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

Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

PURPOSE: Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. METHODS: We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. RESULTS: The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. CONCLUSION: NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype

10 years of CEMARA database in the AnDDI-Rares network: a unique resource facilitating research and epidemiology in developmental disorders in France

Background : In France, the Ministry of Health has implemented a comprehensive program for rare diseases (RD) that includes an epidemiological program as well as the establishment of expert centers for the clinical care of patients with RD. Since 2007, most of these centers have entered the data for patients with developmental disorders into the CEMARA population-based registry, a national online data repository for all rare diseases. Through the CEMARA web portal, descriptive demographic data, clinical data, and the chronology of medical follow-up can be obtained for each center. We address the interest and ongoing challenges of this national data collection system 10 years after its implementation. Methods : Since 2007, clinicians and researchers have reported the “minimum dataset (MDS)” for each patient presenting to their expert center. We retrospectively analyzed administrative data, demographic data, care organization and diagnoses. Results : Over 10 years, 228,243 RD patients (including healthy carriers and family members for whom experts denied any suspicion of RD) have visited an expert center. Among them, 167,361 were patients affected by a RD (median age 11 years, 54% children, 46% adults, with a balanced sex ratio), and 60,882 were unaffected relatives (median age 37 years). The majority of patients (87%) were seen no more than once a year, and 52% of visits were for a diagnostic procedure. Among the 2,869 recorded rare disorders, 1,907 (66.5%) were recorded in less than 10 patients, 802 (28%) in 10 to 100 patients, 149 (5.2%) in 100 to 1,000 patients, and 11 (0.4%) in > 1,000 patients. Overall, 45.6% of individuals had no diagnosis and 6.7% had an uncertain diagnosis. Children were mainly referred by their pediatrician (46%; n = 55,755 among the 121,136 total children referrals) and adults by a medical specialist (34%; n = 14,053 among the 41,564 total adult referrals). Given the geographical coverage of the centers, the median distance from the patient’s home was 25.1 km (IQR = 6.3 km-64.2 km). Conclusions : CEMARA provides unprecedented support for epidemiological, clinical and therapeutic studies in the field of RD. Researchers can benefit from the national scope of CEMARA data, but also focus on specific diseases or patient subgroups. While this endeavor has been a major collective effort among French RD experts to gather large-scale data into a single database, it provides tremendous potential to improve patient care

Fifteen years of research on oral–facial–digital syndromes: from 1 to 16 causal genes

Oral–facial–digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Bases cliniques et génétiques de deux ciliopathies (les syndromes hydrolethalus et acro-calleux)

LYON1-BU Santé (693882101) / SudocSudocFranceF

Etude d une série de 76 patients avec mutation du gène GLI3 et corrélations génotype-phénotype

Le spectre phénotypique des mutations du gène GLI3 comprend les syndromes de Greig (GCPS) et de Pallister-Hall (PHS) de transmission autosomique dominante. Le syndrome de Pallister-Hall initialement décrit comme une affection létale en période néonatale associe des anomalies telles qu'un hamartome de l'hypothalamus, une polydactylie centrale ou postaxiale, une imperforation anale et une épiglotte bifide. Le syndrome de Greig est moins sévère et se caractérise par des polysyndactylies des mains et des pieds et une dysmorphie craniofaciale. Des polydactylies isolées préaxiales et postaxiales ont également été rapportées chez des patients mutés dans le gène GLI3. Une corrélation génotype-phénotype a pu être établie selon la position et la nature de la mutation, corroborant le rôle bifonctionnel de répresseur ou d'activateur du facteur GLI3 au cours du développement. Nous rapportons dans cette étude les résultats cliniques et moléculaires d'une série de 76 patients issus de 55 familles présentant une mutation (49GCPS et 21 PHS) ou une délétion du gène GLI3 (6GCPS). La plupart des mutations sont nouvelles et concorde avec la corrélation précédemment décrite. Nos résultats montrent par ailleurs une corrélation entre la position C-terminale de la mutation et la présence d'anomalies du corps calleux observés chez certains patients GCPS. La description de cas fœtaux présentant un PHS sévère rappelle la possible létalité de ce syndrome et étend le spectre phénotypique à des malformations cranio-faciales et à des dysplasies squelettiques sévères. L'expression de GLI3 étudiée par hybridation in situ au cours du développement humain confirme l'expression précoce de GLI3 dans les tissus cibles. GLI3 est un facteur de transcription cible de la voie Sonic Hedgehog agissant à travers le cil primaire. Le chevauchement phénotypique entre hedgehogopathies et ciliopathes souligne le rôle primordial du cil primaire dans la transduction du signal Sonic Hedgehog, et inversement, le rôle majeur de la dérégulation de la voie SHH dans les malformations observées dans les ciliopathies.The phenotypic spectrum of GLI3 mutations includes Greig cephalopolysyndactyly (GCPS) and Pallister-Hall (PHS) syndromes, both autosomal dominant disorders ascribed to GLI3 mutations. PHS was first described as a neonatally lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. GCPS combines polysyndactyly of hands and feet and craniofacial features. Isolated preaxial and postaxial polydactyly has also been associated with GLI3 mutations. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our resuts also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasise on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations to severe craniofacial and reductional limb defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression n target tissues. GLI3 is a key factor of the Sonic Hedgehog pathways, known to act through the primary cilia in mammals. The phenotypic overlap between this and other hedgehogopathies and ciliopathies highlight the crucial role of the primary cilium in the SHH signalling, and conversely, the major role of SHH misregulation in malformations observed in severe ciliopathies.RENNES1-BU Santé (352382103) / SudocSudocFranceF

Le Syndrome CHARGE (étude foetopathologique et moléculaire d'une série de 50 cas)

Le syndrome CHARGE est une association malformative rare, le plus souvent sporadique et due à une mutation du gène CHD7 dans 60% des cas. Bien que ce syndrome soit désormais bien connu chez l'enfant, seule une série de 10 foetus atteints de ce syndrome a été publiée jusqu'à présent et le diagnostic anténatal reste parfois difficile. Nous avons élargi l'étude précédente à 50 foetus/nouveau nés pour lesquels un examen foetopathologique a été réalisé et le diagnostic de syndrome CHARGE proposé. Une mutation dans le gène CHD7 a été identifiée chez 44 d'entre eux. Nous retiendrons notamment de l'étude de notre série le taux élevé de garçons par rapport aux filles (2,7/1), suggérant des formes plus sévères chez le garçon. Les signes les plus constants sont l'anomalie des oreilles externes, suivie de l'agénésie des canaux semi circulaires et de l'arhinencéphalie. Ces signes sont des éléments prédictifs puissants d'une mutation dans CHD7 dans notre série et ce d'autant plus qu'ils sont présents simultanément. En revanche aucun foetus ne présentait de retard de croissance intra utérin. L'un des foetus muté pour CHD7 de notre série, présentait un phénotype atypique qui avait tout d'abord fait évoquer le diagnostic de syndrome 3C, rappelant que le syndrome CHARGE peut avoir une présentation variable et partage un certain nombre d'éléments communs à d'autres syndromes qui seront discutés. A l'inverse quelques foetus répondant aux critères diagnostiques du syndrome CHARGE ne sont pas mutés pour CHD7 et la recherche de nouveaux gènes impliqués se poursuit. Enfin, à l'exception d'un cas, toutes les mutations identifiées dans notre série étaient des mutations tronquantes suggérant une corrélation phénotype/génotype. L'analyse clinique de notre série nous a permis d'affiner la description du phénotype du syndrome CHARGE chez le foetus, de décrire de nouveaux éléments cliniques et finalement de proposer un ajustement des critères diagnostiques chez le foetus afin d'aider au diagnostic de syndrome CHARGE au décours d'une interruption médicale de grossesse pour syndrome malformatif sévère.POITIERS-BU Médecine pharmacie (861942103) / SudocSudocFranceF

Screening of MITF and SOX10 regulatory regions in Waardenburg syndrome type 2.

Waardenburg syndrome (WS) is a rare auditory-pigmentary disorder that exhibits varying combinations of sensorineural hearing loss and pigmentation defects. Four subtypes are clinically defined based on the presence or absence of additional symptoms. WS type 2 (WS2) can result from mutations within the MITF or SOX10 genes; however, 70% of WS2 cases remain unexplained at the molecular level, suggesting that other genes might be involved and/or that mutations within the known genes escaped previous screenings. The recent identification of a deletion encompassing three of the SOX10 regulatory elements in a patient presenting with another WS subtype, WS4, defined by its association with Hirschsprung disease, led us to search for deletions and point mutations within the MITF and SOX10 regulatory elements in 28 yet unexplained WS2 cases. Two nucleotide variations were identified: one in close proximity to the MITF distal enhancer (MDE) and one within the U1 SOX10 enhancer. Functional analyses argued against a pathogenic effect of these variations, suggesting that mutations within regulatory elements of WS genes are not a major cause of this neurocristopathy