Methylation Defects Research Articles

Recurrent Small Variants in NESP55/NESPAS Associated with Broad GNAS Methylation Defects and Pseudohypoparathyroidism Type 1b.

Pseudohypoparathyroidism type 1B (PHP1B) is associated with epigenetic changes on the maternal allele of the imprinted GNAS gene that inhibit expression of the alpha subunit of Gs (Gsα), thereby leading to parathyroid hormone resistance in renal proximal tubule cells where expression of Gs from the paternal GNAS allele is normally silent. Although all patients with PHP1B show loss of methylation for the exon A/B differentially methylated region (DMR), some patients with autosomal dominant PHP1B (AD-PHP1B) and most patients with sporadic PHP1B have additional methylation defects that affect the DMRs corresponding to exons XL, AS1, and NESP. Because the genetic defect is unknown in most of these patients, we sought to identify the underlying genetic basis for AD-PHP1B in two multigenerational families with broad GNAS methylation defects and negative clinical exomes. Genome sequencing identified small GNAS variants in each family that were also present in unrelated PHP1B subjects in a replication cohort. Maternal transmission of one GNAS microdeletion showed reduced penetrance in some unaffected patients. Expression of AS transcripts was increased, and NESP was decreased, in cells from affected patients. These results suggest that the small deletion activate AS transcription leading to methylation of the NESP DMR with consequent inhibition of NESP transcription, and thereby provide a potential mechanism for PHP1B.

Nuclear localization of MTHFD2 is required for correct mitosis progression.

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs.

Comprehensive molecular and clinical findings in 29 patients with multi-locus imprinting disturbance

BackgroundMulti-locus imprinting disturbance (MLID) with methylation defects in various differentially methylated regions (DMRs) has recently been identified in approximately 150 cases with imprinting disorders (IDs), and deleterious variants have been found in genes related to methylation maintenance of DMRs, such as those encoding proteins constructing the subcortical maternal complex (SCMC), in a small fraction of patients and/or their mothers. However, integrated methylation analysis for DMRs and sequence analysis for MLID-causative genes in MLID cases and their mothers have been performed only in a single study focusing on Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) phenotypes.ResultsOf 783 patients with various IDs we have identified to date, we examined a total of 386 patients with confirmed epimutation and 71 patients with epimutation or uniparental disomy. Consequently, we identified MLID in 29 patients with epimutation confirmed by methylation analysis for multiple ID-associated DMRs using pyrosequencing and/or methylation-specific multiple ligation-dependent probe amplification. MLID was detected in approximately 12% of patients with BWS phenotype and approximately 5% of patients with SRS phenotype, but not in patients with Kagami-Ogata syndrome, Prader-Willi syndrome, or Angelman syndrome phenotypes. We next conducted array-based methylation analysis for 78 DMRs and whole-exome sequencing in the 29 patients, revealing hypomethylation-dominant aberrant methylation patterns in various DMRs of all the patients, eight probably deleterious variants in genes for SCMC in the mothers of patients, and one homozygous deleterious variant in ZNF445 in one patient. These variants did not show gene-specific methylation disturbance patterns. Clinically, neurodevelopmental delay and/or intellectual developmental disorder (ND/IDD) was observed in about half of the MLID patients, with no association with the identified methylation disturbance patterns and genetic variants. Notably, seven patients with BWS phenotype were conceived by assisted reproductive technology (ART).ConclusionsThe frequency of MLID was 7.5% (29/386) in IDs caused by confirmed epimutation. Furthermore, we revealed diverse patterns of hypomethylation-dominant methylation defects, nine deleterious variants, ND/IDD complications in about half of the MLID patients, and a high frequency of MLID in ART-conceived patients.

Molecular mechanisms for DNA methylation defects induced by ICF syndrome-linked mutations in DNMT3B.

