Association of MSX1 c.*6C > T Variant with Nonsyndromic Cleft Lip With or Without Cleft Palate in Turkish Patients

Abstract

Objective: Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common birth defects, with a birth prevalence of 1/1000 in Caucasians. MSX1 (muscle segment homeobox gene 1) is a strong candidate gene for nsCL/P. The aim of this study was to investigate the association between MSX1 variants and nsCL/P in Turkish patients. Patients and Methods: Our study included 80 patients with nsCL/P and 125 age-matched healthy individuals. Genomic DNA was isolated from peripheral blood leukocytes and exon 2 of the MSX1 gene was amplified using polymerase chain reaction (PCR). After PCR, we sequenced the products using an automated sequencer. Results: We found the c.*6C > T variation in the MSX1 gene. This variant in the 3′ untranslated region is located 6 bp downstream of the stop codon (TAG) in exon 2. Forty-eight individuals (60%) of 80 in the case group had the CT genotype. We revealed a statistically significant association between the MSX1 c.*6C > T variant and nsCL/P in Turkey (p = 0.01). Conclusion: Our identification of the c.*6C > T variant appears to be the first reported result associating variants of the MSX1 gene with nsCL/P patients.

Introduction

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, OMIM 119530) is among the most common and significant congenital malformations (Carinci et al., 2003). Its prevalence ranges from 1/700 to 1/1000 among Caucasians (Fraser, 1970). The prevalence rate changes according to geographical origin, sex, racial background, ethnicity, and socioeconomic status (Vanderas, 1987; Croen et al., 1998; Clark et al., 2003). The frequency of nsCL/P is the highest in Asian and the lowest in African populations (Bhaskar et al., 2011). The etiology of nsCL/P is multifactorial, involving both genetic and environmental factors (Murray, 2002).

Numerous association studies have reported candidate genes for nsCL/P, including IRF6, MTHFR, PVRL1, TGFB1, TGFB3, TGFA, BMP4, and CBS, in different populations. The muscle segment homeobox gene 1 (MSX1) is strongly associated with nsCL/P in different populations on a consistent basis (Blanton et al., 2005; Turhani et al., 2005; Carinci et al., 2007; Zhu et al., 2010; Martinelli et al., 2011; Chen et al., 2012; Singh and Ramu, 2012; Stoll et al., 2015).

The MSX1 gene, which encodes a DNA binding sequence, is localized to chromosomal region 4p16.1 (Lace et al., 2006). The gene consists of two exons of 704 and 1229 bp. It has been shown that MSX1 plays a role in the programming of craniofacial morphogenesis and in the development of teeth and the craniofacial skeleton (Pawlowska et al., 2009). MSX1 variants are found in ∼2% of the patients with nsCL/P. Some studies have shown numerous mutations and polymorphisms in the MSX1 gene (Jezewski et al., 2003; Suzuki et al., 2004; Tongkobpetch et al., 2006). One such variation is c.*6C>T (rs8670). It involves a cytosine-to-thymine change in the 3′ untranslated region (UTR) of exon 2. This variation in the 3′ UTR is located 6 bp downstream from the stop codon (TAG) in exon 2. *6C>T does not result in an amino acid substitution in the product of the MSX1 gene (Modesto et al., 2006).

The 3′ UTR of genes plays an important role in the translation efficiency, localization, and stability of mRNA. A single alteration in this region may result in the altered expression of many genes; therefore, variants of 3′ UTR are very significant (Mignone et al., 2002; Chatterjee and Pal, 2009). The aim of this study was to investigate the association between MSX1 variants and nsCL/P in Turkish patients.

Patients and Methods

Study population

We enrolled 80 children aged 0-10 years with nsCL/P (the case group) in this study and admitted them to the Department of Orthodontics at Ankara University Faculty of Dentistry. Blood samples were collected from them. The selection criterion for the nsCL/P group was no association with any other major malformation. All cases were examined and screened for the presence of an association between nsCL/P and other syndromes.

The control group comprised 125 healthy children aged 0-10 years with no reported familial history of orofacial clefts. The mothers of the case and control group children were interviewed using the same questionnaire. They were asked to give their age at pregnancy and periconceptional use of vitamin supplements containing folic acid, as well as their smoking and alcohol consumption. The study was approved by the Clinical Research Ethics Committee of Ankara University Faculty of Dental Medicine.

