Imprinting Defects Research Articles

Hyperphagia in Prader-Willi syndrome with obesity: From development to pharmacological treatment.

Prader-Willi syndrome (PWS) is a rare genetic disorder due to lack of genes expression inherited from the paternal chromosome 15q11-q13 region usually from paternal deletions, maternal uniparental disomy 15 or imprinting defect. There are two different nutritional stages reported in an individual with PWS; first stage during infancy marked by feeding and growth difficulties and second stage where hyperphagia starts and leads to development of obesity. However, the exact mechanism of hyperphagia development, from having difficulties in feeding during early years to insatiable appetite after they grow is still unknown and is the focused in this review. The keywords used for literature search such as "Prader-Willi syndrome", "hyperphagia", "obesity", and "treatment" were used to create the search strings by using synonyms in order to retrieve the relevant records from PubMed, Scopus and Science Direct. The possible mechanism of hyperphagia can be classed into hormonal abnormalities such as increase in ghrelin and leptin from infancy to adulthood. Low level of hormones was observed in the thyroid, insulin and peptide YY at certain ages. Neuronal abnormalities contributed by Orexin A and brain structure alteration was documented at 4-30 years old. Treatment in the form of drugs such as livoletide, topiramate, and diazoxide could potentially alleviate these abnormalities and make hyperphagia less prominent in PWS. The approaches are important to regulate the hormonal changes and neuronal involvement as potentially controlling hyperphagia and obesity.

Rapid flavonoid-focused sub-chemome characterization of Draconis Sanguis using UPLC-Q-TOF-MS in combination with molecular weight imprinting and mass defect filtering

Draconis Sanguis is a precious Chinese medicinal material for activating blood and resolving stasis, and its effective components are flavonoids. However, the structural diversity of flavonoids in Draconis Sanguis brings great challenges to the in-depth chara-cterization of its chemical composition profiles. To clarify the substance basis of Draconis Sanguis, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used in this study to acquire MS data of Draconis Sanguis. The molecular weight imprinting(MWI) and mass defect filtering(MDF) were developed for rapid screening of flavonoids in Draconis Sanguis. Full-scan MS and MS~2 were recorded within the mass range m/z 100-1 000 in positive ion mode. Accor-ding to previous literature, MWI was employed to hunt for reported flavonoids in Draconis Sanguis, and the mass tolerance range of [M+H]~+ was set as ±10×10~(-3). A five-point MDF screening frame was further constructed to narrow the screening range of flavonoids from Draconis Sanguis. Combined with diagnostic fragment ions(DFI) and neutral loss(NL) as well as mass fragmentation pathways, 70 compounds were preliminarily identified from the extract of Draconis Sanguis, including 5 flavan oxidized congeners, 12 flavans, 1 dihydrochalcones, 49 flavonoids dimers, 1 flavonoids trimer and 2 flavonoid derivatives. This study clarified the chemical composition of flavonoids in Draconis Sanguis. Moreover, it also showed that high-resolution MS combined with data post-processing methods such as MWI and MDF could achieve rapid characterization of the chemical composition in Chinese medicinal materials.

Perinatal and neonatal characteristics of Prader-Willi syndrome inJapan.

