Comparison of the TaqMan and LightCycler systems in pharmacogenetic testing: evaluation of the CYP2C9*2/*3 polymorphisms.

Background: Pharmacogenetic testing for drugmetabolizing enzymes is not yet widely used in clinical practice. Methods: In an attempt to facilitate the application of this procedure, we have compared two real-time PCRbased methods, the TaqMan_ and the LightCycler_ for the pharmacogenetic evaluation of CYP2C9*2/*3 polymorphisms. Results and Conclusion: Both procedures are suitable for pharmacogenetic studies. The TaqMan procedure was less expensive in terms of cost per sample, but the TaqMan apparatus is more expensive than the LightCycler apparatus

Sudden cardiac death in young athletes: Literature review of molecular basis

Intense athletic training and competition can rarely result in sudden cardiac death (SCD). Despite the introduction of pre-participation cardiovascular screening, especially among young competitive athletes, sport-related SCD remains a debated issue among medical personnel, sports communities and laypersons alike, and generates significant media attention. The most frequent cause of SCD is a hidden inherited cardiomyopathy, the athletes may not even be aware of. Predictive medicine, by searching the presence of pathogenic alterations in cardiac genes, may be an integrative tool, besides the conventional ones used in cardiology (mainly electro and echocardiogram), to reach a definitive diagnosis in athletes showing signs/symptoms, even borderline, of inherited cardiomyopathy/ channelopathy, and in athletes presenting family history of SCD and/or of hereditary cardiac disease. In this review, we revised the molecular basis of the major cardiac diseases associated to sudden cardiac death and the clinical molecular biology approach that can be used to perform risk assessment at DNA level of sudden cardiac death, contributing to the early implementation of adequate therapy. Alterations can occur in ion channel genes, in genes encoding desmosomal and junctional proteins, sarcomeric and Z-disc proteins, proteins for the cytoskeleton and the nuclear envelope. The advent of next generation sequencing (NGS) technology has provided the means to search for mutations in all these genes, at the same time. Therefore, this molecular approach should be the preferred methodology for the aforementioned purpose

The mtDNA 15497 G/A polymorphism in cytochrome b in severe obese subjects from Southern Italy.

Background and aim: A large number of mitochondrial DNA (mtDNA) mutations have been implicated in degenerative diseases and aging. The aim of this study was to evaluate whether the 15497 G/A mtDNA polymorphism (G251S) in the cytochrome b subunit of respiratory complex III, which has been associated with obesity-related variables and lipid metabolism in a Japanese population, is associated with severe obesity also in adult Caucasians from southern Italy. Methods and results: Unrelated severely obese patients (n Z 317; BMI > 40 kg/m2) and controls (n Z 217; BMI < 25 kg/m2) from Southern Italy were genotyped by allelic discrimination TaqMan assay for the 15497 G/A mtDNA polymorphism. In obese patients fasting serum total cholesterol, triglycerides, HDL-cholesterol and glucose were measured enzymatically and sitting blood pressure and heart rate were also collected. Mean levels of total cholesterol, triglycerides and glucose were below the upper reference limit for healthy subjects. Female obese subjects showed lower levels of blood pressure and heart rate and higher levels of HDL cholesterol than male obese patients (P < 0.001). All the control subjects and 315/317 severely obese patients were homozygous for the G allele (wild type), whereas only 2/317, were females homozygous for the A allele. Conclusions: The mtDNA 15497 G/A polymorphism in cytochrome b was present in 0.6% obese subjects, two females whose lipid parameters and BMI were similar to those of the overall group. Therefore, this mutation may appear to contribute in rare instances to severe obesity but does not explain the majority of cases in our population. A more extensive genetic haplogroup characterization is required to identify associations to obesity in Caucasians

Mitochondrial mutation in adult patient with Hypertrophic Cardiomyopathy and renal failure

