Investigation of Multilocus Imprinting Disturbance (MLID) in 101 Beckwith-Wiedemann Spectrum patients

Investigation of Multilocus Imprinting Disturbance (MLID) in 101 Beckwith-Wiedemann Spectrum patients | 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 Investigation of Multilocus Imprinting Disturbance (MLID) in 101 Beckwith-Wiedemann Spectrum patients Alejandro Parra, Mario Cazalla, Carlos Rodríguez-Antolín, Cristina Silván, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6555801/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Apr, 2026 Read the published version in Clinical Epigenetics → Version 1 posted 10 You are reading this latest preprint version Abstract Beckwith-Wiedemann spectrum (BWSp) is an overgrowth disorder caused by both genetic and epigenetic defects within the 11p15.5 chromosomal region. The most common cause of BWSp is DNA methylation anomalies in two imprinting control regions (ICR1, the telomeric centre that includes H19/IGF2:IG DMR and ICR2, the centromeric centre that includes KCNQ1OT1:TSS-DMR) located within the 11p15.5 locus. Previous studies demonstrated that a subset of BWSp patients had methylation defects extending beyond 11p15.5 to other chromosomal loci, an entity known as multilocus imprinting disturbances (MLID). In this study, the multilocus methylation status of 101 BWSp patients was analysed by both various methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and methylation microarrays. MS-MLPA detected MLID in 15.84% of the patients, which increased to 44.55% using methylation arrays. ICR2 hypomethylation was observed in all MLID cases, and 25 imprinted differentially methylated regions (DMRs) were additionally detected. Recurrent loci associated with the genes such as GNAS , MEST , and DIRAS3 , previously reported in MLID patients, were also observed as hypomethylated in our cohort. As eight of the 45 BWSp-MLID patients were born following assisted reproductive technology (ART), our findings highlight the increased prevalence of MLID in pregnancies conceived through ART. This study underscores the value of genome-wide methylation analyses for uncovering the molecular complexity, enhancing diagnostic accuracy, and improving prenatal care in BWSp with MLID. Future research should investigate the long-term clinical impact of MLID and the molecular mechanisms involved. Beckwith-Wiedemann spectrum methylation Multilocus Imprinting Disturbances MLID array MS-MLPA Figures Figure 1 Figure 2 INTRODUCTION Beckwith-Wiedemann spectrum (BWSp, MIM #130650) is an overgrowth disorder characterized by a highly variable spectrum of clinical features, including macroglossia, macrosomia, abdominal wall defects (such as omphalocele, diastasis recti or umbilical hernia), neonatal hypoglycaemia and predisposition to tumour development, among many others [ 1 ]. The estimated prevalence of BWSp is approximately 1 in 12,000 live births, although this rate is higher in individuals conceived through assisted reproductive techniques (ART) [ 2 ]. BWSp results from both genetic and epigenetic alterations within the 11p15.5 chromosomal region. In 2018, the term Beckwith-Wiedemann spectrum (BWSp) was suggested to cover classical Beckwith-Wiedemann syndrome (BWS) without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly [ 3 ]. The 11p15.5 locus encompasses two distinct imprinting control regions (ICRs; ICR1, the telomeric one which includes H19/IGF2:IG DMR and ICR2, the centromeric one which includes KCNQ1OT1:TSS-DMR), which consist of clusters of genes playing crucial roles in biological processes, especially during embryonic development, such as somatic growth, cell cycle regulation and proliferation [ 1 , 2 , 4 ]. The primary cause of BWSp is DNA methylation defects at these ICRs: over 50% of patients present with loss of methylation (LoM) at the maternal ICR2, 5–10% exhibit gain of methylation (GoM) at the maternal ICR1, and 20–25% shows mosaic paternal uniparental disomy (UPD) within chromosome 11p. Hypomethylation of ICR2 and hypermethylation of ICR1 led to the dysregulation of CDKN1C (Cyclin-Dependent Kinase Inhibitor 1C) and IGF2 (Insulin-Like Growth Factor II), respectively [ 5 , 6 ]. Other rarer causes of BWSp include pathogenic variants in CDKN1C (5–10%) and large genomic rearrangements involving the 11p15.5 locus (~ 2–3%) [ 7 ]. Previous studies demonstrated that some patients with BWSp had methylation abnormalities at other loci additional to the 11p15.5 region [ 8 – 10 ], known as multilocus imprinting disturbance (MLID) [ 11 ]. The additionally affected differentially methylated regions (DMRs) observed in patients with BWSp are usually associated with the imprinted PLAGL1, GNAS, GRB10, MEG3, PEG3 and MEST genes [ 8 , 9 , 12 – 16 ]. The underlying molecular mechanism of MLID is not entirely understood; however, monogenic variants in several genes, including members of the NOD-like receptor protein (NLRP) family ( NLRP2, NLRP4, NLRP5, NLRP7 ), have been identified in mothers of some patients with MLID. These genes encode proteins that are part of the subcortical maternal complex (SCMC) and are essential for early embryonic development [ 11 , 17 ]. The growing advances in methylation analysis, such as methylation microarrays and methyl-seq, provide a comprehensive approach for examining methylation status across the genome [ 18 – 20 ]. These high-throughput technologies offer the potential to uncover novel insights into the complex epigenetic landscape of patients with MLID, allowing the identification of well-known DMRs as well as potentially new regions with aberrant methylation that could influence the phenotype of patients. In this study, we applied a combination of methylation-specific multiplex ligation-dependent probe amplification (multi-locus imprinting MS-MLPA; 11 different imprinted loci within seven chromosomal regions) and genome-wide methylation microarray assays (Infinium MethylationEPIC v2.0 BeadChip, Illumina) in 101 BWSp patients, with the aim of identifying the extent of MLID and looking for additional regions with aberrant methylation. PATIENTS, MATERIALS AND METHODS Patients Patients included in this study were selected from the Spanish Overgrowth Syndromes Registry Initiative (SOGRI), which contains more than 2,500 individuals and relatives with overgrowth disorders. All selected patients (N = 101) had previously been diagnosed with BWSp based on their clinical findings and subsequent detection of abnormal methylation on the 11p15.5 chromosomal region with the SALSA MS-MLPA probemix ME030 BWS/RSS kit (MRC-Holland, Amsterdam, Netherlands). Eighty-five patients presented with hypomethylation of ICR2 (84.1%), 13 cases presented with paternal UPD (12.87%) and three presented with hypermethylation of ICR1 (2.97%) (Table S1 ). This study was approved by the Ethical Committee of the Hospital Universitario La Paz (CEIm PI-446), and informed consent was obtained from all patients and/or their parents/ legal guardians. MS-MLPA analysis The SALSA MS-MLPA probemix ME034 Multi-locus Imprinting kit (MRC-Holland, Amsterdam, Netherlands) was applied to identify samples with MLID. Data analyses were conducted following the manufacturer protocol, defining relative probe signals by dividing each measured peak area by the sum of all peak areas of the control probes for that sample. Each peak’s relative probe area ratio was then compared to a DNA control sample (Promega, UK), using Coffalyser.net (MRC- Holland, Amsterdam, Netherlands). Methylation microarrays DNA was extracted from whole blood collected from 101 BWSp patients and 41 controls. Bisulfite conversion of the DNA was performed using the EZ DNA Methylation-Lightning™ kit (Illumina, San Diego, CA). Methylation levels were assessed using the Infinium MethylationEPIC v2.0 BeadChip (Illumina, San Diego, CA) array, which covers over 930,000 CpG sites. Raw data (. idat files) were imported into R (v4.4.1), processed, and normalized using the R package minfi (v1.50.0) [ 21 ] and subjected to quality control. Probes were filtered out based on the following criteria: detection p-value > 0.01, probes located on the X and Y chromosomes, probes known to contain a single-nucleotide polymorphism (SNP), and probes known to cross-react with other genomic locations. After this filtering step, β-values and M-values, representing the methylation levels at each CpG site, were calculated. Sample labelling verification, including sex and age estimation, was conducted using the R packages wateRmelon (v2.10.0) [ 22 ] and methylclock (v1.10.0) [ 23 ]. Methylation status of the patients was first evaluated at the DMRs reported by Court et al. (2014) [ 24 ]. To assess methylation status, the mean β-value and standard deviation of controls were calculated for each probe. A standardized score was computed, and a probe was considered significantly hypermethylated if the score was ≥ + 3SD and hypomethylated if it was ≤ -3SD. Additionally, we performed a differential methylation analysis to compare methylation profiles between controls and BWSp patients, leading to the identification of differentially methylated positions (DMPs) outside the previously analysed gDMRs. To visualize the DMPs across the genome, the R package ENmix (v1.40.2) [ 25 ] was used. The top DMPs were used to generate a heatmap with the R library ComplexHeatmap (v2.20.0) [ 26 ], allowing the identification of methylation patterns across our cohort beyond the gDMRs. Statistical analysis of clinical features Statistical analysis was conducted using SPSS v.25 (IBM Corporation, United States) to assess differences in clinical features. Descriptive analyses included the mean ± SD for continuous variables (age in this cohort) and frequency tables for categorical variables. Categorical variables were expressed as binary values (1 or 0), categorized as “ever” having a given condition versus “never” having the condition. Comparisons were made between our cohort of patients with BWSp and MLID and the reported frequencies of clinical features in patients with BWSp [ 27 , 28 ]. Chi-square tests and/or Fisher’s exact test were used to evaluate differences, with z-tests applied to compare