Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Importance of Comprehensive Assessment in Brugada Syndrome. Reply to Kataoka, N.; Imamura, T. How to Diagnose and Risk Stratify Brugada Syndrome. Comment on "Matusik et al. Twelve-Lead ECG, Holter Monitoring Parameters, and Genetic Testing in Brugada Syndrome: Insights from Analysis of Multigenerational Family with a History of Sudden Cardiac Arrest during Physical Activity. J. Clin. Med. 2023, 12, 6581".

We would like to thank Dr. Imamura for their interest in our study and their valuable comments on diagnostics and risk stratification in Brugada syndrome (BrS) [...].

Twelve-Lead ECG, Holter Monitoring Parameters, and Genetic Testing in Brugada Syndrome: Insights from Analysis of Multigenerational Family with a History of Sudden Cardiac Arrest during Physical Activity.

Brugada syndrome (BrS) is an arrhythmogenic disorder increasing the risk of syncopal episodes and sudden cardiac death. BrS usually runs through families with reduced penetrance and variable expression. We analyzed the multigenerational family of a patient who died after sudden cardiac arrest with post-mortem diagnosis of BrS. We analyzed clinical history, comprehensive arrhythmic risk, genetic findings, and additional tests, including electrocardiogram (ECG), detailed 24-hour Holter ECG results, and standard echocardiography findings, and followed up the patients in the ambulatory clinic. We analyzed a pedigree of 33 members of four generations of the family (19 male and 14 female patients). In this family, we identified 7 patients with BrS (median Modified Shanghai Score and Sieira model: 4.5 (4-6) and 1 (0-4) points, respectively), including both parents of the deceased patient, and 8 relatives with negative sodium channel blocker drug challenge test. Genetic testing revealed a novel mutation in sodium voltage-gated channel alpha subunit 5 (SCN5A) c.941A>G, (p.Tyr314Cys) inherited from the father of the proband. Patients with BrS were characterized by longer P-wave duration (120 (102-155) vs. 92.5 (88-110) ms, p = 0.013) and longer PR intervals (211.3 ±26.3 vs. 161.6 ± 18.9 ms, p = 0.001), along with more frequent positive aVR sign, but did not differ in terms of QRS duration or T-wave characteristics in resting ECGs. BrS patients were characterized by lower mean, minimal, and maximal (for all p ≤ 0.01) heart rates obtained from Holter ECG monitoring, while there was no difference in arrhythmias among investigated patients. Moreover, visual diurnal variability of ST segment changes and fragmented QRS complexes were observed in patients with BrS in Holter ECG monitoring. There were no major arrhythmic events during median follow-up of 68.7 months of alive BrS patients. These results suggest ECG features which may be associated with a diagnosis of BrS and indicate a novel SCN5A variant in BrS patients. Twelve-lead Holter ECG monitoring, with modified precordial leads placement, may be useful in BrS diagnostics and risk stratification in personalized medicine.

The Utility of Next-Generation Sequencing for Identifying the Genetic Basis of Dementia.

The clinical manifestations of dementia are often rapidly matched to a specific clinical syndrome, but the underlying neuropathology is not always obvious. A genetic factor often plays an important role in early onset dementia, but there are cases in which the phenotype has a different genetic basis than is assumed. Two patients, at different times, presented to the Memory Clinic because of memory problems and difficulty in performing daily activities and work. Neither caregiver complained of marked behavioural or personality changes, except for apathy. Patients underwent standard dementia evaluation procedures including clinical symptoms, family history, neuroimaging, neuropsychological evaluation, and genetic analysis of selected genes. Based on specific clinical phenotypes and genetic analysis of selected genes, both patients were diagnosed with frontal variant of Alzheimer's disease. The presence of a rare polymorphism in PSEN2 in both patients allowed the discovery that they belong to the same family. This fact reinforced the belief that there is a strong genetic factor responsible for causing dementia in the family. Next-generation sequencing based on a panel of 118 genes was performed to identify other potential genetic factors that may determine the background of the disease. A mutation in the GRN gene was identified, and the previous diagnosis was changed to frontotemporal dementia. The described cases show how important it is to combine all diagnostic tests available in the diagnostic centre, including new generation genetic tests, in order to establish/confirm the pathological background of clinical symptoms of dementia. If there is any doubt about the final diagnosis, persistent efforts should be made to verify the cause.

