Hypomethylation of the MEG8:Int2-DMR  in patients with pathogenic PLAG1 variants suggests new role of the chr14q32 imprinting cluster in Silver-Russell syndrome

Hypomethylation of the MEG8:Int2-DMR in patients with pathogenic PLAG1 variants suggests new role of the chr14q32 imprinting cluster in Silver-Russell syndrome | 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 Hypomethylation of the MEG8:Int2-DMR in patients with pathogenic PLAG1 variants suggests new role of the chr14q32 imprinting cluster in Silver-Russell syndrome Emilia D'Angelo, Laura Pignata, Francesco Cecere, Alessandro Vimercati, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7693802/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Nov, 2025 Read the published version in Clinical Epigenetics → Version 1 posted 10 You are reading this latest preprint version Abstract Background Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous imprinting disorder. The most common molecular defects are loss of methylation of the H19/IGF2 :IG-DMR on chromosome 11p15.5, followed by maternal uniparental disomy of chromosome 7. Further molecular lesions are genetic variants in the PLAG1 oncogene, as well as in the transcription factor HMGA2 and the fetal growth factor IGF2 . A phenotypic overlap exists between SRS and Temple syndrome (TS14) that is also characterized by growth restriction but associated with abnormalities in the imprinted chromosome 14q32 gene cluster. In TS14 patients, the germline MEG3/DLK1 : IG-DMR is hypomethylated and the MEG8 :Int2-DMR gains methylation probably as consequence of transcriptional readthrough from the MEG3 promoter on the paternal chromosome. However, the functional role of the MEG8 DMR remains unknown. Results We analysed the DNA methylation of 11-12 imprinted regions in 17 cases with clinical SRS features and heterozygous for a PLAG1 variant. We observed a specific loss of methylation of the MEG8 :Int2-DMR associated with pathogenic PLAG1 variants that result in aberrant proteins. Normal MEG8 methylation was observed in the cases carrying variants of uncertain pathogenicity or gene deletions. Most of the PLAG1 cases are familial and both epigenetic and genetic defects co-segregated within the families. Additionally, we assessed the methylation status of the MEG8 :Int2-DMR in several SRS patients with HMGA2 or IGF2 variants, H19/IGF2 :IG-DMR-LoM and upd(7)mat and all of them showed normal methylation. Conclusions Our results indicate that pathogenic PLAG1 variants leading to stable aberrant PLAG1 proteins and possibly acting in a dominant-negative manner influence methylation of the MEG8 locus. This study suggests a new pathogenetic mechanism of the PLAG1 gene in SRS, involving imprinted genes in the chr14q32 cluster through deregulation of the MEG8 :Int2-DMR and provides an epigenetic signature that may be used to assess the damaging potential of the PLAG1 variants. Silver-Russell syndrome Growth retardation Genomic imprinting DNA methylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Imprinting disorders (ImpDis) are a group of congenital diseases caused by deregulation of imprinted genes and affecting human growth, metabolism and behaviour (1). Most imprinted genes are organized in clusters, where their parent of origin-dependent expression is regulated by cis -acting regions exhibiting differential DNA methylation (Differentially Methylated Regions, DMRs) between the maternal and paternal alleles (2). Imprinted DMRs are categorized into two groups: primary DMRs, also known as germline DMRs, and secondary DMRs, also referred to as somatic DMRs. Germline DMRs acquire their methylation pattern during gametogenesis and are stably maintained through the epigenetic reprogramming occurring during the oocyte-to-embryo transition. In contrast, somatic DMRs acquire their allele-specific methylation post-fertilization and are regulated by the neighbouring germline DMR in a hierarchical manner (3). ImpDis are associated with genetic and epigenetic alterations involving changes in both gene sequences (genetic mutations) and gene regulation (epigenetic mutations) in imprinted gene clusters (1). Epigenetic abnormalities can affect a single or multiple germline DMRs (multi-locus imprinting disturbances, MLID) and can be associated with genetic variants acting in cis or in trans (2,4). Silver-Russell syndrome (SRS; OMIM #180860; prevalence at birth 1:30,000/1:100,000) is a clinically and genetically heterogeneous imprinting disorder, characterised by intrauterine and post-natal growth retardation, relative macrocephaly at birth, feeding difficulties, protruding forehead in early life and body asymmetry, along with numerous additional features at lower frequencies. According to the Netchine-Harbison clinical scoring system (NH-CSS), a clinical diagnosis is considered if a patient scores at least four out of six most frequent criteria (5). However, the definition of Silver-Russell syndrome spectrum (SRSp) has been proposed to include cases with a clinical score <4 yet still exhibiting clinical or molecular features of SRS (6). Despite the complexity of its molecular mechanisms, an underlying molecular cause can currently be identified in around 60% of patients with clinical diagnosis of SRS. The most common molecular alterations are loss of methylation (LoM) at the H19/IGF2 :IG (intergenic)-DMR, also known as IC1, on chr11p15.5, accounting for 30-60% of patients and maternal uniparental disomy of chromosome 7 (upd(7)mat) detected in 5-10% of the cases (5). About 10–20% of SRS cases with IC1-LoM show MLID (7,8). Further molecular lesions associated with SRS phenotype are represented by genetic variants in PLAG1 ( Pleiomorphic Adenoma Gene 1 ), IGF2 ( Insulin Like Growth Factor 2 ), HMGA2 ( High Mobility Group AT-Hook 2 ) and more rarely CDKN1C ( Cyclin Dependent Kinase Inhibitor 1C ) gene (9–12). A phenotypic overlap exists between SRS and Temple syndrome (TS14) that is also characterized by growth restriction but associated with abnormalities in the imprinted chr14q32 gene cluster (Fig. 1). This evolutionary conserved imprinted region contains the paternally expressed genes DLK1, RTL1 and DIO3 and the maternally expressed noncoding RNA genes MEG3/GTL2, MEG8, MEG9, and RTL1AS , as well as two large clusters of maternally expressed microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) (13). The parent-of-origin-specific expression of the imprinted chr14q32 genes is regulated by two DMRs: the germline MEG3/DLK1 :IG-DMR located between the DLK1 and MEG3 genes and the somatic MEG3 :TSS-DMR overlapping the MEG3 promoter region (14). The MEG3/DLK1 :IG-DMR acts upstream of the MEG3 :TSS-DMR and governs it in a hierarchical fashion (15,16). Two further somatic DMRs have been recently described, the MEG8 : Int2-DMR located in intron 2 of the MEG8 gene and the DLK1 :Int1-DMR overlapping the second exon of the DLK1 gene (17–19). The function of these two DMRs is currently unknown. In TS14 patients, the paternally methylated MEG3/DLK1 :IG-DMR and MEG3 :TSS-DMR are hypomethylated leading to upregulation of MEG3 and MEG8 and downregulation of DLK1 . In these cases, the MEG8 :Int2-DMR, normally methylated on the maternal chromosome, gains methylation probably as consequence of transcriptional readthrough derived from the activated paternal MEG3 promoter (17). The overlapping phenotypic features between SRS and TS14 can be attributed to the involvement of shared biological pathways and/or gene co-regulation (1). Several trans-molecular interactions between imprinted genes located on chr11p15 and chr14q32 have been reported. In particular, the concomitant overexpression of MEG3 and MEG8 leads to downregulation of IGF2 in cultured cells (20). PLAG1 , located on human chromosome 8q12, was initially identified as an oncogene associated with certain types of cancer and is the main translocation target in pleomorphic adenomas of the salivary glands (21). It belongs to the PLAG family of zinc finger transcription factors, along with PLAG-like 1 (PLAGL1), which is a tumor suppressor, and PLAG-like 2 (PLAGL2), which acts as an oncogene. PLAG1 protein contains seven canonical C2H2 zinc finger domains (ZF) and a serine-rich COOH terminus that exhibits transactivation capacities suggesting that it may act as a transcriptional regulator. Its DNA binding site is composed of the core sequence GRGGC and a G-cluster RGGK separated by seven random nucleotides. The interaction with the core sequence is mediated by fingers 6 and 7, and that with the G-cluster by finger 3 (22). The three PLAG proteins share the highest degree of homology in their NH₂-terminal zinc finger domain, with PLAGL1 and PLAGL2 showing 73% and 79% identity, respectively. In contrast, the COOH-terminal region is significantly more divergent. The greatest similarity is observed in zinc fingers 6 and 7, suggesting that PLAGL1 and PLAGL2 may also recognize a core motif similar to the PLAG1 core (GRGGC). Zinc fingers 2–5 are less conserved, but key amino acids at critical positions remain preserved (21,22). PLAGL1 dimerization through zinc finger 2 enhances transactivation of target genes (23). PLAG1 plays a critical role in the oncogenic HMGA2–PLAG1–IGF2 pathway as it has been shown to be a transcriptional activator of IGF2 that binds its P3 promoter (22). Genetic defects of this pathway can lead to fetal and postnatal growth restriction (10). Inherited or de novo alterations involving the PLAG1 gene, including whole-gene deletions and intragenic pathogenic variants, were demonstrated in several cases with phenotypic features of SRS (10,11,24–33). It has been proposed that PLAG1 haploinsufficiency leads to IGF2 repression and growth deficiency (10). However, if further genes have a role in mediating the effect of PLAG1 on somatic growth is unknown. To look for a possible dysregulation of imprinted genes linked to PLAG1 defects, we analysed the DNA methylation of 11-12 imprinted regions in 17 cases with clinical SRS features carrying heterozygous PLAG1 variants. We observed a specific loss of methylation of the MEG8 :Int2-DMR in all the cases predicted to generate stable aberrant PLAG1 proteins, indicating a functional interaction between PLAG1 and the chr14q32 imprinting cluster. Patients The study cohort included 17 patients with clinical diagnosis of SRS based on the Netchine-Harbison clinical scoring system (Additional file 1: Table S1). In these patients IC1 LoM and upd(7)mat as well as molecular TS14 were excluded. As detailed in Table 1, 8 cases were reported in previous publications. All the cases carried a variant involving the PLAG1 gene. Table 1. Summary of the molecular features of the probands and their families Cases Proband sex PLAG1 variant Pathogenicity evaluation* Genotype MEG8 :Int2-DMR methylation Publication SRS1 Female NM_002655.2: c.1363del; p.Gln455Serfs*16 novel frameshift mutation-premature stop Likely Pathogenic Proband: het Mother: wt Father: wt Proband: hypomethylation Mother: NA Father: NA Abi Habib W. et al., 2018 SRS2 Female NM_002655.2: c.439del; p.Ser147Valfs*82 novel frameshift mutation-premature stop Pathogenic Proband: het Mother: het Father: wt Proband’s sister: het Proband: hypomethylation Mother: hypomethylation Father: NA Proband’s sister: hypomethylation Abi Habib W. et al., 2018 SRS3 Female NM_002655.2: c.599dup; p.Arg201Profs*52 novel frameshift mutation-premature stop Likely Pathogenic Proband: het Mother: wt Father: wt Proband: normal methylation Mother: NA Father: NA Meyer R., et al. , 2021 SRS4 Female chr8q12.1 deletion including the PLAG1 gene arr [ GRCh37 ] 8q12.1 (56,834,331_ 58,921,491) × 1 Pathogenic Proband: het Mother: wt Father: wt Proband: normal methylation Father: NA Mother: NA Fernández-Fructuoso JR. et al ., 2021 SRS5 Male chr8q12.1 deletion including the PLAG1 gene arr [ GRCh37 ] 8q12.1 (57,079,399_57,155,945) × 1 Pathogenic Proband: het Mother: wt Father: wt Proband: normal methylation Father: NA Mother: NA Baba N., et al ., 2022 SRS6 Male NM_002655.3 : c.545A > T; p.Glu182Val novel missense mutation VUS Low Pathogenic Support Proband: het Mother: NA Father: NA Proband: normal methylation Father: NA Mother: NA Kessler L., et al ., 2024 SRS7 Male NM_002655.3: c.610_612del; p.