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.

ADAR1 mediated regulation of neural crest derived melanocytes and Schwann cell development.

The neural crest gives rise to numerous cell types, dysfunction of which contributes to many disorders. Here, we report that adenosine deaminase acting on RNA (ADAR1), responsible for adenosine-to-inosine editing of RNA, is required for regulating the development of two neural crest derivatives: melanocytes and Schwann cells. Neural crest specific conditional deletion of Adar1 in mice leads to global depigmentation and absence of myelin from peripheral nerves, resulting from alterations in melanocyte survival and differentiation of Schwann cells, respectively. Upregulation of interferon stimulated genes precedes these defects, which are associated with the triggering of a signature resembling response to injury in peripheral nerves. Simultaneous extinction of MDA5, a key sensor of unedited RNA, rescues both melanocytes and myelin defects in vitro, suggesting that ADAR1 safeguards neural crest derivatives from aberrant MDA5-mediated interferon production. We thus extend the landscape of ADAR1 function to the fields of neural crest development and disease.

LKB1 specifies neural crest cell fates through pyruvate-alanine cycling.

Metabolic processes underlying the development of the neural crest, an embryonic population of multipotent migratory cells, are poorly understood. Here, we report that conditional ablation of the Lkb1 tumor suppressor kinase in mouse neural crest stem cells led to intestinal pseudo-obstruction and hind limb paralysis. This phenotype originated from a postnatal degeneration of the enteric nervous ganglia and from a defective differentiation of Schwann cells. Metabolomic profiling revealed that pyruvate-alanine conversion is enhanced in the absence of Lkb1. Mechanistically, inhibition of alanine transaminases restored glial differentiation in an mTOR-dependent manner, while increased alanine level directly inhibited the glial commitment of neural crest cells. Treatment with the metabolic modulator AICAR suppressed mTOR signaling and prevented Schwann cell and enteric defects of Lkb1 mutant mice. These data uncover a link between pyruvate-alanine cycling and the specification of glial cell fate with potential implications in the understanding of the molecular pathogenesis of neural crest diseases.

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.

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.

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.

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.

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.

Gene expression profiling identifies Hes6 as a transcriptional target of ATOH1 in cochlear hair cells.

ATOH1 is a basic Helix-Loop-Helix transcription factor crucial for hair cell (HC) differentiation in the inner ear. In order to identify ATOH1 target genes, we performed a genome-wide expression profiling analysis in cells expressing ATOH1 under the control of a tetracycline-off system and found that HES6 expression is induced by ATOH1. We performed in situ hybridisation and showed that the rise and fall of Hes6 expression closely follow that of Atoh1 in cochlear HC. Moreover, electrophoretic mobility shift assays and luciferase assays show that ATOH1 activates HES6 transcription through binding to three clustered E boxes of its promoter.