Relation of the WNT5A gene and non-syndromic orofacial clefts

Abstract

Orofacial clefts are anomalies arising from defects during the fusion of the craniofacial processes. Non-syndromic orofacial cleft (NSOC) is the most frequent malformation in the craniofacial region. Understanding their etiology is a challenge requiring a multidisciplinary approach and deep understanding of environmental and genetic factors. Polymorphic variants in genes linked to craniofacial development emerge as key pieces in this puzzle.

Aim  This research aimed to deepen the understanding of the genetic mechanisms of the WNT5A gene in the development of NSOC.

Methods  To this end, a broad, comprehensive and updated synthesis of existing literature was carried out, through a narrative literature review.

Results  The WNT5A gene plays a crucial role in regulating cellular functions, significantly contributing to the formation and development of labial and palatal structures. It interacts with multiple gene signaling pathways also strongly linked to craniofacial development. Specifically, the SNP rs566926 has been associated with the occurrence of NSOC in various populations.

Conclusions  This study reinforces the importance of the WNT5A gene and its polymorphic variant in craniofacial development. Understanding these genetic mechanisms may pave the way for new diagnostic strategies, treatment, and improvement in the quality of life of affected individuals and their families.

Keywords
Cleft lip; Cleft palate; Polymorphism, genetic; Wnt-5a protein

Introduction

Orofacial cleft (OC) is the major congenital craniofacial malformation globally¹. This condition it is the result of failure in the fusion of the maxillary, medial nasal and palatal processes between the nasal and oral cavities during embryogenesis¹,². This malformation can present itself independently, without any other cognitive or craniofacial structural abnormalities, or it can be part of a broad spectrum of chromosomal, Mendelian, or teratogenic syndromes³. Approximately 70% of individuals with cleft lip with or without palate involvement (CL±P), and 50% of cases of isolated cleft palate (CP) manifest in the non-syndromic form of non-syndromic oral cleft (NSOC)⁴.

The origin of NSOC is complex, given the multifactorial interaction between genetic elements and environmental exposures³-⁵. As a result, the condition has a variable global prevalence, between 2:1,000 and 0.4:1,000 live births³,⁶. This variability is directly influenced by geographic origin, racial and ethnic groups, environmental conditions and socioeconomic status⁴. It is crucial to understand the combination of genetic and environmental causes in order to develop effective strategies for preventing NSOC²,³.

The most important environmental factors are an advanced age of both parents, consanguinity, as well as maternal active and passive smoking, use of medication, and alcohol consumption²,³,⁷. The genetic contribution to the development of NSOC is attributed to variations in candidate genes that act during craniofacial development. However, the exact mechanism by which this relationship influences the development of the malformation is not yet well established¹.

To understand the genetic etiology related to the development of congenital malformations, it is crucial to consider the familial aggregation of the characteristic in question. This involves acknowledging the presence of one or more genes within families⁸. When it comes to understanding the origin of NSOC, different strategies are employed⁹. These include linkage studies, whole-genome association analyses, gene expression surveys, targeted genetic sequencing, investigations of genetic interactions between polymorphisms in candidate genes for the development of OC, and direct DNA sequencing⁸,⁹. Utilizing these strategies, several genes and genetic loci have been identified⁷,⁸. These participate in signaling pathways associated with the development of the lip and palate and, therefore, may be implicated in the etiology of NSOC⁸.

Among these genes, WNT5A stands out for its role in cell migration and polarity, in addition to regulating developmental pathways during embryogenesis, such as the migration of mesenchymal cells in palatogenesis⁵,¹⁰. Mutations in this gene have been associated with the occurrence of NSOC⁵,⁶,¹¹. Among the several single nucleotide polymorphisms (SNP) located in WNT5A, the rs566926 SNP is notable for its association with the occurrence of NSOC in populations of Europeans, Americans of European descent, and Brazilians⁵,⁶,¹¹,¹². Considering that polymorphic variants in genes associated with craniofacial development may have a significant role in the etiology of NSOC, the present study aims to consolidate current knowledge about the role of the WNT5A gene in the development of NSOC, providing a comprehensive and updated view of the existing literature.

Materials and Methods

A literature search in the PubMed, Web of Science and Google Scholar databases for articles published in English from 1980 to 2024 was performed.

The research strategy included the MeSH terms “Wnt-5a Protein”, “cleft lip” and “cleft palate”. Additional terms commonly used in studies, “wnt5a cleft”, “wnt5a palate” and “wnt5a signaling” were also used in the searches.

Duplicate articles, those investigating syndromic OC, those offering perspectives and opinions, commentary pieces, and book reviews were excluded from the selection.

Results

NSOC are among the congenital craniofacial malformations that occur most frequently in humans³. They result from failed fusion of facial processes during embryogenesis¹. The incidence of NSOC is highly variable, with the highest rates found among Asian and Native American populations (2:1000), followed by individuals of European (1:1000) and African descent (0.4:1000)³,⁶.

NSOCs are etiologically heterogeneous, which means they have multiple and complex causes that depend on the interaction between inherited genetic patterns and environmental factors⁸. In terms of genetic involvement, it is currently impossible to determine exactly how many genes are related to the etiology of NSOC⁷. However, it is estimated that many genes and gene loci participate in signaling pathways associated with the correct embryonic development of the lip and palate and, therefore, could be related to the etiology of malformations⁵,¹³.

Certain gene families are responsible for controlling early embryonic development, as well as the formation and diversification of the facial skeleton in vertebrates¹⁴. The Wingless (WNT) family is included in this group. WNT family genes were discovered in 1980, and since then, have proven to be significant in signaling for the normal development of polarity in vertebrate limbs¹⁵. In 1992, Nusse and Varmus published the most classic study on these genes, indicating evidence that they are evolutionarily conserved. This theory is widely accepted today, as they can be identified in all metazoan organisms, and the 19 genes identified in the human genome are conserved in the genomes of mammals¹⁵,¹⁶.WNT genes are structurally related but have distinct functions¹⁷. The proteins translated by the WNT are secreted glycoproteins, rich in cysteines, modified by lipids that act as ligands for various membrane-bound receptors¹⁸.

The WNT play roles in cellular communication at different stages¹⁹, regulate cell growth, motility and differentiation during the embryonic phase¹⁷,¹⁹and tissue homeostasis in the post-embryonic phase¹⁸,¹⁹. These genes are active in almost all tissues during craniofacial development, allowing proliferation and cell polarity to occur simultaneously and in the same cells⁵,¹⁵,¹⁸. Therefore, alterations in these genes may contribute to a variety of developmental defects, such the OCs⁵,⁶,¹¹-¹³,²⁰.

The WNT5A gene, was isolated for the first time in 1990, in mouse fetal tissue, and in human cells the first isolation occurred in 1993. This gene is member 5 of the WNT family, located in chromosome 3p14.3 in the human genome. Like other WNTs, WNT5A is highly conserved among species. The human WNT5A gene has 98.7% of similar amino acids to the rodent Wnt5a gene²¹. This gene encodes the WNT5A-specific protein that regulates a variety of cellular functions such as proliferation, differentiation, migration, adhesion and polarity²². It is fundamental in the migration of mesenchymal cells during palatogenesis¹⁰, in addition to regulating postnatal tissue and bone homeostasis²³. Mutations in mouse Wnt5a are linked to limb morphogenesis defects with shortened and/or malformed skeletal elements²⁴, as well as reduced skull bone formation²⁵. The deletion of this gene is associated with defective growth of the tail and snout, cleft palate, jaw malformation, tongue malformation, other skeletal defects, and perinatal lethality¹⁰,²⁴,²⁶. In humans, mutations in the WNT5A gene have been associated with poor heart development²², the occurrence of NSOC⁵,⁶,¹¹, and autosomal dominant Robinow syndrome, which frequently includes cleft palate²⁶. These mutations have also been linked to a variety of malignant tumors¹⁰.

The WNT5A protein is a key regulator of ß-catenin-independent WNT signaling, also known as non-canonical pathway. However, it can also influence the ß-catenin-dependent pathway, also known as canonical or classical, in two ways²⁵. The number of expressed receptors, coreceptors and endogenous inhibitors present are responsible for this variation¹⁶,¹⁷.

The main function of the canonical pathway is to encode proteins responsible for regulating vascular, fibroblast and antigen endothelial proliferation; matrix metalloproteinases and some components of the extracellular matrix; cadherins; lineage-specific proteins, particularly those linked to the skin and eyes²⁷; the proliferation, differentiation and maintenance of stem cells²⁸; and the formation, differentiation and maintenance of bone mineral density²⁹. The initiation of signaling occurs after the extracellular WNT protein docks with its receptor Frizzled (FzD) 4 or 9³⁰ juxtaposed with a lipoprotein receptor-related protein (LRP) 5 receptor molecule on the cell surface³¹,³². It forms a heterotrimeric complex responsible for intracellular signaling, it binds to Dishevelled (Dsh) and in the cytoplasmic tail to Axin, glycogen synthase kinase-3β (GSK-3β) and Adenomatous Polyposis Coli (APC)¹⁸,¹⁹,³¹. The migration of the Axin/GSK-3β/APC complex from the cytoplasm to the plasma membrane inactivates the activity of degrading β- catenin³³. Consequently, β-catenin accumulated in the cytoplasm migrates to the nucleus. Once in the nucleus, β-catenin can interact with several proteins and activate the main transcription factors of the canonical, T-cell-specific pathway. transcription factor/lymphoid enhancer-binding factor 1 (TCF/LEF1), to promote the expression of effector genes¹⁸,¹⁹,³³. Mutations in TCF/LEF1 have been correlated with changes in facial formation³⁴.

The non-canonical Ca²⁺ pathway is involved in changes in the arrangement of the cytoskeleton, regulation of cell motility, migration and cell connections. It also inhibits the transcriptional activity of canonical WNT signaling³⁵. This pathway is activated when the extracellular WNT5A protein binds to its receptor FzD 2, 3, 4, 5, 6, or 7¹⁶,³⁰, juxtaposed with LRP 5 or 6, which is binded to heterotrimeric Dsh and G protein. These proteins then stimulate the Phospholipase C enzyme (PLC). Consequently, the messengers Inositol Triphosphate (IP3) and Diacylglycerol (DAG) induce the release of calcium from intracellular stores¹⁶,³². This release activates calcium-sensitive enzymes, such as Protein Kinase C (PKC), Calmodulin-dependent Protein Kinase II (CaMKII), and Calcineurin¹⁶,³⁵. PKC stimulates the reorganization of the cytoskeleton through effector genes, thus it can alter the cell migration process in gastrulation. CaMKII binds to TCF/LEF1 that would bind to β-catenin, thus inhibiting the action of the canonical pathway³²,³⁵,³⁶. Calcineurin, stimulates cellular control through effector genes. Thus, the WNT5A Ca²⁺ pathway can promote or inhibit chondrogenesis¹⁶.

The non-canonical planar cell polarity (PCP) pathway is involved in regulating the modification of actin cytoskeleton structures and cell motility³⁷. Although the PCP pathway may share the use of the FzD and Dsh receptors with other signaling pathways, this pathway offers numerous organizational possibilities, varying according to the tissue in which the cell is located, and may oppose each other’s functions of other pathways or influence neighboring cells to regulate cellular polarity³²,³⁸. WNT5A can activate the PCP pathway by binding to FzD, LRP5/6 and Dsh, which binds to kinases: RhoA or Rac. Both induce the activation of other kinase molecules: Rho kinase (ROCK) and Jun N-terminal kinase (JNK), respectively. Thus, they stimulate effector genes to reorganize the cytoskeleton, which can promote or inhibit chondral tissue formation¹⁶. Another form of organization is WNT5A being directly bound by molecules such as related orphan receptor 2 (ROR2) and receptor tyrosine kinase-like (RYK), with or without the presence of FzD. ROR2 can activate the JNK molecule to stimulate target genes to reorganize the cytoskeleton or stimulate β-catenin degradation through binding with TCF/LEF1³⁷-³⁹. ROR2, is expressed in the mesenchyme of the secondary palate¹⁰. WNT5A mediated by ROR2 regulates cell proliferation and directional cell movement⁴⁰, so lower WNT5A cell proliferation in dental mesenchyme and/or epithelium is associated with delayed tooth growth⁴¹. The gene mutation mediated by the ROR2 receptor is associated with smaller and incorrectly patterned teeth, while the absence of WNT5A can cause defective tooth development⁴⁰,⁴¹. ROR2 knockout mice showed craniofacial defects, including cleft palate²⁶. Mutations in RYK are also associated with cleft palate in mice⁴²and non-syndromic cleft lip and palate (NSCLP) in Vietnamese and Japanese individuals⁴³. Furthermore, deletion of PK1, through WNT5A binding to ROR2/RYK, causes CP and limb defects in mice⁴⁴.

Figure 1
Simplified scheme of the WNT5A signaling pathways. a. Canonical signaling pathway activated by WNT5A: WNT5A binds to FzD4 or 9 and LRP5. These, in turn, bind to Dsh on the plasma membrane. Dsh attracts: Axin, GSK-3β and APC, thus inhibiting the activity of the β-degrading complex-catenin. The β-catenin accumulated in the cytoplasm migrates to the nucleus where it interacts with TCF/LEF1 to promote the expression of effector genes. b. Non-canonical Ca2+ signaling pathway activated by WNT5A: WNT5A binds to FzD 2, 3, 4, 5, 6 or 7 and LRP5/6, which bind to Dsh and G protein. They stimulate PLC, which activates IP3 and DAG, inducing the release of calcium from intracellular stores. Thus, calcium-sensitive enzymes act: calcineurin stimulates cellular control through effector genes, CaMKII binds to TCF/LEF1, inhibiting β- catenin and PKC stimulates cytoskeletal reorganization through effector genes. c. Non-canonical PCP signaling pathway activated by WNT5A: One of the ways this pathway signals is with WNT5A binding to FzD and LRP5/6, which bind to Dsh. The other bindings stimulate effector genes to reorganize the cytoskeleton. d. Direct non-canonical PCP signaling pathway activated by WNT5A: Another way of signaling this pathway is with WNT5A binding to ROR2 or RYK, with or without the presence of FzD. This activates the JNK molecule to stimulate target genes to reorganize the cytoskeleton or stimulate degradation of β-catenin through binding with TCF/LEF1.

Discussion

The WNT5A gene has been associated with the occurrence of NSOC. It is widely linked to several pathways and interacts with many genes¹⁶. This feature makes WNT5A a promising candidate to participate in epistatic interactions, which influence the occurrence of OC¹⁰,⁴⁵. The WNT5A gene was initially proposed as a possible influencer of the risk of NSOC occurrence in multiplex families in the population of the United States¹². The study noted that regions D3S3666 and D3S1547 provided strong evidence for a non-syndromically manifested CL±P-linked locus (NSCL±P) in this region, with WNT5A as a leading candidate¹². Later, the regions D3S3719, D3S2408 and the SNP rs566926 in WNT5A were analyzed¹¹. Associations between the rs566926 variant and the occurrence of NSCL±P in the sample of Americans of European descent were identified¹¹. Gene-gene interactions between the SNPs rs566926-WNT5A and rs1745420-WNT3A in the sample of Americans of Hispanic descent were also observed, but these demonstrated an insufficient p-value to confirm an association with NSOC¹¹. A study conducted in two centers in the Southeast region of Brazil also investigated the SNP rs566926 of the WNT5A gene. Initially, a significant association was found in individuals with unilateral left NSCL±P. However, the significance was lost in both groups after the Bonferroni correction²⁰.

Other variants have also been analyzed in WNT5A. A study using data from an international consortium of parent-child trios from European and Asian European populations found significant epistatic interactions between rs358817 and rs3856709 in WNT5A and s3790604 in WNT2B, among individuals of European ancestry. The rs7640326 (WNT5A) variant as well as the rs2013162 (C1orf107) and rs126280 (IRF6) variants demonstrated associations in Asian trios⁴⁶. Another study also using data from two international consortia and parent-child trios in Denmark, Norway, the United States, Singapore, Taiwan, Philippines, Korea and China identified signs associated with WNT5A. However, the specific SNPs were not mentioned⁴⁷. A study with both parent-child trios and isolated patients investigated the rs1499890-WNT5A variant in the development of NSOC in the Japanese population. However, the methods and limited sample power did not enable either confirming or ruling out the association⁴⁸. A large survey with individuals from different regions of China investigated seven genes of the WNT pathway, with 45 SNPs for NSCL±P and 46 for non-syndromic cleft palate (NSCP) in WNT5A. Three SNPs in WNT5A were found to be associated with epistatic interactions for NSCL±P: rs7618735 (WNT5A) and rs10848543 (WNT5B); rs556874 (WNT5A) and rs631948 (WNT11); rs472631 (WNT5A) and rs631948 (WNT11). Two SNPs were related for NSCP: rs358792 (WNT5A) and rs1402704 (WNT11); rs358793 (WNT5A) and rs1402704 (WNT11)⁴⁹. The SNP rs769829279 in the WNT5A gene was investigated in the Polish population, but it was not possible either to confirm or rule out its association with the development of NSOC. This may have occurred due to the limited sample size and/or the method employed⁵⁰.