(Met204del) novel in-frame deletion Likely pathogenic Proband: het Mother: het Father: wt Proband: hypomethylation Mother: hypomethylation Father: normal methylation Vimercati A., et al. , 2025 SRS8 Male NM_002655.3: c.671G > A; p.(Arg224Gln) novel missense mutation Likely pathogenic Proband: het Mother: het Father: wt Proband: hypomethylation Mother: hypomethylation Father: normal methylation Vimercati A., et al. , 2025 SRS9 Female NM_002655.3: c.1023T>A; p.Tyr341* novel nonsense mutation Likely Pathogenic Proband: het Mother: het Father: wt Proband: hypomethylation Mother: hypomethylation Father: NA This study SRS10 Female NM_002655.3: c.666del; p.Phe222Leufs*7 novel frameshift mutation-premature stop Likely Pathogenic Proband: het Mother: NA Father: NA Proband: hypomethylation Mother: NA Father: NA This study SRS11 Female NM_002655.3: c.527C>A; p.Ser176* novel nonsense mutation Likely Pathogenic Proband: het Mother: wt Father: wt Proband: hypomethylation Mother: normal methylation Father: NA This study SRS12 Female NM_002655.3 : c.1455_1502del ; p.Ser485delinsArgAspSerGlyThrTrpIleHisTyrArgAsnValCysValAlaValPro* novel stop-loss mutation; Change of the C-ter end of the peptide, elongated protein (+1 aa). Likely Pathogenic Proband : het Mother :het Father :wt Proband’s brother : het Proband: hypomethylation Mother: hypomethylation Father: normal methylation Proband’s brother: hypomethylation This study SRS13 Male NM_002655.3: c.779_780del; p.Val260Alafs*16 novel frameshift mutation-premature stop Likely Pathogenic Proband: het Mother: het Father: wt Proband: hypomethylation Mother: hypomethylation Father: normal methylation This study SRS14 Male NM_002655.3: c.770del; p.Asn257Metfs*6 novel frameshift mutation-premature stop Likely Pathogenic Proband: het Mother: het Father: wt Proband: hypomethylation Mother: hypomethylation Father: normal methylation This study SRS15 Female NM_002655.3: c.-117-5del p.? rs1023307529 AF: 0.0005235 non-coding; (intron-exon boundary) VUS >> cl2 Uncertain pathogenicity Proband: het Mother: het Father: wt Maternal grandmother: het Maternal grandfather: wt Porband: normal methylation Mother: normal methylation Father: NA Maternal grandmother: NA Maternal grandfather: NA This study SRS16 Male NM_002655.3: c.1162A>G; p.Ile388Val rs765459935 AF: 0.00001593 missense mutation VUS >> cl2 Uncertain pathogenicity Proband:het Mother:het Father:NA Proband: normal methylation Mother: normal methylation Father: NA This study SRS17 Male chr8q12.1 deletion including the PLAG1 gene arr[ GRCh37 ]8q12.1 (56,986,129_57,169,684)x1 Pathogenic Proband:het Mother:wt Father:wt Proband: normal methylation Father: NA Mother: NA This study * Pathogenicity assessment based on ACMG criteria. In third column AF: Allele frequency in European population. In fifth column: het= heterozygous; wt= wild type. In both fifth and sixth column: NA = not analysed. Materials and Methods DNA extraction Genomic DNA of patients and their relatives was isolated from peripheral blood leukocyte (PBL) by standard procedures. NGS analysis For SRS1-SRS3,SRS6-SRS8 and their relatives, sequencing analysis has been reported previously (10,25,32,33). For SRS9-SRS11, SRS15 and SRS16 families, library preparation was performed using a custom sequencing panel designed by Sophia Genetics. Libraries were sequenced on a MiSeq (Illumina, San Diego, CA, USA). For SRS12-SRS14 families, library preparation, DNA enrichment and sequencing were performed as described in Torella et al. (34). The WES data preprocessing followed the nf-core Sarek pipeline (version 3.3.2) using its default settings (35). The sequencing reads were mapped to the human reference genome GRCh38 using the BWA-MEM algorithm. The resulting variant call format (VCFs) files were then annotated with Franklin by Genoox. Validation and segregation analysis of variants in the family members were performed by Sanger sequencing. Molecular karyotyping Molecular karyotyping for SRS4 and SRS5 was previously reported (27,28). For SRS17, CGH array analysis was carried out by using a 180k microarray (Agilent Technologies, Santa Clara, CA, USA). Raw data were analysed by the CytoGenomics V3.0 software (Agilent Technologies). In silico prediction of variant pathogenicity and protein stability Variants were initially classified based on their predicted impact on the protein. In silico pathogenicity prediction was performed for all SNVs and indels using CADD v1.7 (36). For missense variants, additional analyses were conducted using PolyPhen-2 (37), SIFT (38), AlphaMissense (39) and REVEL (40). Furthermore, all variants, including the three large deletions, were classified according to the ACMG standards and guidelines (41,42). Protein stability was investigated using DEGRONOPEDIA (43), which enables the identification of known degron motifs involved in protein degradation pathways. Methylation analysis Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) targeting 11 or 12 imprinted loci was performed using the SALSA MS-MLPA Kit (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Probemix ME034-C1 or Probemix ME034-D1, designed for multi-locus imprinting analysis, were used. Raw data were analysed using Coffalyser.Net software (MRC Holland, Amsterdam, The Netherlands). Pyrosequencing analysis was carried out as described in Sparago et al. (44) and it was conducted in separate batches of experiments. The 13 analysed CpGs are included in the following coordinates: chr14:100,904,601-100,904,702 (GRCh38/hg38). ImprintCap, a NGS-based methodology for large-scale methylation studies at several imprinted loci was performed as described in Brioude et al. (45) on 13 control samples to explore the methylation of MEG8 :Int2-DMR. The 42 analysed CpGs are included in the following coordinates: chr14: 100,904,423- 100,905,080 (GRCh38/hg38). Results DNA methylation analyses We investigated the DNA methylation of 11-12 imprinted regions in 17 SRS cases carrying rare PLAG1 variants in heterozygosity (Table 1) and 54 control individuals, by using the multi-locus MS-MLPA C1 or D1 kit. Compared to the mean of the controls, we found a partial loss of methylation of the MEG8 :Int2-DMR in the cases SRS1,SRS2,SRS5 and SRS7-SRS14. Most of these cases were familial and the imprinting defect segregated within the family, being detected in all the members who carried the PLAG1 variant (Fig. 2). Conversely, MEG8 :Int2-DMR methylation was normal in SRS3, SRS4, SRS6, and SRS15-SRS17. The MS-MLPA results are reported in the Additional file 1: Table S2. In all cases, the methylation levels of the other tested DMRs, including the MEG3 :TSS-DMR, were comparable to those of the controls (Fig. 2). In addition, we evaluated the methylation status of the MEG8 :Int2-DMR in 7 SRS patients carrying HMGA2 variants and 8 with IGF2 variants and all of them showed normal methylation (Additional file 1: Table S3). To further explore MEG8 methylation in other molecular subgroups of SRS, we performed multi-locus MS-MLPA in 28 patients with IC1 loss of methylation (IC1-LoM), 4 with maternal uniparental disomy of chromosome 7, and 31 idiopathic cases (NH-CSS ≥ 4). All of these also exhibited normal methylation levels (Additional file 1: Table S4). Finally, 2/54 of the healthy control individuals showed lower methylation levels at the MEG8 :Int2-DMR, according to the MS-MLPA results (Additional file 1: Table S5). To validate the MS-MLPA results, we analysed MEG8 :Int2-DMR methylation through the more quantitative pyrosequencing assay in the patients carrying the PLAG1 variants and in several controls. With this assay, we tested the methylation level of 13 CpGs across the MEG8 :Int2-DMR. The results were consistent with the data obtained by MS-MLPA in 10/11 patients and confirmed the co-segregation of the MEG8 :Int2-DMR hypomethylation with the PLAG1 variants (Fig. 3A-G). Furthermore, the pyrosequencing results demonstrated that the hypomethylation affected the entire MEG8 :Int2-DMR. The reduction in methylation levels was between 15% and 33% compared to the mean of the controls (Additional file 1: Table S6; Additional file 2: Fig. S1). Patient SRS5 and the two healthy individuals found hypomethylated with MS-MLPA showed a more modest (3-6%) methylation defect restricted to only a few CpGs located at the 5’ part of the DMR (Fig. 3H; Additional file 1: Table S7; Additional file 3: Fig. S2). From ImprintCap analysis on 13 control samples, those latter 5’-CpGs were shown to be highly variable, making those 5’-CpGs not suitable to define gain or loss-of-methylation in patients (Additional file 1: Table S8; Additional file 4: Fig. S3). In summary, these results indicate a specific loss of methylation of the entire MEG8 :Int2-DMR in 10 out of 17 SRS cases carrying a genetic variant in PLAG1. Genetic alterations in PLAG1 Since the MEG8 methylation defect was not consistently present in all the 17 SRS cases with PLAG1 variants, we assessed the pathogenic potential of the identified genetic variants by in silico analysis (Fig. 4; Table 1). The clinical features and PLAG1 variants of 8 patients (SRS1-SRS8) have been described in previous studies (10,25,27,28,32,33), while the remaining nine are novel cases. The patients SRS1-SRS3 and SRS7-SRS14 (Table 1) carried heterozygous single nucleotide variants (SNVs) or indels of PLAG1 that were classified as pathogenic or likely pathogenic according to the ACMG guidelines and gene variant interpretation (Additional file 1: Table S9). In particular, two nonsense variants were identified in cases SRS9 and SRS11. The variant p.Tyr341* resulting in loss of the C-terminal activation domain was found in both the SRS9 proband and her mother , while the variant p.Ser176* causes the loss of ZF6, ZF7 and the entire C-terminal region of PLAG1 in patient SRS11. Frameshift variants were identified in the cases SRS1-SRS3 (10,25), SRS10, SRS13 and SRS14. These pathogenic variants lead to putative truncated proteins lacking key functional domains (Additional file 5: Fig. S4). In particular, the variant p.Phe222Leufs*7 carried by patient SRS10 causes a frameshift starting from ZF7 and likely resulting in a truncated peptide composed of 227 amino acid residues (221 wild-type and 6 mutated), while the p.Val260Alafs*16 and p.Asn257Metfs*6 variants, inherited maternally by the SRS13 and SRS14 patients, respectively, lead to loss of the entire C-terminal domain. A novel 48 bp deletion was identified in the SRS12 proband, as well as in her mother and brother, but not in her father. This mutation likely results in a protein of 501 amino acids, with 484 wild-type, 16 mutated and one additional residue at the C-terminus (p.Ser485delinsArgAspSerGlyThrTrpIleHisTyrArgAsnValCysValAlaValPro*). Finally, SRS7 and SRS8 carried an in-frame deletion and a missense mutation in the ZF6 and ZF7 sequences, respectively (33). To assess the stability of the 11 predicted aberrant proteins resulting from deleterious PLAG1 mutations, we analysed the presence of known degron motifs involved in degradation pathways (43). Notably, a RxxGxx motif was identified at the C-terminus of the PLAG1 mutant p.Arg201Profs*52 (SRS3). APPBP2, a substrate receptor of CRL2 (Cullin-RING E3 ubiquitin ligase 2) complexes, is known to recognize C-degrons defined by a distinctive C-terminal Arg-x-x-Gly sequence (46). This specific degron motif was absent in the other mutated proteins that were subjected to analysis, suggesting a potential importance of the R-x-x-G motif in substrate recognition and subsequent ubiquitination processes within CRL2-mediated proteolysis for the PLAG1 mutant p.Arg201Profs*52 (Additional file 5: Fig. S4). According to the results of the in-silico analysis and the ACMG criteria, the patients SRS6 (32), SRS15 and SRS16 carried Variants of Uncertain Significance (VUS) in the PLAG1 gene (Table 1, Additional file 1: Table S9). In particular, an intronic variant (NM_002655.3:c.