Transcriptomic analysis reveals sex-specific patterns in the hippocampus in Alzheimer's disease.

Background: The hippocampus, vital for memory and learning, is among the first brain regions affected in Alzheimer's Disease (AD) and exhibits adult neurogenesis. Women face twice the risk of developing AD compare to men, making it crucial to understand sex differences in hippocampal function for comprehending AD susceptibility. Methods: We conducted a comprehensive analysis of bulk mRNA postmortem samples from the whole hippocampus (GSE48350, GSE5281) and its CA1 and CA3 subfields (GSE29378). Our aim was to perform a comparative molecular signatures analysis, investigating sex-specific differences and similarities in the hippocampus and its subfields in AD. This involved comparing the gene expression profiles among: (a) male controls (M-controls) vs. female controls (F-controls), (b) females with AD (F-AD) vs. F-controls, (c) males with AD (M-AD) vs. M-controls, and (d) M-AD vs. F-AD. Furthermore, we identified AD susceptibility genes interacting with key targets of menopause hormone replacement drugs, specifically the ESR1 and ESR2 genes, along with GPER1. Results: The hippocampal analysis revealed contrasting patterns between M-AD vs. M-controls and F-AD vs. F-controls, as well as M-controls vs. F-controls. Notably, BACE1, a key enzyme linked to amyloid-beta production in AD pathology, was found to be upregulated in M-controls compared to F-controls in both CA1 and CA3 hippocampal subfields. In M-AD vs. M-controls, the GABAergic synapse was downregulated, and the Estrogen signaling pathway was upregulated in both subfields, unlike in F-AD vs. F-controls. Analysis of the whole hippocampus also revealed upregulation of the GABAergic synapse in F-AD vs. F-controls. While direct comparison of M-AD vs. F-AD, revealed a small upregulation of the ESR1 gene in the CA1 subfield of males. Conversely, F-AD vs. F-controls exhibited downregulation of the Dopaminergic synapse in both subfields, while the Calcium signaling pathway showed mixed regulation, being upregulated in CA1 but downregulated in CA3, unlike in M-AD vs. M-controls. The upregulated Estrogen signaling pathway in M-AD, suggests a compensatory response to neurodegenerative specifically in males with AD. Our results also identified potential susceptibility genes interacting with ESR1 and ESR2, including MAPK1, IGF1, AKT1, TP53 and CD44. Conclusion: These findings underscore the importance of sex-specific disease mechanisms in AD pathogenesis. Region-specific analysis offers a more detailed examination of localized changes in the hippocampus, enabling to capture sex-specific molecular patterns in AD susceptibility and progression.

RFC1 Repeat Distribution in the Cypriot Population: Study of a Large Cohort of Patients With Undiagnosed Ataxia and Non-Disease Controls.

Background and Objectives: The intronic biallelic AAGGG expansion in the replication factor C subunit 1 (RFC1) gene was recently associated with a phenotype combining cerebellar ataxia, neuropathy, and vestibular areflexia syndrome, as well as with late-onset ataxia. Following this discovery, studies in multiple populations extended the phenotypic and genotypic spectrum of this locus. Multiple benign and additional pathogenic configurations are currently known. Our main objectives were to study the prevalence of the pathogenic AAGGG expansion in the Cypriot population, to further characterize the RFC1 repeat locus allele distribution, and to search for possible novel repeat configurations. Methods: Cypriot undiagnosed patients, in the majority presenting at least with cerebellar ataxia and non-neurologic disease controls, were included in this study. A combination of conventional methods was used, including standard PCR flanking the repeat region, repeat-primed PCR, long-range PCR, and Sanger sequencing. Bioinformatics analysis of already available in-house short-read whole-genome sequencing data was also performed. Results: A large group of undiagnosed patients (n = 194), mainly presenting with pure ataxia or with ataxia accompanied by neuropathy or additional symptoms, as well as a group of non-disease controls (n = 100), were investigated in the current study. Our findings include the diagnosis of 10 patients homozygous for the pathogenic AAGGG expansion and a high percentage of heterozygous AAGGG carriers in both groups. The benign AAAAGn, AAAGGn, and AAGAGn configurations were also identified in our cohorts. We also report and discuss the identification of 2 recently reported novel and possibly benign repeat configurations, AAAGGGn and AAGACn, thus confirming their existence in another distinct population, and we highlight an increased frequency of the AAAGGGn in the patient group, including a single case of homozygosity. Discussion: Our findings indicate the existence of genetic heterogeneity regarding the RFC1 repeat configurations and that the AAGGG pathogenic expansion is a frequent cause of ataxia in the Cypriot population.

