A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms

Chromosomal abnormalities and somatic mutations are found in patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in around 50-80% of cases. The identification of these alterations is important for the accurate diagnosis and prognostic classification of these patients. Often, an apparently normal or failed karyotype might lead to an inadequate estimation of the prognostic risk, and several strategies should be combined to solve these cases. The aim of this study was to introduce a novel next-generation sequencing (NGS)-based strategy for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this approach on a large cohort of patients by comparing our findings with those obtained with standard-of-care methods (i.e., karyotype and SNP-arrays). We show that our platform represents a significant improvement on current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders. Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorder

Dystrophinopathy Phenotypes and Modifying Factors in Exon 45-55 Deletion

Duchenne muscular dystrophy (DMD) exon 45-55 deletion (del45-55) has been postulated as a model that could treat up to 60% of DMD patients, but the associated clinical variability and complications require clarification. We aimed to understand the phenotypes and potential modifying factors of this dystrophinopathy subset. This cross-sectional, multicenter cohort study applied clinical and functional evaluation. Next generation sequencing was employed to identify intronic breakpoints and their impact on the Dp140 promotor, intronic long noncoding RNA, and regulatory splicing sequences. DMD modifiers (SPP1, LTBP4, ACTN3) and concomitant mutations were also assessed. Haplotypes were built using DMD single nucleotide polymorphisms. Dystrophin expression was evaluated via immunostaining, Western blotting, reverse transcription polymerase chain reaction (PCR), and droplet digital PCR in 9 muscle biopsies. The series comprised 57 subjects (23 index) expressing Becker phenotype (28%), isolated cardiopathy (19%), and asymptomatic features (53%). Cognitive impairment occurred in 90% of children. Patients were classified according to 10 distinct index-case breakpoints; 4 of them were recurrent due to founder events. A specific breakpoint (D5) was associated with severity, but no significant effect was appreciated due to the changes in intronic sequences. All biopsies showed dystrophin expression of >67% and traces of alternative del45-57 transcript that were not deemed pathogenically relevant. Only the LTBP4 haplotype appeared associated the presence of cardiopathy among the explored extragenic factors. We confirmed that del45-55 segregates a high proportion of benign phenotypes, severe cases, and isolated cardiac and cognitive presentations. Although some influence of the intronic breakpoint position and the LTBP4 modifier may exist, the pathomechanisms responsible for the phenotypic variability remain largely unresolved. ANN NEUROL 2022;92:793-80

Identification et caractérisation de variants atypiques dans le Syndrome de Usher