-117-5del;p.?) occurring in a homopolymeric region of PLAG1 intron 3 was found in SRS15, her mother and her healthy maternal grandmother, while the missense variants p.Ile388Val and p.Glu182Val were identified in cases SRS6 and SRS16, respectively. In particular, the p.Ile388Val variant involved a non-conserved residue and, in both SRS6 and SRS16 cases, the functional PLAG1 domains were not affected. Thus, their pathogenicity remains uncertain. Finally, patients SRS4 (27), SRS5 (28), and SRS17 carried heterozygous deletions on chromosome 8q (Table 1; Additional file 6: Fig. S5). Among them, patient SRS5 exhibited the smallest deletion, affecting only PLAG1 and CHCHD7 genes. In contrast, SRS4 presented with a larger de novo deletion of approximately 2.1 Mb at 8q12, encompassing 32 genes, including 9 OMIM-annotated genes. In SRS17, molecular karyotyping identified a ~183 kb de novo deletion (arr[GRCh37]8q12.1(56,986,129_57,169,684)x1) involving PLAG1 but also CHCHD7, MOS , and SDR16C5 . In all three cases, PLAG1 was proposed as the primary candidate gene responsible for the observed SRS phenotype (27,28). According to the ACMG criteria, all three deletions are classified as pathogenic. Overall, we collected a wide spectrum of PLAG1 variants: two nonsense variants, six frameshift, one stop-loss, one in-frame deletion, three missense variants, one intronic variant, and three large deletions affecting the entire PLAG1 gene on chromosome 8q. Evaluation of their potential pathogenicity led us to observe that 11 out of 14 SNVs/indels were predicted to be damaging, whereas the remaining 3 were classified as VUS. Among these 11 damaging variants, one harbours a degron motif likely associated with protein degradation. The copy number variants were also classified as pathogenic. Correlation of MEG8 methylation with PLAG1 gene variants To further investigate the relationship between MEG8 :Int2-DMR methylation and PLAG1 function, we compared the methylation of this locus among the 17 SRS patients carrying different PLAG1 variants (Table 1). Interestingly, loss of MEG8 :Int2-DMR methylation was detected in 10 out of 11 cases carrying pathogenic or likely pathogenic single nucleotide variants or indels, with SRS3 (p.Arg201Profs*52) representing the only exception. In particular, this mutated protein uniquely harbours the C-terminal RxxGxx motif, which facilitates protein degradation (46,47), suggesting a distinct impact on protein stability compared with the other pathogenic mutations. Conversely, all the 6 cases carrying VUS or large deletions encompassing the PLAG1 gene showed normal methylation levels. Notably, MEG8 :Int2-DMR hypomethylation was not detected in any of the SRS patients with molecular alterations in other genes of the HMGA2–PLAG1–IGF2 pathway, in those with IC1-LoM, upd(7)mat and in idiopathic SRS cases. In summary, MEG8 hypomethylation is associated with PLAG1 variants that are predicted to produce stable abnormal proteins, but not with whole PLAG1 gene deletions, variants of uncertain pathogenicity or variants leading to protein degradation. Discussion The expression of many imprinted genes impacts pre- and postnatal growth (1). The etiology of SRS has been primarily linked to the imprinted gene clusters located on chr11p15 and chr7, but pathogenic variants of the HMGA2, IGF2 and PLAG1 genes were also found in individuals with SRS features (10). Both HMGA2 and PLAG1 were proposed to control the expression of the IGF2 gene, but their possible interaction with other imprinted genes was not explored. Here, we demonstrate that damaging PLAG1 variants are associated with MEG8 :Int2-DMR hypomethylation, suggesting that PLAG1 may affect MEG8 methylation and thereby expression of chr14q32 genes controlling somatic growth. In the present study, MEG8 :Int2-DMR hypomethylation was demonstrated in cases with pathogenic or likely pathogenic PLAG1 variants that are predicted to produce abnormal proteins. In contrast, normal MEG8 methylation was observed in the cases with missense/intronic variants classified as VUS according to the ACMG guidelines and PLAG1 deletions. In addition, most of the pathogenic PLAG1 cases are familial and both epigenetic and genetic defects co-segregated within the families, further supporting the association between damaging PLAG1 variants and MEG8 hypomethylation. This epigenetic defect appears to be specific for PLAG1 , because it was not found in the cases with HMGA2 and IGF2 variants, patients with IC1-LoM, upd(7)mat cases and idiopathic cases. Apart from MEG8 , the methylation of other 10-11 imprinted loci was found to be normal in the patients studied, suggesting a specific interaction between PLAG1 and MEG8 -DMR. Analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data indicates that PLAG1 normally interacts with the MEG8 :Int2-DMR, although it is unclear if its binding is influenced by DNA methylation (48). Interestingly, PLAGL2, a homologue of PLAG1, is also predicted to bind the same DMR, as suggested by data from JASPAR CORE 2024 - Predicted Transcription Factor Binding Sites (49). Particularly, PLAGL2 shares 79% identity with PLAG1 in its NH₂-terminal zinc finger domain, suggesting that the zinc finger domain may be involved in recognizing the MEG8 :Int2-DMR. In humans, ENCODE ChIP-seq data revealed a conserved CTCF binding site within the MEG8 -DMR (19). In mice, however, CTCF binds to the Meg8 -DMR in a non-allele-specific manner in vivo (50). CTCF (CCCTC-binding factor) is a well-characterized vertebrate protein with eleven zinc fingers, the first ten of which are C2H2-type, similar to those found in PLAG1, while the last one is of C2HC-type (51). Notably, PLAG1 and CTCF recognize similar consensus sequences, as both bind to G-rich regions. In the present SRS patients, the truncating PLAG1 variants map in the last two exons of the gene and should therefore escape the nonsense-mediated decay (NMD) pathway (52). Also, they likely lead to aberrant PLAG1 proteins, which could influence methylation at the MEG8 :Int2-DMR because of reduced DNA binding. Indeed, because zinc finger 2 is retained in all these variants, they may dimerize similar to PLAGL1 and exert a dominant-negative effect on the wildtype allele (23). Consistent with this hypothesis, the single nucleotide deletion (SRS7) and the missense variant (SRS8) in the zinc fingers 6 and 7, that are responsible for specific DNA motif recognition (33), result in MEG8 hypomethylation. In contrast, the SRS3 variant that contains a C-terminal degron motif and likely results in an unstable aberrant protein is associated with normal MEG8 methylation (46,47). Also, the variants of uncertain pathogenicity are unlikely to disrupt the DNA binding function of PLAG1 and thereby maintain normal MEG8 methylation. Nevertheless, the mechanism by which reduced PLAG1 binding may interfere with MEG8 methylation remains to be defined. The absence of methylation defects in the cases with whole gene deletion is consistent with the dominant-negative hypothesis (53). In SRS5 and the two healthy individuals who scored hypomethylated at the MEG8 :Int2-DMR according to the MS-MLPA results, further analysis by pyrosequencing showed that this methylation defect was less severe compared to that detected in all the other patients and restricted to a few CpGs located at the 5’ part of the DMR, which was shown to be highly variable in a control population. This finding suggests that while MS-MLPA may indicate MEG8 :Int2-DMR hypomethylation, it does not always reflect the full methylation pattern, and further confirmation using more sensitive and extensive techniques like pyrosequencing or NGS-based technologies like ImprintCap is essential for its accurate assessment. The observed MEG8 hypomethylation in the present study suggests that chr14q32 genes have a role in PLAG1-dependent pathogenesis of SRS. In the TS14 cases with MEG3 :TSS-DMR hypomethylation, the MEG3 long noncoding RNA is activated and transcription through the MEG8 :Int2-DMR leads to its hypermethylation (17,54). However, in the cases with PLAG1 mutations, MEG3 methylation is unaffected, suggesting that alternative molecular mechanisms lead to MEG8 :Int2-DMR hypomethylation. In a recent study, Baena et al. (19) described a family in which MEG8 but not MEG3 exhibits a methylation pattern dependent on the parental origin of a DLK1 deletion, suggesting an interaction between these two loci. Also, a conserved CTCF binding site is present and therefore an insulator may be formed within the MEG8 -DMR (19). The MEG8 DMR overlaps the MEG8 lncRNA and two clusters of miRNAs and snoRNAs, all of which are deregulated in cancer (55,56). It is possible that the disrupted PLAG1 binding to MEG8 interferes with expression of one or more of these genes. Finally, the normal methylation in the cases with HMGA2 and IGF2 variants suggests that MEG8 works as PLAG1 target upstream of HMGA2 and IGF2 in the PLAG1–HMGA2-IGF2 pathway. Conclusions Diagnostic methylation changes have been recently identified in cohorts of individuals affected by developmental disorders and carrying pathogenic variants in epigenetic modifiers (57). The present study identifies MEG8 :Int2-DMR hypomethylation as a specific epigenetic signature of damaging PLAG1 variants likely causing dominant-negative effect in patients with clinical SRS features. These findings support a novel pathogenetic mechanism whereby impaired PLAG1 function alters the methylation and possibly the expression of imprinted genes within the chr14q32 region. Furthermore, MEG8 :Int2-DMR methylation studies might be useful in the future as a functional test for PLAG1 VUS. Abbreviations DMRs Differentially Methylated Regions ImpDis Imprining Disorders LoM Loss of methylation MLID Multi-locus imprinting disturbances NH-CSS Netchine-Harbison clinical scoring system NMD Nonsense-mediated decay PBL Peripheral blood leukocytes SRS Silver-Russell syndrome SRSp Silver-Russell syndrome spectrum SNVs Single nucleotide variants TS Temple syndrome UPD Uniparental disomy VUS Variant of uncertain significance ZF Zinc Finger Declarations Ethics approval and consent to participate The study was approved by the ethical committees of the University of Campania Luigi Vanvitelli, Istituto Auxologico Italiano, Hôpital Trousseau (Paris), Center for Human Genetics and Genome Medicine (Aachen), Hospital General Universitario de Santa Lucía. Consent for publication The families agreed for publication by signing an informed consent template. Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional files. Competing interests The authors declare no competing interests. Funding This work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC; IG 2020 ID 24405) and Fondazione Telethon (GMR23T1062) awarded to AR and Italian Ministry of University and Research PRIN 2022B2N2BY awarded to AR and MVC. TE is supported by the Deutsche Forschungsgemeinschaft (EG 115/13-1). ImprintCap experiments were funded with the French Agence Nationale pour la Recherche (ANR) grant no. ANR-22-CE14-0021. Authors’ contributions Conceptualization, FB, AR, FlCe; investigation, EDA ,LP ,FrCe ,AV ,MVC, AS, CG, NT, SR, TE,IN, JRF; writing—draft preparation EDA, FlCe, AR, FB; supervision, FB, AR. All authors have read and agreed to the published version of the manuscript. 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Trousseau","correspondingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Brioude","suffix":""}],"badges":[],"createdAt":"2025-09-23 11:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7693802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7693802/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13148-025-02024-6","type":"published","date":"2025-11-23T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93015842,"identity":"752e99f3-9785-487f-82fb-e75f077b4e79","added_by":"auto","created_at":"2025-10-08 08:00:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the imprinted gene cluster on human chromosome 14q32.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7693802/v1/c97e516ba8cf4aa653bd3682.png"},{"id":93015840,"identity":"281b707f-39f2-4b60-bea4-259d3c082db9","added_by":"auto","created_at":"2025-10-08 08:00:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative MS-MLPA results in one of the families.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7693802/v1/d5354ed6f45652d0e9f1a799.png"},{"id":93015841,"identity":"d543e376-bf2f-4bd3-b92e-41f74aa4fdd5","added_by":"auto","created_at":"2025-10-08 08:00:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePyrosequencing analyses of DNA methylation at the MEG8:Int2-DMR in cases with PLAG1 variants.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7693802/v1/7325c29b23daf5055cfeed63.png"},{"id":93016870,"identity":"5fb8917e-dd8d-42e9-b08c-b6805280b0d4","added_by":"auto","created_at":"2025-10-08 08:08:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePLAG1 mutations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7693802/v1/1b6b00d653fec15139b2fc28.png"},{"id":96650142,"identity":"1024b2ba-46d3-49d9-8b9d-7ac5afeaac63","added_by":"auto","created_at":"2025-11-24 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08:00:39","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":84953,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile6Fig.S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7693802/v1/9a5c27936881b451dd8d8f07.pdf"},{"id":93015846,"identity":"da633a30-7401-4fe0-a1d2-f9a47ae556ea","added_by":"auto","created_at":"2025-10-08 08:00:39","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17282,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7693802/v1/73b799152c6171a0e8413533.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hypomethylation of the MEG8:Int2-DMR in patients with pathogenic PLAG1 variants suggests new role of the chr14q32 imprinting cluster in Silver-Russell syndrome ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImprinting disorders (ImpDis) are a group of congenital diseases caused by deregulation of imprinted genes and affecting human growth, metabolism and behaviour (1). Most imprinted genes are organized in clusters, where their parent of origin-dependent expression is regulated by \u003cem\u003ecis\u003c/em\u003e-acting regions exhibiting differential DNA methylation (Differentially Methylated Regions, DMRs) between the maternal and paternal alleles (2). Imprinted DMRs are categorized into two groups: primary DMRs, also known as germline DMRs, and secondary DMRs, also referred to as somatic DMRs. Germline DMRs acquire their methylation pattern during gametogenesis and are stably maintained through the epigenetic reprogramming occurring during the oocyte-to-embryo transition. In contrast, somatic DMRs acquire their allele-specific methylation post-fertilization and are regulated by the neighbouring germline DMR in a hierarchical manner (3). ImpDis are associated with genetic and epigenetic alterations involving changes in both gene sequences (genetic mutations) and gene regulation (epigenetic mutations) in imprinted gene clusters (1). Epigenetic abnormalities can affect a single or multiple germline DMRs (multi-locus imprinting disturbances, MLID) and can be associated with genetic variants acting \u003cem\u003ein cis\u003c/em\u003e or \u003cem\u003ein trans\u0026nbsp;\u003c/em\u003e(2,4).\u003c/p\u003e\n\u003cp\u003eSilver-Russell syndrome (SRS; OMIM #180860; prevalence at birth 1:30,000/1:100,000) is a clinically and genetically heterogeneous imprinting disorder, characterised by intrauterine and post-natal growth retardation, relative macrocephaly at birth, feeding difficulties, protruding forehead in early life and body asymmetry, along with numerous additional features at lower frequencies. According to the Netchine-Harbison clinical scoring system (NH-CSS), a clinical diagnosis is considered if a patient scores at least four out of six most frequent criteria (5). However, the definition of Silver-Russell syndrome spectrum (SRSp) has been proposed to include cases with a clinical score \u0026lt;4 yet still exhibiting clinical or molecular features of SRS (6). Despite the complexity of its molecular mechanisms, an underlying molecular cause can currently be identified in around 60% of patients with clinical diagnosis of SRS. The most common molecular alterations are loss of methylation (LoM) at the \u003cem\u003eH19/IGF2\u003c/em\u003e:IG (intergenic)-DMR, also known as IC1, on chr11p15.5, accounting for 30-60% of patients and maternal uniparental disomy of chromosome 7 (upd(7)mat) detected in 5-10% of the cases (5). About 10\u0026ndash;20% of SRS cases with IC1-LoM show MLID (7,8). Further molecular lesions associated with SRS phenotype are represented by genetic variants in \u003cem\u003ePLAG1\u003c/em\u003e (\u003cem\u003ePleiomorphic Adenoma Gene 1\u003c/em\u003e), \u003cem\u003eIGF2\u003c/em\u003e (\u003cem\u003eInsulin Like Growth Factor 2\u003c/em\u003e), \u003cem\u003eHMGA2\u0026nbsp;\u003c/em\u003e(\u003cem\u003eHigh Mobility Group AT-Hook 2\u003c/em\u003e) and more rarely \u003cem\u003eCDKN1C\u003c/em\u003e (\u003cem\u003eCyclin Dependent Kinase Inhibitor 1C\u003c/em\u003e) gene (9\u0026ndash;12).\u003c/p\u003e\n\u003cp\u003eA phenotypic overlap exists between SRS and Temple syndrome (TS14) that is also characterized by growth restriction but associated with abnormalities in the imprinted chr14q32 gene cluster (Fig. 1). This evolutionary conserved imprinted region contains the paternally expressed genes \u003cem\u003eDLK1, RTL1\u003c/em\u003e and \u003cem\u003eDIO3\u003c/em\u003e and the maternally expressed noncoding RNA genes \u003cem\u003eMEG3/GTL2, MEG8, MEG9,\u003c/em\u003e and \u003cem\u003eRTL1AS\u003c/em\u003e, as well as two large clusters of maternally expressed microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) (13). The parent-of-origin-specific expression of the imprinted chr14q32 genes is regulated by two DMRs: the germline \u003cem\u003eMEG3/DLK1\u003c/em\u003e:IG-DMR located between the \u003cem\u003eDLK1\u003c/em\u003e and \u003cem\u003eMEG3\u003c/em\u003e genes and the somatic \u003cem\u003eMEG3\u003c/em\u003e:TSS-DMR overlapping the \u003cem\u003eMEG3\u0026nbsp;\u003c/em\u003epromoter region (14). The \u003cem\u003eMEG3/DLK1\u003c/em\u003e:IG-DMR acts upstream of the \u003cem\u003eMEG3\u003c/em\u003e:TSS-DMR and governs it in a hierarchical fashion (15,16). Two further somatic DMRs have been recently described, the \u003cem\u003eMEG8\u003c/em\u003e: Int2-DMR located in intron 2 of the \u003cem\u003eMEG8\u003c/em\u003e gene and the \u003cem\u003eDLK1\u003c/em\u003e:Int1-DMR overlapping the second exon of the \u003cem\u003eDLK1\u003c/em\u003e gene (17\u0026ndash;19). The function of these two DMRs is currently unknown. In TS14 patients, the paternally methylated \u003cem\u003eMEG3/DLK1\u003c/em\u003e:IG-DMR and \u003cem\u003eMEG3\u003c/em\u003e:TSS-DMR are hypomethylated leading to upregulation of \u003cem\u003eMEG3\u003c/em\u003e and \u003cem\u003eMEG8\u003c/em\u003e and downregulation of \u003cem\u003eDLK1\u003c/em\u003e. In these cases, the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR, normally methylated on the maternal chromosome, gains methylation probably as consequence of transcriptional readthrough derived from the activated paternal \u003cem\u003eMEG3\u003c/em\u003e promoter (17). The overlapping phenotypic features between SRS and TS14 can be attributed to the involvement of shared biological pathways and/or gene co-regulation (1). Several trans-molecular interactions between imprinted genes located on chr11p15 and chr14q32 have been reported. In particular, the concomitant overexpression of \u003cem\u003eMEG3\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMEG8\u003c/em\u003e leads to downregulation of \u003cem\u003eIGF2\u003c/em\u003e in cultured cells (20).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePLAG1\u003c/em\u003e, located on human chromosome 8q12, was initially identified as an oncogene associated with certain types of cancer and is the main translocation target in pleomorphic adenomas of the salivary glands (21). It belongs to the PLAG family of zinc finger transcription factors, along with PLAG-like 1 (PLAGL1), which is a tumor suppressor, and PLAG-like 2 (PLAGL2), which acts as an oncogene. \u0026nbsp;PLAG1 protein contains seven canonical C2H2 zinc finger domains (ZF) and a serine-rich COOH terminus that exhibits transactivation capacities suggesting that it may act as a transcriptional regulator. Its DNA binding site is composed of the core sequence GRGGC and a G-cluster RGGK separated by seven random nucleotides. The interaction with the core sequence is mediated by fingers 6 and 7, and that with the G-cluster by finger 3 (22). The three PLAG proteins share the highest degree of homology in their NH₂-terminal zinc finger domain, with PLAGL1 and PLAGL2 showing 73% and 79% identity, respectively. In contrast, the COOH-terminal region is significantly more divergent. The greatest similarity is observed in zinc fingers 6 and 7, suggesting that PLAGL1 and PLAGL2 may also recognize a core motif similar to the PLAG1 core (GRGGC). Zinc fingers 2\u0026ndash;5 are less conserved, but key amino acids at critical positions remain preserved (21,22). PLAGL1 dimerization through zinc finger 2 enhances transactivation of target genes (23).