{"paper_id":"09b7c203-e930-4150-ab1c-330db009c4f8","body_text":"Concordant Epigenetic and Discordant Clinical Php1b/ippsd3 Manifestations in Two Monozygotic Adolescent Twins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Concordant Epigenetic and Discordant Clinical Php1b/ippsd3 Manifestations in Two Monozygotic Adolescent Twins Gustavo Perez-Nanclares, Africa Manero-Azua, Gema Grau, Arrate Pereda, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7979825/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Imprinting disorders are caused by epigenetic alterations leading to abnormal gene expression from imprinted loci. Pseudohypoparathyroidism type 1B is characterized by methylation defects at the GNAS locus, affecting hormone responsiveness. Monozygotic twins discordant for imprinting disorders provide a unique opportunity to investigate the timing, maintenance, and tissue-specific distribution of epimutations and their relation to clinical expression. Most previous studies focus on infants, where fetal blood chimerism may influence epigenetic profiles, but data in adolescents remain scarce. Results We studied adolescent monozygotic twin sisters presenting clinical and biochemical discordance for pseudohypoparathyroidism type 1B. Both twins exhibited an identical, partial methylation defect at the GNAS locus in peripheral blood, consistent with a postzygotic epigenetic alteration occurring before embryonic splitting. Exclusion of known genetic causes, including uniparental disomy and structural variants, and absence of multilocus imprinting disturbances or maternal-effect gene mutations, were confirmed through comprehensive genomic and methylation analyses. The affected twin showed involvement of methylation abnormalities across all three germ layers derived tissues, whereas the unaffected twin presented very mild or absent defects in some tissues, possibly explaining the phenotypic divergence. The persistent concordance of partial methylation defects in blood despite phenotypic discordance suggests stable early epimutations maintained in hematopoietic progenitors, while tissue-specific mosaicism and differential methylation maintenance across organs critical to disease expression contribute to clinical differences. Advanced long-read sequencing demonstrated sensitivity for detecting partial and mosaic methylation patterns, surpassing traditional techniques. Conclusions This study underscores the complexity of epigenetic mosaicism in imprinting disorders, highlighting that early postzygotic partial epimutations may yield identical blood methylation profiles but divergent tissue distributions, leading to discordant clinical phenotypes in monozygotic twins. The adolescent age of our subjects challenges the fetal blood chimerism explanation for methylation concordance and emphasizes the importance of multi-tissue evaluation and sensitive molecular assays. These findings have important implications for diagnosis, monitoring, and genetic counseling in imprinting disorders, advocating for integrated approaches to assess epigenetic heterogeneity and phenotypic variability. Pseudohypoparathyroidism Imprinting disorders Monozygotic twins Epigenetic mosaicism GNAS locus Postzygotic epimutation Phenotypic discordance Long-read sequencing Tissue-specific methylation Figures Figure 1 Figure 2 INTRODUCTION Imprinting disorders (ImpDis) are a group of congenital diseases caused by epigenetic and genetic alterations affecting the expression of imprinted genes—genes that are expressed in a parent-of-origin-specific manner. Nowadays thirteen ImpDis have been identified whose molecular basis involves four molecular alterations: uniparental disomy, copy number variants or rare chromosomal rearrangements, (likely) pathogenic point variants and aberrant DNA methylation at imprinted regions (known as imprinting defects (ImpDefs) or epimutations (for review see [ 1 ])). A subgroup of patients with epimutations presents DNA methylation changes at multiple imprinted loci, a condition referred to as multi‑locus imprinting disturbance (MLID). The etiology of MLID has been linked to environmental factors such as assisted reproduction, and to trans‑acting genetic variants, frequently not found in the patients but in their mothers [ 1 , 2 ]. While ImpDis are typically sporadic, their occurrence in monozygotic (MZ) twins has provided a unique window into the role of epigenetics in human disease. Monozygotic twins arise from a single zygote and are considered genetically identical. However, several studies have reported phenotypic discordance in MZ twins affected by ImpDis, where one twin presents with the full clinical phenotype while the co-twin is either mildly affected or entirely asymptomatic [ 3 ]. This phenomenon challenges the assumption of complete epigenetic identity in MZ twins and underscores the dynamic nature of epigenetic regulation during early embryogenesis. One of the most compelling explanations for this discordance is the occurrence of postzygotic epigenetic alterations, particularly in DNA methylation patterns. These changes can arise during the early cleavage stages of embryonic development, leading to mosaicism in the distribution of epimutations across different tissues. One of the ImpDis where cases of discordant monozygotic twins, usually female pairs [ 4 – 9 ] and rarely males [ 5 , 6 , 9 , 10 ], have been most frequently described is BWS caused by mosaic loss of methylation at KCNQ1OT1 :TSS-DMR. It has also been rarely associated with uniparental disomy (UPD) in a male MZ twin pair [ 8 ] and a mosaic trisomy 11p15 including patUPD in a female MZ twin pair [ 11 ]. This discordance among MZ twins has also been reported in transient neonatal diabetes caused by loss of methylation at PLAGL1 :alt-TSS-DMR [ 12 , 13 ], and in Silver-Russell syndrome due to hypomethylation of the H19/IGF2 :IG-DMR [ 4 , 14 – 16 ]. MLID has been reported among MZ twins in BWS with KCNQ1OT1 :TSS-DMR hypomethylation [ 9 ], in SRS with hypomethylation at H19/IGF2 :IG-DMR [ 15 ] and in TNDM with loss of methylation at PLAGL1 :alt-TSS-DMR [ 12 ]. To our knowledge, twinning has been described twice for PHP1B/iPPSD3, with both twins presenting the same methylation pattern and phenotype compatible with the disease. The first pair of \"identical\" twins are two boys diagnosed with sporPHP-1B at 3 and 5 years of age, respectively, with a complete alteration of methylation affecting the four DMRs of the GNAS locus [ 17 ]. The second pair is a 26-year-old pair of female twins (with no information on amnionicity and chorionicity) with asymptomatic sporPHP1B incidentally diagnosed due to the presence of hypocalcemia, hyperphosphatemia, hypocalciuria, and elevated serum intact PTH levels, withoutAHO phenotype. Molecular testing identified almost complete loss of methylation at the GNAS-AS1 :TSS-DMR, GNAS-XL :Ex1-DMR and GNAS A/B :TSS-DMR and gain of methylation at the GNAS-NESP :TSS-DMR. The possibility of MLID was ruled out. Whole genome sequencing did not identify alterations in the coding regions of the GNAS locus, nor deletions, insertions, or inversions at the GNAS and STX16 loci, nor pathogenic variants in candidate genes involved in imprinting control, including genes known to cause MLID. The SNP array study ruled out CNVs and isodisomy (iUPD), however, analysis of 28 consecutive SNPs could not rule out the possibility of paternal heterodisomy (hUPDpat) within a 22 kb span spanning the NESP55 exon and AS exon 5 [ 18 ]. Importantly, the detection of these epigenetic differences depends heavily on the type of tissue analyzed. Peripheral blood is the most commonly used tissue due to its accessibility, but it may not always reflect the methylation status in disease-relevant tissues such as skin, buccal epithelium, or internal organs. Several studies have demonstrated that methylation patterns can vary significantly between tissues, and in some cases, the epimutations are restricted to specific lineages. For instance, in MZ twins discordant for ImpDis, studies performed on DNA obtained from blood usually showed epigenetic alteration in both twins, while in alternative tissues (as saliva, buccal cells, fibroblast) results revealed that only the affected twin carried the epimutation [ 9 , 10 , 12 , 15 , 16 ]. In this study we report two iPPSD3-phenotypically discordant female monozygotic twins, trying to identify the possible (epi)genetic cause that explains this clinical difference. PATIENTS and METHODS Case report The index case is a 14-year-old female, a biamniotic monochorionic twin, born to healthy, unrelated parents with no family history of neurological or endocrinological diseases (mother: 47 years old, height 150.5 cm; father: 47 years old, height 180 cm). The pregnancy occurred naturally, with a history of twins on the maternal side. After an uneventful, well-controlled pregnancy, delivery was by cesarean section at 39.5 weeks of gestation due to lack of progression, with no apparent complications (birth weight 3570 g, 1.03 SD [ 19 ]; length 50 cm, 0.34 SD; APGAR scores 8 and 9). Her twin sister’s anthropometric values were also within the normal range (birth weight 2930 g, -0.68 SD; length 51 cm, 0.94 SD; APGAR scores 8 and 9). At 7.5 years of age, the index was referred to the Neuropediatrics Service due to generalized seizures and was diagnosed with possible benign partial epilepsy. Treatment with levetiracetam was initiated and discontinued at 9 years of age. At 8.5 years old, during follow-up, asymptomatic hypocalcemia was detected, and she was evaluated by the Pediatric Endocrinology Service. At that time, her anthropometric measurements were: weight 26.5 kg (-0.64 SD), height 121.8 cm (-1.58 SD), BMI 17.8. She showed no signs of Albright’s hereditary osteodystrophy. Biochemical evaluation revealed: total calcium 6.5 mg/dL (NV: 8.1–10.4 mg/dL), ionized calcium 2.8 mg/dL (NV: 4.5–5.2 mg/dL), phosphorus 8.7 mg/dL (NV: 2.5–4.7 mg/dL), magnesium 1.84 mg/dL (NV: 1.7–2.5 mg/dL), iPTH 560 pg/mL (NV: 10–75 pg/mL), vitamin D 26 ng/mL. No other hormonal deficiencies were detected (TSH 3.44 mU/L; fT4 1.08 ng/dL; LH 0.1 U/L; FSH 1.5 U/L; estradiol < 12 pg/mL; cortisol 4.9 mg/dL; calcitonin 5 pg/mL; IGF-1 112 ng/mL; IGFBP3 3.6 µg/mL). Cranial CT revealed bilateral basal ganglia calcifications. Treatment was initiated with calcium (1,200 mg daily) and calcitriol (0.25 µg every 12 hours). Her twin sister was asymptomatic and showed no biochemical abnormalities. She started puberty at age 10 and had her first menstruation at 11 years and one month. At present, she has a weight of 57 kg (p75) and a height of 154.3 cm (p25), with complete pubertal development. During follow-up, she developed TSH resistance, so her current treatment consists of calcium 1,000 mg every 12 hours, calcitriol 0.5 µg every 12 hours, and levothyroxine 50 µg daily. Given the clinical suspicion of