Francesca Torricelli

Myofilament protein gene mutation screening and outcome of patients with hypertrophic cardiomyopathy.

OBJECTIVE To determine the influence of a positive genetic test for hypertrophic cardiomyopathy (HCM) on clinical outcome. PATIENTS AND METHODS A cohort of 203 unrelated patients with HCM (mean +/- SD age, 50+/-18 years) was enrolled from January 1, 2002, through December 31, 2003. They were followed up for a mean +/- SD time of 4.0+/-1.7 years after genetic testing of the 8 HCM-susceptibility genes that encode key sarcomeric/myofilament proteins. The clinical phenotype of those with a positive genetic test (myofilament-positive HCM) was compared with those with a negative genetic test (myofilament-negative HCM). RESULTS In this cohort of 203 patients, 87 mutations were identified in 126 patients (myofilament-positive HCM, 62%); the remaining 77 patients (38%) were myofilament-negative. Despite similar baseline features, patients with myofilament-positive HCM showed increased risk of the combined end points of cardiovascular death, nonfatal stroke, or progression to New York Heart Association class III or IV compared with the patients with myofilament-negative HCM (25% vs 7%, respectively; independent hazard ratio, 4.27; P=.008). These end points occurred at any age among patients with myofilament-positive HCM (range, 14-86 years), but only in those aged 65 years and older among patients with myofilament-negative HCM. Moreover, patients with myofilament-positive HCM showed greater probability of severe left ventricular systolic and diastolic dysfunction, defined as an ejection fraction of less than 50% and a restrictive filling pattern (P=.02 and P<.02, respectively, vs myofilament-negative HCM). CONCLUSION Screening for sarcomere protein gene mutations in HCM identifies a broad subgroup of patients with increased propensity toward long-term impairment of left ventricular function and adverse outcome, irrespective of the myofilament (thick, intermediate, or thin) involved.

Clinical Features and Outcome of Hypertrophic Cardiomyopathy Associated With Triple Sarcomere Protein Gene Mutations

OBJECTIVES The aim of this study was to describe the clinical profile associated with triple sarcomere gene mutations in a large hypertrophic cardiomyopathy (HCM) cohort. BACKGROUND In patients with HCM, double or compound sarcomere gene mutation heterozygosity might be associated with earlier disease onset and more severe outcome. The occurrence of triple mutations has not been reported. METHODS A total of 488 unrelated index HCM patients underwent screening for myofilament gene mutations by direct deoxyribonucleic acid sequencing of 8 genes, including myosin binding protein C (MYBPC3), beta-myosin heavy chain (MYH7), regulatory and essential light chains (MYL2, MYL3), troponin-T (TNNT2), troponin-I (TNNI3), alpha-tropomyosin (TPM1), and actin (ACTC). RESULTS Of the 488 index patients, 4 (0.8%) harbored triple mutations, as follows: MYH7-R869H, MYBPC3-E258K, and TNNI3-A86fs in a 32-year-old woman; MYH7-R723C, MYH7-E1455X, and MYBPC3-E165D in a 46-year old man; MYH7-R869H, MYBPC3-K1065fs, and MYBPC3-P371R in a 45-year old woman; and MYH7-R1079Q, MYBPC3-Q969X, and MYBPC3-R668H in a 50-year old woman. One had a history of resuscitated cardiac arrest, and 3 had significant risk factors for sudden cardiac death, prompting the insertion of an implantable cardioverter-defibrillator in all, with appropriate shocks in 2 patients. Moreover, 3 of 4 patients had a severe phenotype with progression to end-stage HCM by the fourth decade, requiring cardiac transplantation (n=1) or biventricular pacing (n=2). The fourth patient, however, had clinically mild disease. CONCLUSIONS Hypertrophic cardiomyopathy caused by triple sarcomere gene mutations was rare but conferred a remarkably increased risk of end-stage progression and ventricular arrhythmias, supporting an association between multiple sarcomere defects and adverse outcome. Comprehensive genetic testing might provide important insights to risk stratification and potentially indicate the need for differential surveillance strategies based on genotype.

Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model.

Recent studies indicate that mesenchymal stem cells (MSC) exhibit a degree of immune privilege due to their ability to suppress T cell mediated responses causing tissue rejection; however, the impact of allogeneic MSC in the setting of organ transplantation has been poorly investigated so far. The aim of our study was to evaluate the effect of intravenous donor MSC infusion for clinical tolerance induction in allogeneic skin graft transplantations in rats. MSC were isolated from Wistar rats and administered in Sprague-Dawley rats receiving Wistar skin graft with or without cyclosporine A (CsA). Graft biopsies were performed at day 10 post transplantation in all experimental groups for histological and gene expression studies. Intravenous infusion with donor MSC in CsA-treated transplanted rats resulted in prolongation of skin allograft survival compared to control animals. Unexpectedly, donor MSC infusion in immunocompetent rats resulted in a faster rejection as compared to control group. Cytokine expression analysis at the site of skin graft showed that CsA treatment significantly decreased pro-inflammatory cytokines IFN-γ and IL-2 and reduced TNF-α gene expression; however, the level of TNF-α is high in MSC-treated and not immunosuppressed rats. Results of our study in a rat tissue transplantation model demonstrated a possible immunogenic role for donor (allogeneic) MSC, confirming the need of adequate preclinical experimentation before clinical use.

