Jasmin Beygo

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.

Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans

Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative trans-acting mutations have been found. Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.

Novel deletions affecting the MEG3-DMR provide further evidence for a hierarchical regulation of imprinting in 14q32

The imprinted region on chromosome 14q32 harbors several maternally or paternally expressed genes as well as two DMRs (differentially methylated regions), the IG-DMR and the MEG3-DMR, which both act as imprinting control centers. Genetic aberrations affecting the imprinted gene cluster in 14q32 result in distinct phenotypes, known as maternal or paternal uniparental disomy 14 phenotypes (upd(14)mat, upd(14)pat). In both syndromes, three types of molecular alterations have been reported: uniparental disomy 14, deletions and epimutations. In contrast to uniparental disomy and epimutations, deletions affecting regulatory elements in 14q32 are associated with a high-recurrence risk. Based on two single deletion cases a functional hierarchy of the IG-DMR as a regulator for the methylation of the MEG3-DMR has been proposed. We have identified two novel deletions of maternal origin spanning the MEG3-DMR, but not the IG-DMR in patients with upd(14)pat syndrome, one de novo deletion of 165 kb and another deletion of 5.8 kb in two siblings. The 5.8 kb deletion was inherited from the phenotypically normal mother, who carries the deletion in a mosaic state on her paternal chromosome 14. The methylation at both DMRs was investigated by quantitative next generation bisulfite sequencing and revealed normal methylation patterns at the IG-DMR in all patients with the exception of certain CpG dinucleotides. Thus, we could confirm that deletions of the MEG3-DMR does not generally influence the methylation pattern of the IG-DMR, which strengthens the hypothesis of a hierarchical structure and distinct functional properties of the two DMRs.

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.

Epigenetic germline mosaicism in infertile men

Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mothers or fathers germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.

Evidence for anticipation in Beckwith–Wiedemann syndrome

Classical Beckwith–Wiedemann syndrome (BWS) was diagnosed in two sisters and their male cousin. The children’s mothers and a third sister were tall statured (178, 185 and 187 cm) and one had mild BWS features as a child. Their parents had average heights of 173 cm (mother) and 180 cm (father). This second generation tall stature and third generation BWS correlated with increased methylation of the maternal H19/IGF2-locus. The results were obtained by bisulphite treatment and subclone Sanger sequencing or next generation sequencing to quantitate the degree of CpG-methylation on three locations: the H19 promoter region and two CTCF binding sites in the H19 imprinting control region (ICR1), specifically in ICR1 repeats B1 and B7. Upon ICR1 copy number analysis and sequencing, the same maternal point variant NCBI36:11:g.1979595T>C that had been described previously as a cause of BWS in three brothers, was found. As expected, this point variant was on the paternal allele in the non-affected grandmother. This nucleotide variant has been shown to affect OCTamer-binding transcription factor-4 (OCT4) binding, which may be necessary for maintaining the unmethylated state of the maternal allele. Our data extend these findings by showing that the OCT4 binding site mutation caused incomplete switching from paternal to maternal ICR1 methylation imprint, and that upon further maternal transmission, methylation of the incompletely demethylated variant ICR1 allele was further increased. This suggests that maternal and paternal ICR1 alleles are treated differentially in the female germline, and only the paternal allele appears to be capable of demethylation.

Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains

ABSTRACT The analysis of DNA methylation has become routine in the pipeline for diagnosis of imprinting disorders, with many publications reporting aberrant methylation associated with imprinted differentially methylated regions (DMRs). However, comparisons between these studies are routinely hampered by the lack of consistency in reporting sites of methylation evaluated. To avoid confusion surrounding nomenclature, special care is needed to communicate results accurately, especially between scientists and other health care professionals. Within the European Network for Human Congenital Imprinting Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission. The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion.

A familial disorder of altered DNA-methylation

Background In a subset of imprinting disorders caused by epimutations, multiple imprinted loci are affected. Familial occurrence of multilocus imprinting disorders is rare. Purpose/objective We have investigated the clinical and molecular features of a familial DNA-methylation disorder. Methods Tissues of affected individuals and blood samples of family members were investigated by conventional and molecular karyotyping. Sanger sequencing and RT-PCR of imprinting-associated genes (NLRP2, NLRP7, ZFP57, KHDC3L, DNMT1o), exome sequencing and locus-specific, array-based and genome-wide technologies to determine DNA-methylation were performed. Results In three offspring of a healthy couple, we observed prenatal onset of severe growth retardation and dysmorphism associated with altered DNA-methylation at paternally and maternally imprinted loci. Array-based analyses in various tissues of the offspring identified the DNA-methylation of 2.1% of the genes in the genome to be recurrently altered. Despite significant enrichment of imprinted genes (OR 9.49), altered DNA-methylation predominately (90.2%) affected genes not known to be imprinted. Sequencing of genes known to cause comparable conditions and exome sequencing in affected individuals and their ancestors did not unambiguously point to a causative gene. Conclusions The family presented herein suggests the existence of a familial disorder of DNA-methylation affecting imprinted but also not imprinted gene loci potentially caused by a maternal effect mutation in a hitherto not identified gene.

The differentially methylated region of MEG8 is hypermethylated in patients with Temple syndrome.

AIM To investigate the DNA-methylation levels in the newly described MEG8 differentially methylated region (DMR) in the imprinted cluster in 14q32 in patients with Temple syndrome. PATIENTS & METHODS We included three patients with Temple syndrome which were studied by Infinium HumanMethylation450 BeadChips, locus-specific bisulfite-pyrosequencing, methylation-specific-MLPA and microsatellite analyses. The tag-CpG of the MEG8-DMR was investigated using the Infinium HumanMethylation450 BeadChip. RESULTS In all three patients, the identical pattern of DNA-hypermethylation of the MEG8-DMR was observed along with DNA-hypomethylation of the IG-DMR and MEG3-DMR. CONCLUSION Based on the observed MEG8-DMR DNA-hypermethylation and previously published data, we conclude that DNA-methylation of the MEG3- and MEG8-DMR is functionally dependent on the DNA-methylation pattern of the IG-DMR. The observed combination of epimutations is predicted to be associated with bi-allelic MEG3 and MEG8 expression in individuals with Temple syndrome.

Phenotypic spectrum and extent of DNA methylation defects associated with multilocus imprinting disturbances.

AIM To characterize the genotypic and phenotypic extent of multilocus imprinting disturbances (MLID). MATERIALS & METHODS We analyzed 37 patients with imprinting disorders (explorative cohort) for DNA methylation changes using the Infinium HumanMethylation450 BeadChip. For validation, three independent cohorts with imprinting disorders or cardinal features thereof were analyzed (84 patients with imprinting disorders, 52 with growth disorder, 81 with developmental delay). RESULTS In the explorative cohort 21 individuals showed array-based MLID with each one displaying an Angelman or Temple syndrome phenotype, respectively. Epimutations in ZDBF2 and FAM50B were associated with severe MLID regarding number of affected regions. By targeted analysis we identified methylation changes of ZDBF2 and FAM50B also in the three validation cohorts. CONCLUSION We corroborate epimutations in ZDBF2 and FAM50B as frequent changes in MLID whereas these rarely occur in other patients with cardinal features of imprinting disorders. Moreover, we show cell lineage specific differences in the genomic extent of FAM50B epimutation.