pseudohypoparathyroidism (PHP) in the patient, genetic testing was requested. Molecular studies DNA extraction Genomic DNA of the family quartet was extracted from peripheral blood leukocytes using the commercial QIAamp DNA Blood Mini kit (Qiagen, Düren, Germany), following the manufacturer's instructions. The saliva samples of both twins were collected using the Oragene DNA kit (OG-500, DNA Genotek, Stittswille, Canada) and DNA was extracted using the prepIT.L2P extraction reagent (DNA Genotek), according to the manufacturer's instructions. Urine samples (20 mL) of both twins were centrifuged at 4,100 × g for 20 minutes. The supernatant was discarded, retaining 1 mL to resuspend the pellet. Samples were transferred to 1.5 mL tubes and subjected to three successive centrifugation steps at 13,000 × g for 5 minutes, each time resuspending the pellet in 1 mL of sterile PBS (1×, pH 7.4). Final pellets were resuspended in 200 µL of PBS (1×, pH 7.4), and DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen) following the manufacturer's protocol. Analysis of GNAS locus The ME031-C1 kit (lot C1-0523) was used to study the dosage and methylation patterns of GNAS locus in DNA extracted from blood of the quartet, and from saliva and urine cells of both twins, following the manufacturer’s recommendations (MRC-Holland, Amsterdam, The Netherlands). In parallel, a custom upd(20q)pat MS-MLPA kit was used to analyze DNA from blood of both twins, as previously described [ 20 ]. Fragment sizes were assessed using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA), according to the manufacturer’s protocols. Data analysis was carried out with Coffalyser.net software version 9.4 (MRC-Holland). The SNP array technique was used to analyze the possible presence of uniparental disomy as an underlying cause of the identified methylation pattern. For this purpose, the CytoScan 750K Array (ThermoFisher Scientific, Massachusetts, USA) and the subsequent analysis was carried out using the recommended Chromosome Analysis Suite (ChAS) software version 4.5 (ThermoFisher Scientific). STR study was performed for the quartet by fluorescent PCR, using different markers along the long arm of chromosome 20 ( D20S857, D20S1044, D20S468, D20S149, D20S430, D20S451, D20S496, D20S459, D20S443, D20S171, D20S173 ) (Fig. 1 ). The fragments’ size was determined with an ABI 3500 Genetic analyzer. The results were analyzed with GeneMapper Software 5 (Applied Biosystems). Analysis of MLID The methylation status and gene dosage of principal clinical DMRs (CA-DMRs) [ 2 ], including PLAGL1 :alt-TSS-DMR (6q24.2), MEST :alt-TSS-DMR (7q32.2), H19/IGF2 :IG-DMR (11p15.5), KCNQ1OT1 :TSS-DMR (11p15.5), MEG3 :TSS-DMR (14q32.2), SNURF :TSS-DMR (15q11.2), PEG3 :TSS-DMR (19q13.43), as well as GNAS-NESP :TSS-DMR, GNAS-AS1 :TSS-DMR, GNAS-XL :Ex1-DMR, and GNAS A/B :TSS-DMR, were studied using MS-MLPA with the ME034-C1 kit (lot C1-0121, MRC-Holland). Looking for genetic underlying causes IR panel To identify genetic variants underlying the sisters' epigenetic alterations, we employed our custom Imprinting Regulators v1 (IR) panel, which covers 104 genes (see supplementary material) including known imprinting regulators, maternal effect genes, and additional candidates implicated in mammalian methylation regulation, zinc finger protein interactions with imprinted DMRs, as well as genes relevant to spermatogenesis and oogenesis. Library Preparation Enzymatic Fragmentation (EF) Kit 2.0 and Twist Target Enrichment Standard Hybridization v2 Protocol (Twist Biosciences, San Francisco, USA) were used for library preparation and enrichment. These libraries were sequenced on a MiniSeq High Output platform (7.5 Gb) (Illumina Inc., San Diego, CA, USA) in 150 pair-end mode. The DNA enrichment analysis module integrated in MiniSeq (bcl2fastq 2.17.1.5 conversion software) was used for primary analysis. Downstream secondary analysis was facilitated by the pipeline of the commercial platform Orchestrator (Euformatics, Espoo, Finland), based on GATK Best Practices ( https://gatk.broadinstitute.org/hc/en-us ). Finally, tertiary bioinformatics analysis of VCF files (variant annotation, filtering and prioritization) and BAM files (for CNV detection) was performed with the commercial software OmnomicsNGS 2.2.1 (Euformatics), followed by interpretation according to standardized guidelines [ 21 ]. In the case of maternal effect genes, the approach was similar to that used previously [ 22 ]. The BAM files were also visualized with Integrative Genomics Viewer (IGV), as a standard practice, to confirm variant calls, reduce false positives, and interpret complex genomic events that may not be readily resolved by automated pipelines. The parameters used for filtering and prioritization were assessed by the commercial tool OmnomicsQ (Euformatics): quality assessment Q > 30; mean coverage > 100x, uniformity 90%. Long read sequencing (including broader MLID analysis) Long-read sequencing was performed on DNA extracted from blood and saliva of both twins using the PromethION P2 platform (Oxford Nanopore Technologies, ONT, Oxford, United Kingdom). Approximately 2 µg of genomic DNA per sample was used to prepare sequencing libraries with the ONT Ligation Sequencing Kit (SQK-LSK114), following the manufacturer's protocol with minor modifications. Bioinformatic processing was conducted using a custom pipeline comprising: (i) basecalling with the Dorado basecaller integrated within ONT’s MinKNOW software; (ii) alignment to the GRCh38 human reference genome using Minimap2; and (iii) structural variant calling using Sniffles. Taking advantage of the native methylation detection provided by nanopore technology, the presence of MLID was evaluated by examining the methylation status of known DMRs [ 2 ]. After Dorado basecalling, the software modbam2bed (version 0.10) and modkit (version 0.4.2) were used to convert the modified BAM files to BED files containing methylation information at each CpG position. The 48 regions of interest (ROI) were visualized using Methylartist tool [ 23 ]. Genome analyses Whole genome sequencing (WGS) of the quartet was performed on DNA extracted from peripheral blood under the IMPaCT-GENóMICA program. WGS libraries were prepared using the PCR-free KAPA HyperPrep kit and sequenced using the NovaSeq 6000 System sequencer (Illumina Inc.). Paired-end reads of 150 bp were generated with an average coverage of at least 30 reads (35.24 for the index twin, 31.74 healthy twin, 32 father and 31.8 mother). Both the alignment of the sequencing data obtained against the GRCh38 reference genome and the detection of SNVs, INDELs, SVs, CNVs and STRs were carried out using the Illumina DRAGEN Bio-IT Platform v4.2 software (including the detection of mitochondrial variants). SNVs and INDELs have been annotated through the GPAP platform, SVs and CNVs with AnnotSV (version 3.3.4.), mitochondrial variants using the MitoMap database (18/02/2023) and STR variants using the STRipy-pipeline (version 2.4). The identified variants have been screened and evaluated on a case-by-case basis, using resources such as inheritance mode, population allele frequency (gnomAD), pathogenicity prediction (SnpEff, SIFT, Polyphen2, CADD, REVEL), splicing effect prediction (SpliceAI), ACMG classification, or current scientific knowledge regarding their pathogenicity (ClinVar 08/12/2024). Current scientific knowledge on candidate variants has been consulted in genetic databases. RESULTS Methylation analysis with MS-MLPA of all family members revealed partial losses of methylation at the GNAS-AS1 :TSS-DMR, GNAS-XL :Ex1-DMR, and GNAS A/B :TSS-DMR and a partial gain of methylation at the GNAS-NESP :TSS-DMR in both twins but not in other family members in blood. The methylation alteration was similar in both twins’ samples. However, the methylation defect in saliva (and more marked in urine) was lower in the healthy sister (Supplementary Fig. 1). SNP array discarded any putative mosaic copy number alteration on chromosome 20 and the possibility of paternal iUPD20q (Supplementary Fig. 2). The STRs study was compatible with biparental inheritance except for the region between D20S496 and D20S443 , encompassing the whole GNAS locus and its regulatory gene STX16 (from, at least, Chr20: 58,661,059 − 58,920,545 according to GRCh38), where microsatellites were not sufficiently informative to rule out hUPD (Fig. 1 ). However, expecting it to be mosaic based on the aforementioned methylation pattern results, the normalized ratios of the intensity of the sisters' alleles in the three microsatellites did not differ from those obtained in their parents (data not shown). The possibility of mosaicism in this region was also ruled out based on the SNP array results, within the detection limits of the technique (approximately 15–20%). Neither the Log R Ratio (LRR) nor the B Allele Frequency (BAF) plots showed intermediate deviations from the normal pattern, and the signal values across chromosome 20q remained stable, with no evidence of aberrant or mosaic profiles (Supplementary Fig. 2). The study of MLID using MS-MLPA in DNA extracted from blood confirmed the presence of partial methylation alteration in GNAS -DMRs in both sisters, without involvement of other additional clinical DMRs. The results obtained from long-read sequencing confirmed this partial alteration, which was similar in both twins in blood and showed less involvement in the healthy sister in the case of the saliva sample (Fig. 2 ). At the genome-wide level, none of the samples showed methylation alterations in any of the additional 48 DMRs, neither in those considered as clinical [ 2 ]. (Supplementary Fig. 3) nor in the non-clinical ones (Supplementary Fig. 4). As part of the analysis of potential genetic defects underlying the epigenetic alteration, we applied a custom NGS panel including genes known or candidates to be involved in methylation establishment and/or maintenance, longread sequencing, and short-read WGS in the quartet. No known deletions, insertions, inversions or retrotransposons were found in the region spanning from the STX16 locus to GNAS , where methylation regulatory alterations have previously been identified in AD-PHP1B patients [ 24 ]. Additionally, a thorough review of the genome for the analysis of SNPs located at Chr20: 58,661,059 − 58,920,545 allowed us to completely rule out hUPDpat (data not shown). On the other hand, 21 heterozygous SVs of moderate impact were identified, all of them affecting transcription binding sites. Eighteen were discarded because they had also been detected in our control cohort; one was discarded because the phasing tool confirmed that, although the VAF was compatible with heterozygosity, they were found in some of the readings of both alleles; and one was discarded because it was detected in both parents. The (hg38) NC_000020.11:g.52142651_52144388del was classified