Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization.

BACKGROUND: Lipoprotein(a) [Lp(a)] level variability, related to atherothrombotic risk increase, is mainly attributed to LPA gene, encoding apolipoprotein(a), with kringle IV type 2 (KIV2) copy number variation (CNV) acting as the primary genetic determinant. Genetic characterization of Lp(a) is in continuous growth; nevertheless, the peculiar structural characteristics of this variant constitute a significant challenge to the development of effective detection methods. The aim of the study was to compare quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR) in the evaluation of KIV2 repeat polymorphism. METHODS: We analysed 100 subjects tested for cardiovascular risk in which Lp(a) plasma levels were assessed. RESULTS: Correlation analysis between CNV values obtained with the two methods was slightly significant (R = 0.413, p = 0.00002), because of the wider data dispersion in qPCR compared with ddPCR. Internal controls C1, C2 and C3 measurements throughout different experimental sessions revealed the superior stability of ddPCR, which was supported by a reduced intra/inter-assay coefficient of variation determined in this method compared to qPCR. A significant inverse correlation between Lp(a) levels and CNV values was confirmed for both techniques, but it was higher when evaluated by ddPCR than qPCR (R = -0.393, p = 0.000053 vs R = -0.220, p = 0.028, respectively). When dividing subjects into two groups according to 500 mg/L Lp(a) cut-off value, a significantly lower number of KIV2 repeats emerged among subjects with greater Lp(a) levels, with stronger evidence in ddPCR than in qPCR (p = 0.000013 and p = 0.001, respectively). CONCLUSIONS: Data obtained support a better performance of ddPCR in the evaluation of KIV2 repeat polymorphism.

The Role of Extracellular Matrix and Inflammation in the Stratification of Bleeding and Thrombotic Risk of Atrial Fibrillation on Oral Anticoagulant Therapy: Insights from Strat-Af Study.

In anticoagulated atrial fibrillation (AF) patients, the validity of models recommended for the stratification of the risk ratio between benefits and hemorrhage risk is limited. We hypothesize that both circulating and neuroimaging-based markers might improve the prediction of bleeding and thrombotic risk in anticoagulated AF patients. The Strat-AF study is an observational, prospective, single-center study enrolling 170 patients with AF; recruited patients are evaluated by means of a comprehensive protocol, with clinical, cerebral magnetic resonance imaging and circulating biomarkers assessment. The main outcome is the evaluation of cerebral microangiopathy related to the levels of circulating biomarkers of inflammation and extracellular matrix (ECM) remodeling. At multivariate logistic regression analysis adjusted for age, sex, CHA2DS2-VASc, HAS-BLED and type of anticoagulant, matrix metalloproteinases (MMP)-2 levels were significantly and positively associated with the presence of cerebral microbleeds (CMBs). A significant association between MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1,-2,-4 levels and white matter hyperintensity was also found. Concerning the small vessel disease (SVD) score, MMP-2 and TIMP-1,-2 levels were associated with the presence of two and three or more signs of SVD, whereas TIMP-4 levels were associated with the presence of three signs of SVD with respect to patients with no instrumental signs of SVD. As regarding the presence of enlarged perivascular spaces (EPVS), a significant association was found for high levels of interleukin (IL)-8 and TIMP 1-2-3. These results demonstrate that patients with AF have evidence of impaired ECM degradation, which is an independent risk factor for thrombotic complications of AF patients on oral anticoagulant therapy. The incorporation of these markers in the prognostic schemes might improve their clinical capability in predicting stroke risk and thrombotic complications.

Nuclear Magnetic Resonance-Based Metabolomics to Predict Early and Late Adverse Outcomes in Ischemic Stroke Treated with Intravenous Thrombolysis.

Metabolic perturbations and inflammatory mediators play a fundamental role in both early and late adverse post-acute ischemic stroke outcomes. Using data from the observational MAGIC (MArker bioloGici nell'Ictus Cerebrale) study, we evaluated the effect of 130 serum metabolic features, using a nuclear magnetic spectroscopy approach, on the following outcomes: hemorrhagic transformation at 24 h after stroke, non-response to intravenous thrombolytic treatment with the recombinant tissue plasminogen activator (rt-PA), and the 3 month functional outcome. Blood circulating metabolites, lipoproteins, and inflammatory markers were assessed at the baseline and 24 h after rt-PA treatment. Adjusting for the major determinants for unfavorable outcomes (i.e., age, sex, time onset-to-treatment, etc.), we found that acetone and 3-hydroxybutyrate were associated with symptomatic hemorrhagic transformation and with non-response to rt-PA; while 24 h after rt-PA, levels of triglycerides high-density lipoprotein (HDL) and triglycerides low-density lipoprotein (LDL) were associated with 3 month mortality. Cholesterol and phospholipids levels, mainly related to smaller and denser very low-density lipoprotein (VLDL) and LDL subfractions were associated with 3 month poor functional outcomes. We also reported associations between baseline 24 h relative variation (Δ) in VLDL subfractions and ΔC-reactive protein, Δinterleukin-10 levels with hemorrhagic transformation. All observed metabolic changes reflect a general condition of energy failure, oxidative stress, and systemic inflammation that characterize the development of adverse outcomes.

