Genetic and Epigenetic Studies in Nonsyndromic Oral Clefts

Abstract

The etiology of non‐syndromic oral clefts (NSOFC) is complex with genetics, genomics, epigenetics and stochastics factors playing a role. Several approaches have been applied to understand the etiology of non‐syndromic oral clefts. These include linkage, candidate gene association studies, genome‐wide association studies, whole genome sequencing, copy number variations and epigenetics. In this review we shared these approaches, genes and loci reported in some studies.

Full text

Oral Diseases. 2022;00:1–12. wileyonlinelibrary.com/journal/odi
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1© 2022 Wiley Periodicals LLC
1 | INTRODUCTIO N
Non- syndromic oral clefts (NSOFC) account for 70% of all clef ts
and are some of the most common birth defects in humans. The
global prevalence is about 1 in 700 live bir ths. (Mossey & Modell,
2012). However, there are population differences in the prevalence
which ranges from 1/500 in Asians to 0.5/1000 in Africans (Mossey
et al., 2009).
The etiology of non- syndromic clefts is complex with genetics,
genomics, epigenetics, and stochastics factors playing a role. Several
studies alluded to the possible genetic causes based on the segrega-
tion of clefts phenot ypes in families with multiple affected individuals.
Trew reported a f amily with several NSOFC affected members in 1751.
Andersen concluded that there is a presence of a strong genetic com-
ponent which did not align with the classic Mendelian dominant/reces-
sive inheritance pattern (Andersen, 1942). Attempts at uncovering the
inheritance pattern led to the suggestion of a multifactorial threshold
model (Fraser, 1976). This was later rejected in favor of a major locus/
gene (Melnick et al., 1986) and a mixed model, that is, major gene and
multifactorial model (Chung et al., 1986; Marazita et al., 1984).
Several a pproaches have be en applied to under stand the eti ology
of non- syndromic clef ts. These include linkage, candidate gene as-
sociation studies, genome- wide association studies, whole- genome
sequencing, copy number variations, and epigenetics (Table 1).
2 | LINKAGE STUDIES
The major gene effect model motivated early linkage studies
to understand the genetic basis of NSOFC . Linkage is a power-
ful method used to identify region(s) of the genome harboring a
disease- causing gene/locus by typing DNA markers to see whether
they co- segregates with the disease phenotype in related individu-
als (Rahimov et al., 2012; Wyszynski, 2002). This design is based
on the concept of linkage and identity by descent (IBD). Two loci
are in linkage if the recombination probability between them is less
than 50% during meiosis. IBD refers to genomic regions with iden-
tical nucleotide sequences inherited from a common ancestor and
not due to recombination. Linkage analysis can either be parametric
or non- parametric. Parametric linkage analyses are used for major
gene disorders with a pre- specified mode of inheritance. In con-
trast, non- parametric methods are used for complex diseases (e.g.,
NSOFC) where several genes contribute to disease risk (Kruglyak
et al., 1996). The non- parametric analysis approach looks for excess
IDB shared allele at any region/locus between affected sibling/rela-
tives pairs to identify regions/loci likely predisposing to disease (Shih
& Whittemore, 2001).
Through linkage analysis, over 20 chromosomal regions have
been linked to NSOFC. Notable ones include chr 1p, 1q21, 1q32-
42.3, 6p, 2p, 4q, and 17q (Carinci et al., 2000; Martinelli et al., 1998;
Received: 21 September 2021
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Revised: 11 January 2022
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Accepted: 20 January 2022
DOI : 10.1111/o di.14146
INVITED REVIEW
Genetic and epigenetic studies in non- syndromic oral clefts
Azeez Alade1,2,3 | Waheed Awotoye1,2 | Azeez Butali1,2
1Depar tment of Oral Patholog y, Radiolog y
and Medi cine, Colle ge of Dentistry,
University of Iowa, I owa City, Iowa, U SA
2Iowa Institute for Or al Health Research,
University of Iowa, I owa City, Iowa, U SA
3Depar tment of Epid emiology, College of
Public Health, University of Iowa, Iowa
City, Iowa , USA
Correspondence
Azeez B utali, Department of Oral
Pathology, Radiology and Medicine,
College of D entistr y, Universit y of Iowa,
Iowa Cit y, IA, USA.
