A Diagnosis for All Rare Genetic Diseases: The Horizon and the Next Frontiers.

The introduction of exome sequencing in the clinic has sparked tremendous optimism for the future of rare disease diagnosis, and there is exciting opportunity to further leverage these advances. To provide diagnostic clarity to all of these patients, however, there is a critical need for the field to develop and implement strategies to understand the mechanisms underlying all rare diseases and translate these to clinical care.

Genomic imprinting disorders: lessons on how genome, epigenome and environment interact.

Genomic imprinting, the monoallelic and parent-of-origin-dependent expression of a subset of genes, is required for normal development, and its disruption leads to human disease. Imprinting defects can involve isolated or multilocus epigenetic changes that may have no evident genetic cause, or imprinting disruption can be traced back to alterations of cis-acting elements or trans-acting factors that control the establishment, maintenance and erasure of germline epigenetic imprints. Recent insights into the dynamics of the epigenome, including the effect of environmental factors, suggest that the developmental outcomes and heritability of imprinting disorders are influenced by interactions between the genome, the epigenome and the environment in germ cells and early embryos.

A case of mosaic deletion of paternally-inherited PLAGL1 and two cases of upd(6)mat add to evidence for PLAGL1 under-expression as a cause of growth restriction.

PLAGL1 is one of a group of imprinted genes, whose altered expression causes imprinting disorders impacting growth, development, metabolism, and behavior. PLAGL1 over-expression causes transient neonatal diabetes mellitus (TNDM type 1) and, based on murine models, under-expression would be expected to cause growth restriction. However, only some reported individuals with upd(6)mat have growth restriction, giving rise to uncertainty about the role of PLAGL1 in human growth. Here we report three individuals investigated for growth restriction, two with upd(6)mat and one with a mosaic deletion of the paternally-inherited allele of PLAGL1. These cases add to evidence of its involvement in pre- and early post-natal human growth.

Whole-genome analysis as a diagnostic tool for patients referred for diagnosis of Silver-Russell syndrome: a real-world study.

BACKGROUND: Silver-Russell syndrome (SRS) is an imprinting disorder characterised by prenatal and postnatal growth restriction, but its clinical features are non-specific and its differential diagnosis is broad. Known molecular causes of SRS include imprinting disturbance, single nucleotide variant (SNV), CNV or UPD affecting several genes; however, up to 40% of individuals with a clinical diagnosis of SRS currently receive no positive molecular diagnosis. METHODS: To determine whether whole-genome sequencing (WGS) could uncover pathogenic variants missed by current molecular testing, we analysed data of 72 participants recruited to the 100,000 Genomes Project within the clinical category of SRS. RESULTS: In 20 participants (27% of the cohort) we identified genetic variants plausibly accounting for SRS. Coding SNVs were identified in genes including CDKN1C, IGF2, IGF1R and ORC1. Maternal-effect variants were found in mothers of five participants, including two participants with imprinting disturbance and one with multilocus imprinting disorder. Two regions of homozygosity were suggestive of UPD involving imprinted regions implicated in SRS and Temple syndrome, and three plausibly pathogenic CNVs were found, including a paternal deletion of PLAGL1. In 48 participants with no plausible pathogenic variant, unbiased analysis of SNVs detected a potential association with STX4. CONCLUSION: WGS analysis can detect UPD, CNV and SNV and is potentially a valuable addition to diagnosis of SRS and related growth-restricting disorders.

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders.

Height and body mass index in molecularly confirmed Silver-Russell syndrome and the long-term effects of growth hormone treatment.

OBJECTIVE: Silver-Russell syndrome (SRS) causes short stature. Growth hormone (GH) treatment aims to increase adult height. However, data are limited on the long-term outcomes of GH in patients with molecularly confirmed SRS. This study evaluated height, body mass index (BMI) and GH treatment in molecularly confirmed SRS. DESIGN: An observational study with retrospective data collection. PATIENTS: Individuals with molecularly confirmed SRS aged ≥13 years. MEASUREMENTS: Data were collected on height, height gain (change in height standard deviation score [SDS] from childhood to final or near-final height), BMI and gain in BMI (from childhood to adulthood) and previous GH treatment. RESULTS: Seventy-one individuals (40 female) were included. The median age was 22.0 years (range 13.2-69.7). The molecular diagnoses: H19/IGF2:IG-DMR LOM in 80.3% (57/71); upd(7)mat in 16.9% (12/71) and IGF2 mutation in 2.8% (2/71). GH treatment occurred in 77.5% (55/71). Total height gain was greater in GH-treated individuals (median 1.53 SDS vs. 0.53 SDS, p = .007), who were shorter at treatment initiation (-3.46 SDS vs. -2.91 SDS, p = .04) but reached comparable heights to GH-untreated individuals (-2.22 SDS vs. -2.74 SDS, p = .7). In GH-treated individuals, BMI SDS was lower at the most recent assessment (median -1.10 vs. 1.66, p = .002) with lower BMI gain (2.01 vs. 3.58, p = .006) despite similar early BMI SDS to GH-untreated individuals (median -2.65 vs. -2.78, p = .3). CONCLUSIONS: These results support the use of GH in SRS for increasing height SDS. GH treatment was associated with lower adult BMI which may reflect improved metabolic health even following discontinuation of therapy.

