Integrated Approach to Highlighting the Molecular Bases of a Deep Vein Thrombosis Event in an Elite Basketball Athlete.

Acute or intense exercise can result in metabolic imbalances, muscle injuries, or reveal hidden disorders. Laboratory medicine in sports is playing an increasingly crucial role in monitoring athletes' health conditions. In this study, we designed an integrated approach to explore the causes of a deep venous thrombosis event in an elite basketball player. Since the complete blood count revealed a marked platelet count (838 × 103 µL), and thrombophilia screening tests did not reveal any significant alteration, we evaluated the thrombin generation, which highlights a state of hypercoagulability. First-level haemostasis exams showed only a slight prolongation of the activated Partial Thromboplastin Time (aPTT). Thus, screening tests for von Willebrand Disease showed a reduction in vWF parameters. Therefore, we directed our hypothesis towards a diagnosis of acquired von Willebrand disease secondary to Essential Thrombocythemia (ET). To confirm this hypothesis and highlight the molecular mechanism underlying the observed phenotype, molecular tests were performed to evaluate the presence of the most common mutations associated with ET, revealing a 52-bp deletion in the coding region of CALR exon 9. This case report highlights the importance of an integrated approach to monitoring the athletes' health status to personalise training and treatments, thus avoiding the appearance of diseases and injuries that, if underestimated, can undermine the athlete's life.

Contribution of Genetic Test to Early Diagnosis of Methylenetetrahydrofolate Reductase (MTHFR) Deficiency: The Experience of a Reference Center in Southern Italy.

BACKGROUND: the deficiency of 5,10-Methylenetetrahydrofolate reductase (MTHFR) constitutes a rare and severe metabolic disease and is included in most expanded newborn screening (NBS) programs worldwide. Patients with severe MTHFR deficiency develop neurological disorders and premature vascular disease. Timely diagnosis through NBS allows early treatment, resulting in improved outcomes. METHODS: we report the diagnostic yield of genetic testing for MTHFR deficiency diagnosis, in a reference Centre of Southern Italy between 2017 and 2022. MTHFR deficiency was suspected in four newborns showing hypomethioninemia and hyperhomocysteinemia; otherwise, one patient born in pre-screening era showed clinical symptoms and laboratory signs that prompted to perform genetic testing for MTHFR deficiency. RESULTS: molecular analysis of the MTHFR gene revealed a genotype compatible with MTHFR deficiency in two NBS-positive newborns and in the symptomatic patient. This allowed for promptly beginning the adequate metabolic therapy. CONCLUSIONS: our results strongly support the need for genetic testing to quickly support the definitive diagnosis of MTHFR deficiency and start therapy. Furthermore, our study extends knowledge of the molecular epidemiology of MTHFR deficiency by identifying a novel mutation in the MTHFR gene.

Clinical, Genetic, and Histological Characterization of Patients with Rare Neuromuscular and Mitochondrial Diseases Presenting with Different Cardiomyopathy Phenotypes.

Cardiomyopathies are mostly determined by genetic mutations affecting either cardiac muscle cell structure or function. Nevertheless, cardiomyopathies may also be part of complex clinical phenotypes in the spectrum of neuromuscular (NMD) or mitochondrial diseases (MD). The aim of this study is to describe the clinical, molecular, and histological characteristics of a consecutive cohort of patients with cardiomyopathy associated with NMDs or MDs referred to a tertiary cardiomyopathy clinic. Consecutive patients with a definitive diagnosis of NMDs and MDs presenting with a cardiomyopathy phenotype were described. Seven patients were identified: two patients with ACAD9 deficiency (Patient 1 carried the c.1240C>T (p.Arg414Cys) homozygous variant in ACAD9; Patient 2 carried the c.1240C>T (p.Arg414Cys) and the c.1646G>A (p.Ar549Gln) variants in ACAD9); two patients with MYH7-related myopathy (Patient 3 carried the c.1325G>A (p.Arg442His) variant in MYH7; Patient 4 carried the c.1357C>T (p.Arg453Cys) variant in MYH7); one patient with desminopathy (Patient 5 carried the c.46C>T (p.Arg16Cys) variant in DES); two patients with mitochondrial myopathy (Patient 6 carried the m.3243A>G variant in MT-TL1; Patient 7 carried the c.253G>A (p.Gly85Arg) and the c.1055C>T (p.Thr352Met) variants in MTO1). All patients underwent a comprehensive cardiovascular and neuromuscular evaluation, including muscle biopsy and genetic testing. This study described the clinical phenotype of rare NMDs and MDs presenting as cardiomyopathies. A multidisciplinary evaluation, combined with genetic testing, plays a main role in the diagnosis of these rare diseases, and provides information about clinical expectations, and guides management.

