Background: Kleefstra syndrome 1(KLEFS1, OMIM#610253) is a rare neurodevelopmental disorder (NDD) instigated by heterozygous variants or microdeletions occurring in the 9q34.4 genomic region of the euchromatic histone methyltransferase-1 (EHMT1) gene and is inherited in an autosomal dominant (AD) manner. The clinical phenotype of KLEFS1 includes moderate to severe intellectual disability (ID), hypotonia, and distinctive facial features and additionally involves other organ systems (heart, renal, genitourinary, sensory) albeit with phenotypic heterogeneity between patients. The purpose of this study is to expand the genotypic spectrum of KLEFS1 and compare phenotypic features of the syndrome of already published cases. Methods: Exome sequencing (ES), chromosomal microarray analysis (CMA), as well as sanger sequencing, for confirmation of the de novo status of the frameshift variant, were used. Results: Here we describe two more cases, both males with a similar age and carriers of novel variants; one with a frameshift variant involving exon 13: p.Val692Glyfs*64 and the other with the smallest so far described, 11 Kb (exons 19-25), 9q34.4 microdeletion: 9q34.3 (140703393-140714454). Both presented with an NDD disorder with one showing more severe ID with significant social disabilities, while the other with the microdeletion had mild ID and following a normal education curriculum. Neither of them were obese nor had any other significant organ system disorder. Conclusions: The observed phenotypic variability due to genotypic differences in the two children contributes to the expanding spectrum of KLEFS1 disease phenotypes.

BACKGROUND:Tuberous Sclerosis Complex (TSC) is a rare, multisystem genetic disorder with highly variable clinical manifestations. While international registries such as TOSCA have provided large-scale data, national-level studies remain limited. This study represents the first national cohort analysis of TSC patients in Greece, providing comprehensive insights into clinical characteristics, genotype-phenotype correlations, and previously underreported rare manifestations. METHODS:A descriptive analysis was conducted on 115 TSC patients diagnosed based on the latest criteria. Clinical, genetic, and treatment-related data were analysed, with a particular focus on neurological, renal, cardiac, dermatological, and pulmonary manifestations, as well as rare or atypical disease presentations. RESULTS:The median age at diagnosis was 1.2 years (range: 0-43 years). Epilepsy was the most frequent initial symptom (70.4 %), with drug-resistant epilepsy (DRE) affecting 39.5 % of cases. Intellectual disability, autism spectrum disorder, and behavioral issues correlated significantly with early seizure onset and TSC2 variants. Common manifestations include cortical tubers (93.9 %), subependymal nodules (92.2 %), angiomyolipomas (48.7 %), and cardiac rhabdomyoma (33 %). Notably, we report several rare manifestations, including high-grade glioma in a pediatric patient, diffuse lipomatosis, pancreatic neuroendocrine tumours, rectal polyps, and erythema nodosum presented in a patient on everolimus therapy, further highlighting the systemic complexity and malignancy risks in TSC. CONCLUSIONS:Our study provides novel epidemiological and clinical data on TSC in Greece, reinforcing genotype-phenotype correlations and expanding the spectrum of rare manifestations. These findings emphasise the need for lifelong surveillance, multidisciplinary management, and early detection strategies to mitigate long-term complications. This study also contributes to the broader understanding of TSC by documenting atypical presentations that may inform future clinical guidelines and patient care strategies.

