Association of low‐frequency genetic variants in regulatory regions with nonsyndromic orofacial clefts

Genome‐wide scans have shown that common risk alleles for orofacial clefts (OFC) tend to be located in noncoding regulatory elements and cumulatively explain only part of the heritability of OFCs. Low‐frequency variants may account for some of the “missing” heritability. Therefore, we scanned low‐frequency variants located within putative craniofacial enhancers to identify novel OFC risk variants and implicate new regulatory elements in OFC pathogenesis. Analyses were performed in a multiethnic sample of 1,995 cases of cleft lip with or without cleft palate (CL/P), 221 cases with cleft palate (CP) only, and 1,576 unaffected controls. One hundred and nineteen putative craniofacial enhancers identified from ChIP‐Seq studies in craniofacial tissues or cell lines contained multiple low‐frequency (0.01–1%) variants, which we genotyped in participants using a custom Illumina panel. Two complementary statistical approaches, sequence kernel association test and combined multivariate and collapsing, were used to test association of the aggregated low‐frequency variants across each enhancer region with CL/P and CP. We discovered a significant association between CP and a branchial arch enhancer near FOXP1 (mm60; p‐value = .0002). Additionally, we observed a suggestive association between CL/P and a forebrain enhancer near FOXE1 (hs1717; p‐value = .001). These findings suggest that low‐frequency variants in craniofacial enhancer regions contribute to the complex etiology of nonsyndromic OFCs.     

Expanding the phenotypic spectrum of TP63‐related disorders including the first set of monozygotic twins

Individuals with Tumor Protein P63 (TP63)‐related disorders are known to present with a range of phenotypic features, including ectrodactyly, ectodermal dysplasia, cleft lip/palate, Rapp‐Hodgkin, Hay–Wells, and limb‐mammary syndromes. We present six individuals from three families, including a set of monozygotic twins, with pathogenic TP63 variants who had novel clinical findings. The twins were discordant for cleft lip and palate, and the type of hand malformations, but concordant for choanal atresia, and bilateral volar nail. Both failed newborn screening for severe combined immunodeficiency (SCID) due to T‐cell lymphopenia. The second family included three family members across two generations. Two of these three family members had orofacial clefting, but the remaining child had a laryngeal web and hydrocele with no clefting or hand anomalies, highlighting the variable expressivity in TP63‐related disorders. The individual from the third family had unilateral cleft lip and palate, hydronephrosis, and bilateral volar nails. Together, these cases illustrate that: there is significant familial variability, including discordant major but concordant minor anomalies in the first ever reported set of molecularly confirmed monozygotic twins with pathogenic variants in TP63; pathogenic variants in TP63 should be considered in individuals with volar nail, which was previously only strongly associated with 4q34 deletion syndrome; and failed SCID newborn screening due to abnormal immune functioning may be part of the phenotypic spectrum of TP63‐related disorders, as it was reported in one prior individual and two of the individuals in our case series.     

Embryonic expression of EphA receptor genes in mice supports their candidacy for involvement in cleft lip and palate

Background: Eph receptors, comprising the A‐ and B‐subfamilies, are the largest family of receptor tyrosine kinases in the mammalian genome, and their function is critical for morphogenesis in a variety of contexts. Whereas signaling through B‐type Ephs has been demonstrated to play a role in cleft lip and palate (CL/P), the involvement of A‐type Ephs has not been examined in this context notwithstanding a recent genome‐wide association study that identified the EPHA3 locus as a candidate for non‐syndromic CL/P. Results: Here, we present a systematic analysis of the gene expression patterns for the nine EphA receptors at progressive stages of mouse development and find that EphA3, EphA4, and EphA7 exhibit restricted overlapping patterns of expression during palate development. We find that homozygous mutation of EphA3 or compound homozygous mutation of EphA3 and EphA4 in mice does not result in defective midfacial development, supporting the possibility of redundant function with EphA7. We also document previously undescribed expression patterns in other tissues of the craniofacial complex including the lacrimal duct and salivary glands. Conclusions: Together, these results are consistent with the hypothesis that mutations in EPHA family genes may cause CL/P and also suggest that functional redundancy between family members may be at play. Developmental Dynamics 243:1470–1476, 2014. © 2014 Wiley Periodicals, Inc.     

