Study of Alzheimer's disease- and frontotemporal dementia-associated genes in the Cretan Aging Cohort.

Using exome sequencing, we analyzed 196 participants of the Cretan Aging Cohort (CAC; 95 with Alzheimer's disease [AD], 20 with mild cognitive impairment [MCI], and 81 cognitively normal controls). The APOE ε4 allele was more common in AD patients (23.2%) than in controls (7.4%; p < 0.01) and the PSEN2 p.Arg29His and p.Cys391Arg variants were found in 3 AD and 1 MCI patient, respectively. Also, we found the frontotemporal dementia (FTD)-associated TARDBP gene p.Ile383Val variant in 2 elderly patients diagnosed with AD and in 2 patients, non CAC members, with the amyotrophic lateral sclerosis/FTD phenotype. Furthermore, the p.Ser498Ala variant in the positively selected GLUD2 gene was less frequent in AD patients (2.11%) than in controls (16%; p < 0.01), suggesting a possible protective effect. While the same trend was found in another local replication cohort (n = 406) and in section of the ADNI cohort (n = 808), this finding did not reach statistical significance and therefore it should be considered preliminary. Our results attest to the value of genetic testing to study aged adults with AD phenotype.

High Hereditary Transthyretin-Related Amyloidosis Prevalence in Crete: Genetic Heterogeneity and Distinct Phenotypes.

Background and Objectives: Our goal was to study hereditary transthyretin-related amyloidosis (hATTR) in Crete, Greece. Methods: We aimed at ascertaining all hATTR cases in Crete, an island of 0.62 million people. For this, we evaluated patients with polyneuropathy, autonomic involvement, cardiomyopathy, and/or ophthalmopathy suggestive of hATTR, who presented to the physicians of this study or were referred to them by other physicians. Genetic analyses were performed on all patients suspected of suffering from hATTR. We included in our observational longitudinal cohort study all individuals, residents of Crete, who, during the study period (1993-2019), were found to carry a pathogenic TTR variant. Results: Over the past 27 years, 30 individuals (15 female patients, 15 male patients), from 12 apparently unrelated families, were diagnosed with hATTR, whereas evaluation of their offspring identified 5 asymptomatic TTR pathogenic variant carriers. The most prevalent TTR variant detected was p.Val50Met, affecting 19 patients (11 female patients, 8 male patients) and causing a rather consistent phenotype characterized by predominant polyneuropathy of early adult onset (median age of symptom onset: 30 years; range: 18-37 years). Specifically, patients affected by the p.Val50Met TTR variant experienced progressive sensorimotor disturbances, involving mainly the lower extremities, associated with autonomic and/or gastrointestinal dysfunction. The second most frequent TTR variant was p.Val114Ala, found in 10 patients (4 female patients, 6 male patients) who were affected at an older age (median age of symptom onset: 70 years; range: 54-78 years). This variant caused a predominantly cardiomyopathic phenotype, manifested by congestive heart failure and associated with peripheral neuropathy, carpal tunnel syndrome, and/or autonomic involvement. In these patients, cardiac amyloid deposition was detected on 99m-technetium pyrophosphate scintigraphy and/or heart biopsy. The third TTR variant (p.Arg54Gly) was found in a 50-year-old male patient with ophthalmopathy due to vitreous opacities and positive family history for visual loss. As 22 patients were alive at the end of the study, we calculated the hATTR prevalence in Crete to be 35 cases per 1 million inhabitants. Discussion: Our study revealed that the prevalence of hATTR in Crete is one of the world's highest. Three different pathogenic TTR variants causing distinct clinical phenotypes were identified in this relatively small population pool.

Genetic cause of epilepsy in a Greek cohort of children and young adults with heterogeneous epilepsy syndromes.

We describe a cohort of 10 unrelated Greek patients (4 females, 6 males; median age 6.5 years, range 2-18 years) with heterogeneous epilepsy syndromes with a genetic basis. In these patients, causative genetic variants, including two novel ones, were identified in 9 known epilepsy-related genes through whole exome sequencing. A patient with glycine encephalopathy was a compound heterozygote for the p.Arg222Cys and the p.Ser77Leu AMT variant. A patient affected with Lafora disease carried the homozygous p.Arg171His EPM2A variant. A de novo heterozygous variant in the GABRG2 gene (p.Pro282Thr) was found in one patient and a pathogenic variant in the GRIN2B gene (p.Gly820Val) in another patient. Infantile-onset lactic acidosis with seizures was associated with the p.Arg446Ter PDHX gene variant in one patient. In two additional epilepsy patients, the p.Ala1662Val and the novel non-sense p.Phe1330Ter SCN1A gene variants were found. Finally, in 3 patients we observed a novel heterozygous missense variant in SCN2A (p.Ala1874Thr), a heterozygous splice site variant in SLC2A1 (c.517-2A>G), as a cause of Glut1 deficiency syndrome, and a pathogenic variant in STXBP1 (p.Arg292Leu), respectively. In half of our cases (patients with variants in the GRIN2B, SCN1A, SCN2A and SLC2A1 genes), a genetic cause with potential management implications was identified.

