Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

Acknowledgements: We thank all patients who contributed to this study. The work was supported by grants from the Instituto de Salud Carlos III (ISCIII, MINECO) (operating grants: PI13/00285 and RD12/0036/0008 awarded to C.L. and PIE13/00022 and RD12/0036/0031 awarded to G.C.) and confunded by FEDER funds/European Regional Development Fund (ERDF) - a way to Build Europe-"// FONDOS FEDER "una manera de hacer Europa", the Generalitat de Catalunya (Government of Catalonia) (operating grant 2014SGR338, awarded to G.C.) and the Asociación Española Contra el Cáncer (operating grants, 2010 Grupos Estables, awarded to G.C.). J.B. received a Spanish Society of Medical Oncology grant. This activity is sponsored by the ISCIII Ministerio de Economía y Competitividad (PT13/0001/0044).Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eightythree genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes

Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases

Altres ajuts: Generalitat de Catalunya, Departament de Salut; Generalitat de Catalunya, Departament d'Empresa i Coneixement i CERCA Program; Ministerio de Ciencia e Innovación; Instituto Nacional de Bioinformática; ELIXIR Implementation Studies (CNAG-CRG); Centro de Investigaciones Biomédicas en Red de Enfermedades Raras; Centro de Excelencia Severo Ochoa; European Regional Development Fund (FEDER).Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%)

Complejo factor VIIa-factor tisular y su papel compensatorio en las disfunciones hemostáticas, El

Tradicionalmente las terapias de corrección de los defectos hemostáticos son sustitutivas y se basan en la administración exógena de derivados sanguíneos o fármacos recombinantes que corrijan la carencia. Los estudios realizados en esta tesis han sido dirigidos a investigar sistemas alternativos, a través del complejo FT/FVIIa, como compensatorios de otros elementos hemostáticos deficitarios. Esta tesis doctoral ha explorado la posibilidad de que potenciar un mecanismo no deficitario, como puede ser la coagulación iniciada por el complejo FT/FVIIa, pueda restablecer una correcta hemostasia tanto en pacientes que presenten coagulopatías como en pacientes que presenten un déficit en la hemostasia primaria.Para estudiar distintos desórdenes hemostáticos con sintomatología clínica se han empleado aproximaciones experimentales in vitro en sistemas que emulan las condiciones fisiológicas que se encuentran en distintos puntos del árbol vascular. Hemos empleado muestras de sangre procedentes de pacientes con trastornos hemostáticos: Coagulopatías (hemofilia A, cirrosis y anticoagulación inducida por acenocumarol), trombocitopatías (síndrome de Bernard-Soulier, trombastenia de Glanzmann y administración de abciximab) y trombocitopenias.El mecanismo por el cual la potenciación de la vía del FT-FVIIa produce su efecto hemostático viene mediado por la presencia de FT en la zona vascular lesionada. Esta vía da lugar a la generación de una gran cantidad de trombina en las inmediaciones de la zona dañada que posibilita la formación de una malla de fibrina estable y la activación de nuevas plaquetas.Esta tesis doctoral presenta evidencias claras de que la potenciación de la vía del FT mediante FVIIa puede tener utilidad clínica para compensar déficits hemostáticos debidos tanto a coagulopatías como a trombocitopatías, y aporta mecanismos de acción específicos que lo apoyan. Asimismo, nuestro trabajo indica que el rFVIIa puede emplearse como fármaco de "rescate" de tratamientos anticoagulantes o antiagregantes plaquetarios. El descubrimiento de que el FT tiene propiedades proadhesivas para las plaquetas abre un nuevo campo de estudio en el entendimiento de las enfermedades cardiovasculares y apunta al FT y al complejo FT-FVIIa como una posible diana terapéutica sobre la que actuar tanto para potenciar su efecto prohemostático o como estrategia inhibidora."The complex Tissue Factor - Factor VIIa and its effect in the compensation of hemostatic disorders"Traditionally, therapies for the correction of hemostatic defects are based in the replacement of exogenous administration of blood derivates or recombinant drugs that correct that deficiency. The studies inclouded in this thesis aimed to investigate alternative systems, through the FT/FVIIa complex, as compensator of other deficitary hemostatic elements. This doctoral thesis has explored the possibility that potentiating a non-deficient mechanism, like the coagulation initiated by the FT/FVIIa complex, can as much restore a correct hemostasis in patients who present coagulopathies like in patients who present a deficit in primary hemostasis.In order to study different hemostatic disorders with clinical symptomatology, in vitro experimental approaches that emulate the physiological conditions found in different points from the vascular tree have been used. We have used blood samples from patients with hemostatic defects: Coagulopathies (hemofilia A, oral anticoagulation induced by acenocumarol and cirrhosis,), thrombocytopathies (Bernard-Soulier syndrome, Glanzmann's thrombastenia and administration of abciximab) and thrombocytopenias.The mechanism by which the potentiation of the complex TF-FVIIa produces its hemostatic effect is mediated by the presence of FT in the injured vascular zone. This route generates a thrombin burst in the environs of the damaged zone, which activates new platelets and allows the formation of a fibrin mesh that stabilizes the hemostatic plug.This doctoral thesis presents clear evidences supporting that the potentiation of the TF by means of FVIIa can have clinical utility to compensate hemostatic disorders (not only coagulopathies but also thrombocytopathies and thrombocytopenies) and the specific mechanisms of action involved in these effects. Besides, our work indicates that rFVIIa can be used as drug of "rescue" of anticoagulating treatments or grugs that inhibit aggregation of platelets. The discovery that the FT has pro-adhesive properties for platelets opens a new field of study in the understanding of the cardiovascular diseases and aims at the TF and the TF-FVIIa complex as a possible therapeutic target on which to act to potentiate its prohemostatic effect as well as to inhibit it. </I

