Clinical and Molecular Characterization of SMAD4 Splicing Variants in Patients with Juvenile Polyposis Syndrome.

Juvenile polyposis syndrome (JPS) is an inherited autosomal dominant condition that predisposes to the development of juvenile polyps throughout the gastrointestinal (GI) tract, and it poses an increased risk of GI malignancy. Germline causative variants were identified in the SMAD4 gene in a subset (20%) of JPS cases. Most SMAD4 germline genetic variants published to date are missense, nonsense, and frameshift mutations. SMAD4 germline alterations predicted to result in aberrant splicing have rarely been reported. Here, we report two unrelated Italian families harboring two different SMAD4 intronic variants, c.424+5G>A and c.425-9A>G, which are clinically associated with colorectal cancer and/or juvenile GI polyps. In silico prediction analysis, in vitro minigene assays, and RT-PCR showed that the identified variants lead to aberrant SMAD4 splicing via the exonization of intronic nucleotides, resulting in a premature stop codon. This is expected to cause the production of a truncated protein. This study expands the landscape of SMAD4 germline genetic variants associated with GI polyposis and/or cancer. Moreover, it emphasizes the importance of the functional characterization of SMAD4 splicing variants through RNA analysis, which can provide new insights into genetic disease variant interpretation, enabling tailored genetic counseling, management, and surveillance of patients with GI polyposis and/or cancer.

Classic Galactosemia: Clinical and Computational Characterization of a Novel GALT Missense Variant (p.A303D) and a Literature Review.

Classic galactosemia is an autosomal recessive inherited liver disorder of carbohydrate metabolism caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT). While a galactose-restricted diet is lifesaving, most patients still develop long-term complications. In this study, we report on a two-week-old female patient who is a compound heterozygote for a known pathogenic variant (p.K285N) and a novel missense variant (p.A303D) in the GALT gene. Segregation analysis showed that the patient inherited the p.K285N pathogenic variant from her father and the p.A303D variant from her mother. A bioinformatics analysis to predict the impact of the p.A303D missense variant on the structure and stability of the GALT protein revealed that it may be pathogenic. Based on this finding, we performed a literature review of all GALT missense variants identified in homozygous and compound heterozygous galactosemia patients carrying the p.K285N pathogenic variant to explore their molecular effects on the clinical phenotype of the disease. Our analysis revealed that these missense variants are responsible for a wide range of molecular defects. This study expands the clinical and mutational spectrum in classic galactosemia and reinforces the importance of understanding the molecular consequences of genetic variants to incorporate genetic analysis into clinical care.

Tumor Testing and Genetic Analysis to Identify Lynch Syndrome Patients in an Italian Colorectal Cancer Cohort.

Lynch syndrome (LS) is an inherited cancer susceptibility syndrome caused by germline mutations in a DNA mismatch repair (MMR) gene or in the EPCAM gene. LS is associated with an increased lifetime risk of colorectal cancer (CRC) and other malignancies. The screening algorithm for LS patient selection is based on the identification of CRC specimens that have MMR loss/high microsatellite instability (MSI-H) and are wild-type for BRAFV600. Here, we sought to clinically and molecularly characterize patients with these features. From 2017 to 2023, 841 CRC patients were evaluated for MSI and BRAFV600E mutation status, 100 of which showed MSI-H. Of these, 70 were wild-type for BRAFV600. Among these 70 patients, 30 were genetically tested for germline variants in hereditary cancer predisposition syndrome genes. This analysis showed that 19 of these 30 patients (63.3%) harbored a germline pathogenic or likely pathogenic variant in MMR genes, 2 (6.7%) harbored a variant of unknown significance (VUS) in MMR genes, 3 (10%) harbored a VUS in other cancer-related genes, and 6 (20%) were negative to genetic testing. These findings highlight the importance of personalized medicine for tailored genetic counseling, management, and surveillance of families with LS and other hereditary cancer syndromes.

Characterization of a rare variant (c.2635-2A>G) of the MSH2 gene in a family with Lynch syndrome.