Prader-Willi syndrome (PWS) is suspected at birth if extreme hypotonia, difficulty in feeding, hypogonadism, and failure to thrive are present. Genetic diagnosis of PWS can generally be made within the first few months of life; however, a delayed diagnosis of PWS is frequently reported. Although the clinical characteristics of perinatal and neonatal patients with PWS have been reported, there are no such reports on the clinical characteristics of these patients in Japan. This retrospective, single-center study involved 177 Japanese patients with PWS and their medical data regarding the perinatal and neonatal periods were evaluated. The median maternal age at birth was 34 years; 12.7% of the mothers had a history of assisted reproductive technology (ART). Of the mothers, 13.5% reported polyhydramnios and 4.3% had oligohydramnios. Decreased fetal movement during pregnancy was reported by 76% of the mothers. A total of 60.5% of patients were born by cesarean section. Genetic subtypes included deletions (66.1%), uniparental disomy (31.0%), imprinting defects (0.6%), and other or unknown subtypes (2.3%). The median birth length was 47.5 cm and the median birthweight was 2476 g. Of the 160 patients, 14 (8.8%) were classified as small for gestational age. Most patients had hypotonia (98.8%), and 89.3% required gavage feeding at birth. Breathing problems, congenital heart disease, and undescended testis were noted in 33.1%, 7.0%, and 93.5% of patients, respectively. In our study, higher rates of ART, polyhydramnios, decreased fetal movements, cesarean section, hypotonia, feeding difficulties, and undescended testis were observed in PWS.

Diagnosis of Prader-Willi syndrome and Angelman syndrome by targeted nanopore long-read sequencing

The CpG island flanking the promoter region of SNRPN on chromosome 15q11.2 contains CpG sites that are completely methylated in the maternally derived allele and unmethylated in the paternally derived allele. Both unmethylated and methylated alleles are observed in normal individuals. Only the methylated allele is observed in patients with Prader-Willi syndrome, whereas only the unmethylated allele is observed in those with Angelman syndrome. Hence, detection of aberrant methylation at the differentially methylated region is fundamental to the molecular diagnosis of Prader-Willi syndrome and Angelman syndromes. Traditionally, bisulfite treatment and methylation-sensitive restriction enzyme treatment or methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) have been used. We here developed a long-read sequencing assay that can distinguish methylated and unmethylated CpG sites at 15q11.2 by the difference in current intensity generated from nanopore reads. We successfully diagnosed 4 Prader-Willi syndrome patients and 3 Angelman syndrome patients by targeting differentially methylated regions. Concurrent copy number analysis, homozygosity analysis, and structural variant analysis also allowed us to precisely delineate the underlying pathogenic mechanisms, including gross deletion, uniparental heterodisomy, uniparental isodisomy, or imprinting defect. Furthermore, we showed allele-specific methylation in imprinting-related differentially methylated regions on chromosomes 6, 7, 11, 14, and 20 in a normal individual together with 4 Prader-Willi patients and 3 Angelman syndrome patients. Hence, presently reported method is likely to be applicable to the diagnosis of imprinting disorders other than Prader-Willi syndrome and Angelman syndrome as well.

Prader-Willi syndrome patient with atypical phenotypes caused by mosaic deletion in the paternal 15q11-q13 region: a case report

BackgroundPrader-Willi syndrome (PWS) is a multisystemic complex genetic disorder caused by the loss of paternally expressed genes in the human chromosome region 15q11.2-q13. It is characterized by severe hypotonia and feeding difficulties in early infancy, followed in later infancy or early childhood by excessive eating and gradual development of morbid obesity. Motor milestones and language development are delayed and most patients have intellectual disability.Case presentationHere we describe a rare PWS case caused by mosaic imprinting defect in the region 15q11.2-q13 of paternal origin. The proband was a male child with a clinical presentation of global developmental delay and hypotonia with specific facial features. Karyotype of the child was noted as mosaic: 45XY,der(15)?t(15;21),-21[26]/46,XY[24]. Whole-exome sequencing (WES) identified a deletion of 22.7 Mb in size at chr15q11.2q21.1 region and a deletion of 2.1 Mb in size at chr21q22.3 region. The Methylation-specific multiplex ligation-dependent probe amplification(MS-MLPA) of the 15q11.2-q13 region showed that the loading ratio of methylated alleles was 70% and that of unmethylated alleles was 30%(50% normal), which confirmed that the loss of mosaic imprinted defects in the paternal allele led to the diagnosis of PWS.ConclusionsWe propose that complete clinical criteria for PWS should not be considered sensitive in diagnosing partial atypical PWS due to mosaic imprinting defects. In contrast, clinical suspicion based on less restrictive criteria followed by multiple techniques is a more powerful approach.