Wnt genes in colonic polyposis predisposition

Data de publicació electrònica: 29-12-2022This research was funded by the Spanish Ministry of Science and Innovation (Agencia Estatal de Investigación), and co-funded by FEDER funds a way to build Europe [Grants No. SAF2016-80888-R (LV), PID2020-112595RB-I00 (LV), and PID2019-111254RB-I00 (GC), and predoctoral fellowship “Formación de Personal Investigador” (IQ)]; Instituto de Salud Carlos III [CIBERONC CB16/12/00234, Sara Borrell Postdoctoral contract (PM)]; Government of Catalonia, Spain [PERIS MedPerCan, AGAUR 2017SGR1282, CERCA Program for institutional support]; Scientific Foundation “Asociación Española Contra el Cáncer” [AECC Investigador (MT)]; Marie Skłodowska-Curie Individual Fellowship [Organ-VIP; Grant agreement No. 897064 (NG-A)]. The Solve-RD project is funded by the European Union's Horizon 2020 research and innovation program under grant agreement No. 779257. This study was supported by the European Reference Network on Genetic Tumor Risk Syndromes (ERN GENTURIS) - Project ID No. 739547 (www.genturis.eu), and the COST action CA17118, supported by COST (European Cooperation in Science and Technology)

Benchmarking of whole exome sequencing and ad hoc designed panels for genetic testing of hereditary cancer

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eighty-three genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes

Background: Mature microRNAs (miRNAs) play an important role in repressing the expression of a wide range of mRNAs. The presence of polymorphic sites in miRNA genes and their corresponding 3'UTR binding sites can disrupt canonical conserved miRNA-mRNA pairings, and thus modify gene expression patterns. However, to date such polymorphic sites in miRNA genes and their association with gene expression phenotypes and complex traits are poorly characterized in pigs. Results: By analyzing whole-genome sequences from 120 pigs and wild boars from Europe and Asia, we identified 285 single nucleotide polymorphisms (SNPs) that map to miRNA loci, and 109,724 SNPs that are located in predicted 7mer-m8 miRNA binding sites within porcine 3'UTR. In porcine miRNA genes, SNP density is reduced compared with their flanking non-miRNA regions. By sequencing the genomes of five Duroc boars, we identified 12 miRNA SNPs that were subsequently genotyped in their offspring (N = 345, Lipgen population). Association analyses of miRNA SNPs with 38 lipid-related traits and hepatic and muscle microarray expression phenotypes recorded in the Lipgen population were performed. The most relevant detected association was between the genotype of the rs319154814 (G/A) SNP located in the apical loop of the ssc-miR-326 hairpin precursor and PPP1CC mRNA levels in the liver (q-value = 0.058). This result was subsequently confirmed by qPCR (P-value = 0.027). The rs319154814 (G/A) genotype was also associated with several fatty acid composition traits. Conclusions: Our findings show a reduced variability of porcine miRNA genes, which is consistent with strong purifying selection, particularly in the seed region that plays a critical role in miRNA binding. Although it is generally assumed that SNPs mapping to the seed region are those with the most pronounced consequences on mRNA expression, we show that a SNP mapping to the apical region of ssc-miR-326 is significantly associated with hepatic mRNA levels of the PPP1CC gene, one of its predicted targets. Although experimental confirmation of such an interaction is reported in humans but not in pigs, this result highlights the need to further investigate the functional effects of miRNA polymorphisms that are located outside the seed region on gene expression in pigs.The research presented in the current publication was funded by Grants AGL2013-48742-C2-1-R and AGL2013-48742-C2-2-R awarded by the Spanish Ministry of Economy and Competitivity. We also acknowledge the support of the Spanish Ministry of Science and Innovation for the Center of Excellence Severo Ochoa 2020–2023 (CEX2019-000902-S) grant awarded to the Centre for Research in Agricultural Genomics (CRAG, Bellaterra, Spain). Emilio Mármol-Sánchez was funded by a FPU Ph.D. grant from the Spanish Ministry of Education (FPU15/01733). María Gracia Luigi-Sierra was funded with a Ph.D. fellowship “Formación de Personal Investigador” (BES-C-2017-0024) awarded by the Spanish Ministry of Economy and Competitivity. Dailu Guan was funded by a Ph.D. fellowship from the Scholarship Council of China (CSC). The authors thank the CERCA Programme of the Generalitat de Catalunya (Barcelona, Spain) for their support and those who provided publicly available dat