Le syndrome de Usher (USH) est une maladie transmise selon le mode autosomique récessif caractérisée par l’association d’une surdité congénitale (HL) et d’une rétinite pigmentaire (RP), et dans certains cas, d’une aréflexie vestibulaire. Une hétérogénéité clinique et génétique est reconnue. Environ 10 % des cas USH restent non résolus après analyse moléculaire exhaustive des différents gènes. Ces cas incluent les patients qui ne portent aucune mutation dans un des gènes USH connus ainsi que les patients porteurs d’une seule mutation dans un gène USH. Au cours de cette thèse, nous nous sommes intéressés à l’étude des patients porteurs d’une seule mutation dans les gènes USH2A et PCDH15.Dans la première partie de la thèse, nous avons analysé une cohorte de patients avec un phénotype USH2A bien défini : 5 patients pour lesquels une seule mutation à l’état hétérozygote avait été identifiée dans le gène USH2A et un patient porteur d’un variant silencieux en trans d’une mutation non-sens.Pour les 5 patients, nous avons émis l’hypothèse que la seconde mutation, restant à être identifiée, pourrait se trouver dans des régions introniques profondes. Pour cela, nous avons développé une approche de séquençage à haut débit (NGS) de l’ADN pour identifier les variants introniques profonds dans le gène USH2A et évaluer leurs conséquences sur l’épissage. Comme preuve de concept et pour valider l’approche, y compris le pipeline bio-informatique et l’évaluation des outils de prédiction de l’épissage, nous avons analysé un patient porteur d’un pseudoexon (PE) connu dans le gène USH2A. Ensuite, les 5 patients ont été étudiés en utilisant le pipeline défini, ce qui a conduit à l’identification de 3 nouveaux variants introniques profonds chez 4 d’entre eux. Tous les variants ont été prédits comme pouvant avoir un impact sur l’épissage et aboutir à l’insertion de PE. Ces prédictions ont été validées par les essais minigènes. Grâce à cette étude, nous présentons une stratégie innovante pour identifier les mutations introniques profondes, lorsque l’analyse des transcrits n’est pas possible. Par ailleurs, le pipeline bio-informatique développé fonctionne indépendamment de la taille du gène analysé, ce qui permet l’application possible de cette approche à n’importe quel gène. Par ailleurs, un oligonucléotide antisens de type morpholino (AMO) a été évalué in vitro afin de rétablir l’altération d’épissage induite par une des mutations identifiées. Les résultats ont montré un taux d’exclusion élevé du transcrit aberrant et suggèrent une application possible en thérapie moléculaire. Nous avons ensuite effectué des études sur le variant USH2A c.1377T>A, un variant silencieux afin d’évaluer son effet sur l’épissage. L’analyse de l’ARN issu de cellules nasales du patient a montré que ce variant conduit au saut de l’exon 8 dans les transcrits USH2A. Ceci a été confirmé par un essai minigène. En outre, des études préliminaires ont été réalisées en utilisant des outils de prédictions et des essais minigènes pour évaluer l’implication des éléments cis-régulateurs dans le défaut d’épissage observé chez le patient. Dans la deuxième partie de la thèse, nous avons analysé une patiente USH1, pour laquelle une seule mutation avait été identifiée dans le gène PCDH15. Dans ce cas, nous avons combiné la culture des cellules épithéliales nasales avec l’analyse des transcrits PCDH15. Celle-ci a été réalisée par séquençage de cinq RT-PCR chevauchantes. Grâce à cette analyse, nous avons réussi à délimiter une région d’intérêt dans le transcrit, dont l’amplification a échoué exclusivement pour l’allèle porteur de la mutation non identifiée. D’autres analyses ont été effectuées dans la région génomique correspondante par capture ciblée couplée au séquençage NGS et LongRange PCR suivi de séquençage Sanger. Cependant, aucun variant candidat n’a été identifié à ce jour. Nous suggérons l’implication de mécanismes moléculaires complexes qui restent à être caractérisés.Usher syndrome (USH) is an autosomal recessive disorder characterized by the association of sensorineural hearing loss (HL) and retinitis pigmentosa (RP), and in some cases, vestibular areflexia. Clinical and genetic heterogeneity are recognised. Indeed, three clinical types can be caused by mutations in one of the 10 known genes and USH2A represents the most frequently involved gene.Approximately 10 % of the USH cases remain genetically unsolved after extensive molecular analysis of the different genes, which includes sequencing of the exons and their intronic boundaries, combined to large rearrangements screening by array CGH. These unsolved cases include patients who do not carry any mutation in any of the known USH genes and patients who carry a single USH mutation. During this thesis we focalised on the study of patients carrying a single mutation in USH2A and PCDH15 gene.First, we have analysed a cohort of well-defined USH2A patients: five patients, for whom a single USH2A heterozygous mutation had been identified and one patient carrying a silent variant in trans to a nonsense mutation. For the 5 patients, we supposed that the second mutation remaining to be found could be localised deep in the introns. Indeed, a deep intronic mutation resulting in the inclusion of a pseudoexon (PE 40) in USH2A transcripts had been identified, following RNA analysis from nasal cells. Unfortunately, analysing USH2A transcripts still represent a challenging approach in a diagnostic settings and it is not always possible. To circumvent this issue, we have developed a DNA-Next Generation Sequencing (NGS) approach to identify deep intronic variants in USH2A and evaluate their consequences on splicing. As a proof of concept and to validate this approach, including the bioinformatics pipeline and the assessment of splicing predictor tools, the patient carrying the PE 40 was analysed at first. Then, the 5 patients were studied using the defined pipeline, which led to the identification of 3 distinct novel deep intronic variants in 4 of them. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. Through this study, we present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. In addition, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Moreover, an antisense morpholino oligonucleotide (AMO) tested in vitro for its ability to restore the splicing alterations caused by one of the identified mutation provided high inhibition rates. These results are indicative of a potential application for molecular therapy.In the second case, we have performed studies on the USH2A c.1377T>A silent variant to investigate its effect on splicing. Analysis of RNA from nasal cells of patients showed that this variant led to the skipping of exon 8 in USH2A transcripts. This was confirmed by minigene assay. Moreover, preliminary studies have been performed using prediction tools and minigene assays to assess the involvement of cis-acting elements in causing the aberrant splicing.In the second part of the thesis, we have analysed an USH1 patient, for whom only one mutation had been identified in the PCDH15 gene. In this case, we combined nasal epithelial cells culture with the analysis of the PCDH15 transcripts. This was performed by sequencing five overlapping RT-PCRs. Through this analysis, we were able to delimit a region within the transcript, which failed to be amplified exclusively in the allele carrying the unidentified mutation. Further analyses have been performed in the corresponding genomic region by NGS-target capture and LongRange PCR associated with Sanger sequencing. However, no evident mutation has been identified so far. Therefore, we suggest the involvement of complex molecular mechanisms that remain to be characterised