The influence of environmental risk factors in the development of ALS in the Mediterranean Island of Cyprus.

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating, uniformly lethal degenerative disease of motor neurons, presenting with relentlessly progressive muscle atrophy and weakness. The etiology of ALS remains unexplained for over 85% of all cases, suggesting that besides the genetic basis of the disease, various environmental factors are implicated in the pathogenesis of ALS. This study aimed to investigate the contribution of known environmental risk factors of ALS in the Cypriot population. Methods: We conducted a case-control study with a total of 56 ALS cases and 56 healthy gender/age-matched controls of Cypriot nationality. Demographic, lifestyle characteristics, medical conditions, and environmental exposures were collected through the use of a detailed questionnaire. Statistical analyses using the R programming language examined the association between the above environmental factors and ALS. Results: A chi-square test analysis revealed a statistically significant (p = 0.000461) difference in smoking status between the two groups. In addition, univariate logistic regression analysis showed a statistically significant association between ALS cases for head trauma/injury (p = 0.0398) and exposure to chemicals (p = 0.00128), compared to controls. Conclusion: This case-control investigation has shed some light on the epidemiological data of ALS in Cyprus, by identifying environmental determinants of ALS, such as smoking, head trauma, and chemical exposure, in the Cypriot population.

Unraveling the transcriptomic signatures of Parkinson's disease and major depression using single-cell and bulk data.

Background: Motor symptoms are well-characterized in Parkinson's disease (PD). However, non-motor symptoms, such as depression, are commonly observed and can appear up to 10 years before motor features, resulting in one-third of individuals being misdiagnosed with a neuropsychiatric disorder. Thus, identifying diagnostic biomarkers is crucial for accurate PD diagnosis during its prodromal or early stages. Methods: We employed an integrative approach, combining single nucleus RNA and bulk mRNA transcriptomics to perform comparative molecular signatures analysis between PD and major depressive disorder (MDD). We examined 39,834 nuclei from PD (GSE202210) and 32,707 nuclei from MDD (GSE144136) in the dorsolateral prefrontal cortex (dlPFC) of Brodmann area 9. Additionally, we analyzed bulk mRNA peripheral blood samples from PD compared to controls (GSE49126, GSE72267), as well as MDD compared to controls (GSE39653). Results: Our findings show a higher proportion of astrocytes, and oligodendrocyte cells in the dlPFC of individuals with PD vs. MDD. The excitatory to inhibitory neurons (E/I) ratio analysis indicates that MDD has a ratio close to normal 80/20, while PD has a ratio of 62/38, indicating increased inhibition in the dlPFC. Microglia displayed the most pronounced differences in gene expression profiles between the two conditions. In PD, microglia display a pro-inflammatory phenotype, while in MDD, they regulate synaptic transmission through oligodendrocyte-microglia crosstalk. Analysis of bulk mRNA blood samples revealed that the COL5A, MID1, ZNF148, and CD22 genes were highly expressed in PD, whereas the DENR and RNU1G2 genes were highly expressed in MDD. CD22 is involved in B-cell activation and the negative regulation of B-cell receptor signaling. Additionally, CD86, which provides co-stimulatory signals for T-cell activation and survival, was found to be a commonly differentially expressed gene in both conditions. Pathway analysis revealed several immune-related pathways common in both conditions, including the complement and coagulation cascade, and B-cell receptor signaling. Discussion: This study demonstrates that bulk peripheral immune cells play a role in both conditions, but neuroinflammation in the dlPFC specifically manifests in PD as evidenced by the analysis of single nucleus dlPFC datasets. Integrating these two omics levels offers a better understanding of the shared and distinct molecular pathophysiology of PD and MDD in both the periphery and the brain. These findings could lead to potential diagnostic biomarkers, improving accuracy and guiding pharmacological treatments.

PheWAS and cross-disorder analysis reveal genetic architecture, pleiotropic loci and phenotypic correlations across 11 autoimmune disorders.

