The Cleft Palate–Craniofacial Journal 52(1) pp. 49–53 January 2015 Ó Copyright 2015 American Cleft Palate–Craniofacial Association
ORIGINAL ARTICLE CBS c.844ins68 Polymorphism Frequencies in Control Populations: Implications on Nonsyndromic Cleft Lip With or Without Cleft Palate Jyotsna Murthy, M.Ch., D.N.B., Saikrishna Lakkakula, M.Sc., Venkatesh Babu Gurramkonda, M.Sc., Ram Mohan Pathapati, M.D., D.M., Rajasekhar Maram, M.Sc., Ph.D., Bhaskar V.K.S. Lakkakula, M.Sc., Ph.D. Introduction: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with substantial clinical and social impact. Folate deficiency is one of the factors that have been associated with increased risk for NSCLP. Polymorphisms in folate and homocysteine pathway genes may act as susceptibility factors. Objective: The objective of this study was to evaluate prevalence estimates of cystathionine beta-synthase (CBS) insertion of 68-bp (c.844ins68) polymorphisms and their correlation with NSCLP. Material and Methods: A total of 236 unrelated individuals from seven Indian populations and an additional 355 cases with NSCLP and 357 controls without NSCLP were included in this study. We investigated the CBS c.844ins68 polymorphism in all samples. Genotyping was performed with polymerase chain reaction and electrophoresis. The data were statistically analyzed using the chi-square test. Results: The CBS c.844ins68 allele is present in six of the seven populations analyzed, and allele frequencies range from 1.5% in Balija to 9.1% in Sugali populations. The CBS c.844ins68 polymorphism showed a significant protective effect on NSCLP at both genotype (WW versus WI: odds ratio [OR] ¼ 0.54, 95% confidence interval [CI] ¼ 0.31 to 0.95, P ¼ .149) and allele levels (W versus I: OR ¼ 0.56, 95% CI ¼ 0.32 to 0.96, P ¼ .033). Conclusions: The current study observed significant differences in the frequency of the CBS 844ins68 allele across populations. There is a significant association between CBS c.844ins68 polymorphism and cleft lip and palate in the Indian population. Additional studies are warranted to identify the functional variants in the genes controlling homocysteine as etiological contributors to the formation of oral clefts. KEY WORDS:
CBS, NSCLP, polymorphism, VNTR
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect involving both genetic and environmental factors (Murthy and Bhaskar, 2009). Higher transsulfuration and transmethylation of methionine that is found, respectively, in the first trimester and third trimester during pregnancy suggested a higher
requirement of methionine and folate during these periods (Dasarathy et al., 2010). Folate metabolism plays a role in the etiopathogenesis of cleft lip and palate, and variants in folate pathway genes may act as predisposition factors (Bhaskar et al., 2011). Cystathionine b-synthase (CBS) is a key enzyme involved in the transsulfuration pathway, which catalyzes condensation of serine and homocysteine to produce cystathionine, a substrate for cysteine synthesis (Ratnam et al., 2002). This transsulfuration pathway governs the cellular methylation and antioxidant potential that is observed in several complex diseases (Prudova et al., 2006). Cystathionine b-synthase uses vitamin B6 to make hydrogen sulfide, a putative signaling molecule that helps to maintain an array of physiological processes (Koutmos et al., 2010). The gene encoding CBS is located on chromosome 21q22.3 (Munke et al., 1988). The CBS gene is built of 17 exons, but only 3 to 17 exons are translated and encode the peptide sequence of 551 amino acids. The c.844ins68 is a 68bp insertion in the coding region of exon 8 of the CBS gene and duplicates the intron 7 acceptor splice site and may lead
Dr. Murthy is Professor, Department of Plastic Surgery, Sri Ramachandra University, Chennai, India. Mr. Lakkakula is Doctoral Fellow, Department of Zoology, Sri Venateswara University, Tirupati, India. Mr. Gurramkonda is Doctoral Fellow, Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India. Dr. Pathapati is Assistant Professor, Department of Pharmacology, Narayana Medical College, Nellore, India. Dr. Maram is Assistant Professor, Department of Zoology, Sri Venateswara University, Tirupati, India. Dr. Lakkakula is Associate Professor, Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India. Submitted March 2013; Revised July 2013; September 2013; Accepted October 2013. Address correspondence to: Dr. Bhaskar V.K.S. Lakkakula, Department of Biomedical Sciences, Sri Ramachandra University, No.1 Ramachandra Nagar, Porur, Chennai 600 116, India. E-mail: [email protected]. DOI: 10.1597/13-051 49
