A recombination-based method to characterize human BRCA1 missense variants

Purpose. Many missense variants in BRCA1 are of unclear clinical significance. Functional and genetic approaches have been proposed for elucidating the clinical significance of such variants. The purpose of the present study was to evaluate BRCA1 missense variants for their effect on both Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). Methods. HR frequency evaluation: HeLaG1 cells, containing a stably integrated plasmid that allows to measure HR events by gene conversion events were transfected with the pcDNA3β expression vector containing the BRCA1-wild type (BRCA1-WT) or the BRCA1-Unclassified Variants (BRCA1-UCVs). The NHEJ was measured by a random plasmid integration assay. Results. This assays suggested a BRCA1 involvement mainly in the NHEJ. As a matter of fact, the Y179C and the A1789T variant altered significantly the NHEJ activity as compared to the wild type, suggesting that they may be related to BRCA1 associated pathogenicity by affecting this function. The variants N550H and I1766S, and the mutation M1775R did not alter the NHEJ frequency. Conclusions. These data, beside proposing a method for the study of BRCA1 variants effect on HR and NHEJ, highlighted the need for a range of functional assays to be performed in order to identify variants with altered function

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Whole-exome analysis in osteosarcoma to identify a personalized therapy

Osteosarcoma is the most common pediatric primary non-hematopoietic bone tumor. Survival of these young patients is related to the response to chemotherapy and development of metastases. Despite many advances in cancer research, chemotherapy regimens for osteosarcoma are still based on non-selective cytotoxic drugs. It is essential to investigate new specific molecular therapies for osteosarcoma to increase the survival rate of these patients. We performed exomic sequence analyses of 8 diagnostic biopsies of patients with conventional high grade osteosarcoma to advance our understanding of their genetic underpinnings and to correlate the genetic alteration with the clinical and pathological features of each patient to identify a personalized therapy. We identified 18,275 somatic variations in 8,247 genes and we found three mutated genes in 7/8 (87%) samples (KIF1B, NEB and KMT2C). KMT2C showed the highest number of variations; it is an important component of a histone H3 lysine 4 methyltransferase complex and it is one of the histone modifiers previously implicated in carcinogenesis, never studied in osteosarcoma. Moreover, we found a group of 15 genes that showed variations only in patients that did not respond to therapy and developed metastasis and some of these genes are involved in carcinogenesis and tumor progression in other tumors. These data could offer the opportunity to get a key molecular target to identify possible new strategies for early diagnosis and new therapeutic approaches for osteosarcoma and to provide a tailored treatment for each patient based on their genetic profile

Reappraisal of the F/M amplitude ratio in carpal tunnel syndrome

The F-wave/M-wave amplitude (F/M-amp) ratio has been shown to be increased in peripheral neuropathies, provided the maximum M-wave is relatively preserved. Reduced M-wave amplitudes and central facilitation of antidromically-induced reactivation of the anterior horn cells’ axon hillocks (F-wave) are believed to contribute to higher F/M-amp ratios. The present study was undertaken to re-evaluate mechanisms responsible for higher F/M-amp ratios in carpal tunnel syndrome (CTS). We enrolled 232 cases affected by CTS and 108 controls. F-and M-wave amplitudes and F-wave chronodispersion were analyzed for the median and ulnar nerves. The F/M-amp ratio of the median nerve in CTS subjects with moderate-severe nerve damage was significantly higher than that of mild CTS subjects and controls. Chronodispersion of the median nerve F-wave increased with increasing CTS severity. We conclude that the relative preservation of the median nerve F-wave is due to damage to the large diameter muscle afferent fibers responsible for the monosynaptic response. Absence of the monosynaptic response makes the small motoneurons, usually inaccessible to the antidromic volley because of its collision with the orthodromic reflex volley, able to fire in the F-wave

c-KIT receptor expression is strictly associated with the biological behaviour of thyroid nodules

<p>Abstract</p> <p>Background</p> <p>A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively.</p> <p>Methods</p> <p>In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene.</p> <p>Results</p> <p>We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%.</p> <p>Conclusion</p> <p>We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first, then the use of the c-KIT expression to ultimately assess the diagnosis of the nodules that otherwise would remain suspicious. The c-KIT expression-based classification is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of thyroid suspicious malignancies.</p

Whole-exome sequencing to identify causative variants in juvenile sudden cardiac death

Background: Juvenile sudden cardiac death (SCD) remains unexplained in approximately 40% of cases, leading to a significant emotional burden for the victims' families and society. Comprehensive investigations are essential to uncover its elusive causes and enable cascade family screening. This study aimed to enhance the identification of likely causative variants in juvenile SCD cases (age ≤ 50&nbsp;years), particularly when autopsy findings are inconclusive. Results: Autopsy revealed diagnostic structural abnormalities in 46%, non-diagnostic findings in 23%, and structurally normal hearts in 31%&nbsp;of cases. Whole-exome sequencing (WES), refined through a customized virtual gene panel was used to identify variants. These variants were then evaluated using a multidisciplinary approach and a structured variant prioritization scheme. Our extended&nbsp;approach identified likely causative variants in 69% of cases, outperforming the diagnostic yields of both the cardio panel and standard susceptibility gene analysis (50% and 16%, respectively). The extended cardio panel achieved an 80% diagnostic yield in cases with structurally normal hearts, demonstrating its efficacy in challenging scenarios. Notably, half of the positive cases harboured a single variant, while the remainder had two or more variants. Conclusion: This study highlights the efficacy of a multidisciplinary approach employing WES and a tailored virtual gene panel to elucidate the aetiology of juvenile SCD. The findings support the&nbsp;expansion of genetic testing using tailored gene panels and prioritization schemes as part of routine autopsy evaluations to improve the identification of causative variants and potentially facilitate early diagnosis in first-degree relatives