Christina Votsi

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.

Dominantly inherited intronic GAA repeat expansions in the fibroblast growth factor 14 gene have recently been shown to cause spinocerebellar ataxia 27B. Currently, the pathogenic threshold of (GAA)≥300 repeat units is considered highly penetrant, while (GAA)250–299 is likely pathogenic with reduced penetrance. This study investigated the frequency of the GAA repeat expansion and the phenotypic profile in a Cypriot cohort with unresolved late-onset cerebellar ataxia. We analysed this trinucleotide repeat in 155 patients with late-onset cerebellar ataxia and 227 non-neurological disease controls. The repeat locus was examined by long-range PCR followed by fragment analysis using capillary electrophoresis, agarose gel electrophoresis and automated electrophoresis. A comprehensive comparison of all three electrophoresis techniques was conducted. Additionally, bidirectional repeat-primed PCRs and Sanger sequencing were carried out to confirm the absence of any interruptions or non-GAA motifs in the expanded alleles. The (GAA)≥250 repeat expansion was present in 12 (7.7%) patients. The average age at disease onset was 60 ± 13.5 years. The earliest age of onset was observed in a patient with a (GAA)287 repeat expansion, with ataxia symptoms appearing at 25 years of age. All patients with spinocerebellar ataxia 27B displayed symptoms of gait and appendicular ataxia. Nystagmus was observed in 41.7% of the patients, while 58.3% exhibited dysarthria. Our findings indicate that spinocerebellar ataxia 27B represents the predominant aetiology of autosomal dominant cerebellar ataxia in the Cypriot population, as this is the first dominant repeat expansion ataxia type detected in this population. Given our results and existing research, we propose including fibroblast growth factor 14 GAA repeat expansion testing as a first-tier genetic diagnostic approach for patients with late-onset cerebellar ataxia.

Background Boucher Neuhäuser Syndrome (BNS) is a rare disease with autosomal recessive inheritance defined by the classical triad; early-onset ataxia, hypogonadism and chorioretinal dystrophy. Case presentation We present two siblings diagnosed with BNS at midlife, identified with homozygous state of a novel PNPLA6 missense mutation. One healthy sibling and the mother were heterozygous carriers of the mutation. The proband presented with the classical triad and the other sibling presented with visual problems at first. The proband was referred to our department by a private Neurologist, in early adulthood, because of hypogonadism, cerebellar ataxia, axonal neuropathy, and chorioretinal dystrophy for further evaluation. The sibling was referred to our department for evaluation, at childhood, due to visual problems. Later, the patient displayed the triad of ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. The unusual medical history of the two siblings led to further examinations and eventually the diagnosis of the first BNS cases in Cyprus. WES-based ataxia in silico gene panel analysis revealed 15 genetic variants and further filtering analysis revealed the PNPLA6 c.3323G > A variant. Segregation analysis in the family with Sanger sequencing confirmed the PNPLA6 homozygous variant c.3323G > A, p.Arg1108Gln in exon 29. Conclusions This highlights the importance of considering rare inherited causes of visual loss, spinocerebellar ataxia, or/and HH in a neurology clinic and the significant role of genetic sequencing in the diagnostic process.

Background: Spastic ataxias (SAs) encompass a group of rare and severe neurodegenerative diseases, characterized by an overlap between ataxia and spastic paraplegia clinical features. They have been associated with pathogenic variants in a number of genes, including GBA2. This gene codes for the non-lysososomal β-glucosylceramidase, which is involved in sphingolipid metabolism through its catalytic role in the degradation of glucosylceramide. However, the mechanism by which GBA2 variants lead to the development of SA is still unclear. Methods: In this work, we perform next-generation RNA-sequencing (RNA-seq), in an attempt to discover differentially expressed genes (DEGs) in lymphoblastoid, fibroblast cell lines and induced pluripotent stem cell-derived neurons derived from patients with SA, homozygous for the GBA2 c.1780G > C missense variant. We further exploit DEGs in pathway analyses in order to elucidate candidate molecular mechanisms that are implicated in the development of the GBA2 gene-associated SA. Results: Our data reveal a total of 5217 genes with significantly altered expression between patient and control tested tissues. Furthermore, the most significant extracted pathways are presented and discussed for their possible role in the pathogenesis of the disease. Among them are the oxidative stress, neuroinflammation, sphingolipid signaling and metabolism, PI3K-Akt and MAPK signaling pathways. Conclusions: Overall, our work examines for the first time the transcriptome profiles of GBA2-associated SA patients and suggests pathways and pathway synergies that could possibly have a role in SA pathogenesis. Lastly, it provides a list of DEGs and pathways that could be further validated towards the discovery of disease biomarkers.