as VUS. These last two SVs are located in regions that appear to be associated with coverage difficulties (Supplementary Fig. 5). Additionally, no (likely) pathogenic alteration (including SNVs, CNVs or SVs) was detected in any of the candidate genes studied through gene panel and genome analysis. Only one VUS was detected in the coding region inherited from the mother [NM_003482.4(KMT2D):c.6497A > G;p.Gln2166Arg], while the rest, both maternally inherited and de novo , were located in deep intronic regions or in UTRs. DISCUSSION Imprinting disorders (ImpDis) are complex diseases caused by disruptions of normal epigenetic regulation at imprinted loci. Among them, pseudohypoparathyroidism type 1B (PHP1B)/iPPSD3 is characterized by aberrant methylation at the GNAS locus, affecting at least GNAS A/B :TSS-DMR that can be accompanied by methylation defect at the other GNAS -DMRs. Clinically results in a PTH hormone resistance phenotype characterized by hypocalcemia and hyperphosphatemia. The study of monozygotic (MZ) twins discordant for clinical phenotype yet sharing molecular findings offers a unique window into epigenetic dynamics, tissue mosaicism, and developmental timing of epimutations [ 1 , 3 , 9 , 10 , 12 , 15 , 16 ]. In the present case, adolescent MZ twin sisters exhibit clear clinical discordance: only one twin presents biochemical and clinical signs consistent with PHP1B/iPPSD3, notably hypocalcemia (even incidentally detected), whereas the other remains asymptomatic. Remarkably, both twins share an identical yet partial methylation defect at the four GNAS -DMRs in peripheral blood DNA, indicating a mosaic methylation state. This partial methylation defect suggests a postzygotic epigenetic alteration arising prior to the embryonic split that gave rise to the twins [ 3 , 9 ]. Such early timing implies that the epimutation originated in a progenitor cell(s) common to both embryos, subsequently propagated and variably distributed during development. Additionally, the phenotypic discordance is likely explained by differential tissue distribution and severity of methylation defects across germ layers and organs. The affected twin shows involvement of all three embryonic germ layers—ectoderm, mesoderm, and endoderm—with consistent methylation abnormalities detected in tissues derived from each layer. Conversely, the clinically unaffected sister exhibits very mild or even absent methylation defects in some of these germ layer derivatives, as reported in other MZ twins with ImpDis [ 10 , 15 ]. This mosaicism in tissue-specific epimutations, with uneven burden across critical cell types such as those in renal, parathyroid, and osseous tissues, likely underlies the clinical divergence between the twins. The degree of epimutation in these disease-relevant tissues may reach pathogenic threshold only in the symptomatic sibling, explaining the manifestation of PHP1B/iPPSD3 symptoms exclusively in one twin. The current work rigorously excludes previously known genetic and epigenetic causes of altered methylation at the GNAS locus including (segmental) paternal uniparental disomy of chromosome 20q (patUPD20q) and deletions, inversions or transposons affecting imprinting control regions [ 24 , 25 ]. SNP array analyses revealed no regions of absence of heterozygosity or copy number alterations consistent with UPD or deletions. Longread sequencing further discarded any known deletion, structural variant or transposon insertion. To address the possibility of MLID, methylation-sensitive MLPA (MS-MLPA) multi-locus methylation analyses were performed, with samples evaluated not just for GNAS but for the panel of DMRs known to be recurrently involved in MLID. The absence of broad imprinting defects in both twins indicates that a classical MLID phenotype can be excluded as the underlying cause in this case [ 2 ]. Traditionally, MLID and methylation defects in ImpDis have been studied using methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA), a robust and widely employed technique that detects alterations in DNA methylation but generally provides a binary or semi-quantitative readout suited to complete or near-complete methylation losses or gains. However, this work underscores the added value and utility of long-read sequencing technologies for epigenetic profiling. Unlike MS-MLPA, long-read sequencing allows for base-resolution mapping of methylation patterns across genome and can detect subtle or partial methylation alterations indicative of mosaicism. In our study, this approach successfully confirmed the partial methylation changes in the twins’ blood samples—an advancement over prior applications of long-read sequencing that mostly targeted cases with complete methylation losses [ 26 – 29 ]. This capability is especially important given the partial and mosaic nature of the epimutations identified here, highlighting the superior sensitivity and resolution of long-read sequencing to uncover epigenetic heterogeneity that might remain obscured or underestimated by conventional methods [ 1 ]. The study of methylation, including non-clinical DMRs, performed in both twins using long-read sequencing completely ruled out MLID. Further, the use of a custom “imprinting regulator” gene panel—covering more than 100 candidate genes with roles in methylation establishment, maintenance, or imprinting regulation—serves to comprehensively interrogate for rare or novel genetic variants, including those associated with maternal-effect MLID [ 1 , 2 , 30 , 31 ]. The negative findings from this rigorous targeted sequencing effort not only exclude known causative variants in maternal-effect genes (such as NLRP7, KHDC3L , and others) but also provide indirect evidence against yet-unknown highly-penetrant genetic regulators in this family. The question then arises: what mechanisms explain the strikingly similar blood methylation profiles in adolescent twins discordant for phenotype? The feto-fetal transfusion effect has been invoked to explain such phenomena in previous studies of younger twins. This effect relies on vascular anastomoses in monochorionic placentas allowing exchange of hematopoietic progenitor cells during fetal life, generating hematopoietic chimerism. This exchange homogenizes the blood epigenotype while permitting tissue mosaicism in other embryonic layers, thus rationalizing phenotypic discordance [ 9 , 10 , 15 ]. However, this case uniquely involves adolescent twins, a temporal context which complicates attributing blood methylation concordance solely to fetal vascular sharing. Hematopoietic stem cells and progenitors undergo constant turnover through life, replenishing the entire blood compartment approximately every few weeks to months [ 32 ]. Over more than a decade, replacement by endogenous hematopoiesis minimizes the persistence of fetal hematopoietic clones derived from vascular chimerism. Thus, fetal transfusion alone is insufficient to explain near-identical blood methylation profiles in twins at this age. One plausible model posits that the epimutation arose very early during embryogenesis—pre-twinning or peri-twinning—in an early progenitor cell populating hematopoietic and other lineages [ 3 , 9 ]. Since DNA methylation imprinting is robustly maintained during somatic cell divisions in hematopoietic stem cells, the aberrant methylation pattern could be perpetuated faithfully throughout hematopoiesis into adolescence and beyond. This scenario aligns with known imprint stability mechanisms, and with reports of similar epigenetic mosaics persisting in somatic cells long after zygotic cleavage [ 3 ]. Concurrently, mosaic distribution in other tissues, critical for PHP1B/iPPSD3 phenotype like parathyroid, bone, and kidney, might differ substantially between twins. The parathyroid gland, bone, and kidney originate from different embryonic germ layers, which define their distinct development and cell renewal patterns. The parathyroids arise from the endoderm with low proliferative activity and stable epigenetic marks [ 33 ]. Bone derives mainly from mesodermal mesenchymal stem cells that support continuous remodeling via dynamic epigenetic regulation [ 34 , 35 ]. The kidney, from intermediate mesoderm, contains specialized epithelial cells with variable renewal and epigenetic dynamics [ 36 ]. Thus, the affected twin’s clinical picture—and the well twin’s biochemical normality—can both be explained by the proportions and tissue locations of epimutation-bearing cells. The possibility remains that only the diseased twin sustained substantial epimutant cell populations in PTH-regulated target tissues, while blood (as a stem cell-dense, high-traffic tissue) became homogenized in both. Consequently, one twin may harbor epimutant cells at pathogenic thresholds within these organs, while the other lacks such burden or distribution, leading to phenotypic discordance despite identical blood epigenotype. Additionally, environmental influences including hormonal shifts during adolescence and uncertainty about epigenetic plasticity in certain tissues could further modulate epigenetic patterns postnatally, potentially triggering or amplifying clinical expression in the affected twin [ 1 , 2 ]. Clinically, these findings emphasize limitations in using peripheral blood as a sole surrogate for epigenetic diagnostic assays in ImpDis, particularly in older or phenotypically discordant MZ twins, mainly when methylations defects resemble mosaicism (partial methylation alterations). Incorporation of multi-tissue methylation analyses, including skin fibroblasts or buccal epithelium, and utilization of advanced techniques such as long-read sequencing enable detection of partial methylation changes and mosaicism, thereby refining diagnosis and interpretation [ 1 , 2 ]. From a genetic counseling perspective, asymptomatic relatives with identical blood methylation defects may warrant longitudinal monitoring, as phenotypic expression can evolve over time due to dynamic or tissue-specific epigenetic processes. CONCLUSIONS This case illustrates a nuanced epigenetic scenario: a postzygotic, early embryonic partial methylation defect shared in blood but displaying variable mosaic distribution across germ layers and tissues, resulting in phenotypic discordance in adolescent MZ twins. This challenges simplistic models relying on feto-fetal transfusion alone and highlights the complexity of epigenetic maintenance, mosaicism, and phenotypic penetrance in imprinting disorders. Future integration of single-cell and multi-tissue epigenomic profiling, alongside longitudinal clinical follow-up, will be essential to unravel the developmental trajectories and clinical significance of such mosaic epimutations. Declarations Ethics approval and consent to participate This study was approved by Ethics committee for clinical research of Euskadi- Basque Country (CEIm-E) (protocol codes PI2017018 and PI2021053). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Consent for publication Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of data included in this article. Competing interests The authors declare that they have no competing interests. Funding The study was supported in part by a grants from the Instituto de Salud Carlos III (ISCIIII) of the Ministry of Economy and Competitiveness (Spain), which was co-financed by the European Regional Development Fund (grant number PI24/01290 to GPdN), the Spanish Ministry of Science, Innovation and Universities (Ministerio de Ciencia, Innovación y Universidades) [grant number PTQ2022-012727] to E.N.). Part of this work was performed by using the data contained in the “Programa Infraestructura de Medicina de Precisión asociada a la Ciencia y la Tecnología en Medicina Genómica (IMPaCT-GENóMICA)”, coordinated by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)” and founded by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación and Unión Europea – European Regional Development Fund (grant number IMP/00009). Author Contribution GPdN conceived and designed the study. GG recruited the family, provided family samples and clinical details. GP-N, AM-A, AP, EN, ACA-D and AG-C performed genetic studies. GP-N, AM-A, AP, JC, EN, BdlM-B and GPdN analyzed and interpreted the genetic data. GG, LC and GPdN integrated the genetic information with clinical manifestations. GPdN wrote the first manuscript draft. All authors reviewed and criticized it, approved the final version as submitted, and agreed to be accountable for all. Acknowledgement Authors would like to thank the family for their willingness to carry out these studies and to publish this work. The authors would also like to acknowledge the work from past laboratory members, that despite not directly involved in the manuscript have contributed along the years to the development of all the methods and techniques currently used. Data Availability Most of the data generated or analysed during this study are included in this published article [and its supplementary information files].The datasets are available in the ENA repository (https://www.ebi.ac.uk/ena): Project: PRJEB95923 for IR panel and long-read sequencing, and PRJEB99000 for short-read genome. References Eggermann T, Monk D, de Nanclares GP, Kagami M, Giabicani E, Riccio A, et al. Imprinting disorders. Nat Rev Dis Prim. 2023;9:1–19. Mackay DJG, Gazdagh G, Monk D, Brioude F, Giabicani E, Krzyzewska IM et al. Multi-locus imprinting disturbance (MLID): interim joint statement for clinical and molecular diagnosis. Clin Epigenetics [Internet]. 2024;16:99. Available from: http://www.ncbi.nlm.nih.gov/pubmed/39090763 Ollikainen M, Craig JM. Epigenetic discordance at imprinting control regions in twins. Epigenomics [Internet]. 2011;3:295–306. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22122339 Yamazawa K, Kagami M, Fukami M, Matsubara K, Ogata T. 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J Mol Endocrinol [Internet]. 2024;72:139–48. Available from: file:///C:/Users/Carla%0ACarolina/Desktop/Artigos%0Apara%0Aacrescentar%0Ana%0Aqualificação/The%0Aimpact%0Aof%0Abirth%0Aweight%0Aon%0Acardiovascular%0Adisease%0Arisk%0Ain Mantovani G, Bastepe M, Monk D, De Sanctis L, Thiele S, Usardi A et al. Diagnosis and management of pseudohypoparathyroidism and related disorders: First international Consensus Statement. Nat Rev Endocrinol [Internet]. 2018 [cited 2022 Aug 10];14:476–500. Available from: http://www.ncbi.nlm.nih.gov/pubmed/29959430 Dada S, Akbari V, Hejla D, Shen Y, Dixon K, Choufani S et al. Using long-read sequencing to detect and subtype a case with Temple syndrome. J Med Genet [Internet]. 2025;62:502–7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/40447311 Akbari V, Dada S, Shen Y, Dixon K, Hejla D, Galbraith A et al. Long-read sequencing for detection and subtyping of Prader-Willi and Angelman syndromes. J Med Genet [Internet]. 2024;62:32–6. 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Available from: https://clinicalepigeneticsjournal.biomedcentral.com/articles/ 10.1186/s13148-022-01292-w Eggermann T, Yapici E, Bliek J, Pereda A, Begemann M, Russo S et al. Trans-acting genetic variants causing multilocus imprinting disturbance (MLID): common mechanisms and consequences. Clin Epigenetics [Internet]. 2022;14:41. Available from: http://www.ncbi.nlm.nih.gov/pubmed/35296332 Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell [Internet]. 2008;135:1118–29. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19062086 Scharpf J, Kyriazidis N, Kamani D, Randolph G. Anatomy and embryology of the parathyroid gland. Oper Tech Otolaryngol Neck Surg [Internet]. 2016;27:117–21. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1043181016300276 Sharma G, Sultana A, Abdullah KM, Pothuraju R, Nasser MW, Batra SK et al. 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Supplementary Files Supplementarymaterialmonozygotic.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Feb, 2026 Reviews received at journal 23 Feb, 2026 Reviews received at journal 22 Feb, 2026 Reviewers agreed at journal 20 Feb, 2026 Reviewers agreed at journal 13 Jan, 2026 Reviews received at journal 27 Nov, 2025 Reviewers agreed at journal 18 Nov, 2025 Reviewers invited by journal 07 Nov, 2025 Editor assigned by journal 07 Nov, 2025 Submission checks completed at journal 07 Nov, 2025 First submitted to journal 29 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Paternal uniparental disomy is excluded, except at the three microsatellite markers highlighted in red, for which either biparental inheritance or paternal uniparental heterodisomy remains possible. These markers flank the GNAS locus. I: index (affected twin); T: twin (healthy).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7979825/v1/2fca31d7f38a0883834f6195.png\"},{\"id\":96283698,\"identity\":\"8aefe735-eb65-413b-b66c-e696f627c6bb\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 11:49:54\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":897682,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eResults of the \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eGNAS\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e locus methylation analysis following long-read sequencing\\u003c/strong\\u003e. In both images, the upper panel displays individual sequencing reads aligned with the methylation state annotated for each relevant base. Blue boxes highlight the four DMRs of the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus. The bottom two panels summarize overall methylation levels across the region, generated using statistical metrics; the plotted curves provide an overview, identifying regions of hypermethylation or hypomethylation and illustrating broader methylation trends at the locus. (A) Methylation profiles obtained from blood samples of the patient (orange), her sister (green), and a control reveal a highly similar pattern between the sisters, with a slight increase in methylation at \\u003cem\\u003eGNAS-NESP:\\u003c/em\\u003eTSS-DMR (partial hypermethylation) and a decrease (partial hypomethylation) in \\u003cem\\u003eGNAS-AS1\\u003c/em\\u003e:TSS-DMR, \\u003cem\\u003eGNAS-XL:\\u003c/em\\u003eEx1-DMR and \\u003cem\\u003eGNAS A/B:\\u003c/em\\u003eTSS-DMR compared to the control. (B) The saliva-derived results for both sisters, compared to an unrelated control, show that the patient's methylation pattern deviates further from normality than that of her healthy sister.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7979825/v1/f5148a83694077c4578b75ee.jpg\"},{\"id\":96369075,\"identity\":\"c50a7a55-4ebe-4691-83c9-428e87375c5a\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 10:19:36\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1652937,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7979825/v1/8d9ddc3b-e601-4d52-9236-bc551b924ce3.pdf\"},{\"id\":96283699,\"identity\":\"dc2bfcde-252d-40a9-8679-5487eea92ced\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 11:49:54\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1940223,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterialmonozygotic.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7979825/v1/4d756494ee6fca0287fa5895.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"\\u003cp\\u003eConcordant Epigenetic and Discordant Clinical Php1b/ippsd3 Manifestations in Two Monozygotic Adolescent Twins\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eImprinting disorders (ImpDis) are a group of congenital diseases caused by epigenetic and genetic alterations affecting the expression of imprinted genes\\u0026mdash;genes that are expressed in a parent-of-origin-specific manner. Nowadays thirteen ImpDis have been identified whose molecular basis involves four molecular alterations: uniparental disomy, copy number variants or rare chromosomal rearrangements, (likely) pathogenic point variants and aberrant DNA methylation at imprinted regions (known as imprinting defects (ImpDefs) or epimutations (for review see [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e])). A subgroup of patients with epimutations presents DNA methylation changes at multiple imprinted loci, a condition referred to as multi‑locus imprinting disturbance (MLID). The etiology of MLID has been linked to environmental factors such as assisted reproduction, and to trans‑acting genetic variants, frequently not found in the patients but in their mothers [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eWhile ImpDis are typically sporadic, their occurrence in monozygotic (MZ) twins has provided a unique window into the role of epigenetics in human disease. Monozygotic twins arise from a single zygote and are considered genetically identical. However, several studies have reported phenotypic discordance in MZ twins affected by ImpDis, where one twin presents with the full clinical phenotype while the co-twin is either mildly affected or entirely asymptomatic [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. This phenomenon challenges the assumption of complete epigenetic identity in MZ twins and underscores the dynamic nature of epigenetic regulation during early embryogenesis.