Syndromic cardiac malformations can result in morbidity, yet their genetic etiology is only understood for a subset of individuals. Genome sequencing efforts in congenital anomaly cohorts may identify disease-associated variants in previously unrecognized genes. Through international matchmaking efforts, we identified eighteen individuals in total with de novo or loss-of-function variants in EIF3A (n = 4) or EIF3B (n = 14). The clinical phenotype varied but predominantly included cardiac defects, craniofacial dysmorphisms, mild developmental delays, and behavioral abnormalities. These genes encode core subunits of the eukaryotic initiation factor 3 (eIF3) complex, which plays a critical role in binding mRNA transcripts to the 40S ribosomal subunit during translation initiation. Both genes are highly constrained against loss of function, and animal models have demonstrated that disruptions in the eIF3 complex result in a range of developmental defects, including cardiovascular malformations. Additionally, EIF3B is located within the minimally overlapping region implicated in cardiac anomalies associated with 7p22.3 microdeletions. We sought to further study the role of these genes in syndromic congenital heart disease. To explore their functional impact, we generated zebrafish models with mutations in the orthologous eif3s10 and eif3ba genes, which resulted in developmental abnormalities, including thin heart tubes, lack of craniofacial cartilage, and embryonic lethality. We propose that pathogenic variants in EIF3A, as well as pathogenic variants or microdeletions involving EIF3B, cause a distinct autosomal-dominant neurodevelopmental syndrome characterized by cardiovascular and craniofacial manifestations.

LDB1 encodes transcriptional regulator protein LIM domain-binding protein 1, which plays an important role in neurogenesis. Few C-terminal likely gene-disrupting (LGD) variants have been reported in the literature in individuals with congenital ventriculomegaly. Through international collaboration, we now assembled a cohort of 16 individuals with de novo variants affecting various regions of LDB1. Eleven variants affect either the whole gene or the N-terminal dimerization domain (including gene deletions, as well as nonsense-mediated mRNA decay (NMD)-sensitive LGD and missense variants), and five variants (missense or NMD-escaping LGD variants) affect only the C terminus of LDB1 containing the LIM interaction domain. All individuals showed variable neurodevelopmental phenotypes, including developmental delay and behavioral anomalies. In line with literature reports, individuals harboring C-terminal variants additionally presented with ventriculomegaly, which suggests a potential genotype-phenotype correlation. In accordance, we found diverging pathomechanisms in vitro: N-terminal missense variants disrupt homodimerization of LDB1, likely leading to a loss of function, while C-terminal variants impair interaction with the essential partner LHX2 in a dominant-negative manner. These findings were confirmed in vivo in Drosophila melanogaster. Toxicity of overexpressed human LDB1 in Drosophila was not observed with N-terminal missense variants but was exacerbated by C-terminal variants. Similarly, phenotypes associated with LDB1/chi loss were rescued by overexpression of wild-type LDB1 but not by LDB1 harboring N-terminal missense variants or by C-terminal variants that even worsened phenotypes. In summary, our findings indicate that de novo variants in LDB1 are linked to two overlapping but distinct neurodevelopmental phenotypes based on variant location and propose two separate pathomechanisms underlying LDB1-related neurodevelopmental disorders.

Objectives: To characterize heterozygous pathogenic COCH variants in a French cohort with non-syndromic sensorineural hearing loss (NSHL) and assess genotype-phenotype correlations in autosomal dominant NSHL (DFNA9). Setting: National Reference Center for Genetic Hearing Loss, Necker-Enfants Malades Hospital, Paris, France. Methods: This retrospective observational study included 69 individuals from 20 unrelated families diagnosed with DFNA9 (2005-2025). All individuals underwent clinical and audiological evaluations and genetic testing via targeted COCH Sanger sequencing or next-generation sequencing (NGS) panels. Variants were interpreted according to ACMG guidelines. Audiometric profiles and vestibular data were collected. Results: Seven known pathogenic COCH variants were found in ten families, and ten novel likely pathogenic variants in the others. Variants in vWFA domains were associated with early or late onset, progressive, bilateral and symmetrical hearing loss. Three variants (p.Gln410Arg, p.Ile450Val, p.Cys542Arg) were associated with congenital or prelingual onset, an atypical DFNA9 presentation. Variants in the LCCL domain were associated with later-onset hearing loss and more frequent vestibular dysfunction. Vestibular abnormalities were observed in about half of early-onset cases. Conclusions: COCH-related hearing loss is a rare cause of autosomal dominant NSHL, with only 20 families identified over two decades within the French network. This study expands the mutational spectrum of COCH by reporting ten novel variants and supports a domain-specific genotype-phenotype correlation. These findings improve the understanding of DFNA9 variability and have direct implications for clinical diagnosis, prognosis, and genetic counseling.

