Carrier frequency of CFTR variants in the non-Caucasian populations by genome aggregation database (gnomAD)-based analysis.

The complexity in the molecular diagnosis of Cystic Fibrosis (CF) also depends on the variable prevalence/incidence of the disease associated with the wide CFTR allelic heterogeneity among different populations. In fact, CF incidence in Asian and African countries is underestimated and the few patients reported so far have rare or unique CFTR pathogenic variants. To obtain insights into CF variants profile and frequency, we used the large population sequencing data in the Genome Aggregation Database (gnomAD). We selected 207 CF-causing/varying clinical consequence variants from CFTR2 database and additional 15 variants submitted to the ClinVar database. Only 14 of these variants were found in the East-Asian population, while for South-Asian and African populations we identified 43 and 52 variants, respectively, confirming the peculiarity of the CFTR allelic spectrum with only few population-specific variants. These data could be used to optimize CFTR carrier screening in non-Caucasian subjects, choosing between the full gene sequencing and cost and time-effective targeted panels.

Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: A 10-year single center experience.

BACKGROUND: Marfan Syndrome (MFS) is a chronic, life-threatening, autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene, coding for fibrillin-1. All organ systems may be affected, but particularly the cardiovascular system, eyes, and skeleton. Mortality generally results from cardiovascular complications, mainly aortic dissection. Currently, the diagnosis of MFS is based on the revised Ghent nosology. Molecular analysis of the FBN1 gene reduces diagnostic uncertainty in patients with suspected MFS or MFS-related disorders (MFS-RD). To date, more than 2700 FBN1 mutations are known. METHODS: Using Next Generation Sequencing (NGS) followed by Multiplex Ligation-dependent Probe Amplification on NGS-negative samples, we screened FBN1 gene on 124 unrelated patients (101 MFS fulfilling revised Ghent criteria, 20 suspected MFS, 3 MFS-RD) enrolled from 2008 to 2018 at the Multidisciplinary Marfan Clinic, Tor Vergata Hospital, Rome. RESULTS: An FBN1 variant was identified in 107/124 (86.3%) patients, including 48 novel variants (46 pathogenic/likely pathogenic, 2 VUS). A pathogenic/likely pathogenic variant was detected in 90/101 (89.1%) MFS patients. Our approach allowed early diagnosis for 10 young patients (age 3-19 years) with suspected MFS. CONCLUSIONS: This study broadens the mutation spectrum of FBN1, providing a full update of the molecular basis of MFS in Italy.

Lp-PLA2, a new biomarker of vascular disorders in metabolic diseases.

Metabolic diseases are chronic disorders correlated to a greater risk of cardiovascular event and death. Recently, many data have sustained the biological link between microvascular dysfunction, oxidative stress, vascular inflammation, and metabolic diseases. The determination of new and specific blood biomarkers of vascular inflammation associated with obesity-related metabolic syndrome (MetS) and diabetes such as lipoprotein-associated phospholipase A2 (Lp-PLA2) could be useful to identify subject with high risk of cardiovascular events. Lp-PLA2 participates by a crucial role in microvascular dysfunction and oxidative stress showing positive association with metabolic disorders. In this review, we will argue the evolving role of Lp-PLA2 in predicting cardiovascular events in metabolic disease patients.

Drug repositioning screening identifies etravirine as a potential therapeutic for friedreich's ataxia.

BACKGROUND: Friedreich's ataxia is an autosomal-recessive cerebellar ataxia caused by mutation of the frataxin gene, resulting in decreased frataxin expression, mitochondrial dysfunction, and oxidative stress. Currently, no treatment is available for Friedreich's ataxia patients. Given that levels of residual frataxin critically affect disease severity, the main goal of a specific therapy for Friedreich's ataxia is to increase frataxin levels. OBJECTIVES: With the aim to accelerate the development of a new therapy for Friedreich's ataxia, we took a drug repositioning approach to identify market-available drugs able to increase frataxin levels. METHODS: Using a cell-based reporter assay to monitor variation in frataxin amount, we performed a high-throughput screening of a library containing 853 U.S. Food and Drug Administration-approved drugs. RESULTS: Among the potentially interesting candidates isolated from the screening, we focused our attention on etravirine, an antiviral drug currently in use as an anti-human immunodeficiency virus therapy. Here, we show that etravirine can promote a significant increase in frataxin levels in cells derived from Friedreich's ataxia patients, by enhancing frataxin messenger RNA translation. Importantly, frataxin accumulation in treated patient cell lines is comparable to frataxin levels in unaffected carrier cells, suggesting that etravirine could be therapeutically relevant. Indeed, etravirine treatment restores the activity of the iron-sulphur cluster containing enzyme aconitase and confers resistance to oxidative stress in cells derived from Friedreich's ataxia patients. CONCLUSIONS: Considering its excellent safety profile along with its ability to increase frataxin levels and correct some of the disease-related defects, etravirine represents a promising candidate as a therapeutic for Friedreich's ataxia. © 2019 International Parkinson and Movement Disorder Society.

