Mycn deficiency underlies the development of orofacial clefts in mice and humans.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common subphenotype of non-syndromic orofacial clefts arising from genetic and/or environmental perturbations during embryonic development. We previously identified 2p24.2 as a risk locus associated with NSCL/P in the Chinese Han population, and MYCN is a candidate risk gene in this region. To understand the potential function of MYCN in craniofacial development, we generated Wnt1-Cre;Mycnflox/flox mice that exhibited cleft palate, microglossia and micrognathia, resembling the Pierre Robin sequence (PRS) in humans. Further analyses indicated that the cleft palate was secondary to the delayed elevation of palatal shelves caused by micrognathia. The micrognathia resulted from impaired chondrogenic differentiation in Merkel's cartilage, which limited tongue development, leading to microglossia. In terms of mechanism, Mycn deficiency in cranial neural crest cells (CNCCs) downregulated Sox9 expression by inhibiting Wnt5a in a CNCC-derived chondrogenic lineage in Merkel's cartilage. To investigate whether MYCN deficiency contributed to NSCL/P, we performed direct sequencing targeting all exons and exon-intron boundaries of MYCN in 104 multiplex families with Mendelian NSCL/P and identified a novel pathogenic variant in MYCN. Taken together, our data indicate that ablation of Mycn in mouse CNCCs could resemble PRS by suppressing the Wnt5a-Sox9 signaling pathway in Merkel's cartilage and that mutations in MYCN may be novel potential causes of NSCL/P.

Knockout of myomaker results in defective myoblast fusion, reduced muscle growth and increased adipocyte infiltration in zebrafish skeletal muscle.

The fusion of myoblasts into multinucleated muscle fibers is vital to skeletal muscle development, maintenance and regeneration. Genetic mutations in the Myomaker (mymk) gene cause Carey-Fineman-Ziter syndrome (CFZS) in human populations. To study the regulation of mymk gene expression and function, we generated three mymk mutant alleles in zebrafish using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and analyzed the effects of mymk knockout on muscle development and growth. Our studies demonstrated that knockout of mymk resulted in defective myoblast fusion in zebrafish embryos and increased mortality at larval stage around 35-45 days post-fertilization. The viable homozygous mutants were smaller in size and weighed approximately one-third the weight of the wild type (WT) sibling at 3 months old. The homozygous mutants showed craniofacial deformities, resembling the facial defect observed in human populations with CFZS. Histological analysis revealed that skeletal muscles of mymk mutants contained mainly small-size fibers and substantial intramuscular adipocyte infiltration. Single fiber analysis revealed that myofibers in mymk mutant were predominantly single-nucleated fibers. However, myofibers with multiple myonuclei were observed, although the number of nuclei per fiber was much less compared with that in WT fibers. Overexpression of sonic Hedgehog inhibited mymk expression in zebrafish embryos and blocked myoblast fusion. Collectively, these studies demonstrated that mymk is essential for myoblast fusion during muscle development and growth.

RBM10: Harmful or helpful-many factors to consider.

RBM10 is an RNA binding motif (RBM) protein expressed in most, if not all, human and animal cells. Interest in RBM10 is rapidly increasing and its clinical importance is highlighted by its identification as the causative agent of TARP syndrome, a developmental condition that significantly impacts affected children. RBM10's cellular functions are beginning to be explored, with initial studies demonstrating a tumor suppressor role. Very recently, however, contradictory results have emerged, suggesting a tumor promoter role for RBM10. In this review, we describe the current state of knowledge on RBM10, and address this dichotomy in RBM10 function. Furthermore, we discuss what may be regulating RBM10 function, particularly the importance of RBM10 alternative splicing, and the relationship between RBM10 and its paralogue, RBM5. As RBM10-related work is gaining momentum, it is critical that the various aspects of RBM10 molecular biology revealed by recent studies be considered moving forward. It is only if these recent advances in RBM10 structure and function are considered that a clearer insight into RBM10 function, and the disease states with which RBM10 mutation is associated, will be gained.

A defect in myoblast fusion underlies Carey-Fineman-Ziter syndrome.

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.

Ablation of the Sox11 Gene Results in Clefting of the Secondary Palate Resembling the Pierre Robin Sequence.

Mouse gene inactivation has shown that the transcription factor Sox11 is required for mouse palatogenesis. However, whether Sox11 is primarily involved in the regulation of palatogenesis still remains elusive. In this study, we explored the role ofSox11in palatogenesis by analyzing the developmental mechanism in cleft palate formation in mutants deficient in Sox11. Sox11 is expressed both in the developing palatal shelf and in the surrounding structures, including the mandible. We found that cleft palate occurs only in the mutant in which Sox11is directly deleted. As in the wild type, the palatal shelves in the Sox11 mutant undergo outgrowth in a downward direction and exhibit potential for fusion and elevation. However, mutant palatal shelves encounter clefting, which is associated with a malpositioned tongue that results in physical obstruction of palatal shelf elevation at embryonic day 14.5 (E14.5). We found that loss of Sox11led to reduced cell proliferation in the developing mandibular mesenchyme via Cyclin D1, leading to mandibular hypoplasia, which blocks tongue descent. Extensive analyses of gene expression inSox11 deficiency identified FGF9 as a potential candidate target of Sox11 in the modulation of cell proliferation both in the mandible and the palatal shelf between E12.5 and E13.5. Finally we show, using in vitro assays, that Sox11 directly regulates the expression of Fgf9 and that application of FGF9 protein to Sox11-deficient palatal shelves restores the rate of BrdU incorporation. Taken together, the palate defects presented in the Sox11 loss mutant mimic the clefting in the Pierre Robin sequence in humans.

A newborn with Pierre Robin sequence after preconceptional mitoxantrone exposure of a female with multiple sclerosis.

Multiple sclerosis is a common disease of young adults in which accidental and unplanned pregnancies under disease modifying or immunosuppressive therapies may occur. The experience with mitoxantrone (MIX) especially in the first trimenon is very limited, until now only one case of a pregnant woman with MS who was exposed to MIX in early pregnancy and delivered a growth restricted but healthy child was published. We report a case of a secondary progressive MS patient who was exposed periconceptionally to MIX and delivered a child with Pierre Robin Sequence (PRS), a syndrome with the main features of glossoptosis, micrognathia, and palate clefts. PRS is a very rare defect and therefore a causal relation with MIX seems possible.

The Pierre Robin syndrome reassessed in the light of recent research.

For many years clinicians have known the coincidental presentation of micrognathia, glossoptosis and cleft palate as the Pierre Robin syndrome. In conferences in 1974 and 1975 the term "Anomalad" was introduced which by definition is a primary malformation with superimposed secondary structural changes and the Pierre Robin syndrome became known as the Robin Anomalad. The concept was based on experimental observations available at that time. However, since that date further studies have demonstrated that administration of drugs to pregnant female rodents can produce coincidental failure of normal development of both mandible and palate. In the light of this work a critical review is made of the evidence upon which the mechanistic view of the condition evolved and an alternative hypothesis developed. From animal experimentation it can be argued that the nature of the condition is not mechanical and is more likely to be metabolic. Indeed, confirmatory evidence in man has recently been presented from Finland. If this is the case it may be erroneous to consider the Robin malformations as an "Anomalad" and mandibular maxillary agenesis would probably be a more accurate term.