\\u003c/p\\u003e\\u003cp\\u003eOne of the most compelling explanations for this discordance is the occurrence of postzygotic epigenetic alterations, particularly in DNA methylation patterns. These changes can arise during the early cleavage stages of embryonic development, leading to mosaicism in the distribution of epimutations across different tissues. One of the ImpDis where cases of discordant monozygotic twins, usually female pairs [\\u003cspan additionalcitationids=\\\"CR5 CR6 CR7 CR8\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] and rarely males [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], have been most frequently described is BWS caused by mosaic loss of methylation at \\u003cem\\u003eKCNQ1OT1\\u003c/em\\u003e:TSS-DMR. It has also been rarely associated with uniparental disomy (UPD) in a male MZ twin pair [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e] and a mosaic trisomy 11p15 including patUPD in a female MZ twin pair [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. This discordance among MZ twins has also been reported in transient neonatal diabetes caused by loss of methylation at \\u003cem\\u003ePLAGL1\\u003c/em\\u003e:alt-TSS-DMR [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], and in Silver-Russell syndrome due to hypomethylation of the \\u003cem\\u003eH19/IGF2\\u003c/em\\u003e:IG-DMR [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR15\\\" citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. MLID has been reported among MZ twins in BWS with \\u003cem\\u003eKCNQ1OT1\\u003c/em\\u003e:TSS-DMR hypomethylation [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], in SRS with hypomethylation at \\u003cem\\u003eH19/IGF2\\u003c/em\\u003e:IG-DMR [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e] and in TNDM with loss of methylation at \\u003cem\\u003ePLAGL1\\u003c/em\\u003e:alt-TSS-DMR [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eTo our knowledge, twinning has been described twice for PHP1B/iPPSD3, with both twins presenting the same methylation pattern and phenotype compatible with the disease. The first pair of \\\"identical\\\" twins are two boys diagnosed with sporPHP-1B at 3 and 5 years of age, respectively, with a complete alteration of methylation affecting the four DMRs of the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. The second pair is a 26-year-old pair of female twins (with no information on amnionicity and chorionicity) with asymptomatic sporPHP1B incidentally diagnosed due to the presence of hypocalcemia, hyperphosphatemia, hypocalciuria, and elevated serum intact PTH levels, withoutAHO phenotype. Molecular testing identified almost complete loss of methylation at the \\u003cem\\u003eGNAS-AS1\\u003c/em\\u003e:TSS-DMR, \\u003cem\\u003eGNAS-XL\\u003c/em\\u003e:Ex1-DMR and \\u003cem\\u003eGNAS A/B\\u003c/em\\u003e:TSS-DMR and gain of methylation at the \\u003cem\\u003eGNAS-NESP\\u003c/em\\u003e:TSS-DMR. The possibility of MLID was ruled out. Whole genome sequencing did not identify alterations in the coding regions of the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus, nor deletions, insertions, or inversions at the \\u003cem\\u003eGNAS\\u003c/em\\u003e and \\u003cem\\u003eSTX16\\u003c/em\\u003e loci, nor pathogenic variants in candidate genes involved in imprinting control, including genes known to cause MLID. The SNP array study ruled out CNVs and isodisomy (iUPD), however, analysis of 28 consecutive SNPs could not rule out the possibility of paternal heterodisomy (hUPDpat) within a 22 kb span spanning the \\u003cem\\u003eNESP55\\u003c/em\\u003e exon and \\u003cem\\u003eAS\\u003c/em\\u003e exon 5 [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eImportantly, the detection of these epigenetic differences depends heavily on the type of tissue analyzed. Peripheral blood is the most commonly used tissue due to its accessibility, but it may not always reflect the methylation status in disease-relevant tissues such as skin, buccal epithelium, or internal organs. Several studies have demonstrated that methylation patterns can vary significantly between tissues, and in some cases, the epimutations are restricted to specific lineages. For instance, in MZ twins discordant for ImpDis, studies performed on DNA obtained from blood usually showed epigenetic alteration in both twins, while in alternative tissues (as saliva, buccal cells, fibroblast) results revealed that only the affected twin carried the epimutation [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn this study we report two iPPSD3-phenotypically discordant female monozygotic twins, trying to identify the possible (epi)genetic cause that explains this clinical difference.\\u003c/p\\u003e\"},{\"header\":\"PATIENTS and METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCase report\\u003c/h2\\u003e\\u003cp\\u003eThe index case is a 14-year-old female, a biamniotic monochorionic twin, born to healthy, unrelated parents with no family history of neurological or endocrinological diseases (mother: 47 years old, height 150.5 cm; father: 47 years old, height 180 cm). The pregnancy occurred naturally, with a history of twins on the maternal side.\\u003c/p\\u003e\\u003cp\\u003eAfter an uneventful, well-controlled pregnancy, delivery was by cesarean section at 39.5 weeks of gestation due to lack of progression, with no apparent complications (birth weight 3570 g, 1.03 SD [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]; length 50 cm, 0.34 SD; APGAR scores 8 and 9). Her twin sister\\u0026rsquo;s anthropometric values were also within the normal range (birth weight 2930 g, -0.68 SD; length 51 cm, 0.94 SD; APGAR scores 8 and 9).\\u003c/p\\u003e\\u003cp\\u003eAt 7.5 years of age, the index was referred to the Neuropediatrics Service due to generalized seizures and was diagnosed with possible benign partial epilepsy. Treatment with levetiracetam was initiated and discontinued at 9 years of age. At 8.5 years old, during follow-up, asymptomatic hypocalcemia was detected, and she was evaluated by the Pediatric Endocrinology Service.\\u003c/p\\u003e\\u003cp\\u003eAt that time, her anthropometric measurements were: weight 26.5 kg (-0.64 SD), height 121.8 cm (-1.58 SD), BMI 17.8. She showed no signs of Albright\\u0026rsquo;s hereditary osteodystrophy. Biochemical evaluation revealed: total calcium 6.5 mg/dL (NV: 8.1\\u0026ndash;10.4 mg/dL), ionized calcium 2.8 mg/dL (NV: 4.5\\u0026ndash;5.2 mg/dL), phosphorus 8.7 mg/dL (NV: 2.5\\u0026ndash;4.7 mg/dL), magnesium 1.84 mg/dL (NV: 1.7\\u0026ndash;2.5 mg/dL), iPTH 560 pg/mL (NV: 10\\u0026ndash;75 pg/mL), vitamin D 26 ng/mL. No other hormonal deficiencies were detected (TSH 3.44 mU/L; fT4 1.08 ng/dL; LH 0.1 U/L; FSH 1.5 U/L; estradiol\\u0026thinsp;\\u0026lt;\\u0026thinsp;12 pg/mL; cortisol 4.9 mg/dL; calcitonin 5 pg/mL; IGF-1 112 ng/mL; IGFBP3 3.6 \\u0026micro;g/mL). Cranial CT revealed bilateral basal ganglia calcifications.\\u003c/p\\u003e\\u003cp\\u003eTreatment was initiated with calcium (1,200 mg daily) and calcitriol (0.25 \\u0026micro;g every 12 hours). Her twin sister was asymptomatic and showed no biochemical abnormalities.\\u003c/p\\u003e\\u003cp\\u003eShe started puberty at age 10 and had her first menstruation at 11 years and one month. At present, she has a weight of 57 kg (p75) and a height of 154.3 cm (p25), with complete pubertal development. During follow-up, she developed TSH resistance, so her current treatment consists of calcium 1,000 mg every 12 hours, calcitriol 0.5 \\u0026micro;g every 12 hours, and levothyroxine 50 \\u0026micro;g daily.\\u003c/p\\u003e\\u003cp\\u003eGiven the clinical suspicion of pseudohypoparathyroidism (PHP) in the patient, genetic testing was requested.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eMolecular studies\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eDNA extraction\\u003c/h2\\u003e\\u003cp\\u003eGenomic DNA of the family quartet was extracted from peripheral blood leukocytes using the commercial QIAamp DNA Blood Mini kit (Qiagen, D\\u0026uuml;ren, Germany), following the manufacturer's instructions. The saliva samples of both twins were collected using the Oragene DNA kit (OG-500, DNA Genotek, Stittswille, Canada) and DNA was extracted using the prepIT.L2P extraction reagent (DNA Genotek), according to the manufacturer's instructions. Urine samples (20 mL) of both twins were centrifuged at 4,100 \\u0026times; g for 20 minutes. The supernatant was discarded, retaining 1 mL to resuspend the pellet. Samples were transferred to 1.5 mL tubes and subjected to three successive centrifugation steps at 13,000 \\u0026times; g for 5 minutes, each time resuspending the pellet in 1 mL of sterile PBS (1\\u0026times;, pH 7.4). Final pellets were resuspended in 200 \\u0026micro;L of PBS (1\\u0026times;, pH 7.4), and DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen) following the manufacturer's protocol.\\u003c/p\\u003e\\u003cp\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003eAnalysis of\\u003c/span\\u003e \\u003cspan type=\\\"ItalicUnderline\\\" class=\\\"ItalicUnderline\\\" name=\\\"Emphasis\\\"\\u003eGNAS\\u003c/span\\u003e \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003elocus\\u003c/span\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe ME031-C1 kit (lot C1-0523) was used to study the dosage and methylation patterns of \\u003cem\\u003eGNAS\\u003c/em\\u003e locus in DNA extracted from blood of the quartet, and from saliva and urine cells of both twins, following the manufacturer\\u0026rsquo;s recommendations (MRC-Holland, Amsterdam, The Netherlands). In parallel, a custom upd(20q)pat MS-MLPA kit was used to analyze DNA from blood of both twins, as previously described [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Fragment sizes were assessed using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA), according to the manufacturer\\u0026rsquo;s protocols. Data analysis was carried out with Coffalyser.net software version 9.4 (MRC-Holland).\\u003c/p\\u003e\\u003cp\\u003eThe SNP array technique was used to analyze the possible presence of uniparental disomy as an underlying cause of the identified methylation pattern. For this purpose, the CytoScan 750K Array (ThermoFisher Scientific, Massachusetts, USA) and the subsequent analysis was carried out using the recommended Chromosome Analysis Suite (ChAS) software version 4.5 (ThermoFisher Scientific).\\u003c/p\\u003e\\u003cp\\u003eSTR study was performed for the quartet by fluorescent PCR, using different markers along the long arm of chromosome 20 (\\u003cem\\u003eD20S857, D20S1044, D20S468, D20S149, D20S430, D20S451, D20S496, D20S459, D20S443, D20S171, D20S173\\u003c/em\\u003e) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The fragments\\u0026rsquo; size was determined with an ABI 3500 Genetic analyzer. The results were analyzed with GeneMapper Software 5 (Applied Biosystems).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eAnalysis of MLID\\u003c/h3\\u003e\\n\\u003cp\\u003eThe methylation status and gene dosage of principal clinical DMRs (CA-DMRs) [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e], including \\u003cem\\u003ePLAGL1\\u003c/em\\u003e:alt-TSS-DMR (6q24.2), \\u003cem\\u003eMEST\\u003c/em\\u003e:alt-TSS-DMR (7q32.2), \\u003cem\\u003eH19/IGF2\\u003c/em\\u003e:IG-DMR (11p15.5), \\u003cem\\u003eKCNQ1OT1\\u003c/em\\u003e:TSS-DMR (11p15.5), \\u003cem\\u003eMEG3\\u003c/em\\u003e:TSS-DMR (14q32.2), \\u003cem\\u003eSNURF\\u003c/em\\u003e:TSS-DMR (15q11.2), \\u003cem\\u003ePEG3\\u003c/em\\u003e:TSS-DMR (19q13.43), as well as \\u003cem\\u003eGNAS-NESP\\u003c/em\\u003e:TSS-DMR, \\u003cem\\u003eGNAS-AS1\\u003c/em\\u003e:TSS-DMR, \\u003cem\\u003eGNAS-XL\\u003c/em\\u003e:Ex1-DMR, and \\u003cem\\u003eGNAS A/B\\u003c/em\\u003e:TSS-DMR, were studied using MS-MLPA with the ME034-C1 kit (lot C1-0121, MRC-Holland).