Identification of a variant hotspot in MYBPC3 and of a novel CSRP3 autosomal recessive alteration in a cohort of Polish patients with hypertrophic cardiomyopathy.

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is a heart disorder caused by autosomal dominant alterations affecting both sarcomeric genes and other nonsarcomeric loci in a minority of cases. However, in some patients, the occurrence of the causal pathogenic variant or variants in homozygosity, compound heterozygosity, or double heterozygosity has also been described. Most of the HCM pathogenic variants are missense and unique, but truncating mutations of the MYBPC3 gene have been reported as founder pathogenic variants in populations from Finland, France, Japan, Iceland, Italy, and the Netherlands. OBJECTIVES: This study aimed to assess the genetic background of HCM in a cohort of Polish patients. PATIENTS AND METHODS: Twenty­nine Polish patients were analyzed by a next generation sequencing panel including 404 cardiovascular genes. RESULTS: Pathogenic variants were found in 41% of the patients, with ultra­ rare MYBPC3 c.2541C>G (p.Tyr847Ter) mutation standing for a variant hotspot and correlating with a lower age at HCM diagnosis. Among the nonsarcomeric genes, the CSRP3 mutation was found in a single case carrying the novel c.364C>T (p.Arg122Ter) variant in homozygosity. With this finding, the total number of known HCM cases with human CSRP3 knockout cases has reached 3. CONCLUSIONS: This report expands the mutational spectrum and the inheritance pattern of HCM.

Case report on novel mutation in SPAST gene in Polish family with spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegia is a large group of degenerative, neurological disorders characterized by progressive lower limb spasticity and weakness. The disease was investigated precisely but still clinicians often make incorrect or late diagnosis. Our aim was to investigate the genetic background and clinical phenotype of spastic paraplegia in large Polish family. CASE PRESENTATION: A 37 years old woman presented with 4-year history of walking difficulties. On neurological examination, she had signs of upper motor lesion in lower extremities. She denied sphincter dysfunction and her cognition was normal. Her family history was positive for individuals with gait problems. The initial diagnosis was familial spastic paraplegia. Genetic testing identified a novel mutation in SPAST gene. All available family members were examined and had genetic testing. The same mutation in SPAST gene was identified in other affected family members. All patients caring the mutation presented with different phenotypes. CONCLUSION: This study presents a family with spastic paraplegia due to a novel mutation c.1390G>T(p.Glu464Term) in SPAST gene. Affected individuals showed a range of phenotypes that varied in their severity. This case report demonstrates, the signs of hereditary spastic paraplegia can be often misdiagnosed with other diseases. Therefore genetic testing should always be considered in patients with lower limb spasticity and positive family history in order to help to establish the correct diagnosis.

High detection rates of antithrombin deficiency and antiphospholipid syndrome in outpatients aged over 50 years using the standardized protocol for thrombophilia screening.

INTRODUCTION: Thrombophilia screening has limited detection efficiency. We assessed the detection rate when a standardized approach to thrombophilia-screened outpatients was used. METHODS: We analyzed 1185 patients (36.5% males, median age: 43 years [IQR 33-54]) referred to a single center from January 2014 to October 2017 with 11 different clinical indications for thrombophilia screening, which was performed in the adherence to published guidelines. Factor V Leiden, prothrombin G20210A mutation, antithrombin (AT), protein C, protein S deficiencies and antiphospholipid syndrome (APS) were determined. RESULTS: The overall positivity rate was 37.1% (95% CI 34.3%-39.7%). The highest positivity rate was found in women following VTE during pregnancy/childbirth (64.1%) and provoked VTE patients with positive family history (52.9%). In patients aged >50 years (32.5%), APS was found at a similar rate as in younger subjects (11.4% vs 10.1%), while AT deficiency was detected more frequently in the older group (5.7% vs 2.4%, p = 0.003). CONCLUSIONS: Standard indications for thrombophilia screening lead to detection rates of 37% or more. Frequent detection of APS and AT deficiency among older patients, which often implies a need for long-term anticoagulation and could impact clinical practice patterns, suggests a benefit of thrombophilia screening in this population in selected clinical circumstances.