Can whole exome sequencing (WES) represent a reliable approach for PGT of monogenic disorders, aneuploidy and structural rearrangements, providing comprehensive genomic information from embryos? WES-based PGT (PGT-WES) has demonstrated high accuracy in detecting inherited and de novo pathogenic variants, aneuploidy, and microdeletion/microduplication syndromes, providing unparalleled insights into embryo genetics. High-throughput sequencing technologies, such as WES, have greatly enhanced diagnostic precision in prenatal testing, leading to the discovery of numerous pathogenic genetic variants. However, its application to PGT—analyzing DNA from few-cells biopsied from embryos—remains challenging. This study aimed to validate and clinically implement a universal WES-based PGT approach enabling all forms of PGT in a single assay. This comprehensive method empowers the simultaneous detection of inherited monogenic conditions and chromosomal abnormalities, including aneuploidies, segmental imbalances, with enhanced resolution for identifying microdeletion/microduplication syndromes. Importantly, it also enables screening for de novo variants and hundreds of severe monogenic diseases. This prospective study involved a double blind parallel evaluation of WES alongside conventional PGT methods (Minisequencing, fluorescent-PCR, shallow Next Generation Sequencing-NGS). A total of 226 blastocysts from 33 clinical PGT-M cycles (with or without PGT-A) were analyzed. The consistency of PGT-WES was assessed by comparing its results with diagnoses obtained using standard PGT methods. The aim was to evaluate the accuracy of PGT-WES in testing embryos generated from couples at risk of monogenic disorders. Blastocyst-stage embryos were biopsied, and DNA from the biopsied cells underwent whole-genome amplification followed by simultaneous analysis via WES and standard PGT methods. A custom bioinformatics pipeline, aligned with GATK4 best practices, was developed for copy number variant (CNV) analysis, variant calling and filtering, and error correction using unique molecular identifiers. This pipeline enabled the detection of aneuploidies, sub-chromosomal CNVs, inherited and de novo single-nucleotide variants causing monogenic disorders, and single-nucleotide polymorphisms for linkage-based assessments. A total of 226 blastocysts were analyzed for 20 genetic conditions and 36 unique variants using PGT-WES. The results showed a high level of concordance with standard PGT methods. Outcomes for PGT-M were fully consistent across all embryos with conclusive results (217/217, 100%) and matched the predicted haplotypes. WES reliably detected the presence or absence of target mutations and uncovered additional genetic variations at numerous loci associated with monogenic disorders, without prior knowledge of parental genetic status. PGT-WES also detected 5 de novo pathogenic mutations linked to severe early-onset genetic conditions and identified 8 embryos affected by inherited diseases caused by mutations from unrecognized carrier parents. Additionally, WES resolved 9 cases where standard PGT methods provided inconclusive results. The CNV detection capability of WES matched the performance of standalone PGT-A assays, accurately identifying both whole-chromosome and segmental aneuploidies. Ploidy status was 100% (58/58) concordant across all embryos screened for PGT-A. Pathogenic CNVs >200 kb associated with parental microdeletion/microduplication syndromes were reliably detected in 24 blastocysts deriving from 4 PGT cases, owing to the enhanced resolution of PGT-WES. To date 10 live births confirmed the results from PGT-WES diagnosis. While this study demonstrates the technical feasibility and accuracy of WES-based PGT in preventing numerous inherited and de novo genetic diseases, further research and broader clinical applications are necessary to refine its role in PGT and validate its clinical effectiveness. This novel PGT approach provides an unprecedented level of insight into embryo genetics. By enabling simultaneous detection of aneuploidy, inherited and de novo gene mutations, and structural chromosomal abnormalities, WES-based PGT has the potential to revolutionize the field. No

The human 16p11.2 chromosomal region is rich in segmental duplications which mediate the formation of recurrent CNVs. CNVs affecting the 16p11.2 region are associated with an increased risk for developing neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia, and intellectual disability (ID), as well as abnormal body weight and head circumference and dysmorphic features, with marked phenotypic variability and reduced penetrance. CNVs affecting the 16p11.2 region mainly affect a distal interval of ~220 Kb, between Breakpoints 2 and 3 (BP2–BP3), and a proximal interval of ~593 Kb (BP4–BP5). Here, we report on 15 patients with recurrent 16p11.2 rearrangements that were identified among a cohort of 1600 patients (0.9%) with neurodevelopmental disorders. A total of 13 deletions and two duplications were identified, of which eight deletions included the proximal 16p11.2 region (BP4–BP5) and five included the distal 16p11.2 region (BP2–BP3). Of the two duplications that were identified, one affected the proximal and one the distal 16p11.2 region; however, both patients had additional CNVs contributing to phenotypic severity. The features observed and their severity varied greatly, even between patients within the same family. This article aims to further delineate the clinical spectrum of patients with 16p11.2 recurrent rearrangements in order to aid the counselling of patients and their families.