Young plasma transfer recovers decreased sperm counts and restores epigenetics in aged testis

Background and aimAging is one of the causes of male infertility, and abnormal global DNA methylation and imprinting defects have been characterized in testis during biological aging. One of the important emerging approaches aims to take advantage of the healing properties of young blood plasma to limit the progression of aging in various organs in the body. We aimed to show whether blood plasma transfer has an effect on DNA methylation and spermatogenetic cell development. In addition, we aimed to show whether the young plasma transfer to old mice has an effect on the rejuvenation of the old and whether the impaired DNA methylation and PCNA expression in old age can be restored. MethodsGroups were (i) young control, (ii) young plasma transfer to aged, (iii) aged control, (iv) aged plasma transfer to young. We utilized IHC and WB in protein level of Dnmts. For the global DNA methylation level, we used 5-methylcytosine staining. We also analyzed PCNA protein expressions in all groups by IHC. ResultsWe found that transfusion of young plasma into the old animal restored DNA methylation and PCNA expression as it did in the young animal. Most importantly, we observed an increase in spermatogonia and spermatid counts in older animals after young blood plasma transfer. ConclusionsOur findings show that young plasma transfer can restore epigenetic disorders that occur with aging and solve infertility problems by increasing the sperm count that decreases. It needs to be supported by different studies, especially human studies.

Concurrent Hepatoblastoma and Wilms Tumor Leading to Diagnosis of Beckwith-Wiedemann Syndrome.

Beckwith-Wiedemann syndrome (BWS) is an epigenetic overgrowth disorder and cancer predisposition syndrome caused by imprinting defects of chromosome 11p15.5-11p15.4. BWS should be considered in children with atypical presentations of embryonal tumors regardless of clinical phenotype. Risk of malignancy correlates with specific molecular subgroups of BWS making molecular subclassification important for appropriate cancer screening. We report the first case of concurrent embryonal tumors in a phenotypically normal child, leading to the diagnosis of BWS with paternal uniparental disomy and describe the molecular classification of BWS as it relates to malignancy risk, along with approach to management.

First step towards a consensus strategy for multi-locus diagnostic testing of imprinting disorders

BackgroundImprinting disorders, which affect growth, development, metabolism and neoplasia risk, are caused by genetic or epigenetic changes to genes that are expressed from only one parental allele. Disease may result from changes in coding sequences, copy number changes, uniparental disomy or imprinting defects. Some imprinting disorders are clinically heterogeneous, some are associated with more than one imprinted locus, and some patients have alterations affecting multiple loci. Most imprinting disorders are diagnosed by stepwise analysis of gene dosage and methylation of single loci, but some laboratories assay a panel of loci associated with different imprinting disorders. We looked into the experience of several laboratories using single-locus and/or multi-locus diagnostic testing to explore how different testing strategies affect diagnostic outcomes and whether multi-locus testing has the potential to increase the diagnostic efficiency or reveal unforeseen diagnoses.ResultsWe collected data from 11 laboratories in seven countries, involving 16,364 individuals and eight imprinting disorders. Among the 4721 individuals tested for the growth restriction disorder Silver–Russell syndrome, 731 had changes on chromosomes 7 and 11 classically associated with the disorder, but 115 had unexpected diagnoses that involved atypical molecular changes, imprinted loci on chromosomes other than 7 or 11 or multi-locus imprinting disorder. In a similar way, the molecular changes detected in Beckwith–Wiedemann syndrome and other imprinting disorders depended on the testing strategies employed by the different laboratories.ConclusionsBased on our findings, we discuss how multi-locus testing might optimise diagnosis for patients with classical and less familiar clinical imprinting disorders. Additionally, our compiled data reflect the daily life experiences of diagnostic laboratories, with a lower diagnostic yield than in clinically well-characterised cohorts, and illustrate the need for systematising clinical and molecular data.

