Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing (WGS) data from nearly 10,000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n=6 from 5 families), AAGGC (n=2 from 1 family), AGAGG (n=1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, here we show a pathogenic role for large AAAGG repeat configuration expansions (n=5). Long read sequencing was used to fully characterise the entire repeat sequence and revealed a pure AGGGC expansion in six patients, whereas the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs seem to have arisen from a common haplotype and are predicted to form highly stable G quadruplexes, which have been previously demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions, since the AAAGG motif when very large (>500 repeats) or in the presence of AAGGG interruptions. Accurate sizing and full sequencing of the satellite repeat with long read is recommended in clinically selected cases, in order to achieve an accurate molecular diagnosis and counsel patients and their families.
Introduction:Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)nrepeat expansions in the second intron of the Replication Factor Complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expands the genotypic spectrum of CANVAS by identifying point mutations inRFC1coding region associated with this condition.Methods:Fifteen individuals diagnosed with CANVAS and carrying only one heterozygous (AAGGG)nexpansion inRFC1underwent WGS or WES to test for the presence of a second variant inRFC1or other unrelated gene. To assess the impact of truncating variants onRFC1expression we tested the level of RFC1 transcript and protein on patients’ derived cell lines.Results:We identified seven patients from five unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)nexpansion together with a second truncating variantin transinRFC1, which included: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6) and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein.Discussion:Our report expands the genotype spectrum of RFC1 disease. FullRFC1sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)nexpansions. Also, it sheds further light on the pathogenesis of RFC1 CANVAS as it supports the existence of a loss of function mechanism underlying this complex neurodegenerative condition.
Cystic fibrosis (CF) is a genetic disease caused by loss-offunction mutations in the CFTR gene, which codes for a defective ion channel. This causes an electrolyte imbalance and results in a spiral of negative effects on multiple organs, most notably the accumulation of thick mucus in the lungs, chronic respiratory tract infections and inflammation leading to pulmonary exacerbation and premature death. Progressive decline of lung function is mainly linked to persistent or recurring infections, mostly caused by bacteria, which require treatments with antibiotics and represent one of the major life-limiting factors in subjects with CF. Treatment of such a complex disease require multiple drugs with a consequent therapeutic burden and complications caused by drug-drug interactions and rapid emergence of bacterial drug resistance. We report herein our recent efforts in developing innovative multifunctional antibiotics specifically tailored to CF by a direct action on bacterial topoisomerases and a potential indirect effect on the pulmonary mucociliary clearance mediated by ΔF508-CFTR correction. The obtained results may pave the way for the development of a simplified therapeutic approach with a single agent acting as multifunctional Antibacterial-Corrector.
Inhibition of c-Src is considered one of the most studied approaches to cancer treatment, with several heterocyclic compounds approved during the last 15 years as chemotherapeutic agents. Starting from the biological evaluation of an in-house collection of small molecules, indolinone was selected as the most promising scaffold. In this work, several functionalised indolinones were synthesised and their inhibitory potency and cytotoxic activity were assayed. The pharmacological profile of the most active compounds, supported by molecular modelling studies, revealed that the presence of an amino group increased the affinity towards the ATP-binding site of c-Src. At the same time, bulkier derivatizations seemed to improve the interactions within the enzymatic pocket. Overall, these data represent an early stage towards the optimisation of new, easy-to-be functionalised indolinones as potential c-Src inhibitors.
The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood–brain barriers led us to select compound 5 for further in vivo assays.
Src is a non-receptor tyrosine kinase (TK) whose involvement in cancer, including glioblastoma (GBM), has been extensively demonstrated. In this context, we started from our in-house library of pyrazolo[3,4-d]pyrimidines that are active as Src and/or Bcr-Abl TK inhibitors and performed a lead optimization study to discover a new generation derivative that is suitable for Src kinase targeting. We synthesized a library of 19 compounds, 2a-s. Among these, compound 2a (SI388) was identified as the most potent Src inhibitor. Based on the cell-free results, we investigated the effect of SI388 in 2D and 3D GBM cellular models. Interestingly, SI388 significantly inhibits Src kinase, and therefore affects cell viability, tumorigenicity and enhances cancer cell sensitivity to ionizing radiation.
During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c-Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c-Myc protein expression, leading to a reduction in the levels of the canonical c-Myc isoform and an increase in the expression of c-Myc 1, a c-Myc isoform translated from an in-frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c-Myc expression as in glutamine-fed cells, with high levels of canonical c-Myc and c-Myc 1 almost undetectable. In asparagine-fed cells, global protein translation was higher than in glutamine-starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine-starved asparagine-fed cells, the inhibition of c-Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c-Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.
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