Performance characteristics of lipoprotein-associated phospholipase A2 activity assay on the Dimension Vista analyser and preliminary study of a healthy Italian population.

INTRODUCTION: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a marker of vascular inflammation associated with coronary heart disease and stroke. We evaluated analytical performance of the PLAC® Activity Test on Siemens Dimension Vista® 1500 analyzer and measured Lp-PLA2 activity in Italian adults to establish reference intervals (RIs) and evaluate correlation with circulating lipids and age. MATERIALS AND METHODS: The evaluation protocol consisted of precision, linearity, sensitivity, method comparison, substrate depletion ("hook") effect and interference assessment. Inhibitor (Darapladib) effect was also evaluated. Lp-PLA2 activity was measured in 250 healthy donors (123 males, 127 females, aged 18-70 years). Central 95% RIs were established using nonparametric statistics. RESULTS: Intra-assay and inter-assay precision showed CVs of 0.6% - 1.4% and 0.9% - 2.0%, respectively. Linearity replicates showed R2 > 0.98. Limit of quantitation was 5.8 U/L (CV = 9.4%). Bland Altman plot showed bias - 0.9, 95% limits of agreement -6.5 - 4.72. Passing-Bablok regression showed excellent correlation (Slope = 1.02, 95% CI: 1.01 to 1.03; Intercept = - 1.86, 95% CI: - 3.08 to - 1.26; R2 = 0.999). No "hook effect" was observed at Lp-PLA2 activities ≤ 1000 U/L. Average Lp-PLA2 activity in 250 healthy donors was 182 ± 44 U/L (mean ± SD). Males showed statistically significant higher activities than females (P < 0.001). RIs were 107 - 265 U/L for males and 84 - 225 U/L for females. Moderate significant correlation (r = 0.29, P < 0.001) was found between Lp-PLA2 activity and total cholesterol. CONCLUSIONS: The PLAC® Activity Test shows very good performance characteristics on Dimension Vista® 1500.

Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC). METHODS: Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis. RESULTS: We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/ß-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of ß-actin was 26.12 ± 13.39 ng/mL for healthy, and 49.61 ± 46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC. CONCLUSIONS: This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results.

Genetic risk factors in typical haemolytic uraemic syndrome.

BACKGROUND: Haemolytic uraemic syndrome (HUS) is a disorder characterized by thrombotic microangiopathy, which is caused in 'typical forms' by gastrointestinal infections with Escherichia Coli species that produce verotoxins. Several studies have identified negative prognostic factors of the disease, among which prolonged oliguria, neurological involvement and increased leukocytosis have been more consistently reported. We have hypothesized that the genetic background may also predispose to the development of typical forms of HUS and may influence the clinical course of the disease. METHODS: Fourteen polymorphisms, known to influence the coagulation pathway or the activity of the renin-angiotensin system, have been selected and studied in 150 Italian children with typical forms of HUS. Two hundred healthy Italian children were used as controls. RESULTS: The risk of developing HUS was strongly associated with the platelet glycoprotein 1balpha 145M allele (OR 3.08; CI: 1.62-5.85) (P < 0.001). A significant association was also found with polymorphisms located in the adipocyte-derived leucine aminopeptidase and factor V genes. A longer duration of dialysis was moderately associated with increased leukocytosis and with the 807T allele of the platelet glycoprotein 1a gene. High white blood cell count was also strongly associated with the risk of long-term sequelae (OR 2.91, CI: 1.21-6.98) (P < 0.02), whereas the 1166C allele of the angiotensin II type 1 receptor had a significant protective effect (OR 0.28, CI: 0.09-0.83) (P < 0.02). CONCLUSIONS: These results highlight the role of glycoprotein 1balpha in the physiopathology of typical forms of HUS and show that the genetic background plays a role in the susceptibility and severity of the disease.