column proportions. A p-value < 0.05 was considered indicative of a statistically significant difference. RESULTS MS-MLPA . DNA of 101 BWSp patients with previously confirmed methylation defects at the 11p15.5 locus were re-investigated using the multi- locus MS-MLPA kit that revealed the presence of MLID in 16 of the patients (15.84%) (Table S2 ). As expected, all these patients identified with MLID exhibited LOM at ICR2, while none of the patients with isolated GoM at ICR1 or with UPD showed MLID. An abnormal methylation patterns were observed exclusively at maternally imprinted loci, specifically PLAGL1 (6q24.2), GRB10 (7p12.1), MEST (7q32.2), SNRPN (15q11.2), GNAS-NESP55 (20q13.32), PEG3 (19q13.43), and GNAS (20q13.32). Among these, MEST and GNAS were the most frequently affected loci, each altered in 43.75% of MLID-BWSp cases (Fig. 1 ). None of the patients showed abnormal methylation of the MEG3 , MEG8 and H19 loci. Eleven patients had LoM at only one additional locus, three had LoM at two additional loci, one patient showed LoM at five additional loci and one patient exhibited LoM at two additional loci and GoM at one (Table S2 ). Methylation microarrays . All the results obtained with the multi-locus MS-MLPA test were confirmed with the methylation microarray, with a few exceptions. In contrast to the MS-MLPA results, no abnormal methylation was detected at PLAGL1 for patient #79, MEST for patient #87 and GNAS for patient #81. In the latter patient, although LoM was observed with the methylation microarray, it did not reach the statistically significant threshold of -3 standard deviation (SD). Additionally, patients #31, #86 and #90, had LoM in the ICR2 region detected by both chromosome 11 specific MS-MLPA and multi-locus MS-MLPA, but was not identified with the methylation microarray. In individuals #86 and #90, the hypomethylation level did not exceed − 3 SD but was detected at -2 SD. Patient #31, however, exhibited methylation levels in the ICR2 region similar to those of the controls. Notably, by studying the DMRs described by Court and colleagues [ 24 ], we identified 29 additional patients with MLID, increasing the occurrence of MLID in 45 out of 101 BWSp patients (44.55%). We detected abnormal methylation status in 25 DMRs of which eight corresponded to regions previously identified as abnormally methylated using multi-locus MS-MLPA: KCNQ1OT1, GNAS, GNAS-NESP55, MEST, GRB10, PLAGL1 , SNRPN and PEG3 . Notably, the methylation array also revealed abnormal methylation in some of these DMRs in patients, who were not detected by MS-MLPA. The remaining 17 DMRs were not covered by the MS-MLPA and include: DIRAS3 (1p31.3), FAM50B (6p25.2), ZDBF2 (2q33.3), L3MBTL1 (20q13.12), NAP1L5 (4q22.1), SNU13 (22q13.2), GET1 (21q22.2), IGF2R (6q25.3), ERLIN2 (8p11.23), MKRN3 (15q11.22), NNAT (20q11.23), PPIEL (1p34.3), RB1 (13q14.2), ZNF331 (19q13.42), ZNF597 (16p13.3), HTR5A (7q36.2) and PEG10 (7q21.3). As shown in Fig. 2 , other regions, such as DIRAS3, FAM50B and ZDBF2 , emerged as having abnormal methylation at similar or even higher frequencies than GNAS or MEST . Table 1 provides detailed results for each of the 45 MLID-BWSp patients identified in this study, along with their clinical features. Table S3 and Figures S1 A-D show detailed information about the methylation status of these patients across the identified DMRs with abnormal methylation status. To further investigate additional epigenetic alterations beyond the canonical imprinted DMRs, we analysed regions with abnormal methylation patterns compared to controls, excluding the previously assessed DMRs. This analysis revealed that 55 out of the 101 BWSp patients exhibited aberrant methylation in such regions (Figures S2 A-C). Of these 55 patients, 25 had been previously classified as MLID-BWSp, while the remaining 30 were BWSp patients without methylation alterations in the assessed DMRs. The affected regions included HR, JAKMIP1, HOXB6, MIRLET7BHG, TRAJ4, ZNF503, BST2, GNG12, OTAIRM1 , SNED1, PRRT1 , TRAJ39, STRA6, APOB, GCNT2, EXD3 and TROAP , most of which were hypomethylated, except for BST2 which showed hypermethylation. Interestingly, JAKMIP1, HOXB6 and SNED1 have been reported to be candidate imprinted DMRs in genome-wide methylation screens using reciprocal maternal and paternal uniparental diploidy samples [ 29 ]. Statistical analysis of clinical features A statistical analysis was conducted to compare the clinical features between patients with isolated BWSp and those with MLID-BWSp (Table S4 ). Most features were significantly more frequent in isolated BWSp compared to MLID-BWSp, including organomegaly (OR = 56.07, p = 8.44×10⁻¹⁸), congenital heart disease (OR = 55, p = 6.63×10⁻¹¹), and prognathism (OR = 91.67, p = 6.77×10⁻¹⁴). Additionally, umbilical hernia (p = 0.0007), inguinal hernia (p = 1.08×10⁻⁵), diastasis recti (OR = 26.91, p = 3.32×10⁻¹¹), nevi (p = 4.26×10⁻¹⁴), omphalocele (p = 2.99×10⁻⁹), and hypoglycaemia (p = 0.0067) were also significantly more prevalent in isolated BWSp. In contrast, macroglossia was the only feature significantly more frequent in MLID- BWSp (p = 0.024), while hemihyperplasia showed no significant difference between the two groups (p = 1.0). Notably, eight out of the 45 BWS-MLID patients identified in this study were conceived through assisted reproductive technologies (ART). Discussion In this study, we investigated the DNA methylation profiles of 101 patients with BWSp using MS-MLPA and methylation arrays, aiming to characterize the presence of MLID and assess potential epigenetic alterations outside of the clinically associated DMRs (CA-DMRs). MLID was identified in 16 patients (15.84%) applying multi-locus MS-MLPA which includes 11 loci, and this figure increased to 44.55% (55 patients) when using methylation microarray analysis. This prevalence is in line with the current literature evidence, where MLID frequencies among BWSp patients range from 10–50%, depending on the methodologies applied. Fontana et al. [ 8 ] reported a prevalence of 50% using a similar methylation analysis technique (MassARRAY methylation platform); while the frequencies reported by Bliek et al. [ 12 ] and Urakawa et al. [ 16 ] were 20% and 12%, respectively. This variation highlights the influence of detection methods on outcomes. Advanced tools such as high-density methylation microarrays offer broader genomic coverage, enabling the detection of subtle methylation abnormalities and additional loci uncovered by classic techniques such as MS-MLPA. For example, Kim et al. [ 19 ] demonstrated that methylation microarrays could identify MLID cases more accurately compared to traditional methods, such as MS-MLPA, which failed to detect MLID in some patients. These findings suggested that comprehensive genome-wide approaches might be integrated into routine diagnostic protocols for more accurate assessments of MLID prevalence in BWSp individuals. Our findings show that all patients with BWSp and MLID exhibited LoM at the ICR2 locus. This observation is consistent with prior studies, which have established ICR2 LoM as a hallmark epigenetic alteration in BWSp patients with MLID. ICR2 regulates the expression of CDKN1C , a gene that encodes a critical protein for growth regulation. LoM at ICR2 leads to the downregulation of CDKN1C , resulting in unrestrained cellular proliferation and contributing to the overgrowth phenotype characteristic of BWSp. International Consensus on the diagnosis and management of BWSp highlights ICR2 LoM as a defining feature in patients with MLID, recommending its use in diagnostic protocols [ 3 , 11 ]. Additionally, Bliek et al. [ 12 ] noted that ICR2 LoM was consistently present in their BWSp cohort with MLID, reinforcing the critical role of this epigenetic alteration in disease pathogenesis. This consistency across studies underlines the importance of ICR2 LoM as a diagnostic marker and a target for further research into therapeutic interventions. These findings also highlight the importance of thoroughly investigating these patients and ruling out MLID when ICR2 LoM is detected. In this study, we also identified several loci with recurrent LoM, including GNAS, MEST, GRB10 , and PLAGL1 , supporting the previous research. Several studies [ 8 , 12 , 15 , 18 ] reported that these loci were frequently affected in patients with MLID as also observed in the present cohort. These loci play a critical role in growth regulation and metabolic processes, and their dysregulation likely exacerbates the overgrowth features of BWSp. For instance, the GNAS locus, which encodes the alpha subunit of the stimulatory G protein, is involved in multiple signalling pathways. LoM at this locus can disrupt these pathways, contributing to the complex phenotype observed in MLID patients [ 18 ]. Similarly, MEST is a maternally imprinted gene involved in foetal development, and its hypomethylation is associated with altered growth patterns [ 30 ]. Our methylation microarray analysis uncovered loci with abnormal methylation, including DIRAS3 , FAM50B , or ZDBF2 , which were not covered by ME034 MS-MLPA. Remarkably, the frequency of abnormal methylation at these novel loci is equal to or even higher than that observed in the most frequent CA-DMRs studied by MS-MLPA, suggesting that future iterations of the MS-MLPA design may incorporate these loci DIRAS3 (DIRAS Family GTPase 3) is a maternally imprinted tumour suppressor gene expressed from the paternal allele, with important regulatory roles in cellular growth, autophagy, and tumour suppression. Hypermethylation of DIRAS3's regulatory CpG islands and loss of heterozygosity are mechanisms that can reduce or silence DIRAS3 expression. DIRAS3 acts as a potent inhibitor of pathways like PI3K/AKT and RAS/MAPK. Through these roles, it maintains cellular homeostasis, suppresses aberrant proliferation, and supports cellular dormancy. The potential disruption of these pathways due to MLID-related LoM or overexpression of DIRAS3 could lead to developmental anomalies. The involvement of DIRAS3 in autophagy and cellular signalling aligns with some of the molecular pathways dysregulated in BWSp, underscoring