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to two CBS mRNA variants (Sperandeo et al., 1996). Since the identification of the CBS c.844ins68 polymorphism, a large number of epidemiologic studies have been conducted to investigative the association of homocystinuria and other disease endpoints with CBS genotype (Pepe et al., 1999; Grossmann et al., 2002; Galbiatti et al., 2010). Mutations in the CBS gene cause homocystinuria, an inherited disorder that affects the central nervous system, eye, skeleton, and vascular systems (Dutta et al., 2009). As there is clear evidence for the potential role of CBS in maintaining the levels of homocysteine through transsulfuration of homocysteine to cysteine (Ratnam et al., 2002), in this study we examined the association between cleft lip and palate and CBS c.844ins68 genotypes in a South Indian population. In addition, we also examined the CBS c.844ins68 polymorphism in 236 unrelated individuals belonging to seven Indian populations to understand the ethnic variations. MATERIALS
AND
METHODS
A total of 236 unrelated individuals belong to seven Indian populations and an additional 355 cases with NSCLP and 357 unrelated control individuals without clefts were included in this study. The population samples included from seven ethnic groups of south India were apparently normal healthy volunteers. The subjects with nonsyndromic clefts and unrelated control individuals without clefts or family history of clefting were ascertained at Sri Ramachandra Cleft and Craniofacial Center, Sri Ramachandra University, Chennai, India. To determine their individual cleft status, all of the subjects with clefts were examined clinically two different times by two surgeons independently, and screening was also done using the subjects’ medical records. All of the subjects in the case group had isolated nonsyndromic clefts, without congenital malformations or major developmental disorders. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University, Chennai, India. As many of the children were younger than 15 years, consent was requested from their parents. A sample of 3 mL of blood was collected from all of the participants after obtaining informed consent. Genomic DNA of the above samples was isolated by standard protocols with phenol-chloroform extraction and ethanol precipitation (Sambrook and Russell, 2001). Genotyping for CBS c.844ins68 polymorphism was performed following a published polymerase chain reaction (PCR)–based method (Mostowska et al., 2010). Amplifications were performed in a 10-lL volume containing 23 Ampliqon master mix, 1 pmol of forward primer, 1 pmol of reverse primer, and 40 ng of genomic DNA. All PCR products were examined by agarose gel electrophoresis, and the genotypes were independently scored by two researchers to minimize error. Allele
frequencies were determined by direct counting of alleles at each locus. The genotype distribution for c.844ins68 in each group was evaluated for Hardy-Weinberg equilibrium (HWE) using the HWSIM program (Cubells et al., 1997). The association between CBS polymorphism and NSCLP was analyzed using the chi-square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The chi-square homogeneity test was employed to assess the minor allele frequency (MAF) difference between ethnic groups. All statistical analyses were performed with SPSS statistical software version 17.0 (SPSS Inc, Chicago, IL) for Windows. RESULTS Population-specific frequencies and counts of CBS c.844ins68 genotypes among different populations are shown in Figure 1. The CBS c.844ins68 allele is present in six of the seven populations analyzed, and allele frequencies range from 1.5% in Balija to 9.1% in Sugali populations. Genotype distributions follow the Hardy-Weinberg equation in all seven Indian populations studied. Figure 1 also depicts the distribution of CBS c.844ins68 genotypes, allele frequencies, and 95% confidence intervals from controls of various published studies around the world. Data showed significant differences in the frequency of the CBS c.844ins68 allele across the populations (P , .0001, df ¼ 34). The CBS c.844ins68 polymorphisms follow the HWE in the control group as well as in cases (Table 1). The c.844ins68 insertion of the CBS gene was found in 37 of 320 control subjects with MAF of 5.2%. In the patient group, 21 subjects had the c.844ins68 insertion with MAF of 3%. Homozygosity for the c.844ins68 insertion of the CBS gene was not observed in either the case or control groups. The CBS c.844ins68 showed a significant association with NSCLP at genotype (WW versus WI: OR ¼ 0.54, 95% CI ¼ 0.31 to 0.95, P ¼ .030) and allele level (W versus I: OR ¼ 0.56, 95% CI ¼ 0.32 to 0.96, P ¼ .033; Table 1). DISCUSSION An analysis of seven populations