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePLAG1\u003c/em\u003e plays a critical role in the oncogenic \u003cem\u003eHMGA2\u0026ndash;PLAG1\u0026ndash;IGF2\u003c/em\u003e pathway as it has been shown to be a transcriptional activator of \u003cem\u003eIGF2\u003c/em\u003e that binds its P3 promoter (22). Genetic defects of this pathway can lead to fetal and postnatal growth restriction (10). Inherited or \u003cem\u003ede novo\u003c/em\u003e alterations involving the \u003cem\u003ePLAG1\u003c/em\u003e gene, including whole-gene deletions and intragenic pathogenic variants, were demonstrated in several cases with phenotypic features of SRS (10,11,24\u0026ndash;33). It has been proposed that \u003cem\u003ePLAG1\u003c/em\u003e haploinsufficiency leads to \u003cem\u003eIGF2\u003c/em\u003e repression and growth deficiency (10). However, if further genes have a role in mediating the effect of \u003cem\u003ePLAG1\u003c/em\u003e on somatic growth is unknown.\u003c/p\u003e\n\u003cp\u003eTo look for a possible dysregulation of imprinted genes linked to \u003cem\u003ePLAG1\u003c/em\u003e defects, we analysed the DNA methylation of 11-12 imprinted regions in 17\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecases with clinical SRS features carrying heterozygous \u003cem\u003ePLAG1\u003c/em\u003e variants. We observed a specific loss of methylation of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR in all the cases predicted to generate stable aberrant PLAG1 proteins, indicating a functional interaction between \u003cem\u003ePLAG1\u003c/em\u003e and the chr14q32 imprinting cluster.\u0026nbsp;\u003c/p\u003e"},{"header":"Patients","content":"\u003cp\u003eThe study cohort included 17 patients with clinical diagnosis of SRS based on the Netchine-Harbison clinical scoring system (Additional file 1: Table S1). In these patients IC1 LoM and upd(7)mat as well as molecular TS14 were excluded. As detailed in Table 1, 8 cases were reported in previous publications. All the cases carried a variant involving the \u003cem\u003ePLAG1\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Summary of the molecular features of the probands and their families\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"756\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCases\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProband sex\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePLAG1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;variant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathogenicity evaluation*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMEG8\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:Int2-DMR methylation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePublication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.2:\u003c/p\u003e\n \u003cp\u003ec.1363del;\u003c/p\u003e\n \u003cp\u003ep.Gln455Serfs*16\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003eframeshift mutation-premature stop\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: wt\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eAbi Habib W. \u003cem\u003eet al.,\u003c/em\u003e 2018\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.2:\u003c/p\u003e\n \u003cp\u003ec.439del;\u003c/p\u003e\n \u003cp\u003ep.Ser147Valfs*82\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003cbr\u003e\u0026nbsp;frameshift mutation-premature stop\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003eProband\u0026rsquo;s sister: het\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003eProband\u0026rsquo;s sister: hypomethylation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eAbi Habib W. \u003cem\u003eet al.,\u003c/em\u003e 2018\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.2:\u003c/p\u003e\n \u003cp\u003ec.599dup;\u003c/p\u003e\n \u003cp\u003ep.Arg201Profs*52\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003eframeshift mutation-premature stop\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: wt\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: normal methylation\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eMeyer R., \u003cem\u003eet al.\u003c/em\u003e, 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003echr8q12.1 deletion including the \u003cem\u003ePLAG1\u003c/em\u003e gene\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003earr [\u003cstrong\u003eGRCh37\u003c/strong\u003e] 8q12.1\u003c/p\u003e\n \u003cp\u003e(56,834,331_\u0026nbsp;58,921,491) \u0026times; 1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: wt\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eFern\u0026aacute;ndez-Fructuoso JR. \u003cem\u003eet al\u003c/em\u003e., 2021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003echr8q12.1 deletion including the \u003cem\u003ePLAG1\u003c/em\u003e gene\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003earr [\u003cstrong\u003eGRCh37\u003c/strong\u003e] 8q12.1\u003c/p\u003e\n \u003cp\u003e(57,079,399_57,155,945) \u0026times; 1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: wt\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eBaba N., \u003cem\u003eet al\u003c/em\u003e., 2022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS6\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3\u0026nbsp;:\u003c/p\u003e\n \u003cp\u003ec.545A\u0026thinsp;\u0026gt;\u0026thinsp;T;\u003c/p\u003e\n \u003cp\u003ep.Glu182Val\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003cbr\u003e\u0026nbsp;missense mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eVUS\u003c/p\u003e\n \u003cp\u003eLow Pathogenic Support\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eKessler L., \u003cem\u003eet al\u003c/em\u003e., 2024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.610_612del;\u003c/p\u003e\n \u003cp\u003ep.(Met204del)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003ein-frame deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely pathogenic\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: normal methylation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eVimercati A., \u003cem\u003eet al.\u003c/em\u003e, 2025\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.671G \u0026gt; A;\u003c/p\u003e\n \u003cp\u003ep.(Arg224Gln)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003emissense mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely pathogenic\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: normal methylation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eVimercati A., \u003cem\u003eet al.\u003c/em\u003e, 2025\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.1023T\u0026gt;A;\u003c/p\u003e\n \u003cp\u003ep.Tyr341* \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003enonsense mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS10\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.666del;\u003c/p\u003e\n \u003cp\u003ep.Phe222Leufs*7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003eframeshift mutation-premature stop\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS11\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.527C\u0026gt;A;\u003c/p\u003e\n \u003cp\u003ep.Ser176*\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003enonsense mutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: wt\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS12\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3\u0026nbsp;:\u003c/p\u003e\n \u003cp\u003ec.1455_1502del\u0026nbsp;;\u003c/p\u003e\n \u003cp\u003ep.Ser485delinsArgAspSerGlyThrTrpIleHisTyrArgAsnValCysValAlaValPro*\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003estop-loss mutation;\u003c/p\u003e\n \u003cp\u003eChange of the C-ter end of the peptide, elongated protein (+1 aa).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband\u0026nbsp;: het\u003c/p\u003e\n \u003cp\u003eMother\u0026nbsp;:het\u003c/p\u003e\n \u003cp\u003eFather\u0026nbsp;:wt\u003c/p\u003e\n \u003cp\u003eProband\u0026rsquo;s brother\u0026nbsp;: het\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: normal methylation\u003c/p\u003e\n \u003cp\u003eProband\u0026rsquo;s brother: hypomethylation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS13\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.779_780del;\u003c/p\u003e\n \u003cp\u003ep.Val260Alafs*16\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003eframeshift mutation-premature stop\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: normal methylation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS14\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.770del;\u003cbr\u003e\u0026nbsp;p.Asn257Metfs*6\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003enovel\u003c/p\u003e\n \u003cp\u003eframeshift mutation-premature stop\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: hypomethylation\u003c/p\u003e\n \u003cp\u003eMother: hypomethylation\u003c/p\u003e\n \u003cp\u003eFather: normal methylation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS15\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.-117-5del\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ep.?\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ers1023307529\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAF: 0.0005235\u003c/p\u003e\n \u003cp\u003enon-coding;\u003cbr\u003e\u0026nbsp;(intron-exon boundary)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eVUS \u0026gt;\u0026gt; cl2\u003cbr\u003eUncertain pathogenicity\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband: het\u003c/p\u003e\n \u003cp\u003eMother: het\u003c/p\u003e\n \u003cp\u003eFather: wt\u003c/p\u003e\n \u003cp\u003eMaternal grandmother: het\u003c/p\u003e\n \u003cp\u003eMaternal grandfather: wt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003ePorband: normal methylation\u003c/p\u003e\n \u003cp\u003eMother: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003eMaternal grandmother: NA\u003c/p\u003e\n \u003cp\u003eMaternal grandfather: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS16\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003eNM_002655.3:\u003c/p\u003e\n \u003cp\u003ec.1162A\u0026gt;G;\u003c/p\u003e\n \u003cp\u003ep.Ile388Val\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ers765459935\u003c/p\u003e\n \u003cp\u003eAF:\u0026nbsp;0.00001593\u003c/p\u003e\n \u003cp\u003emissense mutation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003eVUS \u0026gt;\u0026gt; cl2\u003c/p\u003e\n \u003cp\u003eUncertain pathogenicity\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband:het\u003c/p\u003e\n \u003cp\u003eMother:het\u003c/p\u003e\n \u003cp\u003eFather:NA\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: normal methylation\u003c/p\u003e\n \u003cp\u003eMother: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 7.53968%;\"\u003e\n \u003cp\u003eSRS17\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.73016%;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.7831%;\"\u003e\n \u003cp\u003echr8q12.1 deletion including the \u003cem\u003ePLAG1\u003c/em\u003e gene\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003earr[\u003cstrong\u003eGRCh37\u003c/strong\u003e]8q12.1\u003cbr\u003e\u0026nbsp;(56,986,129_57,169,684)x1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2434%;\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.9471%;\"\u003e\n \u003cp\u003eProband:het\u003c/p\u003e\n \u003cp\u003eMother:wt\u003c/p\u003e\n \u003cp\u003eFather:wt\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22.4868%;\"\u003e\n \u003cp\u003eProband: normal methylation\u003c/p\u003e\n \u003cp\u003eFather: NA\u003c/p\u003e\n \u003cp\u003eMother: NA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.2698%;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e* Pathogenicity assessment based on ACMG criteria. In third column AF: Allele frequency in European population. In fifth column: het= heterozygous; wt= wild type. In both fifth and sixth column: NA = not analysed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA extraction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA of patients and their relatives was isolated from peripheral blood leukocyte (PBL) by standard procedures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNGS analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor SRS1-SRS3,SRS6-SRS8\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand their relatives, sequencing analysis has been reported previously (10,25,32,33). For SRS9-SRS11, SRS15 and SRS16 families, library preparation was performed using a custom sequencing panel designed by Sophia Genetics. Libraries were sequenced on a MiSeq (Illumina, San Diego, CA, USA). For SRS12-SRS14 families, library preparation, DNA enrichment and sequencing were performed as described in Torella et al. (34). The WES data preprocessing followed the nf-core Sarek pipeline (version 3.3.2) using its default settings (35). The sequencing reads were mapped to the human reference genome GRCh38 using the BWA-MEM algorithm. The resulting variant call format (VCFs) files were then annotated with Franklin by Genoox.\u0026nbsp;Validation and segregation analysis of variants in the family members were performed by Sanger sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular karyotyping\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular karyotyping for SRS4 and SRS5 was previously reported (27,28). For SRS17, CGH array analysis was carried out by using a 180k microarray (Agilent Technologies, Santa Clara, CA, USA). Raw data were analysed by the CytoGenomics V3.0 software (Agilent Technologies).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico prediction of variant pathogenicity and protein stability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVariants were initially classified based on their predicted impact on the protein. In silico pathogenicity prediction was performed for all SNVs and indels using CADD v1.7 (36). For missense variants, additional analyses were conducted using PolyPhen-2 (37), SIFT (38), AlphaMissense (39) and REVEL (40). Furthermore, all variants, including the three large deletions, were classified according to the ACMG standards and guidelines (41,42). Protein stability was investigated using DEGRONOPEDIA (43), which enables the identification of known degron motifs involved in protein degradation pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMethylation analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethylation-specific multiple ligation-dependent probe amplification (MS-MLPA) targeting 11 or 12 imprinted loci was performed using the SALSA MS-MLPA Kit (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer\u0026rsquo;s instructions. Probemix ME034-C1 or Probemix ME034-D1, designed for multi-locus imprinting analysis, were used.\u0026nbsp;Raw data were analysed using Coffalyser.Net software (MRC Holland, Amsterdam, The Netherlands). Pyrosequencing analysis was carried out as described in Sparago et al. (44) and it was conducted in separate batches of experiments. The 13 analysed CpGs are included in the following coordinates: chr14:100,904,601-100,904,702 (GRCh38/hg38). ImprintCap, a NGS-based methodology for large-scale methylation studies at several imprinted loci was performed as described in Brioude et al. (45) on 13 control samples to explore the methylation of \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR. The 42 analysed CpGs are included in the following coordinates: chr14: 100,904,423- 100,905,080 (GRCh38/hg38).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA methylation analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the DNA methylation of 11-12 imprinted regions in 17 SRS cases carrying rare \u0026nbsp;\u003cem\u003ePLAG1\u003c/em\u003e variants in heterozygosity (Table 1) and 54 control individuals, by using the multi-locus MS-MLPA C1 or D1 kit. Compared to the mean of the controls, we found a partial loss of methylation of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR in the cases SRS1,SRS2,SRS5 and SRS7-SRS14. Most of these cases were familial and the imprinting defect segregated within the family, being detected in all the members who carried the \u003cem\u003ePLAG1\u003c/em\u003e variant (Fig. 2). Conversely, \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR methylation was normal in SRS3, SRS4, SRS6, and SRS15-SRS17. The MS-MLPA results are reported in the Additional file 1: Table S2. In all cases, the methylation levels of the other tested DMRs, including the \u003cem\u003eMEG3\u003c/em\u003e:TSS-DMR, were comparable to those of the controls (Fig. 2). \u0026nbsp;In addition, we evaluated the methylation status of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR in 7 SRS patients carrying \u003cem\u003eHMGA2\u0026nbsp;\u003c/em\u003evariants and 8 with \u003cem\u003eIGF2\u003c/em\u003e variants and all of them showed normal methylation (Additional file 1: Table S3).\u003c/p\u003e\n\u003cp\u003eTo further explore \u003cem\u003eMEG8\u003c/em\u003e methylation in other molecular subgroups of SRS, we performed multi-locus MS-MLPA in 28 patients with IC1 loss of methylation (IC1-LoM), 4 with maternal uniparental disomy of chromosome 7, and 31 idiopathic cases (NH-CSS \u0026ge; 4). All of these also exhibited normal methylation levels (Additional file 1: Table S4). Finally, 2/54\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eof the\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehealthy control individuals showed lower methylation levels at the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR, according to the MS-MLPA results (Additional file 1: Table S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the MS-MLPA results, we analysed \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR methylation through the more quantitative pyrosequencing assay in the patients carrying the \u003cem\u003ePLAG1\u003c/em\u003e variants and in several controls. With this assay, we tested the methylation level of 13 CpGs across the \u003cem\u003e\u0026nbsp;MEG8\u003c/em\u003e:Int2-DMR. The results were consistent with the data obtained by MS-MLPA in 10/11 patients and confirmed the co-segregation of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation with the \u003cem\u003ePLAG1\u003c/em\u003e variants (Fig. 3A-G).\u003c/p\u003e\n\u003cp\u003eFurthermore, the pyrosequencing results demonstrated that the hypomethylation affected the entire \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR. The reduction in methylation levels was between 15% and 33% compared to the mean of the controls (Additional file 1: Table S6; Additional file 2: Fig. S1). Patient SRS5 and the two healthy individuals found hypomethylated with MS-MLPA showed a more modest (3-6%) methylation defect restricted to only a few CpGs located at the 5\u0026rsquo; part of the DMR (Fig. 3H; Additional file 1: Table S7; Additional file 3: Fig. S2). From ImprintCap analysis on 13 control samples, those latter 5\u0026rsquo;-CpGs were shown to be highly variable, making those 5\u0026rsquo;-CpGs not suitable to define gain or loss-of-methylation in patients (Additional file 1: Table S8; Additional file 4: Fig. S3).\u003c/p\u003e\n\u003cp\u003eIn summary, these results indicate a specific loss of methylation of the entire \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR in 10 out of 17 SRS cases carrying a genetic variant in \u003cem\u003ePLAG1.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGenetic alterations in PLAG1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the \u003cem\u003eMEG8\u003c/em\u003e methylation defect was not consistently present in all the 17 SRS cases with \u003cem\u003ePLAG1\u003c/em\u003e variants, we assessed the pathogenic potential of the identified genetic variants by in silico analysis (Fig. 4; Table 1). The clinical features and \u0026nbsp;\u003cem\u003ePLAG1\u003c/em\u003e variants of 8 patients (SRS1-SRS8) have been described in previous studies (10,25,27,28,32,33), while the remaining nine are novel cases.\u003c/p\u003e\n\u003cp\u003eThe patients SRS1-SRS3 and SRS7-SRS14 (Table 1) carried heterozygous single nucleotide variants (SNVs) or indels of \u003cem\u003ePLAG1\u003c/em\u003e that were classified as pathogenic or likely pathogenic according to the ACMG guidelines and gene variant interpretation (Additional file 1: Table S9). In particular, \u0026nbsp;two nonsense variants were identified in cases SRS9 and SRS11. The variant \u0026nbsp;p.Tyr341* resulting in loss of the C-terminal activation domain was found in both the SRS9 proband and her mother , while the variant p.Ser176* causes the loss of ZF6, ZF7 and the entire C-terminal region of \u003cem\u003ePLAG1\u003c/em\u003e in patient SRS11. Frameshift variants were identified in the cases SRS1-SRS3 (10,25), SRS10, SRS13 and SRS14.\u0026nbsp;These pathogenic variants lead to putative truncated proteins lacking key functional domains (Additional file 5: Fig. S4). In particular, the variant p.Phe222Leufs*7 carried by patient SRS10 causes a frameshift starting from ZF7 and likely resulting in a truncated peptide composed of 227 amino acid residues (221 wild-type and 6 mutated), while the p.Val260Alafs*16 and p.Asn257Metfs*6 variants, inherited maternally by the SRS13 and SRS14 patients, respectively, \u0026nbsp;lead to loss of the entire C-terminal domain. A novel 48 bp deletion was identified in the SRS12 proband, as\u0026nbsp;well as in her mother and brother, but not in her father. This mutation likely results in a protein of 501 amino acids, with 484 wild-type, 16 mutated and one additional residue at the C-terminus (p.Ser485delinsArgAspSerGlyThrTrpIleHisTyrArgAsnValCysValAlaValPro*). Finally, SRS7 and SRS8 carried an in-frame deletion and a missense mutation in the ZF6 and ZF7\u0026nbsp;sequences, respectively\u0026nbsp;(33).