Introduction: Autoimmune disorders (ADs) are a group of about 80 disorders that occur when self-attacking autoantibodies are produced due to failure in the self-tolerance mechanisms. ADs are polygenic disorders and associations with genes both in the human leukocyte antigen (HLA) region and outside of it have been described. Previous studies have shown that they are highly comorbid with shared genetic risk factors, while epidemiological studies revealed associations between various lifestyle and health-related phenotypes and ADs. Methods: Here, for the first time, we performed a comparative polygenic risk score (PRS) - Phenome Wide Association Study (PheWAS) for 11 different ADs (Juvenile Idiopathic Arthritis, Primary Sclerosing Cholangitis, Celiac Disease, Multiple Sclerosis, Rheumatoid Arthritis, Psoriasis, Myasthenia Gravis, Type 1 Diabetes, Systemic Lupus Erythematosus, Vitiligo Late Onset, Vitiligo Early Onset) and 3,254 phenotypes available in the UK Biobank that include a wide range of socio-demographic, lifestyle and health-related outcomes. Additionally, we investigated the genetic relationships of the studied ADs, calculating their genetic correlation and conducting cross-disorder GWAS meta-analyses for the observed AD clusters. Results: In total, we identified 508 phenotypes significantly associated with at least one AD PRS. 272 phenotypes were significantly associated after excluding variants in the HLA region from the PRS estimation. Through genetic correlation and genetic factor analyses, we identified four genetic factors that run across studied ADs. Cross-trait meta-analyses within each factor revealed pleiotropic genome-wide significant loci. Discussion: Overall, our study confirms the association of different factors with genetic susceptibility for ADs and reveals novel observations that need to be further explored.

Investigation of in-phase bilateral exercise effects on corticospinal plasticity in relapsing remitting multiple sclerosis: A registered report single-case concurrent multiple baseline design across five subjects.

Relapsing-remitting Multiple Sclerosis is the most common demyelinating neurodegenerative disease and is characterized by periods of relapses and generation of various motor symptoms. These symptoms are associated with the corticospinal tract integrity, which is quantified by means of corticospinal plasticity which can be probed via transcranial magnetic stimulation and assessed with corticospinal excitability measures. Several factors, such as exercise and interlimb coordination, can influence corticospinal plasticity. Previous work in healthy and in chronic stroke survivors showed that the greatest improvement in corticospinal plasticity occurred during in-phase bilateral exercises of the upper limbs. During in-phase bilateral movement, both upper limbs are moving simultaneously, activating the same muscle groups and triggering the same brain region respectively. Altered corticospinal plasticity due to bilateral cortical lesions is common in MS, yet, the impact of these type of exercises in this cohort is unclear. The aim of this concurrent multiple baseline design study is to investigate the effects of in-phase bilateral exercises on corticospinal plasticity and on clinical measures using transcranial magnetic stimulation and standardized clinical assessment in five people with relapsing-remitting MS. The intervention protocol will last for 12 consecutive weeks (30-60 minutes /session x 3 sessions/week) and include in-phase bilateral movements of the upper limbs, adapted to different sports activities and to functional training. To define functional relation between the intervention and the results on corticospinal plasticity (central motor conduction time, resting motor threshold, motor evoked potential amplitude and latency) and on clinical measures (balance, gait, bilateral hand dexterity and strength, cognitive function), we will perform a visual analysis and if there is a potential sizeable effect, we will perform statistical analysis. A possible effect from our study, will introduce a proof-of-concept for this type of exercise that will be effective during disease progression. Trial registration: ClinicalTrials.gov NCT05367947.

Integrated Bioinformatics Analysis of Shared Genes, miRNA, Biological Pathways and Their Potential Role as Therapeutic Targets in Huntington's Disease Stages.

