Rebecca L Poole

Methylation analysis of 79 patients with growth restriction reveals novel patterns of methylation change at imprinted loci

This study was an investigation of 79 patients referred to the Wessex Regional Genetics Laboratory with suspected Russell–Silver Syndrome or unexplained short stature/intra uterine growth restriction, warranting genetic investigation. Methylation status was analysed at target sequences within eleven imprinted loci (PLAGL1, IGF2R, PEG10, MEST1, GRB10, KCNQ1OT1, H19, IGF2P0, DLK1, PEG3, NESPAS). Thirty seven percent (37%) (29 of 79) of samples were shown to have a methylation abnormality. The commonest finding was a loss of methylation at H19 (23 of 29), as previously reported in Russell–Silver Syndrome. In addition, four of these patients had methylation anomalies at other loci, of whom two showed hypomethylation of multiple imprinted loci, and two showed a complete gain of methylation at IGF2R. This latter finding was also present in five other patients who did not have demonstrable changes at H19. In total, 7 of 79 patients showed a gain of methylation at IGF2R and this was significantly different from a normal control population of 267 individuals (P=0.002). This study in patients with growth restriction shows the importance of widening the epigenetic investigation to include multiple imprinted loci and highlights potential involvement of the IGF2R locus.

Epigenotype–phenotype correlations in Silver–Russell syndrome

Background Silver–Russell syndrome (SRS) is characterised by intrauterine growth restriction, poor postnatal growth, relative macrocephaly, triangular face and asymmetry. Maternal uniparental disomy (mUPD) of chromosome 7 and hypomethylation of the imprinting control region (ICR) 1 on chromosome 11p15 are found in 5–10% and up to 60% of patients with SRS, respectively. As many features are non-specific, diagnosis of SRS remains difficult. Studies of patients in whom the molecular diagnosis is confirmed therefore provide valuable clinical information on the condition. Methods A detailed, prospective study of 64 patients with mUPD7 (n=20) or ICR1 hypomethylation (n=44) was undertaken. Results and conclusions The considerable overlap in clinical phenotype makes it difficult to distinguish these two molecular subgroups reliably. ICR1 hypomethylation was more likely to be scored as ‘classical’ SRS. Asymmetry, fifth finger clinodactyly and congenital anomalies were more commonly seen with ICR1 hypomethylation, whereas learning difficulties and referral for speech therapy were more likely with mUPD7. Myoclonus-dystonia has been reported previously in one mUPD7 patient. The authors report mild movement disorders in three further cases. No correlation was found between clinical severity and level of ICR1 hypomethylation. Use of assisted reproductive technology in association with ICR1 hypomethylation seems increased compared with the general population. ICR1 hypomethylation was also observed in affected siblings, although recurrence risk remains low in the majority of cases. Overall, a wide range of severity was observed, particularly with ICR1 hypomethylation. A low threshold for investigation of patients with features suggestive, but not typical, of SRS is therefore recommended.

Genome-wide DNA methylation analysis of patients with imprinting disorders identifies differentially methylated regions associated with novel candidate imprinted genes

Background Genomic imprinting is allelic restriction of gene expression potential depending on parent of origin, maintained by epigenetic mechanisms including parent of origin-specific DNA methylation. Among approximately 70 known imprinted genes are some causing disorders affecting growth, metabolism and cancer predisposition. Some imprinting disorder patients have hypomethylation of several imprinted loci (HIL) throughout the genome and may have atypically severe clinical features. Here we used array analysis in HIL patients to define patterns of aberrant methylation throughout the genome. Design We developed a novel informatic pipeline capable of small sample number analysis, and profiled 10 HIL patients with two clinical presentations (Beckwith–Wiedemann syndrome and neonatal diabetes) using the Illumina Infinium Human Methylation450 BeadChip array to identify candidate imprinted regions. We used robust statistical criteria to quantify DNA methylation. Results We detected hypomethylation at known imprinted loci, and 25 further candidate imprinted regions (nine shared between patient groups) including one in the Down syndrome critical region (WRB) and another previously associated with bipolar disorder (PPIEL). Targeted analysis of three candidate regions (NHP2L1, WRB and PPIEL) showed allelic expression, methylation patterns consistent with allelic maternal methylation and frequent hypomethylation among an additional cohort of HIL patients, including six with Silver–Russell syndrome presentations and one with pseudohypoparathyroidism 1B. Conclusions This study identified novel candidate imprinted genes, revealed remarkable epigenetic convergence among clinically divergent patients, and highlights the potential of epigenomic profiling to expand our understanding of the normal methylome and its disruption in human disease.