the need for further research [ 31 ]. FAM50B (Family with Sequence Similarity 50 Member B) is a paternally expressed imprinted gene located on chromosome 6. Its biological function is yet largely unknown, but emerging evidence suggests its contribution to MLID and potential associations with BWSp. Previous studies have identified LoM at the FAM50B locus in patients with MLID, indicating its susceptibility to epigenetic alterations. For instance, it has been reported that FAM50B exhibited aberrant methylation patterns in individuals with MLID [ 32 ]. A study [ 33 ] demonstrated that epimutations in FAM50B were frequent among patients with MLID, underscoring its involvement in the disorder's epigenetic dysregulation. Kim and colleagues [ 19 ] identified aberrant methylation at the FAM50B locus in patients with BWSp and MLID, indicating a potential link between FAM50B dysregulation and the BWSp phenotype. All these findings together imply that FAM50B may contribute to the complex epigenetic landscape of BWSp, particularly in cases with multilocus involvement. The exact mechanisms by which FAM50B influences BWSp pathogenesis remain to be elucidated, needing further research to clarify its role and potential as a biomarker or therapeutic target. ZDBF2 (Zinc Finger DBF-Type Containing 2) is a paternally imprinted gene located on 2q33.3 and its function is not yet well understood. Functional studies in mouse models suggest that this gene controls neonatal growth in mice, in a dose-sensitive manner and its expression is directly correlated with IGF-1 (Insulin Growth Factor 1) levels [ 34 ]. In line with previous studies [ 2 , 20 , 32 ], we also detected a GoM at this gene in 10 out of the 45 MLID-BWSp patients. This GoM is likely secondary to LoM of the CMKLR2-AS DMR (previously known as GPR1-AS ), which regulates ZDBF2 in a hierarchical manner [ 35 ]. Our analysis of methylation beyond the CA-DMRs revealed additional differentially methylated loci in a subset of BWSp patients. These findings suggest that MLID-BWSp may not be restricted to imprinting control regions but could involve broader epigenetic dysregulation. Several of the identified loci, including HOXB6, MIRLET7BHG and APOB , are implicated in developmental processes, gene regulation, and metabolism, which may have potential relevance to BWSp pathogenesis [ 36 – 38 ]. Notably, while most of these regions were hypomethylated, BST2 exhibited hypermethylation, suggesting a possible differential regulatory mechanism in MLID. The functional significance of these epigenetic alterations remains unclear, and further studies are required to determine whether they influence gene expression and contribute to the clinical variability observed in MLID-BWSp patients. These findings expand our understanding of the epigenetic landscape in BWSp and highlight the need for additional research to elucidate the broader impact of MLID. In this study, we did not identify significant sex-based differences in MLID prevalence among BWSp patients (23 females, 22 males). However, we observed significant clinical differences between BWSp patients with or without MLID (Table S4 ). All the clinical features were more commonly represented in the isolated BWSp group, with the exception of macroglossia, which was more prevalent in the MLID-BWSp group. No significant differences were found for hemihyperplasia between the two groups. These findings underline the subtle phenotypic differences between patients with BWSp with or without MLID, providing insights into their clinical characterization. While the associations are statistically significant, the small sample size of 45 MLID patients warrants caution and a larger cohort is required to replicate these findings. There is growing evidence that ART are associated with an increased risk of imprinting disorders, including BWSp, due to the increase dysregulation in the methylation patterns. Several studies have implicated ART procedures in disrupting the establishment or maintenance of imprinted methylation, potentially due to in vitro manipulation of gametes/ embryos or ovarian stimulation. In support of this, in our previous studies we found a higher prevalence of BWSp in ART-conceived individuals compared to naturally conceived controls [ 39 ]. Notably, in our current cohort, 8 of 45 MLID-BWSp (17.78%) patients were conceived through ART, which is almost twice the expected rate based on the prevalence of ART-conceived births in the Spanish population, according to data from the Spanish Fertility Society. Thus, these findings may have significant implications for prenatal diagnosis in ART pregnancies. Comprehensive prenatal methylation screening may help early detection of MLID in high-risk pregnancies, in combination with the traditional screening, allowing for tailored monitoring and management. This is particularly pertinent given that ART-conceived pregnancies are at elevated risk for imprinting disorders. Mussa et al. emphasized the importance of integrating epigenetic screening into prenatal care for ART-conceived pregnancies to improve outcomes [ 6 ]. Moreover, understanding and evaluating the epigenetic risks associated with ART can support the development of better reproductive technologies and risk stratification; e.g. optimizing culture conditions and minimizing embryo manipulation could reduce the risk of imprinting defects [ 40 ]. In summary, our study highlights the complexity and clinical significance of MLID in BWSp. By combining MS-MLPA and methylation microarray analyses, we identified MLID in 44.55% of the analysed BWSp cohort. The recurrent involvement of DMRs such as GNAS , MEST , FAM50B , DIRAS3 , and ZDBF2 , alongside other regions like BST2 , GNG12 , GNCT2 , and APOB , suggest a broader epigenetic landscape in BWSp that warrants further investigation. These findings indicate that, beyond the CA-DMRs, additional regions are affected in BWSp patients, offering deeper insight into the intricate epigenetic mechanisms underlying the disease. Thus, the integration of advanced methylation profiling techniques into clinical workflows might improve diagnostic accuracy and enable early interventions, ultimately enhancing outcomes for patients and their families. Future studies should aim to explore the long-term clinical implications of MLID in adulthood and the underlying molecular mechanisms of this phenomenon. This study provides new insights into the epigenetic landscape of MLID in patients with BWSp, emphasizing the universality of ICR2 LoM (as a constant hallmark of these conditions), the variability in MLID prevalence across studies, recurrently affected loci, and the clinical variability among MLID-BWSp patients. Declarations Conflict of interest statement The authors declare no conflicts of interest. Funding This research was funded by the FIS PI20/01053, from the ISCIII with funding from FEDER, Europe; by PMP21/00063 from the ISCIII with funding from the FEDER, Europe, and PMP22/00049 from the ISCIII with funding from the FEDER, Europe. Author Contribution Alejandro Parra: data collection, investigation, conceptualization, writing original draft and review. Mario Cazalla: investigation, conceptualisation, software, writing original draft and review. Carlos Rodriguez-Antolín: software and validation. Cristina Silván, Lucía Miranda-Alcaraz, Mónica Mora-Gómez, Natalia Gallego-Zazo, Manuel Rodríguez-Canó, Juan A. Jiménez-Estrada and Pedro Arias: investigation and data collection. Enrique Galán Gomez, Antonio González-Meneses, Pablo Barbero, Vanesa Lotersztein, Spanish OverGrowth Registry Initiative, Alfredo Santana: resources and data collection. Jair Tenorio-Castano: conceptualization, investigation, review, supervision and funding acquisition. Julián Nevado: conceptualization, investigation and review. Mathis HIldonen, Zeynep Tümer, Victor L Ruiz-Perez, David Monk: investigation and review. Pablo Lapunzina: conceptualization, investigation, review, supervision and funding acquisition. All the authors commented to the manuscript and accepted the final version. Acknowledgement ZT, JT, VLRP, FR, JN and PL are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA (ERN-ITHACA) funded by the European Union, under the grant agreement N°101156387.We would like to thank all the families and especially to the patients who were involved in this project, as well as the participants of the Spanish OverGrowth Registry Initiative (SOGRI) Consortium. The SOGRI (Spanish OverGrowth Registry Initiative) Consortium is comprised of the following researchers: Antonio Plasencia, Alberto L. Rosa, Aleixandre Blanquer, Alfredo Garcı’a-Alix, Alfredo Santana, Alicia Delicado, Almudena Alonso, Amaya Rodriguez, Amparo Sanchis, Ana Moreno, Ana Patiño Gar-cía, Ana Vega, Analía Bredani, Andrea Paula Solari, Andrea Villavicencio, Angelina Acosta, Anibal Nieto, Anna María Cueto González, Antonio Baldellón, Antonio González Meneses, An-tonio Martínez Carrascal, Aranzazu Díaz de Bustamante, Arteche Ocasar, Blanca Gener, Blasco González, Boris Groisman, Bradford Coffee, Carlos Alcalde Martín, Carmen Aragon Fernández, Carmen Benito, Carmen Martin Seisdedos, Carmen Roche, Claudia Arberas, Claudia Perando-nes, Claudio Contessotto, Cristina Olivas, Daniel Armenta, Denise Cavalcanti, Dolores Elorza, Elena Zamora, Elisa Zambrano, Elisabeth Steichen, Enrique Caro Cruz, Enrique Galán Gómez, Enriqueta Román, Ernesto Goldschmidt, Esteban Marfil, Esther Gean, Eugenia Antolín, F. Javier Gascón Jiménez, Feliciano Ramos, Fermina López Grondona, Fernández Córdoba, Fernando Regla Vargas, Francisco Martínez, J. Miguel García Vegada, Giovannucci Uzielli, Gloria Gacio, Carmen González Armengod, Graciela Mercado, Hamilton Cassinelli, Ieda Orioli, Ignacio Arroyo, Ignacio Díez López, Ignacio Onsurbe Ramírez, Ignacio Pascual Castroviejo, Ignacio Pascual Pascual, Ignacio Vázquez Rio, Inés Bueno, Isabel Espejo Portero, Isabel Lorda Sánchez, Jaime Sánchez del Pozo, Jaume Campistol, Javier Arcas, Javier Fernández, Javier García Planells, Javier López Pisón, Jesús Barreiro, Jesús del Valle Nuñez, María José Jiménez Liria, Joaquín Fer-nández Toral, Joaquín Ramírez, Jordi Rosell, Jorge