DNA methyltransferase 3B (DNMT3B) plays a crucial role in DNA methylation during mammalian development. Mutations in DNMT3B are associated with human genetic diseases, particularly immunodeficiency, centromere instability, facial anomalies (ICF) syndrome. Although ICF syndrome-related missense mutations in the DNMT3B have been identified, their precise impact on protein structure and function remains inadequately explored. Here, we delve into the impact of four ICF syndrome-linked mutations situated in the DNMT3B dimeric interface (H814R, D817G, V818M, and R823G), revealing that each of these mutations compromises DNA-binding and methyltransferase activities to varying degrees. We further show that H814R, D817G, and V818M mutations severely disrupt the proper assembly of DNMT3B homodimer, whereas R823G does not. We also determined the first crystal structure of the methyltransferase domain of DNMT3B-DNMT3L tetrameric complex hosting the R823G mutation showing that the R823G mutant displays diminished hydrogen bonding interactions around T775, K777, G823, and Q827 in the protein-DNA interface, resulting in reduced DNA-binding affinity and a shift in sequence preference of +1 to +3 flanking positions. Altogether, our study uncovers a wide array of fundamental defects triggered by DNMT3B mutations, including the disassembly of DNMT3B dimers, reduced DNA-binding capacity, and alterations in flanking sequence preferences, leading to aberrant DNA hypomethylation and ICF syndrome.

A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency.

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+ progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.

Study of Correlation between Serum Vitamin B12 Level and Aberrant DNA Methylation in Infertile Males.

Altered DNA methylation pattern in sperms has been associated with infertility in males demonstrating defective spermatogenesis or low semen quality. Vitamin B-12, by affecting 1-carbon metabolism pathways, might alter the DNA methylation pattern. We aimed to study the correlation of serum vitamin B12 levels with aberrant DNA methylation in infertile male patients. A cross-sectional study was conducted on 17 oligozoospermic infertile males (WHO criteria, 2010) and 10 healthy fertile males. Serum vitamin B12 levels were estimated using the chemiluminescence method. Global methylation was determined using the ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich). The levels of global DNA methylation were calculated and compared relative to the methylated (100%) control DNA provided with the kit. Mean serum vitamin B12 concentration in the control group was higher than that of the case group. This difference in serum vitamin B12 concentration in both groups was found statistically significant. Although the results of this study show that oligozoospermic men have relatively lower global DNA methylation as compared to normozoospermic control, the values could not reach a statistically significant level. A small positive correlation was found between serum vitamin B12 levels and percent methylation defect (r = 0.14) but was statistically insignificant. Our study concludes that oligozoospermic infertile males have a significant deficiency of vitamin B12 as compared to normozoospermic fertile males. This study did not find any significant difference in global DNA methylation between the two groups. The present study does not suggest any correlation between serum vitamin B12 level and percent DNA methylation.

ARGONAUTE1-binding Tudor domain proteins function in small interfering RNA production for RNA-directed DNA methylation.

Transposable elements (TEs) contribute to plant evolution, development, and adaptation to environmental changes, but the regulatory mechanisms are largely unknown. RNA-directed DNA methylation (RdDM) is 1 TE regulatory mechanism in plants. Here, we identified that novel ARGONAUTE 1 (AGO1)-binding Tudor domain proteins Precocious dissociation of sisters C/E (PDS5C/E) are involved in 24-nt siRNA production to establish RdDM on TEs in Arabidopsis thaliana. PDS5 family proteins are subunits of the eukaryote-conserved cohesin complex. However, the double mutant lacking angiosperm-specific subfamily PDS5C and PDS5E (pds5c/e) exhibited different developmental phenotypes and transcriptome compared with those of the double mutant lacking eukaryote-conserved subfamily PDS5A and PDS5B (pds5a/b), suggesting that the angiosperm-specific PDS5C/E subfamily has a unique function in angiosperm plants. Proteome and imaging analyses revealed that PDS5C/E interact with AGO1. The pds5c/e double mutant had defects in 24-nt siRNA accumulation and CHH DNA methylation on TEs. In addition, some lncRNAs that accumulated in the pds5c/e mutant were targeted by AGO1-loading 21-nt miRNAs and 21-nt siRNAs. These results indicate that PDS5C/E and AGO1 participate in 24-nt siRNA production for RdDM in the cytoplasm. These findings indicate that angiosperm plants evolved a new regulator, the PDS5C/E subfamily, to control the increase in TEs during angiosperm evolution.

Sperm DNA methylation defects in a new mouse model of the 5,10-methylenetetrahydrofolate reductase 677C>T variant and correction with moderate dose folic acid supplementation.

5,10-Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that plays a key role in providing methyl groups for DNA methylation, including during spermatogenesis. A common genetic variant in humans (MTHFR 677C>T) results in reduced enzyme activity and has been linked to various disorders, including male infertility. A new animal model has been created by reproducing the human equivalent of the polymorphism in mice using CRISPR/Cas9. Biochemical parameters in the Mthfr 677TT mice recapitulate alterations found in MTHFR 677TT men. Our aims were to characterize the sperm DNA methylome of the Mthfr 677CC and TT mice on a control diet (2 mg folic acid/kg diet) and assess the effects of folic acid supplementation (10 mg/kg diet) on the sperm DNA methylome. Body and reproductive organ weights, testicular sperm counts, and histology were examined. DNA methylation in sperm was assessed using bisulfite pyrosequencing and whole-genome bisulfite sequencing (WGBS). Reproductive parameters and locus-specific imprinted gene methylation were unaffected by genotype or diet. Using WGBS, sperm from 677TT mice had 360 differentially methylated tiles as compared to 677CC mice, predominantly hypomethylation (60% of tiles). Folic acid supplementation mostly caused hypermethylation in sperm of males of both genotypes and was found to partially correct the DNA methylation alterations in sperm associated with the TT genotype. The new mouse model will be useful in understanding the role of MTHFR deficiency in male fertility and in designing folate supplementation regimens for the clinic.

(Epi)genetic and clinical characteristics in 84 patients with pseudohypoparathyroidism type 1B.

Pseudohypoparathyroidism type 1B (PHP1B) caused by methylation defects of differentially methylated regions (DMRs) on the GNAS locus can be categorized into groups according to etiologies and methylation defect patterns of the DMRs. The aim of this study was to clarify the clinical characteristics in each group. Comprehensive molecular analyses consisting of methylation, copy number, and microsatellite analyses. Eighty-four patients with PHP1B were included in this study. We classified them into five groups, namely, autosomal dominant inheritance-PHP1B (Group 1, G1), sporadic-PHP1B (G2), and atypical-PHP1B (G3-G5), based on the methylation defect patterns in four DMRs on the GNAS locus and etiologies and evaluated the clinical findings in each group and compared them among the groups. G2 had the youngest age and the highest serum intact parathyroid hormone levels among the five groups at the time of diagnosis. The most common symptoms at the time of diagnosis were tetany in G1, and seizures or loss of consciousness in G2. Albright's hereditary osteodystrophy and PHP-suggestive features were most frequently observed in the G2 proband. Nine patients had neurodevelopmental disorders (NDs) consisting of mild to borderline intellectual disability and/or developmental delay. There were no significant correlations between the average methylation ratios of seven CpG sites in the GNAS-A/B:TSS-DMR and hormonal and biochemical findings. This study revealed the differences in some clinical characteristics, particularly clinical features and ages at the time of diagnosis between G2 and other groups and detailed NDs observed in some patients with PHP1B.

Rapid and Accurate Remethylation of Dnmt3a Deficient Hematopoietic Cells with Restoration of DNMT3A Activity

