Deep bisulfite sequencing of aberrantly methylated loci in a patient with multiple methylation defects.

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.

The molecular function and clinical phenotype of partial deletions of the IGF2/H19 imprinting control region depends on the spatial arrangement of the remaining CTCF-binding sites.

At chromosome 11p15.5, the imprinting centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4-2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith-Wiedemann syndrome, although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wild-type allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes.