This study provides the first systematic, mutational-level characterization of a Cypriot Mendelian disease cohort, establishing a local baseline diagnostic yield and revealing a high proportion of novel variants that reflect the underrepresentation of Eastern Mediterranean populations in global databases.
Abstract
Background/Objectives: Congenital anomalies and neurodevelopmental disorders frequently co-occur and exhibit substantial genetic and phenotypic heterogeneity, posing a persistent diagnostic challenge. Exome sequencing has become an important first- or second-tier diagnostic tool for these conditions, yet diagnostic yields vary considerably depending on phenotype, ancestry, sequencing strategy, and interpretation, with over half of referrals remaining without a definitive genetic diagnosis. Methods: We analyzed data from 692 patients referred to the Department of Cytogenetics and Genomics at the Cyprus Institute of Neurology and Genetics between January 2021 and December 2025 for clinical exome sequencing or whole-exome sequencing as part of the diagnostic work-up for congenital disorders and/or syndromic or non-syndromic neurodevelopmental disorders. Results: A total of 134 distinct variants were identified, corresponding to an overall diagnostic yield of 17.9% out of which 52 (38.8%) were novel, and 50 variants (37.3%) were de novo, as expected from the high proportion of severe neurodevelopmental presentations. Missense variants were the most prevalent within our cohort, while chromatin and transcriptional regulator genes constituted the largest functional gene category, followed by variants in collagen-encoding genes. Conclusions: This study provides the first systematic, mutational-level characterization of a Cypriot Mendelian disease cohort, establishing a local baseline diagnostic yield and revealing a high proportion of novel variants that reflect the underrepresentation of Eastern Mediterranean populations in global databases. These findings underscore the value of submitting population-specific variants to public repositories and of phenotype-driven reanalysis targeting recurrent gene families, supporting more efficient diagnostics and future precision medicine initiatives in Cyprus.
Chromosomal microarray analysis (CMA) is a first-tier diagnostic tool for children with neurodevelopmental disorders and congenital anomalies; however, interpretation of variants of uncertain significance (VUS) remains challenging. This study aimed to characterize the copy number variant (CNV) spectrum in pediatric patients with congenital anomalies and/or dysmorphic features, focusing on VUS interpretation through segregation analysis and detailed phenotype–genotype correlation. We retrospectively evaluated 28 pediatric patients with abnormal CMA results referred to two tertiary genetics clinics between 2021 and 2022. Indications included neurodevelopmental delay, intellectual disability, dysmorphism, and/or congenital anomalies. CNVs were classified according to 2020 ACMG/ClinGen standards. Parental segregation analysis was performed for 15 CNVs in 13 families, with particular attention to gene disruption caused by CNV breakpoints. The cohort included 16 males and 12 females, with a mean age of 5.28±4.39 years. Thirty-four CNVs were identified, including 14 deletions and 20 duplications; seven were pathogenic/likely pathogenic and 27 were VUS. Segregation analysis identified five de novo, six maternal, and four paternal variants. A 2q22.2 gain disrupting KYNU at intron 12 was identified in a patient with VACTERL-like features. Three patients had additional karyotypic abnormalities, highlighting the complementary role of CMA. A de novo 4p16.3 duplication disrupting both HTT and ADD1 was also identified. Our findings highlight the importance of detailed phenotyping, breakpoint analysis, and segregation studies in interpreting CNVs, particularly VUS and rare intragenic disruptions. Integrating genomic findings with clinical features and inheritance patterns may improve phenotype–genotype correlation and support the identification of candidate loci.
BACKGROUND
Orofacial clefts (OFCs) are the most frequent congenital craniofacial anomalies that occur during embryonic development. The incidence is ~ 1 in 700 live births, and it may occur in isolation or with other abnormalities, such as limb deformities. Congenital limb malformations are the second most prevalent birth defect, affecting 1 per 500 to 1000 live births. It can also occur in isolation or as part of a syndrome. This study investigated the genetic etiology of OFCs co-occurring with limb abnormalities in a Sub-Saharan African cohort.