Diagnostic and Therapeutic Potential for HNP-1, HBD-1 and HBD-4 in Pregnant Women with COVID-19.

Pregnancy is characterized by significant immunological changes and a cytokine profile, as well as vitamin deficiencies that can cause problems for the correct development of a fetus. Defensins are small antimicrobial peptides that are part of the innate immune system and are involved in several biological activities. Following that, this study aims to compare the levels of various cytokines and to investigate the role of defensins between pregnant women with confirmed COVID-19 infection and pregnant women without any defined risk factor. TNF-α, TGF-ß, IL-2 and IL-10, ß-defensins, have been evaluated by gene expression in our population. At the same time, by ELISA assay IL-6, IL-8, defensin alpha 1, defensin beta 1 and defensin beta 4 have been measured. The data obtained show that mothers affected by COVID-19 have an increase in pro-inflammatory factors (TNF-α, TGF-ß, IL-2, IL-6, IL-8) compared to controls; this increase could generate a sort of "protection of the fetus" from virus attacks. Contemporarily, we have an increase in the anti-inflammatory cytokine IL-10 and an increase in AMPs, which highlights how the mother's body is responding to the viral attack. These results allow us to hypothesize a mechanism of "trafficking" of antimicrobial peptides from the mother to the fetus that would help the fetus to protect itself from the infection in progress.

The TNFRSF13C H159Y Variant Is Associated with Severe COVID-19: A Retrospective Study of 500 Patients from Southern Italy.

To identify host genetic determinants involved in humoral immunity and associated with the risk of developing severe COVID-19, we analyzed 500 SARS-CoV-2 positive subjects from Southern Italy. We examined the coding sequences of 10 common variable immunodeficiency-associated genes obtained by the whole-exome sequencing of 121 hospitalized patients. These 10 genes showed significant enrichment in predicted pathogenic point mutations in severe patients compared with the non-severe ones. Moreover, in the TNFRSF13C gene, the minor allele of the p.His159Tyr variant, which is known to increase NF-kB activation and B-cell production, was significantly more frequent in the 38 severe cases compared to both the 83 non-severe patients and the 375 asymptomatic subjects further genotyped. This finding identified a potential genetic risk factor of severe COVID-19 that not only may serve to unravel the mechanisms underlying the disease severity but, also, may contribute to build the rationale for individualized management based on B-cell therapy.

Hepatic Presentation of Late-Onset Multiple Acyl-CoA Dehydrogenase Deficiency (MADD): Case Report and Systematic Review.