Molecular analysis

Genomic DNA was isolated from peripheral blood samples by the conventional phenol-chloroform (Merck) method using proteinase K (MBI Fermentas). Peripheral blood samples were collected in test tubes containing EDTA.

Approximately 200 ng of genomic DNA was used as a template for polymerase chain reaction (PCR) amplification. The primer set was as follows: exon 2 forward primer: 5′-GGCTGATCATGCTCCAATGC-3′ and reverse primer: 5′-CACCAGGGCTGGAGGAA-3′. PCR was carried out in a volume of 50 μL containing 200 ng of template DNA, 1 U Taq polymerase (Fermentas), 1× (NH4)₂SO₄ buffer, 10 pmol of each primer, 0.2 mM of all four dNTPs (Fermentas), and 1.5 mM MgC1₂. The PCR conditions were as follows: 5 min at 95°C, 35 cycles at 94°C for 40 s, 40 s at 58°C, 40 s at 72°C, and a final 10-min elongation step at 72°C. A 556-bp fragment was amplified by PCR. The PCR products were electrophoresed in a 2% agarose gel containing ethidium bromide (Fig. 1). After PCR, we sequenced them using an automated sequencer (Beckman-Coulter CEQ™ 2000XL DNA Analysis System).

FIG. 1.

PCR products of the MSX1 gene in 2% agarose gel. M, 100-bp DNA ladder molecular weight markers. Lanes 1-4, 556-bp PCR products. MSX1, muscle segment homeobox gene 1; PCR, polymerase chain reaction.

Statistical analysis

Genotype and allele frequencies were calculated by counting the genotypes and compared with the predicted values using the chi-square test, based on the assumption of Hardy-Weinberg equilibrium. Statistical significance was accepted at p < 0.05.

Results

In our study, 80 Turkish patients with nsCL/P and 125 healthy individuals were studied. We found a known variation, c.*6C > T (rs8670), which involves a cytosine-to-thymine change in the 3′ UTR of exon 2. Figure 2 shows the c.*6C > T variation in the 3′ UTR of exon 2 in MSX1.

FIG. 2.

Sequencing chromatograms for control (A) and c.*6C>T heterozygote (B) in the 3′ UTR of the MSX1 gene. The polymorphism involves a cytosine-to-thymine transition in the 3′ UTR of exon 2. Arrow indicates the altered nucleotide, and sequencing chromatograms display the forward sequence of the DNA strand. UTR, untranslated region.

The genotype and allele frequencies of the c.*6C > T variant in nsCL/P cases and controls are shown in Table 1. In the case group (n = 80), 32 individuals (40%) had the CC genotype and 48 (60%) were heterozygotes. In the control group (n = 125), 73 individuals (58%) had the CC genotype and 52 (42%) were heterozygotes. The frequency of the CT genotype was significantly higher in the case group than in the control group (odds ratio = 2.10, 95% CI 1.18-3.72, p = 0.01).

Table 1.

Genotype and Allele Frequency of the c.^*6C>T Variant Among nsCL/P Case and Control Groups

Variable	Control group, n = 125 (%)	Case group, n = 80 (%)	OR (95% CI)	p
Genotype
CC	73 (0.58)	32 (0.40)	1
CT	52 (0.42)	48 (0.60)	2.10 (1.18-3.72)	0.01
TT	0 (0.0)	0 (0.0)	2.28 (0.04-117)	0.51
Allele C	198 (0.79)	112 (0.70)	1
Allele T	52 (0.21)	48 (0.30)	1.63 (1.03-2.57)	0.03

nsCL/P, nonsyndromic cleft lip with or without cleft palate.

Regarding the frequency of the TT genotype, the likelihood of nsCL/P was significantly higher in patients than in the control group (odds ratio = 2.28, 95% CI 0.04-117, p = 0.51). The frequency of T alleles was found to be higher in nsCL/P patients than in controls (p = 0.03).

Discussion

nsCL/P is one of the most common birth defects, involving environmental and genetic factors (Stuppia et al., 2011). MSX1 is a strong candidate gene for nsCL/P. In 1996, the MSX1 gene missense mutation was first found in a family with autosomal dominant tooth agenesis by Vastardis et al. (1996). MSX1 was initially described as a candidate based on the CL/P and foreshortened maxilla phenotype in the knockout mouse (Satokata and Maas, 1994). Gong (2001) reported a misregulation of the expression of MSX1 in the embryos of A/WySn mice with cleft palate. Satokata and Maas (1994) also reported that MSX1 homozygotes display a cleft secondary palate, alveolar mandible deficiency, and tooth development failure (Gong, 2001).