Vilaplana, José Carlos Cabral de Almeida, José Ignacio Labarta, José L. Herranz, José Luis Fernández Luna, José Luis Fuster, José M. Díaz, Jose M. Gairi, José Miguel García Sagredo, Juan A. Piñero, Juan Carlos López Gutiérrez, Juan Manuel Fernández, Juan P. López Siguero, JuanTovar, Judith Armstrong, Julián Lara, Leonor Arranz, Laura Rodríguez, Leandro Soriano, Liliana De Alba, Loreta Cimbalistiene, Loreto Mar-torell, Luis González Gutiérrez Solana, Luis Pérez Jurado, M Asunción López Ariztegui, M. An-tonia Molina, M. Cruz García, M. Ferrer Lozano, M. Jesús Alija Merillas, M. Luisa Martínez-Frías, María L. Martínez Fernández, M. Rocío Jadraque, María Asunción García Pérez, María Montse-rrat Rodríguez Pedreira, María Pilar Ribate, María Teresa González López, María Teresa Moral Pumarega, Mabel Segovia, Macarena Lizama, Manuel Pombo, Margarita Martínez, Margarita Tabernero, María Antonia Ramos, Maria Ballesta, María Belar, María Jesús Lautre, Marta Cruz, M. Nieves Martínez Guardia, F. Javier Martínez Sarries, Mercedes Artigas, Mercedes Villanueva, Meritxell Torrabías, Miguel del Campo, Miguel Tomás Vila, Miguel Urioste, Mónica Rosello, Nik Kantaputra, Pablo Prieto Matos, Paloma Dorao, Paula Casano, Paula Lalaguna Mallada, Pe-dro Olivares, Raquel Perez Delgado, Priscila Bernardi, Rafael Camino León, Ramón Cañete, Ramón Gaztañaga, Ramón Velazquez, Ramón Vidal Samahuja, Raquel Sáez Villaverde, Ricardo Gracia, Richard Scott, Rita Valdez, Rosa Arteaga, Rosa Cedeño, Rosario Cazorla, Rosario Marín Iglesias, Rubén Bronberg, Salvador Climent, Santiago Conde Barreiro, Seema Kapoor, Soledad Kleppe, Sonia Santillán, Trinidad García Lopez, Teresa Calvo, Teresa Vendrell, Pilar Tirado, Claudia Toledo Pacheco, Alicia Ureta Huertos, Vanesa Lopez, Vanesa Lotersztein, Vanesa Méndez, Selma Vázquez Martín, Verónica Seidel, Vicente Albiach, Víctor M. Navas López, Virgina Soler, and Viviana Cosentino. 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Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx FigureS1A.png FigureS1B.png FigureS1C.png FigureS1D.png FigureS2A.png FigureS2B.png FigureS2C.png TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.docx Legendssuppmaterial.docx Cite Share Download PDF Status: Published Journal Publication published 26 Apr, 2026 Read the published version in Clinical Epigenetics → Version 1 posted Editorial decision: Revision requested 12 May, 2025 Reviews received at journal 12 May, 2025 Reviews received at journal 11 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviewers invited by journal 30 Apr, 2025 Editor assigned by journal 29 Apr, 2025 Submission checks completed at journal 29 Apr, 2025 First submitted to journal 29 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Blue bars indicate hypomethylation, and the red bar indicates hypermethylation. The chromosomal locations of these loci are listed in Table S2. GOM: gain of methylation; LOM: loss of methylation.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/b27d69b514d7683b44196ab6.png"},{"id":82177622,"identity":"6d4f2d0c-a0db-4eac-bf1c-b626aa0a9075","added_by":"auto","created_at":"2025-05-07 11:21:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28401,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of methylation aberrations across the differentially methylated regions (DMRs) identified using the methylation array. Blue bars represent hypomethylation, and red bars represent hypermethylation.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/c5520122617d0de3162120ef.png"},{"id":107928779,"identity":"73e0a9bf-7934-4a07-ad78-4bb3c609f9a6","added_by":"auto","created_at":"2026-04-27 16:12:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":378805,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/b5b46c57-1437-487f-968d-ebaa887590d3.pdf"},{"id":82177619,"identity":"5d65141c-7c54-4ac5-aba0-8afeffaaa668","added_by":"auto","created_at":"2025-05-07 11:21:22","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13323,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/b2c76a0976907f8f8057341b.xlsx"},{"id":82178857,"identity":"38acc2fe-7cb4-4c79-87df-086179bb39fd","added_by":"auto","created_at":"2025-05-07 11:29:23","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":123286,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1A.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/5346d96eb22598dd087aba1f.png"},{"id":82178861,"identity":"5abbac18-b401-48fd-923b-5d04d70b1410","added_by":"auto","created_at":"2025-05-07 11:29:23","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":123850,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1B.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/1df801506d1a429ce318a445.png"},{"id":82177655,"identity":"46ff326e-27b8-43ef-a422-711286ef89e3","added_by":"auto","created_at":"2025-05-07 11:21:24","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":115547,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1C.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/3bf358ec448eb55f71b914d0.png"},{"id":82178858,"identity":"92c1ec70-537b-4d3a-a54f-8fb0eef57436","added_by":"auto","created_at":"2025-05-07 11:29:23","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":133984,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1D.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/0dc0403163e1d349a03f3441.png"},{"id":82177628,"identity":"b95a8c2c-d9b3-4fb5-9763-ab07ec7db5b3","added_by":"auto","created_at":"2025-05-07 11:21:23","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":119280,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2A.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/553390b997f5b802a3f4da7a.png"},{"id":82179470,"identity":"00c0a0ed-664e-4fc2-ab0f-8afce205fa7d","added_by":"auto","created_at":"2025-05-07 11:37:23","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":126823,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2B.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/25bef1a9e46bfa49ffbd1ae4.png"},{"id":82178871,"identity":"e35c9690-6acc-4fd0-9341-e83e591a059e","added_by":"auto","created_at":"2025-05-07 11:29:24","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":114886,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2C.png","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/4bdc8697ab6aec92ce77a855.png"},{"id":82177653,"identity":"7f985d55-6780-477a-993d-d25862ec3bf5","added_by":"auto","created_at":"2025-05-07 11:21:24","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":10705,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/3086daaaad43246832684a9c.xlsx"},{"id":82178859,"identity":"230c7e77-046d-4e71-9187-09c293a9fa02","added_by":"auto","created_at":"2025-05-07 11:29:23","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":11730,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/0d30e20499a5c1a201a4aa7a.xlsx"},{"id":82177660,"identity":"ad1bcc10-57eb-4711-a3ee-f3135f43b937","added_by":"auto","created_at":"2025-05-07 11:21:25","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":2018140,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/05d9e115983ab31702dfabe4.xlsx"},{"id":82178864,"identity":"caf5fe2c-3ad1-4019-b9e8-0121b07943f3","added_by":"auto","created_at":"2025-05-07 11:29:23","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":17595,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/0ff751765538bef23c69c2bd.docx"},{"id":82179477,"identity":"fc5d27a2-fa84-4f8a-b73c-632753c588df","added_by":"auto","created_at":"2025-05-07 11:37:23","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":13672,"visible":true,"origin":"","legend":"","description":"","filename":"Legendssuppmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555801/v1/bf8acab082e1a8c8f340c23e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of Multilocus Imprinting Disturbance (MLID) in 101 Beckwith-Wiedemann Spectrum patients","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBeckwith-Wiedemann spectrum (BWSp, MIM #130650) is an overgrowth disorder characterized by a highly variable spectrum of clinical features, including macroglossia, macrosomia, abdominal wall defects (such as omphalocele, diastasis recti or umbilical hernia), neonatal hypoglycaemia and predisposition to tumour development, among many others [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The estimated prevalence of BWSp is approximately 1 in 12,000 live births, although this rate is higher in individuals conceived through assisted reproductive techniques (ART) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBWSp results from both genetic and epigenetic alterations within the 11p15.5 chromosomal region. In 2018, the term Beckwith-Wiedemann spectrum (BWSp) was suggested to cover classical Beckwith-Wiedemann syndrome (BWS) without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The 11p15.5 locus encompasses two distinct imprinting control regions (ICRs; ICR1, the telomeric one which includes H19/IGF2:IG DMR and ICR2, the centromeric one which includes KCNQ1OT1:TSS-DMR), which consist of clusters of genes playing crucial roles in biological processes, especially during embryonic development, such as somatic growth, cell cycle regulation and proliferation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The primary cause of BWSp is DNA methylation defects at these ICRs: over 50% of patients present with loss of methylation (LoM) at the maternal ICR2, 5\u0026ndash;10% exhibit gain of methylation (GoM) at the maternal ICR1, and 20\u0026ndash;25% shows mosaic paternal uniparental disomy (UPD) within chromosome 11p. Hypomethylation of ICR2 and hypermethylation of ICR1 led to the dysregulation of \u003cem\u003eCDKN1C\u003c/em\u003e (Cyclin-Dependent Kinase Inhibitor 1C) and \u003cem\u003eIGF2\u003c/em\u003e (Insulin-Like Growth Factor II), respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Other rarer causes of BWSp include pathogenic variants in \u003cem\u003eCDKN1C\u003c/em\u003e (5\u0026ndash;10%) and large genomic rearrangements involving the 11p15.5 locus (~\u0026thinsp;2\u0026ndash;3%) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies demonstrated that some patients with BWSp had methylation abnormalities at other loci additional to the 11p15.5 region [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], known as multilocus imprinting disturbance (MLID) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The additionally affected differentially methylated regions (DMRs) observed in patients with BWSp are usually associated with the imprinted \u003cem\u003ePLAGL1, GNAS, GRB10, MEG3, PEG3\u003c/em\u003e and \u003cem\u003eMEST\u003c/em\u003e genes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The underlying molecular mechanism of MLID is not entirely understood; however, monogenic variants in several genes, including members of the NOD-like receptor protein (NLRP) family (\u003cem\u003eNLRP2, NLRP4, NLRP5, NLRP7\u003c/em\u003e), have been identified in mothers of some patients with MLID. These genes encode proteins that are part of the subcortical maternal complex (SCMC) and are essential for early embryonic development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The growing advances in methylation analysis, such as methylation microarrays and methyl-seq, provide a comprehensive approach for examining methylation status across the genome [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These high-throughput technologies offer the potential to uncover novel insights into the complex epigenetic landscape of patients with MLID, allowing the identification of well-known DMRs as well as potentially new regions with aberrant methylation that could influence the phenotype of patients.