Deep analysis of acquired resistance to FGFR1 inhibitor identifies MET and AKT activation and an expansion of AKT1 mutant cells

The development of acquired resistance (AR) to tyrosine kinase inhibitors (TKIs) of FGFR1 activation is currently not well understood. To gain a deeper insight into this matter in lung cancer, we used the FGFR1-amplified DMS114 cell line and generated multiple clones with AR to an FGFR1-TKI. We molecularly scrutinized the resistant cells, using whole-exome sequencing, RNA sequencing and global DNA methylation analysis. Our results show a de novo activation of AKT and ERK, and a reactivation of mTOR. Furthermore, the resistant cells exhibited strong upregulation and activation of MET, indicating crosstalk between the FGFR1 and MET axes. The resistant cells also underwent a global decrease in promoter hypermethylation of the CpG islands. Finally, we observed clonal expansion of a pre-existing change in AKT1, leading to S266L substitution, within the kinase domain of AKT. Our results demonstrate that AR to FGFR1-TKI involves deep molecular changes that promote the activation of MET and AKT, coupled with common gene expression and DNA methylation profiles. The expansion of a substitution at AKT1 was the only shared genetic change, and this may have contributed to the AR.This work was supported by Spanish grants SAF2014-54571-R (to M Sanchez-Cespedes), AGAUR (Agency for Management of University and Research Grants) (2014SGR641) (to M Sanchez-Cespedes), and PTA2014-09515 (to M Dabad). The CNAG-CRG laboratory is a member of the Spanish National Bioinformatics Institute (INB), PRB2-ISCIII and is supported by grants PT13/0001, from the PE I+D+i 2013-2016, funded by ISCIII and FEDER (to A Esteve-Codina)

The genetic landscape of inherited retinal diseases in a Mexican cohort: genes, mutations and phenotypes

In this work, we aimed to provide the genetic diagnosis of a large cohort of patients affected with inherited retinal dystrophies (IRDs) from Mexico. Our data add valuable information to the genetic portrait in rare ocular diseases of Mesoamerican populations, which are mostly under-represented in genetic studies. A cohort of 144 unrelated probands with a clinical diagnosis of IRD were analyzed by next-generation sequencing using target gene panels (overall including 346 genes and 65 intronic sequences). Four unsolved cases were analyzed by whole-exome sequencing (WES). The pathogenicity of new variants was assessed by in silico prediction algorithms and classified following the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic or likely pathogenic variants were identified in 105 probands, with a final diagnostic yield of 72.9%; 17 cases (11.8%) were partially solved. Eighteen patients were clinically reclassified after a genetic diagnostic test (17.1%). In our Mexican cohort, mutations in 48 genes were found, with ABCA4, CRB1, RPGR and USH2A as the major contributors. Notably, over 50 new putatively pathogenic variants were identified. Our data highlight cases with relevant clinical and genetic features due to mutations in the RAB28 and CWC27 genes, enrich the novel mutation repertoire and expand the IRD landscape of the Mexican population.This research was funded by an anonymous private Mexican donor who generously covered all the expense