Expression and association data strongly support JARID2 involvement in nonsyndromic cleft lip with or without cleft palate

Nonsyndromic cleft lip with or without cleft palate (CL/P) affects approximately 1 in 1,000 births. Genetic studies have provided evidence for the role of several genes and candidate loci in clefting; however, conflicting results have frequently been obtained and much have to be done to unravel the complex genetics of CL/P. In the present investigation we have focused on the candidate region in 6p23, a region that have been found linked to CL/P in several investigations, in the attempt to find out the susceptibility gene provisionally named OFC1. Gene expression experiments in mice embryo of positional candidate genes revealed that JARID2 was highly and specifically expressed in epithelial cells in merging palatal shelves. A family‐based linkage disequilibrium study confirmed the pivotal role of JARID2 in orofacial development and strongly supports a role for this gene in CL/P etiology (multiallelic haplotype test P=6×10^?5). Understanding the molecular role of JARID2 within facial development may offer additional information to further unravel the complex genetics of CL/P. Hum Mutat 31:1–7, 2010. © 2010 Wiley‐Liss, Inc.     

The chromatin remodeling protein CHD7, mutated in CHARGE syndrome,is necessary for proper craniofacial and tracheal development

Background: Heterozygous mutations in the chromatin remodeling gene CHD7 cause CHARGE syndrome, a developmental disorder with variable craniofacial dysmorphisms and respiratory difficulties. The molecular etiologies of these malformations are not well understood. Homozygous Chd7 null mice die by E11, whereas Chd7^Gt/+ heterozygous null mice are a viable and excellent model of CHARGE. We explored skeletal phenotypes in Chd7^Gt/+ and Chd7 conditional knockout mice, using Foxg1‐Cre to delete Chd7 (Foxg1‐CKO) in the developing eye, ear, nose, pharyngeal pouch, forebrain, and gut and Wnt1‐Cre (Wnt1‐CKO) to delete Chd7 in migrating neural crest cells. Results: Foxg1‐CKO mice exhibited postnatal respiratory distress and death, dysplasia of the eye, concha, and frontal bone, hypoplastic maxillary shelves and nasal epithelia, and reduced tracheal rings. Wnt1‐CKO mice exhibited frontal and occipital bone dysplasia, hypoplasia of the maxillary shelves and mandible, and cleft palate. In contrast, heterozygous Chd7^Gt/+ mice had apparently normal skeletal development. Conclusions: Conditional deletion of Chd7 in ectodermal and endodermal derivatives (Foxg1‐Cre) or migrating neural crest cells (Wnt1‐Cre) results in varied and more severe craniofacial defects than in Chd7^Gt/+ mice. These studies indicate that CHD7 has an important, dosage‐dependent role in development of several different craniofacial tissues. Developmental Dynamics 243:1055–1066, 2014. © 2014 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Genetic evidence for the role of loci at 19q13 in cleft lip and palate

Background

Clefts of the lip and palate are common birth defects, affecting approximately 1 in 700 births worldwide. The aetiology of clefting is complex, with multiple genetic and environmental influences.

Methods

Genotype based linkage disequilibrium analysis was conducted using the family based association test (FBAT) and the likelihood ratio test (LRT). We also carried out direct sequencing of the PVR and PVRL2 candidate genes based on their homology to PVRL1, a gene shown previously to cause Margarita Island clefting. Participants included 434 patients with cleft lip with or without cleft palate or cleft palate only and their mothers from eight countries in South America, 205 nuclear triads (father‐mother‐affected child) from Iowa, 541 nuclear triads from Denmark, and 100 patients with cleft lip and palate from the Philippines.

Results

An allelic variant in the PVR gene showed statistically significant association with both South American and Iowa populations (p = 0.0007 and p = 0.0009, respectively). Direct sequencing of PVR and PVRL2 yielded 26 variants, including two rare amino acid changes, one in each gene, which were not seen in controls.

Conclusions

We found an association between a common variant in a gene at 19q and isolated clefting in two heterogeneous populations. However, it is unclear from our data if rare variants in PVR and PVRL2 are sufficient to cause clefting in isolation.     

Cellular and molecular mechanisms in the development of a cleft lip and/or cleft palate; insights from zebrafish (Danio rerio)

Human cleft lip and/or palate (CLP) are immediately recognizable congenital abnormalities of the face. Lip and palate develop from facial primordia through the coordinated activities of ectodermal epithelium and neural crest cells (NCCs) derived from ectomesenchyme tissue. Subtle changes in the regulatory mechanisms of NCC or ectodermal epithelial cells can result in CLP. Genetic and environmental contributions or a combination of both play a significant role in the progression of CLP. Model organisms provide us with a wealth of information in understanding the pathophysiology and genetic etiology of this complex disease. Small teleost, zebrafish (Danio rerio) is one of the popular model in craniofacial developmental biology. The short generation time and large number of optically transparent, easily manipulated embryos increase the value of zebrafish to identify novel candidate genes and gene regulatory networks underlying craniofacial development. In addition, it is widely used to identify the mechanisms of environmental teratogens and in therapeutic drug screening. Here, we discuss the value of zebrafish as a model to understand epithelial and NCC induced ectomesenchymal cell activities during early palate morphogenesis and robustness of the zebrafish in modern research on identifying the genetic and environmental etiological factors of CLP.     