has shown significant difference in allele frequencies among populations, and none of the populations showed the homozygous condition for the c.844ins68 insertion allele. The c.844ins68 insertion allele occurs more frequently among Africans and Europeans than the other populations (Franco, Elion, et al., 1998b). The c.844ins68 insertion allele has been reported in the homozygous condition, with a very low frequency, from few European and African populations (Franco, Elion, et al., 1998b; Relton et al., 2004; Golimbet et al., 2009; Eussen et al., 2010; Galbiatti et al., 2010). The current study suggests that the CBS c.844ins68 insertion polymorphism is associated with cleft lip and palate in the Indian population. The presence of more insertions in controls than in the NSCLP group demon-
Murthy et al., CBS GENE POLYMORPHISMS AND OROFACIAL CLEFTS
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FIGURE 1 The frequency of CBS c.844ins68 polymorphism in the current study compared with the frequency in different ethnic groups in different studies. The points and whiskers in the forest plot represent MAF and the 95% confidence interval (CI), respectively. Whiskers overlap in the forest plot, indicating no significant difference in MAF between populations. Data from previous studies are denoted by the citations. N, sample size; W, wild; I, insertion; HWp, Hardy-Weinberg equilibrium P value.
strates a protective effect of this allele. There is no consistent evidence that the CBS c.844ins68 polymorphism affects CBS function or homocysteine levels (Grobelny et al., 2011). Furthermore, the CBS enzyme assay conducted on individuals carrying different CBS c.844ins68 genotypes showed heterozygous insertion and homozygous insertion activities comparable with the wild-type genotype (Tsai, Garg, et al., 1996). A significant distortion in the transmission of the c.844ins68 allele in mothers of Italian cleft children led to an 18.7-fold increase in the risk of oral clefts due to genomic imprinting or the additive effect of fetal and maternal genotypes (Rubini et al., 2005). The presence of both CBS insertion and the MTHFR c.677T alleles in all c.844ins68 heterozygous cleft children suggested the interaction between the CBS c.844ins68 allele and MTHFR c.677T leading to an increase in the risk of NSCLP (Rubini et al., 2005). A reevaluation study conducted in Central Europeans failed to show such distortion in transmission of CBS c.844ins68 in mothers of the children with cleft or a co-occurrence of the c.844ins68 allele and MTHFR c.677T in children with cleft (Birnbaum et al., 2007). A Norwegian population-based study demonstrated that the offspring of mothers homozygous for CBS c.699C.T (Y233Y; rs234706) showed a reduced risk of cleft lip and palate, but this association was independent of folate status of mothers (Boyles et al., 2008). Although there is no convincing direct evidence for linkage disequilibrium (LD) between c.699C.T and c.844ins68, the presence of a small number of haplotypes carrying these alleles might be indicative of LD between these two variants (Urreizti et al., 2007). Furthermore, a pathway-wide association study in
Norway’s families with orofacial clefts revealed that the multiple tightly linked single-nucleotide polymorphisms of the CBS gene contributed to the incidence of cleft lip with or without cleft palate (Boyles et al., 2009). The CBS c.844ins68 insertion allele was distributed in equal proportion between cases and controls and did not show a significant association with orofacial clefts in Polish samples (Mostowska et al., 2010). Cysteine-rich secretory protein LCCL domain containing 1 and 2 genes (CRISPLD1 and CRISPLD2) plays a role in NSCLP through the interaction with the CBS gene, but this interaction is population specific. CBS interacts with CRISPLD1 in non-Hispanic and with CRISPLD2 in Hispanic populations (Chiquet et al., 2011). Furthermore, the interaction between the CBS gene and several other folate metabolism genes such as BHMT2, FOLR1, FOLR2, MTHFD1, MTHFR, MTRR, NOS3, SLC19A1, and TYMS is found to cause NSCLP pathogenesis (Blanton et al., 2011). A recent family-based association test using Italian cleft lip and palate families suggested that the intergenerational fetal-maternal interaction of CBS genoTABLE 1 Results of Association Tests With CBS c.844ins68 Polymorphisms in Cleft Lip and Palate Genotype WW WI II W* allele I** allele HWp§
Controls, n (%) NSCLP, n (%) 320 (89.6) 37 (10.4) 0 (0) 677 (94.8) 37 (5.2) .301
334 (94.1) 21 (5.9) 0 (0) 689 (97.0) 21 (3.0) .565
OR (95% CI) Reference 0.54 (0.31–0.95) — Reference 0.56 (0.32–0.96)
W*, wild; I**, insertion; HWp§, Hardy-Weinberg equilibrium P value.
