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Transformer2 proteins (Tra2α and Tra2β) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder.Twelve individuals from 11 unrelated families who had predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA-seq and Western blot analyses of Tra2β-1 and Tra2β-3 isoforms from patient-derived cells were performed. Tra2β1-GFP, Tra2β3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells.All variants clustered in the 5' part of TRA2B, upstream of an alternative translation start site responsible for the expression of non-canonical Tra2β-3 isoform. All of affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2β-1 isoform, while they increased the expression of the Tra2β-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2β-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2β-1.Predicted loss-of-function variants clustered in the 5' portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2β protein.

Smith-Kingsmore Syndrome (SKS) is a rare genetic syndrome associated with megalencephaly, a variable intellectual disability, autism spectrum disorder, and MTOR gain of function variants. Only 30 patients with MTOR missense variants are published, including 14 (47%) with the MTOR c.5395G>A p.(Glu1799Lys) variant. Limited phenotypic data impacts the quality of information delivered to families and the robustness of interpretation of novel MTOR missense variation. This study aims to improve our understanding of the SKS phenotype through the investigation of 16 further patients with the MTOR c.5395G>A p.(Glu1799Lys) variant. Through the careful phenotypic evaluation of these 16 patients and integration with data from 14 previously reported patients, we have defined major (100% patients) and frequent (>15%) SKS clinical characteristics and, using these data, proposed guidance for evidence-based management. In addition, in the absence of functional studies, we suggest that the combination of the SKS major clinical features of megalencephaly (where the head circumference is at least 3SD) and an intellectual disability with a de novo MTOR missense variant (absent from population databases) should be considered diagnostic for SKS.

Smith-Kingsmore Syndrome (SKS) is a rare genetic syndrome associated with megalencephaly, a variable intellectual disability, autism spectrum disorder, and MTOR gain of function variants. Only 30 patients with MTOR missense variants are published, including 14 (47%) with the MTOR c.5395GA p.(Glu1799Lys) variant. Limited phenotypic data impacts the quality of information delivered to families and the robustness of interpretation of novel MTOR missense variation. This study aims to improve our understanding of the SKS phenotype through the investigation of 16 further patients with the MTOR c.5395GA p.(Glu1799Lys) variant. Through the careful phenotypic evaluation of these 16 patients and integration with data from 14 previously reported patients, we have defined major (100% patients) and frequent (15%) SKS clinical characteristics and, using these data, proposed guidance for evidence-based management. In addition, in the absence of functional studies, we suggest that the combination of the SKS major clinical features of megalencephaly (where the head circumference is at least 3SD) and an intellectual disability with a de novo MTOR missense variant (absent from population databases) should be considered diagnostic for SKS.

BackgroundGenomic 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.DesignWe 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.ResultsWe 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.ConclusionsThis 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.

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.

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.

We present an infant with a de novo cytogenetically visible interstitial deletion of approximately 21.9 Mb involving chromosome bands 7p15.1–7p12.1, with the loss of 119 genes confirmed by array CGH. The infant had a ventricular septal defect, hand and skull anomalies, and hyperglycaemia compatible with haploinsufficiency of TBX20, GLI3, and GCK genes, respectively. In addition, the infant had some features reminiscent of Beckwith Wiedemann syndrome including macroglossia, umbilical hernia, and a relatively large birth weight and we speculate that this is due to the deletion of GRB10, an imprinted gene on chromosome 7. This report illustrates how knowledge of genes within a deleted interval facilitates optimal medical management, can explain observed phenotypes, and stimulates research questions.

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.

Transient neonatal diabetes (TND) is associated with overexpression of genes within a critical region on 6q24. This study aims to refine the boundaries of this region to reduce the number of potential candidate genes for 6q24 TND.Fifteen patients with transient neonatal diabetes and submicroscopic chromosome 6 duplications were investigated. The duplications were confirmed by microsatellite analysis and subsequently mapped using tiled chromosome 6 array Comparative Genomic Hybridisation (aCGH) and MLPA. Duplication boundaries were compared to identify the minimal shared region of duplication. These data were then used with available clinical data to identify associations between size of 6q24 duplication and severity of TND phenotype.Alignment of the minimal region of duplication to the human genome reduced the minimal TND critical region, formerly estimated at 440 kb, to 160-173 kb, revealing PLAGL1 (pleiomorphic adenoma gene-like 1) and HYMAI (imprinted in hydatidiform mole) to be the only genes wholly included therein. Additionally, the complete paternal duplication of a region containing the theoretical protein FAM164B was associated with the severe growth restriction observed in 6q24 duplication patients.This study has significantly reduced the critical region associated with 6q24 TND. It has eliminated several previous TND candidate genes, leaving the overlapping imprinted genes PLAGL1 and HYMAI as the only remaining complete candidate genes for 6q24 TND. Moreover, these data provide the first evidence that an additional region, encompassing the theoretical protein FAM164B, may have a critical role in the growth restriction phenotype observed in many 6q24 TND patients.