Huntington's Disease (HD) is a progressive neurodegenerative disease caused by CAG repeat expansion in the huntingtin gene (HTT). The HTT gene was the first disease-associated gene mapped to a chromosome, but the pathophysiological mechanisms, genes, proteins or miRNAs involved in HD remain poorly understood. Systems bioinformatics approaches can divulge the synergistic relationships of multiple omics data and their integration, and thus provide a holistic approach to understanding diseases. The purpose of this study was to identify the differentially expressed genes (DEGs), HD-related gene targets, pathways and miRNAs in HD and, more specifically, between the pre-symptomatic and symptomatic HD stages. Three publicly available HD datasets were analysed to obtain DEGs for each HD stage from each dataset. In addition, three databases were used to obtain HD-related gene targets. The shared gene targets between the three public databases were compared, and clustering analysis was performed on the common shared genes. Enrichment analysis was performed on (i) DEGs identified for each HD stage in each dataset, (ii) gene targets from the public databases and (iii) the clustering analysis results. Furthermore, the hub genes shared between the public databases and the HD DEGs were identified, and topological network parameters were applied. Identification of HD-related miRNAs and their gene targets was obtained, and a miRNA-gene network was constructed. Enriched pathways identified for the 128 common genes revealed pathways linked to multiple neurodegeneration diseases (HD, Parkinson's disease, Spinocerebellar ataxia), MAPK and HIF-1 signalling pathways. Eighteen HD-related hub genes were identified based on network topological analysis of MCC, degree and closeness. The highest-ranked genes were FoxO3 and CASP3, CASP3 and MAP2 were found for betweenness and eccentricity and CREBBP and PPARGC1A were identified for the clustering coefficient. The miRNA-gene network identified eleven miRNAs (mir-19a-3p, mir-34b-3p, mir-128-5p, mir-196a-5p, mir-34a-5p, mir-338-3p, mir-23a-3p and mir-214-3p) and eight genes (ITPR1, CASP3, GRIN2A, FoxO3, TGM2, CREBBP, MTHFR and PPARGC1A). Our work revealed that various biological pathways seem to be involved in HD either during the pre-symptomatic or symptomatic stages of HD. This may offer some clues for the molecular mechanisms, pathways and cellular components underlying HD and how these may act as potential therapeutic targets for HD.

Investigating the Dietary Intake Using the CyFFQ Semi-Quantitative Food Frequency Questionnaire in Cypriot Huntington's Disease Patients.

Huntington's disease (HD) is a rare progressive neurodegenerative disease characterised by autosomal dominant inheritance. The past decade saw a growing interest in the associations between the Mediterranean Diet (MD) and HD risk and outcomes. The aim of this case-control study was to assess the dietary intake and habits of Cypriot HD patients, comparing them to gender and age-matched controls, using the Cyprus Food Frequency Questionnaire (CyFFQ) and to assess adherence to the MD by disease outcomes. The method relied on the validated CyFFQ semi-quantitative questionnaire to assess energy, macro- and micronutrient intake over the past year in n = 36 cases and n = 37 controls. The MedDiet Score and the MEDAS score were used to assess adherence to the MD. Patients were grouped based on symptomatology such as movement and cognitive and behavioral impairment. The two-sample Wilcoxon rank-sum (Mann-Whitney) test was used to compare cases vs. controls. Statistically significant results were obtained for energy intake (kcal/day) (median (IQR): 4592 (3376) vs. 2488 (1917); p = 0.002) from cases and controls. Energy intake (kcal/day) (median (IQR): 3751 (1894) vs. 2488 (1917); p = 0.044) was also found to be significantly different between asymptomatic HD patients and controls. Symptomatic patients were also different from controls in terms of energy intake (kcal/day) (median (IQR): 5571 (2907) vs. 2488 (1917); p = 0.001); % energy monounsaturated fatty acids (median (IQR): 13.4 (5.2) vs. 15.5 (5.7); p = 0.0261) and several micronutrients. A significant difference between asymptomatic and symptomatic HD patients was seen in the MedDiet score (median (IQR): 31.1 (6.1) vs. 33.1 (8.1); p = 0.024) and a significant difference was observed between asymptomatic HD patient and controls (median (IQR): 5.5 (3.0) vs. 8.2 (2.0); p = 0.014) in the MEDAS score. This study confirmed previous findings that HD cases have a significantly higher energy intake than controls, revealing differences in macro and micronutrients and adherence to the MD by both patients and controls and by HD symptom severity. These findings are important as they are an effort to guide nutritional education within this population group and further understand diet-disease associations.

Genetic Study of Early Onset Parkinson's Disease in Cyprus.

Parkinson's Disease (PD) is a multifactorial neurodegenerative disease characterized by motor and non-motor symptoms. The etiology of PD remains unclear. However, several studies have demonstrated the interplay of genetic, epigenetic, and environmental factors in PD. Early-onset PD (EOPD) is a subgroup of PD diagnosed between the ages of 21 and 50. Population genetic studies have demonstrated great genetic variability amongst EOPD patients. Hence, this study aimed to obtain a genetic landscape of EOPD in the Cypriot population. Greek-Cypriot EOPD patients (n = 48) were screened for variants in the six most common EOPD-associated genes (PINK1, PRKN, FBXO7, SNCA, PLA2G6, and DJ-1). This included DNA sequencing and Multiplex ligation-dependent probe amplification (MLPA). One previously described frameshift variant in PINK1 (NM_032409.3:c.889del) was detected in five patients (10.4%)-the largest number to be detected to date. Copy number variations in the PRKN gene were identified in one homozygous and 3 compound heterozygous patients (8.3%). To date, the pathogenic variants identified in this study have explained the PD phenotype for 18.8% of the EOPD cases. The results of this study may contribute to the genetic screening of EOPD in Cyprus.

Serum miRNAs as biomarkers for the rare types of muscular dystrophy.

Muscular dystrophies are a group of disorders that cause progressive muscle weakness. There is an increasing interest for the development of biomarkers for these disorders and specifically for Duchene Muscular Dystrophy. Limited research however, has been performed on the biomarkers' development for the most rare muscular dystrophies, like the Facioscapulohumeral Muscular Dystrophy, Limb-Girdle Muscular Dystrophy and Myotonic Dystrophy type 2. Here, we aimed to identify novel serum-based miRNA biomarkers for these rare muscular dystrophies, through high-throughput next-generation RNA sequencing. We identified many miRNAs that associate with muscular dystrophy patients compared to controls. Based on a series of selection criteria, the two best candidate miRNAs for each of these disorders were chosen and validated in a larger number of patients. Our results showed that miR-223-3p and miR-206 are promising serum-based biomarkers for Facioscapulohumeral Muscular Dystrophy type 1, miR-143-3p and miR-486-3p for Limb-Girdle Muscular Dystrophy type 2A whereas miR-363-3p and miR-25-3p associate with Myotonic Dystrophy type 2. Some of the identified miRNAs were significantly elevated in the serum of the patients compared to controls, whereas some others were lower. In conclusion, we provide new evidence that certain circulating miRNAs may be used as biomarkers for three types of rare muscular dystrophies.

Circulating small RNA signatures differentiate accurately the subtypes of muscular dystrophies: small-RNA next-generation sequencing analytics and functional insights.

Muscular dystrophies are a group of rare and severe inherited disorders mainly affecting the muscle tissue. Duchene Muscular Dystrophy, Myotonic Dystrophy types 1 and 2, Limb Girdle Muscular Dystrophy and Facioscapulohumeral Muscular Dystrophy are some of the members of this family of disorders. In addition to the current diagnostic tools, there is an increasing interest for the development of novel non-invasive biomarkers for the diagnosis and monitoring of these diseases. miRNAs are small RNA molecules characterized by high stability in blood thus making them ideal biomarker candidates for various diseases. In this study, we present the first genome-wide next-generation small RNA sequencing in serum samples of five different types of muscular dystrophy patients and healthy individuals. We identified many small RNAs including miRNAs, lncRNAs, tRNAs, snoRNAs and snRNAs, that differentially discriminate the muscular dystrophy patients from the healthy individuals. Further analysis of the identified miRNAs showed that some miRNAs can distinguish the muscular dystrophy patients from controls and other miRNAs are specific to the type of muscular dystrophy. Bioinformatics analysis of the target genes for the most significant miRNAs and the biological role of these genes revealed different pathways that the dysregulated miRNAs are involved in each type of muscular dystrophy investigated. In conclusion, this study shows unique signatures of small RNAs circulating in five types of muscular dystrophy patients and provides a useful resource for future studies for the development of miRNA biomarkers in muscular dystrophies and for their involvement in the pathogenesis of the disorders.

Transcriptomic characterization of tissues from patients and subsequent pathway analyses reveal biological pathways that are implicated in spastic ataxia.

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 ß-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.

