Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

Mowat-Wilson syndrome (MOWS) is a rare congenital disease caused by haploinsufficiency of ZEB2, encoding a transcription factor required for neurodevelopment. MOWS is characterized by intellectual disability, epilepsy, typical facial phenotype and other anomalies, such as short stature, Hirschsprung disease, brain and heart defects. Despite some recognizable features, MOWS rarity and phenotypic variability may complicate its diagnosis, particularly in the neonatal period. In order to define a novel diagnostic biomarker for MOWS, we determined the genome-wide DNA methylation profile of DNA samples from 29 individuals with confirmed clinical and molecular diagnosis. Through multidimensional scaling and hierarchical clustering analysis, we identified and validated a DNA methylation signature involving 296 differentially methylated probes as part of the broader MOWS DNA methylation profile. The prevalence of hypomethylated CpG sites agrees with the main role of ZEB2 as a transcriptional repressor, while differential methylation within the ZEB2 locus supports the previously proposed autoregulation ability. Correlation studies compared the MOWS cohort with 56 previously described DNA methylation profiles of other neurodevelopmental disorders, further validating the specificity of this biomarker. In conclusion, MOWS DNA methylation signature is highly sensitive and reproducible, providing a useful tool to facilitate diagnosis.

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.

Summary: Sézary syndrome (SS) is an aggressive cutaneous T‐cell lymphoma, characterized by erythroderma, lymphadenopathy and circulating malignant CD4 ⁺ T cells. Despite therapeutic advances, SS remains an incurable disease. Early diagnosis is therefore essential for timely therapeutic intervention; however, reliable biomarkers to identify circulating SS cells are still lacking. Recent studies highlighted dysregulation of the ectoenzymes CD39, CD73 and CD38 in SS, suggesting a potential role for these molecules in the disease. Thirty‐three patients were enrolled in this study, and CD39/CD73/CD38 expression was analysed on circulating CD4 ⁺ T cells by multiparametric flow cytometry, enabling discrimination between malignant and non‐malignant T‐cell subsets. SS cells exhibited marked CD39 or CD73 overexpression together with consistent CD38 downregulation, a pattern also confirmed in paired skin biopsy, whereas non‐malignant CD4 ⁺ T cells largely maintained expression profiles comparable to healthy controls. Genotyping further identified ENTPD1 single nucleotide polymorphism rs10748643 as a contributor to CD39 dysregulation, defining a CD39‐permissive subgroup characterized by CD39 ⁺ SS cells. In conclusion, the combined overexpression of CD39 or CD73 and loss of CD38 defines a distinct tumour‐restricted immunophenotypic signature in SS. Integrating these ectoenzymes into flow cytometry panels may improve detection of malignant CD4 ⁺ T cells across blood and skin, eventually strengthening diagnostic accuracy.

Background: SATB2-associated syndrome (SAS), also known as Glass syndrome, is a neurodevelopmental disorder (NDD) characterized by intellectual disability, developmental delay, absent or limited speech, and distinctive craniofacial and dental anomalies. It is caused by autosomal dominant pathogenic variants in the SATB2 gene, which plays a crucial role in brain, dental, and jaw development. Due to its variable phenotype, clinical diagnosis can be challenging, necessitating genetic confirmation. Methods: We present six new cases of SAS with SATB2 germline variants identified through next generation sequencing (NGS) technologies, expanding the known genetic and clinical spectrum of the syndrome. Detailed clinical phenotyping was performed for all patients. Results: Our cohort exhibits a broad range of clinical manifestations consistent with SAS, encompassing severe intellectual disability, profound speech delay, various palatal and dental abnormalities. We report the oldest adult patient (56 years old) carrying an in-frame duplication, and a pediatric patient with a missense variant who presented a significant reduction in visual acuity, likely of neurological or cortical origin, in the absence of ophthalmological abnormalities. SATB2 variants include three missenses, two in-frame deletion/duplication and one frameshift variant, several of which are novel and classified as likely pathogenic or pathogenic according to ACMG guidelines. Conclusions: This report provides new clinical and genetic insights into the landscape of SAS. Our findings confirm the phenotypic heterogeneity of SAS and highlight the critical role of comprehensive genetic testing for accurate diagnosis in NDD patients.

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.