The MTHFD1 Gene is not Involved in Cleft Lip with or Without Palate Onset Among the Italian Population

Nonsyndromic cleft lip with or without cleft palate (CL/P) is the most common orofacial malformation, having a non‐Mendelian and multifactorial aetiology. It has been shown that polymorphic variants of genes encoding key proteins of folate and methionine metabolism might be important maternal risk factors for having a child with these craniofacial anomalies. The aim of this study was to evaluate the role of two polymorphisms of the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene, the A1958G and the G401A variants, on the risk of CL/P in the Italian population. A1958G and G401A polymorphism genotyping of MTHFD1 was performed on 216 CL/P triads, (patient and parents), for this study by restriction endonuclease digestion of PCR products. Linkage disequilibrium between markers and disease was tested using both pairwise and haplotype analyses. In our case‐parents triad design no significant association between MTHFD1 and the disease is evident. Our data do not support MTHFD1 involvement in CL/P onset among the Italian population.     

Association between alleles of the transforming growth factor alpha locus and cleft lip and palate in the chilean population

Two RFLPs at the TGFA locus were studied in 39 unrelated Chilean (Caucasoid-Mongoloid) patients with non-syndromic cleft lip/palate [CL(P)] and 51 control individuals. A highly significant association between BamHI A2 allele and CL(P) was detected (χ² = 6.00; P = 0.014), while no association was found between TaqI RFLPs and clefting. No significant differences were found when comparing genotypes by type of cleft and a positive or negative family history of clefting. Our results seem to support rather definitively the association between TGFA and clefting but do not support the hypothesis that TGFA is a major causal gene of CL(P). © 1995 Wiley-Liss, Inc.     

Mapping cellular processes in the mesenchyme during palatal development in the absence of Tbx1 reveals complex proliferation changes and perturbed cell packing and polarity

The 22q11 deletion syndromes represent a spectrum of overlapping conditions including cardiac defects and craniofacial malformations. Amongst the craniofacial anomalies that are seen, cleft of the secondary palate is a common feature. Haploinsufficiency of TBX1 is believed to be a major contributor toward many of the developmental structural anomalies that occur in these syndromes, and targeted deletion of Tbx1 in the mouse reproduces many of these malformations, including cleft palate. However, the cellular basis of this defect is only poorly understood. Here, palatal development in the absence of Tbx1 has been analysed, focusing on cellular properties within the whole mesenchymal volume of the palatal shelves. Novel image analyses and data presentation tools were applied to quantify cell proliferation rates, including regions of elevated as well as reduced proliferation, and cell packing in the mesenchyme. Also, cell orientations (nucleus–Golgi axis) were mapped as a potential marker of directional cell movement. Proliferation differed only subtly between wild‐type and mutant until embryonic day (E)15.5 when proliferation in the mutant was significantly lower. Tbx1^?/? palatal shelves had slightly different cell packing than wild‐type, somewhat lower before elevation and higher at E15.5 when the wild‐type palate has elevated and fused. Cell orientation is biased towards the shelf distal edge in the mid‐palate of wild‐type embryos but is essentially random in the Tbx1^?/? mutant shelves, suggesting that polarised processes such as directed cell rearrangement might be causal for the cleft phenotype. The implications of these findings in the context of further understanding Tbx1 function during palatogenesis and of these methods for the more general analysis of genotype–phenotype functional relationships are discussed.     

