Clinical and Functional Characterization of the Recurrent TUBA1A p.(Arg2His) Mutation

The TUBA1A gene encodes tubulin alpha-1A, a protein that is highly expressed in the fetal brain. Alpha- and beta-tubulin subunits form dimers, which then co-assemble into microtubule polymers: dynamic, scaffold-like structures that perform key functions during neurogenesis, neuronal migration, and cortical organisation. Mutations in TUBA1A have been reported to cause a range of brain malformations. We describe four unrelated patients with the same de novo missense mutation in TUBA1A, c.5G>A, p.(Arg2His), as found by next generation sequencing. Detailed comparison revealed similar brain phenotypes with mild variability. Shared features included developmental delay, microcephaly, hypoplasia of the cerebellar vermis, dysplasia or thinning of the corpus callosum, small pons, and dysmorphic basal ganglia. Two of the patients had bilateral perisylvian polymicrogyria. We examined the effects of the p.(Arg2His) mutation by computer-based protein structure modelling and heterologous expression in HEK-293 cells. The results suggest the mutation subtly impairs microtubule function, potentially by affecting inter-dimer interaction. Based on its sequence context, c.5G>A is likely to be a common recurrent mutation. We propose that the subtle functional effects of p.(Arg2His) may allow for other factors (such as genetic background or environmental conditions) to influence phenotypic outcome, thus explaining the mild variability in clinical manifestations

AI-based diagnosis in mandibulofacial dysostosis with microcephaly using external ear shapes

IntroductionMandibulo-Facial Dysostosis with Microcephaly (MFDM) is a rare disease with a broad spectrum of symptoms, characterized by zygomatic and mandibular hypoplasia, microcephaly, and ear abnormalities. Here, we aimed at describing the external ear phenotype of MFDM patients, and train an Artificial Intelligence (AI)-based model to differentiate MFDM ears from non-syndromic control ears (binary classification), and from ears of the main differential diagnoses of this condition (multi-class classification): Treacher Collins (TC), Nager (NAFD) and CHARGE syndromes.MethodsThe training set contained 1,592 ear photographs, corresponding to 550 patients. We extracted 48 patients completely independent of the training set, with only one photograph per ear per patient. After a CNN-(Convolutional Neural Network) based ear detection, the images were automatically landmarked. Generalized Procrustes Analysis was then performed, along with a dimension reduction using PCA (Principal Component Analysis). The principal components were used as inputs in an eXtreme Gradient Boosting (XGBoost) model, optimized using a 5-fold cross-validation. Finally, the model was tested on an independent validation set.ResultsWe trained the model on 1,592 ear photographs, corresponding to 1,296 control ears, 105 MFDM, 33 NAFD, 70 TC and 88 CHARGE syndrome ears. The model detected MFDM with an accuracy of 0.969 [0.838–0.999] (p < 0.001) and an AUC (Area Under the Curve) of 0.975 within controls (binary classification). Balanced accuracies were 0.811 [0.648–0.920] (p = 0.002) in a first multiclass design (MFDM vs. controls and differential diagnoses) and 0.813 [0.544–0.960] (p = 0.003) in a second multiclass design (MFDM vs. differential diagnoses).ConclusionThis is the first AI-based syndrome detection model in dysmorphology based on the external ear, opening promising clinical applications both for local care and referral, and for expert centers

Identification, functional characterization and phenotypic spectrum of genes responsible for neurodevelopmental anomalies