P Value .030
.033
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types mediates the development of orofacial structures (Martinelli et al., 2011). In conclusion, our results support the involvement of the CBS c.844ins68 polymorphism in NSCLP. It is well known that the multiple risk factors contribute to NSCLP, and the effect of each factor may be pretty small in itself, but together they multiply into a much bigger interaction between them (Murthy and Bhaskar, 2009). Furthermore, limited available data on folate metabolism does not support their direct role in NSCLP pathogenesis. Hence, additional studies are warranted to identify the potential functional variants in the genes controlling homocysteine as contributors to the formation of oral clefts. Acknowledgments. L.V.K.S. Bhaskar acknowledges funding from the Indian Council of Medical Research (ICMR), Government of India (Project Ref. No. 56/15/2007-BMS).
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Ott N, Geddert H, Sarbia M. Polymorphisms in methionine synthase (A2756G) and cystathionine beta-synthase (844ins68) and susceptibility to carcinomas of the upper gastrointestinal tract. J Cancer Res Clin Oncol. 2008;134:405–410. Pepe G, Vanegas OC, Rickards O, Giusti B, Comeglio P, Brunelli T, Marcucci R, Prisco D, Gensini GF, Abbate R. World distribution of the T833C/844INS68 CBS in cis double mutation: a reliable anthropological marker. Hum Genet. 1999;104:126–129. Popp R, Crisan T, Rotar I, Farcas M, Trifa A, Militaru M, Pop I. Cystathionine b-synthase 844ins68 genetic polymorphism in spontaneous abortion susceptibility. Appl Med Inform. 2011;29:34–40. Prudova A, Bauman Z, Braun A, Vitvitsky V, Lu SC, Banerjee R. Sadenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity. Proc Natl Acad Sci U S A. 2006;103:6489–6494. Ratnam S, Maclean KN, Jacobs RL, Brosnan ME, Kraus JP, Brosnan JT. Hormonal regulation of cystathionine beta-synthase expression in liver. J Biol Chem. 2002;277:42912–42918. Relton CL, Wilding CS, Pearce MS, Laffling AJ, Jonas PA, Lynch SA, Tawn EJ, Burn J. Gene-gene interaction in folate-related genes and risk of neural tube defects in a UK population. J Med Genet. 2004;41:256–260. Rubini M, Brusati R, Garattini G, Magnani C, Liviero F, Bianchi F, Tarantino E, Massei A, Pollastri S, Carturan S, et al. Cystathionine beta-synthase c.844ins68 gene variant and non-syndromic cleft lip and palate. Am J Med Genet A. 2005;136A:368–372. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001. Senemar S, Doroudchi M, Pezeshki AM, Bazrgar M, Torab-Jahromi A, Ghaderi A. Frequency of cystathionine beta-synthase 844INS68 polymorphism in Southern Iran. Mol Biol Rep. 2009;36:353–356. Shannon B, Gnanasampanthan S, Beilby J, Iacopetta B. A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability. Gut. 2002;50:520–524.
