Exploration génomique de la déficience intellectuelle

La déficience intellectuelle (DI) définit un groupe de conditions génétiquement hétérogènes caractérisées par l’apparition de troubles cognitifs précoces chez l’enfant. Elle affecte 1-3% de la population dans les pays industrialisés. La prévalence de la DI est beaucoup plus élevée ailleurs dans le monde, en raison de facteurs sociodémographiques comme le manque de ressources dans le système de santé, la pauvreté et la consanguinité. Des facteurs non-génétiques sont mis en cause dans l’étiologie de la DI ; on estime qu’environ 25% des cas de DI sont d’origine génétique. Traditionnellement, les bases moléculaires de la DI ont été investiguées par des analyses cytogénétiques, les approches de cartographie génétique et le séquençage de gènes candidats ; ces techniques de génétiques classiques sont encore mises à rude épreuve dans l’analyse de maladies complexes comme la DI. La DI liée à l’X a été particulièrement étudiée, avec plus d’une centaine de gènes identifiés uniquement sur le chromosome X. Des mutations hétérozygotes composites sont mises en évidence dans la DI autosomique, dans le contexte d’unions non-consanguines. L’occurrence de ce type de mutations est rare, chez des individus non-apparentés, de sorte que les mutations dominantes de novo sont plus courantes. Des mutations homozygotes sont attendues dans les populations consanguines ou marquées par un effet fondateur. En fait, les bases moléculaires de la DI autosomique ont été presqu’exclusivement étudiées dans le contexte de populations avec des forts taux de consanguinité. L’origine de la DI demeure encore inconnue dans environ 60 % des cas diagnostiqués. En l’absence de facteurs environnementaux associés à la DI chez ces individus, il est possible d’envisager que des facteurs génétiques non identifiés entrent en jeu dans ces cas de DI inexpliqués. Dans ce projet de recherche, nous voulions explorer l’origine génétique de la DI, dans vingt familles, où une transmission de la maladie selon un mode autosomique récessif est suspectée. Nous avons mis de l’avant les techniques de séquençage de nouvelle génération, afin de mettre en évidence les déterminants génétiques de la DI, à l’échelle du génome humain. En fait, nous avons priorisé la capture et le séquençage de l’exome; soient la totalité des régions codantes du génome humain et leurs sites d’épissage flanquants. Dans nos analyses, nous avons ciblé les variants qui ne sont pas rapportés trop fréquemment dans différentes bases de données d’individus contrôles, ces mutations rares cadrent mieux avec une condition comme la DI. Nous avons porté une attention particulière aux mutations autosomiques récessives (homozygotes et hétérozygotes composites) ; nous avons confirmé que ces mutations ségréguent avec une transmission récessive dans la famille à l’étude. Nous avons identifié des mutations dans des gènes pouvant être à l’origine de la DI, dans certaines des familles analysées ; nous avons validé biologiquement l'impact fonctionnel des mutations dans ces gènes candidats, afin de confirmer leur implication dans la pathophysiologie de la DI. Nous avons élucidé les bases moléculaires de la DI dans huit des familles analysées. Nous avons identifié le second cas de patients avec syndrome de cassure chromosomique de Varsovie, caractérisé par des dysfonctions de l’ARN hélicase DDX11. Nous avons montré qu’une perte de l’activité de TBC1D7, une des sous-unités régulatrice du complexe TSC1-TSC2, est à l’origine de la pathologie dans une famille avec DI et mégalencéphalie. Nous avons mis en évidence des mutations pathogéniques dans le gène ASNS, codant pour l’Asparagine synthétase, chez des patients présentant une microcéphalie congénitale et une forme progressive d’encéphalopathie. Nous avons montré que des dysfonctions dans la protéine mitochondriale MAGMAS sont mises en cause dans une condition caractérisée par un retard prononcé dans le développement associé à une forme sévère de dysplasie squelettique. Nous avons identifié une mutation tronquant dans SPTBN2, codant pour la protéine spinocerebellar ataxia 5, dans une famille avec DI et ataxie cérébelleuse. Nous avons également mis en évidence une mutation dans PIGN, un gène impliqué dans la voie de biosynthèse des ancres de glycosylphosphatidylinositol , pouvant être à l’origine de la maladie chez des individus avec épilepsie et hypotonie. Par ailleurs, nous avons identifié une mutation - perte de fonction dans CLPB, codant pour une protéine chaperonne mitochondriale, dans une famille avec encéphalopathie néonatale, hyperekplexie et acidurie 3-méthylglutaconique. Le potentiel diagnostic des techniques de séquençage de nouvelle génération est indéniable ; ces technologies vont révolutionner l’univers de la génétique moléculaire, en permettant d’explorer les bases génétiques des maladies complexes comme la DI.Intellectual disability (ID) regroups greatly heterogeneous conditions that are characterized by early-onset cognitive impairment. ID affects about 1-3% of Western populations; but its prevalence is much higher in deprived regions of the world where socio-demographic factors like poor healthcare, lack of resources and parental consanguinity prevail. Non-genetic factors are involved in the etiology of ID; approximately 25% of ID cases are of genetic origin. Traditionally, the molecular basis of ID have been assessed through cytogenetic analyses, genetic mapping and candidate gene approaches. These classical genetic tools are still put to the test in the study of complex diseases like ID. Until recently, X-linked ID cases were the main focus of studies on ID with more than hundred ID genes identified only on the X chromosome. Compound heterozygous mutations are identified in autosomal forms of ID, in the context of non-consanguineous unions. However, the occurrence of such mutations is rare in outbred populations, so that dominant de novo mutations are most common in unrelated individuals. Homozygous mutations are expected in consanguineous unions or in populations marked by a founder effect. In fact, the molecular bases of autosomic recessive ID have been almost exclusively studied in populations with high consanguinity rates. ID remains unsolved in more than 60% of patients. In the absence of environmental factors associated with ID in these individuals, it is possible to consider that unidentified genetic factors are involved in these unexplained ID cases. In this research project, we used next generation sequencing technologies to highlight the genetic causes of ID in twenty families were an autosomal recessive mode of inheritance is expected. We prioritized the use of whole-exome sequencing, namely all coding exons in the genome of this individual. In our analyses, we filtered out variants that were too common in control individuals to describe a rare condition like ID. We focussed our attention on rare autosomic recessive varaiants (homozygous and compound heterozygous), these mutations were confirmed by Sanger re-sequencing to segregate with an autosomal recessive mode of inheritance in the family. We identified mutations in candidate genes for ID in some of the family analysed, we validated the functional impact of the mutations in these genes to confirm their involvement in the pathophysiology of ID in the family studied. We explained the molecular basis of ID in eight of the families studied. We identified the second case of Warsaw-Breakage-Syndrome, a rare genetic disorder characterised by dysfunction of the RNA helicase DDX11. We showed that disruption in TBC1D7, a functional subunit of the TSC1-TSC2 protein complex, cause ID and megalencephaly. We demonstrated that ASNS, the Asparagine Synthetase gene, is defective in patients with congenital microcephaly and progressive encephalopathy. We showed that the gene coding for the mitochondrial protein MAGMAS is involved in the pathophysiology of a condition characterised by developmental delay and severe skeletal dysplasia. We identified a truncating mutation in SPTBN2, encoding for the spinocerebellar ataxia 5 proteins, in a family with ID and spinocerellar ataxia. We also identified a mutation in a gene involved in the biosynthetic pathway of glycosylphosphatidylinositol anchors; the mutation in PIGN may cause the epilepsy and hypotonia features observed in the affected individuals of that family. Finally, we identified a loss of function mutation in CLPB, coding for a mitochondrial chaperone, in individuals with severe encephalopathy, hypereklexia and 3-methylglutaconic aciduria. The diagnostic potential of next generation sequencing technologies is undeniable. These technologies will revolutionize the world of molecular genetics; they will help deciphering the molecular basis of complex diseases like ID

Biallelic disruption of DDX41 activity is associated with distinct genomic and immunophenotypic hallmarks in acute leukemia

IntroductionInherited DDX41 mutations cause familial predisposition to hematologic malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), with the majority of DDX41 mutated MDS/AMLs described to date harboring germline DDX41 and co-occurring somatic DDX41 variants. DDX41-AMLs were shown to share distinguishing clinical features such as a late AML onset and an indolent disease associated with a favorable outcome. However, genotype-phenotype correlation in DDX41-MDS/AMLs remain poorly understood.MethodsHere, we studied the genetic profile, bone marrow morphology and immunophenotype of 51 patients with DDX41 mutations. We further assessed the functional impact of ten previously uncharacterized DDX41 variants of uncertain significance.ResultsOur results demonstrate that MDS/AML cases harboring two DDX41 variants share specific clinicopathologic hallmarks that are not seen in other patients with monoallelic DDX41 related hematologic malignancies. We further showed that the features seen in these individuals with two DDX41 variants were concordant with biallelic DDX41 disruption.DiscussionHere, we expand on previous clinicopathologic findings on DDX41 mutated hematologic malignancies. Functional analyses conducted in this study unraveled previously uncharacterized DDX41 alleles and further illustrate the implication of biallelic disruption in the pathophysiology of this distinct AML entity

