Molecular dissection of structural variations involved in antithrombin deficiency

Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type.Supported by the National Institute for Health Research (NIHR) for the NIHR BioResource project (grant numbers RG65966 and RG94028), by the Instituto de Salud Carlos III grant; Fondo Europeo de Desarrollo Regional (FEDER) grant PI18/00598; and Fundación Séneca 19873/GERM/15. M.E.d.l.M.-B. has a postdoctoral contract from University of Murcia, Murcia, Spain. C.B.-P. has a Río Hortega fellowship. B.d.l.M.-B. has a postdoctoral fellowship from Fundación Séneca. J.C.-G. has a predoctoral fellowship from the Ministry of Universities FPU19/03662

Unraveling the effect of silent, intronic and missense mutations on VWF splicing : contribution of next generation sequencing in the study of mRNA

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. identifier:02869074

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES) : comprehensive genetic analysis by next-generation sequencing of 480 patients

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population

Aplicació de les noves tecnologies de seqüenciació massiva al diagnòstic molecular de la malaltia de von Willebrand. Estudi de grans cohorts i anàlisi de la correlació genotip-fenotipº

[cat] La malaltia de von Willebrand (VWD) és la patologia hemorràgica hereditària més freqüent a la població i està causada per dèficits quantitatius o qualitatius del factor von Willebrand (VWF). Segons el defecte, es distingeixen tres grans categories: la VWD de tipus 1 caracteritzada per una reducció parcial del VWF, la VWD de tipus 3 per una absència total de factor i la VWD de tipus 2 per defectes funcionals en aquesta proteïna. Actualment el diagnòstic d’aquesta patologia es basa en manifestacions clíniques de sagnat, una història familiar positiva i la realització d’un conjunt extens de proves de coagulació. Per altra banda, el diagnòstic genètic de la VWD s’ha considerat un test de segona línia degut a l’elevat cost que suposa realitzar l’anàlisi molecular del gen del VWF (VWF) mitjançant la tecnologia de Sanger. Els avenços realitzats en els últims anys en les eines de seqüenciació massiva (NGS) han promogut un interès creixent per la seva aplicació al diagnòstic clínic de malalties causades per gens de gran mida i altament polimòrfics com és el cas de la VWD. L’objectiu general d’aquesta tesi doctoral consisteix en aprofitar la tecnologia de NGS pel desenvolupament i optimització d’un protocol versàtil i econòmic per abordar l’estudi molecular de la VWD. Es pretén aplicar el nou protocol a grans cohorts de pacients per aprofundir en la investigació dels mecanismes genètics de la VWD i determinar la implicació de les variants identificades al VWF en diferents aspectes del fenotip amb rellevància en el curs clínic de la malaltia. En aquest sentit, s’ha dissenyat i optimitzat un protocol eficaç i econòmic que permet amplificar el VWF en 48 pacients simultàniament i seqüenciar-los posteriorment per NGS. El desenvolupament d’aquesta estratègia ha tingut especial rellevància dins del projecte “Perfil Clínic i Molecular de la malaltia de von Willebrand: Registre Espanyol” (PCM-EVW-ES), l’objectiu del qual és el d'establir un registre nacional per a la VWD i en el qual han participat la majoria dels grans hospitals del país reclutant 556 individus. L’aplicació del procediment en aquest registre ha permès identificar un elevat número de mutacions diferents al VWF i ha estat essencial per a la confirmació i la classificació de la VWD en 480 pacients dels 556 inicialment inclosos, establint-se una correlació genotip-fenotip en el 94,6% dels casos. Tanmateix, l’aplicació del protocol en l’estudi retrospectiu d’una cohort portuguesa de 92 pacients amb VWD, ha demostrat la seva potència, eficàcia, rapidesa i reducció de costos, en comparació amb la tecnologia de Sanger, pel diagnòstic molecular d’aquesta patologia. A continuació, derivat de l’estudi genètic d’aquestes cohorts s’han seleccionat varies mutacions potencials d’splicing (PSSM) per a investigar el seu efecte patogènic mitjançant estudis transcripcionals in vivo. Els resultats obtinguts han permès caracteritzar l’efecte patogènic en el processament de l’mRNA del VWF de 8 PSSM i posen de manifest que mutacions de tipus missense i sinònimes també poden actuar alternat l’splicing. Per últim, s’ha investigat la participació dels polimorfismes en la modulació de la gravetat i les manifestacions clíniques de la VWD. D’aquesta manera s’ha estimat que quatre polimorfismes poden arribar a explicar de l’1,5% al 2,6% de la variància fenotípica de diferents mesures del VWF (VWF:Ag, VWF:RCo i/o VWF:CB). En resum, l’anàlisi genètica del VWF mitjançant la tecnologia NGS ha demostrat la seva vàlua en el diagnòstic de la VWD i ha aportat dades per a comprendre i esclarir la base molecular d’aquesta patologia.[eng] Von Willebrand's disease (VWD) is the most common inherited bleeding disorder and is caused by a deficiency or dysfunction of von Willebrand factor (VWF). There are three major categories: type 1 resulting from a partial reduction of the VWF, VWD type 3 resulting from a complete or near complete absence of this factor and VWD type 2 characterized by qualitative deficiency. Currently, the diagnosis of this pathology is based on clinical and phenotypic information. The molecular diagnosis of VWD has been considered as a second line test due to the high cost to perform the molecular analysis of the VWF gene (VWF) by Sanger. Recently, the advances in high throughput sequencing methologies (NGS) have promoted a growing interest in its application to the clinical diagnosis of monogenic diseases. The main objective of this doctoral thesis is to develop and optimize a versatile and economic protocol based on NGS to address the molecular study of the VWD. It is intended to apply the new protocol to large patient cohorts to study in depth the molecular mechanisms of the VWD and determine the implication of VWF variants in the VWD phenotype. In this sense, an effective and economical protocol has been designed and optimized that allows the amplification of the VWF in 48 patients simultaneously and sequencing by NGS. The development of this strategy has been of particular relevance in the "Clinical and Molecular Profile of von Willebrand's Disease: Spanish Registry" (PCM-EVW-ES), whose purpose is to establish a national registry of the VWD. The majority of Spanish hospitals participated in this project and recruited a total of 556 individuals. The application of the developed procedure in this registry has prompted the identification of a high number of VWF mutations and has been essential for confirmation and classification of the VWD in 480 patients of the 556 initially included, establishing a genotype-phenotype correlation in 94.6% of cases. Additionally, the application of this protocol in the retrospective study of a Portuguese cohort of 92 VWD patients has demonstrated its power, efficiency, and cost reduction, compared to Sanger's technology, in molecular diagnosis of VWD. Derived from the genetic study of these cohorts, several potential splice site mutations (PSSM) have been selected to investigate their pathogenic effect through transcriptional studies in vivo. The results obtained demonstrate the pathogenic effect of 8 VWF PSSM in the mRNA processing and prove that missense and synonymous mutations could also affect splicing. Finally, it has been investigated the participation of VWF polymorphisms in the modulation of the severity and the clinical manifestations of the VWD. In this way, it has been estimated that four polymorphisms can account from 1.5% to 2.6% of the phenotypic variance of different VWF measurements (VWF: Ag, VWF: RCo and / or VWF: CB). In summary, genetic analysis of the VWF by means of NGS technology has proven its worth in the diagnosis of the VWD and has provided data to understand and elucidate the molecular basis of this pathology

Aplicació de les noves tecnologies de seqüenciació massiva al diagnòstic molecular de la malaltia de von Willebrand. Estudi de grans cohorts i anàlisi de la correlació genotip-fenotip

La malaltia de von Willebrand (VWD) és la patologia hemorràgica hereditària més freqüent a la població i està causada per dèficits quantitatius o qualitatius del factor von Willebrand (VWF). Segons el defecte, es distingeixen tres grans categories: la VWD de tipus 1 caracteritzada per una reducció parcial del VWF, la VWD de tipus 3 per una absència total de factor i la VWD de tipus 2 per defectes funcionals en aquesta proteïna. Actualment el diagnòstic d’aquesta patologia es basa en manifestacions clíniques de sagnat, una història familiar positiva i la realització d’un conjunt extens de proves de coagulació. Per altra banda, el diagnòstic genètic de la VWD s’ha considerat un test de segona línia degut a l’elevat cost que suposa realitzar l’anàlisi molecular del gen del VWF (VWF) mitjançant la tecnologia de Sanger. Els avenços realitzats en els últims anys en les eines de seqüenciació massiva (NGS) han promogut un interès creixent per la seva aplicació al diagnòstic clínic de malalties causades per gens de gran mida i altament polimòrfics com és el cas de la VWD. L’objectiu general d’aquesta tesi doctoral consisteix en aprofitar la tecnologia de NGS pel desenvolupament i optimització d’un protocol versàtil i econòmic per abordar l’estudi molecular de la VWD. Es pretén aplicar el nou protocol a grans cohorts de pacients per aprofundir en la investigació dels mecanismes genètics de la VWD i determinar la implicació de les variants identificades al VWF en diferents aspectes del fenotip amb rellevància en el curs clínic de la malaltia. En aquest sentit, s’ha dissenyat i optimitzat un protocol eficaç i econòmic que permet amplificar el VWF en 48 pacients simultàniament i seqüenciar-los posteriorment per NGS. El desenvolupament d’aquesta estratègia ha tingut especial rellevància dins del projecte “Perfil Clínic i Molecular de la malaltia de von Willebrand: Registre Espanyol” (PCM-EVW-ES), l’objectiu del qual és el d'establir un registre nacional per a la VWD i en el qual han participat la majoria dels grans hospitals del país reclutant 556 individus. L’aplicació del procediment en aquest registre ha permès identificar un elevat número de mutacions diferents al VWF i ha estat essencial per a la confirmació i la classificació de la VWD en 480 pacients dels 556 inicialment inclosos, establint-se una correlació genotip-fenotip en el 94,6% dels casos. Tanmateix, l’aplicació del protocol en l’estudi retrospectiu d’una cohort portuguesa de 92 pacients amb VWD, ha demostrat la seva potència, eficàcia, rapidesa i reducció de costos, en comparació amb la tecnologia de Sanger, pel diagnòstic molecular d’aquesta patologia. A continuació, derivat de l’estudi genètic d’aquestes cohorts s’han seleccionat varies mutacions potencials d’splicing (PSSM) per a investigar el seu efecte patogènic mitjançant estudis transcripcionals in vivo. Els resultats obtinguts han permès caracteritzar l’efecte patogènic en el processament de l’mRNA del VWF de 8 PSSM i posen de manifest que mutacions de tipus missense i sinònimes també poden actuar alternat l’splicing. Per últim, s’ha investigat la participació dels polimorfismes en la modulació de la gravetat i les manifestacions clíniques de la VWD. D’aquesta manera s’ha estimat que quatre polimorfismes poden arribar a explicar de l’1,5% al 2,6% de la variància fenotípica de diferents mesures del VWF (VWF:Ag, VWF:RCo i/o VWF:CB). En resum, l’anàlisi genètica del VWF mitjançant la tecnologia NGS ha demostrat la seva vàlua en el diagnòstic de la VWD i ha aportat dades per a comprendre i esclarir la base molecular d’aquesta patologia.Von Willebrand's disease (VWD) is the most common inherited bleeding disorder and is caused by a deficiency or dysfunction of von Willebrand factor (VWF). There are three major categories: type 1 resulting from a partial reduction of the VWF, VWD type 3 resulting from a complete or near complete absence of this factor and VWD type 2 characterized by qualitative deficiency. Currently, the diagnosis of this pathology is based on clinical and phenotypic information. The molecular diagnosis of VWD has been considered as a second line test due to the high cost to perform the molecular analysis of the VWF gene (VWF) by Sanger. Recently, the advances in high throughput sequencing methologies (NGS) have promoted a growing interest in its application to the clinical diagnosis of monogenic diseases. The main objective of this doctoral thesis is to develop and optimize a versatile and economic protocol based on NGS to address the molecular study of the VWD. It is intended to apply the new protocol to large patient cohorts to study in depth the molecular mechanisms of the VWD and determine the implication of VWF variants in the VWD phenotype. In this sense, an effective and economical protocol has been designed and optimized that allows the amplification of the VWF in 48 patients simultaneously and sequencing by NGS. The development of this strategy has been of particular relevance in the "Clinical and Molecular Profile of von Willebrand's Disease: Spanish Registry" (PCM-EVW-ES), whose purpose is to establish a national registry of the VWD. The majority of Spanish hospitals participated in this project and recruited a total of 556 individuals. The application of the developed procedure in this registry has prompted the identification of a high number of VWF mutations and has been essential for confirmation and classification of the VWD in 480 patients of the 556 initially included, establishing a genotype-phenotype correlation in 94.6% of cases. Additionally, the application of this protocol in the retrospective study of a Portuguese cohort of 92 VWD patients has demonstrated its power, efficiency, and cost reduction, compared to Sanger's technology, in molecular diagnosis of VWD. Derived from the genetic study of these cohorts, several potential splice site mutations (PSSM) have been selected to investigate their pathogenic effect through transcriptional studies in vivo. The results obtained demonstrate the pathogenic effect of 8 VWF PSSM in the mRNA processing and prove that missense and synonymous mutations could also affect splicing. Finally, it has been investigated the participation of VWF polymorphisms in the modulation of the severity and the clinical manifestations of the VWD. In this way, it has been estimated that four polymorphisms can account from 1.5% to 2.6% of the phenotypic variance of different VWF measurements (VWF: Ag, VWF: RCo and / or VWF: CB). In summary, genetic analysis of the VWF by means of NGS technology has proven its worth in the diagnosis of the VWD and has provided data to understand and elucidate the molecular basis of this pathology

Novel Double Factor PGT strategy analyzing blastocyst stage embryos in a single NGS procedure

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies

Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next-generation sequencing

BACKGROUND: The 2 main forms of thrombotic microangiopathy (TMA) are thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). Deficiency of ADAMTS13 and dysregulation of the complement pathway result in TTP and aHUS, respectively; however, overlap of their clinical characteristics makes differential diagnosis challenging. OBJECTIVES AND METHODS: We aimed to develop a TMA diagnosis workflow based on ADAMTS13 activity and screening of ADAMTS13 and complement genes using a custom next-generation sequencing (NGS) gene panel. PATIENTS: For this, from a cohort of 154 Portuguese patients with acute TMA, the genotype-phenotype correlations were analyzed in 7 hereditary TTP (ADAMTS13 activity <10%, no inhibitor), 36 acquired TTP (ADAMTS13 activity <10%, presence of an inhibitor), and in 34 presumable aHUS. RESULTS: In total, 37 different rare variants, 8 of which novel (in ADAMTS13,CFH, and CD46), were identified across 7 genes. Thirteen TTP patients were homozygous (n=6), compound heterozygous (n=2), and heterozygous (n=5) for 11 ADAMTS13 variants (6 pathogenic mutations). Among the 34 aHUS patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause aHUS only when combined with other variants and/or CFH and CD46 risk haplotypes or CFHR1-3 deletion. CONCLUSIONS: Our study provides evidence of the usefulness of the NGS panel as an excellent technology that enables more rapid diagnosis of TMA, and is a valuable asset in clinical practice to discriminate between TTP and aHUS.info:eu-repo/semantics/publishedVersio

Combined study of 13 and complement genes in the diagnosis of thrombotic microangiopathies using next-generation sequencing

The 2 main forms of thrombotic microangiopathy () are thrombotic thrombocytopenic purpura () and atypical hemolytic uremic syndrome (). Deficiency of 13 and dysregulation of the complement pathway result in and , respectively; however, overlap of their clinical characteristics makes differential diagnosis challenging. We aimed to develop a diagnosis workflow based on 13 activity and screening of ADAMTS13 and complement genes using a custom next-generation sequencing () gene panel. For this, from a cohort of 154 Portuguese patients with acute , the genotype-phenotype correlations were analyzed in 7 hereditary (13 activity <10%, no inhibitor), 36 acquired (13 activity <10%, presence of an inhibitor), and in 34 presumable . In total, 37 different rare variants, 8 of which novel (in ADAMTS13, , and CD46), were identified across 7 genes. Thirteen patients were homozygous (n=6), compound heterozygous (n=2), and heterozygous (n=5) for 11 ADAMTS13 variants (6 pathogenic mutations). Among the 34 patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause only when combined with other variants and/or and CD46 risk haplotypes or CFHR1-3 deletion. Our study provides evidence of the usefulness of the panel as an excellent technology that enables more rapid diagnosis of , and is a valuable asset in clinical practice to discriminate between and