INTRODUCTION: Lynch syndrome is caused by germline mutations in one of the mismatch repair genes ( MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. Lynch syndrome is defined on the basis of clinical, pathological, and genetic findings. Accordingly, the identification of predisposing genes allows for accurate risk assessment and tailored screening protocols. CASE DESCRIPTION: Here, we report a family case with three family members manifesting the Lynch syndrome phenotype, all of which harbor the rare variant c.2635-2A>G affecting the splice site consensus sequence of intron 15 of the MSH2 gene. This mutation was previously described only in one family with Lynch syndrome, in which mismatch repair protein expression in tumor tissues was not assessed. In this study, we report for the first time the molecular characterization of the MSH2 c.2635-2A>G variant through in silico prediction analysis, microsatellite instability, and mismatch repair protein expression experiments on tumor tissues of Lynch syndrome patients. The potential effect of the splice site variant was revealed by three splicing prediction bioinformatics tools, which suggested the generation of a new cryptic splicing site. The potential pathogenic role of this variant was also revealed by the presence of microsatellite instability and the absence of MSH2/MSH6 heterodimer protein expression in the tumor cells of cancer tissues of the affected family members. CONCLUSIONS: We provide compelling evidence in favor of the pathogenic role of the MSH2 variant c.2635-2A>G, which could induce an alteration of the canonical splice site and consequently an aberrant form of the protein product (MSH2).

Association of beta-adrenergic receptor polymorphisms and progression to heart failure in patients with idiopathic dilated cardiomyopathy.

PURPOSE: Increased sympathetic nervous system activation via the beta-adrenergic pathway influences the evolution of idiopathic dilated cardiomyopathy. We assessed the effects of beta-adrenergic receptor variants on heart failure in idiopathic dilated cardiomyopathy. METHODS: We prospectively analyzed 171 consecutive patients (mean [+/- SD] age, 49 +/- 14 years; 129 men) with idiopathic dilated cardiomyopathy who were receiving conventional treatment. All were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis for Ser49Gly and Arg389Gly in the beta1-adrenergic receptor; the 5' leader cistron (LC) Arg19Cys, Arg16Gly, Gln27Glu, and Thr164Ile in the beta2-adrenergic receptor; and Arg64Trp in the beta3-adrenergic receptor. The endpoint was heart failure, defined as a worsening of clinical condition leading to hospitalization for heart failure, cardiac transplantation, or death from heart failure. RESULTS: During a median follow-up of 33 months, 24 patients had heart failure. In a Cox univariate analysis, the beta1Gly49 and beta2 5'LC-Cys19, Arg16, and Gln27 alleles were associated with a lower risk of heart failure. In a multivariate analysis that considered age, functional class, left ventricular ejection fraction, and beta-blocker use, three beta2-adrenergic receptor alleles were associated with lower risk: 5'LC-Cys19 (hazard ratio [HR]: 0.15; 95% confidence interval [CI]: 0.05 to 0.42), Arg16 (HR: 0.12; 95% CI: 0.04 to 0.35), and Gln27 (HR: 0.15; 95% CI: 0.05 to 0.42). CONCLUSION: The Gly49 allele in the beta1-adrenergic receptor and the 5' LC-Cys19, Arg16, and Gln27 alleles in the beta2-adrenergic receptor were associated with a lower risk of heart failure in idiopathic dilated cardiomyopathy, suggesting that the beta1- and beta2-adrenergic receptor genes are modifier genes.

QT-interval prolongation in right precordial leads: an additional electrocardiographic hallmark of Brugada syndrome.

OBJECTIVES: The aim of this study was to evaluate whether the occurrence of the Brugada Syndrome typical electrocardiogram (ECG) pattern (i.e., right bundle branch block, coved-type ST-segment elevation, and T-wave inversion in the right precordial leads) is characterized by a concomitant lengthening of QT intervals in the right precordial leads. BACKGROUND: It has been suggested that the typical ECG pattern of Brugada syndrome is due to a decreased net inward current during phase 1 of the action potential, which also leads to its prolongation in the right epicardium. METHODS: Thirty-two subjects (19 males) age 37 +/- 15 years with a suspicious baseline ECG, or who were relatives of Brugada syndrome patients, underwent 12-lead ECG before and after the administration of flecainide. RESULTS: The flecainide test was negative in 14 and positive in 18 subjects. After flecainide administration, the positive ECGs were characterized by a greater QT interval corrected for heart rate (QTc) prolongation in the right precordial leads than that in the negative ECGs (78.2 +/- 35.5 ms vs. 22.0 +/- 28.4 ms in V(1) and 107.1 +/- 43.8 ms vs. 26.7 +/- 30.1 ms in V(2); p < 0.01), whereas there was no difference in the QTc prolongation in the left precordial leads (55.2 +/- 25.3 ms vs. 35.1 +/- 28.1 ms in V(5) and 53.1 +/- 32.8 ms vs. 27.3 +/- 22.4 ms in V(6); p = NS). CONCLUSIONS: In accordance with the electrophysiological background, the typical ECG pattern of Brugada syndrome is also characterized by a considerable prolongation of the QT interval in right precordial leads.