The MSX1 regulatory proteins function as transcriptional repressors and are involved in the development of the craniofacial, limb, and nervous systems. All variants can potentially disrupt the gene function by altering gene activation or transcriptional regulation (Alappat et al., 2003). Some studies have shown the presence of an association between candidate genes and nsCL/P (van den Boogaard et al., 2000; Otero et al., 2007; Singh and Ramu, 2012). However, one study has shown an association between the 3′ UTR of MSX1 and nsCL/P. Modesto et al. (2006) observed an association between the MSX1 gene *6C > T variant and CL/P. They searched for mutations in the MSX1 gene in 33 individuals with CL/P and reported that the *6C > T variant was more common in individuals with CL/P than in controls (p = 0.001) (Modesto et al., 2006).

In this study, we detected c.*6C > T (g.8485C > T) in the 3′ UTR of exon 2 in MSX1. This variation in the 3′ UTR is located 6 bp downstream of the stop codon (TAG) in exon 2. *6C > T does not result in an amino acid substitution in the product of the MSX1 gene.

Ceyhan et al. (2014), who researched MSX1 gene mutations in Turkish children with nonsyndromic tooth agenesis and other dental anomalies, showed that there was a *6C > T base change in the 3′ UTR of the second exon of this gene.

In the present study, the CT genotype of the MSX1 *6C > T variant was significantly more common in Turkish patients with nsCL/P than in controls (p = 0.01). In addition, the likelihood of having the TT genotype was 2.28-fold higher in Turkish patients with nsCL/P compared with that of having the CC genotype.

Our findings are supported by the study of Modesto et al. (2006), who showed that the *6C > T variant was more common in CL/P patients (p = 0.01). The current report on the *6C > T variant appears to be the first report on the MSX1 gene in nsCL/P patients.

The 3′ UTR of genes plays an important role in the translation efficiency, localization, and mRNA stability because it contains regulatory regions that affect the gene expression through post-transcriptional regulation (Mignone et al., 2002). Variations in the 3′ UTR can be very consequential as only one alteration can be responsible for the altered expression of many genes (Chatterjee and Pal, 2009).

In addition, the 3′ UTR is described by secondary structures that have regulatory functions. Chen et al. (2006) performed a study on 83 disease-related variants found in the 3′ UTR of various mRNAs and showed an association between the functional roles of known variants and changes in the secondary structure. In conclusion, these findings may be helpful in understanding the involvement of the MSX1 c.*6C > T gene in the etiology and pathogenesis of nsCL/P. Studies on populations with different ethnic backgrounds with a larger number of nsCL/P patients may be beneficial to verify this association and to elucidate the role of MSX1 *6C > T in nsCL/P.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Alappat

, Zhang

, Chen

(2003) Msx homeobox gene family and craniofacial development. Cell Res, 13:429-442.

Bhaskar

, Murthy

, Venkatesh Babu

(2011) Polymorphisms in genes involved in folate metabolism and orofacial clefts. Arch Oral Biol, 56:723-737.

Blanton

, Cortez

, Stal

, et al. (2005) Variation in IRF6 contributes to nonsyndromic cleft lip and palate. Am J Med Genet A, 137A:259-262.

Carinci

, Pezzetti

, Scapoli

, et al. (2003) Recent developments in orofacial cleft genetics. J Craniofac Surg, 14:30-143.

Carinci

, Scapoli

, Palmieri

, et al. (2007) Human genetic factors in nonsyndromic cleft lip: an update. Int J Pediatr Otorhinolaryngol, 71:1509-1519.

Ceyhan

, Kirzioglu

, Sahin Calapoglu

(2014) Mutations in the MSX1 gene in Turkish children with non-syndromic tooth agenesis and other dental anomalies. Indian J Dent, 5:172-182.

Chatterjee

, Pal

(2009) Role of 5′- and 3′-untranslated regions of mRNAs in human diseases. Biol Cell, 101:251-262.

Chen

, Férec

, Cooper

(2006) A systematic analysis of disease-associated variants in the 3′ regulatory regions of human protein-coding genes II: the importance of mRNA secondary structure in assessing the functionality of 3′ UTR variants. Hum Genet, 120:301-333.