Correction: Liquori et al. Acute Promyelocytic Leukemia: A Constellation of Molecular Events around a Single PML-RARA Fusion Gene. Cancers 2020, 12, 624

The authors wish to make the following corrections to this paper [...

Whole USH2A Gene Sequencing Identifies Several New Deep Intronic Mutations

International audienceDeep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%-19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next-generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959-4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy

Case Report: Partial Uniparental Disomy Unmasks a Novel Recessive Mutation in the LYST Gene in a Patient With a Severe Phenotype of Chédiak-Higashi Syndrome

Síndrome de Chédiak-Higashi; LYST; Disomia uniparentalSíndrome de Chédiak-Higashi; LYST; Disomía uniparentalChédiak-Higashi syndrome; LYST; Uniparental disomyChédiak-Higashi syndrome (CHS) is a rare autosomal recessive (AR) immune disorder that has usually been associated to missense, nonsense or indels mutations in the LYST gene. In this study, we describe for the first time the case of a CHS patient carrying a homozygous mutation in the LYST gene inherited as a result of a partial uniparental isodisomy (UPiD) of maternal origin. Sanger sequencing of the LYST cDNA and single nucleotide polymorphism (SNP)-arrays were performed to identify the causative mutation and to explain the molecular mechanism of inheritance, respectively. Partial-UPiD leads to a copy neutral loss of heterozygosity (CN-LOH) of the telomeric region of chromosome 1 (1q41q44), unmasking the potential effect of the mutation detected. The mutation (c.8380dupT) is an insertion located in exon 32 of the LYST gene resulting in a premature stop codon and leading to the loss of all the conserved domains at the C-terminal of the LYST protein. This would account for the severe phenotype observed. We also reviewed the only two previously reported cases of CHS as a result of a uniparental disomy. In this study, we show that the combination of different strategies, including the use of SNP-arrays, is pivotal to fine-tune the diagnosis of rare AR disorders, such as CHS. Moreover, this case highlights the relevance of uniparental disomy as a potential mechanism of CHS expression in non-consanguineous families.This study was supported by research funding from FEDER funds (CIBERONC, CB16/12/00284), Instituto de Salud Carlos III grants PI16/01113, PI17/00660, PI18/1472, PI19/00812 cofinanced by the European Regional Development Fund (ERDF); as well as from the “Conselleria de Educación, Cultura y Deporte” GV/2019/084. MB-N and AL are recipients of a fellowship from the “Asociación Española Contra El Cáncer” and the “Fundación Española de Hematología y Hemoterapia”, respectively