Diagnosis of pediatric steatohepatitis is a challenging issue due to a vast number of established and novel causes. Here, we report a child with Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) presenting with an underrated muscle weakness, exercise intolerance and an atypically severe steatotic liver involvement. A systematic literature review of liver involvement in MADD was performed as well. Our patient is a 11-year-old otherwise healthy, non-obese, male child admitted for some weakness/asthenia, vomiting and recurrent severe hypertransaminasemia (aspartate and alanine aminotransferases up to ×20 times upper limit of normal). Hepatic ultrasound showed a bright liver. MRI detected mild lipid storage of thighs muscles. A liver biopsy showed a micro-macrovacuolar steatohepatitis with minimal fibrosis. Main causes of hypertransaminasemia were ruled out. Serum aminoacids (increased proline), acylcarnitines (increased C4-C18) and a large excretion of urinary glutaric acid, ethylmalonic, butyric, isobutyric, 2-methyl-butyric and isovaleric acids suggested a diagnosis of MADD. Serum acylcarnitines and urinary organic acids fluctuated overtime paralleling serum transaminases during periods of illness/catabolic stress, confirming their recurrent nature. Genetic testing confirmed the diagnosis [homozygous c.1658A > G (p.Tyr553Cys) in exon 12 of the ETFDH gene]. Lipid-restricted diet and riboflavin treatment rapidly ameliorated symptoms, hepatic ultrasonography/enzymes, and metabolic profiles. Literature review (37 retrieved eligible studies, 283 patients) showed that liver is an extramuscular organ rarely involved in late-onset MADD (70 patients), and that amongst 45 patients who had fatty liver only nine had severe presentation. Conclusion: MADD is a disorder with a clinically heterogeneous phenotype. Our study suggests that MADD warrants consideration in the work-up of obesity-unrelated severe steatohepatitis.

Effects of the COVID-19 Pandemic on Job Activity, Dietary Behaviours and Physical Activity Habits of University Population of Naples, Federico II-Italy.

Coronaviruses (CoVs) are a large family of respiratory viruses that can cause mild to moderate illness. The new variant COVID-19 has started to spread rapidly since December 2019, posing a new threat to global health. To counter the spread of the virus, the Italian government forced the population to close all activities starting from 9 March 2020 to 4 May 2020. In this scenario, we conducted a cross-sectional study on a heterogeneous sample (average age of 28 ± 12 years, 62.6% females) of the University of Naples Federico II (Italy). The aim of the study was to describe the lifestyle change in the university population during quarantine for the COVID 19 pandemic. Participants compiled an online survey consisting of 3 sections: socio-demographic data, dietary behaviours, physical activity habits and psychological aspects. The different results by gender are: 90.8% of females continued to work from home (81.9% were students); 34.8% increased their physical activity; and, only 0.8% prefer ready meals. Whereas, the same percentage of men continued to work from home (90%), but only 72.1% were students (p < 0.001 vs. females), only 23.9% increased physical activity (p < 0.001) and 1.7% favous ready meals. Our data shows that the male population was more affected by isolation and quarantine reporting more unfavourable behavioural changes.

Impact of Physical Activity on Cognitive Functions: A New Field for Research and Management of Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease inherited by an autosomal recessive mechanism and characterized by a progressive and severe multi-organ failure. Mutations in Cystic Fibrosis Conductance Regulator (CFTR) protein cause duct obstructions from dense mucus secretions and chronic inflammation related to organ damage. The progression of the disease is characterized by a decline of lung function associated with metabolic disorders and malnutrition, musculoskeletal disorders and thoracic deformities, leading to a progressive decrement of the individual's quality of life. The World Health Organization (WHO) qualifies Physical Activity (PA) as a structured activity produced by skeletal muscles' movements that requires energy consumption. In the last decade, the number of studies on PA increased considerably, including those investigating the effects of exercise on cognitive and brain health and mental performance. PA is recommended in CF management guidelines, since it improves clinic outcomes, such as peripheral neuropathy, oxygen uptake peak, bone health, glycemic control and respiratory functions. Several studies regarding the positive effects of exercise in patients with Cystic Fibrosis were carried out, but the link between the effects of exercise and cognitive and brain health in CF remains unclear. Animal models showed that exercise might improve learning and memory through structural changes of brain architecture, and such a causal relationship can also be described in humans. Indeed, both morphological and environmental factors seem to be involved in exercise-induced neural plasticity. An increase of gray matter volume in specific areas is detectable as a consequence of regular training in humans. Neurobiological processes associated with brain function improvements include biochemical modifications, such as neuromodulator or neurohormone release, brain-derived neurotrophic factor (BDNF) production and synaptic activity changes. From a functional point of view, PA also seems to be an environmental factor enhancing cognitive abilities, such as executive functions, memory and processing speed. This review describes the current state of research regarding the impacts of physical activity and exercise on cognitive functions, introducing a possible novel field of research for optimizing the management of Cystic Fibrosis.