Chen

, Wang

, Hetmanski

, et al. (2012) BMP4 was associated with NSCL/P in an Asian population. PLoS One, 7:e35347.

10.

Clark

, Mossey

, Sharp

, et al. (2003) Socioeconomic status and orofacial clefts in Scotland, 1989 to 1998. Cleft Palate Craniofac J, 40:481-485.

11.

Croen

, Shaw

, Wasserman

, et al. (1998) Racial and ethnic variations in the prevalence of orofacial clefts in California, 1983-1992. Am J Med Genet, 79:42-47.

12.

Fraser

(1970) The genetics of cleft lip and cleft palate. Am J Hum Genet, 22:336-352.

13.

Gong

(2001) Phenotypic and molecular analyses of A/WySn mice. Cleft Palate Craniofac J, 38:486-491.

14.

Jezewski

, Vieira

, Nishimura

, et al. (2003) Complete sequencing shows a role for MSX1 in non-syndromic cleft lip and palate. J Med Genet, 40:399-407.

15.

Lace

, Vasiljeva

, Dundure

, et al. (2006) Mutation analysis of the MSX1 gene exons and intron in patients with nonsyndromic cleft lip and palate. Stomatologija, 8:21-24.

16.

Martinelli

, Masiero

, Carinci

, et al. (2011) New evidence for the role of cystathionine beta-synthase in non-syndromic cleft lip with or without cleft palate. Eur J Oral Sci, 119:193-197.

17.

Mignone

, Gissi

, Liuni

, et al. (2002) Untranslated regions of mRNAs. Genome Biol, 3:REVIEWS0004.

18.

Modesto

, Moreno

, Krahn

, et al. (2006) MSX1 and orofacial clefting with and without tooth agenesis. J Dent Res, 85:542-546.

19.

Murray

(2002) Gene/environment causes of cleft lip and/or palate. Clin Genet, 61:248-256.

20.

Otero

, Gutiérrez

, Cháves

, et al. (2007) Association of MSX1 with nonsyndromic cleft lip and palate in a Colombian population. Cleft Palate Craniofac J, 44:653-656.

21.

Pawlowska

, Janik-Papis

, Wisniewska-Jarosinska

, et al. (2009) Mutations in the human homeobox MSX1 gene in the congenital lack of permanent teeth. Tohoku J Exp Med, 217:307-312.

22.

Satokata

, Maas

(1994) Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet, 6:348-356.

23.

Singh

, Ramu

(2012) Association of MSX1 799G>T variant with nonsyndromic cleft lip/palate in South Indian adolescent patients. Int J Paediatr Dent, 22:228-231.

24.

Stoll

, Mengsteab

, Stoll

, et al. (2015) Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults. BMC Med Genet, 22:5-15.

25.

Stuppia

, Capogreco

, Marzo

, et al. (2011) Genetics of syndromic and nonsyndromic cleft lip and palate. J Craniofac Surg, 22:1722-1726.

26.

Suzuki

, Jezewski

, Machida

, et al. (2004) In a Vietnamese population, MSX1 variants contribute to cleft lip and palate. Genet Med, 6:117-125.

27.

Tongkobpetch

, Siriwan

, Shotelersuk

(2006) MSX1 mutations contribute to nonsyndromic cleft lip in a Thai population. J Hum Genet, 51:671-676.

28.

Turhani

, Item

, Watzinger

, et al. (2005) Mutation analysis of CLPTM 1 and PVRL 1 genes in patients with non-syndromic clefts of lip, alveolus and palate. J Craniomaxillofac Surg, 33:301-306.

29.

van den Boogaard

, Dorland

, Beemer

, et al. (2000) MSX1 mutation is associated with orofacial clefting and tooth agenesis in humans. Nat Genet, 24:342-343.

30.

Vanderas

(1987) Incidence of cleft lip, cleft palate, and cleft lip and palate among races: a review. Cleft Palate J, 24:216-225.

31.

Vastardis

, Karimbux

, Guthua

, et al. (1996) A human MSX1 homeodomain missense mutation causes selective tooth agenesis. Nat Genet, 3:417-421.

32.

Zhu

, Hao

, Li

, et al. (2010) MTHFR, TGFB3, and TGFA polymorphisms and their association with the risk of non-syndromic cleft lip and cleft palate in China. Am J Med Genet, 152A:291-298.