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Abstract Background Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder caused by defects at the 11p15.5 imprinted region. Many cases of female monozygotic (MZ) twins discordant for BWS have been reported, but no definitive conclusions have been drawn regarding the link between epigenetic defects, twinning process, and gender. Here, we report a comprehensive characterization and follow‐up of female MZ twins discordant for BWS. Methods Methylation pattern at 11p15.5 and multilocus methylation disturbance (MLID) profiling were performed by pyrosequencing and MassARRAY in placental/umbilical cord samples and postnatal tissues. Whole‐exome sequencing was carried out to identify MLID causative mutations. X‐chromosome inactivation (XCI) was determined by HUMARA test. Results Both twins share KCNQ1OT1:TSS‐DMR loss of methylation (LOM) and MLID in blood and the epigenetic defect remained stable in the healthy twin over time. KCNQ1OT1:TSS‐DMRLOM was nonhomogeneously distributed in placental samples and the twins showed the same severely skewed XCI pattern. No MLID‐causative mutations were identified. Conclusion This is the first report on BWS‐discordant twins with methylation analyses extended to extraembryonic tissues. The results suggest that caution is required when attempting prenatal diagnosis in similar cases. Although the causative mechanism underlying LOM remains undiscovered, the XCI pattern and mosaic LOM suggest that both twinning and LOM/MLID occurred after XCI commitment.

Background: The VATER/VACTERL association (VACTERL) is defined as the non-random occurrence of the following congenital anomalies: Vertebral, Anal, Cardiac, Tracheal-Esophageal, Renal, and Limb anomalies. As no unequivocal candidate gene has been identified yet, patients are diagnosed phenotypically. The aims of this study were to identify patients with monogenic disorders using a genetics-first approach, and to study whether variants in candidate genes are involved in the etiology of VACTERL or the individual features of VACTERL: Anorectal malformation (ARM) or esophageal atresia with or without trachea-esophageal fistula (EA/TEF).Methods: Using molecular inversion probes, a candidate gene panel of 56 genes was sequenced in three patient groups: VACTERL (n = 211), ARM (n = 204), and EA/TEF (n = 95). Loss-of-function (LoF) and additional likely pathogenic missense variants, were prioritized and validated using Sanger sequencing. Validated variants were tested for segregation and patients were clinically re-evaluated.Results: In 7 out of the 510 patients (1.4%), pathogenic or likely pathogenic variants were identified in SALL1, SALL4, and MID1, genes that are associated with Townes-Brocks, Duane-radial-ray, and Opitz-G/BBB syndrome. These syndromes always include ARM or EA/TEF, in combination with at least two other VACTERL features. We did not identify LoF variants in the remaining candidate genes.Conclusions: None of the other candidate genes were identified as novel unequivocal disease genes for VACTERL. However, a genetics-first approach allowed refinement of the clinical diagnosis in seven patients, in whom an alternative molecular-based diagnosis was found with important implications for the counseling of the families.