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.

Figure 1. Results of the pseudoproband approach.

The diagram shows the number of CpGs in all investigated individuals passing the threshold of delta β ≥ 0.3 when assigned as a pseudoproband. All imprinted genes were excluded. a - t – healthy controls; M – mother of the patient; F – father of the patient; average – average number of CpGs showing a deviation in normal controls and the parents.

Figure 2. Results of methylation and expression analyses of FAM50B in blood.

a) Methylation analysis. The figure shows the results of the methylation analyses by deep bisulfite sequencing of two normal controls (NC 4 and NC 15) and the patient (top). Heterozygosity for a SNP in both normal controls allowed to separate the alleles (bottom). Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information; * – CpG investigated on the 27k array (CpG 17). b) Expression analysis. Expression analysis in four normal controls heterozygous for a SNP (rs6597007 C/G). For each normal control (NC) sequences obtained from peripheral blood DNA and RNA are shown. In the RNA only one allele is present, which indicates monoallelic expression.

Figure 3. Results of methylation and expression analyses of TRPC3 in blood.

a) Methylation patterns of TRPC3. Results of the methylation analyses of 24 CpG dinucleotides obtained by deep bisulfite sequencing. Peripheral blood samples of eight normal controls (NC) and the patient were investigated. The present SNP at CpG 6 indicates the distribution of methylation across the two alleles. As it disrupts the CpG site 6, the white squares refer to the C allele, while filled squares refer to the G allele. Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information, at CpG 6 due to a SNP (rs13121031) that disrupts the CpG dinucleotide; * – CpG investigated on the 27k array (CpG 21). b) Expression patterns of TRPC3 in blood. Expression analyses in three normal controls heterozygous for a SNP (rs11732666 G/A). For each normal control (NC) sequences obtained from peripheral blood DNA and RNA are shown. Expression varies from biallelic to skewed.

Figure 4. Methylation patterns of AKR1C3.

For this locus the patient shows hypermethylation, whereas the two normal controls (NC) display very low levels of methylation. Mean methylation levels and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information; * – CpG investigated on the 27k array (CpG 7).

Figure 5. Methylation patterns of ACTN3.

The two normal controls investigated display a high variability in methylation levels of adjacent CpGs. The same pattern is visible in the patient in a hypomethylated form. Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information; * – CpG investigated on the 27k array (CpG 3).

Figure 6. Methylation patterns of HKR1.

Methylation patterns of three normal controls in blood displaying inter-individual variability. The methylation level in the patient is very low. In NC 3 the alleles could be separated (see lower part of the figure). Mean methylation levels, SNP allele and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares – unmethylated CpGs; red squares – methylated CpGs; white squares – missing sequence information; * – CpG investigated on the 27k array (CpG 5).