Potocki-Shaffer syndrome (PSS) is a rare non-recurrent contiguous gene deletion syndrome involving chromosome 11p11.2. Current literature implies a minimal region with haploinsufficiency of three genes, ALX4 (parietal foramina), EXT2 (multiple exostoses), and PHF21A (craniofacial anomalies, and intellectual disability). The rest of the PSS phenotype is still not associated with a specific gene. We report a systematic review of the literature and included two novel cases. Because deletions are highly variable in size, we defined three groups of patients considering the PSS-genes involved. We found 23 full PSS cases (ALX4, EXT2, and PHF21A), 14 cases with EXT2-ALX4, and three with PHF21A only. Among the latter, we describe a novel male child showing developmental delay, café-au-lait spots, liner postnatal overgrowth and West-like epileptic encephalopathy. We suggest PSS cases may have epileptic spasms early in life, and PHF21A is likely to be the causative gene. Given their subtle presentation these may be overlooked and if left untreated could lead to a severe type or deterioration in the developmental plateau. If our hypothesis is correct, a timely therapy may ameliorate PSS phenotype and improve patients’ outcomes. Our analysis also shows PHF21A is a candidate for the overgrowth phenotype.

Blepharophimosis with intellectual disability (BIS) is a recently recognized disorder distinct from Nicolaides‐Baraister syndrome that presents with distinct facial features of blepharophimosis, developmental delay, and intellectual disability. BIS is caused by pathogenic variants in SMARCA2 , that encodes the catalytic subunit of the superfamily II helicase group of the BRG1 and BRM‐associated factors (BAF) forming the BAF complex, a chromatin remodeling complex involved in transcriptional regulation. Individuals bearing variants within the bipartite nuclear localization (BNL) signal domain of ADNP present with the neurodevelopmental disorder known as Helsmoortel‐Van Der Aa Syndrome (HVDAS). Distinct DNA methylation profiles referred to as episignatures have been reported in HVDAS and BAF complex disorders. Due to molecular interactions between ADNP and BAF complex, and an overlapping craniofacial phenotype with narrowing of the palpebral fissures in a subset of patients with HVDAS and BIS, we hypothesized the possibility of a common phenotype‐specific episignature. A distinct episignature was shared by 15 individuals with BIS‐causing SMARCA2 pathogenic variants and 12 individuals with class II HVDAS caused by truncating pathogenic ADNP variants. This represents first evidence of a sensitive phenotype‐specific episignature biomarker shared across distinct genetic conditions that also exhibit unique gene‐specific episignatures.

Rubinstein–Taybi syndrome (RSTS) is a rare autosomal dominant neurodevelopmental disorder linked to haploinsufficiency of CREBBP (RSTS1) and EP300 (RSTS2) genes. Characteristic features often include distinctive facial traits, broad thumbs and toes, short stature, and various degrees of intellectual disability. The clinical presentation of RSTS is notably variable, making it challenging to establish a clear genotype–phenotype correlation, except for specific variants which cause the allelic Menke–Hennekam syndrome. Trio exome analysis, data collection via networking and GeneMatcher platforms, transcript processing analysis, and DNA methylation profiling were performed. We identified two unrelated patients with de novo variants in EP300 (NM_001429.4: c.3671+5G>C; c.3671+5_3671+8delGTAA) predicted to cause in‐frame exon 20 skipping, confirmed in one patient. In silico 3D protein modeling suggested that exon 20 deletion (comprising 27 amino acids) likely alters the structural conformation between the RING_CBP‐p300 and HAT‐KAT11 domains. Clinically, both patients displayed severe RSTS2‐like clinical features, including autism spectrum disorder, speech delay, hearing loss, microcephaly, developmental delay, and intellectual disability, alongside ocular, respiratory, and cardiovascular abnormalities. Additionally, one patient developed early‐onset colorectal cancer. DNA methylation profiling in Subject #1 confirmed RSTS but did not align with the specific episignatures for RSTS1 or RSTS2. We propose that skipping of exon 20 in EP300 is associated with a distinct form of Rubinstein–Taybi syndrome featuring clinical characteristics not fully aligning with RSTS1 or RSTS2. Our findings increase the understanding of RSTS genetic and molecular basis and stress the need for further research to establish definitive genotype–phenotype correlations.

Exome sequencing (ES) has become a primary tool for diagnosing neurodevelopmental disorders (NDDs), yet the interpretation of genetic variants in large, heterogeneous cohorts presents significant challenges that automated pipelines often fail to resolve. This study showcases the complexities and novel findings derived from a decade-long analysis of 419 Italian NDD patient-parent trios. While ES established a molecular diagnosis in 36.5% of cases (53.8% in syndromic presentations), our investigation moves beyond diagnostic yield to highlight the critical value of manual curation integrated with deep phenotyping. We demonstrate how this rigorous approach uncovers complex disease mechanisms, revealing that variants initially misclassified as missense or stop-gain are, in fact, pathogenic splicing defects confirmed by functional analysis. Furthermore, we resolve the paradox of pathogenic truncating PPM1D variants in control databases by demonstrating their somatic mosaic nature, a crucial insight for population data interpretation. Our work also refines gene-disease correlations by challenging the established role of MID2 in NDDs, providing further evidence for DSCAM as a high-confidence risk gene, and expanding the known phenotypic spectrum of disorders, such as a novel GNAI2 -related syndrome lacking expected immune dysfunction. This study underscores that navigating the complexities of large NDD cohorts requires a detailed, expert-driven approach to not only enhance diagnostic yield but also to advance our fundamental understanding of rare disease genetics.

Neurodevelopmental disorders (NDDs) are among the most genetically complex human conditions, yet even in the era of routine exome sequencing (ES), a large fraction of patients remains without a molecular diagnosis. Here, we analyzed 203 unrelated families from NeuroWES‐Macedonia, the first national genomic initiative in North Macedonia, using a fully integrated diagnostic strategy that combined trio‐based ES, copy number variant (CNV) analysis, RNA studies, and DNA methylation (episignature) profiling. Rather than focusing solely on diagnostic yield, we systematically examined recurrent interpretative failure modes that lead to missed, delayed, or incomplete diagnoses in routine clinical genomics. Likely pathogenic or pathogenic variants were identified in 29.6% (60/203) of probands, with diagnostic yield rising to 62.3% (38/61) in syndromic NDDs. However, many of the most instructive cases lay beyond conventional Mendelian expectations. We uncovered cryptic splicing defects evade in silico prediction, low‐level parental and proband mosaicism, multilocus genomic disease, and inherited pathogenic variants concealed by subtle or unrecognized parental phenotypes. Epigenomic profiling proved particularly powerful in resolving cases involving chromosomal abnormalities, mosaic aneuploidies, and overlapping syndromes, allowing us to disentangle primary from secondary molecular drivers. Beyond improving diagnostic understanding, analysis of this cohort expanded the mutational and phenotypic spectra of known NDD genes and yielded novel candidate disease genes, several of which were independently validated through international data sharing. Importantly, multiple cases demonstrated how rigid inheritance assumptions, reliance on de novo filtering, and compartmentalized analysis of SNVs and CNVs can actively obscure true disease mechanisms—even when high‐quality sequencing data are available. Together, our findings show that the persistent diagnostic gap in NDDs is driven not only by missing genes but also by systematic blind spots in variant interpretation. Integrating multiomic data with phenotype‐driven, flexible analytic frameworks is essential to unlock the full potential of genomic medicine in NDDs.