International Journal of Pediatric Otorhinolaryngology 79 (2015) 1081–1084

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Evidence of the involvement of the polymorphisms near MSX1 gene in non-syndromic cleft lip with or without cleft palate Venkatesh Babu Gurramkonda a, Altaf Hussain Syed b, Jyotsna Murthy b, Bhaskar V.K.S. Lakkakula a,c,* a b c

Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India Department of Plastic Surgery, Sri Ramachandra University, Chennai, India Sickle Cell Institute Chhattisgarh, Raipur, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 March 2015 Received in revised form 18 April 2015 Accepted 21 April 2015 Available online 29 April 2015

Objective: Non-syndromic cleft lip, with or without cleft palate (NSCL/P) is a common craniofacial birth defect, characterised by an incomplete separation between nasal and oral cavities without any other congenital anomaly in humans. Several genes which play a role in cell differentiation, migration, growth and apoptosis, have been associated with clefting. The purpose of this study was to investigate the association between single-nucleotide polymorphisms (SNPs) near MSX1 gene and NSCL/P among South Indian population. Methods: A case-control analysis of five single nucleotide polymorphisms near MSX1 gene (rs11726039, rs868257, rs6446693, rs1907998 and rs6832405) was carried out in 173 patients with NSCL/P and 176 unaffected controls to determine their association with NSCL/P. Results: All SNPs were polymorphic in the study population. Comparisons of allele and genotype frequencies revealed that the C variant allele and the TC/CC genotypes of rs11726039 was significantly higher in controls than in the NSCL/P group (OR: 0.63; 95% CI: 0.41  0.097; p = 0.037). However, neither of these findings remained significant after Bonferroni correction for multiple comparisons. The frequencies of rs868257, rs6446693, rs1907998 and rs6832405 minor alleles and genotypes were similar between the control and NSCL/P groups. No significant linkage disequilibrium (LD) was observed. Genotype-genotype interaction and the haplotype analysis did not reveal any significant association with NSCL/P. Conclusions: The study results were suggestive of a positive association between MSX1 rs11726039 and NSCL/P in the South Indian population. ß 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: MSX1 Orofacial clefts SNP India

1. Introduction Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common craniofacial birth defect characterised by an incomplete separation between nasal and oral cavities without any other congenital anomaly in humans [1]. The normal embryologic development of the lip and palate involves a series of closely coordinated events such as cell differentiation, migration, growth and apoptosis in the facial primordia. Any disturbance in these events by genetic and environmental factors may affect the normal

* Corresponding author at: Sickle Cell Institute Chhattisgarh, Raipur 492 001, Chhattisgarh, India. Tel.: +91 9940524037. E-mail addresses: [email protected], [email protected] (Bhaskar V.K.S. Lakkakula). http://dx.doi.org/10.1016/j.ijporl.2015.04.034 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.

morphology of the facial structures [2]. By using different approaches, a number of potential susceptible candidate regions and genes have been identified [1]. Muscle segment homeobox 1 (MSX1) is a member of the mammalian MSX gene family which consists of MSX1, MSX2 and MSX3 [3]. Of these MSX1 and MSX2 are widely expressed in an overlapping manner in many organs of developing vertebrate embryos, particularly in the craniofacial regions, indicating a role of these genes in craniofacial development [4,5]. Studies in animal models have revealed role of MSX1 in null mutant mice embryos. In this mechanism, the palate do shelves elevate normally, but failed to fuse and manifest a cleft in secondary palate which is the primordium of the hard and soft parts of the palate [6]. Muscle segment homeobox 1, has been considered as one of the susceptible genes for NSCL/P. The gene coding for human MSX1 maps to human chromosome 4p16.1 [7], and exhibits homology of