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the stability of the 11 predicted aberrant proteins resulting from deleterious \u003cem\u003ePLAG1\u0026nbsp;\u003c/em\u003emutations, we analysed the presence of known degron motifs involved in degradation pathways (43). Notably, a RxxGxx motif was identified at the C-terminus of the PLAG1 mutant p.Arg201Profs*52 (SRS3). APPBP2, a substrate receptor of CRL2 (Cullin-RING E3 ubiquitin ligase 2) complexes, is known to recognize C-degrons defined by a distinctive C-terminal Arg-x-x-Gly sequence (46). This specific degron motif was absent in the other mutated proteins that were subjected to analysis, suggesting a potential importance of the R-x-x-G motif in substrate recognition and subsequent ubiquitination processes within CRL2-mediated proteolysis for the PLAG1 mutant p.Arg201Profs*52 (Additional file 5: Fig. S4).\u003c/p\u003e\n\u003cp\u003eAccording to the results of the in-silico analysis and the ACMG criteria, the patients SRS6 (32), SRS15 and SRS16 carried Variants of Uncertain Significance (VUS) in the \u003cem\u003ePLAG1\u003c/em\u003e gene (Table 1, Additional file 1: Table S9). In particular, \u0026nbsp;an intronic variant (NM_002655.3:c.-117-5del;p.?) occurring in a homopolymeric region of \u003cem\u003ePLAG1\u003c/em\u003e intron 3 was found in\u0026nbsp;SRS15,\u0026nbsp;her mother and her healthy maternal grandmother, while the missense variants \u0026nbsp;p.Ile388Val and p.Glu182Val were identified in cases SRS6 and SRS16, respectively. In particular, the p.Ile388Val variant involved a non-conserved residue and, in both SRS6 and SRS16\u0026nbsp;cases, the functional \u003cem\u003ePLAG1\u003c/em\u003e domains were not affected. Thus, their pathogenicity remains uncertain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, patients SRS4 (27), SRS5 (28), and SRS17 carried heterozygous deletions on chromosome 8q (Table 1; Additional file 6: Fig. S5). Among them, patient SRS5 exhibited the smallest deletion, affecting only \u003cem\u003ePLAG1\u003c/em\u003e and \u003cem\u003eCHCHD7\u0026nbsp;\u003c/em\u003egenes. In contrast, SRS4 presented with a larger \u003cem\u003ede novo\u003c/em\u003e deletion of approximately 2.1 Mb at 8q12, encompassing 32 genes, including 9 OMIM-annotated genes. In SRS17, molecular karyotyping identified a ~183 kb \u003cem\u003ede novo\u003c/em\u003e deletion (arr[GRCh37]8q12.1(56,986,129_57,169,684)x1) involving \u003cem\u003ePLAG1\u003c/em\u003e but also \u003cem\u003eCHCHD7, MOS\u003c/em\u003e, and \u003cem\u003eSDR16C5\u003c/em\u003e. In all three cases, \u003cem\u003ePLAG1\u003c/em\u003e was proposed as the primary candidate gene responsible for the observed SRS phenotype (27,28). According to the ACMG criteria, all three deletions are classified as pathogenic.\u003c/p\u003e\n\u003cp\u003eOverall, we collected a wide spectrum of \u003cem\u003ePLAG1\u003c/em\u003e variants: two nonsense variants, six frameshift, one stop-loss, one in-frame deletion, three missense variants, one intronic variant, and three large deletions affecting the entire \u003cem\u003ePLAG1\u003c/em\u003e gene on chromosome 8q. Evaluation of their potential pathogenicity led us to observe that 11 out of 14 SNVs/indels were predicted to be damaging, whereas the remaining 3 were classified as VUS. Among these 11 damaging variants, one harbours a degron motif likely associated with protein degradation. The copy number variants were also classified as pathogenic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorrelation of MEG8 methylation with PLAG1 gene variants\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the relationship between \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR methylation and PLAG1 function, we compared the methylation of this locus among the 17 SRS patients carrying different \u003cem\u003ePLAG1\u003c/em\u003e variants (Table 1). Interestingly, loss of \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR methylation was detected in 10 out of 11 cases carrying pathogenic or likely pathogenic single nucleotide variants or indels, with SRS3 (p.Arg201Profs*52) representing the only exception. In particular, this mutated protein uniquely harbours the C-terminal RxxGxx motif, which facilitates protein degradation (46,47), suggesting a distinct impact on protein stability compared with the other pathogenic mutations. \u0026nbsp;Conversely, all the 6 cases carrying VUS or large deletions encompassing the \u003cem\u003ePLAG1\u003c/em\u003e gene showed normal methylation levels. Notably, \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation was not detected in any of the SRS patients with molecular alterations in other genes of the \u003cem\u003eHMGA2\u0026ndash;PLAG1\u0026ndash;IGF2\u003c/em\u003e pathway, in those with IC1-LoM, upd(7)mat and in idiopathic SRS cases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, \u003cem\u003eMEG8\u003c/em\u003e hypomethylation is associated with \u003cem\u003ePLAG1\u003c/em\u003e variants that are predicted to produce stable abnormal proteins, but not with whole \u003cem\u003ePLAG1\u003c/em\u003e gene deletions, variants of uncertain pathogenicity or variants leading to protein degradation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe expression of many imprinted genes impacts pre- and postnatal growth\u0026nbsp;(1). The etiology of SRS has been primarily linked to the imprinted gene clusters located on chr11p15 and chr7, but pathogenic variants of the \u003cem\u003eHMGA2, IGF2\u003c/em\u003e and\u003cem\u003e\u0026nbsp;PLAG1\u003c/em\u003e genes were also found in individuals with SRS features (10). Both \u003cem\u003eHMGA2\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;PLAG1\u003c/em\u003e were proposed to control the expression of the \u003cem\u003eIGF2\u003c/em\u003e gene, but their possible interaction with other imprinted genes was not explored. Here, we demonstrate that damaging \u003cem\u003ePLAG1\u003c/em\u003e variants are associated with \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation, suggesting that \u003cem\u003ePLAG1\u003c/em\u003e may affect \u003cem\u003eMEG8\u003c/em\u003e methylation and thereby expression of chr14q32 genes controlling somatic growth.\u003c/p\u003e\n\u003cp\u003eIn the present study, \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation was demonstrated in cases with pathogenic or likely pathogenic\u003cem\u003e\u0026nbsp;PLAG1\u003c/em\u003e variants that are predicted to produce abnormal proteins. In contrast, normal \u003cem\u003eMEG8\u003c/em\u003e methylation was observed in the cases with missense/intronic variants classified as VUS according to the ACMG guidelines and \u003cem\u003ePLAG1\u003c/em\u003e deletions. In addition, most of the pathogenic \u003cem\u003ePLAG1\u003c/em\u003e cases are familial and both epigenetic and genetic defects co-segregated within the families, further supporting the association between damaging \u003cem\u003ePLAG1\u003c/em\u003e variants and \u003cem\u003eMEG8\u003c/em\u003e hypomethylation. This epigenetic defect appears to be specific for \u003cem\u003ePLAG1\u003c/em\u003e, because it was not found in the cases with \u003cem\u003eHMGA2\u003c/em\u003e and \u003cem\u003eIGF2\u003c/em\u003e variants, patients with IC1-LoM, upd(7)mat cases and idiopathic cases.\u003c/p\u003e\n\u003cp\u003eApart from \u003cem\u003eMEG8\u003c/em\u003e, the methylation of other 10-11 imprinted loci was found to be normal in the patients studied, suggesting a specific interaction between PLAG1 and \u003cem\u003eMEG8\u003c/em\u003e-DMR. Analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data indicates that PLAG1 normally interacts with the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR, although it is unclear if its binding is influenced by DNA methylation (48).\u0026nbsp; \u0026nbsp;Interestingly, PLAGL2, a homologue of PLAG1, is also predicted to bind the same DMR, as suggested by data from JASPAR CORE 2024 - Predicted Transcription Factor Binding Sites\u0026nbsp;(49). Particularly, PLAGL2 shares 79% identity with PLAG1 in its NH₂-terminal zinc finger domain, suggesting that the zinc finger domain may be involved in recognizing the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR. In humans, ENCODE ChIP-seq data revealed a conserved CTCF binding site within the \u003cem\u003eMEG8\u003c/em\u003e-DMR\u0026nbsp;(19). In mice, however, CTCF binds to the \u003cem\u003eMeg8\u003c/em\u003e-DMR in a non-allele-specific manner \u003cem\u003ein vivo\u003c/em\u003e (50). CTCF (CCCTC-binding factor) is a well-characterized vertebrate protein with eleven zinc fingers, the first ten of which are C2H2-type, similar to those found in PLAG1, while the last one is of \u0026nbsp;C2HC-type\u0026nbsp;(51). Notably, PLAG1 and CTCF recognize similar consensus sequences, as both bind to G-rich regions.\u003c/p\u003e\n\u003cp\u003eIn the present SRS patients, the truncating \u003cem\u003ePLAG1\u0026nbsp;\u003c/em\u003evariants map in the last two exons of the gene and should therefore escape the nonsense-mediated decay (NMD) pathway (52). Also, they likely lead to aberrant PLAG1 proteins, which could influence methylation at the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR because of reduced DNA binding. Indeed, because zinc finger 2 is retained in all these variants, they may dimerize similar to PLAGL1 and exert a dominant-negative effect on the wildtype allele (23). Consistent with this hypothesis, the single nucleotide deletion (SRS7) and the missense variant (SRS8) in the zinc fingers 6 and 7, that are responsible for specific DNA motif recognition (33), result in \u003cem\u003eMEG8\u003c/em\u003e hypomethylation. In contrast, the SRS3 variant that contains a C-terminal degron motif and likely results in an unstable aberrant protein is associated with normal \u003cem\u003eMEG8\u003c/em\u003e methylation (46,47). Also, the variants of uncertain pathogenicity are unlikely to disrupt the DNA binding function of PLAG1 and thereby maintain normal \u003cem\u003eMEG8\u003c/em\u003e methylation. Nevertheless, the mechanism by which reduced PLAG1 binding may interfere with \u003cem\u003eMEG8\u0026nbsp;\u003c/em\u003emethylation remains to be defined. The absence of methylation defects in the cases with whole gene deletion is consistent with the dominant-negative hypothesis (53).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn SRS5 and the two healthy individuals who scored hypomethylated at the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR according to the MS-MLPA results, further analysis by pyrosequencing showed that this methylation defect was less severe compared to that detected in all the other patients and restricted to a few CpGs located at the 5\u0026rsquo; part of the DMR, which was shown to be highly variable in a control population. \u0026nbsp;This finding suggests that while MS-MLPA may indicate \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation, it does not always reflect the full methylation pattern, and further confirmation using more sensitive and extensive techniques like pyrosequencing or NGS-based technologies like ImprintCap is essential for its accurate assessment.