A patient with multilocus imprinting disturbance involving hypomethylation at 11p15 and 14q32, and phenotypic features of Beckwith-Wiedemann and Temple syndromes.

Beckwith-Wiedemann syndrome (BWS) and Temple syndrome (TS) are classical imprinting disorders (IDs) with nonconfluent clinical features. We report here on a patient with clinical features of both syndromes, in whom epimutations were found at the BWS and TS imprinted regions, consistent with multilocus imprinting disturbance (MLID). This is the first case report of a patient with clinical features of both conditions who was found to have loss of methylation (LOM) of KCNQ1OT1: TSS-DMR (ICR2) in the 11p15 imprinted region associated with BWS and LOM of MEG3: TSS-DMR in the 14q32 imprinted region associated with TS. The report draws attention to the importance of testing for MLID as a cause of atypical clinical presentations of patients with IDs.

Phenotype of genetically confirmed Silver-Russell syndrome beyond childhood.

BACKGROUND: Silver-Russell syndrome is an imprinting disorder that restricts growth, resulting in short adult stature that may be ameliorated by treatment. Approximately 50% of patients have loss of methylation of the imprinting control region (H19/IGF2:IG-DMR) on 11p15.5 and 5%-10% have maternal uniparental disomy of chromosome 7. Most published research focuses on the childhood phenotype. Our aim was to describe the phenotypic characteristics of older patients with SRS. METHODS: A retrospective cohort of 33 individuals with a confirmed molecular diagnosis of SRS aged 13 years or above were carefully phenotyped. RESULTS: The median age of the cohort was 29.6 years; 60.6% had a height SD score (SDS) ≤-2 SDS despite 70% having received growth hormone treatment. Relative macrocephaly, feeding difficulties and a facial appearance typical of children with SRS were no longer discriminatory diagnostic features. In those aged ≥18 years, impaired glucose tolerance in 25%, hypertension in 33% and hypercholesterolaemia in 52% were noted. While 9/33 accessed special education support, university degrees were completed in 40.0% (>21 years). There was no significant correlation between quality of life and height SDS. 9/25 were parents and none of the 17 offsprings had SRS. CONCLUSION: Historical treatment regimens for SRS were not sufficient for normal adult growth and further research to optimise treatment is justified. Clinical childhood diagnostic scoring systems are not applicable to patients presenting in adulthood and SRS diagnosis requires molecular confirmation. Metabolic ill-health warrants further investigation but SRS is compatible with a normal quality of life including normal fertility in many cases.

Genetic diagnosis of subfertility: the impact of meiosis and maternal effects.

During reproductive age, approximately one in seven couples are confronted with fertility problems. While the aetiology is diverse, including infections, metabolic diseases, hormonal imbalances and iatrogenic effects, it is becoming increasingly clear that genetic factors have a significant contribution. Due to the complex nature of infertility that often hints at a multifactorial cause, the search for potentially causal gene mutations in idiopathic infertile couples has remained difficult. Idiopathic infertility patients with a suspicion of an underlying genetic cause can be expected to have mutations in genes that do not readily affect general health but are only essential in certain processes connected to fertility. In this review, we specifically focus on genes involved in meiosis and maternal-effect processes, which are of critical importance for reproduction and initial embryonic development. We give an overview of genes that have already been linked to infertility in human, as well as good candidates which have been described in other organisms. Finally, we propose a phenotypic range in which we expect an optimal diagnostic yield of a meiotic/maternal-effect gene panel.

Causes and Consequences of Multi-Locus Imprinting Disturbances in Humans.