ODP117 Pseudohypoparathyroidism Type IB: A Rare Cause of Hypocalcemia

Abstract Introduction Pseudohypoparathyroidism (PHP) describes disorders of hypoparathyroidism that are the result of resistance of target tissues to the actions of parathyroid hormone (PTH). Studies have estimated the prevalence of PHP to be 0.34 in 100,000 in Japan and 1.1 in 100,000 in Denmark. The first biochemical abnormalities to become apparent are elevated serum levels of PTH and elevated serum levels of phosphorus, followed by hypocalcaemia. We present a patient diagnosed with PHP Type 1B (PHP1B), which was presented to us many years after her initial diagnosis, with other major comorbidities now present. Case description This patient is an 81 year old female presenting to the clinic to discuss her previously diagnosed PHP1B. Her past medical history revealed she began having symptoms of intermittent muscle cramping at the age of 22, followed by carpopedal spasms and periorbital paresthesia a year later. She has also had a physical examination significant for a positive Trousseau sign, but no phenotypical signs of Albright hereditary Osteodystrophy (AHO). Lab evidence from 1977 was recovered showing urinary cyclic AMP level <20 nmol/mg after administration of PTH, consistent with PHP1B. The family history in her parents and children has revealed no similar genetic history so far. Other comorbidities she has developed since her diagnosis are hypokalemia, hypothyroidism, osteoporosis with a femur fracture, obesity, chronic kidney disease and nephrolithiasis. She has been continuing treatment throughout her life with calcium, calcitriol and vitamin D supplementation for hypocalcemia. She has been mostly eucalcemic since then, and complains of occasional carpopedal spasms and perioral numbness. Discussion PHP1B is isolated resistance to PTH in the kidney and normal PTH response in bone without features of AHO. PHP1A and 1C have PTH resistance in bone, and both express AHO. These various forms of PHP are due to defects in the GNAS gene that lead to decreased expression of the alpha-subunit of G protein (Gsa). In the absence of molecular analysis, the clinical and biochemical overlap between PHP and related disorders can lead to challenges in diagnostic classification. There are 2 types of PHP1B cases- autosomal dominant (AD) and sporadic. However, the majority of Patients with PHP1B do not have mutations within the GNAS but they have methylation and imprinting defects that result in absence of maternal Gsa. To emphasize, epimutation study rather than sequencing of the coding region in GNAS should be performed for genetic investigation, which has now become standard practice. Although there is no difference in clinical presentation between AD PHP1B and sporadic PHP1B, their differentiation early during the disease onset can help providers give patients more reliable genetic and reproductive counseling and provide optimal medical management of complications such as hypocalcemia. Presentation: No date and time listed

Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.

An Analysis of Phenotype and Genotype in a Large Cohort of Chinese Children with Angelman Syndrome.

Angelman syndrome (AS) is a neurodevelopmental genetic disorder, but there has been limited analysis of a large cohort of Chinese children with Angelman syndrome. This study aims to assess the phenotype and genotype of Chinese children with Angelman syndrome. We retrospectively analyzed data through a detailed online survey combined with an on-site study. Furthermore, phenotype analysis stratified by deletion and non-deletion groups was carried out. The responses of family members of 695 individuals with AS revealed that 577 patients (83.02%) had maternal deletions, 65 patients (9.35%) carried UBE3A mutations, 31 (4.46%) patients had UPD15pat (one patient with UPD15pat constituted by a mosaic), 10 patients (1.44%) had imprinting defects and 12 (1.58%) patients only showed abnormal methylation without further detection. We identified 50 different pathogenic variants in this cohort, although 18 of these variants were unreported. Recurrent variant c.2507_2510del (p.K836Rfs*4) was found in 7 patients. In the deletion group, patients were diagnosed at an earlier age, had a more severe clinical phenotype, a higher rate of epilepsy with more multiple seizure types, and more frequently combined medication. Strabismus and sleep disturbances were both common in deletion and non-deletion groups. The top three resources invested in caring for AS children are: daily involvement in patient care, rehabilitation cost, and anti-epileptic treatment. Our study showed the genetic composition of Chinese children with 83.02% of maternal deletions, and the mutation spectrum for UBE3A variants was expanded. Developmental outcomes are associated with genotype, and this was confirmed by deletion patients having a worse clinical phenotype and complex epilepsy.