Alpha-2-antiplasmin Arg407Lys polymorphism and cryptogenic ischemic cerebrovascular events: Association with neurological deficit.

OBJECTIVE: Genetic background of cryptogenic ischemic stroke (IS) and transient ischemic attack (TIA) remains uncertain. Alpha-2-antiplasmin (α2AP) Arg407Lys polymorphism has been shown to be less common in patients with abdominal aortic aneurysm (AAA) compared with healthy controls. We investigated associations of α2AP Arg407Lys polymorphism with cryptogenic IS and TIA. METHODS: We studied 165 consecutive Caucasian patients who experienced cryptogenic IS (n=123) or TIA (n=42). Neurological outcomes were assessed using the modified Rankin Scale (mRS) in the acute phase of cerebral ischemia and 8 (6-12) months after the index episode. Patients were genotyped for α2AP Arg407Lys polymorphism (rs1057335) using real time PCR technique. RESULTS: The allele frequency of Arg407Lys polymorphism was: 0.82/0.18. The 407Lys allele was more frequent in TIA patients compared to the IS group (0.29 vs. 0.14, p=0.003). In the whole group, as well as in IS and TIA patients analyzed separately, possession of the 407Lys allele was associated with excellent outcome (mRS 0-1) during follow-up (p<0.05) but not in the acute phase of ischemic events both in thrombolyzed and nonthrombolyzed IS patients. The multivariate logistic regression model showed that the excellent outcome (mRS 0-1) assessed after 8 (6-12) months since the index cerebral ischemia was predicted by the occurrence of Lys407 allele (OR 6.18, 95% CI, 2.01-18.98, p=0.001). CONCLUSION: The presence of 407Lys allele is associated with better prognosis in cryptogenic cerebrovascular events. Our findings suggest that the α2AP Arg407Lys polymorphism could be involved in the pathogenesis of cerebral ischemia and its outcomes.

Venous thromboembolism associated with protein S deficiency due to Arg451* mutation in PROS1 gene: a case report and a literature review.

Protein S (PS) is a vitaminK-dependent glycoproteinwhich plays an important role in the regulation of blood coagulation. PS deficiency has been found in 1.5-7% of thrombophilic patients. Here, we report the first Polish case with PS deficiency caused by the p.Arg451* in the PROS1 gene detected in a 21-year-old man with trauma-induced venous thromboembolism. To our knowledge, we provided the review of all the available data on this mutation (a total of 56 cases). The proband, his mother and his sister were screened for thrombophilia. To elucidate genetic background of PS deficiency, all PROS1 genes were subjected to direct sequencing. The free PS levels were 35% in the proband, 21% in the proband's mother and 28% in the proband's sister and their PS total levels were 37.1, 47.5 and 55.1%, respectively. Type I PS deficiency was diagnosed. In all patients, genetic analysis revealed the presence of heterozygous nonsense mutation (c.1351C>T; p.Arg451*) located in exon 12 of PROS1 gene. This mutation interrupts the reading frame by premature termination codon at position 451 and may lead to the production of truncated protein. The present case combined with the review of the literature suggests that p.Arg451* in the PROS1 gene mainly leads to clinically evident thrombosis following trauma, surgery or serious comorbidities especially malignancy.

Algorithmic Perspectives of Network Transitive Reduction Problems and their Applications to Synthesis and Analysis of Biological Networks.