\u003c/p\u003e \u003cp\u003eIn this study, we applied a combination of methylation-specific multiplex ligation-dependent probe amplification (multi-locus imprinting MS-MLPA; 11 different imprinted loci within seven chromosomal regions) and genome-wide methylation microarray assays (Infinium MethylationEPIC v2.0 BeadChip, Illumina) in 101 BWSp patients, with the aim of identifying the extent of MLID and looking for additional regions with aberrant methylation.\u003c/p\u003e"},{"header":"PATIENTS, MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003ePatients included in this study were selected from the Spanish Overgrowth Syndromes Registry Initiative (SOGRI), which contains more than 2,500 individuals and relatives with overgrowth disorders. All selected patients (N\u0026thinsp;=\u0026thinsp;101) had previously been diagnosed with BWSp based on their clinical findings and subsequent detection of abnormal methylation on the 11p15.5 chromosomal region with the SALSA MS-MLPA probemix ME030 BWS/RSS kit (MRC-Holland, Amsterdam, Netherlands). Eighty-five patients presented with hypomethylation of ICR2 (84.1%), 13 cases presented with paternal UPD (12.87%) and three presented with hypermethylation of ICR1 (2.97%) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This study was approved by the Ethical Committee of the Hospital Universitario La Paz (CEIm PI-446), and informed consent was obtained from all patients and/or their parents/ legal guardians.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMS-MLPA analysis\u003c/h3\u003e\n\u003cp\u003eThe SALSA MS-MLPA probemix ME034 Multi-locus Imprinting kit (MRC-Holland, Amsterdam, Netherlands) was applied to identify samples with MLID. Data analyses were conducted following the manufacturer protocol, defining relative probe signals by dividing each measured peak area by the sum of all peak areas of the control probes for that sample. Each peak\u0026rsquo;s relative probe area ratio was then compared to a DNA control sample (Promega, UK), using Coffalyser.net (MRC- Holland, Amsterdam, Netherlands).\u003c/p\u003e\n\u003ch3\u003eMethylation microarrays\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from whole blood collected from 101 BWSp patients and 41 controls. Bisulfite conversion of the DNA was performed using the EZ DNA Methylation-Lightning\u0026trade; kit (Illumina, San Diego, CA). Methylation levels were assessed using the Infinium MethylationEPIC v2.0 BeadChip (Illumina, San Diego, CA) array, which covers over 930,000 CpG sites. Raw data (.\u003cem\u003eidat\u003c/em\u003e files) were imported into R (v4.4.1), processed, and normalized using the R package \u003cem\u003eminfi\u003c/em\u003e (v1.50.0) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and subjected to quality control. Probes were filtered out based on the following criteria: detection p-value\u0026thinsp;\u0026gt;\u0026thinsp;0.01, probes located on the X and Y chromosomes, probes known to contain a single-nucleotide polymorphism (SNP), and probes known to cross-react with other genomic locations. After this filtering step, β-values and M-values, representing the methylation levels at each CpG site, were calculated. Sample labelling verification, including sex and age estimation, was conducted using the R packages \u003cem\u003ewateRmelon\u003c/em\u003e (v2.10.0) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and \u003cem\u003emethylclock\u003c/em\u003e (v1.10.0) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMethylation status of the patients was first evaluated at the DMRs reported by Court et al. (2014) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To assess methylation status, the mean β-value and standard deviation of controls were calculated for each probe. A standardized score was computed, and a probe was considered significantly hypermethylated if the score was \u0026ge;\u0026thinsp;+\u0026thinsp;3SD and hypomethylated if it was \u0026le; -3SD. Additionally, we performed a differential methylation analysis to compare methylation profiles between controls and BWSp patients, leading to the identification of differentially methylated positions (DMPs) outside the previously analysed gDMRs. To visualize the DMPs across the genome, the R package ENmix (v1.40.2) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] was used. The top DMPs were used to generate a heatmap with the R library ComplexHeatmap (v2.20.0) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], allowing the identification of methylation patterns across our cohort beyond the gDMRs.\u003c/p\u003e\n\u003ch3\u003eStatistical analysis of clinical features\u003c/h3\u003e\n\u003cp\u003eStatistical analysis was conducted using SPSS v.25 (IBM Corporation, United States) to assess differences in clinical features. Descriptive analyses included the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for continuous variables (age in this cohort) and frequency tables for categorical variables. Categorical variables were expressed as binary values (1 or 0), categorized as \u0026ldquo;ever\u0026rdquo; having a given condition versus \u0026ldquo;never\u0026rdquo; having the condition. Comparisons were made between our cohort of patients with BWSp and MLID and the reported frequencies of clinical features in patients with BWSp [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Chi-square tests and/or Fisher\u0026rsquo;s exact test were used to evaluate differences, with z-tests applied to compare column proportions. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered indicative of a statistically significant difference.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMS-MLPA\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eDNA of 101 BWSp patients with previously confirmed methylation defects at the 11p15.5 locus were re-investigated using the multi- locus MS-MLPA kit that revealed the presence of MLID in 16 of the patients (15.84%) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). As expected, all these patients identified with MLID exhibited LOM at ICR2, while none of the patients with isolated GoM at ICR1 or with UPD showed MLID. An abnormal methylation patterns were observed exclusively at maternally imprinted loci, specifically \u003cem\u003ePLAGL1\u003c/em\u003e (6q24.2), \u003cem\u003eGRB10\u003c/em\u003e (7p12.1), \u003cem\u003eMEST\u003c/em\u003e (7q32.2), \u003cem\u003eSNRPN\u003c/em\u003e (15q11.2), \u003cem\u003eGNAS-NESP55\u003c/em\u003e (20q13.32), \u003cem\u003ePEG3\u003c/em\u003e (19q13.43), and \u003cem\u003eGNAS\u003c/em\u003e (20q13.32). Among these, MEST and GNAS were the most frequently affected loci, each altered in 43.75% of MLID-BWSp cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). None of the patients showed abnormal methylation of the \u003cem\u003eMEG3\u003c/em\u003e, \u003cem\u003eMEG8\u003c/em\u003e and \u003cem\u003eH19\u003c/em\u003e loci. Eleven patients had LoM at only one additional locus, three had LoM at two additional loci, one patient showed LoM at five additional loci and one patient exhibited LoM at two additional loci and GoM at one (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMethylation microarrays\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAll the results obtained with the multi-locus MS-MLPA test were confirmed with the methylation microarray, with a few exceptions. In contrast to the MS-MLPA results, no abnormal methylation was detected at \u003cem\u003ePLAGL1\u003c/em\u003e for patient #79, \u003cem\u003eMEST\u003c/em\u003e for patient #87 and \u003cem\u003eGNAS\u003c/em\u003e for patient #81. In the latter patient, although LoM was observed with the methylation microarray, it did not reach the statistically significant threshold of -3 standard deviation (SD). Additionally, patients #31, #86 and #90, had LoM in the ICR2 region detected by both chromosome 11 specific MS-MLPA and multi-locus MS-MLPA, but was not identified with the methylation microarray. In individuals #86 and #90, the hypomethylation level did not exceed \u0026minus;\u0026thinsp;3 SD but was detected at -2 SD. Patient #31, however, exhibited methylation levels in the ICR2 region similar to those of the controls.\u003c/p\u003e \u003cp\u003eNotably, by studying the DMRs described by Court and colleagues [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], we identified 29 additional patients with MLID, increasing the occurrence of MLID in 45 out of 101 BWSp patients (44.55%). We detected abnormal methylation status in 25 DMRs of which eight corresponded to regions previously identified as abnormally methylated using multi-locus MS-MLPA: \u003cem\u003eKCNQ1OT1, GNAS, GNAS-NESP55, MEST, GRB10, PLAGL1\u003c/em\u003e, \u003cem\u003eSNRPN\u003c/em\u003e and \u003cem\u003ePEG3\u003c/em\u003e. Notably, the methylation array also revealed abnormal methylation in some of these DMRs in patients, who were not detected by MS-MLPA. The remaining 17 DMRs were not covered by the MS-MLPA and include: \u003cem\u003eDIRAS3\u003c/em\u003e (1p31.3), \u003cem\u003eFAM50B\u003c/em\u003e (6p25.2), \u003cem\u003eZDBF2\u003c/em\u003e (2q33.3), \u003cem\u003eL3MBTL1\u003c/em\u003e (20q13.12), \u003cem\u003eNAP1L5\u003c/em\u003e (4q22.1), \u003cem\u003eSNU13\u003c/em\u003e (22q13.2), \u003cem\u003eGET1\u003c/em\u003e (21q22.2), \u003cem\u003eIGF2R\u003c/em\u003e (6q25.3), \u003cem\u003eERLIN2\u003c/em\u003e (8p11.23), \u003cem\u003eMKRN3\u003c/em\u003e (15q11.22), \u003cem\u003eNNAT\u003c/em\u003e (20q11.23), \u003cem\u003ePPIEL\u003c/em\u003e (1p34.3), \u003cem\u003eRB1\u003c/em\u003e (13q14.2), \u003cem\u003eZNF331\u003c/em\u003e (19q13.42), \u003cem\u003eZNF597\u003c/em\u003e (16p13.3), \u003cem\u003eHTR5A\u003c/em\u003e (7q36.2) and \u003cem\u003ePEG10\u003c/em\u003e (7q21.3). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, other regions, such as \u003cem\u003eDIRAS3, FAM50B\u003c/em\u003e and \u003cem\u003eZDBF2\u003c/em\u003e, emerged as having abnormal methylation at similar or even higher frequencies than \u003cem\u003eGNAS\u003c/em\u003e or \u003cem\u003eMEST\u003c/em\u003e. Table\u0026nbsp;1 provides detailed results for each of the 45 MLID-BWSp patients identified in this study, along with their clinical features. Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e and Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A-D show detailed information about the methylation status of these patients across the identified DMRs with abnormal methylation status.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate additional epigenetic alterations beyond the canonical imprinted DMRs, we analysed regions with abnormal methylation patterns compared to controls, excluding the previously assessed DMRs. This analysis revealed that 55 out of the 101 BWSp patients exhibited aberrant methylation in such regions (Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e A-C). Of these 55 patients, 25 had been previously classified as MLID-BWSp, while the remaining 30 were BWSp patients without methylation alterations in the assessed DMRs. The affected regions included \u003cem\u003eHR, JAKMIP1, HOXB6, MIRLET7BHG, TRAJ4, ZNF503, BST2, GNG12, OTAIRM1\u003c/em\u003e, \u003cem\u003eSNED1, PRRT1\u003c/em\u003e, \u003cem\u003eTRAJ39, STRA6, APOB, GCNT2, EXD3\u003c/em\u003e and \u003cem\u003eTROAP\u003c/em\u003e, most of which were hypomethylated, except for \u003cem\u003eBST2\u003c/em\u003e which showed hypermethylation. Interestingly, \u003cem\u003eJAKMIP1, HOXB6\u003c/em\u003e and \u003cem\u003eSNED1\u003c/em\u003e have been reported to be candidate imprinted DMRs in genome-wide methylation screens using reciprocal maternal and paternal uniparental diploidy samples [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis of clinical features\u003c/h2\u003e \u003cp\u003eA statistical analysis was conducted to compare the clinical features between patients with isolated BWSp and those with MLID-BWSp (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Most features were significantly more frequent in isolated BWSp compared to MLID-BWSp, including organomegaly (OR\u0026thinsp;=\u0026thinsp;56.07, p\u0026thinsp;=\u0026thinsp;8.44\u0026times;10⁻\u0026sup1;⁸), congenital heart disease (OR\u0026thinsp;=\u0026thinsp;55, p\u0026thinsp;=\u0026thinsp;6.63\u0026times;10⁻\u0026sup1;\u0026sup1;), and prognathism (OR\u0026thinsp;=\u0026thinsp;91.67, p\u0026thinsp;=\u0026thinsp;6.77\u0026times;10⁻\u0026sup1;⁴). Additionally, umbilical hernia (p\u0026thinsp;=\u0026thinsp;0.0007), inguinal hernia (p\u0026thinsp;=\u0026thinsp;1.08\u0026times;10⁻⁵), diastasis recti (OR\u0026thinsp;=\u0026thinsp;26.91, p\u0026thinsp;=\u0026thinsp;3.32\u0026times;10⁻\u0026sup1;\u0026sup1;), nevi (p\u0026thinsp;=\u0026thinsp;4.26\u0026times;10⁻\u0026sup1;⁴), omphalocele (p\u0026thinsp;=\u0026thinsp;2.99\u0026times;10⁻⁹), and hypoglycaemia (p\u0026thinsp;=\u0026thinsp;0.0067) were also significantly more prevalent in isolated BWSp. In contrast, macroglossia was the only feature significantly more frequent in MLID- BWSp (p\u0026thinsp;=\u0026thinsp;0.024), while hemihyperplasia showed no significant difference between the two groups (p\u0026thinsp;=\u0026thinsp;1.0). Notably, eight out of the 45 BWS-MLID patients identified in this study were conceived through assisted reproductive technologies (ART).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the DNA methylation profiles of 101 patients with BWSp using MS-MLPA and methylation arrays, aiming to characterize the presence of MLID and assess potential epigenetic alterations outside of the clinically associated DMRs (CA-DMRs). MLID was identified in 16 patients (15.84%) applying multi-locus MS-MLPA which includes 11 loci, and this figure increased to 44.55% (55 patients) when using methylation microarray analysis. This prevalence is in line with the current literature evidence, where MLID frequencies among BWSp patients range from 10\u0026ndash;50%, depending on the methodologies applied. Fontana et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] reported a prevalence of 50% using a similar methylation analysis technique (MassARRAY methylation platform); while the frequencies reported by Bliek et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and Urakawa et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] were 20% and 12%, respectively. This variation highlights the influence of detection methods on outcomes. Advanced tools such as high-density methylation microarrays offer broader genomic coverage, enabling the detection of subtle methylation abnormalities and additional loci uncovered by classic techniques such as MS-MLPA. For example, Kim et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] demonstrated that methylation microarrays could identify MLID cases more accurately compared to traditional methods, such as MS-MLPA, which failed to detect MLID in some patients. These findings suggested that comprehensive genome-wide approaches might be integrated into routine diagnostic protocols for more accurate assessments of MLID prevalence in BWSp individuals.\u003c/p\u003e \u003cp\u003eOur findings show that all patients with BWSp and MLID exhibited LoM at the ICR2 locus. This observation is consistent with prior studies, which have established ICR2 LoM as a hallmark epigenetic alteration in BWSp patients with MLID. ICR2 regulates the expression of \u003cem\u003eCDKN1C\u003c/em\u003e, a gene that encodes a critical protein for growth regulation. LoM at ICR2 leads to the downregulation of \u003cem\u003eCDKN1C\u003c/em\u003e, resulting in unrestrained cellular proliferation and contributing to the overgrowth phenotype characteristic of BWSp. International Consensus on the diagnosis and management of BWSp highlights ICR2 LoM as a defining feature in patients with MLID, recommending its use in diagnostic protocols [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, Bliek et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] noted that ICR2 LoM was consistently present in their BWSp cohort with MLID, reinforcing the critical role of this epigenetic alteration in disease pathogenesis. This consistency across studies underlines the importance of ICR2 LoM as a diagnostic marker and a target for further research into therapeutic interventions. These findings also highlight the importance of thoroughly investigating these patients and ruling out MLID when ICR2 LoM is detected.\u003c/p\u003e \u003cp\u003eIn this study, we also identified several loci with recurrent LoM, including \u003cem\u003eGNAS, MEST, GRB10\u003c/em\u003e, and \u003cem\u003ePLAGL1\u003c/em\u003e, supporting the previous research. Several studies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported that these loci were frequently affected in patients with MLID as also observed in the present cohort. These loci play a critical role in growth regulation and metabolic processes, and their dysregulation likely exacerbates the overgrowth features of BWSp. For instance, the \u003cem\u003eGNAS\u003c/em\u003e locus, which encodes the alpha subunit of the stimulatory G protein, is involved in multiple signalling pathways. LoM at this locus can disrupt these pathways, contributing to the complex phenotype observed in MLID patients [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, \u003cem\u003eMEST\u003c/em\u003e is a maternally imprinted gene involved in foetal development, and its hypomethylation is associated with altered growth patterns [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur methylation microarray analysis uncovered loci with abnormal methylation, including \u003cem\u003eDIRAS3\u003c/em\u003e, \u003cem\u003eFAM50B\u003c/em\u003e, or \u003cem\u003eZDBF2\u003c/em\u003e, which were not covered by ME034 MS-MLPA. Remarkably, the frequency of abnormal methylation at these novel loci is equal to or even higher than that observed in the most frequent CA-DMRs studied by MS-MLPA, suggesting that future iterations of the MS-MLPA design may incorporate these loci\u003c/p\u003e \u003cp\u003e \u003cem\u003eDIRAS3\u003c/em\u003e (DIRAS Family GTPase 3) is a maternally imprinted tumour suppressor gene expressed from the paternal allele, with important regulatory roles in cellular growth, autophagy, and tumour suppression. Hypermethylation of DIRAS3's regulatory CpG islands and loss of heterozygosity are mechanisms that can reduce or silence DIRAS3 expression. DIRAS3 acts as a potent inhibitor of pathways like PI3K/AKT and RAS/MAPK. Through these roles, it maintains cellular homeostasis, suppresses aberrant proliferation, and supports cellular dormancy. The potential disruption of these pathways due to MLID-related LoM or overexpression of DIRAS3 could lead to developmental anomalies. The involvement of DIRAS3 in autophagy and cellular signalling aligns with some of the molecular