Atypical 15q11.2-q13 Deletions and the Prader-Willi Phenotype.

Background: Prader-Willi syndrome (PWS) is a rare genetic disorder resulting from the lack of expression of the PWS region (locus q11-q13) on the paternally derived chromosome 15, as a result of a type I or II paternal deletion (50%), maternal uniparental disomy (43%), imprinting defect (4%) or translocation (<1%). In very rare cases, atypical deletions, smaller or larger than the typical deletion, are identified. These patients may have distinct phenotypical features and provide further information regarding the genotype–phenotype correlation in PWS. Methods: A prospective study in eight patients (six males and two females) with an atypical deletion in the PWS region accompanies an overview of reported cases. Results: All patients had hypotonia (100%) and many had typical PWS facial characteristics (75%), social and emotional developmental delays (75%), intellectual disabilities (50%), neonatal feeding problems and tube feeding (63%), history of obesity (50%), hyperphagia (50%) and scoliosis (50%). All males had cryptorchidism. Two patients had two separate deletions in the PWS critical region. Conclusions: Our findings provide further insight into PWS genotype–phenotype correlations; our results imply that inclusion of both SNURF-SNPRN and SNORD-116 genes in the deletion leads to a more complete PWS phenotype. A larger deletion, extending further upstream and downstream from these genes, does not cause a more severe phenotype. Conventional PWS methylation testing may miss small deletions, which can be identified using targeted next generation sequencing. PWS’s phenotypic diversity might be caused by differentially methylated regions outside the 15q11.2 locus.

Proteins and proteases of Prader-Willi syndrome: a comprehensive review and perspectives.

Prader–Willi Syndrome (PWS) is a rare complex genetic disease that is associated with pathological disorders that include endocrine disruption, developmental, neurological, and physical problems as well as intellectual, and behavioral dysfunction. In early stage, PWS is characterized by respiratory distress, hypotonia, and poor sucking ability, causing feeding concern and poor weight gain. Additional features of the disease evolve over time. These include hyperphagia, obesity, developmental, cognitive delay, skin picking, high pain threshold, short stature, growth hormone deficiency, hypogonadism, strabismus, scoliosis, joint laxity, or hip dysplasia. The disease is associated with a shortened life expectancy. There is no cure for PWS, although interventions are available for symptoms management. PWS is caused by genetic defects in chromosome 15q11.2-q13, and categorized into three groups, namely Paternal deletion, Maternal uniparental disomy, and Imprinting defect. PWS is confirmed through genetic testing and DNA-methylation analysis. Studies revealed that at least two key proteins namely MAGEL-2 and NECDIN along with two proteases PCSK1 and PCSK2 are linked to PWS. Herein, we summarize our current understanding and knowledge about the role of these proteins and enzymes in various biological processes associated with PWS. The review also describes how loss and/or impairment of functional activity of these macromolecules can lead to hormonal disbalance by promoting degradation of secretory granules and via inhibition of proteolytic maturation of precursor-proteins. The present review will draw attention of researchers, scientists, and academicians engaged in PWS study and will help to identify potential targets and molecular pathways for PWS intervention and treatment.

