Ludmila Kousoulidou

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.

Background/Objectives: X-linked intellectual disability (XLID) is a highly heterogeneous disorder accounting for ~10% of all males with ID. Next-generation sequencing (NGS) has revolutionized the discovery of causal XLID genes and variants; however, many cases remain unresolved. We present a four-generation syndromic XLID family with multiple males exhibiting variable degree of ID, focal dystonia and epilepsy. Methods: Extensive cytogenetic and targeted genetic testing was initially performed, followed by whole-exome sequencing (WES) and short-read whole-genome sequencing (WGS). Apart from the routine NGS analysis pipelines, sequencing data was revisited by focusing on poorly covered/mapped regions on chromosome X (chrX), to potentially reveal unidentified clinically relevant variants. Candidate variant validation and family segregation analysis were performed with Sanger sequencing. Results: All initial diagnostic testing was negative. Subsequently, 300 previously reported “dark” chrX coding DNA sequences, overlapping 97 genes, were cross-checked against 29 chrX genes highly associated (p < 0.05) with ID and focal dystonia, according to Phenomizer. Manual inspection of the existing NGS data in two low-coverage regions, chrX:25013469-25013696 and chrX:111744737-111744820 (hg38), revealed a recurrent pathogenic ARX variant NM_139058.3:c.441_464dup p.(Ala148_Ala155dup) (ARXdup24) associated with non-syndromic or syndromic XLID, including Partington syndrome. Sanger sequencing confirmed ARXdup24 in all affected males, with carrier status in their unaffected mothers, and absence in other unaffected relatives. Conclusions: After several years of diagnostic odyssey, the pathogenic ARXdup24 variant was unmasked, supporting a genotype–phenotype correlation in the first Partington syndrome family in Cyprus. This study highlights that re-examining underrepresented genomic regions and using phenotype-driven tools can provide critical diagnostic insights in unresolved XLID cases.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in the brain and muscle. Pathogenic variants in genes encoding VGSCs have been associated with severe disorders including epileptic encephalopathies and congenital myopathies. In this study, we identified pathogenic variants in genes encoding the α subunit of VGSCs in the fetuses of two unrelated families with the use of trio-based whole exome sequencing, as part of a larger cohort study. Sanger sequencing was performed for variant confirmation as well as parental phasing. The fetus of the first family carried a known de novo heterozygous missense variant in the SCN2A gene (NM_001040143.2:c.751G>A p.(Val251Ile)) and presented intrauterine growth retardation, hand clenching and ventriculomegaly. Neonatally, the proband also exhibited refractory epilepsy, spasms and MRI abnormalities. The fetus of the second family was a compound heterozygote for two parentally inherited novel missense variants in the SCN4A gene (NM_000334.4:c.4340T>C, p.(Phe1447Ser), NM_000334.4:c.3798G>C, p.(Glu1266Asp)) and presented a severe prenatal phenotype including talipes, fetal hypokinesia, hypoplastic lungs, polyhydramnios, ear abnormalities and others. Both probands died soon after birth. In a subsequent pregnancy of the latter family, the fetus was also a compound heterozygote for the same parentally inherited variants. This pregnancy was terminated due to multiple ultrasound abnormalities similar to the first pregnancy. Our results suggest a potentially crucial role of the VGSC gene family in fetal development and early lethality.

Pericentric inversions are among the known polymorphisms detected in the general population at a frequency of 1–2%. Despite their generally benign nature, pericentric inversions affect the reproductive potential of carriers by increasing the risk for unbalanced live-born offspring, miscarriages, or other fertility problems. Here we present a novel large pericentric inversion of chromosome 9, inv(9)(p23q22.3), detected in 30 heterozygote carriers, 24 from seven apparently unrelated families and 6 isolated patients, where the probands were mainly referred for fertility and prenatal problems. The inversion carries a significant risk for recombinant abnormal chromosomes, as in two families one supernumerary rec(9)dup(9p) and one rec(9)dup(9q) were identified, leading to neonatal death and miscarriage, respectively. The inversion carriers were identified by three different laboratories in Greece, Cyprus and Germany respectively, however all carriers have Southeast European origin. The inversion appears to be more frequent in the Greek population, as the majority of the carriers were identified in Greece. We were able to determine that the inversion is identical in all individuals included in the study by applying a combination of several methodologies, such as karyotype, fluorescence in situ hybridization (FISH), chromosomal microarrays (CMA) and haplotype analysis. In addition, haplotype analysis supports that the present inversion is identical by descent (IBD) inherited from a single common ancestor. Our results are, therefore, highly indicative of a founder effect of this inversion, presumably reflecting an event that was present in a small number of individuals that migrated to the current Southeast Europe/Northern Greece from a larger population.

Key Clinical Message: We describe a female with Rett syndrome carrying a rare de novo mosaic nonsense mutation on MECP2 gene, with random X‐chromosome inactivation. Rett syndrome severity in females depends on mosaicism level and tissue specificity, X‐chromosome inactivation, epigenetics and environment. Rett syndrome should be considered in both males and females.

Autism spectrum disorders (ASDs) comprise a distinct entity of neurodevelopmental disorders with a strong genetic component. Despite the identification of several candidate genes and causative genomic copy number variations (CNVs), the majority of ASD cases still remain unresolved. We have applied microarray-based comparative genomic hybridization (array-CGH) using Agilent 400K custom array in the first Cyprus population screening for identification of ASD-associated CNVs. A cohort of 50 ASD patients (G1), their parents (G2), 50 ethnically matched normal controls (G3), and 80 normal individuals having children with various developmental and neurological conditions (G4) were tested. As a result, 14 patients were found to carry 20 potentially causative aberrations, two of which werede novo. Comparison of the four population groups revealed an increased rate of rare disease-associated variants in normal parents of children with autism. The above data provided additional evidence, supporting the complexity of ASD aetiology in comparison to other developmental disorders involving cognitive impairment. Furthermore, we have demonstrated the rationale of a more targeted approach combining accurate clinical description with high-resolution population-oriented genomic screening for defining the role of CNVs in autism and identifying meaningful associations on the molecular level.

Genomic imbalances in locus copy-number are highly significant for the diagnosis and prognosis of cancer. Rapidly progressing DNA microarray technologies detect such pathogenic copy-number changes in the genome with high throughput, efficiency, and resolution. A variety of different microarray-based approaches have emerged, with array comparative genomic hybridization (array-CGH) being the method of choice in current clinical practice. Here we describe an alternative microarray-based technique called array-MAPH, derived from conventional Multiplex Amplifiable Probe Hybridization (MAPH). The main novelty of array-MAPH is the directed reduction of test DNA complexity prior to hybridization, yielding a mixture of specific probes, identical to target sequences on the microarray and thus increasing hybridization specificity. Unique amplifiable 400–600 bp fragments can be designed for any genomic region of interest, PCR-amplified, and spotted onto arrays as targets. The same sequences are combined into a probe mixture and hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered by quantitative PCR and hybridized to the array. Array-MAPH can be used for the detection of small-scale copy-number changes, thereby providing new insights into the genetic basis of several diseases, including cancer.

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.