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synteny with the murine MSX1 gene that located on mouse chromosome 5 [8]. Complete sequencing of MSX1 gene in NSCL/P cases of different ethnicities have demonstrated several potentially etiologic mutations such as missense, point and rare mutations [9]. Association studies among different populations have also shown that the variants within and nearby MSX1 gene are involved in the pathogenesis of oral clefts [10–12]. Functional and structural studies on MSX1 gene have identified BP (basal promoter) and other boxes in the upstream region where several consensus sequences are present for transcription factors binding [13–15]. Single nucleotide polymorphisms in these regions may alter gene expression by influencing the binding affinity of transcription factors. Therefore, such SNPs could play an important role in an individual’s susceptibility to complex diseases. Previous studies have also shown the strong association of variants nearby MSX1 with NSCL/P [12,16]. Our aim in the present study was to investigate the role of the variants near the MSX1 gene (Table 1) in the pathogenesis of NSCL/P among South Indian population. 2. Materials and methods 2.1. Subjects The study was conducted on altogether 349 individuals, comprising 173 NSCL/P cases and 176 controls. All the subjects were recruited at Sri Ramachandra cleft and craniofacial centre, Sri Ramachandra University, Chennai, India. The case groups were examined by two plastic surgeons to exclude syndromes known to be associated with any type of orofacial clefting. Cases with possible specific malformations and those with mental retardation or other anomalies were excluded from the study. Individuals without clefts or family history of clefting were recruited as controls in this study. The study was approved by Institutional Ethics Committee of Sri Ramachandra University and informed consent was obtained from parents or legal guardians of all subjects. 2.2. Genotyping Peripheral blood samples (3 ml) were collected from all the subjects. DNA was isolated from the peripheral venous blood lymphocytes by the phenol–chloroform method following standard procedure [17]. Genotyping of all the 5 SNPs was performed by Kbioscience (Hoddesdon Herts, United Kingdom) by using KASPar chemistry, which uses a competitive allele-specific PCR SNP genotyping system and FRET quencher cassette oligos. On the basis of the fluorescence obtained, the allele call data were viewed graphically as a scatter plot for each marker using the SNPViewer (http://www.lgcgenomics.com). 2.3. Statistical analysis First, Hardy–Weinberg equilibrium (HWE) was assessed for all the polymorphisms among case and control groups by using Chi-square test. Allele frequencies were estimated by the gene

Table 1 Marker information of five MSX1 SNPs. SNP

Marker position

Alleles

rs11726039 rs868257 rs6446693 rs1907998 rs6832405

11.9 kb upstream from transcription start 9.2 kb upstream from transcription start 6.3 kb upstream from transcription start 4.8 kb upstream from transcription start 18.0 kb downstream from transcription end

T/C C/G T/C A/G G/T

counting method. The genotype and allele frequencies of five SNPs were in agreement with HWE in both cases and controls. Comparison of genotypes and allele frequencies among case and control group was performed by the Chi-square test. Odds ratio and 95% confidence intervals were calculated using wild type genotypes or alleles as reference group. Bonferroni’s multiple adjustments was applied to address the multiple comparisons problem, the level of significance was set at p < 0.01 (0.05/5) for genotypes and alleles [18]. Pairwise linkage disequilibrium (LD) was computed as both of D0 and r2 for all SNPs by using the Haploview [19]. SNP-SNP interactions among variants near MSX1 gene were assessed by using Multifactor Dimensionality Reduction software (MDR) 3.0.2 [20]. 3. Results The genotype and allele frequencies of studied MSX1 near variants (rs11726039, rs868257, rs6446693, rs1907998 and rs6832405) have been presented in Table 2. The genotype frequencies of all the variants were in Hardy–Weinberg equilibrium in controls, except for rs11726039 SNP (Table 2). The rs11726039 minor allele frequency (MAF) was 16.8% and 23.9% in NSCL/P and control groups respectively and showed a decreased risk for oral clefts in different models indicating its protective role in the occurrence of oral clefts (Table 2). However, neither of these findings remained significant after Bonferroni correction for multiple comparisons. Pairwise linkage disequilibrium between the studied SNPs near MSX1 gene revealed no significant LD between SNPs (Table 3). Haplotype analysis revealed only six haplotypes at a frequency of 5% with their cumulative frequency comprised 75% of the total haplotypes, whereas remaining 25% constituted 17 haplotypes at a frequency of less than 5%. Haplotype phenotype association was not informative (data not shown). MDR software was used to analyze the SNP–SNP interaction and the results were documented in Table 4. The best model predicted for 1, 2 and 3 locus interactions was not significant. The entropy graph showed higher degree of synergy between rs11726039 and rs6832405 with a positive information gain (IG) of 0.42%. Additionally the redundancy interaction was found between rs6832405 and rs1907998 with a negative information gain (IG) of 0.02% (Fig. 1). 4. Discussion Analysis of SNPs located upstream and downstream of MSX1 revealed that most individual SNPs were not significantly different between the NSCL/P and control groups without SNP-SNP interaction. Only, rs11726039 SNP was found to be significantly associated with NSCL/P. No significant LD was observed between these SNPs, and the haplotype phenotype association was not informative. The role of MSX1 in the susceptibility of NSCL/P was previously shown in MSX1 knock-out mice [6]. The MSX1 encoded protein has been involved in the regulation of expression of BMP4 in the palatal mesenchyme and SHH in the medial edge epithelium. A heterozygous nonsense mutation (Ser104stop) in exon 1 of MSX1 gene has been identified in a three-generation study on Dutch family exhibiting various combinations of oral clefts [21]. In a follow-up study, complete sequencing of the MSX1 gene in 1000 unrelated individuals with CL/P showed that mutations in MSX1 alone accounted for 2% of isolated CL/P [9]. The CA dinucleotide repeat microsatellite polymorphism located in the MSX1 intron has been extensively investigated for NSCL/P. The CA 169-bp repeat allele polymorphism showed association with NSCL/P [9,22–27]. On the contrary, transmission distortion analysis in Brazilian parent–child triads revealed no