Beckwith–Wiedemann syndrome caused by maternally inherited mutation of an OCT-binding motif in the IGF2/H19-imprinting control region, ICR1

The imprinted expression of the IGF2 and H19 genes is controlled by the imprinting control region 1 (ICR1) located at chromosome 11p15.5. DNA methylation defects involving ICR1 result in two growth disorders with opposite phenotypes: an overgrowth disorder, the Beckwith–Wiedemann syndrome (maternal ICR1 hypermethylation in 10% of BWS cases) and a growth retardation disorder, the Silver–Russell syndrome (paternal ICR1 loss of methylation in 60% of SRS cases). In familial BWS, hypermethylation of ICR1 has been found in association with microdeletion of repetitive DNA motifs within ICR1 that bind the zinc finger protein CTCF; but more recently, ICR1 point mutations were described in BWS pedigrees. We present a case report of two brothers with BWS and prolonged post-pubertal growth resulting in very large stature. A maternally inherited point mutation was identified in ICR1 in both brothers, which altered binding of OCT transcription factors. The same mutation was present on the paternally inherited allele of their unaffected mother. This is a second report of a point mutation causing ICR1 hypermethylation by altering an OCT-binding motif. The atypical growth phenotype of the brothers may be connected to the unusual underlying cause of their BWS.

Targeted methylation testing of a patient cohort broadens the epigenetic and clinical description of imprinting disorders

Imprinting disorders are associated with mutations and epimutations affecting imprinted genes, that is those whose expression is restricted by parent of origin. Their diagnosis is challenging for two reasons: firstly, their clinical features, particularly prenatal and postnatal growth disturbance, are heterogeneous and partially overlapping; secondly, their underlying molecular defects include mutation, epimutation, copy number variation, and chromosomal errors, and can be further complicated by somatic mosaicism and multi‐locus methylation defects. It is currently unclear to what extent the observed phenotypic heterogeneity reflects the underlying molecular pathophysiology; in particular, the molecular and clinical diversity of multilocus methylation defects remains uncertain. To address these issues we performed comprehensive methylation analysis of imprinted genes in a research cohort of 285 patients with clinical features of imprinting disorders, with or without a positive molecular diagnosis. 20 of 91 patients (22%) with diagnosed epimutations had methylation defects of additional imprinted loci, and the frequency of developmental delay and congenital anomalies was higher among these patients than those with isolated epimutations, indicating that hypomethylation of multiple imprinted loci is associated with increased diversity of clinical presentation. Among 194 patients with clinical features of an imprinting disorder but no molecular diagnosis, we found 15 (8%) with methylation anomalies, including missed and unexpected molecular diagnoses. These observations broaden the phenotypic and epigenetic definitions of imprinting disorders, and show the importance of comprehensive molecular testing for patient diagnosis and management.

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.

Deletions and rearrangements of the H19/IGF2 enhancer region in patients with Silver–Russell syndrome and growth retardation