Genotype-Phenotype Correlation: A Triple DNA Mutational Event in a Boy Entering Sport Conveys an Additional Pathogenicity Risk.

The purpose of this paper is to present a clinical and laboratory study of a family, in which a 12-year-old boy was examined to assess his health status before starting competitive sports. A variety of clinical and instrumental tests were used to evaluate the status of the heart and its functions. Using Sanger sequencing (SS), we sequenced six related genes to verify suspected arrhythmogenic right ventricular cardiomyopathy (ARVC) hypothesized at the cardiac assessment and, subsequently, by a next-generation sequencing (NGS)-based multi-gene panel for more paramount genetic risk of sudden cardiac death (SCD) assessment. SS revealed two variants in the PKP2 gene, one was inherited from the father and the other from the mother. The analysis on a large panel of genes (n = 138), putatively associated with sudden cardiac death, revealed, in the proband, a third variant in a different gene (DES) that encodes the protein desmin. Our results indicate that: i) NGS revealed a mutational event in a gene not conventionally screened as a first-line test in the presence of clinical suspicion of the arrhythmic disease; ii) a plurality of variants in different genes in the same subject (the proband) may increase the risk of heart disease; iii) in silico analysis with various methodological software and bioinformatic prediction tools indicates that the cumulative effects of the three variants in the same subject constitute an additional risk factor. This case report indicates that more pathogenic variants or likely pathogenic variants can contribute to the clinical phenotype of an individual, thereby contributing to the diagnosis and prognosis of inherited heart diseases.

Hypermethioninemia in Campania: Results from 10 years of newborn screening.

In the last years tandem mass spectrometry (MS/MS) has become a leading technology used for neonatal screening purposes. Newborn screening by MS/MS on dried blood spot samples (DBS) has one of its items in methionine levels: the knowledge of this parameter allows the identification of infant affected by homocystinuria (cystathionine ß-synthase, CBS, deficiency) but can also lead, as side effect, to identify cases of methionine adenosyltransferase (MAT) type I/III deficiency. We started an expanded newborn screening for inborn errors of metabolism in Campania region in 2007. Here we report our ten years experience on expanded newborn screening in identifying patients affected by hypermethioninemia. During this period we screened approximately 77,000 infants and identified two cases: one case of classical homocystinuria and one patient affected by defect of MAT I/III. In this paper we describe these patients and their biochemical follow-up and review the literature concerning worldwide newborn screening reports on incidence of CBS and MAT deficiency.

Mild dehydration in dyspeptic athletes is able to increase gastrointestinal symptoms: Protective effects of an appropriate hydration.

BACKGROUND: Water balance influences gastrointestinal (GI) activity. Our aim was to evaluate how dehydration and rehydration with different types of water are able to affect GI activity in healthy and dyspeptic athletes. METHODS: Twenty non-competitive athletes, respectively 10 healthy and 10 dyspeptic subjects, were enrolled. All subjects underwent three test sessions (0, A, B) of 6 hours. Dehydration was achieved with a walking/jogging exercise test on a treadmill. After exercising, 500 mL of calcium-bicarbonate (Test A) or soft water (Test B) were administered, while no rehydration was provided during Test 0; thereafter, all subjects consumed a light lunch. GI symptoms were evaluated during each test and an electrocardiogram (ECG) Holter recording was performed at the end of the exercise. KEY RESULTS: Dyspeptic subjects exhibited higher overall symptoms during Test 0 (VAS: 30.8 ± 0.8 mm) compared to Test A (18.4 ± 1.1, P < 0.001) and Test B (24.4 ± 1.3, P < 0.001). However, analyzing GI symptoms, only subjects receiving calcium-bicarbonate water (Test A) showed significantly lower symptomatic scores compared to Test 0 or Test B. Moreover, heart rate variability analyses revealed that only in Test A dyspeptic patients exhibit a trend to a decrease in the post-prandial low/high frequency (LF/HF) ratio, similarly to healthy subjects, while in Test 0 and Test B, post-prandial LF/HF ratio was increased compared to the pre-prandial phase. CONCLUSIONS AND INFERENCES: Our results show that mild dehydration in dyspeptic athletes is able to increase GI symptoms but an adequate rehydration, with calcium-bicarbonate water, is able to improve post-exercise disturbances restoring sympathovagal imbalance.