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Table 2 Association test results for MSX1 gene polymorphisms in NSCLP and control groups. SNP

Genotype

Control

NSCL/P

OR(95%CI)

p Value

rs11726039

TT TC CC HWP TC + CC T C

100 68 8 0.401 76 268(76.14) 84(23.86)

117 54 2 0.118 56 288(83.24) 58(16.76)

Reference 0.69(0.43–1.06) 0.22(0.04–1.03)

0.038*

0.63(0.41–0.97)

0.037

0.64(0.44–0.93)

0.019

CC CG GG HWP CG + GG C G

69 86 21 0.433 107 224(63.64) 128(36.36)

69 80 24 0.915 104 218(63.01) 128(36.99)

Reference 0.93(0.60–1.55) 1.14(0.67–2.24)

0.822*

0.97(0.63–1.49)

0.896

1.02(0.85–1.49)

0.862

TT TC CC HWP TC + CC T C

51 92 33 0.453 125 194(55.11) 158(44.59)

40 101 32 0.025 133 181(52.31) 165(47.69)

Reference 1.40(0.85–2.31) 1.23(0.65–2.34)

0.066*

1.36(0.84–2.19)

0.212

1.12(0.83–1.50)

0.457

AA AG GG HWP AG + GG A G

46 86 44 0.764 130 178(50.57) 174(49.43)

50 91 32 0.403 123 191(55.20) 155(44.80)

Reference 0.97(0.68–1.60) 0.67(0.36–1.23)

0.336*

0.87(0.54–1.48)

0.562

0.83(0.63–1.12)

0.22

GG GT TT HWP GT + TT G T

113 53 10 0.265 63 279(79.26) 73(20.74)

113 50 10 0.169 60 276(79.77) 70(20.23)

Reference 0.94(0.68–1.50) 1.00(0.40–2.59)

0.969*

0.95(0.61–1.57)

0.827

0.57(0.75–1.40)

0.868

ra868257

rs6446693

rs1907998

rs6832405

*

p Value by x2 test (df = 2), p value by x2 test (df = 1), HWP: Hardy–Weinberg p value.

significant association between intronic CA repeat polymorphisms and NSCL/P [28]. The MSX1 P147Q polymorphism which was not evaluated in our study, found only in the Asian population with very less frequency, showed contrasting results on the association with NSCL/P [29,30]. Several rare variants have been reported in either families or specific populations with NSCL/P [9,30]. The rs3821949 located in 50 UTR region of MSX1 gene has not been found to be associated with NSCL/P in Han Chinese population [31], but its A allele had significantly increased the risk of NSCL/P in the Korean population [32]. Analysis of several SNPs near to MSX1 gene showed conflicting results regarding the association with NSCL/P. The rs6446693 SNP located 6.3 kb upstream to the transcription start site was not associated with NSCL/P in the present study and this is in concurrence with the previous studies [33,34]. But this polymorphism showed

Table 3 Pairwise linkage disequilibrium measures for MSX1.

