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RESEARCH ARTICLE

Novel TBX22 mutations in Chinese no-syndromic Cleft Lip/Palate families

Running title: TBX22 mutations in Chinese CL/P families

Key words: no-syndromic; cleft lip; cleft palate; TBX22; Chinese

Jiewen Dai1, Chen Xu2, Guomin Wang1, Yun Liang1, Teng Wan1, Yong Zhang1,

Xiaofeng Xu1, Lebin Yu2, Zonggang Che2, Qiqing Han2, Dandan Wu1*, Yusheng

Yang1*

Jiewen Dai and Chen Xu are co-first authors.

1Department of Oral & Cranio-maxillofacial Surgery, Shanghai ninth People’s

Hospital, Shanghai JiaoTong University, School of Medicine, Shanghai, 200011,P.R.

China

2Department of Oral and Maxillofacial Surgery, Central Hospital of ZiBo, Shandong

Zibo 255036, P.R. China

*Co-corresponding authors. Yusheng Yang & Dandan Wu. Department of Oral &

Cranio-maxillofacial Surgery, Shanghai ninth People’s Hospital, Shanghai JiaoTong

University, School of Medicine, NO. 639 Zhizaoju Road, Shanghai, 200011, P.R.

China. E-Mail: yysdj4829@yahoo.com.cn (Y.S.Y). 179554314@qq.com (D.D.W)

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Abstract

TBX22 is a contribute gene of Cleft Lip/Palate, and many mutations have been

reported. However, the exact role of TBX22 mutation in Chinee no-syndromic Cleft

Lip/Palate (NSCL/P) family has not been clearly explored. In this study, we tried to

investigate the profiles and effects of TBX22 mutation in Chinese NSCL/P family.

Members in two Chinese NSCL/P families, and 200 normal controls were enrolled in

this study. Then DNA sequence and bioinformatic analysis for TBX22 were performed.

The results showed that a novel and essential splicing site mutation, IVS6-1G>C, was

detected in a family with cleft palate. The bioinformatic analysis results showed that

this mutation would lead to abnormal transcription or translation, followed by leading

to a loss of function of TBX22. In addition, A hemizygous missense mutation,

c.874G>A (p.D292N), was firstly showed in another Chinese family, which may

exhibit aggravated effects on the phenotypes of CL/P. Taking these findings together,

this study provided a profile of TBX22 mutation in Chinese NSCL/P families, and

further confirmed the important role of TBX22 in familial cases with X-linked cleft

palate.

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Introduction

Orofacial clefts, including cleft lip/palate (CL/P) are common congenital birth

defects in human, and the reported prevalence is about 1/700 to 1/2000 for live births

in worldwide (Murray 2002), as well as about 1.64‰ in China. The etiology of

orofacial clefts is greatly unclear, and a generally accepted consensus is multiple

factors, including genetic and environmental factors, contribute to orofacial clefts

(Wu et al. 2012). Many efforts have been carried out to identify these genetic

determinants of orofacial clefts in different regions and different populations, and a

number of genes, such as IRF6, p63, PVRL1, MSX1 and TBX22, have been reported to

be related to syndromic or Mendelian clefts (Dai et al. 2014; Dai et al. 2015). TBX22,

a transcription factor of T-box gene family that shares a highly conserved

DNA-binding domain which encodes 180 amino acids, mainly expressed in palatal

shelves and the base of tongue during craniofacial development (Braybrook et al.

2001). Previous genetic models also showed that TBX22 played crucial role in

palatogenesis and glossogenesis (Haenig et al. 2002; Pauws et al. 2009; Fuchs et al.

2010).

X-linked cleft palate (CPX) is a semidominant disorder characterized by a strong

X linked Mendelian inheritance, and an isolated cleft of the secondary palate,

sometimes accompanied with ankyloglossia (tongue-tie). Its phenotypes exhibit great

varies from a highly arched palate to a complete cleft of the secondary palate.

Previously, several CPX families were described in different populations (Bjornsson

et al. 1989; Gorski et al. 1992), and showed that CPX was caused by mutations in

TBX22 (Braybrook et al. 2001). Many mutations of TBX22 have been reported,

including nonsense, frame-shift, splice-site, or missense mutation (Pauws et al. 2013).

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Except for familial cases with well-defined CPX, TBX22 mutations also have been

found in many sporadic on-syndromic cleft palate cases (Marcano et al. 2004;

Suphapeetiporn et al. 2007), as well as cleft lip with palate, hypodontia and limb

anomaly (Kantaputra et al. 2011; Kaewkhampa et al. 2012). All these findings

inspired us to investigate the possible roles of TBX22 in Chinese NSCL/P families.