METHODS
Nine unrelated probands with concurrent OFC and limb anomalies were recruited, including one multiplex family involving an affected mother and proband. Whole exome sequencing (WES) was performed at 100X on DNA samples from affected families, utilising a paired-end configuration on the Illumina HiSeq platform. Variant calling utilized the Sentieon workflow. Rare, deleterious variants were identified in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines on variant classification. De novo and other variants predicted as pathogenic were prioritized based on all possible Mendelian inheritance patterns, including variable penetrance and expressivity. Pathway enrichment analysis, protein-protein interactions, and gene expression analysis were undertaken to decipher the biological functions of implicated genes.
RESULTS
All cases were syndromic, presenting with preaxial and postaxial limb anomalies along with other craniofacial features. WES revealed plausible pathogenic variants in pleiotropic genes (TP63, NIPBL, MYH3, and FGFR2) in four simplex cases. In four other simplex probands, multiple rare variants were identified in developmentally relevant genes (e.g., RGPD5, FAM90A26, FOXD4L1, FAM170A, TRIM74, TRIM73, and PRDM9) necessary for normal craniofacial and limb development. The multiplex family had two affected individuals (the mother and the proband), both carrying a TP63 variant, consistent with autosomal dominant inheritance with variable expressivity. Most of the observed variants were de novo, with some being novel. Functional genomic analysis supported the craniofacial relevance of the implicated genes.
CONCLUSION
While some cases can be attributed to single-gene syndromes (e.g., NIPBL-associated Cornelia de Lange Syndrome), others may result from multiple co-occurring syndromes. These findings may inform recurrence risk estimates, genetic counselling, and clinical management if validated across multiple populations.
Edna Tackie, S. Obiri-Yeboah, Gideon Okyere Mensah et al.· BMC Medical Genomics· 0 citations
In FTD and ALS, these results support universal access to genetic testing independent of age at onset or family history, and provide a clear diagnostic advantage in NDDs marked by substantial clinical and genetic overlap.
Emma Ehn, H. Thonberg, Inger Nennesmo et al.· Human Molecular Genetics· 0 citations
Autism spectrum disorder (ASD) is genetically heterogeneous, involving rare and common variants that disrupt neurodevelopmental pathways. To explore this complexity, we performed whole-exome sequencing in children with ASD. Clinical phenotypes were systematically recorded, and severity was classified according to DSM-5 criteria. Variants were interpreted using ACMG guidelines, with recurrence analysis to identify genes shared across individuals and cohort enrichment testing against gnomAD. To examine genotype-phenotype relationships, we applied SKAT/SKAT-O across 16 phenotypes after covariate adjustment. Among 25 included individuals, pathogenic or likely pathogenic variants were found in 9, yielding a diagnostic yield of 36%. These involved genes linked to neurodevelopmental, epileptic, metabolic, and syndromic disorders. Recurrence analysis identified 586 genes present in at least two individuals, with PABPC1, GTF2I, PCLO, PKD1, and EP400 being the most frequent. SKAT/SKAT-O revealed the strongest burden associations for motor delay, aggressive behavior, mutism, anxiety, unresponsiveness to spoken voice, digestive disorder, and sleep disturbances, with limited overlap across phenotypes. Several recurrent genes also showed phenotype-specific associations. Overall, this integrative WES study provides clinically actionable diagnoses, highlights recurrent genes, and uncovers phenotype-specific signals, supporting convergent pathways with gene-level heterogeneity.
Zainab Gaouzi, Giulia Spoto, F. Polito et al.· International Journal of Mol...· 0 citations
This study showcases the complexities and novel findings derived from a decade-long analysis of 419 Italian NDD patient-parent trios, and underscores that navigating the complexities of large NDD cohorts requires a detailed, expert-driven approach to enhance diagnostic yield.
Simona Cardaropoli, Lisa Pavinato, Slavica Trajkova et al.· Human Genetics· 0 citations
A high prevalence of rare (mainly de novo) genetic variants in individuals with severe and sporadic DLD is revealed, and extensive molecular overlap with other neurodevelopmental disorders is demonstrated.
Milou G. P. Kennis, Leenke van Haaften, Karen van Hulst et al.· European Journal of Human Ge...· 0 citations