\u003c/p\u003e\n\u003cp\u003eThe observed \u003cem\u003eMEG8\u003c/em\u003e hypomethylation in the present study suggests that chr14q32 genes have a role in PLAG1-dependent pathogenesis of SRS. In the TS14 cases with \u003cem\u003eMEG3\u003c/em\u003e:TSS-DMR hypomethylation, the \u003cem\u003eMEG3\u003c/em\u003e long noncoding RNA is activated and transcription through the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR leads to its hypermethylation (17,54). However, in the cases with \u003cem\u003ePLAG1\u003c/em\u003e mutations, \u003cem\u003eMEG3\u003c/em\u003e methylation is unaffected, suggesting that alternative molecular mechanisms lead to \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation. In a recent study, Baena et al. (19) described a family in which \u003cem\u003eMEG8\u003c/em\u003e but not \u003cem\u003eMEG3\u003c/em\u003e exhibits a methylation pattern dependent on the parental origin of a \u003cem\u003eDLK1\u003c/em\u003e deletion, suggesting an interaction between these two loci. Also, a conserved CTCF binding site is present and therefore an insulator may be formed within the \u003cem\u003eMEG8\u003c/em\u003e-DMR (19). The \u003cem\u003eMEG8\u003c/em\u003e DMR overlaps the \u003cem\u003eMEG8\u003c/em\u003e lncRNA and \u0026nbsp;two clusters of miRNAs and snoRNAs, all of which are deregulated in cancer (55,56). It is possible that the disrupted PLAG1 binding to \u003cem\u003eMEG8\u003c/em\u003e interferes with expression of one or more of these genes. Finally, the normal methylation in the cases with \u003cem\u003eHMGA2\u003c/em\u003e and \u003cem\u003eIGF2\u003c/em\u003e variants suggests that \u003cem\u003eMEG8\u003c/em\u003e works as \u003cem\u003ePLAG1\u003c/em\u003e target upstream of \u003cem\u003eHMGA2\u003c/em\u003e and \u003cem\u003eIGF2\u0026nbsp;\u003c/em\u003ein the \u003cem\u003ePLAG1\u0026ndash;HMGA2-IGF2\u003c/em\u003e pathway.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDiagnostic methylation changes have been recently identified in cohorts of individuals affected by developmental disorders and carrying pathogenic variants in epigenetic modifiers (57). The present study identifies \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR hypomethylation as a specific epigenetic signature of damaging \u003cem\u003ePLAG1\u0026nbsp;\u003c/em\u003evariants likely causing dominant-negative effect in patients with clinical SRS features. These findings support a novel pathogenetic mechanism whereby impaired PLAG1 function alters the methylation and possibly the expression of imprinted genes within the chr14q32 region. Furthermore, \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR methylation studies might be useful in the future as a functional test for \u003cem\u003ePLAG1\u003c/em\u003e VUS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eDMRs\u0026nbsp;\u003cbr\u003e\u003c/strong\u003eDifferentially Methylated Regions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpDis\u003c/strong\u003e\u003cbr\u003eImprining Disorders\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLoM\u003c/strong\u003e\u003cbr\u003eLoss of methylation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLID\u003c/strong\u003e\u003cbr\u003eMulti-locus imprinting disturbances\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNH-CSS\u003c/strong\u003e\u003cbr\u003eNetchine-Harbison clinical scoring system\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMD\u003c/strong\u003e\u003cbr\u003eNonsense-mediated decay\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBL\u003c/strong\u003e\u003cbr\u003ePeripheral blood leukocytes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSRS\u003c/strong\u003e\u003cbr\u003eSilver-Russell syndrome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSRSp\u003c/strong\u003e\u003cbr\u003eSilver-Russell syndrome spectrum\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSNVs\u003c/strong\u003e\u003cbr\u003eSingle nucleotide variants\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTS\u003c/strong\u003e\u003cbr\u003eTemple syndrome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUPD\u003c/strong\u003e\u003cbr\u003eUniparental disomy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVUS\u003c/strong\u003e\u003cbr\u003eVariant of uncertain significance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZF\u003c/strong\u003e\u003cbr\u003eZinc Finger\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethical committees of the University of Campania Luigi Vanvitelli, Istituto Auxologico Italiano, H\u0026ocirc;pital Trousseau (Paris), Center for Human Genetics and Genome Medicine (Aachen), Hospital General Universitario de Santa Luc\u0026iacute;a.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe families agreed for publication by signing an informed consent template.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Associazione Italiana Ricerca sul Cancro (AIRC; IG 2020 ID 24405) and Fondazione Telethon (GMR23T1062) awarded to AR and Italian Ministry of University and Research PRIN 2022B2N2BY awarded to AR and MVC. TE is supported by the Deutsche Forschungsgemeinschaft (EG 115/13-1). \u0026nbsp;ImprintCap experiments were funded with the French Agence Nationale pour la Recherche (ANR) grant no. ANR-22-CE14-0021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, FB, AR, FlCe; investigation, EDA ,LP ,FrCe ,AV ,MVC, AS, CG, NT, SR, TE,IN, JRF; writing\u0026mdash;draft preparation EDA, FlCe, AR, FB; supervision, FB, AR. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank the patients and their families for their valuable participation in this research project. We also thank Sophie Rondeau (Paris), Genevieve Baujat (Paris), Sylvie Rossignol (Strasbourg), Salima El Chehadeh (Strasbourg), Cecile Teinturier (Le Kremlin Bic\u0026ecirc;tre), Eloise Giabicani (Paris), Jean-Luc Alessandri (Saint Denis La R\u0026eacute;union), Albane Simon (Versailles), Marie-Aliette Dommergues (Versailles), Madeleine Harbison (New York), Godelieve Morel \u0026nbsp; (Saint Denis La R\u0026eacute;union), Bruno Hay Mele (Italy).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEggermann T, Monk D, De Nanclares GP, Kagami M, Giabicani E, Riccio A, et al. Imprinting disorders. Nat Rev Dis Primer. 2023 Jun 29;9(1):33. \u003c/li\u003e\n\u003cli\u003eMonk D, Mackay DJG, Eggermann T, Maher ER, Riccio A. Genomic imprinting disorders: lessons on how genome, epigenome and environment interact. 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Genet Med. 2024 May;26(5):101075. \u003c/li\u003e\n\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":"[email protected]","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":"Silver-Russell syndrome, Growth retardation, Genomic imprinting, DNA methylation","lastPublishedDoi":"10.21203/rs.3.rs-7693802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7693802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous imprinting disorder. The most common molecular defects are loss of methylation of the \u003cem\u003eH19/IGF2\u003c/em\u003e:IG-DMR on chromosome 11p15.5, followed by maternal uniparental disomy of chromosome 7. Further molecular lesions are genetic variants in the \u003cem\u003ePLAG1 \u003c/em\u003eoncogene, as well as in the transcription factor \u003cem\u003eHMGA2 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003ethe fetal growth factor \u003cem\u003eIGF2\u003c/em\u003e. A phenotypic overlap exists between SRS and Temple syndrome (TS14) that is also characterized by growth restriction but associated with abnormalities in the imprinted chromosome 14q32 gene cluster. In TS14 patients, the germline \u003cem\u003eMEG3/DLK1\u003c/em\u003e: IG-DMR is hypomethylated and the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR gains methylation probably as consequence of transcriptional readthrough from the \u003cem\u003eMEG3\u003c/em\u003e promoter on the paternal chromosome. However, the functional role of the \u003cem\u003eMEG8\u003c/em\u003e DMR remains unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e We analysed the DNA methylation of 11-12 imprinted regions in 17\u003cstrong\u003e \u003c/strong\u003ecases with clinical SRS features and heterozygous for a \u003cem\u003ePLAG1\u003c/em\u003e variant. We observed a specific loss of methylation of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR associated with pathogenic \u003cem\u003ePLAG1 \u003c/em\u003evariants that result in aberrant proteins. Normal \u003cem\u003eMEG8 \u003c/em\u003emethylation was observed in the cases carrying variants of uncertain pathogenicity or gene deletions. Most of the \u003cem\u003ePLAG1\u003c/em\u003e cases are familial and both epigenetic and genetic defects co-segregated within the families. Additionally, we assessed the methylation status of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR in several SRS patients with \u003cem\u003eHMGA2 \u003c/em\u003eor\u003cem\u003e IGF2\u003c/em\u003e variants, \u003cem\u003eH19/IGF2\u003c/em\u003e:IG-DMR-LoM and upd(7)mat and all of them showed normal methylation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eOur results indicate that pathogenic \u003cem\u003ePLAG1\u003c/em\u003evariants leading to stable aberrant PLAG1 proteins and possibly acting in a dominant-negative manner influence methylation of the \u003cem\u003eMEG8\u003c/em\u003e locus. This study suggests a new pathogenetic mechanism of the \u003cem\u003ePLAG1\u003c/em\u003e gene in SRS, involving imprinted genes in the chr14q32 cluster through deregulation of the \u003cem\u003eMEG8\u003c/em\u003e:Int2-DMR and provides an epigenetic signature that may be used to assess the damaging potential of the \u003cem\u003ePLAG1\u003c/em\u003e variants.\u003c/p\u003e","manuscriptTitle":"Hypomethylation of the MEG8:Int2-DMR in patients with pathogenic PLAG1 variants suggests new role of the chr14q32 imprinting cluster in Silver-Russell syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 08:00:34","doi":"10.21203/rs.3.rs-7693802/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T10:05:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-04T03:45:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T09:20:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138746137347566468331475146451218769375","date":"2025-09-29T14:03:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225385277229193732211768486088426216892","date":"2025-09-25T23:18:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94586436050236740014025936856564645090","date":"2025-09-25T03:54:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T03:36:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-25T03:28:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-25T02:25:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Epigenetics","date":"2025-09-23T11:22:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"578f6fd8-1742-4c9e-8076-5d9a93eb1558","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:02:09+00:00","versionOfRecord":{"articleIdentity":"rs-7693802","link":"https://doi.org/10.1186/s13148-025-02024-6","journal":{"identity":"clinical-epigenetics","isVorOnly":false,"title":"Clinical Epigenetics"},"publishedOn":"2025-11-23 15:57:56","publishedOnDateReadable":"November 23rd, 2025"},"versionCreatedAt":"2025-10-08 08:00:34","video":"","vorDoi":"10.1186/s13148-025-02024-6","vorDoiUrl":"https://doi.org/10.1186/s13148-025-02024-6","workflowStages":[]},"version":"v1","identity":"rs-7693802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7693802","identity":"rs-7693802","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}