Ioannis Zaganas

Introduction/Aims: Muscle‐specific kinase myasthenia gravis (MuSK‐MG) is an autoimmune neuromuscular disorder predominantly mediated by IgG4 autoantibodies disrupting MuSK signaling. The contribution of other isotypes remains incompletely defined. We characterized the serological profile of a Greek cohort of MuSK‐MG patients. Methods: Baseline ( n = 140) and longitudinal (available n = 99) samples from 140 patients, positive for anti‐MuSK by radioimmunoprecipitation (RIPA) were analyzed using a live cell‐based assay (L‐CBA) to detect total IgG, IgG1–4 subclasses, IgM, and IgA. Also, disease and healthy controls ( n = 102) were included. Selected samples were additionally analyzed by flow cytometry. Clinical data were available for specific patients. Results: Our analysis revealed 15 distinct immunoglobulin combinations and provided insights into the presence of IgA isotype. IgG4 was the most common subclass in 120/140 of patients, followed by IgG1 in 79/140, IgG3 in 59/140, and IgG2 in 42/140 patients, while IgM positivity was detected in selected patients. Anti‐MuSK IgA(1) immunoreactivity was detected in 54/140 patients, frequently co‐occurring with IgG, and in four patients at baseline with only‐IgA positivity. IgA was detected both near disease onset and at later stages, and persisted over time in rituximab‐treated patients. Controls tested largely negative for IgA, although low‐intensity signals were observed in six samples and were considered non‐specific. L‐CBA detected anti‐MuSK IgA more frequently than flow cytometry. Discussion: Our findings expand the serology of MuSK‐MG beyond IgG subclasses, identifying IgA as an additional component of the anti‐MuSK response. These results enhance further investigation into the clinical significance, pathogenic potential, and treatment‐associated fluctuations of anti‐MuSK IgA antibodies.

Background: NOTCH3 gene variants are associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). In this study we aimed to examine the presence of pathogenic NOTCH3 variants in individuals with suspected CADASIL on the Greek island of Crete. This represents the first report of CADASIL patients in Crete. Methods: We reviewed the medical records of the University Hospital of Heraklion and identified three patients with the clinical diagnosis of CADASIL. In these patients pathogenic NOTCH3 variants were identified through targeted or whole‐exome sequencing (WES). Results: A novel heterozygous variant in exon 4 of the NOTCH3 gene (p.Cys206Trp; NM_000435.3:c.618C>G) was found in a 67‐year‐old woman who suffered from recurrent ischemic strokes, cognitive impairment, depression, and headache, as well as her son, who presented with headache, anxiety disorder, and insomnia. Brain MRI for both patients revealed white matter disease, including the anterior temporal lobes. The characteristics of this variant (a Cys‐related variant in the epidermal growth factor repeats area) support its pathogenicity. We also identified a 72‐year‐old patient affected by CADASIL and carrying a previously described p.Arg607Cys (NM_000435.3:c.1819C>T) NOTCH3 variant. Conclusions: This report extends the geographic and genotypic spectrum of pathogenic NOTCH3 variants and documents the first CADASIL cases on the island of Crete, Greece.

Background: Missense variants in TTN pose a major challenge in genetic diagnostics due to their high frequency in the general population, the large size of the gene, and the complex multidomain architecture of the titin protein. While the contribution of truncating variants (TTNtv) to titinopathies is well established, the role of rare TTN missense variants remains poorly defined. Advances in computational prediction and functional testing offer new tools to assess their potential pathogenicity, which however are currently not fully utilized for clinical application. Methods: We analyzed an international cohort of unsolved myopathy cases selected based on the presence of a rare missense variant in trans with a TTNtv. Clinical data were collected from neuromuscular centers worldwide. In silico predictions were generated using AlphaMissense and complemented by MAF and exon usage information. Additional inclusion criteria were based on a Minor Allele Frequency < 0.010 and an AlphaMissense score ≥ 0.792 for the missense variant, in accordance with the latest ClinGen guidelines. Selected missense variants were characterized in vitro through protein expression and cell imaging assays to assess their effects on domain solubility and aggregation. Results: Thirty patients with TTNtv/missense combinations were identified, presenting with heterogeneous myopathic phenotypes, ranging from congenital to adult onset. An in-depth analysis on AlphaMissense predictions highlighted those changes most frequently predicted as possibly pathogenic. Functional assays showed that three selected variants with changes to proline, located in β-sheets of Ig domains, led to impaired folding, cytoplasmic aggregation and co-localisation with proteostasis markers. In our cohort, all non-proline mutations occurred at buried sites, while some proline substitutions affected exposed residues. Notably, the variant p.(Gln7023Pro) was identified in 5 unrelated families sharing a conserved haplotype, indicating a common ancestor. This variant and the previously reported p.(Arg25480Pro) variant have been reclassified as likely pathogenic. Conclusions: By integrating clinical, computational, and functional evidence, we propose a framework for interpreting TTN missense variants. Combining multiple lines of evidence is essential for variants’ classification and interpretation, especially given TTN complexity. Advancing diagnostic accuracy will require tailored interpretation guidelines and a global effort in data sharing and functional validation.