In this study, we sequenced TBX22 in members from two Chinese NSCL/P

families and 200 normal controls, and bioinformatic analysis for TBX22 mutation was

performed to explore the profiles and effects of TBX22 mutation in Chinese NSCL/P

family.

Materials and methods

Patients

The protocols were approved by the Ethics Committee of Shanghai Ninth People’s

Hospital, Shanghai Jiaotong University School of Medicine, and written informed

consents were obtained from all patients or their parents. Members in two Chinese

NSCL/P families and 200 controls were included in this study, and the blood samples

were collected from them.

Family one (Fig. 1A) with CPX characteristics and features was included to

analyze TBX22 mutations. The female proband was presented with isolated cleft

palate (CPI). Her father and two uncles also had CPI. Her family was Han Chinese

origin, and also included another two affected males and two affected females.

Inconsistent with previously reported classic CPX families, family history taking or

inspection excluded ankyloglossia in all these affected members, as well as female

carriers.

Family two, the male proband was presented with cleft lip, cleft palate and cleft

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alveolar, and his father exhibited cleft lip and slight cleft alveolar, whereas his mother

exhibited no CL/P and cleft alveolar phenotypes.

200 control samples were peoples who admitted to our hospital due to trauma

and had no orofacial clefts or others congenital diseases, and had no history of oral

clefts in their family members.

Sequence and mutation analysis

Genomic DNA was extracted from blood samples according to the standard

protocols. Primer 3 (version 0.4.0, http://frodo.wi.mit.edu/) was used for primer

design. The polymerase chain reaction (PCR) was conducted in a 10L reaction

system. PCR products were purified with ExoSAP-IT (USP Corp., Cleveland, OH)

and sequenced using BigDye terminator chemistry and ABI 3700 DNA sequencer

according to the standard procedure. The results were analyzed using Sequenche

(version 4.2; Gene Codes Corp., Ann Arbor, MI), and were compared to the

corresponding normal mRNA (NCBI Reference Sequence: NM_001109878.1), cDNA

and genomic DNA sequences. For variation sites, 200 controls from unaffected

ethnic-matched people were screened.

Putative missense mutations were analyzed in silico by 3 Web-based programs:

PANTHER (http://www.pantherdb.org/), PolyPhen

(http://genetics.bwh.harvard.edu/pph/) and SIFT (http://sift.jcvi.org/). Augustus

(http://bioinf.uni-greifswald.de/augustus/submission) and Netgene2

(http://www.cbs.dtu.dk/services/NetGene2/) were used to predict the effect of splicing

site mutations. Phyer2 was used to predict the 3D structure of TBX22 protein (Kelley

et al. 2015).

Protein sequence comparison

Protein sequence alignments were completed by BioEdit Sequence Alignment

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Editor. The complete TBX22 protein sequences and corresponding proteins were first

downloaded from the Ensembl genome browser (http://asia.ensembl.org/) and saved

as FASTA format. Then protein sequence was imported into the BioEdit Sequence

Alignment Editor software. The human TBX22 protein sequence was aligned with

mouse, rat, monkey and rabbit TBX22 protein sequence, as well as human TBX1,

TBX4, TBX15 and TBX21 protein sequence using the function of ClustalW Multiple

alignment.

Results

IVS6-1G>C mutation in family one

An IVS6-1G>C transversion mutation was detected (Fig. 1B) in family one. The

proband was marked with an arrow in Fig. 1A. The same mutation also was found in

female carriers, as well as the affected people in this family, but absent in the

unaffected members. Although the typical CPX and ankyloglossia were not observed

based on examination and family history, a semi-dominant X linked inheritance was

clearly established. To our knowledge, this was the first CPX family reported in

Chinese population about the splicing site mutation in TBX22 according to Human

Gene Mutation Database. This mutation was absent in all the 200 controls.