association with NSCL/P among Estonian population [16]. In the present study, the rs11726039 SNP was found to be associated with NSCL/P with the lesser minor allele frequency in the cases (16.8%) than the controls (23.9%). The protective effect on NSCL/P in South Indian population could be attributed to the above mentioned association. The present findings consistent with other studies that have also observed decreased risk for NSCL/P with rs11726039 in an Estonian sample [16]. A transgenic analysis of 13 kb of DNA around the MSX1 locus revealed that a 4.9 kb fragment upstream of the translational start site is sufficient to generate the nearly complete expression pattern of the gene [35]. Structural and functional analyses of a 4.9-kb segment of the 50 -flanking region of MSX1 gene in mouse revealed, that 4 kb upstream of the transcription start site (contains positive and negative elements) was responsible for controlling gene expression [15]. Further sequence analysis of this region identified a small fragment of 130 bp showed extensive conservation and consensus binding sites compared to human MSX1 gene promoter,

MSX1 SNP

rs11726039

rs11726039 rs868257 rs6446693 rs1907998 rs6832405

0.127 0.072 0.029 0.008

rs868257

rs6446693

rs1907998

rs6832405

0.927

0.491 0.868

0.355 0.649 0.86

0.088 0.26 0.464 0.32

0.376 0.274 0.01

0.568 0.065

0.024

D0 values are located above the diagonal; r2 values are below the diagonal.

Table 4 Interaction models by MDR analysis. Factors

CVC

TA

p Value

rs11726039 rs11726039, rs6832405 rs11726039, rs1907998, rs6832405

10/10 6/10 8/10

0.55 0.53 0.51

0.510 0.701 0.907

CVC: Cross validation consistency; TA: testing accuracy.

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References

Fig. 1. Interaction entropy graph for variations near MSX1 gene and NSCL/P risk. Positive percent entropy indicates information gain (IG) or synergy; whereas, negative percent indicates redundancy or lack of information gain (IG). The red colour represent a high degree of synergy (positive information gain), and gold representing independence and a midway point between synergy and redundancy.

whereas its removal completely abolished gene expression in cultured cells [15]. Evolutionary conservation of the endogenous MSX1 antisense RNA (MSX1–AS RNA) mediated regulation of MSX1 gene expression in both mouse and human, suggested the need to search non-coding and nearby regions of the MSX1 gene [36]. However, very few studies have investigated the role of MSX1 gene nearby variants in the pathogenesis of NSCL/P. Future research should therefore concentrate on the investigation of MSX1 variants that are involved in the pathogenesis of NSCL/P. In conclusion, the present study demonstrated a significant association between MSX1 SNPs rs11726039 and NSCL/P in the South Indian population. Authors contribution LVKSB, SAH and JM defined the research theme. LVKSB and GVB designed methods and experiments, carried out the laboratory experiments. LVKSB and GVB analyzed the data, interpreted the results and wrote the paper. All authors have contributed to, seen and approved the manuscript. Acknowledgements L.V.K.S. Bhaskar acknowledges funding from the Indian Council of Medical Research (ICMR), Government of India (Project Ref. No. 54/15/2007-BMS and No. 45/3/2013-Hum/BMS).