Genotyping and Plasma/Cerebrospinal Fluid Profiling of a Cohort of Frontotemporal Dementia-Amyotrophic Lateral Sclerosis Patients.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of the same pathophysiological spectrum and have common genetic and cerebrospinal fluid (CSF) biomarkers. Our aim here was to identify causative gene variants in a cohort of Greek patients with FTD, ALS and FTD-ALS, to measure levels of CSF biomarkers and to investigate genotype-phenotype/CSF biomarker associations. In this cohort of 130 patients (56 FTD, 58 ALS and 16 FTD-ALS), we performed C9orf72 hexanucleotide repeat expansion analysis, whole exome sequencing and measurement of "classical" (Aß42, total tau and phospho-tau) and novel (TDP-43) CSF biomarkers and plasma progranulin. Through these analyses, we identified 14 patients with C9orf72 repeat expansion and 11 patients with causative variants in other genes (three in TARDBP, three in GRN, three in VCP, one in FUS, one in SOD1). In ALS patients, we found that levels of phospho-tau were lower in C9orf72 repeat expansion and MAPT c.855C>T (p.Asp285Asp) carriers compared to non-carriers. Additionally, carriers of rare C9orf72 and APP variants had lower levels of total tau and Aß42, respectively. Plasma progranulin levels were decreased in patients carrying GRN pathogenic variants. This study expands the genotypic and phenotypic spectrum of FTD/ALS and offers insights in possible genotypic/CSF biomarker associations.

Apolipoprotein E ɛ4 (APOE ɛ4) Allele is Associated with Long Sleep Duration Among Elderly with Cognitive Impairment.

BACKGROUND: Apolipoprotein E gene (APOE) ɛ4 allele increases the risk for Alzheimer's disease (AD). Furthermore, among patients with cognitive impairment, longer sleep duration is associated with worse cognitive performance. To date, literature examining the associations between APOE ɛ4 allele and objective sleep duration is limited. OBJECTIVE: Our aim was to assess the association between APOE ɛ4 and objective sleep duration, among patients with mild cognitive impairment (MCI) and AD. A sub-sample of 89 patients with AD (n = 49) and MCI (n = 40) were recruited from a large, population-based cohort of 3,140 elders (>60 years) residing on Crete, Greece. METHODS: All participants underwent medical history/physical examination, extensive neuropsychiatric and neuropsychological evaluation, 3-day 24 h actigraphy and APOE ɛ4 allele genotyping. Comparisons of sleep duration variables between APOE ɛ4 allele carriers and non-carriers were assessed using ANCOVA, controlling for confounders. RESULTS: The sample included 18 APOE ɛ4 carriers and 71 non-carriers, aged 78.6±6.6 and 78.2±6.5 years, respectively. Comparisons between the APOE ɛ4 carriers and non-carriers revealed no significant differences in terms of demographic and clinical variables. In terms of objective sleep duration across the two groups, APOE ɛ4 carriers compared to non-carriers had significantly longer nighttime Total Sleep Time (nTST) (7.7±1.4 versus 7.2±1.3  h, respectively, p = 0.011), as well as 24 h TST (8.5±1.6 versus 7.8±1.5  h, respectively, p = 0.012). CONCLUSION: Among patients with MCI and AD, APOE ɛ4 carriers have longer objective nighttime and 24 h sleep duration compared to non-carriers. These findings further support that objective long sleep duration is a genetically-driven pre-clinical marker associated with worse prognosis in elderly with cognitive impairment.

Cognitive Impairment and Dementia in Primary Care: Current Knowledge and Future Directions Based on Findings From a Large Cross-Sectional Study in Crete, Greece.

