There is growing body of evidence supporting the role of germline mutations in the pathogenesis of pediatric central nervous system (CNS) tumors, and the widespread use of next‐generation sequencing (NGS) panels facilitates their detection. Variants of the MUTYH gene are increasingly recognized as suspected germline background of various extraintestinal malignancies, besides their well‐characterized role in the polyposis syndrome associated with biallelic mutations. Using a multigene NGS panel (Illumina TruSight Oncology 500), we detected one H3 G34V‐ and one H3 K27M‐mutant pediatric high‐grade diffuse glioma, in association with c.1178G>A (p.G393D) and c.916C>T (p.R306C) MUTYH variants, respectively. Both MUTYH mutations were germline, heterozygous and inherited, according to the subsequent genetic testing of the patients and their first‐degree relatives. In the H3 K27M‐mutant glioma, amplifications affecting the 4q12 region were also detected, in association with KDR‐PDGFRA, KIT‐PDGFRA, and KDR‐CHIC2 fusions, previously unreported in this entity. Among 47 other CNS tumors of various histological types tested with the same NGS panel in our institution, only one adult glioblastoma harbored MUTYH mutation. Together with a single previous report, our data raises the possibility of an association between germline MUTYH mutations and CNS malignancies, particularly in pediatric histone H3‐mutant gliomas.

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Administration of targeted therapies provides a promising treatment strategy for rare cancers such as urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC), however, the selection of appropriate drugs remains difficult. Therefore, in the present study, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. Next-generation sequencing using a 161 cancer driver gene panel was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered by four publicly available databases. Therapeutic interpretation has been performed by in silico evaluation of drug-gene interactions using an evidence-based decision support tool. After data processing, 45/54 samples (33 UrC and 12 PBAC) passed the quality control. The sequencing analyses revealed a total of 191 pathogenic SNVs in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), MYC (15%), EGFR (9%) and ERBB2 (9%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways in both tumour types were related to cell cycle regulation, DNA damage control and the MAPK/RAS pathway. Actionable mutations with at least one available, regulatory agency-approved drug could be identified for 31/33 (94%) of UrC and 8/12 (67%) of PBAC patients. In this study, we used a commercially available assay and developed a data processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, including EGFR, BRCA, CCND1/2/3, ERBB2, METex14 suggesting a potentially feasible strategy for both UrC and PBAC treatment.

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Heterozygous (HET) truncating mutations in the TTN gene (TTNtv) encoding the giant titin protein are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here we investigated 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene-sequence predictions were detected in the majority of the TTNtv samples. The total amount of expressed titin, which includes the truncated fragments, was comparable in the TTNtv+ and TTNtv- samples, indicating that titin haploinsufficiency is not the leading cause of the molecular pathogenesis. Proteomic analysis of washed cardiac myofibrils and Stimulated Emission Depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin is structurally integrated in the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape and intensity analysis pointed at structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which may contribute, via faulty mechanosensor function, to the development of manifest DCM.