Allelic Complexity in Long QT Syndrome: A Family-Case Study.

Congenital long QT syndrome (LQTS) is associated with high genetic and allelic heterogeneity. In some cases, more than one genetic variant is identified in the same (compound heterozygosity) or different (digenic heterozygosity) genes, and subjects with multiple pathogenic mutations may have a more severe disease. Standard-of-care clinical genetic testing for this and other arrhythmia susceptibility syndromes improves the identification of complex genotypes. Therefore, it is important to distinguish between pathogenic mutations and benign rare variants. We identified four genetic variants (KCNQ1-p.R583H, KCNH2-p.C108Y, KCNH2-p.K897T, and KCNE1-p.G38S) in an LQTS family. On the basis of in silico analysis, clinical data from our family, and the evidence from previous studies, we analyzed two mutated channels, KCNQ1-p.R583H and KCNH2-p.C108Y, using the whole-cell patch clamp technique. We found that KCNQ1-p.R583H was not associated with a severe functional impairment, whereas KCNH2-p.C108Y, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type. Notably, the common variants KCNH2-p.K897T and KCNE1-p.G38S were previously reported to produce more severe phenotypes when combined with disease-causing alleles. Our results indicate that the novel KCNH2-C108Y variant can be a pathogenic LQTS mutation, whereas KCNQ1-p.R583H, KCNH2-p.K897T, and KCNE1-p.G38S could be LQTS modifiers.

Mulibrey nanism: Two novel mutations in a child identified by Array CGH and DNA sequencing.

In childhood, several rare genetic diseases have overlapping symptoms and signs, including those regarding growth alterations, thus the differential diagnosis is sometimes difficult. The proband, aged 3 years, was suspected to have Silver-Russel syndrome because of intrauterine growth retardation, postnatal growth retardation, typical facial dysmorphic features, macrocephaly, body asymmetry, and bilateral fifth finger clinodactyly. Other features were left atrial and ventricular enlargement and patent foramen ovale. Total X-ray skeleton showed hypoplasia of the twelfth rib bilaterally and of the coccyx, slender long bones with thick cortex, and narrow medullary channels. The genetic investigation did not confirm Silver-Russel syndrome. At the age of 5 the patient developed an additional sign: hepatomegaly. Array CGH revealed a 147 kb deletion (involving TRIM 37 and SKA2 genes) on one allele of chromosome 17, inherited from his mother. These results suggested Mulibrey nanism. The clinical features were found to fit this hypothesis. Sequencing of the TRIM 37 gene showed a single base change at a splicing locus, inherited from his father that provoked a truncated protein. The combined use of Array CGH and DNA sequencing confirmed diagnosis of Mulibrey nanism. The large deletion involving the SKA2 gene, along with the increased frequency of malignant tumours in mulibrey patients, suggests closed monitoring for cancer of our patient and his mother. Array CGH should be performed as first tier test in all infants with multiple anomalies. The clinician should reconsider the clinical features when the genetics suggests this. © 2016 Wiley Periodicals, Inc.

The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches.