Introduction: Dementia severely affects the quality of life of patients and their caregivers; however, it is often not adequately addressed in the context of a primary care consultation, especially in patients with multi-morbidity. Study Population and Methods: A cross-sectional study was conducted between March-2013 and December-2014 among 3,140 consecutive patients aged >60 years visiting 14 primary health care practices in Crete, Greece. The Mini-Mental-State-Examination [MMSE] was used to measure cognitive status using the conventional 24-point cut-off. Participants who scored low on MMSE were matched with a group of elders scoring >24 points, according to age and education; both groups underwent comprehensive neuropsychiatric and neuropsychological assessment. For the diagnosis of dementia and Mild-Cognitive-Impairment (MCI), the Diagnostic and Statistical Manual-of-Mental-Disorders (DSM-IV) criteria and the International-Working-Group (IWG) criteria were used. Chronic conditions were categorized according to ICD-10 categories. Logistic regression was used to provide associations between chronic illnesses and cognitive impairment according to MMSE scores. Generalized Linear Model Lasso Regularization was used for feature selection in MMSE items. A two-layer artificial neural network model was used to classify participants as impaired (dementia/MCI) vs. non-impaired. Results: In the total sample of 3,140 participants (42.1% men; mean age 73.7 SD = 7.8 years), low MMSE scores were identified in 645 (20.5%) participants. Among participants with low MMSE scores 344 (54.1%) underwent comprehensive neuropsychiatric evaluation and 185 (53.8%) were diagnosed with Mild-Cognitive-Impairment (MCI) and 118 (34.3%) with dementia. Mental and behavioral disorders (F00-F99) and diseases of the nervous system (G00-G99) increased the odds of low MMSE scores in both genders. Generalized linear model lasso regularization indicated that 7/30 MMSE questions contributed the most to the classification of patients as impaired (dementia/MCI) vs. non-impaired with a combined accuracy of 82.0%. These MMSE items were questions 5, 13, 19, 20, 22, 23, and 26 of the Greek version of MMSE assessing orientation in time, repetition, calculation, registration, and visuo-constructive ability. Conclusions: Our study identified certain chronic illness-complexes that were associated with low MMSE scores within the context of primary care consultation. Also, our analysis indicated that seven MMSE items provide strong evidence for the presence of dementia or MCI.

Genetic cause of heterogeneous inherited myopathies in a cohort of Greek patients.

Inherited muscle disorders are caused by pathogenic changes in numerous genes. Herein, we aimed to investigate the etiology of muscle disease in 24 consecutive Greek patients with myopathy suspected to be genetic in origin, based on clinical presentation and laboratory and electrophysiological findings and absence of known acquired causes of myopathy. Of these, 16 patients (8 females, median 24 years-old, range 7 to 67 years-old) were diagnosed by Whole Exome Sequencing as suffering from a specific type of inherited muscle disorder. Specifically, we have identified causative variants in 6 limb-girdle muscular dystrophy genes (6 patients; ANO5, CAPN3, DYSF, ISPD, LAMA2, SGCA), 3 metabolic myopathy genes (4 patients; CPT2, ETFDH, GAA), 1 congenital myotonia gene (1 patient; CLCN1), 1 mitochondrial myopathy gene (1 patient; MT-TE) and 3 other myopathy-associated genes (4 patients; CAV3, LMNA, MYOT). In 6 additional family members affected by myopathy, we reached genetic diagnosis following identification of a causative variant in an index patient. In our patients, genetic diagnosis ended a lengthy diagnostic process and, in the case of Multiple acyl-CoA dehydrogenase deficiency and Pompe's disease, it enabled specific treatment to be initiated. These results further expand the genotypic and phenotypic spectrum of inherited myopathies.

Localization of Human Glutamate Dehydrogenases Provides Insights into Their Metabolic Role and Their Involvement in Disease Processes.

Glutamate dehydrogenase (GDH) catalyzes the reversible deamination of L-glutamate to α-ketoglutarate and ammonia. In mammals, GDH contributes to important processes such as amino acid and carbohydrate metabolism, energy production, ammonia management, neurotransmitter recycling and insulin secretion. In humans, two isoforms of GDH are found, namely hGDH1 and hGDH2, with the former being ubiquitously expressed and the latter found mainly in brain, testis and kidney. These two iso-enzymes display highly divergent allosteric properties, especially concerning their basal activity, ADP activation and GTP inhibition. On the other hand, both enzymes are thought to predominantly localize in the mitochondrial matrix, even though alternative localizations have been proposed. To further study the subcellular localization of the two human iso-enzymes, we created HEK293 cell lines stably over-expressing hGDH1 and hGDH2. In these cell lines, immunofluorescence and enzymatic analyses verified the overexpression of both hGDH1 and hGDH2 iso-enzymes, whereas subcellular fractionation followed by immunoblotting showed their predominantly mitochondrial localization. Given that previous studies have only indirectly compared the subcellular localization of the two iso-enzymes, we co-expressed them tagged with different fluorescent dyes (green and red fluorescent protein for hGDH1 and hGDH2, respectively) and found them to co-localize. Despite the wealth of information related to the functional properties of hGDH1 and hGDH2 and the availability of the hGDH1 structure, there is still an ongoing debate concerning their metabolic role and their involvement in disease processes. Data on the localization of hGDHs, as the ones presented here, could contribute to better understanding of the function of these important human enzymes.