Email: Azeez-butali@uiowa.edu
Funding information
Nationa l Institute of Dental and
Craniofacial Research, Grant/Award
Number: DE028300
Abstract
The etiology of non- syndromic oral clefts (NSOFC) is complex with genetics, genom-
ics, epigenetics, and stochastics factors playing a role. Several approaches have been
applied to understand the etiology of non- syndromic oral cleft s. These include linkage,
candidate gene association studies, genome- wide association studies, whole- genome
sequencing, copy number variations, and epigenetics. In this review, we shared these
approaches, genes, and loci reported in some studies.
KEY WORDS
epigenetics, genetics, Non- syndromic clefts

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A LADE Et AL .
Mitchell & Risch, 1992). Some of these have been widely investi-
gated across different populations with varying results. For example,
Carinci et al. (2000) showed evidence for chr 2, 4, and 6 with cleft
lip with or without cleft palate (CL/P) in the Italian population, while
Wong et al. (2000) found no evidence for these regions in Swedish
multiplex CL/P families, suggesting a population- specific effect for
these loci— a phenomenon that has become increasingly apparent
with current knowledge. Although linkage studies have successfully
mapped genes for rare monogenic disorders, limited success was re-
corded with NSOFC. This is because NSOFC are complex disorders
caused by genes/loci with small effect sizes (Rahimov et al., 2012).
In line with the multifactorial inheritance model, linkage studies
were later ex tended to cover the whole genome via the use of mark-
ers placed at regular inter vals covering the whole genome. Prescott
et al. (2000) were the first to report a genome- wide significant link-
age. They carried out a two- stage genome- wide scan of 92 sibling
pairs with CL/P and identified 11 loci on eight chromosomes. Albeit,
these were not statistically significant genome wide. Two of the loci
had been previously reported as susceptible loci for CL/P, and one of
them harbors the TGFA gene. Other genes associated with NSOFCs
via linkage studies include COL11A1 , IRF6, EGF, MSX1, PTCH, TGFB1,
ROR2, FOXE1, TGFB3, RARA, APOC2, BCL3, and PVRL2.(Ardinger
et al., 1989; Chenevix- Trench et al.,1992; Kurihara et al., 1994; Lidral
et al., 1998; Nottoli et al.,1998; van den Boogaard et al., 20 00). The
major limitation to this approach is the broadness of the linked ge-
nomic region, making it difficult to pinpoint the disease- causing
gene or locus within the region (Rahimov et al., 2012).
3 | CANDIDATE GENES ASSOCIATION
STUDIES
While linkage studies require multiplex families with multiple af-
fected relatives/siblings, candidate gene association studies can be
carried out on isolated cleft cases without affected family members.
In general, association studies look for statistically significant differ-
ence in the frequency of an allele or haplotype between individuals
with the phenotype of interest and those without the phenotype in
order to identify the likely causative variant/marker (Kwon & Goate,
2000). Early candidate gene approaches leveraged on the premise
that common variants with less deleterious effec ts in known syndro-
mic cleft genes predispose to NSOFC (Stanier & Moore, 2004). This
hypothesis was tested for I RF6 gene implicated in Van der woude
syndrome and FOXE1 gene implicated in Bamforth Lazarus syn-
drome (Clif ton- Bligh et al., 1998; Zucchero et al., 20 04). Both genes
implicated in syndromic clefts were shown to be associated with
NSOFCs via candidate gene association studies. Other genes that
have been identified following their role in other craniofacial defects
(e.g., craniocynostosis) syndromes include FGFR1 and FGFR2 (Riley &
Murray, 2007). These observations were in line with the hypothesis
that variants in the non- coding regions of these genes are associated
with NSOFC while variants in the coding regions are responsible for
syndromic OFC (Stanier & Moore, 2004).
Candidate genes for NSOFC are also selected based on func-
tional properties, expression pattern, and or informative mouse
model. Several other candidate genes have been identified using
animal models. For instance, a MSX1 gene mouse knock- out model
showed clef t palate phenotype (Satokata & Maas, 1994) and can-
didate gene studies found an association between variants in this
gene and human NSOFC (Butali et al., 2011; Jezewski et al., 2003; Li
et al., 2017). Cleft palate phenotypes were observed for BMP mice
and chick embryos as well (Foppiano et al., 2007; Liu et al., 2005).