pathways dysregulated in BWSp, underscoring the need for further research [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eFAM50B\u003c/em\u003e (Family with Sequence Similarity 50 Member B) is a paternally expressed imprinted gene located on chromosome 6. Its biological function is yet largely unknown, but emerging evidence suggests its contribution to MLID and potential associations with BWSp. Previous studies have identified LoM at the \u003cem\u003eFAM50B locus\u003c/em\u003e in patients with MLID, indicating its susceptibility to epigenetic alterations. For instance, it has been reported that \u003cem\u003eFAM50B\u003c/em\u003e exhibited aberrant methylation patterns in individuals with MLID [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] demonstrated that epimutations in \u003cem\u003eFAM50B\u003c/em\u003e were frequent among patients with MLID, underscoring its involvement in the disorder's epigenetic dysregulation. Kim and colleagues [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] identified aberrant methylation at the \u003cem\u003eFAM50B\u003c/em\u003e locus in patients with BWSp and MLID, indicating a potential link between \u003cem\u003eFAM50B\u003c/em\u003e dysregulation and the BWSp phenotype. All these findings together imply that \u003cem\u003eFAM50B\u003c/em\u003e may contribute to the complex epigenetic landscape of BWSp, particularly in cases with multilocus involvement. The exact mechanisms by which \u003cem\u003eFAM50B\u003c/em\u003e influences BWSp pathogenesis remain to be elucidated, needing further research to clarify its role and potential as a biomarker or therapeutic target.\u003c/p\u003e \u003cp\u003e \u003cem\u003eZDBF2\u003c/em\u003e (Zinc Finger DBF-Type Containing 2) is a paternally imprinted gene located on 2q33.3 and its function is not yet well understood. Functional studies in mouse models suggest that this gene controls neonatal growth in mice, in a dose-sensitive manner and its expression is directly correlated with IGF-1 (Insulin Growth Factor 1) levels [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In line with previous studies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we also detected a GoM at this gene in 10 out of the 45 MLID-BWSp patients. This GoM is likely secondary to LoM of the \u003cem\u003eCMKLR2-AS\u003c/em\u003e DMR (previously known as \u003cem\u003eGPR1-AS\u003c/em\u003e), which regulates \u003cem\u003eZDBF2\u003c/em\u003e in a hierarchical manner [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur analysis of methylation beyond the CA-DMRs revealed additional differentially methylated loci in a subset of BWSp patients. These findings suggest that MLID-BWSp may not be restricted to imprinting control regions but could involve broader epigenetic dysregulation. Several of the identified loci, including \u003cem\u003eHOXB6, MIRLET7BHG\u003c/em\u003e and \u003cem\u003eAPOB\u003c/em\u003e, are implicated in developmental processes, gene regulation, and metabolism, which may have potential relevance to BWSp pathogenesis [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Notably, while most of these regions were hypomethylated, \u003cem\u003eBST2\u003c/em\u003e exhibited hypermethylation, suggesting a possible differential regulatory mechanism in MLID. The functional significance of these epigenetic alterations remains unclear, and further studies are required to determine whether they influence gene expression and contribute to the clinical variability observed in MLID-BWSp patients. These findings expand our understanding of the epigenetic landscape in BWSp and highlight the need for additional research to elucidate the broader impact of MLID.\u003c/p\u003e \u003cp\u003eIn this study, we did not identify significant sex-based differences in MLID prevalence among BWSp patients (23 females, 22 males). However, we observed significant clinical differences between BWSp patients with or without MLID (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). All the clinical features were more commonly represented in the isolated BWSp group, with the exception of macroglossia, which was more prevalent in the MLID-BWSp group. No significant differences were found for hemihyperplasia between the two groups. These findings underline the subtle phenotypic differences between patients with BWSp with or without MLID, providing insights into their clinical characterization. While the associations are statistically significant, the small sample size of 45 MLID patients warrants caution and a larger cohort is required to replicate these findings.\u003c/p\u003e \u003cp\u003eThere is growing evidence that ART are associated with an increased risk of imprinting disorders, including BWSp, due to the increase dysregulation in the methylation patterns. Several studies have implicated ART procedures in disrupting the establishment or maintenance of imprinted methylation, potentially due to \u003cem\u003ein vitro\u003c/em\u003e manipulation of gametes/ embryos or ovarian stimulation. In support of this, in our previous studies we found a higher prevalence of BWSp in ART-conceived individuals compared to naturally conceived controls [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Notably, in our current cohort, 8 of 45 MLID-BWSp (17.78%) patients were conceived through ART, which is almost twice the expected rate based on the prevalence of ART-conceived births in the Spanish population, according to data from the Spanish Fertility Society. Thus, these findings may have significant implications for prenatal diagnosis in ART pregnancies. Comprehensive prenatal methylation screening may help early detection of MLID in high-risk pregnancies, in combination with the traditional screening, allowing for tailored monitoring and management. This is particularly pertinent given that ART-conceived pregnancies are at elevated risk for imprinting disorders. Mussa et al. emphasized the importance of integrating epigenetic screening into prenatal care for ART-conceived pregnancies to improve outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, understanding and evaluating the epigenetic risks associated with ART can support the development of better reproductive technologies and risk stratification; \u003cem\u003ee.g.\u003c/em\u003e optimizing culture conditions and minimizing embryo manipulation could reduce the risk of imprinting defects [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, our study highlights the complexity and clinical significance of MLID in BWSp. By combining MS-MLPA and methylation microarray analyses, we identified MLID in 44.55% of the analysed BWSp cohort. The recurrent involvement of DMRs such as \u003cem\u003eGNAS\u003c/em\u003e, \u003cem\u003eMEST\u003c/em\u003e, \u003cem\u003eFAM50B\u003c/em\u003e, \u003cem\u003eDIRAS3\u003c/em\u003e, and \u003cem\u003eZDBF2\u003c/em\u003e, alongside other regions like \u003cem\u003eBST2\u003c/em\u003e, \u003cem\u003eGNG12\u003c/em\u003e, \u003cem\u003eGNCT2\u003c/em\u003e, and \u003cem\u003eAPOB\u003c/em\u003e, suggest a broader epigenetic landscape in BWSp that warrants further investigation. These findings indicate that, beyond the CA-DMRs, additional regions are affected in BWSp patients, offering deeper insight into the intricate epigenetic mechanisms underlying the disease. Thus, the integration of advanced methylation profiling techniques into clinical workflows might improve diagnostic accuracy and enable early interventions, ultimately enhancing outcomes for patients and their families. Future studies should aim to explore the long-term clinical implications of MLID in adulthood and the underlying molecular mechanisms of this phenomenon. This study provides new insights into the epigenetic landscape of MLID in patients with BWSp, emphasizing the universality of ICR2 LoM (as a constant hallmark of these conditions), the variability in MLID prevalence across studies, recurrently affected loci, and the clinical variability among MLID-BWSp patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest statement\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the FIS PI20/01053, from the ISCIII with funding from FEDER, Europe; by PMP21/00063 from the ISCIII with funding from the FEDER, Europe, and PMP22/00049 from the ISCIII with funding from the FEDER, Europe.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAlejandro Parra: data collection, investigation, conceptualization, writing original draft and review. Mario Cazalla: investigation, conceptualisation, software, writing original draft and review. Carlos Rodriguez-Antol\u0026iacute;n: software and validation. Cristina Silv\u0026aacute;n, Luc\u0026iacute;a Miranda-Alcaraz, M\u0026oacute;nica Mora-G\u0026oacute;mez, Natalia Gallego-Zazo, Manuel Rodr\u0026iacute;guez-Can\u0026oacute;, Juan A. Jim\u0026eacute;nez-Estrada and Pedro Arias: investigation and data collection. Enrique Gal\u0026aacute;n Gomez, Antonio Gonz\u0026aacute;lez-Meneses, Pablo Barbero, Vanesa Lotersztein, Spanish OverGrowth Registry Initiative, Alfredo Santana: resources and data collection. Jair Tenorio-Castano: conceptualization, investigation, review, supervision and funding acquisition. Juli\u0026aacute;n Nevado: conceptualization, investigation and review. Mathis HIldonen, Zeynep T\u0026uuml;mer, Victor L Ruiz-Perez, David Monk: investigation and review. Pablo Lapunzina: conceptualization, investigation, review, supervision and funding acquisition. All the authors commented to the manuscript and accepted the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e ZT, JT, VLRP, FR, JN and PL are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA (ERN-ITHACA) funded by the European Union, under the grant agreement N\u0026deg;101156387.We would like to thank all the families and especially to the patients who were involved in this project, as well as the participants of the Spanish OverGrowth Registry Initiative (SOGRI) Consortium. The SOGRI (Spanish OverGrowth Registry Initiative) Consortium is comprised of the following researchers: Antonio Plasencia, Alberto L. Rosa, Aleixandre Blanquer, Alfredo Garcı\u0026rsquo;a-Alix, Alfredo Santana, Alicia