Tracking an Elusive Killer: State of the Art of Molecular-Genetic Knowledge and Laboratory Role in Diagnosis and Risk Stratification of Thoracic Aortic Aneurysm and Dissection.

The main challenge in diagnosing and managing thoracic aortic aneurysm and dissection (TAA/D) is represented by the early detection of a disease that is both deadly and "elusive", as it generally grows asymptomatically prior to rupture, leading to death in the majority of cases. Gender differences exist in aortic dissection in terms of incidence and treatment options. Efforts have been made to identify biomarkers that may help in early diagnosis and in detecting those patients at a higher risk of developing life-threatening complications. As soon as the hereditability of the TAA/D was demonstrated, several genetic factors were found to be associated with both the syndromic and non-syndromic forms of the disease, and they currently play a role in patient diagnosis/prognosis and management-guidance purposes. Likewise, circulating biomarker could represent a valuable resource in assisting the diagnosis, and several studies have attempted to identify specific molecules that may help with risk stratification outside the emergency department. Even if promising, those data lack specificity/sensitivity, and, in most cases, they need more testing before entering the "clinical arena". This review summarizes the state of the art of the laboratory in TAA/D diagnostics, with particular reference to the current and future role of molecular-genetic testing.

Analysis of Metabolite and Lipid Association Networks Reveals Molecular Mechanisms Associated with 3-Month Mortality and Poor Functional Outcomes in Patients with Acute Ischemic Stroke after Thrombolytic Treatment with Recombinant Tissue Plasminogen Activator.

Here, we present an integrated multivariate, univariate, network reconstruction and differential analysis of metabolite-metabolite and metabolite-lipid association networks built from an array of 18 serum metabolites and 110 lipids identified and quantified through nuclear magnetic resonance spectroscopy in a cohort of 248 patients, of which 22 died and 82 developed a poor functional outcome within 3 months from acute ischemic stroke (AIS) treated with intravenous recombinant tissue plasminogen activator. We explored differences in metabolite and lipid connectivity of patients who did not develop a poor outcome and who survived the ischemic stroke from the related opposite conditions. We report statistically significant differences in the connectivity patterns of both low- and high-molecular-weight metabolites, implying underlying variations in the metabolic pathway involving leucine, glycine, glutamine, tyrosine, phenylalanine, citric, lactic, and acetic acids, ketone bodies, and different lipids, thus characterizing patients' outcomes. Our results evidence the promising and powerful role of the metabolite-metabolite and metabolite-lipid association networks in investigating molecular mechanisms underlying AIS patient's outcome.

Two-Dimensional Aortic Size Normalcy: A Novelty Detection Approach.

Background: To develop a tool for assessing normalcy of the thoracic aorta (TA) by echocardiography, based on either a linear regression model (Z-score), or a machine learning technique, namely one-class support vector machine (OC-SVM) (Q-score). Methods: TA diameters were measured in 1112 prospectively enrolled healthy subjects, aging 5 to 89 years. Considering sex, age and body surface area we developed two calculators based on the traditional Z-score and the novel Q-score. The calculators were compared in 198 adults with TA > 40 mm, and in 466 patients affected by either Marfan syndrome or bicuspid aortic valve (BAV). Results: Q-score attained a better Area Under the Curve (0.989; 95% CI 0.984-0.993, sensitivity = 97.5%, specificity = 95.4%) than Z-score (0.955; 95% CI 0.942-0.967, sensitivity = 81.3%, specificity = 93.3%; p < 0.0001) in patients with TA > 40 mm. The prevalence of TA dilatation in Marfan and BAV patients was higher as Z-score > 2 than as Q-score < 4% (73.4% vs. 50.09%, p < 0.00001). Conclusions: Q-score is a novel tool for assessing TA normalcy based on a model requiring less assumptions about the distribution of the relevant variables. Notably, diameters do not need to depend linearly on anthropometric measurements. Additionally, Q-score can capture the joint distribution of these variables with all four diameters simultaneously, thus accounting for the overall aortic shape. This approach results in a lower rate of predicted TA abnormalcy in patients at risk of TA aneurysm. Further prognostic studies will be necessary for assessing the relative effectiveness of Q-score versus Z-score.

Chiari 1 malformation and exome sequencing in 51 trios: the emerging role of rare missense variants in chromatin-remodeling genes.

Type 1 Chiari malformation (C1M) is characterized by cerebellar tonsillar herniation of 3-5 mm or more, the frequency of which is presumably much higher than one in 1000 births, as previously believed. Its etiology remains undefined, although a genetic basis is strongly supported by C1M presence in numerous genetic syndromes associated with different genes. Whole-exome sequencing (WES) in 51 between isolated and syndromic pediatric cases and their relatives was performed after confirmation of the defect by brain magnetic resonance image (MRI). Moreover, in all the cases showing an inherited candidate variant, brain MRI was performed in both parents and not only in the carrier one to investigate whether the defect segregated with the variant. More than half of the variants were Missense and belonged to the same chromatin-remodeling genes whose protein truncation variants are associated with severe neurodevelopmental syndromes. In the remaining cases, variants have been detected in genes with a role in cranial bone sutures, microcephaly, neural tube defects, and RASopathy. This study shows that the frequency of C1M is widely underestimated, in fact many of the variants, in particular those in the chromatin-remodeling genes, were inherited from a parent with C1M, either asymptomatic or with mild symptoms. In addition, C1M is a Mendelian trait, in most cases inherited as dominant. Finally, we demonstrate that modifications of the genes that regulate chromatin architecture can cause localized anatomical alterations, with symptoms of varying degrees.

When should a rare inherited connective tissue disorder be suspected in bicuspid aortic valve by primary-care internists and cardiologists? Proposal of a score.

Size threshold for aortic surgery in bicuspid aortic valve (BAV) is debated. Connective tissue disorders (CTDs) are claimed as a clinical turning point, suggesting early surgery in BAV patients with CTD. Thus, we aimed at developing a score to detect high risk of carrying CTDs in consecutive BAVs from primary care. Ninety-eight BAVs without ectopia lentis or personal/family history of aortic dissection were studied at the Marfan syndrome Tuscany Referral Center. Findings were compared with those detected in 84 Marfan patients matched for sex and age. We selected traits with high statistical difference between MFS and BAV easily obtainable by cardiologists and primary-care internists: mitral valve prolapse, myopia ≥ 3DO, pectus carenatum, pes planus, wrist and thumb signs, and difference between aortic size at root and ascending aorta ≥ 4 mm. Clustering of ≥ 3 of these manifestations were more frequent in Marfan patients than in BAVs (71.4% vs 6.1%, p < 0.0001) resulting into an Odds Ratio to be affected by MFS of 38.3 (95% confidence intervals 14.8-99.3, p < 0.0001). We propose a score assembling simple clinical and echocardiographic variables resulting in an appropriate referral pattern of BAVs from a primary-care setting to a tertiary center to evaluate the presence of a potential, major CTD.

Sanger Validation of High-Throughput Sequencing in Genetic Diagnosis: Still the Best Practice?

Next-generation sequencing (NGS)'s crucial role in supporting genetic diagnosis and personalized medicine leads to the definition of Guidelines for Diagnostic NGS by the European Society of Human Genetics. Factors of different nature producing false-positive/negative NGS data together with the paucity of internationally accepted guidelines providing specified NGS quality metrics to be followed for diagnostics purpose made the Sanger validation of NGS variants still mandatory. We reported the analysis of three cases of discrepancy between NGS and Sanger sequencing in a cohort of 218 patients. NGS was performed by Illumina MiSeq® and Haloplex/SureSelect protocols targeting 97 or 57 or 10 gene panels usually applied for diagnostics. Variants called following guidelines suggested by the Broad Institute and identified according to MAF <0.01 and allele balance >0.2 were Sanger validated. Three out of 945 validated variants showed a discrepancy between NGS and Sanger. In all three cases, a deep evaluation of the discrepant gene variant results and methodological approach allowed to confirm the NGS datum. Allelic dropout (ADO) occurrence during polymerase chain or sequencing reaction was observed, mainly related to incorrect variant zygosity. Our study extends literature data in which almost 100% "high quality" NGS variants are confirmed by Sanger; moreover, it demonstrates that in case of discrepancy between a high-quality NGS variant and Sanger validation, NGS call should not be a priori assumed to represent the source of the error. Actually, difficulties (i.e., ADO, unpredictable presence of private variants on primer-binding regions) of the so-called gold standard direct sequencing should be considered especially in light of the constantly implemented and accurate high-throughput technologies. Our data along with literature raise a discussion on the opportunity to establish a standardized quality threshold by International Guidelines for clinical NGS in order to limit Sanger confirmation to borderline conditions of variant quality parameters and verification of correct gene variant call/patient coupling on a different blood sample aliquot.

Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease.

BACKGROUND: The genetic basis of stroke susceptibility remains to be elucidated. STR1 quantitative trait locus (STR1/QTL) was identified on rat chromosome 1 of stroke-prone spontaneously hypertensive rat (SHRSP) upon Japanese-style stroke-permissive diet (JD), and it contributes to 20% of the stroke phenotype variance. METHODS AND RESULTS: Nine hundred eighty-six probe sets mapping on STR1 were selected from the Rat RAE230A array and screened through a microarray differential expression analysis in brains of SHRSP and stroke-resistant SHR (SHRSR) fed with either regular diet or JD. The gene encoding Ndufc2 (NADH dehydrogenase [ubiquinone] 1 subunit), mapping 8 Mb apart from STR1/QTL Lod score peak, was found significantly down-regulated under JD in SHRSP compared to SHRSR. Ndufc2 disruption altered complex I assembly and activity, reduced mitochondrial membrane potential and ATP levels, and increased reactive oxygen species production and inflammation both in vitro and in vivo. SHRSR carrying heterozygous Ndufc2 deletion showed renal abnormalities and stroke occurrence under JD, similarly to SHRSP. In humans, T allele variant at NDUFC2/rs11237379 was associated with significant reduction in gene expression and with increased occurrence of early-onset ischemic stroke by recessive mode of transmission (odds ratio [OR], 1.39; CI, 1.07-1.80; P=0.012). Subjects carrying TT/rs11237379 and A allele variant at NDUFC2/rs641836 had further increased risk of stroke (OR=1.56; CI, 1.14-2.13; P=0.006). CONCLUSIONS: A significant reduction of Ndufc2 expression causes complex I dysfunction and contributes to stroke susceptibility in SHRSP. Moreover, our current evidence may suggest that Ndufc2 can contribute to an increased occurrence of early-onset ischemic stroke in humans.

Genetic Contributions to the Development of Complications in Preterm Newborns.

AIM: We aimed to identify specific polymorphisms of genes encoding for vascular endothelial growth factor A (VEGFA), endothelial nitric oxide synthase (eNOS), renin-angiotensin system (angiotensinogen gene [AGT], angiotensinogen type 1 receptor [AGTR1], angiotensin-converting enzyme [ACE]), and heme oxygenase-1 (HMOX-1) in a cohort of preterm infants and correlate their presence with the development of respiratory distress syndrome (RDS) requiring mechanical ventilation (MV), bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH) and retinopathy of prematurity (ROP). STUDY DESIGN: We carried out a retrospective study to evaluate the allele frequency and genotype distribution of polymorphisms of VEGFA, eNOS, AGT, AGTR1, ACE, and HMOX-1 in a population of preterm neonates (n=342) with a gestational age ≤28 weeks according to the presence or absence of RDS requiring MV, BPD, IVH, or ROP. Moreover, we evaluated through the haplotype reconstruction analysis whether combinations of the selected polymorphisms are related to the occurrence of RDS, BPD, IVH and ROP. RESULTS: In our population 157 infants developed RDS requiring MV, 71 BPD, 70 IVH, and 43 ROP. We found that TC+CC rs2070744 eNOS (41.7 vs. 25.4%, p=0.01) and GT+TT rs1799983 eNOS (51.8 vs. 35.2%, p=0.01) polymorphisms are independent risk factors for BPD. Haplotype reconstruction showed that haplotypes in VEGF and eNOS are significantly associated with different effects on RDS, BPD, IVH, and ROP in our population. CONCLUSIONS: We found that TC+CC rs2070744 eNOS and GT+TT rs1799983 eNOS polymorphisms are independent predictors of an increased risk of developing BPD. Haplotypes of VEGFA and eNOS may be independent protective or risk markers for prematurity complications.