Cleft Palate Gene Research Articles

Genes and microRNAs associated with mouse cleft palate: A systematic review and bioinformatics analysis

Cleft palate (CP) is the most prevalent craniofacial deformity, with ethnic and geographic variation in prevalence in humans. Mice have been used as an animal model to study the cause(s) of CP by several approaches, including genetic and chemical-induced approaches. Mouse genetic approaches revealed that significant amounts of genes are involved in the CP pathology. The aim of this study was to identify common features of CP-associated genes and to explore the roles of microRNAs (miRNAs) as important post-transcriptional regulators that may be involved in the regulation of CP genes. To generate an accurate list of genes associated with CP, we first conducted systematic literature searches through main databases such as Medline, Embase, and PubMed, as well as other sources such as Scopus and Mouse Genome Informatics. We found that 195 mouse strains with single-gene mutations and 140 mouse strains with compound-gene mutations were reported to have CP. The CP genes were categorized by functions and pathways using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology annotations, highlighting the contribution of cellular metabolism to CP. A total of 18 miRNAs were involved in the regulation of multiple CP genes. Human genotype-phenotype analysis revealed that variants in five human homologous CP genes (IRF6, FOXE1, VAX1, WNT9B, and GAD1) significantly contributed to the human CP phenotype. Thus, our results suggest that cellular metabolism and miRNAs play an important role in the regulation of genetic pathways and networks crucial for palatal formation.

Shared molecular networks in orofacial and neural tube development.

Single genetic variants can affect multiple tissues during development. Thus it is possible that disruption of shared gene regulatory networks might underlie syndromic presentations. In this study, we explore this idea through examination of two critical developmental programs that control orofacial and neural tube development and identify shared regulatory factors and networks. Identification of these networks has the potential to yield additional candidate genes for poorly understood developmental disorders and assist in modeling and perhaps managing risk factors to prevent morbidly and mortality. We reviewed the literature to identify genes common between orofacial and neural tube defects and development. We then conducted a bioinformatic analysis to identify shared molecular targets and pathways in the development of these tissues. Finally, we examine publicly available RNA-Seq data to identify which of these genes are expressed in both tissues during development. We identify common regulatory factors in orofacial and neural tube development. Pathway enrichment analysis shows that folate, cancer and hedgehog signaling pathways are shared in neural tube and orofacial development. Developing neural tissues differentially express mouse exencephaly and cleft palate genes, whereas developing orofacial tissues were enriched for both clefting and neural tube defect genes. These data suggest that key developmental factors and pathways are shared between orofacial and neural tube defects. We conclude that it might be most beneficial to focus on common regulatory factors and pathways to better understand pathology and develop preventative measures for these birth defects. Birth Defects Research 109:169-179, 2017. © 2016 Wiley Periodicals, Inc.

Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFβ3-knockout mice alleles: RNA-Seq analysis of TGFβ3 Mice

BackgroundIn humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFβ3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFβ3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFβ3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFβ3 knockout mice to identify TGFβ3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFβ3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5).ResultsThe overall transcriptome analysis of TGFβ3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFβ3+/− and TGFβ3−/− were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFβ3−/− mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFβ3−/− mice. When the significantly altered CP genes were overlaid with TGFβ signaling, all of these genes followed the Smad-dependent pathway.ConclusionsOur study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFβ3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGFβ3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.

The Mn1 transcription factor acts upstream ofTbx22and preferentially regulates posterior palate growth in mice

The mammalian secondary palate exhibits morphological, pathological and molecular heterogeneity along the anteroposterior axis. Although the cell proliferation rates are similar in the anterior and posterior regions during palatal outgrowth, previous studies have identified several signaling pathways and transcription factors that specifically regulate the growth of the anterior palate. By contrast, no factor has been shown to preferentially regulate posterior palatal growth. Here, we show that mice lacking the transcription factor Mn1 have defects in posterior but not anterior palatal growth. We show that Mn1 mRNA exhibits differential expression along the anteroposterior axis of the developing secondary palate, with preferential expression in the middle and posterior regions during palatal outgrowth. Extensive analyses of palatal gene expression in wild-type and Mn1(-/-) mutant mice identified Tbx22, the mouse homolog of the human X-linked cleft palate gene, as a putative downstream target of Mn1 transcriptional activation. Tbx22 exhibits a similar pattern of expression with that of Mn1 along the anteroposterior axis of the developing palatal shelves and its expression is specifically downregulated in Mn1(-/-) mutants. Moreover, we show that Mn1 activated reporter gene expression driven by either the human or mouse Tbx22 gene promoters in co-transfected NIH3T3 cells. Overexpression of Mn1 in NIH3T3 cells also increased endogenous Tbx22 mRNA expression in a dose-dependent manner. These data indicate that Mn1 and Tbx22 function in a novel molecular pathway regulating mammalian palate development.

Taking it to the max: the genetic and developmental mechanisms coordinating midfacial morphogenesis and dysmorphology.