Background/Objectives: Waardenburg syndrome (WS) is a genetic disorder characterized by sensorineural hearing loss (SNHL) and pigmentation anomalies. While hearing impairment is a well-established feature of WS, vestibular dysfunction is also reported. This study aimed to investigate vestibular deficits in pediatric WS patients with SNHL, correlating these findings with molecular, audiometric, and radiological data to establish distinct phenotypic profiles for each WS subtype and associated pathogenic variants. Methods: This retrospective study included children with a genetically confirmed diagnosis of WS who underwent vestibular, auditory, and inner ear radiological assessments as part of their routine medical care between July 2000 and May 2022. Data were collected from medical records, including medical history, clinical findings, and assessment results. Results: Vestibular dysfunction was found to be highly prevalent, affecting 64% of the cohort, often impacting the canal sensory organ (89%) and occasionally the otolithic function (33%). Patients with SOX10 pathogenic variations exhibited a markedly higher risk of vestibular dysfunction, highlighting the unique role of SOX10 in inner ear development. Notably, inner ear malformations were identified in all SOX10-mutated subjects, whereas such anomalies were rare among individuals with other WS gene variants, occurring in only two additional cases with minor malformations. Conclusions: This study reveals a significant prevalence of vestibular deficits in pediatric WS patients with SNHL, emphasizing the need for routine vestibular assessments. The higher prevalence and severity of vestibular impairments in SOX10-mutated patients underscore the importance of molecular analysis in clinical diagnosis and management.

Feingold syndrome type 1 (FS1) (OMIM 164280) is an autosomal dominant condition due to heterozygous loss of function variants in MYCN gene or to 2p24 deletion encompassing MYCN gene. The core features of FS1 are digital anomalies, microcephaly, facial dysmorphism, short stature, esophageal/duodenal atresia, and mild learning disabilities. Additional features are reported in a minority of patients, such as cardiac and renal anomalies. Sensorineural deafness is reported in 7 % of the patients. Other features can be associated with classical features of FS1 in patients with 2p deletion including MYCN and other genes. Recently, absence of the flexor pollicis longus tendon has been reported as a new skeletal feature in a pedigree segregating a MYCN variant. Here, we reported on three patients having FS1 without gastrointestinal atresia and unusual features: laryngeal cleft, congenital deafness, agenesis of the corpus callosum, and radio ulnar-synostosis (RUS). After the extension of the genetic screening, RUS was considered as an independent condition linked to SMAD6 variant. Diagnosis of FS1 can be challenging when there are unusual features without digestive malformations drawing attention. In this situation, the diagnostic approach may be based on major criteria of FS1: i) brachymesophalangy of the 2nd and 5th fingers, brachydactyly of fingers and toes with or without 2/3 and/or 4/5 toe syndactylies, ii) microcephaly, and iii) radiographs of the feet to look for amesophalangy of toes. Extension of the genetic screening is required to eliminate the possibility of two independent conditions. In addition to the previous recommendations, we advocate for a set of recommendations for evaluation of FS1 patients following initial diagnosis: systematic search of deafness, verification of the flexion of the interphalangeal joints of the thumbs, laryngoscopy in case of stridor or swallowing disorders, and finally systematic cerebral MRI.

KITLG pathogenic variants have been associated to three distinct clinical presentations with different combinations of hearing loss and/or pigmentation abnormalities. However, its involvement in isolated hearing loss has not been confirmed since its initial description in two families. Besides, KITLG is so far the only gene prevailingly involved in unilateral isolated hearing loss. We therefore conducted a retrospective study of patients with KITLG alterations in the French national Reference Network for Genetic Hearing Loss and one case was added through the Genematcher exchange platform. We describe a series of monoallelic KITLG deletions and variations in a cohort of 14 symptomatic patients from eight unrelated families. All patients presented with unilateral, bilateral symmetric or asymmetric sensorineural hearing loss. When not profound, hearing loss was predominant on low frequencies. Most KITLG alterations are likely to result in loss-of-function and aggregate in the extracellular region, disrupting the KIT-binding domain or its structure. Penetrance is not complete, and unspecific pigmentation alterations were observed in only three patients. The present study confirms KITLG involvement in isolated unilateral, bilateral symmetric or asymmetric hearing loss. This confirmation indicates that genetic testing can be relevant in early-onset, non-sudden, isolated unilateral hearing loss.

Hirschsprung disease (HD) can be associated with congenital central hypoventilation syndrome (CCHS). CCHS is mostly due to PHOX2B pathogenic variants. First report of HD on fetal autopsy leading to CCHS diagnosis. Highlights the role of fetal autopsy in stillborn babies, to guide genetic investigation and refine genetic counseling.

Oculo-auriculo-vertebral spectrum (OAVS) is characterized by abnormal development of the 1st and 2nd branchial arches. Despite arguments against a monogenic condition, a few genes have been involved in a minority of cases. We now report heterozygous, presumably loss-of function variants in the CHAF1A gene in 8 individuals, including 3 members of the same family. Four cases fulfill stringent diagnostic criteria for OAVS, including asymmetric ear dysplasia, preauricular tags, mandibular asymmetry +/− vertebral malformations. Two patients also presented with kidney malformations. CHAF1A encodes a subunit of CAF-1 (chromatin assembly factor-1), a heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3-H4 onto the newly synthetized DNA strand during replication. The identification of loss-of-unction variants in CHAF1A is consistent with the hypothesis of early developmental genes dysregulation driving OAVS and other associations recently lumped under the acronym Recurrent Constellations of Embryonic Malformations (RCEM).

De novo variants adjacent to the canonical splicing sites or in the well-defined splicing-related regions are more likely to impair splicing but remain under-investigated in autism spectrum disorder (ASD). By analyzing large, recent ASD genome sequencing cohorts, we find a significant burden of de novo potential splicing-disrupting variants (PSDVs) in 5048 probands compared to 4090 unaffected siblings. We identified 55 genes with recurrent de novo PSDVs that were highly intolerant to variation. Forty-six of these genes have not been strongly implicated in ASD or other neurodevelopmental disorders previously, including GSK3B. Through international, multicenter collaborations, we assembled genotype and phenotype data for 15 individuals with GSK3B variants and identified common phenotypes including developmental delay, ASD, sleeping disturbance, and aggressive behavior. Using available single-cell transcriptomic data, we show that GSK3B is enriched in dorsal progenitors and intermediate forms of excitatory neurons in the developing brain. We showed that Gsk3b knockdown in mouse excitatory neurons interferes with dendrite arborization and spine maturation which could not be rescued by de novo missense variants identified from affected individuals. In summary, our findings suggest that PSDVs may play an important role in the genetic etiology of ASD and allow for the prioritization of new ASD candidate genes. Importantly, we show that genetic variation resulting in GSK3B loss-of-function can lead to a neurodevelopmental disorder with core features of ASD and developmental delay.

Objective: Here we trained an automatic phenotype assessment tool to recognize syndromic ears in two syndromes in fetuses-=CHARGE and Mandibulo-Facial Dysostosis Guion Almeida type (MFDGA)-versus controls. Method: We trained an automatic model on all profile pictures of children diagnosed with genetically confirmed MFDGA and CHARGE syndromes, and a cohort of control patients, collected from 1981 to 2023 in Necker Hospital (Paris) with a visible external ear. The model consisted in extracting landmarks from photographs of external ears, in applying geometric morphometry methods, and in a classification step using machine learning. The approach was then tested on photographs of two groups of fetuses: controls and fetuses with CHARGE and MFDGA syndromes. Results: The training set contained a total of 1489 ear photographs from 526 children. The validation set contained a total of 51 ear photographs from 51 fetuses. The overall accuracy was 72.6% (58.3%-84.1%, p < 0.001), and 76.4%, 74.9%, and 86.2% respectively for CHARGE, control and MFDGA fetuses. The area under the curves were 86.8%, 87.5%, and 90.3% respectively for CHARGE, controls, and MFDGA fetuses. Conclusion: We report the first automatic fetal ear phenotyping model, with satisfactory classification performances. Further validations are required before using this approach as a diagnostic tool. Key points What's already known about this topic? center dot Facial analysis of fetuses is crucial for antenatal and post-mortem diagnosis. center dot Prenatal abnormalities of the external ear are common in many syndromes. What does this study add? center dot The aim of this study was to train an AI-based tool on photographs of the external ears of control children and children with two genetic syndromes-CHARGE syndrome and Mandibulo-Facial Dysostosis Guion Almeida type-and to test this tool on photographs of fetal ears, with the aim of supporting the medical genetics diagnosis.

Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

The genes EIF3A (HGNC:3271) and EIF3B (HGNC:3280) encode individual subunits constituting the eukaryotic initiation factor 3 (eIF3) complex, a pivotal regulator of protein synthesis. Their interaction forms the nucleation core of the eIF3 complex, a crucial stage in its assembly. Reports have identified interstitial and terminal deletions on chromosome 7p22.3 in patients exhibiting a variable phenotype, encompassing neurodevelopmental delay, distinct facial features, and congenital heart defects (CHD), notably tetralogy of Fallot (TOF).

Mutations of G protein–coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here, we establish p.E303K in the gene encoding the endothelin receptor type A (ETAR/EDNRA) as a recurrent mutation causing mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibited a partial maxillary-to-mandibular transformation, which was rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ETAR mutations as gain of function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increased flexibility of this portion of the helix, thus favoring G protein binding and leading to G protein–mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduced the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, which may provide the basis for drug design targeting GPCRs.

CHARGE syndrome, due to CHD7 pathogenic variations, is an autosomal dominant disorder characterized by a large spectrum of severity. Despite the great number of variations reported, no clear genotype-to-phenotype correlation has been reported. Unsupervised machine learning and clustering was undertaken using a retrospective cohort of 42 patients, after deep radiologic and clinical phenotyping, to establish genotype-phenotype correlation for CHD7-related CHARGE syndrome. It resulted in three clusters showing phenotypes of different severities. While no clear genotype-phenotype correlation appeared within the first two clusters, a single patient was outlying the cohort data (cluster 3) with the most atypical phenotype and the most distal frameshift variant in the gene. We added two other patients with similar distal pathogenic variants and observed a tendency toward mild and/or atypical phenotypes. We hypothesized that this finding could potentially be related to escaping nonsense mediated RNA decay, but found no evidence of such decay in vivo for any of the CHD7 pathogenic variation tested. This indicates that this milder phenotype may rather result from the production of a protein retaining all functional domains.

Waardenburg syndrome type 1 (WS1), a rare genetic disease characterized by pigmentation defects and mild craniofacial anomalies often associated with congenital deafness is caused by heterozygous mutations in the PAX3 gene (2q36.1). We have generated two induced pluripotent stem cell lines (PCli029-A and PCli031-A) from two patients from the same family both carrying the same heterozygous deletion in PAX3 exon 1 (c.-70_85 + 366del). These cells are pluripotent as they can differentiate into ectoderm, mesoderm and endoderm. They also can activate the early neural crest marker SNAI2. These cells will be useful for studying the human neural crest-derived pigment cells.

Waardenburg syndrome (WS) is characterized by the association of sensorineural hearing loss and pigmentation abnormalities. Among the four types, WS Type 2 (WS2) is the only one without a remarkable distinguishing feature. Here, we report a patient initially diagnosed with WS2 who exhibits a 446 kb mosaic duplication in chromosome 22q13.1, encompassing SOX10, and detected using whole genome sequencing in a trio. The patient, a 46,XY boy, presents with profound bilateral sensorineural hearing loss, right heterochromia iridium, left bright blue iris, and skin-depigmented areas in the abdomen and limbs. Vestibular and imaging tests are normal, without inner ear or olfactory bulb malformations. Bilateral cochlear implantation did not prevent language and speech delays. Moderate congenital chronic constipation and neurodevelopmental difficulties were also present. Given the few genes included in this duplicated region (only one OMIM gene with dominant inheritance), this report provides further delineation of the phenotype related to duplications encompassing the entire SOX10 gene.

Waardenburg syndrome (WS) is characterized by hearing loss and pigmentary abnormalities of the eyes, hair, and skin. The condition is genetically heterogeneous, and is classified into four clinical types differentiated by the presence of dystopia canthorum in type 1 and its absence in type 2. Additionally, limb musculoskeletal abnormalities and Hirschsprung disease differentiate types 3 and 4, respectively. Genes PAX3, MITF, SOX10, KITLG, EDNRB, and EDN3 are already known to be associated with WS. In WS, a certain degree of molecularly undetected patients remains, especially in type 2. This study aims to pinpoint causative variants using different NGS approaches in a cohort of 26 Brazilian probands with possible/probable diagnosis of WS1 (8) or WS2 (18). DNA from the patients was first analyzed by exome sequencing. Seven of these families were submitted to trio analysis. For inconclusive cases, we applied a targeted NGS panel targeting WS/neurocristopathies genes. Causative variants were detected in 20 of the 26 probands analyzed, these being five in PAX3, eight in MITF, two in SOX10, four in EDNRB, and one in ACTG1 (type 2 Baraitser-Winter syndrome, BWS2). In conclusion, in our cohort of patients, the detection rate of the causative variant was 77%, confirming the superior detection power of NGS in genetically heterogeneous diseases.