\\u003c/p\\u003e\\n\\u003ch3\\u003eLooking for genetic underlying causes\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eIR panel\\u003c/h2\\u003e\\u003cp\\u003eTo identify genetic variants underlying the sisters' epigenetic alterations, we employed our custom Imprinting Regulators v1 (IR) panel, which covers 104 genes (see supplementary material) including known imprinting regulators, maternal effect genes, and additional candidates implicated in mammalian methylation regulation, zinc finger protein interactions with imprinted DMRs, as well as genes relevant to spermatogenesis and oogenesis.\\u003c/p\\u003e\\u003cp\\u003eLibrary Preparation Enzymatic Fragmentation (EF) Kit 2.0 and Twist Target Enrichment Standard Hybridization v2 Protocol (Twist Biosciences, San Francisco, USA) were used for library preparation and enrichment. These libraries were sequenced on a MiniSeq High Output platform (7.5 Gb) (Illumina Inc., San Diego, CA, USA) in 150 pair-end mode.\\u003c/p\\u003e\\u003cp\\u003eThe DNA enrichment analysis module integrated in MiniSeq (bcl2fastq 2.17.1.5 conversion software) was used for primary analysis. Downstream secondary analysis was facilitated by the pipeline of the commercial platform Orchestrator (Euformatics, Espoo, Finland), based on GATK Best Practices (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://gatk.broadinstitute.org/hc/en-us\\u003c/span\\u003e\\u003cspan address=\\\"https://gatk.broadinstitute.org/hc/en-us\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Finally, tertiary bioinformatics analysis of VCF files (variant annotation, filtering and prioritization) and BAM files (for CNV detection) was performed with the commercial software OmnomicsNGS 2.2.1 (Euformatics), followed by interpretation according to standardized guidelines [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. In the case of maternal effect genes, the approach was similar to that used previously [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. The BAM files were also visualized with Integrative Genomics Viewer (IGV), as a standard practice, to confirm variant calls, reduce false positives, and interpret complex genomic events that may not be readily resolved by automated pipelines.\\u003c/p\\u003e\\u003cp\\u003eThe parameters used for filtering and prioritization were assessed by the commercial tool OmnomicsQ (Euformatics): quality assessment Q\\u0026thinsp;\\u0026gt;\\u0026thinsp;30; mean coverage\\u0026thinsp;\\u0026gt;\\u0026thinsp;100x, uniformity 90%.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eLong read sequencing (including broader MLID analysis)\\u003c/h3\\u003e\\n\\u003cp\\u003eLong-read sequencing was performed on DNA extracted from blood and saliva of both twins using the PromethION P2 platform (Oxford Nanopore Technologies, ONT, Oxford, United Kingdom). Approximately 2 \\u0026micro;g of genomic DNA per sample was used to prepare sequencing libraries with the ONT Ligation Sequencing Kit (SQK-LSK114), following the manufacturer's protocol with minor modifications. Bioinformatic processing was conducted using a custom pipeline comprising: (i) basecalling with the Dorado basecaller integrated within ONT\\u0026rsquo;s MinKNOW software; (ii) alignment to the GRCh38 human reference genome using Minimap2; and (iii) structural variant calling using Sniffles.\\u003c/p\\u003e\\u003cp\\u003eTaking advantage of the native methylation detection provided by nanopore technology, the presence of MLID was evaluated by examining the methylation status of known DMRs [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. After Dorado basecalling, the software modbam2bed (version 0.10) and modkit (version 0.4.2) were used to convert the modified BAM files to BED files containing methylation information at each CpG position. The 48 regions of interest (ROI) were visualized using Methylartist tool [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e].\\u003c/p\\u003e\\n\\u003ch3\\u003eGenome analyses\\u003c/h3\\u003e\\n\\u003cp\\u003eWhole genome sequencing (WGS) of the quartet was performed on DNA extracted from peripheral blood under the IMPaCT-GEN\\u0026oacute;MICA program. WGS libraries were prepared using the PCR-free KAPA HyperPrep kit and sequenced using the NovaSeq 6000 System sequencer (Illumina Inc.). Paired-end reads of 150 bp were generated with an average coverage of at least 30 reads (35.24 for the index twin, 31.74 healthy twin, 32 father and 31.8 mother).\\u003c/p\\u003e\\u003cp\\u003eBoth the alignment of the sequencing data obtained against the GRCh38 reference genome and the detection of SNVs, INDELs, SVs, CNVs and STRs were carried out using the Illumina DRAGEN Bio-IT Platform v4.2 software (including the detection of mitochondrial variants).\\u003c/p\\u003e\\u003cp\\u003eSNVs and INDELs have been annotated through the GPAP platform, SVs and CNVs with AnnotSV (version 3.3.4.), mitochondrial variants using the MitoMap database (18/02/2023) and STR variants using the STRipy-pipeline (version 2.4).\\u003c/p\\u003e\\u003cp\\u003eThe identified variants have been screened and evaluated on a case-by-case basis, using resources such as inheritance mode, population allele frequency (gnomAD), pathogenicity prediction (SnpEff, SIFT, Polyphen2, CADD, REVEL), splicing effect prediction (SpliceAI), ACMG classification, or current scientific knowledge regarding their pathogenicity (ClinVar 08/12/2024). Current scientific knowledge on candidate variants has been consulted in genetic databases.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cp\\u003eMethylation analysis with MS-MLPA of all family members revealed partial losses of methylation at the \\u003cem\\u003eGNAS-AS1\\u003c/em\\u003e:TSS-DMR, \\u003cem\\u003eGNAS-XL\\u003c/em\\u003e:Ex1-DMR, and \\u003cem\\u003eGNAS A/B\\u003c/em\\u003e:TSS-DMR and a partial gain of methylation at the \\u003cem\\u003eGNAS-NESP\\u003c/em\\u003e:TSS-DMR in both twins but not in other family members in blood. The methylation alteration was similar in both twins\\u0026rsquo; samples. However, the methylation defect in saliva (and more marked in urine) was lower in the healthy sister (Supplementary Fig.\\u0026nbsp;1).\\u003c/p\\u003e\\u003cp\\u003eSNP array discarded any putative mosaic copy number alteration on chromosome 20 and the possibility of paternal iUPD20q (Supplementary Fig.\\u0026nbsp;2). The STRs study was compatible with biparental inheritance except for the region between \\u003cem\\u003eD20S496\\u003c/em\\u003e and \\u003cem\\u003eD20S443\\u003c/em\\u003e, encompassing the whole \\u003cem\\u003eGNAS\\u003c/em\\u003e locus and its regulatory gene \\u003cem\\u003eSTX16\\u003c/em\\u003e (from, at least, Chr20: 58,661,059\\u0026thinsp;\\u0026minus;\\u0026thinsp;58,920,545 according to GRCh38), where microsatellites were not sufficiently informative to rule out hUPD (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). However, expecting it to be mosaic based on the aforementioned methylation pattern results, the normalized ratios of the intensity of the sisters' alleles in the three microsatellites did not differ from those obtained in their parents (data not shown). The possibility of mosaicism in this region was also ruled out based on the SNP array results, within the detection limits of the technique (approximately 15\\u0026ndash;20%). Neither the Log R Ratio (LRR) nor the B Allele Frequency (BAF) plots showed intermediate deviations from the normal pattern, and the signal values across chromosome 20q remained stable, with no evidence of aberrant or mosaic profiles (Supplementary Fig.\\u0026nbsp;2).\\u003c/p\\u003e\\u003cp\\u003eThe study of MLID using MS-MLPA in DNA extracted from blood confirmed the presence of partial methylation alteration in \\u003cem\\u003eGNAS\\u003c/em\\u003e-DMRs in both sisters, without involvement of other additional clinical DMRs. The results obtained from long-read sequencing confirmed this partial alteration, which was similar in both twins in blood and showed less involvement in the healthy sister in the case of the saliva sample (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). At the genome-wide level, none of the samples showed methylation alterations in any of the additional 48 DMRs, neither in those considered as clinical [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. (Supplementary Fig.\\u0026nbsp;3) nor in the non-clinical ones (Supplementary Fig.\\u0026nbsp;4).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eAs part of the analysis of potential genetic defects underlying the epigenetic alteration, we applied a custom NGS panel including genes known or candidates to be involved in methylation establishment and/or maintenance, longread sequencing, and short-read WGS in the quartet. No known deletions, insertions, inversions or retrotransposons were found in the region spanning from the \\u003cem\\u003eSTX16\\u003c/em\\u003e locus to \\u003cem\\u003eGNAS\\u003c/em\\u003e, where methylation regulatory alterations have previously been identified in AD-PHP1B patients [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Additionally, a thorough review of the genome for the analysis of SNPs located at Chr20: 58,661,059\\u0026thinsp;\\u0026minus;\\u0026thinsp;58,920,545 allowed us to completely rule out hUPDpat (data not shown). On the other hand, 21 heterozygous SVs of moderate impact were identified, all of them affecting transcription binding sites. Eighteen were discarded because they had also been detected in our control cohort; one was discarded because the phasing tool confirmed that, although the VAF was compatible with heterozygosity, they were found in some of the readings of both alleles; and one was discarded because it was detected in both parents. The (hg38) NC_000020.11:g.52142651_52144388del was classified as VUS. These last two SVs are located in regions that appear to be associated with coverage difficulties (Supplementary Fig.\\u0026nbsp;5).\\u003c/p\\u003e\\u003cp\\u003eAdditionally, no (likely) pathogenic alteration (including SNVs, CNVs or SVs) was detected in any of the candidate genes studied through gene panel and genome analysis. Only one VUS was detected in the coding region inherited from the mother [NM_003482.4(KMT2D):c.6497A\\u0026thinsp;\\u0026gt;\\u0026thinsp;G;p.Gln2166Arg], while the rest, both maternally inherited and \\u003cem\\u003ede novo\\u003c/em\\u003e, were located in deep intronic regions or in UTRs.