Concurrent Zrsr2 mutation and Tet2 loss promote myelodysplastic neoplasm in mice

10 Pág.RNA splicing and epigenetic gene mutations are the most frequent genetic lesions found in patients with myelodysplastic neoplasm (MDS). About 25% of patients present concomitant mutations in such pathways, suggesting a cooperative role in MDS pathogenesis. Importantly, mutations in the splicing factor ZRSR2 frequently associate with alterations in the epigenetic regulator TET2. However, the impact of these concurrent mutations in hematopoiesis and MDS remains unclear. Using CRISPR/Cas9 genetically engineered mice, we demonstrate that Zrsr2m/mTet2-/- promote MDS with reduced penetrance. Animals presented peripheral blood cytopenia, splenomegaly, extramedullary hematopoiesis, and multi-lineage dysplasia, signs consistent with MDS. We identified a myelo-erythroid differentiation block accompanied by an expansion of LT-HSC and MPP2 progenitors. Transplanted animals presented a similar phenotype, thus indicating that alterations were cell-autonomous. Whole-transcriptome analysis in HSPC revealed key alterations in ribosome, inflammation, and migration/motility processes. Moreover, we found the MAPK pathway as the most affected target by mRNA aberrant splicing. Collectively, this study shows that concomitant Zrsr2 mutation and Tet2 loss are sufficient to initiate MDS in mice. Understanding this mechanistic interplay will be crucial for the identification of novel therapeutic targets in the spliceosome/epigenetic MDS subgroup.This investigation was supported by the grant SAF2017-82171-R to ASP from the Spanish Ministry of Economy, Industry, and Competitiveness. CGR is founded by JAP Valencia AECC predoctoral fellowships. CMV is sponsored by a predoctoral fellowship from Generalitat Valenciana (ACIF/2018/255). AL is funded by Generalitat Valenciana (APOSTD/2021/212). AGA was funded by grant RTI2018-093548-B-I00 from the Spanish Ministry of Science and Innovation. ASP is supported by the Ramón y Cajal Program (RYC2015-17534)Peer reviewe

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes

16 Pág. Departamento de Reproducción Animal​Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.This work is supported by SAF2017-82171-R from the Spanish Ministry of Economy, Industry, and Competitiveness. C.M.-V. is funded by a predoctoral fellowship from Generalitat Valenciana (ACIF/2018/255). C.G.-R. is funded by AECC predoctoral fellowship. A.S.-P. is supported by the Ramón y Cajal Program (RYC2015-17534).Peer reviewe

A 4.6 Mb Inversion Leading to PCDH15-LINC00844 and BICC1-PCDH15 Fusion Transcripts as a New Pathogenic Mechanism Implicated in Usher Syndrome Type 1

International audienceUsher type 1 syndrome is a rare autosomal recessive disorder involving congenital severe-to-profound hearing loss, development of vision impairment in the first decade, and severe balance difficulties. The PCDH15 gene, one of the five genes implicated in this disease, is involved in 8-20% of cases. In this study, we aimed to identify and characterize the two causal variants in a French patient with typical Usher syndrome clinical features. Massively parallel sequencing-based gene panel and screening for large rearrangements were used, which detected a single multi-exon deletion in the PCDH15 gene. As the second pathogenic event was likely localized in the unscreened regions of the gene, PCDH15 transcripts from cultured nasal cells were analyzed and revealed a loss of junction between exon 13 and exon 14. This aberration could be explained by the identification of two fusion transcripts, PCDH15-LINC00844 and BICC1-PCDH15, originating from a 4.6 Mb inversion. This complex chromosomal rearrangement could not be detected by our diagnostic approach but was instead characterized by long-read sequencing, which offers the possibility of detecting balanced structural variants (SVs). This finding extends our knowledge of the mutational spectrum of the PCDH15 gene with the first ever identification of a large causal paracentric inversion of chromosome 10 and illustrates the utility of screening balanced SVs in an exhaustive molecular diagnostic approach

Malattia oncologica e telenursing. nuove prospettive infermieristiche

A livello internazionale si è osservato un florido sviluppo della tecnologia digitale nel settore sanitario, anche in risposta alla pandemia, con impatto positivo in termini di qualità delle cure, soddisfazione dei pazienti e outcomes clinici. Pur essendo noto che il contributo infermieristico nel percorso diagnostico-terapeutico dei pazienti oncologici migliora gli outcomes di cura, la diffusione del telenursing è ancora scarsa in Italia e sostanzialmente limitata a triage e follow-up telefonico. Lo sviluppo e l’implementazione di strumenti e interventi infermieristici nell’assistenza e nel monitoraggio da remoto sono fondamentali nella promozione della salute e della continuità delle cure dei pazienti oncologic