Myasthenia gravis genome-wide association study implicates AGRN as a risk locus.

BACKGROUND: Myasthenia gravis (MG) is a rare autoimmune disorder affecting the neuromuscular junction (NMJ). Here, we investigate the genetic architecture of MG via a genome-wide association study (GWAS) of the largest MG data set analysed to date. METHODS: We performed GWAS meta-analysis integrating three different data sets (total of 1401 cases and 3508 controls). We carried out human leucocyte antigen (HLA) fine-mapping, gene-based and tissue enrichment analyses and investigated genetic correlation with 13 other autoimmune disorders as well as pleiotropy across MG and correlated disorders. RESULTS: We confirmed the previously reported MG association with TNFRSF11A (rs4369774; p=1.09×10-13, OR=1.4). Furthermore, gene-based analysis revealed AGRN as a novel MG susceptibility gene. HLA fine-mapping pointed to two independent MG loci: HLA-DRB1 and HLA-B. MG onset-specific analysis reveals differences in the genetic architecture of early-onset MG (EOMG) versus late-onset MG (LOMG). Furthermore, we find MG to be genetically correlated with type 1 diabetes (T1D), rheumatoid arthritis (RA), late-onset vitiligo and autoimmune thyroid disease (ATD). Cross-disorder meta-analysis reveals multiple risk loci that appear pleiotropic across MG and correlated disorders. DISCUSSION: Our gene-based analysis identifies AGRN as a novel MG susceptibility gene, implicating for the first time a locus encoding a protein (agrin) that is directly relevant to NMJ activation. Mutations in AGRN have been found to underlie congenital myasthenic syndrome. Our results are also consistent with previous studies highlighting the role of HLA and TNFRSF11A in MG aetiology and the different risk genes in EOMG versus LOMG. Finally, we uncover the genetic correlation of MG with T1D, RA, ATD and late-onset vitiligo, pointing to shared underlying genetic mechanisms.

A Novel CLN6 Variant Associated With Juvenile Neuronal Ceroid Lipofuscinosis in Patients With Absence of Visual Loss as a Presenting Feature.

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of autosomal recessive lysosomal storage disorders that are characterized by neurodegeneration, progressive cognitive decline, motor impairment, ataxia, loss of vision, seizures, and premature death. To date, pathogenic variants in more than 13 genes have been associated with NCLs. CLN6 encodes an endoplasmic reticulum non-glycosylated transmembrane protein, which is involved in lysosomal acidification. Mutations in CLN6 cause late-infantile juvenile NCL (JNCL) adult-onset NCL, and Kufs disease. Members from two available families with JNCL were clinically evaluated, and samples were collected from consenting individuals. The molecular investigation was performed by whole-exome sequencing, Sanger sequencing, and family segregation analysis. Furthermore, in silico prediction analysis and structural modeling of the identified CLN6 variants were performed. We report clinical and genetic findings of three patients from two Greek-Cypriot families (families 915 and 926) with JNCL. All patients were males, and the first symptoms appeared at the age of 6 years. The proband of family 926 presented with loss of motor abilities, ataxia, spasticity, seizure, and epilepsy. The proband of family 915 had ataxia, spasticity, dysarthria, dystonia, and intellectual disability. Both probands did not show initial signs of vision and/or hearing loss. Molecular analysis of family 926 revealed two CLN6 biallelic variants: the novel, de novo p.Tyr295Cys and the known p.Arg136His variants. In family 915, both patients were homozygous for the p.Arg136His CLN6 variant. Prediction analysis of the two CLN6 variants characterized them as probably damaging and disease-causing. Structural modeling of the variants predicted that they probably cause protein structural differentiation. In conclusion, we describe two unrelated Cypriot families with JNCL. Both families had variants in the CLN6 gene; however, they presented with slightly different symptoms, and notably none of the patients has loss of vision. In silico prediction and structural analyses indicate that both variants are most likely pathogenic.

miR-223-3p and miR-24-3p as novel serum-based biomarkers for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, primarily characterized by muscle wasting and weakness. Many biomarkers already exist in the rapidly developing biomarker research field that aim to improve patients' care. Limited work, however, has been performed on rare diseases, including DM1. We have previously shown that specific microRNAs (miRNAs) can be used as potential biomarkers for DM1 progression. In this report, we aimed to identify novel serum-based biomarkers for DM1 through high-throughput next-generation sequencing. A number of miRNAs were identified that are able to distinguish DM1 patients from healthy individuals. Two miRNAs were selected, and their association with the disease was validated in a larger panel of patients. Further investigation of miR-223-3p, miR-24-3p, and the four previously identified miRNAs, miR-1-3p, miR-133a-3p, miR-133b-3p, and miR-206-3p, showed elevated levels in a DM1 mouse model for all six miRNAs circulating in the serum compared to healthy controls. Importantly, the levels of miR-223-3p, but not the other five miRNAs, were found to be significantly downregulated in five skeletal muscles and heart tissues of DM1 mice compared to controls. This result provides significant evidence for its involvement in disease manifestation.

Prediction of Parkinson's Disease Risk Based on Genetic Profile and Established Risk Factors.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder, and literature suggests that genetics and lifestyle/environmental factors may play a key role in the triggering of the disease. This study aimed to evaluate the predictive performance of a 12-Single Nucleotide Polymorphisms (SNPs) polygenic risk score (PRS) in combination with already established PD-environmental/lifestyle factors. METHODS: Genotypic and lifestyle/environmental data on 235 PD-patients and 464 controls were obtained from a previous study carried out in the Cypriot population. A PRS was calculated for each individual. Univariate logistic-regression analysis was used to assess the association of PRS and each risk factor with PD-status. Stepwise-regression analysis was used to select the best predictive model for PD combining genetic and lifestyle/environmental factors. RESULTS: The 12-SNPs PRS was significantly increased in PD-cases compared to controls. Furthermore, univariate analyses showed that age, head injury, family history, depression, and Body Mass Index (BMI) were significantly associated with PD-status. Stepwise-regression suggested that a model which includes PRS and seven other independent lifestyle/environmental factors is the most predictive of PD in our population. CONCLUSIONS: These results suggest an association between both genetic and environmental factors and PD, and highlight the potential for the use of PRS in combination with the classical risk factors for risk prediction of PD.

A Novel SPG7 Gene Pathogenic Variant in a Cypriot Family With Autosomal Recessive Spastic Ataxia.

The SPG7 gene encodes the paraplegin protein, an inner mitochondrial membrane-localized protease. It was initially linked to pure and complicated hereditary spastic paraplegia with cerebellar atrophy, and now represents a frequent cause of undiagnosed cerebellar ataxia and spastic ataxia. We hereby report the molecular characterization and the clinical features of a large Cypriot family with five affected individuals presenting with spastic ataxia in an autosomal recessive transmission mode, due to a novel SPG7 homozygous missense variant. Detailed clinical histories of the patients were obtained, followed by neurological and neurophysiological examinations. Whole exome sequencing (WES) of the proband, in silico gene panel analysis, variant filtering and family segregation analysis of the candidate variants with Sanger sequencing were performed. RNA and protein expression as well as in vitro protein localization studies and mitochondria morphology evaluation were carried out towards functional characterization of the identified variant. The patients presented with typical spastic ataxia features while some intrafamilial phenotypic variation was noted. WES analysis revealed a novel homozygous missense variant in the SPG7 gene (c.1763C > T, p. Thr588Met), characterized as pathogenic by more than 20 in silico prediction tools. Functional studies showed that the variant does not affect neither the RNA or protein expression, nor the protein localization. However, aberrant mitochondrial morphology has been observed thus indicating mitochondrial dysfunction and further demonstrating the pathogenicity of the identified variant. Our study is the first report of an SPG7 pathogenic variant in the Cypriot population and broadens the spectrum of SPG7 pathogenic variants.

Investigating the Transition of Pre-Symptomatic to Symptomatic Huntington's Disease Status Based on Omics Data.

