ANXA1 with Anti-Inflammatory Properties Might Contribute to Parkinsonism.

We here describe the identification of a novel variant in the anti-inflammatory Annexin A1 protein likely to be the cause of disease in two siblings with autosomal recessive parkinsonism. The disease-segregating variant was ascertained through a combination of homozygosity mapping and whole genome sequencing and was shown to impair phagocytosis in zebrafish mutant embryos. The highly conserved variant, absent in healthy individuals and public SNP databases, affected a functional domain of the protein with neuroprotective properties. This study supports the hypothesis that damaged microglia might lead to impairments in the clearance of accumulated and aggregated proteins resulting in parkinsonism. ANN NEUROL 2021;90:319-323.

Phenotypic and genotypic characterization of families with complex intellectual disability identified pathogenic genetic variations in known and novel disease genes.

Intellectual disability (ID), which presents itself during childhood, belongs to a group of neurodevelopmental disorders (NDDs) that are clinically widely heterogeneous and highly heritable, often being caused by single gene defects. Indeed, NDDs can be attributed to mutations at over 1000 loci, and all type of mutations, ranging from single nucleotide variations (SNVs) to large, complex copy number variations (CNVs), have been reported in patients with ID and other related NDDs. In this study, we recruited seven different recessive NDD families with comorbidities to perform a detailed clinical characterization and a complete genomic analysis that consisted of a combination of high throughput SNP-based genotyping and whole-genome sequencing (WGS). Different disease-associated loci and pathogenic gene mutations were identified in each family, including known (n = 4) and novel (n = 2) mutations in known genes (NAGLU, SLC5A2, POLR3B, VPS13A, SYN1, SPG11), and the identification of a novel disease gene (n = 1; NSL1). Functional analyses were additionally performed in a gene associated with autism-like symptoms and epileptic seizures for further proof of pathogenicity. Lastly, detailed genotype-phenotype correlations were carried out to assist with the diagnosis of prospective families and to determine genomic variation with clinical relevance. We concluded that the combination of linkage analyses and WGS to search for disease genes still remains a fruitful strategy for complex diseases with a variety of mutated genes and heterogeneous phenotypic manifestations, allowing for the identification of novel mutations, genes, and phenotypes, and leading to improvements in both diagnostic strategies and functional characterization of disease mechanisms.

Molecular characterization of PRKN structural variations identified through whole-genome sequencing.

BACKGROUND: Early-onset Parkinson's disease (PD) is the most common inherited form of parkinsonism, with the PRKN gene being the most frequently identified mutated. Exon rearrangements, identified in about 43.2% of the reported PD patients and with higher frequency in specific ethnicities, are the most prevalent PRKN mutations reported to date in PD patients. METHODS: In this study, three consanguineous families with early-onset PD were subjected to whole-genome sequencing (WGS) analyses that were followed by Sanger sequencing and droplet digital PCR to validate and confirm the disease segregation of the identified genomic variations and to determine their parental origin. RESULTS: Five different PRKN structural variations (SVs) were identified. Because the genomic sequences surrounding the break points of the identified SVs might hold important information about their genesis, these were also characterized for the presence of homology and repeated sequences. CONCLUSION: We concluded that all identified PRKN SVs might originate through retrotransposition events.

Pla2g6 Deficiency in Zebrafish Leads to Dopaminergic Cell Death, Axonal Degeneration, Increased ß-Synuclein Expression, and Defects in Brain Functions and Pathways.

This study aimed to gain insights into the pathophysiology underlying PLA2G6-associated neurodegeneration that is implicated in three different neurological disorders, suggesting that other, unknown genetic or environmental factors might contribute to its wide phenotypic expression. To accomplish this, we downregulated the function of pla2g6 in the zebrafish nervous system, performed parkinsonism-related phenotypic characterization, and determined the effects of gene regulation upon the loss of pla2g6 function by using RNA sequencing and downstream analyses. Pla2g6 deficiency resulted in axonal degeneration, dopaminergic and motor neuron cell loss, and increased ß-synuclein expression. We also observed that many of the identified, differentially expressed genes were implicated in other brain disorders, which might explain the variable phenotypic expression of pla2g6-associated disease, and found that top enriched canonical pathways included those already known or suggested to play a major role in the pathogenesis of Parkinson's disease. Our data support that pla2g6 is relevant for cranial motor development with significant implications in the pathophysiology underlying Parkinson's disease.

A Clinical and Molecular Genetic Study of 50 Families with Autosomal Recessive Parkinsonism Revealed Known and Novel Gene Mutations.

In this study, the role of known Parkinson's disease (PD) genes was examined in families with autosomal recessive (AR) parkinsonism to assist with the differential diagnosis of PD. Some families without mutations in known genes were also subject to whole genome sequencing with the objective to identify novel parkinsonism-related genes. Families were selected from 4000 clinical files of patients with PD or parkinsonism. AR inheritance pattern, consanguinity, and a minimum of two affected individuals per family were used as inclusion criteria. For disease gene/mutation identification, multiplex ligation-dependent probe amplification, quantitative PCR, linkage, and Sanger and whole genome sequencing assays were carried out. A total of 116 patients (50 families) were examined. Fifty-four patients (46.55%; 22 families) were found to carry pathogenic mutations in known genes while a novel gene, not previously associated with parkinsonism, was found mutated in a single family (2 patients). Pathogenic mutations, including missense, nonsense, frameshift, and exon rearrangements, were found in Parkin, PINK1, DJ-1, SYNJ1, and VAC14 genes. In conclusion, variable phenotypic expressivity was seen across all families.

Genetic screening in two Iranian families with early-onset Alzheimer's disease identified a novel PSEN1 mutation.

A subset of early-onset Alzheimer's disease is inherited as an autosomal-dominant trait and is associated with mutations in the genes encoding ß-amyloid precursor protein, presenilin 1, or presenilin 2. In this study, we identified 2 PSEN1 mutations (1 novel and 1 known) in 2 unrelated Iranian families with autosomal-dominant Alzheimer's disease. The disease progressed rapidly with a mean age at onset of 33 and 42 years and an age at death ranging from 43 to 48 years.

Whole genome sequencing identifies a novel homozygous exon deletion in the NT5C2 gene in a family with intellectual disability and spastic paraplegia.

Hereditary spastic paraplegias are a rare group of clinically and genetically heterogeneous neurodegenerative diseases, with upper motor neuron degeneration and progressive lower limb spasticity as their main phenotypic features. Despite that 76 distinct loci have been reported and some casual genes identified, most of the underlying causes still remain unidentified. Moreover, a wide range of clinical manifestations is present in most hereditary spastic paraplegias subtypes, adding further complexity to their differential clinical diagnoses. Here, we describe the first exon rearrangement reported in the SPG45/SPG65 (NT5C2) loci in a family featuring a complex hereditary spastic paraplegias phenotype. This study expands both the phenotypic and mutational spectra of the NT5C2-associated disease.

Whole-exome sequencing associates novel CSMD1 gene mutations with familial Parkinson disease.

OBJECTIVE: Despite the enormous advancements made in deciphering the genetic architecture of Parkinson disease (PD), the majority of PD is idiopathic, with single gene mutations explaining only a small proportion of the cases. METHODS: In this study, we clinically evaluated 2 unrelated Spanish families diagnosed with PD, in which known PD genes were previously excluded, and performed whole-exome sequencing analyses in affected individuals for disease gene identification. RESULTS: Patients were diagnosed with typical PD without relevant distinctive symptoms. Two different novel mutations were identified in the CSMD1 gene. The CSMD1 gene, which encodes a complement control protein that is known to participate in the complement activation and inflammation in the developing CNS, was previously shown to be associated with the risk of PD in a genome-wide association study. CONCLUSIONS: We conclude that the CSMD1 mutations identified in this study might be responsible for the PD phenotype observed in our examined patients. This, along with previous reported studies, may suggest the complement pathway as an important therapeutic target for PD and other neurodegenerative diseases.

A Novel p.Glu298Lys Mutation in the ACMSD Gene in Sporadic Parkinson's Disease.

BACKGROUND: Common genetic variability in the ACMSD gene has been associated with increased risk for Parkinson's disease (PD) but ACMSD mutations in clinical cases of PD have so far not been reported. OBJECTIVE: To describe a case of sporadic PD carrying a novel ACMSD mutation. METHODS: As part of a genetic study to identify potential pathogenic gene defects related to PD in the Mediterranean island Menorca, an initial group of 62 PD patients underwent mutational screening using a panel-based sequencing approach. RESULTS: We report a 74-years-old man with sporadic PD who developed tremor in his right hand and slowness. On examination, moderate rigidity, asymmetric bradykinesia, and bilateral action tremor were present. He was started on levodopa with significant improvement. Two years later, he developed wearing off phenomena. The genetic study in the patient identified a novel ACMSD mutation resulting in p.Glu298Lys amino-acid change which was not present in neurologically normal population. CONCLUSIONS: Our data suggest that not only common genetic variability but also rare variants in ACMSD alone or in combination with other risk factors might increase the risk of PD.

PTRHD1 (C2orf79) mutations lead to autosomal-recessive intellectual disability and parkinsonism.

INTRODUCTION: Atypical parkinsonism is a neurodegenerative disease that includes diverse neurological and psychiatric manifestations. OBJECTIVES: We aimed to identify the disease-cauisng mutations in a consanguineous family featuring intellectual disability and parkinsonism. METHODS: Full phenotypic characterization, followed by genome-wide single-nucleotide polymorphism genotyping and whole-genome sequencing, was carried out in all available family members. RESULTS: The chromosome, 2p23.3, was identified as the disease-associated locus, and a homozygous PTRHD1 mutation (c.157C>T) was then established as the disease-causing mutation. The pathogenicity of this PTRHD1 mutation was supported by its segregation with the disease status, its location in a functional domain of the encoding protein, as well as its absence in public databases and ethnicity-matched control chromosomes. CONCLUSION: Given the role of 2p23 locus in patients with intellectual disability and the previously reported PTRHD1 mutation (c.155G>A) in patients with parkinsonism and cognitive dysfunction, we concluded that the PTRHD1 mutation identified in this study is likely to be responsible for the phenotypic features of the family under consideration. © 2016 International Parkinson and Movement Disorder Society.

First And Second-Generation Cryoballoon Ablation Efficacy Restoring And Maintaining Sinus Rhythm In Patients Electrically Selected And Treated For Long-Standing Persistent Atrial Fibrillation After Acute Complete Electrical Disconnection Of Pulmonary Veins From The Left Atrium Demonstrated.

Introduction Cryoballoon ablation (CB) has proven effective to treatment of patients (pts) with atrial fibrillation (AF). However, the isolated efficacy of CB to treat pts with long-standing persistent atrial fibrillation (LSPAF) is less know. We analyzed the acute results and the long-term follow-up of our pts suffering LSPAF and initially treated with CB. Methods A cohort of 44 pts, 37 male (84%) mean age (60±10 year) suffering LSPAF were treated with first (CB1): 15pts, and second (CB2): 29 pts, generation CB. Eight pts (18.1%) had structural heart disease. Prior to CB, all pts were previously electrically cardioverted (CV) and sinus node and A-V nodal function evaluated at electrophysiological study (EP) once in sinus rhythm (SR) before antiarrhythmic drugs (AAD) load. CB ablation procedure was performed after three months waiting period on AAD following CV/EP drug testing. Result CB procedure was performed in 27 (61.4%) in AF, restoring SR in 8 (18.2%). PV isolation (PVI) was achieved in 95.2%. On follow-up of 30±39 months, 16 pts (40%) had AF recurrence. Second procedure (Redo) was performed in 7 pts. After a single procedure, 24 pts (60%) remain in SR without AAD, after Redo, 29 pts (72.5%), and when AAD added, 31 pts (77.5%) remain in SR. Phrenic nerve palsy (PNP) occurred in 9% of pts (75% with CB2). Conclusion CB technique is safe and useful tool to treat pts with LSPAF with 60% success rate maintaining SR without AAD in a long-term follow-up (30±39 months), up to 72.5% after Redo, and to 77.5% when AAD are added. In the majority of pts maintaining SR (77.5%) CB2 was used in 87% of the cases. Patients without structural heart disease along with those who SR was restored during CB showed the best result.

Long-Term Evolution of Patients Treated for Paroxysmal Atrial Fibrillation with First and Second Generation Cryoballoon Catheter Ablation with a Prospective Protocol Guided by Complete Bidirectional Left Atrium-Pulmonary Veins Disconnection after Adenosine as Main Target end Point to achieved. Seven Years Follow-up of Patients with a rough estimation profile of Low ALARMEc Score. A Single Center Report.

INTRODUCTION: Cryoballoon ablation (CB) has proven effective for treating patients with paroxysmal atrial fibrillation (PAF). We analyzed our seven year follow-up of patients, treated for PAF with first (CB1) and second generation (CB2), with demonstration of LA-PV disconnection with bidirectional block (BB) after adenosine (AD). METHODS: Since November 2008 to May 2015, 128 patients, 97 male (58±7 years), without heart disease, highly symptomatic, refractory to antiarrhythmic drugs (AAD) were treated, and follow-up (1411 ±727 days). Left atrial size: 37±6 mm. RESULTS: A total of 439 PV were successfully isolated (91.9%). Acute reconduction: 44 PV (9%): 16 after CB; 16 unmasked by AD; 12 extrapulmonary muscular connections (EMC). Main complication was phrenic nerve palsy (PNP): 9 (7 %). On follow-up, 114 patients (89%) remain asymptomatic in sinus rhythm (SR), free of medication. Fourteen patients (11%) had arrhythmia recurrence: 12 male (52±8 years). Early recurrences occurred in 9 male. Late recurrences presented 3 male at 24, 27 and 60 months, and 2 female at 7 and 40 months respectively. All recurrence patients were Redo, and remain in SR without medication during follow-up. CONCLUSIONS: CB alone is very effective and safe for the definitive treatment of patients suffering PAF with 72.6% success rate, increasing up to 89.1% when this protocol is applied in a single procedure. After Redo, all population group (100%), remain in sinus rhythm, freedom of arrhythmia, without AAD, in this very long term follow-up. Checking for BB, AD protocol, and ruling out EMC allowed-us to identified 14.8% of patients with underlying substrate for potential arrhythmia recurrence. CB2 applications entail a highest risk of PNP. Patients with a rough estimated profile of low ALARMEc score (≤ 1) have an excellent long term outcome, being this series the largest follow-up described so far, for patients treated for PAF with CB.

Nitric oxide from mononuclear cells may be involved in platelet responsiveness to aspirin.

BACKGROUND: Several mechanisms have been proposed to explain why some platelets have a reduced response to aspirin (ASA). Among them, it was reported an increased circulating level of vitamin-D-binding protein (DBP). In addition, nitric oxide (NO) released from mononuclear cells was involved in the antiplatelet effects of ASA. The aim was to analyse the relationship between platelet response to ASA and both NO generation and vitamin-D-binding protein content in mononuclear cells. MATERIALS AND METHODS: Mononuclear cells were obtained from patients with stable coronary artery disease that were divided by a platelet functionality test (PFA-100) as ASA-sensitive (n=23) and ASA resistant (n=27). RESULTS: Both the release of NO (determined by nitrite+nitrate concentration) and the expression of endothelial-type NO synthase (eNOS) were higher in mononuclear cells from ASA sensitive as compared with those from ASA-resistant patients. There was a positive correlation between either the release of NO and the expression of eNOS protein in mononuclear cells with the ability of ASA to inhibit platelet activity. DBP content in mononuclear cells was higher in ASA resistant than in ASA sensitive. The level of DBP content in mononuclear cells was negatively associated with the ability of ASA to inhibit platelets. However, in vitro experiments suggested that there was no association between DBP and NO production by mononuclear cells. CONCLUSIONS: Mononuclear cells from patients with platelets with lower responsiveness to ASA showed a reduced ability to produce NO.

Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO). PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100). RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786) → C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)(1177), an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177) phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser(1177). On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177). During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177).

Impact of clopidogrel and aspirin treatment on the expression of proteins in platelets from type-2 diabetic patients with stable coronary ischemia.

The purpose of this study was to compare the effect of dual antiplatelet therapy [clopidogrel + aspirin (ASA)] with respect to ASA on the protein expression of platelets from controlled type-2 diabetic patients with stable coronary ischemia. Patients had been taking ASA (100 mg day) and they were randomized to receive (n = 29) or not (n = 28) 75 mg day clopidogrel for 12 ± 2 weeks in a blind form. Protein expression was analyzed by two-dimensional electrophoresis and mass spectrometry. The protein expression of a limited number of proteins such as actin-binding protein isotypes 2 and 5, lactate dehydrogenase, serotransferrin isotype 4, protein disulfide isomerase-A3 isotype 1, fibrinogen beta chain isotype 5, Ras-related protein Rab-7b isotypes 1 and 6, and immunoglobulin heavy chain was changed after dual antiplatelet therapy. Plasma level of platelet factor 4 (PF4), an in vivo marker of platelet activity, was not different between both groups. These changes suggest lower platelet reactivity after dual antiplatelet therapy in the studied patients. However, the variation in platelet proteome was lower than it would be initially expected, taking into account the apparent clinical beneficial effects of dual antiplatelet therapy. PF4 plasma level was not further decreased in the platelets treated for a longer time than 9-12 months with ASA + clopidogrel, as compared with ASA alone.

Proteomic changes related to "bewildered" circulating platelets in the acute coronary syndrome.

Acute coronary syndromes (ACS) are associated with platelet activation. The aim of the present study was to study the protein expression level associated with glycolysis, oxidative stress, cytoskeleton and cell survival in platelets obtained during an ACS. Platelets from 42 coronary ischemic patients, divided into patients admitted within 24 h after the onset of chest pain (ACS group; n=16) and patients with stable coronary ischemic disease (CAD, n=26), were analyzed using proteomics. The expression levels of proteins involved in cellular cytoskeleton (F-actin capping, ß-tubulin, α-tubulin isotypes 1 and 2, vinculin, vimentin and two Ras-related protein Rab-7b isotypes), glycolysis pathway (glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and two pyruvate kinase isotypes) and cellular-related antioxidant system (manganese superoxide dismutase) and even the expression and activity of glutathione-S-transferase were significantly reduced in platelets from ACS patients compared to CAD patients. Moreover, reduction in the expression of proteins associated with cell survival such as proteasome subunit ß type 1 was also observed in ACS platelets compared with CAD platelets. Principal component and logistic regression analysis suggested the existence of factors (proteins) expressed in the platelets inversely associated with acute coronary ischemia. In summary, these results suggest the existence of circulating antioxidant, cytoskeleton and glycolytic-"bewildered" platelets during the acute phase of a coronary event.

Can resistance to aspirin be reversed after an additional dose?

Aspirin resistance or aspirin non-responsiveness is a recently described phenomenon which has been consistently associated with an increased risk of cardiovascular events. This study was designed to determine the effects of an additional dose of 100 mg of aspirin on platelet function and proportion of aspirin non-responders using the platelet function analyzer-100 (PFA-100), in a well characterized population of stable coronary heart disease patients already on long-term aspirin treatment. Platelet function was assessed using PFA-100 in 141 patients (64.8 ± 10.1 years, 87.9% men) on long-term aspirin treatment (100 mg/day) before and 1 h after "in site" oral aspirin administration (100 mg). Prevalence of aspirin non-responders using PFA-100 was 50.7% (95% confidence interval 42.4-59). One hour after 100 mg of oral aspirin, reassessment of aspirin effects showed a prevalence of non-responders using PFA of 35.0% (95% CI 27.3-43.2) (P < 0.001 vs. pre-dose proportion). Using the PFA-100 system, reassessment of platelet function following oral administration of daily aspirin dosage significantly reduces the number of stable coronary disease patients considered to be non-responders to such treatment.

Old and new molecular mechanisms associated with platelet resistance to antithrombotics.

Current available data show that about 5 to 40% of coronary patients treated with conventional doses of antithrombotic drugs do not display adequate antiplatelet response. Nowadays, aspirin remains the main antiplatelet therapy. However, a significant number of patients show platelet resistance to aspirin therapy, and recurrent thrombotic events occur. Combined antithrombotic therapies with thienopyridines, such as clopidogrel have been used to resolve this problem. However, clopidogrel treatment has been also associated with wide response variability, and non-responsiveness to clopidogrel also occurs in some patients. Therefore, the main question arising about the antithrombotic therapy is why particular patients do not benefit from the therapy and how they might be identified to improve their treatment. Different hypotheses have been suggested, including genetic factors, platelet heterogeneity, non-compliance and others. However, it is probably that many molecular mechanisms involved in platelet resistance to antithrombotic therapies still remains unknown. New technologies, such as proteomics and genetic, are beginning to show new unknown biological biomarkers and molecular mechanisms which may be associated with platelet antithrombotic drug resistance.