Identification and subcellular localization of a new cystinosin isoform.

Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [(35)S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.

Impaired activity of the gamma-glutamyl cycle in nephropathic cystinosis fibroblasts.

Cystinotic patients have been shown to excrete in their urine high levels of pyroglutamate, an intermediate metabolite of the adenosine triphosphate (ATP)-dependent gamma-glutamyl cycle, which is responsible for glutathione (GSH) synthesis. Human fibroblasts were used to study the mechanisms leading to pyroglutamate accumulation in nephropathic cystinosis (NC). We show that inhibition of ATP synthesis caused a marked intracellular accumulation of pyroglutamate, reflecting decreased GSH synthesis. Despite similar degrees of ATP depletion, pyroglutamate increased more in cystinotic fibroblasts than in controls, while GSH decreased to lower levels. In addition, cystinotic cells exposed to oxidative stress (hydrogen peroxide) were unable to increase their GSH concentration above baseline. These results could not be attributed to differences in mitochondrial oxidative activity or to increased apoptotic cell death. Together, these results support the hypothesis that cysteine derived from lysosomal cystine efflux limits the activity of the gamma-glutamyl cycle and GSH synthesis.

Serum homocysteine, methylenetetrahydrofolate reductase gene polymorphism and cardiovascular disease in heterozygous familial hypercholesterolemia.

Mild hyperhomocysteinemia is considered an important risk factor for vascular disease. A common polymorphism (677C-->T) in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased enzyme activity and consequent higher circulating levels of homocysteine. We hypothesized that the serum levels of homocysteine and/or the MTHFR polymorphism could influence the risk for coronary artery disease (CAD) in patients with heterozygous familial hypercholesterolemia (FH), who are genetically prone to atherosclerosis. We determined the MTHFR genotype and fasting total serum homocysteine level in 249 adult patients (103 males and 146 females) with heterozygous FH. MTHFR polymorphism was a major determinant of serum homocysteine in adult FH of both sexes. The logistic regression analysis showed that in FH patients a high level of homocysteine (> 12 micromol/l, corresponding to the upper quartile of serum distribution) was the most significant predictor of CAD (n=99) in all the groups considered (all CAD, previous myocardial infarction, myocardial infarction plus angiographically confirmed CAD). The adjusted odds ratio (OR (95% CI)) for the homocysteine-associated risk of CAD (upper quartile versus lower quartiles) was 3.27 (1.60-6.62) in males and females considered together, 5.67 (1.50-21.3) in males and 2.78 (1.17-6.62) in females. LDL cholesterol (upper quartile versus lower quartiles) and hypertension were the other variables independently associated with CAD. In both sexes MTHFR polymorphism was not an independent predictor of CAD. Plasma concentration of serum homocysteine, but not MTHFR genotype, is associated with an increased risk of CAD in male and female patients with heterozygous FH.

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography.

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.

Rapid determination of mycophenolic acid in plasma by reversed-phase high-performance liquid chromatography.

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure plasma concentrations of mycophenolic acid. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a Zorbax Eclipse C(8) reversed-phase column with a flow-rate of 1.5 ml/min, and monitoring the absorbance at 250 nm. Throughput was up to 100 samples in 24 h. Within the investigated concentration ranges of mycophenolic acid (0-100 mg/l), good linearity (r>0.99) was obtained. The method is sensitive (the limit of detection was about 20 microg/l) and precise (for 0.49 mg/l added to plasma, within-run C.V. was 2% and between-run was 4.2%; for 2.88 mg/l, within-run C.V. was 0.35% and between-run C.V. was 0.69%; for 24.38 mg/l, within-run C.V. was 0.77% and between-run C.V. was 3.1%). Analytical recoveries were 96% for 0.5 mg/l mycophenolic acid added to plasma, 100% for 12 mg/l and 102.5% for 24 mg/l.