Huntington's disease is a rare neurodegenerative disease caused by a cytosine-adenine-guanine (CAG) trinucleotide expansion in the Huntingtin (HTT) gene. Although Huntington's disease (HD) is well studied, the pathophysiological mechanisms, genes and metabolites involved in HD remain poorly understood. Systems bioinformatics can reveal synergistic relationships among different omics levels and enables the integration of biological data. It allows for the overall understanding of biological mechanisms, pathways, genes and metabolites involved in HD. The purpose of this study was to identify the differentially expressed genes (DEGs), pathways and metabolites as well as observe how these biological terms differ between the pre-symptomatic and symptomatic HD stages. A publicly available dataset from the Gene Expression Omnibus (GEO) was analyzed to obtain the DEGs for each HD stage, and gene co-expression networks were obtained for each HD stage. Network rewiring, highlights the nodes that change most their connectivity with their neighbors and infers their possible implication in the transition between different states. The CACNA1I gene was the mostly highly rewired node among pre-symptomatic and symptomatic HD network. Furthermore, we identified AF198444 to be common between the rewired genes and DEGs of symptomatic HD. CNTN6, DEK, LTN1, MST4, ZFYVE16, CEP135, DCAKD, MAP4K3, NUPL1 and RBM15 between the DEGs of pre-symptomatic and DEGs of symptomatic HD and CACNA1I, DNAJB14, EPS8L3, HSDL2, SNRPD3, SOX12, ACLY, ATF2, BAG5, ERBB4, FOCAD, GRAMD1C, LIN7C, MIR22, MTHFR, NABP1, NRG2, OTC, PRAMEF12, SLC30A10, STAG2 and Y16709 between the rewired genes and DEGs of pre-symptomatic HD. The proteins encoded by these genes are involved in various biological pathways such as phosphatidylinositol-4,5-bisphosphate 3-kinase activity, cAMP response element-binding protein binding, protein tyrosine kinase activity, voltage-gated calcium channel activity, ubiquitin protein ligase activity, adenosine triphosphate (ATP) binding, and protein serine/threonine kinase. Additionally, prominent molecular pathways for each HD stage were then obtained, and metabolites related to each pathway for both disease stages were identified. The transforming growth factor beta (TGF-ß) signaling (pre-symptomatic and symptomatic stages of the disease), calcium (Ca2+) signaling (pre-symptomatic), dopaminergic synapse pathway (symptomatic HD patients) and Hippo signaling (pre-symptomatic) pathways were identified. The in silico metabolites we identified include Ca2+, inositol 1,4,5-trisphosphate, sphingosine 1-phosphate, dopamine, homovanillate and L-tyrosine. The genes, pathways and metabolites identified for each HD stage can provide a better understanding of the mechanisms that become altered in each disease stage. Our results can guide the development of therapies that may target the altered genes and metabolites of the perturbed pathways, leading to an improvement in clinical symptoms and hopefully a delay in the age of onset.

Dietary Intake, Mediterranean Diet Adherence and Caloric Intake in Huntington's Disease: A Review.

Decades of research and experimental studies have investigated Huntington's disease (HD), a rare neurodegenerative disease. Similarly, several studies have investigated whether high/moderate adherence to the Mediterranean Diet and specific macro and micronutrients can decrease cognitive loss and provide a neuroprotective function to neurons. This review systematically identifies and examines studies that have investigated Mediterranean Diet adherence, micro- and macronutrients, supplementation and caloric intake in people with HD, in order to identify if dietary exposures resulted in improvement of disease symptoms, a delay in age of onset or if they contributed to an earlier age of onset in people with HD. A systematic search of PubMed, Directory of open access journal and HubMed was performed independently by two reviewers using specific search terms criteria for studies. The identified abstracts were screened and the studies were included in the review if they satisfied predetermined inclusion criteria. Reference screening of included studies was also performed. A total of 18 studies were included in the review. A few studies found that patients who had high/moderate adherence to Mediterranean Diet showed a slight improvement in their Unified Huntington's Disease Rating Scale and Total Functional Capacity. In addition, people with HD who had high Mediterranean Diet adherence showed an improvement in both cognitive and motor scores and had a better quality of life compared to patients who had low Mediterranean Diet adherence. Furthermore, a few studies showed that supplementation with specific nutrients, such as triheaptanoin, L-acetyl-carnitine and creatine, had no beneficial effect on the patients' Unified Huntington's Disease Rating Scale score. A few studies suggest that the Mediterranean Diet may confer a motor and cognitive benefit to people with HD. Unfortunately, there was little consistency among study findings. It is important for more research to be conducted to have a better understanding of which dietary exposures are beneficial and may result delaying age of onset or disease progression in people with HD.