Dental pulp stem cells as a promising model to study imprinting diseases

Parental imprinting is an epigenetic process leading to monoallelic expression of certain genes depending on their parental origin. Imprinting diseases are characterized by growth and metabolic issues starting from birth to adulthood. They are mainly due to methylation defects in imprinting control region that drive the abnormal expression of imprinted genes. We currently lack relevant animal or cellular models to unravel the pathophysiology of growth failure in these diseases. We aimed to characterize the methylation of imprinting regions in dental pulp stem cells and during their differentiation in osteogenic cells (involved in growth regulation) to assess the interest of this cells in modeling imprinting diseases. We collected dental pulp stem cells from five controls and four patients (three with Silver-Russell syndrome and one with Beckwith-Wiedemann syndrome). Methylation analysis of imprinting control regions involved in these syndromes showed a normal profile in controls and the imprinting defect in patients. These results were maintained in dental pulp stem cells cultured under osteogenic conditions. Furthermore, we confirmed the same pattern in six other loci involved in imprinting diseases in humans. We also confirmed monoallelic expression of H19 (an imprinted gene) in controls and its biallelic expression in one patient. Extensive imprinting control regions methylation analysis shows the strong potential of dental pulp stem cells in modeling imprinting diseases, in which imprinting regions are preserved in culture and during osteogenic differentiation. This will allow to perform in vitro functional and therapeutic tests in cells derived from dental pulp stem cells and generate other cell-types.

EP290: A mosaic Angelman syndrome case with language apraxia and symptoms overlapping Prader-Willi syndrome

Human imprinting disorders are caused by the disruption of the balanced monoallelic parent-of-origin specific expression of imprinted genes. Cycles of erasing, establishing, and maintaining CpG methylation at critical genomic regions in spermatogenesis and oogenesis and following fertilization are the major mechanisms for these genes to be imprinted and to regulate metabolism, growth, development, and behavior. Due to opposite imprinting defects, Prader-Willi syndrome (PWS) and typical Angelman syndrome (AS) are two related disorders with distinct neurodevelopmental phenotype.

Methylation analysis and developmental profile of two individuals with Angelman syndrome due to mosaic imprinting defects

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternally-inherited UBE3A on chromosome 15q11.2. In AS due to a chromosomal deletion that encompasses UBE3A, paternal uniparental disomy of chromosome 15, or imprinting defects (ImpD), the SNRPN locus is unmethylated, while in neurotypical individuals, it is ∼50% methylated. We present the developmental profile of two adults with mild AS assessed using standardized behavioral and neurodevelopmental measures. Both had intellectual disability with unusually advanced verbal communication skills compared to other individuals with AS. Methylation of the SNRPN locus was examined using Methylation Specific Quantitative Melt Analysis (MS-QMA) in different tissues at one time point for participant A (22 years) and two time points for participant B (T1: 22 years, T2: 25 years), and these levels were compared to a typical AS cohort. While participant A showed methylation levels comparable to the typical AS cohort, participant B showed methylation mosaicism in all tissues at both time points and changes in methylation levels from T1 to T2. AS should be considered in individuals with intellectual disability and verbal speech who may not have the typical symptoms of AS.

BrewerIX enables allelic expression analysis of imprinted and X-linked genes from bulk and single-cell transcriptomes

Genomic imprinting and X chromosome inactivation (XCI) are two prototypical epigenetic mechanisms whereby a set of genes is expressed mono-allelically in order to fine-tune their expression levels. Defects in genomic imprinting have been observed in several neurodevelopmental disorders, in a wide range of tumours and in induced pluripotent stem cells (iPSCs). Single Nucleotide Variants (SNVs) are readily detectable by RNA-sequencing allowing the determination of whether imprinted or X-linked genes are aberrantly expressed from both alleles, although standardised analysis methods are still missing. We have developed a tool, named BrewerIX, that provides comprehensive information about the allelic expression of a large, manually-curated set of imprinted and X-linked genes. BrewerIX does not require programming skills, runs on a standard personal computer, and can analyze both bulk and single-cell transcriptomes of human and mouse cells directly from raw sequencing data. BrewerIX confirmed previous observations regarding the bi-allelic expression of some imprinted genes in naive pluripotent cells and extended them to preimplantation embryos. BrewerIX also identified misregulated imprinted genes in breast cancer cells and in human organoids and identified genes escaping XCI in human somatic cells. We believe BrewerIX will be useful for the study of genomic imprinting and XCI during development and reprogramming, and for detecting aberrations in cancer, iPSCs and organoids. Due to its ease of use to non-computational biologists, its implementation could become standard practice during sample assessment, thus raising the robustness and reproducibility of future studies.