Nablus mask-like facial syndrome (NMLFS) is a rare condition characterized by unique facial features, initially described in a 4-year-old boy from Nablus, Palestine. These features include expressionless facial appearance, tight facial skin, blepharophimosis, sparse eyebrows, and a flat nose. Genetic studies have identified a deletion of 8q22.1 as the cause of the syndrome, however while 26 patients have been reported with the deletion, only 13 displayed the characteristic facial features. Here we report on a 35-year-old male with 8q21.3-q22.1 deletion identified by whole exome sequencing and Chromosomal microarray analysis (CMA) that presents with typical and atypical features, including neurodevelopmental disorder, mild facial features, and myopathy, which has not been described in a patient with NMLFS to date. Further research will be required to understand the underlying pathogenetic mechanism of this rare genetic disorder.

OBJECTIVES:Genetics of epilepsy are highly heterogeneous and complex. Lesions detected involve genes encoding various types of channels, transcription factors, and other proteins implicated in numerous cellular processes, such as synaptogenesis. Consequently, a wide spectrum of clinical presentations and overlapping phenotypes hinders differential diagnosis and highlights the need for molecular investigations toward delineation of underlying mechanisms and final diagnosis. Characterization of defects may also contribute valuable data on genetic landscapes and networks implicated in epileptogenesis. METHODS:This study reports on genetic findings from exome sequencing (ES) data of 107 patients with variable types of seizures, with or without additional symptoms, in the context of neurodevelopmental disorders. RESULTS:Multidisciplinary evaluation of ES, including ancillary detection of copy number variants (CNVs) with the ExomeDepth tool, supported a definite diagnosis in 59.8% of the patients, reflecting one of the highest diagnostic yields in epilepsy. CONCLUSION:Emerging advances of next-generation technologies and 'in silico' analysis tools offer the possibility to simultaneously detect several types of variations. Wide assessment of variable findings, specifically those found to be novel and least expected, reflects the ever-evolving genetic landscape of seizure development, potentially beneficial for increased opportunities for trial recruitment and enrollment, and optimized, even personalized, medical management.

Whole-Exome Sequencing (WES) has proven valuable in the characterization of underlying genetic defects in most rare diseases (RDs). Copy Number Variants (CNVs) were initially thought to escape detection. Recent technological advances enabled CNV calling from WES data with the use of accurate and highly sensitive bioinformatic tools. Amongst 920 patients referred for WES, 454 unresolved cases were further analysed using the ExomeDepth algorithm. CNVs were called, evaluated and categorized according to ACMG/ClinGen recommendations. Causative CNVs were identified in 40 patients, increasing the diagnostic yield of WES from 50.7% (466/920) to 55% (506/920). Twenty-two CNVs were available for validation and were all confirmed; of these, five were novel. Implementation of the ExomeDepth tool promoted effective identification of phenotype-relevant and/or novel CNVs. Among the advantages of calling CNVs from WES data, characterization of complex genotypes comprising both CNVs and SNVs minimizes cost and time to final diagnosis, while allowing differentiation between true or false homozygosity, as well as compound heterozygosity of variants in AR genes. The use of a specific algorithm for calling CNVs from WES data enables ancillary detection of different types of causative genetic variants, making WES a critical first-tier diagnostic test for patients with RDs.