Human variants have also been reported for BMP4 in multiple pop-
ulations and in subclinical forms of clefts (Suzuki et al., 2004, 2009).
Other genes have been reported using several approaches such as
genome- wide scans for CRISPLD2 (Chiquet et al., 2007), fluorescent
in situ hybridization (FISH) of a balanced reciprocal translocation
for SUMO1 (Alkuraya et al., 2006), and case– control analyses for
TFGβ (Ardinger et al.1989).
The association of some candidate genes is inconsistent across
populations. The association with some genes has not been repli-
cated, while others show opposite effects. For instance, increased
risk for NSOFC was reported for BMP4 rs17563 polymorphism in
Asians and Caucasians and reduced risk was observed in Brazilian
population. Similarly, inconsistent association has been reported for
MTHFR. Some studies reported increased risk for CP (Mills et al.,
2008; Zhu et al., 2006), some reported a reduced risk for CL(P)
(Jugessur et al., 2003; Little et al., 2008), while another did not find
any associated risk with maternal MTHFR CT or TT genotypes for
either CL(P) or CP (Boyles et al., 2008).
4 | GENOME- WIDE ASSOCIATION
STUDIES
The need to identify novel risk loci/genes contributing to the risk
of NSOFCs ne cessitated the shi ft toward genome- wide as sociation
studies (GWAS). Unlike candidate gene association studies which
are hypothesis driven, GWAS is an unbiased and hypothesis free
approach used to identify genotype– phenotype associations by
testing multiple genetic variants across the genome (Christensen
& Murray, 2007). Since inception, It has revealed multiple loci con-
tributing to several complex traits (Shaffer et al., 2012). The first
GWAS on NSOFC was published by Birnbaum et al. (2009). They
reported a genome- wide significant association between NSCL/P
and three markers in 8q24- 21; a gene deser t devoid of any known
protein- coding gene (Birnbaum et al., 2009). That same year, Gant
et al. found a genome- wide association with the same region in
an independent cohor t of NSCL/P patients (Grant et al., 2009).
Beaty et al. (2010), conducted the first case- parent trios GWAS
on NSOFC. This study replicated the findings from the previous
GWAS and identified two new loci near MAFB and ABCA4 genes.
Further, Mangold et al. (2010) reported two additional loci in
17q22 and 10q25.3 with NOG and VAX1 identified as the possi-
ble candidate genes. Sun et al. (2015) conducted the first Chinese
GWAS on NSCL/P. This study replicated four previously published

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ALAD E Et AL.
Candidate genes
and genomic loci Technology and Methodology
SHH Association, Mutation screen, Targeted sequencing
TP63 Mutation screen, Targeted sequencing, Whole- exome sequencing
GLI2 Targeted sequencing
MSX2 Mutation screen, Targeted sequencing, Linkage
SPRY2 Mutation screen, Targeted sequencing
SPRY1 GWAS
SU LT2A 1 GWAS
CTNNA2 GWA S
PDGFRA Mutation screen, Targeted sequencing, Epigenetics
TBX1 Mutation screen, Copy number variation, Epigenetics
CTNNB1 Association, Mutation screen
PAX9 Targeted sequencing
PVRL1 Targeted sequencing, Mutation screen
TBX22 Targeted sequencing, Mutation screen
CTNND1 Mutation screen, Exome sequencing, Targeted sequencing
RARA Association
FGF10 GWAS, Mutation screen
WNT9B GWAS, Mutation screen
KRT18 G WAS
TFAP 2A GWAS, Mutation screen, Whole- genome sequencing
IRF6 Linkage, Association, Targeted sequencing, GWAS, Exome sequencing,
Copy number variation, whole- genome sequencing
FOXE1 Linkage, Association, Targeted sequencing, GWAS
MSX1 Animal models, Targeted sequencing
BMP4 Animal models, Targeted sequencing
FGFR1 GWAS, Targeted sequencing
FGFR2 Targeted sequencing
CRISPLD2 Linkage and Association
SUMO1 FISH
TFGβAssociation
MAFB GWA S
PAX7 GWAS
VAX1 GWAS
ARHGAP29 GWAS, Mutation Screen, Whole- exome sequencing
Chr8q.24 GWAS
Chr16p13.3 GWAS
VAX1, GWAS
NOG GWAS
GRHL3 Linkage, Exome sequencing
CDH1 Exome sequencing, Targeted sequencing
MGAM Copy number variation
ADAM3A Copy number variation
ZFHX4 Whole- genome sequencing
Chr21q22 Whole- genome sequencing
ADAM5A Copy number variation
TABLE 1 Some candidate genes and
loci that play roles in the etiology of non-
syndromic clefts and the techniques used
in their discoveries

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A LADE Et AL .