The rapid proliferative expansion and complex morphogenetic events that coordinate the development of the face underpin the sensitivity of this structure to genetic and environmental insult and provide an explanation for the high incidence of midfacial malformation. Most notable of these malformations is cleft lip with or without cleft palate (CLP) that, with an incidence of between one in 600 and one in 1000 live births, is the fourth most common congenital disorder in humans. Despite the obvious global impact of the disorder and some recent progress in identifying causative genes for some prominent syndromal forms, our knowledge of the key genetic factors contributing to the more common isolated cases of CLP is still remarkably patchy. The current understanding of the molecular and cellular processes that orchestrate morphogenesis of the midface, with emphasis on events leading to fusion of the lip and primary palate, is detailed in this review. The roles of crucial factors identified from relevant animal model systems, including BMP4 and SHH, and the likely events perturbed by key genes pinpointed in human studies [such as PVRL1, IRF6p63, MID1, MSX1, and PTCH1] are discussed in this light. New candidates for human CLP genes are also proposed.

Isolation and developmental expression analysis of Tbx22, the mouse homolog of the human X-linked cleft palate gene.

Mutations in the TBX22 gene have been identified recently in patients with the X-linked cleft palate and ankyloglossia syndrome, suggesting that the TBX22 transcription factor plays an important role in palate development. However, because ankyloglossia has been reported in the majority of patients with TBX22 mutations, it has been speculated that the cleft palate phenotype is secondary to defective fetal tongue movement. To understand the role of TBX22 in disease pathogenesis and in normal development, it is necessary to carry out a detailed temporal and spatial gene expression analysis. We report here the isolation and developmental expression analysis of the mouse homolog Tbx22. The mouse Tbx22 gene encodes a putative protein of 517 amino acid residues, which shares 72% overall amino acid sequence identity with the human TBX22 protein. By using interspecific backcross analysis, we have localized the Tbx22 gene to mouse chromosome X, in a region syntenic to human chromosome Xq21, where the TBX22 gene resides, indicating that Tbx22 is the ortholog of human TBX22. Our in situ hybridization analysis shows that Tbx22 is expressed in a temporally and spatially highly restricted pattern during mouse palate and tongue development. Together with the mutant phenotypes in human patients, our data indicate a primary role for Tbx22 in both palate and tongue development.

Genetic Studies of a Novel Cleft Palate Gene

Genetic Studies of a Novel Cleft Palate Gene

Vitamin A-enhanced cleft palate susceptibility gene maps between C4 and B144 within the H-2 complex.

Pregnant mice congenic with C57BL/10 in the region of H-2, the major histocompatibility complex on Chromosome 17 (B10.BR, B10.A, B10.A(1R), B10.A(2R), B10.A(15R), B10.A(18R), B10.OL, or crosses between them) were fed Purina Laboratory Chow or the same diet plus 400 IU of vitamin A daily from conception and given dexamethasone (80 or 160 mg/kg) intraperitoneally on the twelfth day of pregnancy. It was found that the added vitamin A increased the frequency of isolated cleft palate only in strains that had b alleles between C4 and B144. The enhancement of susceptibility to glucocorticosteroid-induced cleft palate by vitamin A appears to be a recessive trait and the locus, called Acp, maps centromeric to another corticosteroid-induced cleft palate gene (Dcp-2) that also is in the C4:B144 interval.

Vitamin A-enhanced cleft palate susceptibility associated with H-2.

Pregnant mice from the congenic strains C57BL/10Sn, B10.BR, B10.A/SgSn, B10.A(5R)/SgSn, B10.A(2R)SgSn and B10.A(18R)Sg were fed Purina Laboratory Chow or the same diet plus approximately 400 IU vitamin A daily and given 80 mg/kg dexamethasone intra-peritoneally or a sham injection on the 12th day of pregnancy. It was found that only strains with b alleles between H-2S and H-2D had significantly higher frequencies of isolated cleft palate among their progeny when fed the supplemental vitamin A. The locus appears to be on the centromeric side of a dexamethasone-induced cleft palate gene which has been mapped to the same general area.

Linkage of an X-chromosome cleft palate gene.

Many congenital malformations, such as cleft palate and neural tube defects, have a multifactorial origin involving both environmental and genetic factors. Conditions such as these may be exclusively monogenic, polygenic or environmental, but in most cases both genetic and environmental factors are involved. This study describes the sub-chromosomal localization of a single gene defect causing cleft palate and ankyloglossia (tongue-tied) in a large Icelandic family. This defect is a model for the analysis of other neural-crest malformations that show a more complex multifactorial inheritance pattern.

Glucocorticoid-induced cleft palate genes in chromosome 17: genetic linkage and mapping analyses.

Genes that influence susceptibility to dexamethasone-induced cleft palate and tentatively designated Dep are linked to the major histocompatibility complex H-2 in chromosome 17 of the mouse. Experiments presented refine the map of genes. The results show two or three Dep loci. The two-locus model maps Dep genes to the class II gene E beta and to the chromosomal region between the S and D genes. The three-locus model maps the Dep genes to the chromosomal regions from the centromere to E beta, from E beta to S, and from D to Pgk-2. Experiments were done by comparing the dexamethasone-induced cleft palate dose response of congenic strains with H-2 haplotypes that are recombinants of H-2a and H-2b. The analysis of genetic linkage between H-2 and Dep was expanded to include reciprocal backcrosses. A maternal factor was found to influence the frequency of dexamethasone-induced cleft palate in the backcross fetuses. The factor's origin is associated with the H-2 haplotype of the outcross mother, so the effect is actually a "grandmother effect" that probably is transmitted horizontally. Finally, the sexes were distributed unevenly between the fetuses with cleft palate in two of the congenic strains. This suggests interaction between the H-2-linked Dep genes and a Dep sex-associated gene that modulates susceptibility to dexamethasone-induced cleft palate.