Microvascular Function Is Selectively Impaired in Patients With Hypertrophic Cardiomyopathy and Sarcomere Myofilament Gene Mutations

OBJECTIVES The purpose of this study was to assess myocardial blood flow (MBF) using positron emission tomography in patients with hypertrophic cardiomyopathy (HCM) according to genetic status. BACKGROUND Coronary microvascular dysfunction is an important feature of HCM, associated with ventricular remodeling and heart failure. We recently demonstrated the increased prevalence of systolic dysfunction in patients with HCM with sarcomere myofilament gene mutations and postulated an association between genetic status and coronary microvascular dysfunction. METHODS Maximum MBF (intravenous dipyridamole, 0.56 mg/kg; Dip-MBF) was measured using (13)N-labeled ammonia in 61 patients with HCM (age 38 ± 14 years), genotyped by automatic DNA sequencing of 8 myofilament-encoding genes (myosin-binding protein C, beta-myosin heavy chain, regulatory and essential light chains, troponin T, troponin I, troponin C, alpha-tropomyosin, and alpha-actin). In 35 patients, cardiac magnetic resonance imaging was performed. RESULTS Fifty-three mutations were identified in 42 of the 61 patients (genotype positive; 69%). Despite similar clinical profiles, genotype-positive patients with HCM showed substantially lower Dip-MBF compared with that of genotype-negative patients (1.7 ± 0.6 ml/min/g vs. 2.4 ± 1.2 ml/min/g; p < 0.02). A Dip-MBF <1.5 ml/min/g had 81% positive predictive value for genotype-positive status and implied a 3.5-fold independent increase in likelihood of carrying myofilament gene mutations (hazard ratio: 3.52; 95% confidence interval: 1.05 to 11.7; p = 0.04). At cardiac magnetic resonance imaging, the prevalence of late gadolinium enhancement was greater in genotype-positive patients (22 of 23 [96%] compared with 8 of 12 [67%] genotype-negative patients; p = 0.038). CONCLUSIONS Patients with HCM with sarcomere myofilament mutations are characterized by more severe impairment of microvascular function and increased prevalence of myocardial fibrosis, compared with genotype-negative individuals. These findings suggest a direct link between sarcomere gene mutations and adverse remodeling of the microcirculation in HCM, accounting for the increased long-term prevalence of ventricular dysfunction and heart failure in genotype-positive patients.

Bioinformatics for Next Generation Sequencing Data

The emergence of next-generation sequencing (NGS) platforms imposes increasing demands on statistical methods and bioinformatic tools for the analysis and the management of the huge amounts of data generated by these technologies. Even at the early stages of their commercial availability, a large number of softwares already exist for analyzing NGS data. These tools can be fit into many general categories including alignment of sequence reads to a reference, base-calling and/or polymorphism detection, de novo assembly from paired or unpaired reads, structural variant detection and genome browsing. This manuscript aims to guide readers in the choice of the available computational tools that can be used to face the several steps of the data analysis workflow.

A molecular screening strategy based on β-myosin heavy chain, cardiac myosin binding protein C and troponin T genes in Italian patients with hypertrophic cardiomyopathy

Background Mutations causing hypertrophic cardiomyopathy (HCM) have been described in nine different genes of the sarcomere. Three genes account for most known mutations: β-myosin heavy chain (MYH7), cardiac myosin binding protein C (MYBPC3) and cardiac troponin T (TNNT2). Their prevalence in Italian HCM patients is unknown. Thus, we prospectively assessed a molecular screening strategy of these three genes in a consecutive population with HCM from two Italian centres. Methods Comprehensive screening of MYBPC3, MYH7 and TNNT2 was performed in 88 unrelated HCM patients by denaturing high-performance liquid chromatography and automatic sequencing. Results We identified 32 mutations in 50 patients (57%); 16 were novel. The prevalence rates for MYBPC3, MYH7 and TNNT2 were 32%, 17% and 2%, respectively. MYBPC3 mutations were 18, including two frameshift, five splice-site and two nonsense. All were ‘private’ except insC1065 and R502Q, present in three and two patients, respectively. Moreover, E258K was found in 14% of patients, suggesting a founder effect. MYH7 mutations were 12, all missense; seven were novel. In TNNT2, only two mutations were found. In addition, five patients had a complex genotype [i.e. carried a double MYBPC3 mutation (n = 2), or were double heterozygous for mutations in MYBPC3 and MYH7 (n = 3)]. Conclusions The first comprehensive evaluation of MYBPC3, MYH7 and TNNT2 in an Italian HCM population allowed a genetic diagnosis in 57% of the patients. These data support a combined analysis of the three major sarcomeric genes as a rational and cost-effective initial approach to the molecular screening of HCM.