Les anomalies de développement cérébral sont à l'origine de troubles neurologiques de sévérité variable parmi lesquels les déficits cognitifs, l'épilepsie, les troubles moteurs et du comportement. Si l'avènement des techniques d'analyse globale du génome et notamment l'analyse chromosomique sur puce à ADN (ACPA) ou plus récemment le séquençage haut débit ont permis d'augmenter de façon exponentielle l'identification des bases moléculaires d'anomalies neuro-développementales, nombre d'entre elles demeurent inexpliquées et les mécanismes physiopathologiques sous-jacents mal compris. Mon projet de thèse s'inscrit dans le cadre de l'étude d'une cohorte multicentrique de patients et fœtus atteints de malformations cérébrales telles les malformations du cortex cérébral, les malformations du corps calleux et les hypoplasies cérébelleuses. Je me suis en particulier focalisée sur les anomalies neurodéveloppementales les plus évocatrices d'anomalies du cil primaire, un organelle nécessaire à toutes les étapes du développement cérébral, de la fermeture du tube neural, à la prolifération et à la différenciation des progéniteurs neuraux, jusqu'à la migration neuronale et à la mise en place des connexions synaptiques. Mon projet de thèse a ainsi porté sur plusieurs types d'anomalies cérébrales : 1) la microcéphalie associée à des mutations du gène RTTN codant une protéine centrosomale, travail qui nous a permis de rapporter la première description neuro-pathologique des conséquences de l'invalidation de ce gène chez l'homme, 2) les malformations du corps calleux de découverte anténatale dans le cadre d'une ciliopathie par mutations d'OFD1 ou d'une maladie métabolique avec en particulier l'identification d'un nouveau gène codant une protéine d'assemblage du complexe I de la chaîne respiratoire mitochondriale (TIMMDC1), et 3) l'hypoplasie cérébelleuse associée ou non à une malformation du cortex cérébral et pour lesquelles nous avons identifié un nouveau gène candidat, ASTN1, impliqué dans la migration neuronale. Au-delà du séquençage haut débit, et en plus des études neurohistopathologiques et fonctionnelles que j'ai pu réaliser sur tissus de fœtus humains, j'ai choisi de profiter des avancées technologiques en biologie moléculaire et cellulaire pour développer des approches de modélisation pertinentes permettant de mieux comprendre les mécanismes physiopathologiques à l'origine de ces anomalies neurodéveloppementales. Ainsi, la reprogrammation de cellules somatiques en cellules pluripotentes induites (human Induced Pluripotent Stemcells, hIPSC) couplée à l'édition du génome (CRISPR/CAS9) m'a permis de générer des clones hIPSC mutés ainsi que leurs contrôles isogéniques. J'ai de plus pu confirmer que les modèles cellulaires 2D (rosettes neurales) et 3D (organoïdes cérébraux) de développement du cortex cérébral humain générés à partir d'hIPSC permettent de disséquer les divers mécanismes qui contrôlent le développement du cortex cérébral et en particulier la fonction du cil primaire en conditions normale et pathologique. Parallèlement, et afin de tester l'hypothèse d'un gain de fonction secondaire aux mutations d'ASTN1, j'ai choisi une approche complémentaire in vivo, par électroporation in utero d'embryons murins, permettant de modéliser la surexpression d'ASNT1 sauvage et muté afin d'en étudier les conséquences sur le développement néocortical. Dans l'ensemble, ce travail a permis l'identification de 2 nouveaux gènes dont les mutations sont responsables d'anomalies de développement cérébral, de décrire les anomalies neuro-histologiques associées à l'invalidation de 6 gènes (RTTN, OFD1, CTSD, TIMMDC1, EARS2, MRPS22) par l'étude de cas fœtaux humains. Concernant ASTN1, les approches de modélisation in vivo et in vitro que j'ai développées ouvrent des hypothèses quant aux mécanismes physiopathologiques associés aux mutations de cet excellent gène candidat.Cerebral developmental anomalies lead to various neurodevelopmental disorders including cognitive deficiency, epilepsy, motor and behavioral disorders. If the advent of global genome analysis techniques and in particular molecular caryotyping or more recently high throughput sequencing have allowed an exponential increase in the identification of the genetic basis of neurodevelopmental abnormalities, many remain unexplained and the underlying pathophysiological mechanisms poorly understood. My phD project relies on a multicentric cohort of patients and fetuses with various neurodevelopmental anomalies such as cerebral cortical malformations, corpus callosum malformations and cerebellar hypoplasia. In particular, I focused on neurodevelopmental anomalies reminiscent of abnormal biogenesis or function of the primary cilium, an organelle required for many fundamental processes of brain development, from the closure of the neural tube up to the neuronal migration and the establishment of synaptic connections, including proliferation and fate determination of neural progenitors. My thesis project thus focused on several types of cerebral anomalies: 1) microcephaly due to mutations in RTTN, a gene encoding a centrosomal protein, allowing us to report the first neuropathological description associated to RTTN mutations in human fetal cases, 2) malformations of the corpus callosum in the context of a ciliopathy due to OFD1 mutations or of a metabolic disease with the identification of a novel gene encoding a protein required for the assembly of the mitochondrial complex I (TIMMDC1), and 3) cerebellar hypoplasia associated or not with a malformation of the cerebral cortex and for which we have identified a new candidate gene, ASTN1, involved in neuronal migration. Beyond high throughput sequencing, and in addition to the neurohistopathological and functional analysis that I was able to perform on human fetal tissues, I chose to take advantage of technological advances in molecular and cellular biology to develop relevant modeling approaches to better understand the pathophysiological mechanisms underlying these neurodevelopmental anomalies. Thus, the reprogramming of somatic cells into human Induced Pluripotent Stem Cells (hIPSC) coupled with genome editing (CRISPR/CAS9) allowed me to generate mutated hIPSC clones as well as their isogenic controls. I was also able to confirm that the 2D (neural rosettes) and 3D (cerebral organoids) hIPSC-based models of human cerebral cortical development allow to dissect the various mechanisms that control the development of the cerebral cortex and in particular the function of the primary cilium in normal and pathological conditions. In parallel, and in order to test the hypothesis of a gain of function mechanism of ASTN1 mutations, I chose a complementary in vivo approach, by in utero electroporation of murine embryos, allowing to model the overexpression of wild-type and mutated ASTN1 in order to study their consequences on neocortical development. Overall, this work allowed the identification of two new genes whose mutations are responsible for brain developmental abnormalities and to describe the neuro-histological abnormalities associated with the invalidation of 6 genes (RTTN, OFD1, CTSD, TIMMDC1, EARS2, MRPS22) by studying human fetal cases. Concerning ASTN1, the in vivo and in vitro modeling approaches that I developed open interesting hypotheses as to the pathophysiological mechanisms associated with mutations of this excellent candidate gene