TP53 Mutations in AML Patients Are Associated with Dismal Clinical Outcome Irrespective of Frontline Induction Regimen and Allogeneic Hematopoietic Cell Transplantation

TP53 mutations are associated with extremely poor outcomes in acute myeloid leukemia (AML). The outcomes of patients with TP53-mutated (TP53MUT) AML after different frontline treatment modalities are not well established. Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative procedure for AML; however, long-term outcomes among patients with TP53MUT AML after allo-HCT are dismal, and the benefit of allo-HCT remains controversial. We sought to evaluate the outcomes of patients with TP53MUT AML after treatment with different frontline induction therapies and allo-HCT. A total of 113 patients with TP53MUT AML were retrospectively evaluated. Patients with TP53MUT AML who received intensive or azacitidine-venetoclax induction had higher complete remission rates compared to patients treated with other hypomethylating-agent-based induction regimens. However, OS and EFS were not significantly different among the induction regimen groups. Allo-HCT was associated with improved OS and EFS among patients with TP53MUT AML; however, allo-HCT was not significantly associated with improved OS or EFS in time-dependent or landmark analysis. While the outcomes of all patients were generally poor irrespective of therapeutic strategy, transplanted patients with lower TP53MUT variant allele frequency (VAF) at the time of diagnosis had superior outcomes compared to transplanted patients with higher TP53 VAF. Our study provides further evidence that the current standards of care for AML confer limited therapeutic benefit to patients with TP53 mutations

The Impairment of MAGMAS Function in Human Is Responsible for a Severe Skeletal Dysplasia

International audienceImpairment of the tightly regulated ossification process leads to a wide range of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated protein involved in preprotein translocation into the matrix. We show that MAGMAS is specifically expressed in trabecular bone and cartilage at early developmental stages and that the mutation leads to an instability of the protein. We further demonstrate that the mutation described here confers to yeast strains a temperature-sensitive phenotype, impairs the import of mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a key role for this mitochondrial protein in the ossification process

MAGMAS localization in control growth plates of mice.

<p>Distal femurs of wild-type mice at different developmental stages; (<b>A</b>) 16.5-day embryo; (<b>B</b>) Newborn and (<b>C</b>) 2 week-old; were fixed and stained with anti-MAGMAS antibodies. Sections were also stained with anti-CoX (marker of differentiated hypertrophic chondrocytes) and with HES. MAGMAS expression is reflected by the brown precipitate resulting from the peroxidase reaction. MAGMAS was detected mainly in hypertrophic chondrocytes (black arrows) and osteocytes of control growth plates at the different developmental stages. Its expression was also detected in some type X collagen –negative chondrocytes that are initiating the process of differentiation.</p

Radiological features of the patients F2-IV.3 and F1-IV.3.

<p>Radiographs of patient <b>F2-IV.3</b> at 9 months (<b>A, C, E</b>) and <b>F1-IV.3</b> at birth (<b>B, F</b>) and at 3 months (<b>D</b>) show platyspondyly, square iliac bones, and delayed epiphyseal ossification.</p

Yeast strains and plasmids used in this study.

<p>Yeast strains and plasmids used in this study.</p

Pedigrees of the families F1 and F2 included in this study.

<p>Blackened symbols indicate affected individuals.</p

Transcript expression analysis of <i>MAGMAS</i>, <i>CORO7</i> and <i>CORO7-MAGMAS</i>.

<p>(A) Expression analysis of <i>MAGMAS</i>, <i>CORO7</i> and <i>CORO7-MAGMAS</i> by RT-PCR in control fibroblasts, chondrocytes and osteoblasts, showing that, contrary to <i>MAGMAS</i>, neither <i>CORO7</i> nor <i>CORO7-MAGMAS</i> was significantly expressed in chondrocytes and osteoblasts. (<b>B</b>) Quantitative expression analysis of <i>MAGMAS</i>, <i>CORO7</i> and <i>CORO7-MAGMAS</i> showing a very low level of expression of <i>CORO7</i> and <i>CORO7-MAGMAS</i> in chondrocytes compared to that of <i>MAGMAS</i> in the same sample.</p