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.

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., Genomic imprinting disturbance can give rise to complex congenital disorders affecting growth, metabolism and behaviour. Here the authors report mutations in NLRP5, which suggests a connection between imprinting, maternal reproductive fitness and zygotic development.

Background The 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. Results Here 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. Conclusions This method has potential clinical utility for human disorders where DNA methylation change may be a biomarker of disease. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0081-5) contains supplementary material, which is available to authorized users.

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.

3-M syndrome is an autosomal recessive disorder characterised by pre- and post-natal growth restriction, facial dysmorphism, normal intelligence and radiological features (slender long bones and tall vertebral bodies). It is known to be caused by mutations in the genes encoding cullin 7, obscurin-like 1 and coiled-coil domain containing 8. The mechanisms through which mutations in these genes impair growth are unclear. The aim of this study was to identify novel pathways involved in the growth impairment in 3-M syndrome. RNA was extracted from fibroblast cell lines derived from four 3-M syndrome patients and three control subjects, hybridised to Affymetrix HU 133 plus 2.0 arrays with quantitative real-time PCR used to confirm changes found on microarray. IGF-II protein levels in conditioned cell culture media were measured by ELISA. Of the top 10 downregulated probesets, three represented IGF2 while H19 was identified as the 23rd most upregulated probeset. QRT-PCR confirmed upregulation of H19 (PIGF2 (PPIGF2 expression and increased H19 expression similar to that found in Silver–Russell syndrome. Loss of autocrine IGF-II in the growth plate may be associated with the short stature seen in children with 3-M syndrome.

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.

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.

This study was an investigation of 90 patients referred to the Wessex Regional Genetics Laboratory for and negative by molecular cytogenetic analysis using array comparative genomic hybridization. This patient cohort represents typical referrals to a regional genetic centre. Methylation analysis was performed at 13 imprinted loci [PLAGL1, IGF2R, MEST, GRB10, H19, IGF2 DMR2 (IGF2P0), KCNQ1OT1 (KvDMR), MEG3, SNRPN, PEG3, GNAS (GNAS exon 1a and NESP55) and GNASAS]. In total 6/90 (6.67%) were shown to have a methylation defect, 2 of which were associated with known imprinting disorders: 1 patient had isolated hypomethylation at IGF2P0, an atypical epigenotype associated with Russell-Silver syndrome, and 1 showed hypomethylation at KvDMR consistent with a diagnosis of Beckwith-Wiedemann syndrome. A further 4 patients, 3 exhibiting complete hypermethylation, and 1 partial hypomethylation, had aberrations at IGF2R, the clinical significance of which remains unclear. This study demonstrates the potential utility of epigenetic investigation in routine diagnostic testing.

The Arabidopsis Information Resource (TAIR) is a highly sophisticated, extensive, user friendly, Web-based resource for researchers working on the model plant Arabidopsis thaliana. The main gateway to this resource is through TAIR's homepage http://www.arabidopsis.org. It is a repository of large amounts of data including gene, mapping, protein, expression and community data in the form of a relational database. These data can be searched, downloaded and analysed using the tools provided. Here, the simple search (for retrieval of information), Seq Viewer (for the visualization of the five Arabidopsis chromosomes and associated annotations) and AraCyc (database of Arabidopsis biochemical pathways, with a graphical overview onto which large data sets, such as gene expression data, can be overlaid) tools are described with examples.

The Arabidopsis Information Resource (TAIR) is a highly sophisticated, extensive, user friendly, Web-based resource for researchers working on the model plant Arabidopsis thaliana. The main gateway to this resource is through TAIR's homepage http://www.arabidopsis.org. It is a repository of large amounts of data including gene, mapping, protein, expression and community data in the form of a relational database. These data can be searched, downloaded and analysed using the tools provided. Here, the simple search (for retrieval of information), Seq Viewer (for the visualization of the five Arabidopsis chromosomes and associated annotations) and AraCyc (database of Arabidopsis biochemical pathways, with a graphical overview onto which large data sets, such as gene expression data, can be overlaid) tools are described with examples.