Background: Mitochondrial diseases (MDs) (1:5000-10000) represents a wide group of human disorders associated with mitochondrial DNA (mtDNA) variations causing defect of oxidative phosphorylation system, whereas nuclear genome mutations are somewhat rare. The extremely heterogeneous clinical phenotype, extending from oligosymptomatic condition to complex syndromes involving neurological, ophtalmological, gastroenterological and endocrine features, depends to the involved tissue well as to the specific mtDNA mutations and their heteroplasmic level. Diabetes and deafness are common features of mitochondrial diseases, while renal alterations are rarely reported, especially in adults, probably because of lack of association to mitochondrial conventional phenotypes. Case Presentation: We investigated a 62 years old male affected by hypertrophic cardiomyopathy (HCM) and renal failure that caused already a bilateral transplantation. Pathological anamnesis revealed also diabetes, deafness and Crohn disease. Family history of cardiomyopathy showed a strong mitochondrial involvement. The proband's mother, three brothers (one of which died of renal failure at 26 years), the sister and her child were affected. Materials and Methods: Genomic DNA from peripheral blood and buccal cells was extracted with the Kit-Nucleon-BACC2 (Illustra DNA-Extraction Kit-BACC2-GE Healthcare, UK) and the whole mitochondrial genome was amplified by two pair of primers designed in our laboratory to generate two overlapping fragments. The PCR products were then sequenced and compared to mitochondrial reference sequence (rCRS NC_012920). Results and Discussion: In both biological samples the mtDNA analysis showed the heteroplasmic A3243G mutation in the tRNALeu (UUR), frequently associated with MDs. A cardiological involvement leading to hypertrophic remodelling, caused to mitochondria intermyofibrillar proliferation, occurs up to 40% of patients with mtDNA disease. Molecular backgrounds of mitochondrial cardiomyopathy of adult age are still quite poorly known and the A3243G mutation in tRNA Leu(UUR) of mtDNA has been reported in 40-60% of patients with HCM. The interesting finding presented here support the knowledge that mitochondrial gene altertation represents a possible etiology in cardiological patients with unexplained renal failure. This is particularly true, as in this case, when other associated symtoms linked with dysfunctional oxidative phosphorylation are present. The case presented in this report further suggests that a differential diagnosis in presence of HCM should be solved by a multidisciplinary approach together with mutation analysis of mitochondrial DNA

De novo mitochondrial DNA alteration in child with complex neurilogical compromission.

neuromuscular human diseases have been associated with mitochondrial DNA (mtDNA) variations, causing defects of oxidative phosphorylation. These dysfunctions affect preferentially tissues with high energy demands and give arise to several degenerative disorders such as optic neuropathy, cerebellar ataxia, movement disorders, dementia, muscle weakness and deafness. The extremely heterogeneous clinical phenotype is due to the involved tissue, to specific mtDNA mutations and their heteroplasmic level, but also to nuclear DNA alterations, environmental and epigenetic factors. In this study we investigated a child affected by a complex neurological disease whose clinical features were suggestive of a mitochondrial involvement. Methods: mtDNA from proband, her healthy relatives (grandmother, mother and two sisters) and 80 controls were collected and studied by sequencing. The enzymatic activity of specific respiratory chain complex was tested on lymphocytes by spectrophotometric assay. Bioinformatic analysis was performed to predict the pathogenicity of the detected variants. Results: In all subjects we detected 11 known polymorphisms, whereas 1 novel heteroplasmic variant in complex I [ND5:12514G>A (E60K)] was present only in the proband and in her grandmother and absent in controls. The bioinformatics predicted the novel variant to be deleterious. Further, spectrophotometric assay of complex I activity was lower both in the proband and in her relatives than in the controls. Conclusions: We report a novel mtDNA variant detected in a patient affected by a complex neurological disease. The reduction of complex I respiratory chain activity associated to this variant suggests it could exert a pathogenic role in the disease

Protein Thermodynamic Destabilization in the Assessment of Pathogenicity of a Variant of Uncertain Significance in Cardiac Myosin Binding Protein C.

In the era of next generation sequencing (NGS), genetic testing for inherited disorders identifies an ever-increasing number of variants whose pathogenicity remains unclear. These variants of uncertain significance (VUS) limit the reach of genetic testing in clinical practice. The VUS for hypertrophic cardiomyopathy (HCM), the most common familial heart disease, constitute over 60% of entries for missense variants shown in ClinVar database. We have studied a novel VUS (c.1809T>G-p.I603M) in the most frequently mutated gene in HCM, MYBPC3, which codes for cardiac myosin-binding protein C (cMyBPC). Our determinations of pathogenicity integrate bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. In silico prediction and mRNA analysis indicated no alteration of RNA splicing induced by the variant. At the protein level, the p.I603M mutation maps to the C4 domain of cMyBPC. Although the mutation does not perturb much the overall structure of the C4 domain, the stability of C4 I603M is severely compromised as detected by circular dichroism and differential scanning calorimetry experiments. Taking into account the highly destabilizing effect of the mutation in the structure of C4, we propose reclassification of variant p.I603M as likely pathogenic. Looking into the future, the workflow described here can be used to refine the assignment of pathogenicity of variants of uncertain significance in MYBPC3.J.A.C. was funded by the Ministerio de Ciencia, Innovación y Universidades (MCNU) through grants BIO2017-83640-P (AEI/FEDER, UE) and RYC-2014-16604, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, MINOTAUR, AC16/00045), the Comunidad de Madrid (P2018/NMT-4443) and the CNIC-Severo Ochoa intramural grant program (03-2016 IGP). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). G.F. was funded by the Ministero dell’Istruzione, dell’Università e della Ricerca-Rome PS35-126/IND.S