The possible effect of this splicing site mutation was predicted by Augustus and

Netgene2, and the Augustus analysis results showed that the stop codon appeared in

advance, and only 5 CDS sequence were obtained (Fig.2A). The Netgene2 analysis

results showed that the normal splicing site “tccatagattac” disappeared when the

mutation existed (Fig.2B). Only 265 amino acids would be obtained based on the

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translation from the residue 5 CDS sequence (Fig.2C), whereas there were 520 amino

acids in normal human TBX22 protein, and the structures of the mutant TBX22

protein also were disturbed when compared with normal protein (Fig.3). These

findings implied that IVS6-1G>C transversion mutation may lead to a loss of function

of TBX22 protein.

c.874G>A mutation in family two

A single base substitution (c.874G>A; p.D292N) was identified in exon 7 in

family two (Fig. 4A, B). The proband was a boy with unilateral complete cleft lip and

palate. The unaffected mother carried the same mutation. His father exhibited

unilateral cleft lip and alveolar, but did not carry this mutation. This change was not

found in 200 controls and was not present in the SNP database or 1000 Genomes

database, which also was firstly reported in Chinese population. This mutation located

outside the T-box domain, resulting in a substitution of aspartic acid residue to

asparagine residue (p.D292N). Aspartic acid residue at the position 292 of the

polypeptide was conserved across species, while not conserved in the TBX1, TBX4,

TBX15 and TBX21 subfamily of T-Box protein (Fig. 4C). The SIFT analysis score was

0.15, meaning the change might be tolerated. PANTHER and Polyphen predicted this

variation may be harmful. The 3D structure predicted by Phyer2 software showed that

this mutation would modify the secondary structures of TBX22 protein, and a new

alpha helix followed the T-Box region was observed (Fig.5).

Discussion

The important role of TBX22 in the development of cleft palate has been greatly

confirmed in recent researches. Previous study showed that up to 4% of unselected

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cleft palate cases have TBX22 mutations (Marcano et al. 2004). A study in the Thai

population identified four potentially pathogenic mutations in 53 non-syndromic cleft

palate cases (Suphapeetiporn et al. 2007). Unlike these positive reports, no new

mutation was detected in sporadic CPI patients in china. This heterogeneity may be

explained by the difference of ethnic or geographical origin.

A Chinese family with X-linked CP was reported in this study. The IVS6-1G>C

mutation happened in the essential splicing junction that should be strictly conserved

according to the “GT-AG rule”, and leaded to the missing of one transcription

acceptor, followed by disturbing the cut off of intron 5, which may disturb the

function of TBX22 through three most possible mechanisms: a premature transcription

termination, an exon skipping or an intron retention. All of these changes would result

in truncated or unstable TBX22 mRNA, and nonsense-mediated decay may occur to

avoid producing an aberrant protein by removing the entire transcript. Braybrook et al

reported an IVS6+1 mutation in an Icelandic family (Braybrook et al. 2002), and did

not detect any PCR product across the mutation site, as well as other portions of the

transcript, which supported the theory of nonsense-mediated decay. In addition, even

a truncated protein was obtained from the truncated mRNA, the structure and function

of the protein may be disturbed, which was confirmed in this study by our prediction

of protein structure, and may affect DNA binding, sumoylation or transcriptional

repression that were reported in previous studies (Andreou et al. 2007; Fu et al.

2015).

This reported Chinese CPX family exhibited two features. One feature was

although the pedigree size and family genogram implied X-linked inheritance, the

typical incidental symptom, ankyloglossia (Kantaputra et al. 2011), was not found in

any affected, unaffected or carried family members. Previous researchers analyzed

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CPX families and concluded that ankyloglossia was a common phenotype associated

with TBX22 mutations (Marcano et al. 2004), but its occurrence was not a prerequisite

for cleft palate. In addition, the cleft palate and ankyloglossia were independent

effects of loss of TBX22 function. A previous study on Finnish population also

showed that mutations in TBX22 was not a major cause of ankyloglossia (Klockars et

al. 2012). All of these findings implied that semidominant tendency characterized by a

strong X-linked Mendelian inheritance of cleft palate, with or without ankyloglossia,

may be an indicator for a diagnosis of CPX. The other feature was the diversity of

phenotypes of the heterozygous females in this family. Females who harbor this

mutation have either cleft soft palate or exhibited no defect. A possible mechanism

may due to the non-random X inactivation.