loci and identified a novel locus in Chr 16p13.3 between CREBBP
and ADCY9 genes. With a ver y large sample size consisting of
multiethnic groups, Yu et al. (2017) reported 26 GWAS signifi-
cant loci, 14 of which were known with several new potential cleft
candidate genes (RAD54B, T MEM19, KRT18, WNT9B, GSC/DICER1,
PTCH1, RPS26, OFCC1/TFAP 2A, TAF1B, FGF10, MSX1, LINC00640,
FGFR1, and S PRY1).
With an increasing number of independent GWAS studies, the
first GWAS meta- analysis was conducted by Ludwig et al. (2012).
The study replicated most of the previously identified loci and
identified six novel regions. Following the success recorded by
this meta- analysis, several other meta- analyses were conducted
on NSCL/P (Leslie et al., 2017a; Yang et al., 2020; Yu et al., 2017).
All these studies (discovery and meta- analyses) put together led
to the identification of over 40 risk loci associated with NSCL/P.
The first study to report genome- wide significant loci associated
with non- syndromic cleft palate only was by Leslie et al., (2016).
They identified three novel loci with GRHL3, Yap1, and PARK2 as
possible candidate genes. The signal in GRHL3 was replicated by
another GWAS for CPO by Mangold et al. (2016). In 2019, The
African only GWAS by Butali et al. (2019) and the Chinese GWAS
by Huang et al. (2019), respectively, identified additional NSCPO
risk loci near CTNNA2, SULT2A 1, DLK1, DOCK9, FOXC2/FOXL1,
IRF6, MAU2, PAX9, POMG, NT2, and WHSC1 genes (Butali et al.,
2019; Huang et al., 2019). Despite the tremendous successes re-
corded by GWAS in identifying NSOFCs susceptible genes, all the
genes/loci identified so far only account for 20%– 30% of the tot al
heritability of NSOFC (Leslie et al., 2017a).
The missing heritability could be explained by low frequency/
rare variants, gene– gene interactions, and epigenetics among
others. In a bid to determine the missing heritability, rare variant
studies of NSOFC are becoming increasingly popular. Initially,
through candidate genes resequencing in small samples to iden-
tify private variants and subsequently via genome- wide rare vari-
ants analysis. The first genome- scale study of rare variants was by
Bureau et al in 2014 (Bureau et al., 2014). They performed whole-
exome sequencing and looked for shared rare variants in 348 es-
tablished cleft candidate genes among NSOFC affected relatives
from 55 multiplex families. The study reported five novels shared
variants in the CDH1 gene and showed statistically significant ev-
idence of co- segregation in an Indian family. Leslie et al. (2017b)
examined the role of rare coding variants applying a genome- wide
rare variants aggregate association test and found a statistically
significant association with low frequency and rare variants in
N4BPH gene in the Latin American population. With a multieth-
nic sample of 11,727 participants, Shaffer et al., 2019 focused on
the rare non- coding variants in previously identified craniofacial
enhancer regions. They found a significant association between
mm60 on chr 3p13 (an enhancer element located bet ween MITF
and FOXP1 gene) and CPO (Shaffer et al., 2019). The candidate
gene approach is still ver y relevant following GWAS, especially
as replication for novel genes identified through next- generation
omics approaches.
5 | WHOLE- EXOME SEQUENCING
Whole- exome sequencing (WES) study has long been applied
in the investigation of the genetic risk of common birth defects
(Feliciano et al., 2019; Yu et al., 2013). This next- generation se-
quencing technique has helped to identify pathogenic genetic
mutations that were undetected by earlier screening approaches.
Earlier use of this technique in gene discover y focused on rare
Mendelian disorders (Antonarakis & Beckmann, 2006); however, it
has also been used for complex traits. This sequencing approach is
usually used to identify genetic variants within the coding regions
of the human genome (exome). A large number of mutations within
the exome af fect the amino acid sequence which have a direct ef-
fect on the protein structures and function (Kryukov et al., 2007).
This knowledge forms the foundation of the exome sequencing
studies designed to identify protein- altering mutations that led to
the disorders.
This next- generation sequencing technique has been applied
in the study of NSOFC across multiple populations(Aylward et al.,
2016; Basha et al., 2018; Cai et al., 2017; Liu et al., 2017; Machado
et al., 2021; Pengelly et al., 2016). One of such studies used WES
approach to screen for damaging mutations segregating with the
phenotype across multiple pedigrees in a family with cleft pal-
ate. This approach was used to identify a novel loss- of- function
mutation in ARHGAP29 as the etiologic factor in the family (Liu
et al., 2017). Families with multiple pedigrees (multiplex) with
affected relatives provide a huge opportunity for the identifica-
tion of the genetic risk driving birth defect or trait (Bureau et al.,
2019; D'Netto et al., 2009). This is because the unaffected rel-
atives ser ve as more accurate controls compared to the use of
population- based controls. Thus, this requires a deep phenotyp-
ing of all individuals within the pedigree and using the appropriate
inheritance model for the genetic screens. Other factors that must
be considered when studying the Mendelian disorders and com-
plex traits using WES are penetrance, identification of disease-
causing mutation, and heterogeneity of genetic variants to the risk
of the disorder or trait.
The use of WES in family- based study design helps to inves-
tigate the numerous inheritance models possible in the genetic
risk of cleft. This sequencing strategy can also be applied to case–
control study design while applying the different inheritance
model in the genetic screens. Although WES helps to investigate
high impact mutations that are potentially pathogenic, it is only
limited to discovery of protein- altering mutations within a re-
stricted 1%– 2% of the entire human genome also known as the
protein- coding region of the genome. Nonetheless, this miniscule
region harbors 85% of the disease- causing mutations (Majewski
et al., 2011). Regardless, there is a missing 15% disease- causing
mutation which are potentially harbored within the non- coding
region (~99% of the human genome). This uninvestigated region
(as well as the exome) can, however, be studied by using a whole-
genome sequencing approach with better precision and accuracy
(Belkadi et al., 2015).

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6 | WHOLE- GENOME SEQUENCING
STUDIES
Whole- genome sequencing (WGS) is one of the next- generation
sequencing approaches that is now being used to study the eti-
opathogenesis of genetic disorders. Unlike whole- exome sequenc-
ing (WES), another next- generation sequencing that detects genetic
variants within the coding region (exome) of the genome, WGS can
be used to detect variant s within the non- coding and coding regions.
These non- coding regions account for 99% of the entire genome
and contain regulatory elements that control the gene expression.
Recent studies have investigated and improved our understanding
of the roles of these non- coding variants in the etiopathogenesis of
genetic disorders (French & Edwards, 2020; Li & Montgomery, 2013;
Wells et al., 2019; Zhang & Lupski, 2015).
A report that compared the performance of WGS and WES in
the detec tion of coding variants which are easy to interpret found
that WGS outperforms WES. The broader coverage of WGS gives
us the power to detect potential variants that would have been
missed by using WES (Gilissen et al., 2014; Saunders et al., 2012).
The study showed that WGS could detect about 3% coding vari-
ants that are missed by WES (Belkadi et al., 2015). Additionally,
WGS is more reliable in the detection of structural variants within
the coding region when compared to WES (Belkadi et al., 2015).
The comparable costs of WES and WGS, coupled with the other
factors, make WGS a more reliable choice in genetic variants
screening. As diagnostic tool, trio based WGS has a diagnostic
yield of about 42% which is comparable to that of WES 40% diag-
nostic yield (Wright et al., 2018).
In our quest to underst and the source and identify the missing
heritability of NSOFC, we have begun to explore next- generation
sequencing studies. A larger proportion of the missing heritability is
due to rare variants (Wainschtein et al., 2021). Prior investigation of
these rare pathogenic variants has been limited to candidate gene
sequencing approach which investigated the mutations in known
cleft genes and whole- exome sequencing which identifies only cod-
ing mutations. Whole- genome sequencing studies provide us with
the ability to investigate the entire genome for both coding and non-
coding, common and rare mutations that can explain the genetic risk
of NSOFC. In a single genomic data per individual, we can query the
entire 3 billion base pairs for variations that are pathogenic and can
affect the normal develop of the lip and palate. This genomic se-
quence data can be used to answer every research question as it
concerns the genetics of NSOFC.
One strategy that has been reported recently used the genomic
sequence to identify a new risk locus associated with NSOFC in a
multiethnic population (Mukhopadhyay et al., 2020). Unlike the
traditional GWAS, this strategy does not require imputation and
provides a deeper and more reliable sequence. Another strategy
is the investigation of de novo mutations (DNMs) that increase
the risk of this defect. DNMs are those germline mutations that
are found in the offspring but not carried by either parent. These
DNMs have been reported to increase the risk of congenital defects
(Alonso- Gonzalez et al., 2018; Homsy et al., 2015; Ji et al., 2020),
but limited studies are available for NSOFC. Majorit y of cleft cases
are sporadic thus explains the role of DNMs in the etiopathogen-
esis. One study that investigated the DNMs using whole- genome
sequence data in a NSOFC case- parent trios study design identified
excess loss- of- function DNMs in genes (IRF6, TFAP2A, and ZFHX4)
involved in craniofacial development and identified ZFHX4 as a novel
cleft candidate gene (Bishop et al., 2020).
Thus, with the WGS data and powerful study designs, we can
identify structural variants, common and rare variations that in-
crease the genetic risk of NSOFC. As we race toward improved
management, this genetic study approach will help reduce the
knowledge gap in disease etiology and take us a step closer to-
ward translation.
7 | COPY NUMBER VARIATIONS
In addition to the other methods for identifying risk variants for
orofacial clefts, copy number variations (CNVs) have been reported
in the etiology of complex traits like NSOFC. CNVs modif y gene
expression, disrupt gene, and alter gene dosage leading to syndro-
mic and NSOFC (Maarse et al., 2012). A genome- wide deletion-
association analysis identified a locus near ch7p14.1 associated with
NSOFC (Younkin et al., 2014). Using the same GWAS data set that
was used for the Beaty et al. (2010) study, genome- wide inherited
deletions identified a 67 kb deletion on Chr7q34 and a 200 kb de-
letion on chr8p11 (Younkin et al. 2015). In another CNV analyses
of 23 unrelated individuals with clefts and 200 controls, TCEB3 and
KIF7 were identified as new clef t candidate genes (Simioni et al.,
2015). A large cohort of 312 OFC patients analyzed 249 genomic
deletions and 226 duplications in two publicly accessible databases
(DECIPHER and ECARUCA) of chromosome imbalance. The follow-
ing known cleft candidate genes were identified in these deleted
and duplicated regions: SATB2, MEIS2, DGCR6, FGF2, FRZB, LETM1,
MAPK3, SPRY1, THB S1, TSHZ1, TTC28, TULP4, WHSC1, and WHSC2
(Conte et al., 2018).
Isthmin 1 was identified as a new OFC candidate gene when CNV
analyses was done for 97 NSOFC and 43 cases with CPO. Lansdon
et al. (2018) identified a heterozygous deletion of in one affected
case and deletion in a second case. The deletion removed the puta-
tive 3’ regulatory information from Isthmin 1 (Lansdon et al., 2018).
Another study replicated the findings in the 62kb deletion region
of 7p14 in 399 patients and 1318 controls and reported an asso-
ciation with NSOFC (p = 0.024). They also identified de novo de-
letion in three sporadic families, and both incomplete segregation
and incomplete penetrance in multiplex families. An investigation of
whole- genome high- resolution SNP in 33 patients with syndromic
and NSOFC with in normal karyotypes identified six novel CNVs
(3 in patients with NSOFC and 3 in patients with syndromic clefts).
The authors also identified two novel candidate genes for NSOFC;
K AT6 B and MACROD2 and confirmed the role of CNVs in the etiol-
ogy of NSOFC (Lei et al., 2016).

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8 | EPIGENETICS
Epigenetics is the reversible change in the expression of genes with-
out changes in the DNA sequence. These include DNA methylation,
histone modifications, and non- coding RNA. The role of epigenetics
changes has been well documented in the etiology of several dis-
eases including birth defects (Keil & Vezina, 2015; Wahlqvist et al.,
2015).
8.1 | DNA methylation and orofacial clefts
There are several studies that have conducted epigenetic- wide
association studies (EWAS) for orofacial clefts. A study in the UK
conducted EWAS using blood and lip tissues to test the association
between methylation at each site and cleft subtype (cleft lip only
(CLO) n = 50; cleft palate only (CPO) n = 50; cleft lip and palate (CLP)
n = 50). They found four genomic regions in blood differentially
methylated in CLO compared to CLP, 17 in CPO compared to CLP
and 294 in CPO compared to CLO. Interestingly, they found regions
that mapped to known clefts candidate genes like TBX1, COL11A 2,
HOXA2, and PDGFRA. In addition, they reported 250 novel differen-
tially methylated loci. They also found similar methylated regions in
blood and lip tissues (Sharp et al., 2017).
Another study used a Brazilian cohor t (67 NSCLP, 59 controls)
for EWAS and found 578 methylation variable positions (MVPs) or
differentially methylated regions were significantly associated with
NSCLP. They observed that these MVPs were enriched in regula-
tory and active regions of the genome and in known pathways for
craniofacial development. Four of the 11 MVP were replicated in an
independent UK cohor t (171 NSCLP, 177 controls). Like the study
by Sharp et al. (2017), they demonstrated a significant positive
correlation between blood and lip tissue DNA methylation suppor t-
ing the suitability of blood tissue for NSCLP methylation studies
(Alvizi et al., 2017). In a study that compared whole- genome DNA
methylation in six MZ twin pairs discordant for NSCLP, they found
differential methylation in MAFB and ZEB2 in two independent MZ
twin pairs. They also found common dif ferential methylation in
genes belonging to the Hippo signaling pathway.
Long interspersed nucleotide element- 1 (LINE- 1) is a marker of
global DNA methylation. Studies have reported differential global
DNA methylation patterns between NSOFC and controls using the
methylation levels of LINE- 1 (Cáceres- Rojas et al., 2020; Khan et al.,
2018, 2019; Li et al., 2019). There is evidence to suggest that ge-
netic variations (c.C677T and c.A1298C) in the MTHFR gene lead to
low DNA methylation levels (Frosst et al., 1995; van der Put et al.,
1998). Further evidence for the role of MTHFR gene variant in DNA
methylation has been reported for NSOFC where higher levels of
LINE- 1 methylation were obser ved on the medial side of cleft lip in
individuals with c.C677T genotypes (Khan et al., 2019).
Differentially methylated regions maybe enriched in clefts co-
hort, and these could explain the missing heritability for clefts.
While there may be similar methylated regions across populations,
there possibility of population- specific regions is high considering
the various types of environmental exposures within each popula-
tion. Therefore, exploring the methylation profiles of cleft families in
populations across the world will lead to a clear unders tanding of the
role of epigenetics in the etiology of orofacial clefts.
8.2 | Histone modification and microRNAs
The evidence supporting the role of histone modification in the
etiolog y of orofacial clefts in humans is sparse. Few studies have
FIGURE 1 Critical pathways and the
IRF6 gene- regulatory network relevant to
craniofacial development and reported for
non- syndromic orofacial clefts (NSOFCs).
Some clef t candidate genes in black fonts
within the pathways and net work. The
gene list, pathways, and network are
not exhaustive but represent current
knowledge and a starting point for further
investigations

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ALAD E Et AL.
explored the role in the development of cleft palate in the presence
of drugs and other exposures in mice (Cuiping et al., 2014; Yuan
et al., 2016). In contrast, the role of long non- coding RNAs (lncR-
NAs) and microRNAs in the etiology of orofacial clefts in humans
is becoming clearer and the evidence is growing. For instance, a
study identified 36 lncRNAs, 1,341 mRNAs, and 60 miRNAs to be
differentially expressed in the CL/P group compared to the control
group, and 57 lncRNAs, 1,255 mRNAs, and 162 miRNAs to be dif fer-
entially expressed in the CPO group compared to the control group.
These were identified when they conducted next- generation RNA
sequencing (RNA- seq) to identify mRNAs, lncRNAs, and miRNAs in
patients with CL/P and CPO (Gao et al., 2019). Another study com-
bined the DEMs during mouse embryonic palatal development with
DEMs in NSCL/P patients and identified a let- 7c- 5p- PIGA and miR-
193 a- 3p - TGFB2 net work that may play important roles in the devel-
opment of NSCL/P (Fu et al., 2021).
As we explore the missing heritabilit y in the etiology of orofa-
cial clefts, a comprehensive understanding of the role of histone
modifications, lncRNAs, and microRNAs will be extremely useful
and potentially translational toward the development of prevention
strategies.
9 | PATHWAYS AND GENE- REGULATORY
NETWORKS
Several pathways have been reported to play a role in palate devel-
opment, and genes in these pathways are potential clefting genes.
The WNT pathway is critical for craniofacial development, and WNT
pathway genes such as AXIN1 and WNT9B (Figure 1) have been
associated with NSOFC (Mani et al., 2010; Menezes et al., 2010).
Fgf10/Fgfr2/Shh signaling pathway is also essential for palate de-
velopment (Rice et al., 2004). Genes in this pathway such as FGFR1
and FGF2 have been associated with NSOFC (Riley & Murray, 2007).
Other pathways that have been reported to be important for pal-
ate development include the MSX1 pathway, Folate pathway, and
TGFB pathway (Figure 1). The IRF6 gene- regulatory network is also
critical in the development of the palate (Figure 1), and studies have
shown genes in this network to be associated with NSOFC (Kousa &
Schutte, 2016; Liu et al., 2016).
10 | POPULATION
Accurate phenotyping is the foundation for identif ying genetics
and genomics risks for NSOFC. Furthermore, population and geo-
graphical location of affected individuals with NSOFC are also very
important factors for considerations. Evidence from some genetic
studies shows that some loci are population- specific while other
loci have associations with multiple populations (Beat y et al., 2010).
This is mainly due to differences in allele frequencies and population
specificity between ancestral populations which was demonstrated
by the identification of risk loci at the chr8q24 locus in Caucasians
in Europe and North America and MAFB in Asians. Furthermore,
population specificity has been demonstrated for 15q22, which
reached genome- wide significance in Europeans and Mexicans only
but not in Asians,16p13.3, significant only in the Chinese population
and 2p13.1 where a significant association was identified in Native
Americans and not in Europeans or Asians (Ludwig et al., 2012,
2014; Masotti et al., 2018; Sun et al., 2015).
Finally, the inheritance mode needs to be clearly understood
for any investigation to be meaningful. For instance, a multiplex
family appears to be Mendelian, and approach will prioritize seg-
regation of risk variants. For sporadic cases, priority should be
placed on identifying de novo variants. However, investigators
and the community need to be aware of incomplete penetrance
of the phenotypes and variants as well as the polygenic and multi-
factorial inheritance for NSOFC. Bringing all these into the design
and strategies will improve our understanding and opportunities
for discover y.
11 | CHALLENGES AND FUTURE
DIRECTIONS
Discovery is the foundation for a successful clinical translational
process. For NSOFC, just like other polygenic disorders, the main
challenges will be the identification of the critical pathways for devel-
opment where variants / polymorphisms in candidate genes within
these pathways will lead to the disruptions at critical time points dur-
ing the development of the lip and palate. The challenges in discov-
ery may include access to populations around the world and accurate
phenotyping in resource limited populations. Future directions for
NSOFC should be focused on taking the knowledge from discovery
to translation. This will involve the development of diagnostic tools
for pregnancy screening to enable counseling and pregnancy plan-
ning. It should also involve in utero interventions to rescue the clefts.
While this is theoretically possible and showing promise in animal
models, the success in humans will be a game changer.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of
Health (NIH DE028300).
CONFLICT OF INTEREST
None.
AUTHOR CONTRIBUTIONS
Azeez Alade: Writing – original draft; Writing – review & editing.
Waheed Awotoye: Writing – original draft; Writing – review & edit-
ing. Azeez Butali: Conceptualization; Methodology; Project adminis-
tration; Writing – original draft; Writing – review & editing.
DATA AVAIL ABILI TY STATEMENT
Data sharing not applicable to this article as no datasets were gener-
ated or analyzed during the current study.

8
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ORCID
Azeez Butali https://orcid.org/0000-0002-1229-5964
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