Importance des études de jumeaux en génétique (application aux bases moléculaires de l'atrésie de l'oesophage)

PARIS-BIUP (751062107) / SudocSudocFranceF

Cilia in hereditary cerebral anomalies

International audienceCiliopathies are complex genetic multi-system disorders causally related to abnormal assembly or function of motile or non-motile cilia. While most human cells possess a non-motile sensory/primary cilium (PC) during development and/or in adult tissues, motile cilia are restricted to specialised cells. As a result, PC-associated ciliopathies are characterised by high phenotypic variability with extensive clinical and genetic overlaps. In the present review, we have focused on cerebral developmental anomalies, which are commonly found in PC-associated ciliopathies and which have mostly been linked to Hedgehog signalling defects. In addition, we have reviewed emerging evidence that PC dysfunctions could be directly or indirectly involved in the mechanisms underlying malformations of cerebral cortical development including primary microcephaly

Novel CDK10 variants with multicystic dysplastic kidney, left ventricular non‐compaction, and a solitary median maxillary central incisor

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Diagnosis of Menke‐Hennekam syndrome by prenatal whole exome sequencing and review of prenatal signs

Abstract Introduction CREBBP truncating mutations and deletions are responsible for the well‐known Rubinstein‐Taybi syndrome. Recently, a new, distinct CREBBP‐linked syndrome has been described: missense mutations located at the 3′ end of exon 30 and the 5′ portion of exon 31 induce Menke‐Hennekam syndrome. Patients with this syndrome present a recognizable facial dysmorphism, intellectual disability of variable severity, microcephaly, short stature, autism, epilepsy, visual and hearing impairments, feeding problems, upper airway infections, scoliosis, and/or kyphosis. To date, all diagnoses were made postnatally. Method and Case Report Trio‐whole exome sequencing (WES) was performed in a fetus showing increased nuchal translucency persistence and aorta abnormalities at 28 weeks of gestation (WG). Results WES revealed a CREBBP de novo missense mutation (c.5602C>T; p.Arg1868Trp) in exon 31, previously reported as the cause of Menke‐Hennekam syndrome. Termination of pregnancy was performed at 32 WG. We further reviewed the prenatal signs of Menke‐Hennekam syndrome already reported. Among the 35 patients reported and diagnosed postnatally up to this day, 15 presented recognizable prenatal signs, the most frequent being intra‐uterine growth retardation, brain, and cardiovascular anomalies. Conclusion Menke‐Hennekam is a rare syndrome with unspecific, heterogeneous, and inconstant prenatal symptoms occurring most frequently with the c.5602C>T, p.(Arg1868Trp) mutation. Therefore, the prenatal diagnosis of Menke‐Hennekam syndrome is only possible by molecular investigation. Moreover, this case report and review reinforce the importance of performing prenatal WES when unspecific signs are present on imaging

Bi-allelic variations in CRB2, encoding the crumbs cell polarity complex component 2, lead to non-communicating hydrocephalus due to atresia of the aqueduct of sylvius and central canal of the medulla

International audienceAbstract Congenital hydrocephalus is a common condition caused by the accumulation of cerebrospinal fluid in the ventricular system. Four major genes are currently known to be causally involved in hydrocephalus, either isolated or as a common clinical feature: L1CAM , AP1S2 , MPDZ and CCDC88C. Here, we report 3 cases from 2 families with congenital hydrocephalus due to bi-allelic variations in CRB2, a gene previously reported to cause nephrotic syndrome, variably associated with hydrocephalus. While 2 cases presented with renal cysts, one case presented with isolated hydrocephalus. Neurohistopathological analysis allowed us to demonstrate that, contrary to what was previously proposed, the pathological mechanisms underlying hydrocephalus secondary to CRB2 variations are not due to stenosis but to atresia of both Sylvius Aqueduct and central medullar canal. While CRB2 has been largely shown crucial for apico-basal polarity, immunolabelling experiments in our fetal cases showed normal localization and level of PAR complex components (PKCι and PKCζ) as well as of tight (ZO-1) and adherens (β-catenin and N-Cadherin) junction molecules indicating a priori normal apicobasal polarity and cell–cell adhesion of the ventricular epithelium suggesting another pathological mechanism. Interestingly, atresia but not stenosis of Sylvius aqueduct was also described in cases with variations in MPDZ and CCDC88C encoding proteins previously linked functionally to the Crumbs (CRB) polarity complex, and all 3 being more recently involved in apical constriction, a process crucial for the formation of the central medullar canal. Overall, our findings argue for a common mechanism of CRB2 , MPDZ and CCDC88C variations that might lead to abnormal apical constriction of the ventricular cells of the neural tube that will form the ependymal cells lining the definitive central canal of the medulla. Our study thus highlights that hydrocephalus related to CRB2 , MPDZ and CCDC88C constitutes a separate pathogenic group of congenital non-communicating hydrocephalus with atresia of both Sylvius aqueduct and central canal of the medulla

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

International audiencePrimary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function

Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications