Artificial intelligence-based diagnosis in fetal pathology using external ear shapes.

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.

Investigating genotype-to-phenotype correlation in CHARGE syndrome by deep phenotyping and multiparametric clustering.

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.

A 22q13.1 duplication in mosaicism including SOX10.

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.

Retrospective evaluation of clinical and molecular data of 148 cases of esophageal atresia.

Developmental abnormalities provide a unique opportunity to seek for the molecular mechanisms underlying human organogenesis. Esophageal development remains incompletely understood and elucidating causes for esophageal atresia (EA) in humans would contribute to achieve a better comprehension. Prenatal detection, syndromic classification, molecular diagnosis, and prognostic factors in EA are challenging. Some syndromes have been described to frequently include EA, such as CHARGE, EFTUD2-mandibulofacial dysostosis, Feingold syndrome, trisomy 18, and Fanconi anemia. However, no molecular diagnosis is made in most cases, including frequent associations, such as Vertebral-Anal-Cardiac-Tracheo-Esophageal-Renal-Limb defects (VACTERL). This study evaluates the clinical and genetic test results of 139 neonates and 9 fetuses followed-up at the Necker-Enfants Malades Hospital over a 10-years period. Overall, 52 cases were isolated EA (35%), and 96 were associated with other anomalies (65%). The latter group is divided into three subgroups: EA with a known genomic cause (9/148, 6%); EA with Vertebral-Anal-Cardiac-Tracheo-Esophageal-Renal-Limb defects (VACTERL) or VACTERL/Oculo-Auriculo-Vertebral Dysplasia (VACTERL/OAV) (22/148, 14%); EA with associated malformations including congenital heart defects, duodenal atresia, and diaphragmatic hernia without known associations or syndromes yet described (65/148, 44%). Altogether, the molecular diagnostic rate remains very low and may underlie frequent non-Mendelian genetic models.

SOX10: 20 years of phenotypic plurality and current understanding of its developmental function.

SOX10 belongs to a family of 20 SRY (sex-determining region Y)-related high mobility group box-containing (SOX) proteins, most of which contribute to cell type specification and differentiation of various lineages. The first clue that SOX10 is essential for development, especially in the neural crest, came with the discovery that heterozygous mutations occurring within and around SOX10 cause Waardenburg syndrome type 4. Since then, heterozygous mutations have been reported in Waardenburg syndrome type 2 (Waardenburg syndrome type without Hirschsprung disease), PCWH or PCW (peripheral demyelinating neuropathy, central dysmyelination, Waardenburg syndrome, with or without Hirschsprung disease), intestinal manifestations beyond Hirschsprung (ie, chronic intestinal pseudo-obstruction), Kallmann syndrome and cancer. All of these diseases are consistent with the regulatory role of SOX10 in various neural crest derivatives (melanocytes, the enteric nervous system, Schwann cells and olfactory ensheathing cells) and extraneural crest tissues (inner ear, oligodendrocytes). The recent evolution of medical practice in constitutional genetics has led to the identification of SOX10 variants in atypical contexts, such as isolated hearing loss or neurodevelopmental disorders, making them more difficult to classify in the absence of both a typical phenotype and specific expertise. Here, we report novel mutations and review those that have already been published and their functional consequences, along with current understanding of SOX10 function in the affected cell types identified through in vivo and in vitro models. We also discuss research options to increase our understanding of the origin of the observed phenotypic variability and improve the diagnosis and medical care of affected patients.

Further delineation of auriculocondylar syndrome based on 14 novel cases and reassessment of 25 published cases.

Auriculocondylar syndrome (ACS) is a rare craniofacial disorder characterized by mandibular hypoplasia and an auricular defect at the junction between the lobe and helix, known as a "Question Mark Ear" (QME). Several additional features, originating from the first and second branchial arches and other tissues, have also been reported. ACS is genetically heterogeneous with autosomal dominant and recessive modes of inheritance. The mutations identified to date are presumed to dysregulate the endothelin 1 signaling pathway. Here we describe 14 novel cases and reassess 25 published cases of ACS through a questionnaire for systematic data collection. All patients harbor mutation(s) in PLCB4, GNAI3, or EDN1. This series of patients contributes to the characterization of additional features occasionally associated with ACS such as respiratory, costal, neurodevelopmental, and genital anomalies, and provides management and monitoring recommendations.

EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion-Almeida type.

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.

News from the endothelin-3/EDNRB signaling pathway: Role during enteric nervous system development and involvement in neural crest-associated disorders.

The endothelin system is a vertebrate-specific innovation with important roles in regulating the cardiovascular system and renal and pulmonary processes, as well as the development of the vertebrate-specific neural crest cell population and its derivatives. This system is comprised of three structurally similar 21-amino acid peptides that bind and activate two G-protein coupled receptors. In 1994, knockouts of the Edn3 and Ednrb genes revealed their crucial function during development of the enteric nervous system and melanocytes, two neural-crest derivatives. Since then, human and mouse genetics, combined with cellular and developmental studies, have helped to unravel the role of this signaling pathway during development and adulthood. In this review, we will summarize the known functions of the EDN3/EDNRB pathway during neural crest development, with a specific focus on recent scientific advances, and the enteric nervous system in normal and pathological conditions.

Differentiation of Mouse Enteric Nervous System Progenitor Cells Is Controlled by Endothelin 3 and Requires Regulation of Ednrb by SOX10 and ZEB2.

BACKGROUND & AIMS: Maintenance and differentiation of progenitor cells in the developing enteric nervous system are controlled by molecules such as the signaling protein endothelin 3 (EDN3), its receptor (the endothelin receptor type B [EDNRB]), and the transcription factors SRY-box 10 (SOX10) and zinc finger E-box binding homeobox 2 (ZEB2). We used enteric progenitor cell (EPC) cultures and mice to study the roles of these proteins in enteric neurogenesis and their cross regulation. METHODS: We performed studies in mice with a Zeb2 loss-of-function mutation (Zeb2Δ) and mice carrying a spontaneous recessive mutation that prevents conversion of EDN3 to its active form (Edn3ls). EPC cultures issued from embryos that expressed only wild-type Zeb2 (Zeb2+/+ EPCs) or were heterozygous for the mutation (Zeb2Δ/+ EPCs) were exposed to EDN3; we analyzed the effects on cell differentiation using immunocytochemistry. In parallel, Edn3ls mice were crossed with Zeb2Δ/+mice; intestinal tissues were collected from embryos for immunohistochemical analyses. We investigated regulation of the EDNRB gene in transactivation and chromatin immunoprecipitation assays; results were validated in functional rescue experiments using transgenes expression in EPCs from retroviral vectors. RESULTS: Zeb2Δ/+ EPCs had increased neuronal differentiation compared to Zeb2+/+ cells. When exposed to EDN3, Zeb2+/+ EPCs continued expression of ZEB2 but did not undergo any neuronal differentiation. Incubation of Zeb2Δ/+ EPCs with EDN3, on the other hand, resulted in only partial inhibition of neuronal differentiation. This indicated that 2 copies of Zeb2 are required for EDN3 to prevent neuronal differentiation. Mice with combined mutations in Zeb2 and Edn3 (double mutants) had more severe enteric anomalies and increased neuronal differentiation compared to mice with mutations in either gene alone. The transcription factors SOX10 and ZEB2 directly activated the EDNRB promoter. Overexpression of EDNRB in Zeb2Δ/+ EPCs restored inhibition of neuronal differentiation, similar to incubation of Zeb2+/+ EPCs with EDN3. CONCLUSIONS: In studies of cultured EPCs and mice, we found that control of differentiation of mouse enteric nervous system progenitor cells by EDN3 requires regulation of Ednrb expression by SOX10 and ZEB2.

EDNRB mutations cause Waardenburg syndrome type II in the heterozygous state.

Waardenburg syndrome (WS) is a genetic disorder characterized by sensorineural hearing loss and pigmentation anomalies. The clinical definition of four WS types is based on additional features due to defects in structures mostly arising from the neural crest, with type I and type II being the most frequent. While type I is tightly associated to PAX3 mutations, WS type II (WS2) remains partly enigmatic with mutations in known genes (MITF, SOX10) accounting for only 30% of the cases. We performed exome sequencing in a WS2 index case and identified a heterozygous missense variation in EDNRB. Interestingly, homozygous (and very rare heterozygous) EDNRB mutations are already described in type IV WS (i.e., in association with Hirschsprung disease [HD]) and heterozygous mutations in isolated HD. Screening of a WS2 cohort led to the identification of an overall of six heterozygous EDNRB variations. Clinical phenotypes, pedigrees and molecular segregation investigations unraveled a dominant mode of inheritance with incomplete penetrance. In parallel, cellular and functional studies showed that each of the mutations impairs the subcellular localization of the receptor or induces a defective downstream signaling pathway. Based on our results, we now estimate EDNRB mutations to be responsible for 5%-6% of WS2.

Subnuclear re-localization of SOX10 and p54NRB correlates with a unique neurological phenotype associated with SOX10 missense mutations.

SOX10 is a transcription factor with well-known functions in neural crest and oligodendrocyte development. Mutations in SOX10 were first associated with Waardenburg-Hirschsprung disease (WS4; deafness, pigmentation defects and intestinal aganglionosis). However, variable phenotypes that extend beyond the WS4 definition are now reported. The neurological phenotypes associated with some truncating mutations are suggested to be the result of escape from the nonsense-mediated mRNA decay pathway; but, to date, no mechanism has been suggested for missense mutations, of which approximately 20 have now been reported, with about half of the latter shown to be redistributed to nuclear bodies of undetermined nature and function in vitro. Here, we report that p54NRB, which plays a crucial role in the regulation of gene expression during many cellular processes including differentiation, interacts synergistically with SOX10 to regulate several target genes. Interestingly, this paraspeckle protein, as well as two other members of the Drosophila behavior human splicing (DBHS) protein family, co-localize with SOX10 mutants in nuclear bodies, suggesting the possible paraspeckle nature of these foci or re-localization of the DBHS members to other subnuclear compartments. Remarkably, the co-transfection of wild-type and mutant SOX10 constructs led to the sequestration of wild-type protein in mutant-induced foci. In contrast to mutants presenting with additional cytoplasmic re-localization, those exclusively found in the nucleus alter synergistic activity between SOX10 and p54NRB. We propose that such a dominant negative effect may contribute to or be at the origin of the unique progressive and severe neurological phenotype observed in affected patients.

Unilateral Sensorineural Hearing Loss: Medical Context and Etiology.

OBJECTIVE: Unilateral sensorineural hearing loss (USNHL) is known to impact on school performance and social skills during childhood, but the etiologies remain unclear. The aim of this study was to assess various etiologies and to study the clinical contexts in this population. METHODS: The study is a retrospective review. Characteristics of hearing loss (HL), audiometric parameters, imaging, and genetic and medical contexts were analyzed. RESULTS: Eighty children were included. USNHL was profound in 68%, could be progressive in 19%, and become bilateral in 7.5% of cases. Inner ear malformations were identified in 41% of cases; cochlear nerve deficiency (CND) was frequent (33%). Cytomegalovirus (CMV) infection and genetic syndromes were confirmed in 10 and 6% of cases, respectively. CONCLUSION: Long-term hearing follow-up remains useful in USNHL as it can become bilateral. Looking to etiology, MRI should be the gold standard, as CND is frequently observed and screening for CMV infection should be systematic. Genetic etiologies appear to be different compared to bilateral HL. Further genetic research in this domain is needed.

Loss-of-function mutations in SOX10 cause Kallmann syndrome with deafness.

Transcription factor SOX10 plays a role in the maintenance of progenitor cell multipotency, lineage specification, and cell differentiation and is a major actor in the development of the neural crest. It has been implicated in Waardenburg syndrome (WS), a rare disorder characterized by the association between pigmentation abnormalities and deafness, but SOX10 mutations cause a variable phenotype that spreads over the initial limits of the syndrome definition. On the basis of recent findings of olfactory-bulb agenesis in WS individuals, we suspected SOX10 was also involved in Kallmann syndrome (KS). KS is defined by the association between anosmia and hypogonadotropic hypogonadism due to incomplete migration of neuroendocrine gonadotropin-releasing hormone (GnRH) cells along the olfactory, vomeronasal, and terminal nerves. Mutations in any of the nine genes identified to date account for only 30% of the KS cases. KS can be either isolated or associated with a variety of other symptoms, including deafness. This study reports SOX10 loss-of-function mutations in approximately one-third of KS individuals with deafness, indicating a substantial involvement in this clinical condition. Study of SOX10-null mutant mice revealed a developmental role of SOX10 in a subpopulation of glial cells called olfactory ensheathing cells. These mice indeed showed an almost complete absence of these cells along the olfactory nerve pathway, as well as defasciculation and misrouting of the nerve fibers, impaired migration of GnRH cells, and disorganization of the olfactory nerve layer of the olfactory bulbs.

MITF mutations associated with pigment deficiency syndromes and melanoma have different effects on protein function.

The basic-helix-loop-helix-leucine zipper (bHLHZip) protein MITF (microphthalmia-associated transcription factor) is a master regulator of melanocyte development. Mutations in the MITF have been found in patients with the dominantly inherited hypopigmentation and deafness syndromes Waardenburg syndrome type 2A (WS2A) and Tietz syndrome (TS). Additionally, both somatic and germline mutations have been found in MITF in melanoma patients. Here, we characterize the DNA-binding and transcription activation properties of 24 MITF mutations found in WS2A, TS and melanoma patients. We show that most of the WS2A and TS mutations fail to bind DNA and activate expression from melanocyte-specific promoters. Some of the mutations, especially R203K and S298P, exhibit normal activity and may represent neutral variants. Mutations found in melanomas showed normal DNA-binding and minor variations in transcription activation properties; some showed increased potential to form colonies. Our results provide molecular insights into how mutations in a single gene can lead to such different phenotypes.