Expression and Function of Gonadotropin-releasing Hormone (GnRH) Receptor in Human Olfactory GnRH-secreting Neurons AN AUTOCRINE GnRH LOOP UNDERLIES NEURONAL MIGRATION

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmanns syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3–4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype

This study reports a wide spectrum of factor 8 mutations in the large Italian database. Findings of the study indicate hat the type of mutations is a strong predictor of the clinical phenotype. Background The high mutational heterogeneity of hemophilia A is a challenge for the provision of genetic services. We plan to identify the mutation in patients with hemophilia A in order to create a confidential national database of mutations for the optimization of genetic services in Italy. Design and Methods The factor VIII gene (F8) was analyzed in 1296 unrelated patients with hemophilia A using screening methods for intron 22 and 1 inversions and rare mutations (denaturing high performance liquid chromatography, conformation sensitive gel electrophoresis) and/or direct sequencing. Results F8 mutations were identified in 874 (89%), 146 (89%), and 133 (94%) families with severe, moderate, or mild hemophilia A, respectively. Mutations predicting a null allele were responsible for 80%, 15%, and less than 1% of cases of severe, moderate, or mild hemophilia A, respectively. About 40% of missense and nonsense mutations occurred at a CpG site, arginines being most frequently affected. Of the small deletions or insertions, 29% occurred at one of two stretches of adenines, codons 1191–1194 (8As) and 1439–1441 (9As). Overall, these “hotspots” accounted for 31% of the point mutations in the patients with hemophilia A. Inhibitors developed in 22% of the patients with severe hemophilia A, 8% of those with moderate disease and in 4% of patients with mild hemophilia A. Patients who had severe hemophilia A and mutations predicting a null allele developed inhibitors more frequently (22 to 67%) than patients with missense mutations (5%). Conclusions We report a wide spectrum of mutations in a large national database. The type of mutation was a strong predictor of the clinical phenotype. This database is expected to considerably improve the genetic counselling and medical care of families with hemophilia A in Italy.

Read count approach for DNA copy number variants detection

MOTIVATION The advent of high-throughput sequencing technologies is revolutionizing our ability in discovering and genotyping DNA copy number variants (CNVs). Read count-based approaches are able to detect CNV regions with an unprecedented resolution. Although this computational strategy has been recently introduced in literature, much work has been already done for the preparation, normalization and analysis of this kind of data. RESULTS Here we face the many aspects that cover the detection of CNVs by using read count approach. We first study the characteristics and systematic biases of read count distributions, focusing on the normalization methods designed for removing these biases. Subsequently, we compare the algorithms designed to detect the boundaries of CNVs and we investigate the ability of read count data to predict the exact number of DNA copy. Finally, we review the tools publicly available for analysing read count data. To better understand the state of the art of read count approaches, we compare the performance of the three most widely used sequencing technologies (Illumina Genome Analyzer, Roche 454 and Life Technologies SOLiD) in all the analyses that we perform.

Genetic history of cystic fibrosis mutations in italy. I. Regional distribution

Earlier analysis of the Italian population showed patterns of genetic differentiation that were interpreted as being the result of population settlements going back to pre‐Roman times. DNA disease mutations may be a powerful tool in further testing this hypothesis since the analysis of diseased individuals can detect variants too rare to be resolved in normal individuals. We present data on the relative frequencies of 60 cystic fibrosis (CF) mutations in Italy and the geographical distribution of the 12 most frequent CF mutations screened in 3492 CF chromosomes originating in 13 Italian regions. The 12 most frequent mutations characterize about 73% of the Italian CF chromosomes. The most common mutation, ΔF508, has an average frequency of 51%, followed by N1303K and G542X, both with average frequencies around 5%. Multivariate analyses show that the relative frequencies of CF mutations are heterogeneous among Italian regions, and that this heterogeneity is weakly correlated with the geographical pattern of non‐DNA ‘classical’ genetic markers. The northern regions are well differentiated from the central‐southern regions and within the former group the western and eastern regions are remarkably distinct. Moreover, Sardinia shows the presence of mutation T338I, which seems absent in any other European CF chromosome. The north‐western regions of Italy, characterized by the mutation 1717‐1G→A, were under Celtic influence, while the north‐east regions, characterized by the mutations R1162X, 2183AA→G and 711 + 5G→A, were under the influence of the Venetic culture.