In this survey paper, we will present a number of core algorithmic questions concerning several transitive reduction problems on network that have applications in network synthesis and analysis involving cellular processes. Our starting point will be the so-called minimum equivalent digraph problem, a classic computational problem in combinatorial algorithms. We will subsequently consider a few non-trivial extensions or generalizations of this problem motivated by applications in systems biology. We will then discuss the applications of these algorithmic methodologies in the context of three major biological research questions: synthesizing and simplifying signal transduction networks, analyzing disease networks, and measuring redundancy of biological networks.

[Remote repeatability of tilt test]. / Powtarzalnosc odlegla testu pionizacyjnego.

The aim of this study was the assessment of long-term repeatability of tilt-table test and parameters of heart rate variability analysis. Westminster protocol tilt-table test extended with nitroglycerin test was combined with analysis of heart rate variability. Five-minute intervals of ECG record were evaluated before and after the upright tilting, before syncope and 24-hour record was analysed. The test was performed in duplicate in 27 persons including 14 men (mean age 33 +/- 13.5) at interval of 29 +/- 13 months. The patients were then observed for 14 +/- 11.4 months. The study subjects were divided into groups with and without the recurrence of syncope. Repeatability was observed in 76.5% in positive test result and in 70% in negative test result. A low repeatability of positive test was observed. Blood pressure, heart rate and heart rate variability analysis parameters demonstrated a high repeatability during both tests. The recurrence of syncope was observed in 10 (37%) persons. The patients with both tilt tests positive, demonstrated recurrence of syncope two times more frequently what makes that the identification of the patients at risk for syncope return is easier.

[Diagnostic and prognostic significance of homocysteine determined in acute phase of myocardial infarction]. / Znaczenie rozpoznawczo-prognostyczne homocysteiny oznaczanej w ostrej fazie zawalu.

THE AIM: Of this study was to assess the serum homocysteine concentration in subjects with acute myocardial infarction and its correlation with the course of infarction and further prognosis considering particularly left ventricle dysfunction, heart rate and conduction disorders as well as to assess the usefulness of metionin load test as a prognostic test in patients with myocardial infarction. MATERIAL AND METHODS: 66 patients were studied: 36 with recent myocardial infarction and 30 healthy individuals as a control group. Fasting serum homocysteine and its concentration two hours after metionin load were determined in all patients. They all underwent echocardiographic examination, stress test and 24-hour Holter monitoring. The study revealed a significant positive correlation between increased serum homocysteine concentration in patients with myocardial infarction and worsening of contractility parameters, extent of infarction area, and negative correlation between homocysteine concentration and ejection fraction. CONCLUSIONS: On the basis of the study outcome we can make a statement that increased homocysteine concentration in patients with acute phase of myocardial infarction indicates its more severe course, more extensive disorders of myocardium kinetics, more significant left ventricle diastolic and systolic dysfunction. Increased serum homocysteine in metionin load test indicates higher death risk in patients with myocardial infarction.

[Selected inflammatory markers in patients with acute coronary syndrome]. / Wybrane wskazniki zapalenia u chorych z ostrymi zespolami wiencowymi.

The aim of the study was the assessment of selected inflammatory markers in patients with stable and unstable angina pectoris, in comparison to patients with dyslipidemia without coronary artery disease. The study group included 61 patients (37-79 years old), divided into three subgroups: group I. 26 (43%) with unstable angina, group 2. 19 (26%) with stable angina, group III. 16 (26%) dyslipidemia without coronary artery disease. We measured serum levels of cytokines (IL-1B, IL-1Ra, IL-2, IL-6, TNF-alpha), immunoglobulins (IgG, IgE, IgM), fibrinogen. C-reactive protein and subclass of lymphocytes T CD4 and T CD8. In stable and unstable angina pectoris group we found lower percentage of T CD4, T CD8 and higher level of TNF-alpha. In unstable angina group the level of IL-1 beta was lower and the concentration of C-reactive protein, IgE was higher in comparison to group without coronary artery disease. Observed immunoregulatory disorders confirm immune mechanism in the origin of unstable angina pectoris.