Silver–Russell syndrome (SRS) is characterised by prenatal and postnatal growth retardation, dysmorphic facial features, and body asymmetry. In 35–60% of SRS cases the paternally methylated imprinting control region (ICR) upstream of the H19 gene (H19-ICR) is hypomethylated, leading to downregulation of IGF2 and bi-allelic expression of H19. H19 and IGF2 are reciprocally imprinted genes on chromosome 11p15. The expression is regulated by the imprinted methylation of the ICR, which modulates the transcription of H19 and IGF2 facilitated by enhancers downstream of H19. A promoter element of IGF2, IGF2P0, is differentially methylated equivalently to the H19-ICR, though in a small number of SRS cases this association is disrupted—that is, hypomethylation affects either H19-ICR or IGF2P0. Three pedigrees associated with hypomethylation of IGF2P0 in the probands are presented here, two with paternally derived deletions, and one with a balanced translocation of inferred paternal origin. They all have a breakpoint within the H19/IGF2 enhancer region. One proband has severe growth retardation, the others have SRS. This is the first report of paternally derived structural chromosomal mutations in 11p15 causing SRS. These cases define a novel aetiology of the growth retardation in SRS, namely, dissociation of IGF2 from its enhancers.

An atypical case of hypomethylation at multiple imprinted loci.

Angelman syndrome (AS) and Prader–Willi syndrome (PWS) are caused by genetic and epigenetic mutations of the imprinted gene cluster on chromosome 15q13. Although the imprinting mutations causing PWS and AS are essentially opposite in nature, remarkably, a small number of patients have been reported with clinical features of PWS but epigenetic mutations consistent with AS. We report here a patient who presented with clinical features partially consistent with both PWS and Beckwith–Wiedemann syndrome (BWS). Epimutations were found at both the AS/PWS and BWS loci, and additionally at the H19, PEG3, NESPAS and GNAS loci. This patient is therefore the first described case with a primary epimutation consistent with AS accompanied by hypomethylation of other imprinted loci.

Very small deletions within the NESP55 gene in pseudohypoparathyroidism type 1b

Pseudohypoparathyroidism (PHP) is caused by reduced expression of genes within the GNAS cluster, resulting in parathormone resistance. The cluster contains multiple imprinted transcripts, including the stimulatory G protein α subunit (Gs-α) and NESP55 transcript preferentially expressed from the maternal allele, and the paternally expressed XLas, A/B and antisense transcripts. PHP1b can be caused by loss of imprinting affecting GNAS A/B alone (associated with STX16 deletion), or the entire GNAS cluster (associated with deletions of NESP55 in a minority of cases). We performed targeted genomic next-generation sequencing (NGS) of the GNAS cluster to seek variants and indels underlying PHP1b. Seven patients were sequenced by hybridisation-based capture and fourteen more by long-range PCR and transposon-mediated insertion and sequencing. A bioinformatic pipeline was developed for variant and indel detection. In one family with two affected siblings, and in a second family with a single affected individual, we detected maternally inherited deletions of 40 and 33 bp, respectively, within the deletion previously reported in rare families with PHP1b. All three affected individuals presented with atypically severe PHP1b; interestingly, the unaffected mother in one family had the detected deletion on her maternally inherited allele. Targeted NGS can reveal sequence changes undetectable by current diagnostic methods. Identification of genetic mutations underlying epigenetic changes can facilitate accurate diagnosis and counselling, and potentially highlight genetic elements critical for normal imprint setting.

A statistical method for single sample analysis of HumanMethylation450 array data: genome-wide methylation analysis of patients with imprinting disorders.

BackgroundThe Illumina Infinium HumanMethylation450 BeadChip is an array-based technology for analysing DNA methylation at approximately 475,000 differentially methylated cytosines across the human genome. Hitherto, the array has been used for case-control studies, where sample numbers can be sufficient to yield statistically robust data on a genome-wide basis. We recently reported an informatic pipeline capable of yielding statistically and biologically significant results using only five cases, which expanded the use of this technology to rare disease studies. However, the clinical application of these technologies requires the ability to perform robust analysis of individual patients.ResultsHere we report a novel informatic approach for methylation array analysis of single samples, using the Crawford-Howell t-test. We tested our approach on patients with ultra-rare imprinting disorders with aberrant DNA methylation at multiple locations across the genome, which was previously detected by targeted testing. However, array analysis outperformed targeted assays in three ways: it detected loci not normally analysed by targeted testing, detected methylation changes too subtle to detect by the targeted testing and reported broad and consistent methylation changes across genetic loci not captured by point testing.ConclusionsThis method has potential clinical utility for human disorders where DNA methylation change may be a biomarker of disease.