Open-Source Bioinformatic Pipeline to Improve PMS2 Genetic Testing Using Short-Read NGS Data.

The molecular diagnosis of mismatch repair-deficient cancer syndromes is hampered by difficulties in sequencing the PMS2 gene, mainly owing to the PMS2CL pseudogene. Next-generation sequencing short reads cannot be mapped unambiguously by standard pipelines, compromising variant calling accuracy. This study aimed to provide a refined bioinformatic pipeline for PMS2 mutational analysis and explore PMS2 germline pathogenic variant prevalence in an unselected hereditary cancer (HC) cohort. PMS2 mutational analysis was optimized using two cohorts: 192 unselected HC patients for assessing the allelic ratio of paralogous sequence variants, and 13 samples enriched with PMS2 (likely) pathogenic variants screened previously by long-range genomic DNA PCR amplification. Reads were forced to align with the PMS2 reference sequence, except those corresponding to exon 11, where only those intersecting gene-specific invariant positions were considered. Afterward, the refined pipeline's accuracy was validated in a cohort of 40 patients and used to screen 5619 HC patients. Compared with our routine diagnostic pipeline, the PMS2_vaR pipeline showed increased technical sensitivity (0.853 to 0.956, respectively) in the validation cohort, identifying all previously PMS2 pathogenic variants found by long-range genomic DNA PCR amplification. Fifteen HC cohort samples carried a pathogenic PMS2 variant (15 of 5619; 0.285%), doubling the estimated prevalence in the general population. The refined open-source approach improved PMS2 mutational analysis accuracy, allowing its inclusion in the routine next-generation sequencing pipeline streamlining PMS2 screening.

eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

SpadaHC: a database to improve the classification of variants in hereditary cancer genes in the Spanish population.

Accurate classification of genetic variants is crucial for clinical decision-making in hereditary cancer. In Spain, genetic diagnostic laboratories have traditionally approached this task independently due to the lack of a dedicated resource. Here we present SpadaHC, a web-based database for sharing variants in hereditary cancer genes in the Spanish population. SpadaHC is implemented using a three-tier architecture consisting of a relational database, a web tool and a bioinformatics pipeline. Contributing laboratories can share variant classifications and variants from individuals in Variant Calling Format (VCF) format. The platform supports open and restricted access, flexible dataset submissions, automatic pseudo-anonymization, VCF quality control, variant normalization and liftover between genome builds. Users can flexibly explore and search data, receive automatic discrepancy notifications and access SpadaHC population frequencies based on many criteria. In February 2024, SpadaHC included 18 laboratory members, storing 1.17 million variants from 4306 patients and 16 343 laboratory classifications. In the first analysis of the shared data, we identified 84 genetic variants with clinically relevant discrepancies in their classifications and addressed them through a three-phase resolution strategy. This work highlights the importance of data sharing to promote consistency in variant classifications among laboratories, so patients and family members can benefit from more accurate clinical management. Database URL: https://spadahc.ciberisciii.es/.

Utilidad clínica de la secuenciación de nueva generación en el diagnóstico etiológico de la hipoacusia neurosensorial en una Unidad de Hipoacusia Infantil / Clinical utility of next-generation sequencing in the aetiological diagnosis of sensorineural hearing loss in a Childhood Hearing Loss Unit

INTRODUCCIÓN: La hipoacusia neurosensorial (HNS) es el déficit sensorial más prevalente en nuestro medio. La secuenciación genómica de nueva generación (NGS) permite obtener un diagnóstico etiológico en un alto porcentaje de pacientes. Nuestro estudio piloto muestra los resultados de la aplicación sistemática de la NGS en una Unidad de Hipoacusia Infantil, así como sus implicaciones en el manejo clínico de los pacientes y sus familiares. MATERIAL Y MÉTODO: Se incluyó a 27 pacientes diagnosticados de HNS entre 2014 y 2017 en los que se descartó una causa ambiental. El test genético consistió en un panel de genes analizados mediante NGS (panel OTOgenicsTM). Este panel ha sido diseñado para incluir genes asociados con hipoacusia neurosensorial o mixta, de inicio precoz o tardío, sindrómica y no sindrómica, independientemente de su patrón de herencia. RESULTADOS: Se obtuvo un diagnóstico genético en el 56% (15/27) de los pacientes (62% en el caso de las HNS bilaterales); 5/27 (19%) presentaron variantes patogénicas en el gen GJB2 y el resto variantes patogénicas o probablemente patogénicas en otros genes asociados con HNS aislada (PR2X2, TECTA y STRC), con HNS sindrómicas (CHD7, GATA3, COL4A5, MITF y SOX10) o con HNS sindrómicas y no sindrómicas (BSND, ACTG1 y CDH23). DISCUSIÓN: El diagnóstico etiológico de la HNS supone un desafío en la práctica clínica. Nuestra serie demuestra que es posible implementar el diagnóstico genético en la rutina asistencial y que esta información tiene implicaciones pronósticas y terapéuticas

INTRODUCTION: Sensorineural hearing loss (SNL) is the most prevalent sensory deficit in our environment. Next generation genomic sequencing (NGS) enables an aetiological diagnosis in a high percentage of patients. Our pilot study shows the results of the systematic application of NGS in a Childhood Hearing Loss Unit, as well as its implications for the clinical management of patients and their families. MATERIAL AND METHOD: We included 27 patients diagnosed with SNL between 2014 and 2017, in which an environmental cause was ruled out. The genetic test consisted of a panel of genes analyzed by NGS (OTOgenicsTM panel). This panel has been designed to include genes associated with sensorineural or mixed hearing loss, early onset or late, syndromic and non-syndromic, regardless of their inheritance pattern. RESULTS: A genetic diagnosis was obtained in 56% (15/27) of the patients (62% in the case of bilateral SNL). Of the patients, 5/27 (19%) presented pathogenic variants in the GJB2 gene and the rest pathogenic and / or probably pathogenic variants in other genes associated with isolated SNL (PR2X2, TECTA and STRC), with syndromic SNL (CHD7, GATA3, COL4A5, MITF and SOX10) or with syndromic and non-syndromic SNL (BSND, ACTG1 and CDH23). DISCUSSION: The aetiological diagnosis of SNL is a challenge in clinical practice. Our series demonstrates that it is possible to implement genetic diagnosis in the care routine and that this information has prognostic and therapeutic implications