Recently, two studies focused on the rs566926 variant. The relationship between NSCLP and the rs566926 (WNT5A) and rs1530364 (WNT9B) SNPs were analyzed in a population of 50 individuals from southern India. However, the limited sample size may not have enabled the determination of existing associations⁵¹. A study with 1,955 Brazilians confirmed the relationship between the SNP rs566926- WNT5A and NSCL±P, particularly in individuals with non-syndromic cleft lip (NSCL) and high European ancestry. This study also identified epistatic interactions involving rs566926-WNT5A and the BMP4 SNPs rs2071047 and rs2761887; rs566926- WNT5A and the SNPs rs16969681 and rs16969862 of GREM1 and the association with NSCL±P; and interaction between rs566926- WNT5A and the FGFR1 SNP rs7829058 which revealed a protective association for NSCLP⁵. A meta-analysis that evaluated case-control studies in populations of European descent confirmed rs566926- WNT5A as strongly associated with NSOC⁶.

Thus, the only SNP in the WNT5A gene associated with the occurrence of NSOC in different populations is rs566926⁵,⁶. It is located in a transcription factor binding site that has an intronic enhancer function. These sites are involved in regulating embryonic development and determining cell fate²⁰. Factors common to the studies may explain the discrepancies in the results found for the rs566926-WNT5A variant. First, genetic heterogeneity is intensified by epistatic and gene-environment interactions, affecting different populations differently⁵². Ethnic diversity should also be considered⁵³, as the prevalence of the different types of NSOC can vary significantly among populations⁵,⁵⁴. This diversity significantly influences genetic susceptibility in cases of NSOC⁵³. However, in the assessment of the rs566926-WNT5A variant, only one study performed genetic ancestry analyses for each individual⁵, although some studies separated individuals by self-reported ancestry¹¹,²⁰, or only included studies that investigated the association between genetic factors and NSOC in populations of European descent⁶. Second, the frequently observed variability in penetrance makes it difficult to gain a full understanding of the phenotype⁵². Third, there are difficulties in analyzing and comparing studies, which can compromise the accuracy of the results. One example of this is the difference in the way orofacial cleft subtypes are classified. While some studies adopt a detailed division, such as NSCL, NSCLP and NSCP⁵¹, some combine the NSCL and NSCLP categories into a single group: NSCL±P¹¹ and others use both classifications⁵-⁶,²⁰. This lack of standardization in the classification makes it difficult to compare and interpret data, in addition to limiting the power of specific subgroups. Fourth, all the studies analyzed had a small sample size⁵,¹¹,²⁰,⁵¹, which raises concerns that the results may not adequately reflect reality.

This demonstrates the importance of future studies analyzing the rs566926-WNT5A variant among individuals from all regions of the planet. Additionally, these studies should strive to incorporate ancestry analyses. Furthermore, including a robust sample will allow exploration of interactions between genes and environment, making possible a more detailed investigation of genetic associations with the occurrence of NSOC in humans.

The present review has limitations, as it included studies with different designs, such as case-control⁵,²⁰,⁵¹, multiplex family studies¹¹,¹², parent-child family trio screening¹¹,⁴⁶,⁴⁸,⁴⁹, GWAS⁴⁷,⁴⁸,⁵⁰ and meta-analysis⁶. Differences in methodological quality among the studies and the difficulty in fully exploring interactions between genetic and environmental factors may affect the reliability of our conclusions.

It is also essential to consider that genetic modulation through epigenetic changes is influenced by environmental stimuli, affecting gene expression and the inheritance of phenotypic characteristics⁵⁵. Therefore, the diagnosis of NSOC should include a clinical assessment and genetic testing to identify specific abnormalities and improve the understanding of the malformation⁵²,⁵⁶. Identifying such abnormalities can benefit treatment, enabling more effective personalized interventions⁵⁷. Therefore, a multidisciplinary approach, involving clinical treatment and genetic counseling, is important⁵⁸. Counseling can predict recurrences in future pregnancies and enable access to procedures that prevent the birth of an affected child⁵⁷.

In conclusion, WNT5A gene emerges as a potential influencer in occurrence of NSOC among world populations. It plays a crucial role in regulating cellular functions, contributing significantly to the formation and development of lip and palate structures. Furthermore, WNT5A is widely connected to several signaling pathways and interacts with a series of genes associated with craniofacial development. Notably, the rs566926 SNP has been consistently associated with the occurrence of NSOC in several populations.

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• Data availability:
  Datasets related to this article will be available to the corresponding author upon request.

Edited by

• Editor:
  Dr. Altair A. Del Bel Cury

Data availability

Datasets related to this article will be available to the corresponding author upon request.

Publication Dates

• Publication in this collection
  20 Apr 2026
• Date of issue
  2026

History

• Received
  14 Aug 2024
• Accepted
  02 Feb 2025