Background: Cardiac amyloidosis (CA) is a progressive disorder characterized by amyloid fibril deposition in the heart, leading to heart failure and arrhythmias. Arterial stiffness, assessed by pulse wave velocity (PWV), is recognized as an adverse consequence of amyloidosis, yet its progression and relationship with myocardial dysfunction remain inadequately explored. This study examines the progression of PWV and its potential association with the deterioration of global longitudinal strain (GLS) in CA patients over a 6-month follow-up period. Methods: This prospective study enrolled 31 patients who were diagnosed with CA, including both the immunoglobulin light chain (AL) and transthyretin (ATTR) forms. All participants underwent a full echocardiographic study and PWV measurements (carotid-femoral [c-f] and carotid-radial [c-r] PWV) at baseline and 6-month follow-up. Age- and sex-matched individuals with similar cardiovascular risk factors were included as a control group. Results: In the CA group, the left ventricular mass index (LVMI) increased significantly from 119.4 ± 52.1 to 124 ± 53.2 g/m² (p = 0.002). Both c-f and c-r PWV showed significant increases at the 6-month follow-up (p < 0.001 and p = 0.005, respectively). The GLS deteriorated significantly from −14 ± 4.4% to −12.8 ± 4.9% (p = 0.018). No significant changes were observed in the control group. A weak correlation (r = 0.3; p = 0.095) was found between increases in PWV and GLS deterioration. Conclusions: Both arterial stiffness and myocardial dysfunction worsen rapidly in CA patients. However, the weak correlation between PWV and GLS suggests that they may evolve through independent mechanisms, necessitating further research to understand their complex interplay in CA.

Human evolution is characterized by rapid brain enlargement and the emergence of unique cognitive abilities. Besides its distinctive cytoarchitectural organization and extensive inter-neuronal connectivity, the human brain is also defined by high rates of synaptic, mainly glutamatergic, transmission, and energy utilization. While these adaptations’ origins remain elusive, evolutionary changes occurred in synaptic glutamate metabolism in the common ancestor of humans and apes via the emergence of GLUD2, a gene encoding the human glutamate dehydrogenase 2 (hGDH2) isoenzyme. Driven by positive selection, hGDH2 became adapted to function upon intense excitatory firing, a process central to the long-term strengthening of synaptic connections. It also gained expression in brain astrocytes and cortical pyramidal neurons, including the CA1-CA3 hippocampal cells, neurons crucial to cognition. In mice transgenic for GLUD2, theta-burst-evoked long-term potentiation (LTP) is markedly enhanced in hippocampal CA3-CA1 synapses, with patch-clamp recordings from CA1 pyramidal neurons revealing increased sNMDA receptor currents. D-lactate blocked LTP enhancement, implying that glutamate metabolism via hGDH2 potentiates L-lactate-dependent glia–neuron interaction, a process essential to memory consolidation. The transgenic (Tg) mice exhibited increased dendritic spine density/synaptogenesis in the hippocampus and improved complex cognitive functions. Hence, enhancement of neuron–glia communication, via GLUD2 evolution, likely contributed to human cognitive advancement by potentiating synaptic plasticity and inter-neuronal connectivity.