In family two, a boy with cleft lip and palate had an inherited mutation of

c.874G>A (p.D292N), and was obtained from his healthy mother. His father had cleft

lip and cleft alveolar, but did not carry the mutated TBX22. The position of mutation

located outside the T-box domain, and resulted in substitution of an aspartic acid

residue by an asparagine residue. The mutation altered the charge of TBX22 protein,

but did not alter the chemical polarity of the amino acid, and the silico prediction

results showed low scores and a weak functional impact. However, 3D structure

predicted by Phyer2 software showed that the mutation would modify the secondary

structures of TBX22 protein. Unlike the T-box domain, little was known about the

effect of C-terminal domain where possible functional domain may exist. In addition,

whether the change of charge would influence the transcription regulation and

protein-protein interaction also needed to be further investigated. Although given the

phenotypes of the proband and his father, we can not exclude the involvement of

others autosomal gene that results in the cleft phenotypes, this mutation of TBX22

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may aggravate the phenotypes of the proband when compared to his father, and the

cleft lip and cleft alveolar may be caused by others mutant gene that obtained from his

father, whereas cleft palate was caused by mutation of TBX22 that obtained from his

mother. Of course, the mechanism need to be further investigated in future. A similar

phenomenon also had been reported in a previous study (Kaewkhampa et al. 2012).

In summary, this study reported a novel and essential splicing site mutation,

IVS6-1G>C, in a family with cleft palate, which would lead to abnormal transcription

or translation, followed by resulting in a loss of function of TBX22 protein. In

addition, a hemizygous missense mutation, c.874G>A (p.D292N), was firstly showed

in another Chinese family, which may exhibit aggravated effects on the phenotype of

CL/P. All of these findings provided a profile of TBX22 mutation in Chinese NSCL/P

families, and further confirmed the important role of TBX22 in familial cases with

X-linked cleft palate. Functional study and screening in larger sample sizes are

recommended to enrich the molecular mechanisms of TBX22 that contribute to CL/P.

Acknowledgments

We thanks the patients and their families for participating in our study, and being

gracious with their time and effort. We also thank the research teams of Birth Defect

Research Center & Pathology Research Center, Fudan University, Shanghai, China,

who helped with this research.

The study is supported by Interdisciplinary Program of Shanghai Jiaotong University

(Nos.YG2016MS08, YG2016QN11), National key research and development

program (No. 2016YFC1000502), National Natural Science Foundation of China (No.

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81300842).

Conflict of interest

The authors declare no competing financial interests.

References

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Figure legends

Figure.1. Pedigree and gene sequencing results of members in family one. (A) Three

generation members in a Chinese family with cleft palate. Filled symbols indicate a

cleft palate phenotype; circles with dots indicate obligate female carriers. (B)The

sequence traces showed the IVS6-1G>C mutation in affected male, as well as carried

and affected female.

Figure.2. Analysis of IVS6-1G>C mutation of TBX22 using Augustus and Netgene2

software. (A) Sequence analysis of normal TBX22 (above the black line) and TBX22

IVS6-1G>C mutant (under the black line) by Augustus software showed that normal

TBX22 mRNA had 8 CDS and 7 introns which was similar with the actual situation,

whereas TBX22 with IVS6-1G>C mutation had only 5 exons, and the stop codon

appeared in advance. (B) Netgene2 analysis showed that the normal splicing site

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“tccatagattac” was recognized as a transcription acceptor (number 13100, red

straight line)， whereas the acceptor disappeared (dotted line) when the IVS6-1G>C

mutation existed. (C) 265 amino acids were obtained based on the translation from the

residue 5 CDS sequence using the translate tool in ExPASy on-line software.

Figure.3. The 3D structure of normal and IVS6-1G>C mutant TBX22 protein that

predicted by Phyer2 software.

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Figure.4. Information and sequencing results of TBX22 in family two. (A) The

proband exhibited cleft lip and palate, and father exhibited cleft lip and cleft alveolar

but without cleft palate, whereas his mother exhibited no CL/P phenotypes. A

mutation of c.874G>A (p.D292N) was detected in the proband and his mother, but did

not be detected in his father. (B) Sequence traces showed the proband (hemizygote)

and his mother (heterozygote) have c.874G>A (p.D292N) variant. (C) Aspartic acid

residue at the position 292 of TBX22 protein chain is conserved across species, while

not conserved in the TBX1, TBX4, TBX15 and TBX21 subfamily of T-Box protein.

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Figure.5. The 3D structure of normal and c.874G>A (p.D292N) mutant TBX22

protein that predicted by Phyer2 software. (A) The 3D structure of TBX22 protein

showed that the mutation of c.874G>A (p.D292N) would not obviously disturb the

T-Box domain (rectangle region), but would lead to appearance of a new alpha helix

followed the T-Box region (arrow head). (B) Analysis of the secondary structure of

TBX22 protein also showed appearance of a new alpha helix around the position 292.

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