Heterozygous loss-of-function mutations in the DNMT3A gene are the most common cause of clonal hematopoiesis, and among the most common initiating events for Acute Myeloid Leukemia (AML). A reduction of DNMT3A activity causes a canonical, focal hypomethylation phenotype in hematopoietic cells, which is associated with immortalization of hematopoietic stem cells, and blockage of myeloid differentiation. The methylation defect can be partially reversed with restoration of physiologic levels of DNMT3A expression over a period of several months (Ketkar et al., PNAS, 2020; PMID 35313694).To identify a more efficient remethylation strategy, we first characterized the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic cell deficiency of Dnmt3a, Dnmt3b (or both enzymes), or expressing the dominant negative Dnmt3aR878H mutation (equivalent to R882H in humans; the most common mutation found in AML patients). Using these different mouse models as substrates, we then defined the patterns and completeness of DNA remethylation after “adding back” supraphysiologic levels of wild type DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive “chaperone” that augments the activity of DNMT3A and DNMT3B in early embryogenesis), with MSCV-based retroviruses transduced into primary mouse hematopoietic stem/progenitor cell s. Overexpression of WT DNMT3A for 2 weeks in vitro (~3 fold) can accurately reverse the differentially methylated regions (DMRs, which are >99% hypomethylated) of Dnmt3a deficient hematopoietic cells, or cells expressing the R878H mutation. The DMRs of Dnmt3b deficient mouse bone marrow cells can be corrected by overexpression of DNMT3A, DNMT3B1, or DNMT3B3; however, DNMT3B3 failed to remethylate the DMRs in Dnmt3a/Dnmt3b double knockout mouse bone marrow cells. Since DNMT3B3 is an inactive enzyme that does not cause remethylation in the absence of DNMT3A, these data suggest that DNMT3B3 functions in a DNMT3A-dependent manner in hematopoietic cells. Importantly, both DNMT3B3 and DNMT3L have been shown to facilitate DNA methylation by acting as a chaperone for DNMT3A (Duymich et al., Nat. Communication, 2016; PMID 27121154). In vitro, DNMT3L copurified with DNMT3A leads to an increase in DNMT3A methyltransferase activity that is ~5 times greater than DNMT3B3 copurified with DNMT3A. Remarkably, overexpression of DNMT3L (which is not expressed in adult hematopoietic cells, or AML cells) can completely correct the hypomethylation phenotype of Dnmt3aR878H/+ bone marrow cells within 2 weeks of in vitro overexpression, or 4 weeks of in vivo overexpression ( Figure 1A and 1B), probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele in these heterozygous mutant cells. Two weeks of overexpression of DNMT3A or DNMT3L in Dnmt3aR878H/+ bone marrow cells (cultured in vitro with IL-3, SCF, FLT3L, and TPO to maintain HSPC populations) can induce a differentiation response, increasing the fraction of mature myeloid cells in the cultures by ~4-fold. This finding was recapitulated in vivo where one month of overexpression of DNMT3A or DNMT3L in transplanted Dnmt3aR878H/+ mouse bone marrow cells increased the fraction of mature myeloid cells by 2-fold by DNMT3L and 4-fold by DNMT3A. Together,these data show that the focal, canonical DNA hypomethylation phenotype of Dnmt3a R878H/+ hematopoietic cells can be accurately and efficiently corrected by overexpressing WT DNMT3A or DNMT3L for several weeks. We have also shown that the restoration of methylation in Dnmt3a R878H/+ mutant cells alters the developmental fate of progenitors, increasing the proportion of mature myeloid cells. These data suggest that DNMT3L expression may represent a novel approach for restoring DNMT3A activity in AMLs initiated by DNMT3A mutations, and could possibly alter AML cell fate. Additional studies are underway to determine whether the correction of DNMT3A activity in Dnmt3aR878H/+ -initiated AML cells with retroviral addback of DNMT3A or DNMT3L will cause a differentiation response that alters AML cell fate.

DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss

ABSTRACT Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0–1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

Capturing sex-specific and hypofertility-linked effects of assisted reproductive technologies on the cord blood DNA methylome

BackgroundChildren conceived through assisted reproduction are at an increased risk for growth and genomic imprinting disorders, often linked to DNA methylation defects. It has been suggested that assisted reproductive technology (ART) and underlying parental infertility can induce epigenetic instability, specifically interfering with DNA methylation reprogramming events during germ cell and preimplantation development. To date, human studies exploring the association between ART and DNA methylation defects have reported inconsistent or inconclusive results, likely due to population heterogeneity and the use of technologies with limited coverage of the epigenome. In our study, we explored the epigenetic risk of ART by comprehensively profiling the DNA methylome of 73 human cord blood samples of singleton pregnancies (n = 36 control group, n = 37 ART/hypofertile group) from a human prospective longitudinal birth cohort, the 3D (Design, Develop, Discover) Study, using a high-resolution sequencing-based custom capture panel that examines over 2.4 million autosomal CpGs in the genome.ResultsWe identified evidence of sex-specific effects of ART/hypofertility on cord blood DNA methylation patterns. Our genome-wide analyses identified ~ 46% more CpGs affected by ART/hypofertility in female than in male infant cord blood. We performed a detailed analysis of three imprinted genes which have been associated with altered DNA methylation following ART (KCNQ1OT1, H19/IGF2 and GNAS) and found that female infant cord blood was associated with DNA hypomethylation. When compared to less invasive procedures such as intrauterine insemination, more invasive ARTs (in vitro fertilization, intracytoplasmic sperm injection, embryo culture) resulted in more marked and distinct effects on the cord blood DNA methylome. In the in vitro group, we found a close to fourfold higher proportion of significantly enriched Gene Ontology terms involved in development than in the in vivo group.ConclusionsOur study highlights the ability of a sensitive, targeted, sequencing-based approach to uncover DNA methylation perturbations in cord blood associated with hypofertility and ART and influenced by offspring sex and ART technique invasiveness.