Chromosomal microarray analysis (CMA) is considered a first-tier test for patients with developmental disabilities and congenital anomalies and is also routinely applied in prenatal diagnosis. The current consensus size cut-off for reporting copy number variants (CNVs) in the prenatal setting ranges from 200 Kb to 400 Kb, with the intention of minimizing the impact of variants of uncertain significance (VUS). Very limited data are currently available on the application of higher resolution platforms prenatally. The aim of this study is to investigate the feasibility and impact of applying high-resolution CMA in the prenatal setting. To that end, we report on the outcomes of applying CMA with a size cut-off of 20 Kb in 250 prenatal samples and discuss the findings and diagnostic yield and also provide follow-up for cases with variants of uncertain significance. Overall, 19.6% (49) showed one or more chromosomal abnormalities, with the findings classified as Pathogenic (P) or Likely Pathogenic (LP) in 15.6% and as VUS in 4%. When excluding the cases with known familial aberrations, the diagnostic yield was 12%. The smallest aberration detected was a 32 Kb duplication of the 16p11.2 region. In conclusion, this study demonstrates that prenatal diagnosis with a high-resolution aCGH platform can reliably detect smaller CNVs that are often associated with neurodevelopmental phenotypes while providing an increased diagnostic yield, regardless of the indication for testing, with only a marginal increase in the VUS incidence. Thus, it can be an important tool in the prenatal setting.

Persistent hyperCKemia results from muscle dysfunction often attributed to genetic alterations of muscle-related genes, such as the dystrophin gene (DMD). Retrospective assessment of findings from DMD analysis, in association with persistent HyperCKemia, was conducted. Evaluation of medical records from 1354 unrelated cases referred during the period 1996–2021. Assessment of data concerning the detection of DMD gene rearrangements and nucleotide variants. A total of 730 individuals (657 cases, 569 of Greek and 88 of Albanian origins) were identified, allowing an overall estimation of dystrophinopathy incidence at ~1:3800 live male births. The heterogeneous spectrum of 275 distinct DMD alterations comprised exon(s) deletions/duplications, nucleotide variants, and rare events, such as chromosome translocation {t(X;20)}, contiguous gene deletions, and a fused gene involving the DMD and the DOCK8 genes. Ethnic-specific findings include a common founder variant in exon 36 (‘Hellenic’ variant). Some 50% of hyperCKemia cases were characterized as dystrophinopathies, highlighting that DMD variants may be considered the most common cause of hyperCKemia in Greece. Delineation of the broad genetic and clinical heterogeneity is fundamental for actionable public health decisions and theragnosis, as well as the establishment of guidelines addressing ethical considerations, especially related to the mild asymptomatic patient subgroup.

PURPOSE:To investigate whether preimplantation genetic testing for aneuploidy (PGT-A) improves the clinical outcome in patients with advanced maternal age (AMA), recurrent miscarriages (RM), and recurrent implantation failure (RIF).METHODS:Retrospective cohort study from a single IVF center and a single genetics laboratory. One hundred seventy-six patients undergoing PGT-A were assigned to three groups: an AMA group, an RM group, and a RIF group. Two hundred seventy-nine patients that did not undergo PGT-A were used as controls and subgrouped similarly to the PGT-A cohort. For the PGT-A groups, trophectoderm biopsy was performed and array comparative genomic hybridization was used for PGT-A. Clinical outcomes were compared with the control groups.RESULTS:In the RM group, we observed a significant decrease of early pregnancy loss rates in the PGT-A group (18.1% vs 75%) and a significant increase in live birth rate per transfer (50% vs 12.5%) and live birth rate per patient (36% vs 12.5%). In the RIF group, a statistically significant increase in the implantation rate per transfer (69.5% vs 33.3%) as well as the live birth rate per embryo transfer (47.8% vs 19%) was observed. In the AMA group, a statistically significant reduction in biochemical pregnancy loss was observed (3.7% vs 31.5%); however, live birth rates per embryo transfer and per patient were not significantly higher than the control group.CONCLUSION:Our results agree with recently published studies, which suggest caution in the universal application of PGT-A in women with infertility. Instead, a more personalized approach by choosing the right candidates for PGT-A intervention should be followed.