Mesenchymal fibroblast growth factor receptor signaling regulates palatal shelf elevation during secondary palate formation

Background : Palatal shelf elevation is an essential morphogenetic process during secondary palate closure and failure or delay of palatal shelf elevation is a common cause of cleft palate, one of the most common birth defects in humans. Here, we studied the role of mesenchymal fibroblast growth factor receptor (FGFR) signaling during palate development by conditional inactivation of Fgfrs using a mesenchyme‐specific Dermo1‐Cre driver. Results : We showed that Fgfr1 is expressed throughout the palatal mesenchyme and Fgfr2 is expressed in the medial aspect of the posterior palatal mesenchyme overlapping with Fgfr1. Mesenchyme‐specific disruption of Fgfr1 and Fgfr2 affected palatal shelf elevation and resulted in cleft palate. We further showed that both Fgfr1 and Fgfr2 are expressed in mesenchymal tissues of the mandibular process but display distinct expression patterns. Loss of mesenchymal FGFR signaling reduced mandibular ossification and lower jaw growth resulting in abnormal tongue insertion in the oral‐nasal cavity. Conclusions : We propose a model to explain how redundant Fgfr1 and Fgfr2 expression in the palatal and mandibular mesenchyme regulates shelf medial wall protrusion and growth of the mandible to coordinate the craniofacial tissue movements that are required for palatal shelf elevation. Developmental Dynamics 244:1427–1438, 2015. © 2015 Wiley Periodicals, Inc.     

Contribution of variants in and near the IRF6 gene to the risk of nonsyndromic cleft lip with or without cleft palate in a Malay population

Several studies have shown evidence for the contribution of interferon regulatory factor 6 (IRF6) variants to the risk of nonsyndromic oral clefts in Asians; however, this has not included the Malay population. The current study attempts to address this research gap using allele and haplotype transmission disequilibrium analyses. The results showed a strong transmission distortion for multiple haplotypes to patients with nonsyndromic cleft lip with or without cleft palate. Haplotypes carrying the 243 bp allele of D1S2136 and common alleles at the rs861019 and rs2235371 were over‐transmitted to patients. By contrast, haplotypes consisting of the 251 bp allele of D1S2136 and the rare allele at rs2235371 were more under‐transmitted. Furthermore, several variants and haplotypes showed excess maternal transmission, but none of them attained statistical significance in maternal relative risk analyses. In contrast, a significant child genotype effect was observed for several haplotypes, indicating fetal genotype could be the major genetic contribution rather than maternal genotype. The present study therefore further supports a role for IRF6 variants in clefting in this Southeast Asian population. Overall, Asian genetic backgrounds are most likely more susceptible to the haploinsufficiency of IRF6 variants. These variants may contribute to the condition either themselves, or they may be in linkage disequilibrium with other casual variants. © 2011 Wiley‐Liss, Inc.     

Patterns of orofacial clefting in the facial morphology of bats: a possible naturally occurring model of cleft palate

A normal feature of the facial anatomy of many species of bat is the presence of bony discontinuities or clefts, which bear a remarkable similarity to orofacial clefts that occur in humans as a congenital pathology. These clefts occur in two forms: a midline cleft between the two premaxillae (analogous to the rare midline craniofacial clefts in humans) and bilateral paramedian clefts between the premaxilla and the maxillae (analogous to the typical cleft lip and palate in humans). Here, we describe the distribution of orofacial clefting across major bat clades, exploring the relationship of the different patterns of clefting to feeding mode, development of the vomeronasal organ, development of the nasolacrimal duct and mode of emission of the echolocation call in different bat groups. We also present the results of detailed radiographic and soft tissue dissections of representative examples of the two types of cleft. The midline cleft has arisen independently multiple times in bat phylogeny, whereas the paramedian cleft has arisen once and is a synapomorphy uniting the Rhinolophidae and Hipposideridae. In all cases examined, the bony cleft is filled in by a robust fibrous membrane, continuous with the periosteum of the margins of the cleft. In the paramedian clefts, this membrane splits to enclose the premaxilla but forms a loose fold laterally between the premaxilla and maxilla, allowing the premaxilla and nose‐leaf to pivot dorsoventrally in the sagittal plane under the action of facial muscles attached to the nasal cartilages. It is possible that this is a specific adaptation for echolocation and/or aerial insectivory. Given the shared embryological location of orofacial clefts in bats and humans, it is likely that aspects of the developmental control networks that produce cleft lip and palate in humans may also be implicated in the formation of these clefts as a normal feature in some bats. A better understanding of craniofacial development in bats with and without clefts may therefore suggest avenues for research into abnormal craniofacial development in humans.     

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting,cardiac anomalies,and tall stature

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157–161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157–161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.     

Malpuech facial clefting syndrome in a Japanese boy with cardiac defects

Summary We reported on a Japanese boy similar to the patients previously reported by Malpuechet al. (1983) with mental and growth retardation, hypertelorism, bilateral cleft lips, cleft palate, and urogenital anomalies. He also had undescribed cardiac defects. This is probably the second case report of Malpuech facial clefting syndrome.