Eight syndromes are associated with the loss of methylation at specific imprinted loci. There has been increasing evidence that these methylation defects in patients are not isolated events occurring at a given disease-associated locus but that some of these patients may have multi-locus imprinting disturbances (MLID) affecting additional imprinted regions. With the recent advances in technology, methylation profiling has revealed that imprinted loci represent only a small fraction of the methylation differences observed between the gametes. To figure out how imprinting anomalies occur at multiple imprinted domains, we have to understand the interplay between DNA methylation and histone modifications in the process of selective imprint protection during pre-implantation reprogramming, which, if disrupted, leads to these complex imprinting disorders (IDs).

DNA Methylation Profiling of Uniparental Disomy Subjects Provides a Map of Parental Epigenetic Bias in the Human Genome.

Genomic imprinting is a mechanism in which gene expression varies depending on parental origin. Imprinting occurs through differential epigenetic marks on the two parental alleles, with most imprinted loci marked by the presence of differentially methylated regions (DMRs). To identify sites of parental epigenetic bias, here we have profiled DNA methylation patterns in a cohort of 57 individuals with uniparental disomy (UPD) for 19 different chromosomes, defining imprinted DMRs as sites where the maternal and paternal methylation levels diverge significantly from the biparental mean. Using this approach we identified 77 DMRs, including nearly all those described in previous studies, in addition to 34 DMRs not previously reported. These include a DMR at TUBGCP5 within the recurrent 15q11.2 microdeletion region, suggesting potential parent-of-origin effects associated with this genomic disorder. We also observed a modest parental bias in DNA methylation levels at every CpG analyzed across ∼1.9 Mb of the 15q11-q13 Prader-Willi/Angelman syndrome region, demonstrating that the influence of imprinting is not limited to individual regulatory elements such as CpG islands, but can extend across entire chromosomal domains. Using RNA-seq data, we detected signatures consistent with imprinted expression associated with nine novel DMRs. Finally, using a population sample of 4,004 blood methylomes, we define patterns of epigenetic variation at DMRs, identifying rare individuals with global gain or loss of methylation across multiple imprinted loci. Our data provide a detailed map of parental epigenetic bias in the human genome, providing insights into potential parent-of-origin effects.

Discrepant molecular and clinical diagnoses in Beckwith-Wiedemann and Silver-Russell syndromes.

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are two imprinting disorders associated with opposite molecular alterations in the 11p15.5 imprinting centres. Their clinical diagnosis is confirmed by molecular testing in 50-70% of patients. The authors from different reference centres for BWS and SRS have identified single patients with unexpected and even contradictory molecular findings in respect to the clinical diagnosis. These patients clinically do not fit the characteristic phenotypes of SRS or BWS, but illustrate their clinical heterogeneity. Thus, comprehensive molecular testing is essential for accurate diagnosis and appropriate management, to avoid premature clinical diagnosis and anxiety for the families.

Maternal variants in NLRP and other maternal effect proteins are associated with multilocus imprinting disturbance in offspring.

BACKGROUND: Genomic imprinting results from the resistance of germline epigenetic marks to reprogramming in the early embryo for a small number of mammalian genes. Genetic, epigenetic or environmental insults that prevent imprints from evading reprogramming may result in imprinting disorders, which impact growth, development, behaviour and metabolism. We aimed to identify genetic defects causing imprinting disorders by whole-exome sequencing in families with one or more members affected by multilocus imprinting disturbance. METHODS: Whole-exome sequencing was performed in 38 pedigrees where probands had multilocus imprinting disturbance, in five of whom maternal variants in NLRP5 have previously been found. RESULTS: We now report 15 further pedigrees in which offspring had disturbance of imprinting, while their mothers had rare, predicted-deleterious variants in maternal effect genes, including NLRP2, NLRP7 and PADI6. As well as clinical features of well-recognised imprinting disorders, some offspring had additional features including developmental delay, behavioural problems and discordant monozygotic twinning, while some mothers had reproductive problems including pregnancy loss. CONCLUSION: The identification of 20 putative maternal effect variants in 38 families affected by multilocus imprinting disorders adds to the evidence that maternal genetic factors affect oocyte fitness and thus offspring development. Testing for maternal-effect genetic variants should be considered in families affected by atypical imprinting disorders.

Genetics, molar pregnancies and medieval ideas of monstrous births: the lump of flesh in The King of Tars.

The medieval English romance The King of Tars gives an account of a birth of a lump of flesh. This has been considered as fantastic and monstrous in past literature, the horrific union of a Christian and Saracen. However, while the text certainly speaks to miscegenation, we propose that this lump of flesh is actually a hydatidiform mole. We trace the hydatidiform mole from antiquity, surrounding it with contextual medieval examples, from theology, history and medicine, that also describe abnormal births as 'lumps of flesh'. By discussing medieval ideas of monsters as a warning sign, we interpret the lump of flesh in terms of abnormal births, seed transmission, parental contribution and sin. Ideas of warning, blame and intervention present themselves as a response to moles both in medieval texts as well as in modern reactions to hydatidiform moles. We explore the epigenetics of hydatidiform moles and relate them to the medieval text. In The King of Tars, the fault for the lump of flesh could reside with either parent; we find that this is also the case in the genetic formation of the hydatidiform mole; we also argue that the epigenetics supports medieval theories of seed transmission.

The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: an international cohort study.

BACKGROUND: Traditional genetic testing focusses on analysis of one or a few genes according to clinical features; this approach is changing as improved sequencing methods enable simultaneous analysis of several genes. Neonatal diabetes is the presenting feature of many discrete clinical phenotypes defined by different genetic causes. Genetic subtype defines treatment, with improved glycaemic control on sulfonylurea treatment for most patients with potassium channel mutations. We investigated the effect of early, comprehensive testing of all known genetic causes of neonatal diabetes. METHODS: In this large, international, cohort study, we studied patients with neonatal diabetes diagnosed with diabetes before 6 months of age who were referred from 79 countries. We identified mutations by comprehensive genetic testing including Sanger sequencing, 6q24 methylation analysis, and targeted next-generation sequencing of all known neonatal diabetes genes. FINDINGS: Between January, 2000, and August, 2013, genetic testing was done in 1020 patients (571 boys, 449 girls). Mutations in the potassium channel genes were the most common cause (n=390) of neonatal diabetes, but were identified less frequently in consanguineous families (12% in consanguineous families vs 46% in non-consanguineous families; p<0·0001). Median duration of diabetes at the time of genetic testing decreased from more than 4 years before 2005 to less than 3 months after 2012. Earlier referral for genetic testing affected the clinical phenotype. In patients with genetically diagnosed Wolcott-Rallison syndrome, 23 (88%) of 26 patients tested within 3 months from diagnosis had isolated diabetes, compared with three (17%) of 18 patients referred later (>4 years; p<0·0001), in whom skeletal and liver involvement was common. Similarly, for patients with genetically diagnosed transient neonatal diabetes, the diabetes had remitted in only ten (10%) of 101 patients tested early (<3 months) compared with 60 (100%) of the 60 later referrals (p<0·0001). INTERPRETATION: Patients are now referred for genetic testing closer to their presentation with neonatal diabetes. Comprehensive testing of all causes identified causal mutations in more than 80% of cases. The genetic result predicts the best diabetes treatment and development of related features. This model represents a new framework for clinical care with genetic diagnosis preceding development of clinical features and guiding clinical management. FUNDING: Wellcome Trust and Diabetes UK.

Maternal uniparental disomy of chromosome 20: a novel imprinting disorder of growth failure.

PURPOSE: Maternal uniparental disomy of chromosome 20 (UPD(20)mat) has been reported in only four patients, three of whom also had mosaicism for complete or partial trisomy of chromosome 20. We sought to evaluate the clinical significance of isolated UPD(20)mat in eight individuals. METHODS: We evaluated phenotypic and genomic findings of a series of eight new patients with UPD(20)mat. RESULTS: All eight individuals with UPD(20)mat had intrauterine growth restriction, short stature, and prominent feeding difficulties with failure to thrive. As a common feature, they often required gastric tube feeds. Genomic data in most patients are indicative of UPD as a result of trisomy rescue after meiosis II nondisjunction. CONCLUSION: We describe the first natural history of the disorder and the results of therapeutic interventions, including the frequent requirement of direct gastric feedings only during the first few years of life, and propose that growth hormone supplementation is probably safe and effective for this condition. We suggest that UPD(20)mat can be regarded as a new imprinting disorder and its identification requires specialized molecular testing, which should be performed in patients with early-onset idiopathic isolated growth failure.Genet Med 18 4, 309-315.

Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57.