Goal: Most common diseases are influenced by multiple gene interactions and interactions with the environment. Performing an exhaustive search to identify such interactions is computationally expensive and needs to address the multiple testing problem. A four-step framework is proposed for the efficient identification of n-Way interactions. Methods: The framework was applied on a Multiple Sclerosis dataset with 725 subjects and 147 tagging SNPs. The first two steps of the framework are quality control and feature selection. The next step uses clustering and binary encodes the features. The final step performs the n-Way interaction testing. Results: The feature space was reduced to 7 SNPs and using the proposed binary encoding, more 2-SNP and 3-SNP interactions were identified compared to using the initial encoding. Conclusions: The framework selects informative features and with the proposed binary encoding it is able to identify more n-way interactions by increasing the power of the statistical analysis.

Spastic ataxia (SA) is a group of rare neurodegenerative diseases, characterized by mixed features of generalized ataxia and spasticity. The pathogenetic mechanisms that drive the development of the majority of these diseases remain unclear, although a number of studies have highlighted the involvement of mitochondrial and lipid metabolism, as well as calcium signaling. Our group has previously published the GBA2 c.1780G > C (p.Asp594His) missense variant as the cause of spastic ataxia in a Cypriot consanguineous family, and more recently the biochemical characterization of this variant in patients’ lymphoblastoid cell lines. GBA2 is a crucial enzyme of sphingolipid metabolism. However, it is unknown if GBA2 has additional functions and therefore additional pathways may be involved in the disease development. The current study introduces bioinformatics approaches to better understand the pathogenetic mechanisms of the disease. We analyzed publicly available human gene expression datasets of diseases presented with ‘ataxia’ or ‘spasticity’ in their clinical phenotype and we performed pathway analysis in order to: (a) search for candidate perturbed pathways of SA; and (b) evaluate the role of sphingolipid signaling pathway and sphingolipid metabolism in the disease development, through the identification of differentially expressed genes in patients compared to controls. Our results demonstrate consistent differential expression of genes that participate in the sphingolipid pathways and highlight alterations in the pathway level that might be associated with the disease phenotype. Through enrichment analysis, we discuss additional pathways that are connected to sphingolipid pathways, such as PI3K-Akt signaling, MAPK signaling, calcium signaling, and lipid and carbohydrate metabolism as the most enriched for ataxia and spasticity phenotypes.

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C₁₆ lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.

The advancement of scientific and medical research over the past years has generated a wealth of experimental data from multiple technologies, including genomics, transcriptomics, proteomics and other forms of -omics data, which are available for a number of diseases. The integration of such multisource data is a key component towards the success of precision medicine. In this work we are investigating a multisource data integration method developed by our group, regarding its ability to drive to clusters of connected pathways under two different approaches: (a) a disease-centric approach, where we want to integrate data around a disease, and (b) a gene-centric approach, where we want to integrate data around a gene. We have used as a paradigm for the first approach Huntington's disease (HD), a disease with a plethora of available data, whereas for the second approach the GBA2, a gene that is related to Spastic Ataxia (SA), a phenotype with sparse availability of data. Our work shows that valuable information at the level of disease-related pathway clusters can be obtained for both HD and SA. New pathways that classical pathway analysis methods were unable to reveal, are emerging as necessary “connectors” to build connected pathway stories formed as pathway clusters.

Type 2 diabetes (T2D) has been the subject of numerous genetic studies in recent years which revealed associations of the disease with a large number of susceptibility loci. We hereby initiate the evaluation of T2D susceptibility loci in the Greek-Cypriot population by performing a replication case-control study. One thousand and eighteen individuals (528 T2D patients, 490 controls) were genotyped at 21 T2D susceptibility loci, using the allelic discrimination method. Statistically significant associations of T2D with five of the tested single nucleotide polymorphisms (SNPs) (TCF7L2 rs7901695, FTO rs8050136, HHEX rs5015480, SLC30A8 rs13266634 and IGF2BP2 rs4402960) were observed in this study population. Furthermore, 14 of the tested SNPs had odds ratios (ORs) in the same direction as the previously published studies, suggesting that these variants can potentially be used in the Greek-Cypriot population for predictive testing of T2D. In conclusion, our findings expand the genetic assessment of T2D susceptibility loci and reconfirm five of the worldwide established loci in a distinct, relatively small, newly investigated population.