Ribosomal DNA methylation in human and mouse oocytes increases with age.

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages.

Clinical Utility of Methylation-Specific Multiplex Ligation-Dependent Probe Amplification for the Diagnosis of Prader-Willi Syndrome and Angelman Syndrome.

BackgroundPrader–Willi syndrome (PWS) and Angelman syndrome (AS) are genomic imprinting disorders that are mainly caused by a deletion on 15q11-q13, the uniparental disomy of chromosome 15, or an imprinting defect. We evaluated the utility of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a diagnostic tool and for demonstrating the relationship between molecular mechanisms and clinical presentation.MethodsWe performed MS-MLPA using DNA samples from 93 subjects (45 PWS, 24 AS, and 24 non-PWS/AS controls) who had previously undergone MS-PCR for the diagnosis of PWS/AS. We compared the results of both assays, and patients’ clinical phenotypes were reviewed retrospectively.ResultsMS-MLPA showed a 100% concordance rate with MS-PCR. Among the 45 PWS patients, 26 (57.8%) had a deletion of 15q11-q13, and the others (42.2%) had uniparental disomy 15 or an imprinting defect. Among the 24 AS patients, 16 (66.7%) had a deletion of 15q11-q13, 7 AS patients (29.2%) had uniparental disomy 15 or an imprinting defect, and one AS patient (4.2%) showed an imprinting center deletion.ConclusionsMS-MLPA has clinical utility for the diagnosis of PWS/AS, and it is superior to MS-PCR in that it can identify the molecular mechanism underlying the disease.

Lack of GNAS Remethylation During Oogenesis May Be a Cause of Sporadic Pseudohypoparathyroidism Type Ib.

Pseudohypoparathyroidism type Ib (PHP1B) is characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone resistance in the proximal renal tubules. Maternal pathogenic STX16/GNAS variants leading to maternal epigenetic GNAS changes impair expression of the stimulatory G protein alpha-subunit (Gsα) thereby causing autosomal dominant PHP1B. In contrast, genetic defects responsible for sporadic PHP1B (sporPHP1B) remain mostly unknown. Determine whether PHP1B encountered after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) causes GNAS remethylation defects similar to those in sporPHP1B. Retrospective analysis. Nine among 36 sporPHP1B patients investigated since 2000, all with loss of methylation (LOM) at the 3 maternal GNAS differentially methylated regions (DMRs) and gain of methylation at the paternal NESP DMR, had been conceived through IVF or ICSI. Besides abnormal GNAS methylation, IVF/ICSI PHP1B cases revealed no additional imprinting defects. Three of these PHP1B patients have dizygotic twins, and 4 have IVF/ICSI-conceived siblings, all with normal GNAS methylation; 2 unaffected younger siblings were conceived naturally. Sporadic and IVF/ICSI-conceived PHP1B patients revealed indistinguishable epigenetic changes at all 4 GNAS DMRs, thus suggesting a similar underlying disease mechanism. Given that remethylation at the 3 maternal DMRs occurs during oogenesis, male factors are unlikely to cause LOM postfertilization. Instead, at least some of the sporPHP1B variants could be caused by a defect or defects in an oocyte-expressed gene that is required for fertility and for re-establishing maternal GNAS methylation imprints. It remains uncertain, however, whether the lack of GNAS remethylation alone and the resulting reduction in Gsα expression is sufficient to impair oocyte maturation.