\\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eImprinting disorders (ImpDis) are complex diseases caused by disruptions of normal epigenetic regulation at imprinted loci. Among them, pseudohypoparathyroidism type 1B (PHP1B)/iPPSD3 is characterized by aberrant methylation at the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus, affecting at least \\u003cem\\u003eGNAS A/B\\u003c/em\\u003e:TSS-DMR that can be accompanied by methylation defect at the other \\u003cem\\u003eGNAS\\u003c/em\\u003e-DMRs. Clinically results in a PTH hormone resistance phenotype characterized by hypocalcemia and hyperphosphatemia. The study of monozygotic (MZ) twins discordant for clinical phenotype yet sharing molecular findings offers a unique window into epigenetic dynamics, tissue mosaicism, and developmental timing of epimutations [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn the present case, adolescent MZ twin sisters exhibit clear clinical discordance: only one twin presents biochemical and clinical signs consistent with PHP1B/iPPSD3, notably hypocalcemia (even incidentally detected), whereas the other remains asymptomatic. Remarkably, both twins share an identical yet partial methylation defect at the four \\u003cem\\u003eGNAS\\u003c/em\\u003e-DMRs in peripheral blood DNA, indicating a mosaic methylation state. This partial methylation defect suggests a postzygotic epigenetic alteration arising prior to the embryonic split that gave rise to the twins [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Such early timing implies that the epimutation originated in a progenitor cell(s) common to both embryos, subsequently propagated and variably distributed during development.\\u003c/p\\u003e\\u003cp\\u003eAdditionally, the phenotypic discordance is likely explained by differential tissue distribution and severity of methylation defects across germ layers and organs. The affected twin shows involvement of all three embryonic germ layers\\u0026mdash;ectoderm, mesoderm, and endoderm\\u0026mdash;with consistent methylation abnormalities detected in tissues derived from each layer. Conversely, the clinically unaffected sister exhibits very mild or even absent methylation defects in some of these germ layer derivatives, as reported in other MZ twins with ImpDis [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. This mosaicism in tissue-specific epimutations, with uneven burden across critical cell types such as those in renal, parathyroid, and osseous tissues, likely underlies the clinical divergence between the twins. The degree of epimutation in these disease-relevant tissues may reach pathogenic threshold only in the symptomatic sibling, explaining the manifestation of PHP1B/iPPSD3 symptoms exclusively in one twin.\\u003c/p\\u003e\\u003cp\\u003eThe current work rigorously excludes previously known genetic and epigenetic causes of altered methylation at the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus including (segmental) paternal uniparental disomy of chromosome 20q (patUPD20q) and deletions, inversions or transposons affecting imprinting control regions [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. SNP array analyses revealed no regions of absence of heterozygosity or copy number alterations consistent with UPD or deletions. Longread sequencing further discarded any known deletion, structural variant or transposon insertion.\\u003c/p\\u003e\\u003cp\\u003eTo address the possibility of MLID, methylation-sensitive MLPA (MS-MLPA) multi-locus methylation analyses were performed, with samples evaluated not just for \\u003cem\\u003eGNAS\\u003c/em\\u003e but for the panel of DMRs known to be recurrently involved in MLID. The absence of broad imprinting defects in both twins indicates that a classical MLID phenotype can be excluded as the underlying cause in this case [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eTraditionally, MLID and methylation defects in ImpDis have been studied using methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA), a robust and widely employed technique that detects alterations in DNA methylation but generally provides a binary or semi-quantitative readout suited to complete or near-complete methylation losses or gains. However, this work underscores the added value and utility of long-read sequencing technologies for epigenetic profiling. Unlike MS-MLPA, long-read sequencing allows for base-resolution mapping of methylation patterns across genome and can detect subtle or partial methylation alterations indicative of mosaicism. In our study, this approach successfully confirmed the partial methylation changes in the twins\\u0026rsquo; blood samples\\u0026mdash;an advancement over prior applications of long-read sequencing that mostly targeted cases with complete methylation losses [\\u003cspan additionalcitationids=\\\"CR27 CR28\\\" citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. This capability is especially important given the partial and mosaic nature of the epimutations identified here, highlighting the superior sensitivity and resolution of long-read sequencing to uncover epigenetic heterogeneity that might remain obscured or underestimated by conventional methods [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. The study of methylation, including non-clinical DMRs, performed in both twins using long-read sequencing completely ruled out MLID.\\u003c/p\\u003e\\u003cp\\u003eFurther, the use of a custom \\u0026ldquo;imprinting regulator\\u0026rdquo; gene panel\\u0026mdash;covering more than 100 candidate genes with roles in methylation establishment, maintenance, or imprinting regulation\\u0026mdash;serves to comprehensively interrogate for rare or novel genetic variants, including those associated with maternal-effect MLID [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. The negative findings from this rigorous targeted sequencing effort not only exclude known causative variants in maternal-effect genes (such as \\u003cem\\u003eNLRP7, KHDC3L\\u003c/em\\u003e, and others) but also provide indirect evidence against yet-unknown highly-penetrant genetic regulators in this family.\\u003c/p\\u003e\\u003cp\\u003eThe question then arises: what mechanisms explain the strikingly similar blood methylation profiles in adolescent twins discordant for phenotype? The feto-fetal transfusion effect has been invoked to explain such phenomena in previous studies of younger twins. This effect relies on vascular anastomoses in monochorionic placentas allowing exchange of hematopoietic progenitor cells during fetal life, generating hematopoietic chimerism. This exchange homogenizes the blood epigenotype while permitting tissue mosaicism in other embryonic layers, thus rationalizing phenotypic discordance [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eHowever, this case uniquely involves adolescent twins, a temporal context which complicates attributing blood methylation concordance solely to fetal vascular sharing. Hematopoietic stem cells and progenitors undergo constant turnover through life, replenishing the entire blood compartment approximately every few weeks to months [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Over more than a decade, replacement by endogenous hematopoiesis minimizes the persistence of fetal hematopoietic clones derived from vascular chimerism. Thus, fetal transfusion alone is insufficient to explain near-identical blood methylation profiles in twins at this age.\\u003c/p\\u003e\\u003cp\\u003eOne plausible model posits that the epimutation arose very early during embryogenesis\\u0026mdash;pre-twinning or peri-twinning\\u0026mdash;in an early progenitor cell populating hematopoietic and other lineages [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Since DNA methylation imprinting is robustly maintained during somatic cell divisions in hematopoietic stem cells, the aberrant methylation pattern could be perpetuated faithfully throughout hematopoiesis into adolescence and beyond. This scenario aligns with known imprint stability mechanisms, and with reports of similar epigenetic mosaics persisting in somatic cells long after zygotic cleavage [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eConcurrently, mosaic distribution in other tissues, critical for PHP1B/iPPSD3 phenotype like parathyroid, bone, and kidney, might differ substantially between twins. The parathyroid gland, bone, and kidney originate from different embryonic germ layers, which define their distinct development and cell renewal patterns. The parathyroids arise from the endoderm with low proliferative activity and stable epigenetic marks [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Bone derives mainly from mesodermal mesenchymal stem cells that support continuous remodeling via dynamic epigenetic regulation [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. The kidney, from intermediate mesoderm, contains specialized epithelial cells with variable renewal and epigenetic dynamics [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Thus, the affected twin\\u0026rsquo;s clinical picture\\u0026mdash;and the well twin\\u0026rsquo;s biochemical normality\\u0026mdash;can both be explained by the proportions and tissue locations of epimutation-bearing cells. The possibility remains that only the diseased twin sustained substantial epimutant cell populations in PTH-regulated target tissues, while blood (as a stem cell-dense, high-traffic tissue) became homogenized in both. Consequently, one twin may harbor epimutant cells at pathogenic thresholds within these organs, while the other lacks such burden or distribution, leading to phenotypic discordance despite identical blood epigenotype.\\u003c/p\\u003e\\u003cp\\u003eAdditionally, environmental influences including hormonal shifts during adolescence and uncertainty about epigenetic plasticity in certain tissues could further modulate epigenetic patterns postnatally, potentially triggering or amplifying clinical expression in the affected twin [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eClinically, these findings emphasize limitations in using peripheral blood as a sole surrogate for epigenetic diagnostic assays in ImpDis, particularly in older or phenotypically discordant MZ twins, mainly when methylations defects resemble mosaicism (partial methylation alterations). Incorporation of multi-tissue methylation analyses, including skin fibroblasts or buccal epithelium, and utilization of advanced techniques such as long-read sequencing enable detection of partial methylation changes and mosaicism, thereby refining diagnosis and interpretation [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. From a genetic counseling perspective, asymptomatic relatives with identical blood methylation defects may warrant longitudinal monitoring, as phenotypic expression can evolve over time due to dynamic or tissue-specific epigenetic processes.