A de novo FOXP1 truncating mutation in a patient originally diagnosed as C syndrome

De novo FOXP1 mutations have been associated with intellectual disability (ID), motor delay, autistic features and a wide spectrum of speech difficulties. C syndrome (Opitz C trigonocephaly syndrome) is a rare and genetically heterogeneous condition, characterized by trigonocephaly, craniofacial anomalies and ID. Several different chromosome deletions and and point mutations in distinct genes have been associated with the disease in patients originally diagnosed as Opitz C. By whole exome sequencing we identified a de novo splicing mutation in FOXP1 in a patient, initially diagnosed as C syndrome, who suffers from syndromic intellectual disability with trigonocephaly. The mutation (c.1428 + 1 G > A) promotes the skipping of exon 16, a frameshift and a premature STOP codon (p.Ala450GLyfs*13), as assessed by a minigene strategy. The patient reported here shares speech difficulties, intellectual disability and autistic features with other FOXP1 syndrome patients, and thus the diagnosis for this patient should be changed. Finally, since trigonocephaly has not been previously reported in FOXP1 syndrome, it remains to be proved whether it may be associated with the FOXP1 mutation.The authors thank the patient and his family for wholehearted collaboration. They are also grateful to M. Cozar for technical assistance, and to CNAG for exome sequencing within the “300 exomes to elucidate rare diseases” program. Funding was from Associació Síndrome Opitz C, Terrassa, Spain; Spanish Ministerio de Economía y Competitividad (SAF2014-56562-R; SAF2016-75948-R, FECYT, crowdfunding PRECIPITA); ISCIII Ministerio de Economía y Competitividad (PT13/0001/0044), Catalan Government (2014SGR932) and from CIBERER (U720)

Fine-scale population structure in five rural populations from the Spanish Eastern Pyrenees using high-coverage whole-genome sequence data

Data de publicació electrònica: 09-04-2021The area of the Spanish Pyrenees is particularly interesting for studying the demographic dynamics of European rural areas given its orography, the main traditional rural condition of its population and the reported higher patterns of consanguinity of the region. Previous genetic studies suggest a gradient of genetic continuity of the area in the West to East axis. However, it has been shown that micro-population substructure can be detected when considering high-quality NGS data and using spatial explicit methods. In this work, we have analyzed the genome of 30 individuals sequenced at 40× from five different valleys in the Spanish Eastern Pyrenees (SEP) separated by less than 140 km along a west to east axis. Using haplotype-based methods and spatial analyses, we have been able to detect micro-population substructure within SEP not seen in previous studies. Linkage disequilibrium and autozygosity analyses suggest that the SEP populations show diverse demographic histories. In agreement with these results, demographic modeling by means of ABC-DL identify heterogeneity in their effective population sizes despite of their close geographic proximity, and suggests that the population substructure within SEP could have appeared around 2500 years ago. Overall, these results suggest that each rural population of the Pyrenees could represent a unique entity.OL, IM, RT, JC, and SB acknowledge the support from Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, CERCA Program/Generalitat de Catalunya, Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement, Co-financing with funds from the European Regional Development Fund by the Spanish Ministry of Science and Innovation corresponding to the Programa Operativo FEDER Plurirregional de España (POPE) 2014–2020 and by the Secretaria d’Universitats i Recerca, Departament d’Empresa i Coneixement of the Generalitat de Catalunya corresponding to the Programa Operatiu FEDER de Catalunya 2014–2020. OL gratefully acknowledges the financial support from Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)—RYC-2013-14797, BFU2015-68759-P and PGC2018-098574-B-I00 and Generalitat de Catalunya (Government of Catalonia)—GRC 2017 SGR 937. IM gratefully acknowledges the financial support from the Government of Catalonia | Agència de Gestió d’Ajuts Universitaris i de Recerca (Agency for Management of University and Research Grants)—GRC 2014 SGR 615. MMA was supported by a FPU15/01251 from Ministerio de Ciencia, innovación y Universidades. PM and MMA acknowledge the financial support from Precipita-FECYT (FBG 309307 project). The samples analyzed in this study are part of a much larger sample whose collection was supported by the project CGL2011-27866 of the Ministerio de Ciencia y Tecnologia, the Fundació Moret i Marguí, and the invaluable collaboration of the Hospital San Bernabé (Berguedà), Fundació Sant Hospital (Alt Urgell), Hospital d’Olot i Comarcal (Garrotxa), Hospital de Campdevànol (Ripollès), and Hospital Comarcal (Pallars