Georg Bartisch, a 16th century barber surgeon, published in 1583 his treatise entitled "Ophthalmodouleia" (Greek term meaning eye service), a work in German to promote ophthalmology for his countrymen barber surgeons. He did not have an academic education nor knew classical languages, he had used a triplet in terminology by using German, Hellenic and Latin nomination in his anatomical descriptions. Various accurate illustrations and a peculiar system of presentation with flap to liken an in-depth presentation were demonstrated to add prestige in his work. This raises concerns for another medical surgeon to have helped for this publication. Bartisch innovative approaches introduced various surgical tools. Cataract surgery was the epitome of his work. Anatomy of both the eye and the eyelids was there for only to serve the operation. Religion, magic and Galenic views presented barriers for Bartisch's scientific development. However, he is considered as the patron of German School of Ophthalmology.

Glutamate dehydrogenase (GDH) interconverts glutamate to a-ketoglutarate and ammonia, interconnecting amino acid and carbohydrate metabolism. In humans, two functional GDH genes, GLUD1 and GLUD2, encode for hGDH1 and hGDH2, respectively. GLUD2 evolved from retrotransposition of the GLUD1 gene in the common ancestor of modern apes. These two isoenzymes are involved in the pathophysiology of human metabolic, neoplastic, and neurodegenerative disorders. The 3D structures of hGDH1 and hGDH2 have been experimentally determined; however, no information is available about the path of GDH2 structure changes during primate evolution. Here, we compare the structures predicted by the AlphaFold Colab method for the GDH2 enzyme of modern apes and their extinct primate ancestors. Also, we analyze the individual effect of amino acid substitutions emerging during primate evolution. Our most important finding is that the predicted structure of GDH2 in the common ancestor of apes was the steppingstone for the structural evolution of primate GDH2s. Two changes with a strong functional impact occurring at the first evolutionary step, Arg443Ser and Gly456Ala, had a destabilizing and stabilizing effect, respectively, making this step the most important one. Subsequently, GDH2 underwent additional modifications that fine-tuned its enzymatic properties to adapt to the functional needs of modern-day primate tissues.

Glutamate dehydrogenase (GDH) interconverts glutamate to a-ketoglutarate and ammonia, interconnecting amino acid and carbohydrate metabolism. In humans, two functional GDH genes, GLUD1 and GLUD2, encode for hGDH1 and hGDH2, respectively. GLUD2 evolved from retro-transponsition of the GLUD1 gene in the common ancestor of modern apes. These two isoenzymes are involved in the pathophysiology of neoplastic, neurodegenerative, and metabolic disorders. The 3D structures of hGDH1 and hGDH2 have been experimentally determined; however, no information is available about the path of GDH2 structure changes during primate evolution. Here, we compare the structures predicted by the AlphaFold Colab method for the GDH2 enzyme of modern apes and their extinct primate ancestors. Also, we analyze the individual effect of amino acid substitutions emerging during primate evolution. Our most important finding is that the predicted structure of GDH2 in the common ancestor of apes was the steppingstone for the structural evolution of primate GDH2s. Two changes with a strong functional impact occurring at the first evolutionary step, Arg443Ser and Gly456Ala, had a destabilizing and stabilizing effect, respectively, making this step the most important one. Subsequently, GDH2 underwent additional modifications that fine-tuned its enzymatic properties to adapt to the functional needs of modern-day primate tissues.

There is growing evidence that high basal cortisol levels and systemic inflammation independently contribute to cognitive decline among older people without dementia. The present cross-sectional study examined (a) the potential synergistic effect of cortisol levels and systemic inflammation on executive function and (b) whether this effect is more prominent among older people with mild cognitive impairment (MCI). A sub-sample of 99 patients with MCI and 84 older people without cognitive impairment (CNI) (aged 73.8 ± 7.0 years) were recruited from a large population-based cohort in Crete, Greece, and underwent comprehensive neuropsychiatric and neuropsychological evaluation and a single morning measurement of cortisol and IL-6 plasma levels. Using moderated regression models, we found that the relation between cortisol and executive function in the total sample was moderated by IL-6 levels (b = −0.994, p = 0.044) and diagnostic group separately (b = −0.632, p < 0.001). Moreover, the interaction between cortisol and IL-6 levels was significant only among persons with MCI (b = −0.562, p < 0.001). The synergistic effect of stress hormones and systemic inflammation on cognitive status appears to be stronger among older people who already display signs of cognitive decline. Targeting hypercortisolemia and inflammation may be a promising strategy toward improving the course of cognitive decline.

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 world9s highest. Three different pathogenic TTR variants causing distinct clinical phenotypes were identified in this relatively small population pool.