\\u003c/p\\u003e\"},{\"header\":\"CONCLUSIONS\",\"content\":\"\\u003cp\\u003eThis case illustrates a nuanced epigenetic scenario: a postzygotic, early embryonic partial methylation defect shared in blood but displaying variable mosaic distribution across germ layers and tissues, resulting in phenotypic discordance in adolescent MZ twins. This challenges simplistic models relying on feto-fetal transfusion alone and highlights the complexity of epigenetic maintenance, mosaicism, and phenotypic penetrance in imprinting disorders. Future integration of single-cell and multi-tissue epigenomic profiling, alongside longitudinal clinical follow-up, will be essential to unravel the developmental trajectories and clinical significance of such mosaic epimutations.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003cp\\u003eThis study was approved by Ethics committee for clinical research of Euskadi- Basque Country (CEIm-E) (protocol codes PI2017018 and PI2021053). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants\\u0026rsquo; legal guardians/next of kin.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003cp\\u003eWritten informed consent was obtained from the individual(s), and minor(s)\\u0026rsquo; legal guardian/next of kin, for the publication of data included in this article.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\u003cp\\u003eThe study was supported in part by a grants from the Instituto de Salud Carlos III (ISCIIII) of the Ministry of Economy and Competitiveness (Spain), which was co-financed by the European Regional Development Fund (grant number PI24/01290 to GPdN), the Spanish Ministry of Science, Innovation and Universities (Ministerio de Ciencia, Innovaci\\u0026oacute;n y Universidades) [grant number PTQ2022-012727] to E.N.). Part of this work was performed by using the data contained in the \\u0026ldquo;Programa Infraestructura de Medicina de Precisi\\u0026oacute;n asociada a la Ciencia y la Tecnolog\\u0026iacute;a en Medicina Gen\\u0026oacute;mica (IMPaCT-GEN\\u0026oacute;MICA)\\u0026rdquo;, coordinated by the Centro de Investigaci\\u0026oacute;n Biom\\u0026eacute;dica en Red de Enfermedades Raras (CIBERER)\\u0026rdquo; and founded by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovaci\\u0026oacute;n and Uni\\u0026oacute;n Europea \\u0026ndash; European Regional Development Fund (grant number IMP/00009).\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eGPdN conceived and designed the study. GG recruited the family, provided family samples and clinical details. GP-N, AM-A, AP, EN, ACA-D and AG-C performed genetic studies. GP-N, AM-A, AP, JC, EN, BdlM-B and GPdN analyzed and interpreted the genetic data. GG, LC and GPdN integrated the genetic information with clinical manifestations. GPdN wrote the first manuscript draft. All authors reviewed and criticized it, approved the final version as submitted, and agreed to be accountable for all.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eAuthors would like to thank the family for their willingness to carry out these studies and to publish this work. The authors would also like to acknowledge the work from past laboratory members, that despite not directly involved in the manuscript have contributed along the years to the development of all the methods and techniques currently used.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eMost of the data generated or analysed during this study are included in this published article [and its supplementary information files].The datasets are available in the ENA repository (https://www.ebi.ac.uk/ena): Project: PRJEB95923 for IR panel and long-read sequencing, and PRJEB99000 for short-read genome.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eEggermann T, Monk D, de Nanclares GP, Kagami M, Giabicani E, Riccio A, et al. Imprinting disorders. Nat Rev Dis Prim. 2023;9:1\\u0026ndash;19.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMackay DJG, Gazdagh G, Monk D, Brioude F, Giabicani E, Krzyzewska IM et al. Multi-locus imprinting disturbance (MLID): interim joint statement for clinical and molecular diagnosis. Clin Epigenetics [Internet]. 2024;16:99. Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/39090763\\u003c/span\\u003e\\u003cspan address=\\\"http://www.ncbi.nlm.nih.gov/pubmed/39090763\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eOllikainen M, Craig JM. Epigenetic discordance at imprinting control regions in twins. Epigenomics [Internet]. 2011;3:295\\u0026ndash;306. 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Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/36379849\\u003c/span\\u003e\\u003cspan address=\\\"http://www.ncbi.nlm.nih.gov/pubmed/36379849\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eYang Y. Skeletal morphogenesis during embryonic development. Crit Rev Eukaryot Gene Expr [Internet]. 2009;19:197\\u0026ndash;218. Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/19883365\\u003c/span\\u003e\\u003cspan address=\\\"http://www.ncbi.nlm.nih.gov/pubmed/19883365\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCostantini F, Kopan R. Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev Cell [Internet]. 2010;18:698\\u0026ndash;712. Available from: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/20493806\\u003c/span\\u003e\\u003cspan address=\\\"http://www.ncbi.nlm.nih.gov/pubmed/20493806\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"clinical-epigenetics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"clep\",\"sideBox\":\"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)\",\"snPcode\":\"13148\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13148/3\",\"title\":\"Clinical Epigenetics\",\"twitterHandle\":\"@OAgenetics\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Pseudohypoparathyroidism, Imprinting disorders, Monozygotic twins, Epigenetic mosaicism, GNAS locus, Postzygotic epimutation, Phenotypic discordance, Long-read sequencing, Tissue-specific methylation\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7979825/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7979825/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e\\u003cp\\u003eImprinting disorders are caused by epigenetic alterations leading to abnormal gene expression from imprinted loci. Pseudohypoparathyroidism type 1B is characterized by methylation defects at the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus, affecting hormone responsiveness. Monozygotic twins discordant for imprinting disorders provide a unique opportunity to investigate the timing, maintenance, and tissue-specific distribution of epimutations and their relation to clinical expression. Most previous studies focus on infants, where fetal blood chimerism may influence epigenetic profiles, but data in adolescents remain scarce.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e\\u003cp\\u003eWe studied adolescent monozygotic twin sisters presenting clinical and biochemical discordance for pseudohypoparathyroidism type 1B. Both twins exhibited an identical, partial methylation defect at the \\u003cem\\u003eGNAS\\u003c/em\\u003e locus in peripheral blood, consistent with a postzygotic epigenetic alteration occurring before embryonic splitting. Exclusion of known genetic causes, including uniparental disomy and structural variants, and absence of multilocus imprinting disturbances or maternal-effect gene mutations, were confirmed through comprehensive genomic and methylation analyses. The affected twin showed involvement of methylation abnormalities across all three germ layers derived tissues, whereas the unaffected twin presented very mild or absent defects in some tissues, possibly explaining the phenotypic divergence. The persistent concordance of partial methylation defects in blood despite phenotypic discordance suggests stable early epimutations maintained in hematopoietic progenitors, while tissue-specific mosaicism and differential methylation maintenance across organs critical to disease expression contribute to clinical differences. Advanced long-read sequencing demonstrated sensitivity for detecting partial and mosaic methylation patterns, surpassing traditional techniques.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e\\u003cp\\u003eThis study underscores the complexity of epigenetic mosaicism in imprinting disorders, highlighting that early postzygotic partial epimutations may yield identical blood methylation profiles but divergent tissue distributions, leading to discordant clinical phenotypes in monozygotic twins. The adolescent age of our subjects challenges the fetal blood chimerism explanation for methylation concordance and emphasizes the importance of multi-tissue evaluation and sensitive molecular assays. These findings have important implications for diagnosis, monitoring, and genetic counseling in imprinting disorders, advocating for integrated approaches to assess epigenetic heterogeneity and phenotypic variability.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Concordant Epigenetic and Discordant Clinical Php1b/ippsd3 Manifestations in Two Monozygotic Adolescent Twins\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-19 11:49:49\",\"doi\":\"10.21203/rs.3.rs-7979825/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-02-23T13:44:49+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-23T12:54:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-02-22T19:29:13+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"93600317992047054790150736076290026216\",\"date\":\"2026-02-20T17:18:40+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"284077053441383889243766693631301149642\",\"date\":\"2026-01-13T14:30:58+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-11-28T01:15:30+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"94586436050236740014025936856564645090\",\"date\":\"2025-11-18T19:06:50+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-11-07T08:48:40+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-11-07T05:48:14+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-11-07T05:48:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Clinical Epigenetics\",\"date\":\"2025-10-29T12:20:17+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"clinical-epigenetics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"clep\",\"sideBox\":\"Learn more about [Clinical Epigenetics](http://clinicalepigeneticsjournal.biomedcentral.com/)\",\"snPcode\":\"13148\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13148/3\",\"title\":\"Clinical Epigenetics\",\"twitterHandle\":\"@OAgenetics\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"e652b050-c0e5-401a-b639-b967bc133e12\",\"owner\":[],\"postedDate\":\"November 19th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-07T11:41:08+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-11-19 11:49:49\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7979825\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7979825\",\"identity\":\"rs-7979825\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}