BACKGROUND: Accurate and sensitive detection of BRCA1/2 germ-line mutations is crucial for the clinical management of women affected by breast cancer, for prevention and, notably, also for the identification of at-risk healthy relatives. The most widely used methods for BRCA1/2 molecular analysis are Sanger sequencing, and denaturing high performance liquid chromatography (dHPLC) followed by the Sanger method. However, recent findings suggest that next-generation sequencing (NGS)-based approaches may be an efficient tool for diagnostic purposes. In this context, we evaluated the effectiveness of NGS for BRCA gene analysis compared with dHPLC/Sanger sequencing. METHODS: Seventy women were screened for BRCA1/2 mutations by both dHPLC/Sanger sequencing and NGS, and the data were analyzed using a bioinformatic pipeline. RESULTS: Sequence data analysis showed that NGS is more sensitive in detecting BRCA1/2 variants than the conventional procedure, namely, dHPLC/Sanger. CONCLUSION: Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA1/2 genes.

Targeted metabolomics in the expanded newborn screening for inborn errors of metabolism.

Inborn errors of metabolism are genetic disorders due to impaired activity of enzymes, transporters, or cofactors resulting in accumulation of abnormal metabolites proximal to the metabolic block, lack of essential products or accumulation of by-products. Many of these disorders have serious clinical consequences for affected neonates, and an early diagnosis allows presymptomatic treatment which can prevent severe permanent sequelae and in some cases death. Expanded newborn screening for these diseases is a promising field of targeted metabolomics. Here we report the application, between 2007 and 2014, of this approach to the identification of newborns in southern Italy at risk of developing a potentially fatal disease. The analysis of amino acids and acylcarnitines in dried blood spots by tandem mass spectrometry revealed 24 affected newborns among 45,466 infants evaluated between 48 and 72 hours of life (overall incidence: 1 : 1894). Diagnoses of newborns with elevated metabolites were confirmed by gas chromatography-mass spectrometry, biochemical studies, and genetic analysis. Five infants were diagnosed with medium-chain acyl CoA dehydrogenase deficiency, 1 with methylmalonic acidemia with homocystinuria type CblC, 2 with isolated methylmalonic acidemia, 1 with propionic acidemia, 1 with isovaleric academia, 1 with isobutyryl-CoA dehydrogenase deficiency, 1 with beta ketothiolase deficiency, 1 with short branched chain amino acid deficiency, 1 with 3-methlycrotonyl-CoA carboxylase deficiency, 1 with formimino-transferase cyclodeaminase deficiency, and 1 with cystathionine-beta-synthase deficiency. Seven cases of maternal vitamin B12 deficiency and 1 case of maternal carnitine uptake deficiency were detected. This study supports the widespread application of metabolomic-based newborn screening for these genetic diseases.

Prenatal diagnosis of inherited diseases: 20 years' experience of an Italian Regional Reference Centre.

BACKGROUND: The demand for molecular prenatal diagnosis (PD) of inherited diseases to help high-risk couples make informed reproductive decisions has increased in the past decade. METHODS: We provided multidisciplinary pre-test counselling to 1248 couples at high risk of having a child affected by an inherited disease. RESULTS: After multidisciplinary counselling, 1171 couples requested PD for one of 73 inherited diseases. Of these, 995 (85.0%) were performed on DNA from chorionic villi (CV) and 176 (15.0%) on samples from amniocentesis. The occurrence of pregnancy loss (0.6%) and major complications did not differ significantly between the two groups. We made a diagnosis in all cases (including 8 twin pregnancies) except in 4/995 cases of CV sampling (0.4%) and in 3/176 of amniocentesis (1.7%) due to insufficient DNA. In 15 cases, molecular analysis revealed non-paternity. CONCLUSIONS: PD by analysis of foetal DNA from CV is a reliable aid in reproduction decision-making for couples at high risk of inherited diseases. The complexity of experimental procedures and the specific expertise required for the pre- and post-test multidisciplinary counselling suggest that PD be performed in reference centres also within the framework of supranational networks.