The Effects of 5 Years of Growth Hormone Treatment on Growth and Body Composition in Patients with Temple Syndrome

Introduction: Temple syndrome (TS14) is a rare imprinting disorder caused by maternal uniparental disomy of chromosome 14, paternal deletion of 14q32.2, or an isolated methylation defect. Most patients with TS14 develop precocious puberty. Some patients with TS14 are treated with growth hormone (GH). However, evidence for the effectiveness of GH treatment in patients with TS14 is limited. Methods: This study describes the effect of GH treatment in 13 children and provides a subgroup analysis of 5 prepubertal children with TS14. We studied height, weight, body composition by dual-energy X-ray absorptiometry, resting energy expenditure (REE), and laboratory parameters during 5 years of GH treatment. Results: In the entire group, mean (95% CI) height SDS increased significantly during 5 years of GH treatment from −1.78 (−2.52; −1.04) to 0.11 (−0.66; 0.87). Fat mass percentage SDS decreased significantly during the first year of GH, and lean body mass (LBM) SDS and LBM index increased significantly during 5 years of treatment. IGF-1 and IGF-BP3 levels rose rapidly during GH treatment, and the IGF-1/IGF-BP3 molar ratio remained relatively low. Thyroid hormone levels, fasting serum glucose, and insulin levels remained normal. In the prepubertal group, median (interquartile range [IQR]) height SDS, LBM SDS, and LBM index also increased. REE was normal at start and did not change during 1 year of treatment. Five patients reached adult height and their median (IQR) height SDS was 0.67 (−1.83; −0.01). Conclusion: GH treatment in patients with TS14 normalizes height SDS and improves body composition. There were no adverse effects or safety concerns during GH treatment.

Investigation of 11p15.5 Methylation Defects Associated with Beckwith-Wiedemann Spectrum and Embryonic Tumor Risk in Lateralized Overgrowth Patients.

The Beckwith-Wiedemann spectrum (BWSp) ranges from isolated lateralized overgrowth (ILO) to classic phenotypes. In this broad clinical spectrum, an epigenetic alteration on chromosome 11p15.5 can be detected. The risk for embryonal tumors is high, especially in patients with lateralized overgrowth (LO). The aim of this study is to investigate epigenetic alterations in 11p15.5 and tumor risk in 87 children with LO. The methylation level of 11p15.5 was examined in the blood of all patients and in skin samples or buccal swabs from 40 patients with negative blood tests; 63.2% of patients were compatible with the ILO phenotype, 18.4% were atypical, and 18.4% were classic. The molecular diagnosis rate was 81.2% for the atypical and classic phenotypes, and 10.9% for the ILO phenotype. In patients with epigenetic alterations, LO was statistically significantly more severe than in test negatives. Tumors developed in six (6.9%) of the total 87 patients with LO; four belonged to the atypical or classical phenotype (12.5%) and two to ILO (3.5%). Three of the four patients with atypical/classical phenotypes had pUPD11, one had IC1-GOM alteration, and two ILO patients were negative. We conclude that LO patients should be monitored for tumor risk even if their epigenetic tests are negative.

The aberrant epigenome of DNMT3B-mutated ICF1 patient iPSCs is amenable to correction, with the exception of a subset of regions with H3K4me3- and/or CTCF-based epigenetic memory.

Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to immunodeficiency, centromeric instability, facial anomalies syndrome, type 1 (ICF1). Although several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-derived induced pluripotent stem cells (iPSCs) and their CRISPR-Cas9-corrected clones to determine whether DNMT3B correction can globally overcome DNA methylation defects and related changes in the epigenome. Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1 patient peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are premarked during early development. CRISPR-corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions reacquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increases in H3K4me3 levels and/or abnormal CTCF binding, the epigenetic memory persists, and hypomethylation remains uncorrected. Overall, we demonstrate that restoring the catalytic activity of DNMT3B can reverse the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this rescue, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle.

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.

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

Temple Syndrome: Clinical Findings, Body Composition and Cognition in 15 Patients.

Temple syndrome (TS14) is an imprinting disorder caused by a maternal uniparental disomy of chromosome 14 (UPD(14)mat), paternal deletion of 14q32 or an isolated methylation defect of the MEG3-DMR. Studies on phenotypical characteristics in TS14 are scarce and patients with TS14 often experience delay in diagnosis, which has adverse effects on their health. TS14 is often characterized as either Prader-Willi-like, Silver-Russell-like or as a Silver-Russell spectrum disorder. This study describes 15 patients with TS14 who visited the Dutch Reference Center for Prader-Willi-like from December 2018 to January 2022. Eight patients had UPD(14)mat and seven a methylation defect. The most common symptoms were intra-uterine growth retardation (IUGR) (100%), hypotonia (100%), precocious puberty (89%), small for gestational age (SGA) birth (67%), tube feeding after birth (53%) and psycho-behavioral problems (53%). Median (interquartile range (IQR)) IQ was 91.5 (84.25; 100.0), whilst many patients were enrolled in special education (54%). The median (IQR) fat mass % (FM%) SDS was 2.53 (2.26; 2.90) and lean body mass (LBM) SDS -2.03 (-3.22; -1.28). There were no significant differences in clinical characteristics between patients with a UPD(14)mat and a methylation defect. Our patients share a distinct phenotype consisting of IUGR, SGA birth, precocious puberty, hypotonia, tube feeding after birth, psycho-behavioral problems and abnormal body composition with a high FM% and low LBM. Whilst similarities with Prader-Willi syndrome (PWS) and Silver-Russell syndrome (SRS) exist, TS14 is a discernible syndrome, deserving a tailored clinical approach. Testing for TS14 should be considered in patients with a PWS or SRS phenotype in infancy if PWS/SRS testing is negative.

Usefulness of the MS-MLPA technique in the diagnosis of Beckwith-Wiedemann syndrome and Silver-Russell syndrome.

Epigenetic and genomic imprinting alterations of the 11p15.5 region cause excessive or deficient growth, which result in Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS), respectively. To evaluate the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methylation analysis technique in the diagnosis of BWS and SRS. 11p15.5 methylation and variants were evaluated in patients with clinical diagnosis of BWS and SRS using the MS-MLPA technique in peripheral blood DNA. Paternal uniparental disomy and loss of maternal IC2 methylation were identified in two patients with BWS who had omphalocele and macroglossia, respectively. Paternal IC1hypomethylation was recorded in two patients with SRS of classic phenotype. Adequate genotype-phenotype correlation was observed with the methylation defects that were identified, which confirms the usefulness of MLPA as a first-line study in patients diagnosed with BWS and SRS.

Approaches to long-read sequencing in a clinical setting to improve diagnostic rate

Over the past decade, advances in genetic testing, particularly the advent of next-generation sequencing, have led to a paradigm shift in the diagnosis of molecular diseases and disorders. Despite our present collective ability to interrogate more than 90% of the human genome, portions of the genome have eluded us, resulting in stagnation of diagnostic yield with existing methodologies. Here we show how application of a new technology, long-read sequencing, has the potential to improve molecular diagnostic rates. Whole genome sequencing by long reads was able to cover 98% of next-generation sequencing dead zones, which are areas of the genome that are not interpretable by conventional industry-standard short-read sequencing. Through the ability of long-read sequencing to unambiguously call variants in these regions, we discovered an immunodeficiency due to a variant in IKBKG in a subject who had previously received a negative genome sequencing result. Additionally, we demonstrate the ability of long-read sequencing to detect small variants on par with short-read sequencing, its superior performance in identifying structural variants, and thirdly, its capacity to determine genomic methylation defects in native DNA. Though the latter technical abilities have been demonstrated, we demonstrate the clinical application of this technology to successfully identify multiple types of variants using a single test.