An impairment of long distance SOX10 regulatory elements underlies isolated Hirschsprung disease.

A deletion encompassing several SOX10 enhancers was recently identified in a patient presenting with Waardenburg syndrome type 4 (WS4), which is defined as a combination of Hirschsprung disease (HSCR, intestinal aganglionosis) and WS (deafness and pigmentation defects). The expression patterns of some of the known SOX10 enhancers in animal models led to the speculation that endophenotypes of WS4 may be linked to mutations within some of these sequences. The present study investigated deletions and point mutations within four SOX10 enhancers in 144 unexplained isolated HSCR cases. One deletion and two point mutations affecting binding sites for known neural crest transcription factors were identified. In vitro functional analysis revealed that the first point mutation disrupts autoregulation by SOX10, whereas the second affects AP2a and SOX10 synergistic activity. The present findings suggest that the mutations within SOX10 enhancers contribute to isolated HSCR.

Sox10 and Itgb1 interaction in enteric neural crest cell migration.

SOX10 involvement in syndromic form of Hirschsprung disease (intestinal aganglionosis, HSCR) in humans as well as developmental defects in animal models highlight the importance of this transcription factor in control of the pool of enteric progenitors and their differentiation. Here, we characterized the role of SOX10 in cell migration and its interactions with ß1-integrins. To this end, we crossed the Sox10(lacZ/+) mice with the conditional Ht-PA::Cre; beta1(neo/+) and beta1(fl/fl) mice and compared the phenotype of embryos of different genotypes during enteric nervous system (ENS) development. The Sox10(lacZ/+); Ht-PA::Cre; beta1(neo/fl) double mutant embryos presented with increased intestinal aganglionosis length and more severe neuronal network disorganization compared to single mutants. These defects, detected by E11.5, are not compensated after birth, showing that a coordinated and balanced interaction between these two genes is required for normal ENS development. Use of video-microscopy revealed that defects observed result from reduced migration speed and altered directionality of enteric neural crest cells. Expression of ß1-integrins upon SOX10 overexpression or in Sox10(lacZ/+) mice was also analyzed. The modulation of SOX10 expression altered ß1-integrins, suggesting that SOX10 levels are critical for proper expression and function of this adhesion molecule. Together with previous studies, our results strongly indicate that SOX10 mediates ENCC adhesion and migration, and contribute to the understanding of the molecular and cellular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 mutations.

Phenotypic similarities and differences in patients with a p.Met112Ile mutation in SOX10.

Waardenburg syndrome (WS) is characterized by an association of pigmentation abnormalities and sensorineural hearing loss. Four types, defined on clinical grounds, have been delineated, but this phenotypic classification correlates imperfectly with known molecular anomalies. SOX10 mutations have been found in patients with type II and type IV WS (i.e., with Hirschsprung disease), more complex syndromes, and partial forms of the disease. The phenotype induced by SOX10 mutations is highly variable and, except for the neurological forms of the disease, no genotype-phenotype correlation has been characterized to date. There is no mutation hotspot in SOX10 and most cases are sporadic, making it particularly difficult to correlate the phenotypic and genetic variability. This study reports on three independent families with SOX10 mutations predicted to result in the same missense mutation at the protein level (p.Met112Ile), offering a rare opportunity to improve our understanding of the mechanisms underlying phenotypic variability. The pigmentation defects of these patients are very similar, and the neurological symptoms showed a somewhat similar evolution over time, indicating a potential partial genotype-phenotype correlation. However, variability in gastrointestinal symptoms suggests that other genetic factors contribute to the expression of these phenotypes. No correlation between the rs2435357 polymorphism of RET and the expression of Hirschsprung disease was found. In addition, one of the patients has esophageal achalasia, which has rarely been described in WS.

Two induced pluripotent stem cell (iPSC) lines derived from patients affected by Waardenburg syndrome type 1 retain potential to activate neural crest markers.

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.

Mandibulofacial dysostosis with alopecia results from ETAR gain-of-function mutations via allosteric effects on ligand binding.

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.

Waardenburg Syndrome: The Contribution of Next-Generation Sequencing to the Identification of Novel Causative Variants.

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.