtained using deep-bisulphite sequencing (DBS; Laurentino et al 2015). Due to the requirement for a high number of individual sequences analysed from each single sample, the detection and measurement of epigenetic heterogeneity in sperm as well as in other tissues only became feasible now, with the advent of high-resolution analysis methodologies such as nextgeneration sequencing/DBS (Mikeska et al 2010, Meaburn & Schulz 2012, Beygo et al 2013, Gries et al 2013, Smallwood et al 2014, Putnik et al 2015. The use of novel and highly sensitive DNA isolation and pyrosequencing-based sequencing techniques, such as OSMA (oligo-sperm methylation assay; Laurentino et al 2015) and limiting dilution (LD; Kuhtz et al 2014), allowed the detection of DNA methylation heterogeneity in the sperm of infertile men.…”

Genome-wide methylation analysis of retrocopy-associated CpG islands and their genomic environment

“…The following steps including generation of the bisulfite amplicon libraries, sample preparation and sequencing on the Roche 454 GS Junior system were realized as previously described. 44 Primer sequences and annealing temperatures can be found in the supplement (supplemental material, Table S9). The Python-based amplikyzer software was used to analyze the bisulfite sequencing data generated by the Roche 454 GS junior.…”