Delicado, Almudena Alonso, Amaya Rodriguez, Amparo Sanchis, Ana Moreno, Ana Pati\u0026ntilde;o Gar-c\u0026iacute;a, Ana Vega, Anal\u0026iacute;a Bredani, Andrea Paula Solari, Andrea Villavicencio, Angelina Acosta, Anibal Nieto, Anna Mar\u0026iacute;a Cueto Gonz\u0026aacute;lez, Antonio Baldell\u0026oacute;n, Antonio Gonz\u0026aacute;lez Meneses, An-tonio Mart\u0026iacute;nez Carrascal, Aranzazu D\u0026iacute;az de Bustamante, Arteche Ocasar, Blanca Gener, Blasco Gonz\u0026aacute;lez, Boris Groisman, Bradford Coffee, Carlos Alcalde Mart\u0026iacute;n, Carmen Aragon Fern\u0026aacute;ndez, Carmen Benito, Carmen Martin Seisdedos, Carmen Roche, Claudia Arberas, Claudia Perando-nes, Claudio Contessotto, Cristina Olivas, Daniel Armenta, Denise Cavalcanti, Dolores Elorza, Elena Zamora, Elisa Zambrano, Elisabeth Steichen, Enrique Caro Cruz, Enrique Gal\u0026aacute;n G\u0026oacute;mez, Enriqueta Rom\u0026aacute;n, Ernesto Goldschmidt, Esteban Marfil, Esther Gean, Eugenia Antol\u0026iacute;n, F. Javier Gasc\u0026oacute;n Jim\u0026eacute;nez, Feliciano Ramos, Fermina L\u0026oacute;pez Grondona, Fern\u0026aacute;ndez C\u0026oacute;rdoba, Fernando Regla Vargas, Francisco Mart\u0026iacute;nez, J. Miguel Garc\u0026iacute;a Vegada, Giovannucci Uzielli, Gloria Gacio, Carmen Gonz\u0026aacute;lez Armengod, Graciela Mercado, Hamilton Cassinelli, Ieda Orioli, Ignacio Arroyo, Ignacio D\u0026iacute;ez L\u0026oacute;pez, Ignacio Onsurbe Ram\u0026iacute;rez, Ignacio Pascual Castroviejo, Ignacio Pascual Pascual, Ignacio V\u0026aacute;zquez Rio, In\u0026eacute;s Bueno, Isabel Espejo Portero, Isabel Lorda S\u0026aacute;nchez, Jaime S\u0026aacute;nchez del Pozo, Jaume Campistol, Javier Arcas, Javier Fern\u0026aacute;ndez, Javier Garc\u0026iacute;a Planells, Javier L\u0026oacute;pez Pis\u0026oacute;n, Jes\u0026uacute;s Barreiro, Jes\u0026uacute;s del Valle Nu\u0026ntilde;ez, Mar\u0026iacute;a Jos\u0026eacute; Jim\u0026eacute;nez Liria, Joaqu\u0026iacute;n Fer-n\u0026aacute;ndez Toral, Joaqu\u0026iacute;n Ram\u0026iacute;rez, Jordi Rosell, Jorge Vilaplana, Jos\u0026eacute; Carlos Cabral de Almeida, Jos\u0026eacute; Ignacio Labarta, Jos\u0026eacute; L. Herranz, Jos\u0026eacute; Luis Fern\u0026aacute;ndez Luna, Jos\u0026eacute; Luis Fuster, Jos\u0026eacute; M. D\u0026iacute;az, Jose M. Gairi, Jos\u0026eacute; Miguel Garc\u0026iacute;a Sagredo, Juan A. Pi\u0026ntilde;ero, Juan Carlos L\u0026oacute;pez Guti\u0026eacute;rrez, Juan Manuel Fern\u0026aacute;ndez, Juan P. L\u0026oacute;pez Siguero, JuanTovar, Judith Armstrong, Juli\u0026aacute;n Lara, Leonor Arranz, Laura Rodr\u0026iacute;guez, Leandro Soriano, Liliana De Alba, Loreta Cimbalistiene, Loreto Mar-torell, Luis Gonz\u0026aacute;lez Guti\u0026eacute;rrez Solana, Luis P\u0026eacute;rez Jurado, M Asunci\u0026oacute;n L\u0026oacute;pez Ariztegui, M. An-tonia Molina, M. Cruz Garc\u0026iacute;a, M. Ferrer Lozano, M. Jes\u0026uacute;s Alija Merillas, M. Luisa Mart\u0026iacute;nez-Fr\u0026iacute;as, Mar\u0026iacute;a L. Mart\u0026iacute;nez Fern\u0026aacute;ndez, M. Roc\u0026iacute;o Jadraque, Mar\u0026iacute;a Asunci\u0026oacute;n Garc\u0026iacute;a P\u0026eacute;rez, Mar\u0026iacute;a Montse-rrat Rodr\u0026iacute;guez Pedreira, Mar\u0026iacute;a Pilar Ribate, Mar\u0026iacute;a Teresa Gonz\u0026aacute;lez L\u0026oacute;pez, Mar\u0026iacute;a Teresa Moral Pumarega, Mabel Segovia, Macarena Lizama, Manuel Pombo, Margarita Mart\u0026iacute;nez, Margarita Tabernero, Mar\u0026iacute;a Antonia Ramos, Maria Ballesta, Mar\u0026iacute;a Belar, Mar\u0026iacute;a Jes\u0026uacute;s Lautre, Marta Cruz, M. Nieves Mart\u0026iacute;nez Guardia, F. Javier Mart\u0026iacute;nez Sarries, Mercedes Artigas, Mercedes Villanueva, Meritxell Torrab\u0026iacute;as, Miguel del Campo, Miguel Tom\u0026aacute;s Vila, Miguel Urioste, M\u0026oacute;nica Rosello, Nik Kantaputra, Pablo Prieto Matos, Paloma Dorao, Paula Casano, Paula Lalaguna Mallada, Pe-dro Olivares, Raquel Perez Delgado, Priscila Bernardi, Rafael Camino Le\u0026oacute;n, Ram\u0026oacute;n Ca\u0026ntilde;ete, Ram\u0026oacute;n Gazta\u0026ntilde;aga, Ram\u0026oacute;n Velazquez, Ram\u0026oacute;n Vidal Samahuja, Raquel S\u0026aacute;ez Villaverde, Ricardo Gracia, Richard Scott, Rita Valdez, Rosa Arteaga, Rosa Cede\u0026ntilde;o, Rosario Cazorla, Rosario Mar\u0026iacute;n Iglesias, Rub\u0026eacute;n Bronberg, Salvador Climent, Santiago Conde Barreiro, Seema Kapoor, Soledad Kleppe, Sonia Santill\u0026aacute;n, Trinidad Garc\u0026iacute;a Lopez, Teresa Calvo, Teresa Vendrell, Pilar Tirado, Claudia Toledo Pacheco, Alicia Ureta Huertos, Vanesa Lopez, Vanesa Lotersztein, Vanesa M\u0026eacute;ndez, Selma V\u0026aacute;zquez Mart\u0026iacute;n, Ver\u0026oacute;nica Seidel, Vicente Albiach, V\u0026iacute;ctor M. Navas L\u0026oacute;pez, Virgina Soler, and Viviana Cosentino.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeksberg, R., Shuman, C., \u0026amp; Beckwith, J. B. (2010). Beckwith\u0026ndash;Wiedemann syndrome. European journal of human genetics, \u003cem\u003e18\u003c/em\u003e(1), 8\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTenorio, J., Romanelli, V., Martin-Trujillo, A., Fern\u0026aacute;ndez, G. M., Segovia, M., Perandones, C., \u0026hellip; Lapunzina, P. (2016). Clinical and molecular analyses of Beckwith\u0026ndash;Wiedemann syndrome: comparison between spontaneous conception and assisted reproduction techniques. 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R., van Bockxmeer, F. M., \u0026amp; Burnett, J. R. (2004). Lipid disorders and mutations in the APOB gene. Clinical chemistry, 50(10), 1725\u0026ndash;1732.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTenorio J, et al. Clinical and molecular analyses of Beckwith\u0026ndash;Wiedemann syndrome: Comparison between spontaneous conception and assisted reproduction techniques. Am J Med Genet A. 2016;170(10):2740\u0026ndash;9. DOI:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ajmg.a.37857\u003c/span\u003e\u003cspan address=\"10.1002/ajmg.a.37857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoherty, A. S., Mann, M. R., Tremblay, K. D., Bartolomei, M. S., \u0026amp; Schultz, R. M. (2000). Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biology of reproduction, \u003cem\u003e62\u003c/em\u003e(6), 1526\u0026ndash;1535.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"Beckwith-Wiedemann spectrum, methylation, Multilocus Imprinting Disturbances, MLID, array, MS-MLPA","lastPublishedDoi":"10.21203/rs.3.rs-6555801/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6555801/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBeckwith-Wiedemann spectrum (BWSp) is an overgrowth disorder caused by both genetic and epigenetic defects within the 11p15.5 chromosomal region. The most common cause of BWSp is DNA methylation anomalies in two imprinting control regions (ICR1, the telomeric centre that includes H19/IGF2:IG DMR and ICR2, the centromeric centre that includes KCNQ1OT1:TSS-DMR) located within the 11p15.5 locus. Previous studies demonstrated that a subset of BWSp patients had methylation defects extending beyond 11p15.5 to other chromosomal loci, an entity known as multilocus imprinting disturbances (MLID).\u003c/p\u003e \u003cp\u003eIn this study, the multilocus methylation status of 101 BWSp patients was analysed by both various methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and methylation microarrays. MS-MLPA detected MLID in 15.84% of the patients, which increased to 44.55% using methylation arrays. ICR2 hypomethylation was observed in all MLID cases, and 25 imprinted differentially methylated regions (DMRs) were additionally detected. Recurrent loci associated with the genes such as \u003cem\u003eGNAS\u003c/em\u003e, \u003cem\u003eMEST\u003c/em\u003e, and \u003cem\u003eDIRAS3\u003c/em\u003e, previously reported in MLID patients, were also observed as hypomethylated in our cohort. As eight of the 45 BWSp-MLID patients were born following assisted reproductive technology (ART), our findings highlight the increased prevalence of MLID in pregnancies conceived through ART. This study underscores the value of genome-wide methylation analyses for uncovering the molecular complexity, enhancing diagnostic accuracy, and improving prenatal care in BWSp with MLID. Future research should investigate the long-term clinical impact of MLID and the molecular mechanisms involved.\u003c/p\u003e","manuscriptTitle":"Investigation of Multilocus Imprinting Disturbance (MLID) in 101 Beckwith-Wiedemann Spectrum patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 11:21:18","doi":"10.21203/rs.3.rs-6555801/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-12T14:13:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T09:00:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T01:33:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55349814033762240092013624077541243642","date":"2025-05-05T06:54:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107494866075745993680947614627350674047","date":"2025-05-02T10:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210649443423863126347481161162860842187","date":"2025-04-30T22:27:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-30T13:24:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-30T03:26:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-30T02:00:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Epigenetics","date":"2025-04-29T11:05:02+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":"f956e16d-d1ac-42b2-b8e2-a5bfad86427c","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:07:31+00:00","versionOfRecord":{"articleIdentity":"rs-6555801","link":"https://doi.org/10.1186/s13148-026-02077-1","journal":{"identity":"clinical-epigenetics","isVorOnly":false,"title":"Clinical Epigenetics"},"publishedOn":"2026-04-26 16:00:16","publishedOnDateReadable":"April 26th, 2026"},"versionCreatedAt":"2025-05-07 11:21:18","video":"","vorDoi":"10.1186/s13148-026-02077-1","vorDoiUrl":"https://doi.org/10.1186/s13148-026-02077-1","workflowStages":[]},"version":"v1","identity":"rs-6555801","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6555801","identity":"rs-6555801","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}