BACKGROUND: Transient neonatal diabetes mellitus 1 (TNDM1) is a rare imprinting disorder characterized by intrautering growth retardation and diabetes mellitus usually presenting within the first six weeks of life and resolves by the age of 18 months. However, patients have an increased risk of developing diabetes mellitus type 2 later in life. Transient neonatal diabetes mellitus 1 is caused by overexpression of the maternally imprinted genes PLAGL1 and HYMAI on chromosome 6q24. One of the mechanisms leading to overexpression of the locus is hypomethylation of the maternal allele of PLAGL1 and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers. METHODS: Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound heterozygous ZFP57 mutations, three relatives with heterozygous ZFP57 mutations and five controls. Methylation status of selected regions showing aberrant methylation in the patients was verified using bisulfite-sequencing. RESULTS: We found large variability among the patients concerning the number and identity of the differentially methylated regions, but more than 60 regions were aberrantly methylated in two or more patients and a novel region within PPP1R13L was found to be hypomethylated in all the patients. The hypomethylated regions in common between the patients are enriched for the ZFP57 DNA binding motif. CONCLUSIONS: We have expanded the epimutational spectrum of TNDM1 associated with ZFP57 mutations and found one novel region within PPP1R13L which is hypomethylated in all TNDM1 patients included in this study. Functional studies of the locus might provide further insight into the etiology of the disease.

Temple syndrome as a result of isolated hypomethylation of the 14q32 imprinted DLK1/MEG3 region.

We present a Caucasian female, who was diagnosed at 13 years of age with Temple syndrome (formerly referred to as "maternal UPD 14 phenotype") due to an epigenetic loss of methylation at IG-DMR/MEG3-DMR at the chromosome 14q32 imprinted locus. Clinical features were typical and included intra-uterine growth retardation (IUGR), low birth weight, hypotonia, and poor feeding in the neonatal period; and failure to thrive and developmental delay--particularly in relation to speech--in early childhood. Premature puberty, with short stature and truncal obesity, but normal intelligence, were the key features in teenage years. To date only eight patients with Temple syndrome due to an epigenetic error have been described and the etiology of the methylation defect is currently undetermined. In view of a tendency towards central obesity, patients are at potential risk of early-onset type 2 diabetes mellitus, as well as cardiovascular disease and they, therefore, require appropriate monitoring.

Congenital imprinting disorders: Application of multilocus and high throughput methods to decipher new pathomechanisms and improve their management.

Imprinting disorders (IDs) are a group of congenital diseases affecting growth, development and metabolism. They are caused by changes in the allele-specific regulation ("epigenetic mutation") or in the genomic sequence ("genetic mutation") of imprinted genes. Currently molecular tests in ID patients are generally restricted to single loci classically associated with the disease, but this approach limits diagnostic yield, because of the molecular and clinical heterogeneity between IDs. From the technical point of view, these limitations are aggravated by the lack of standardization in testing methodology, in the DNA sequences tested, and in clinical inclusion criteria prompting testing. However, an increasing number of new studies show that these problems can be addressed by the use of new tests targeting multiple loci and/or a total exome and genome analysis. The rapid development of efficient and high-throughput molecular techniques and their applications in research and diagnostics in the last decade have led to an impressive increase of knowledge on IDs and their basic pathomechanisms. In combination with the improvement of data recording and documentation, the diagnostic strategies are increasingly based on standardized protocols, and thereby provide the backbone for directed counseling, more personalized management, and new therapeutic approaches.

Temple syndrome: improving the recognition of an underdiagnosed chromosome 14 imprinting disorder: an analysis of 51 published cases.

Chromosome 14 harbours an imprinted locus at 14q32. Maternal uniparental disomy of chromosome 14, paternal deletions and loss of methylation at the intergenic differentially methylated region (IG-DMR) result in a human phenotype of low birth weight, hypotonia, early puberty and markedly short adult stature. The analysis of the world literature of 51 cases identifies the key features that will enhance diagnosis and potentially improve treatment. We found a median birth weight SD score (SDS) of -1.88 and median adult final height of -2.04 SDS. Hypotonia and motor delay were reported in 93% and 83% of cases, respectively. Early puberty was reported in 86% of cases with the mean age of menarche at 10 years and 2 months of age. Small hands and feet were reported frequently (87% and 96%, respectively). Premature birth was common (30%) and feeding difficulties frequently reported (n = 22). There was evidence of mildly reduced intellectual ability (measured IQ 75-95). Obesity was reported in 49% of cases, and three patients developed type 2 diabetes mellitus. Two patients were reported to have recurrent hypoglycaemia, and one of these patients was subsequently demonstrated to be growth hormone deficient and started replacement therapy. We propose the use of the name 'Temple syndrome' for this condition and suggest that improved diagnosis and long-term monitoring, especially of growth and cardiovascular risk factors, is required.