[1] J. Murthy, L. Bhaskar, Indian J. Plast. Surg. 42 (2009) 68–81. [2] P.A. Mossey, J. Little, R.G. Munger, M.J. Dixon, W.C. Shaw, Lancet 374 (2009) 1773–1785. [3] D. Davidson, Trends Genet. 11 (1995) 405–411. [4] A. MacKenzie, M.W. Ferguson, P.T. Sharpe, Development 115 (1992) 403–420. [5] M. Mina, J. Gluhak, W.B. Upholt, E.J. Kollar, B. Rogers, Dev. Dyn. 202 (1995) 195–214. [6] I. Satokata, R. Maas, Nat. Genet. 6 (1994) 348–356. [7] A. Ivens, N. Flavin, R. Williamson, M. Dixon, G. Bates, M. Buckingham, et al. Hum. Genet. 84 (1990) 473–476. [8] B. Robert, D. Sassoon, B. Jacq, W. Gehring, M. Buckingham, EMBO J. 8 (1989) 91–100. [9] P.A. Jezewski, A.R. Vieira, C. Nishimura, B. Ludwig, M. Johnson, S.E. O’Brien, et al. J. Med. Genet. 40 (2003) 399–407. [10] T.H. Beaty, J.B. Hetmanski, J.S. Zeiger, Y.T. Fan, K.Y. Liang, C.A. VanderKolk, et al. Genet. Epidemiol. 22 (2002) 1–11. [11] M.D. Fallin, J.B. Hetmanski, J. Park, A.F. Scott, R. Ingersoll, H.A. Fuernkranz, et al. Genet. Epidemiol. 25 (2003) 168–175. [12] J. Suazo, J.L. Santos, H. Carreno, L. Jara, R. Blanco, J. Dent. Res. 83 (2004) 782–785. [13] R. Binato, C.E. Alvarez Martinez, L. Pizzatti, B. Robert, E. Abdelhay, Biochem. J. 393 (2006) 141–150. [14] J.E. Hewitt, L.N. Clark, A. Ivens, R. Williamson, Genomics 11 (1991) 670–678. [15] S.M. Gonzalez, L.H. Ferland, B. Robert, E. Abdelhay, DNA Cell Biol. 17 (1998) 561–572. [16] T. Jagomagi, T. Nikopensius, K. Krjutskov, V. Tammekivi, T. Viltrop, M. Saag, et al. Eur. J. Oral. Sci. 118 (2010) 213–220. [17] J. Sambrook, E.F. Fritsch, T. Maniatis, Cold Spring Harbor laboratory Press, 1989. [18] J.M. Bland, D.G. Altman, BMJ 310 (1995) 170. [19] J.C. Barrett, B. Fry, J. Maller, M.J. Daly, Bioinformatics 21 (2005) 263–265. [20] L.W. Hahn, M.D. Ritchie, J.H. Moore, Bioinformatics 19 (2003) 376–382. [21] M.J. van den Boogaard, M. Dorland, F.A. Beemer, H.K. van Amstel, Nat. Genet. 24 (2000) 342–343. [22] A.C. Lidral, P.A. Romitti, A.M. Basart, T. Doetschman, N.J. Leysens, S. Daack-Hirsch, et al. Am. J. Hum. Genet. 63 (1998) 557–568. [23] A.R. Vieira, I.M. Orioli, E.E. Castilla, M.E. Cooper, M.L. Marazita, J.C. Murray, J. Dent. Res. 82 (2003) 289–292. [24] R.E. Schultz, M.E. Cooper, S. Daack-Hirsch, M. Shi, B. Nepomucena, K.A. Graf, et al. Am. J. Med. Genet. A 125A (2004) 17–22. [25] L.E. Mitchell, J.C. Murray, S. O’Brien, K. Christensen, Am. J. Epidemiol. 153 (2001) 1007–1015. [26] K. Ulucan, A. Akcay, N. Tasky´n, T. Akcay, T. Konuk, Dis. Mol. Med. 1 (2013) 68–71. [27] L.M. Moreno, M. Arcos-Burgos, M.L. Marazita, K. Krahn, B.S. Maher, M.E. Cooper, et al. Am. J. Med. Genet. A 125A (2004) 135–144. [28] A.L.d. Silva, L.A. Ribeiro, M.E. Cooper, M.L. Marazita, D. Moretti-Ferreira, Genet. Mol. Biol. 29 (2006) 439–442. [29] I. Salahshourifar, A.S. Halim, W.A. Wan Sulaiman, B.A. Zilfalil, J. Hum. Genet. 56 (2011) 755–758. [30] Y. Suzuki, P.A. Jezewski, J. Machida, Y. Watanabe, M. Shi, M.E. Cooper, et al. Genet. Med. 6 (2004) 117–125. [31] Y.Q. Huang, J. Ma, M. Ma, Y. Deng, Y.D. Li, H.W. Ren, et al. DNA Cell Biol. 30 (2011) 1057–1061. [32] N.Y. Kim, Y.H. Kim, J.W. Park, S.H. Baek, J. Korean Med. Sci. 28 (2013) 522–526. [33] M.L. Cardoso, J.F. Bezerra, G.H. Oliveira, C.D. Soares, S.R. Oliveira, K.S. de Souza, et al. Oral Dis. 19 (2013) 507–512. [34] J. Suazo, J.L. Santos, L. Jara, R. Blanco, Am. J. Med. Genet. A. 152A (2010) 2011–2016. [35] A. MacKenzie, L. Purdie, D. Davidson, M. Collinson, R.E. Hill, Mech. Dev. 62 (1997) 29–40. [36] C. Blin-Wakkach, F. Lezot, S. Ghoul-Mazgar, D. Hotton, S. Monteiro, C. Teillaud, et al. Proc. Natl. Acad. Sci. USA. 98 (2001) 7336–7341.