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Song KS, Choi JR, Kim HK, Gee SH, Shim WH. Presence of 844ins68 in the cystathionine beta-synthase gene in Asians (Koreans). Thromb Haemost. 2001;86:1130. Speer MC, Nye J, McLone D, Worley G, Melvin EC, Viles KD, Franklin A, Drake C, Mackey J, George TM. Possible interaction of genotypes at cystathionine beta-synthase and methylenetetrahydrofolate reductase (MTHFR) in neural tube defects. NTD Collaborative Group. Clin Genet. 1999;56:142–144. Sperandeo MP, de Franchis R, Andria G, Sebastio G. A 68-bp insertion found in a homocystinuric patient is a common variant and is skipped by alternative splicing of the cystathionine betasynthase mRNA. Am J Hum Genet. 1996;59:1391–1393. Summers CM, Hammons AL, Mitchell LE, Woodside JV, Yarnell JW, Young IS, Evans A, Whitehead AS. Influence of the cystathionine beta-synthase 844ins68 and methylenetetrahydrofolate reductase 677C.T polymorphisms on folate and homocysteine concentrations. Eur J Hum Genet. 2008;16:1010–1013. Tsai MY, Bignell M, Schwichtenberg K, Hanson NQ. High prevalence of a mutation in the cystathionine beta-synthase gene. Am J Hum Genet. 1996;59:1262–1267. Tsai MY, Garg U, Key NS, Hanson NQ, Suh A, Schwichtenberg K. Molecular and biochemical approaches in the identification of heterozygotes for homocystinuria. Atherosclerosis. 1996;122:69–77. Urreizti R, Asteggiano C, Vilaseca MA, Corbella E, Pinto X, Grinberg D, Balcells S. A CBS haplotype and a polymorphism at the MSR gene are associated with cardiovascular disease in a Spanish casecontrol study. Clin Biochem. 2007;40:864–868. Weiner AS, Boyarskikh UA, Voronina EN, Selezneva IA, Sinkina TV, Lazarev AF, Petrova VD, Filipenko ML. Polymorphisms in the folate-metabolizing genes MTR, MTRR, and CBS and breast cancer risk. Cancer Epidemiol. 2012;36:e95–e100. Yakub M, Moti N, Parveen S, Chaudhry B, Azam I, Iqbal MP. Polymorphisms in MTHFR, MS and CBS genes and homocysteine levels in a Pakistani population. PLoS ONE. 2012;7:e33222. Zhang G, Dai C. Gene polymorphisms of homocysteine metabolismrelated enzymes in Chinese patients with occlusive coronary artery or cerebral vascular diseases. Thromb Res. 2001;104:187–195.
ORIGINAL ARTICLE CBS c.844ins68 Polymorphism Frequencies in Control Populations: Implications on Nonsyndromic Cleft Lip With or Without Cleft Palate Jyotsna Murthy, M.Ch., D.N.B., Saikrishna Lakkakula, M.Sc., Venkatesh Babu Gurramkonda, M.Sc., Ram Mohan Pathapati, M.D., D.M., Rajasekhar Maram, M.Sc., Ph.D., Bhaskar V.K.S. Lakkakula, M.Sc., Ph.D. Introduction: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with substantial clinical and social impact. Folate deficiency is one of the factors that have been associated with increased risk for NSCLP. Polymorphisms in folate and homocysteine pathway genes may act as susceptibility factors. Objective: The objective of this study was to evaluate prevalence estimates of cystathionine beta-synthase (CBS) insertion of 68-bp (c.844ins68) polymorphisms and their correlation with NSCLP. Material and Methods: A total of 236 unrelated individuals from seven Indian populations and an additional 355 cases with NSCLP and 357 controls without NSCLP were included in this study. We investigated the CBS c.844ins68 polymorphism in all samples. Genotyping was performed with polymerase chain reaction and electrophoresis. The data were statistically analyzed using the chi-square test. Results: The CBS c.844ins68 allele is present in six of the seven populations analyzed, and allele frequencies range from 1.5% in Balija to 9.1% in Sugali populations. The CBS c.844ins68 polymorphism showed a significant protective effect on NSCLP at both genotype (WW versus WI: odds ratio [OR] ¼ 0.54, 95% confidence interval [CI] ¼ 0.31 to 0.95, P ¼ .149) and allele levels (W versus I: OR ¼ 0.56, 95% CI ¼ 0.32 to 0.96, P ¼ .033). Conclusions: The current study observed significant differences in the frequency of the CBS 844ins68 allele across populations. There is a significant association between CBS c.844ins68 polymorphism and cleft lip and palate in the Indian population. Additional studies are warranted to identify the functional variants in the genes controlling homocysteine as etiological contributors to the formation of oral clefts. KEY WORDS:
CBS, NSCLP, polymorphism, VNTR
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect involving both genetic and environmental factors (Murthy and Bhaskar, 2009). Higher transsulfuration and transmethylation of methionine that is found, respectively, in the first trimester and third trimester during pregnancy suggested a higher
requirement of methionine and folate during these periods (Dasarathy et al., 2010). Folate metabolism plays a role in the etiopathogenesis of cleft lip and palate, and variants in folate pathway genes may act as predisposition factors (Bhaskar et al., 2011). Cystathionine b-synthase (CBS) is a key enzyme involved in the transsulfuration pathway, which catalyzes condensation of serine and homocysteine to produce cystathionine, a substrate for cysteine synthesis (Ratnam et al., 2002). This transsulfuration pathway governs the cellular methylation and antioxidant potential that is observed in several complex diseases (Prudova et al., 2006). Cystathionine b-synthase uses vitamin B6 to make hydrogen sulfide, a putative signaling molecule that helps to maintain an array of physiological processes (Koutmos et al., 2010). The gene encoding CBS is located on chromosome 21q22.3 (Munke et al., 1988). The CBS gene is built of 17 exons, but only 3 to 17 exons are translated and encode the peptide sequence of 551 amino acids. The c.844ins68 is a 68bp insertion in the coding region of exon 8 of the CBS gene and duplicates the intron 7 acceptor splice site and may lead
Dr. Murthy is Professor, Department of Plastic Surgery, Sri Ramachandra University, Chennai, India. Mr. Lakkakula is Doctoral Fellow, Department of Zoology, Sri Venateswara University, Tirupati, India. Mr. Gurramkonda is Doctoral Fellow, Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India. Dr. Pathapati is Assistant Professor, Department of Pharmacology, Narayana Medical College, Nellore, India. Dr. Maram is Assistant Professor, Department of Zoology, Sri Venateswara University, Tirupati, India. Dr. Lakkakula is Associate Professor, Department of Biomedical Sciences, Sri Ramachandra University, Chennai, India. Submitted March 2013; Revised July 2013; September 2013; Accepted October 2013. Address correspondence to: Dr. Bhaskar V.K.S. Lakkakula, Department of Biomedical Sciences, Sri Ramachandra University, No.1 Ramachandra Nagar, Porur, Chennai 600 116, India. E-mail: [email protected]. DOI: 10.1597/13-051 49
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to two CBS mRNA variants (Sperandeo et al., 1996). Since the identification of the CBS c.844ins68 polymorphism, a large number of epidemiologic studies have been conducted to investigative the association of homocystinuria and other disease endpoints with CBS genotype (Pepe et al., 1999; Grossmann et al., 2002; Galbiatti et al., 2010). Mutations in the CBS gene cause homocystinuria, an inherited disorder that affects the central nervous system, eye, skeleton, and vascular systems (Dutta et al., 2009). As there is clear evidence for the potential role of CBS in maintaining the levels of homocysteine through transsulfuration of homocysteine to cysteine (Ratnam et al., 2002), in this study we examined the association between cleft lip and palate and CBS c.844ins68 genotypes in a South Indian population. In addition, we also examined the CBS c.844ins68 polymorphism in 236 unrelated individuals belonging to seven Indian populations to understand the ethnic variations. MATERIALS
AND
METHODS
A total of 236 unrelated individuals belong to seven Indian populations and an additional 355 cases with NSCLP and 357 unrelated control individuals without clefts were included in this study. The population samples included from seven ethnic groups of south India were apparently normal healthy volunteers. The subjects with nonsyndromic clefts and unrelated control individuals without clefts or family history of clefting were ascertained at Sri Ramachandra Cleft and Craniofacial Center, Sri Ramachandra University, Chennai, India. To determine their individual cleft status, all of the subjects with clefts were examined clinically two different times by two surgeons independently, and screening was also done using the subjects’ medical records. All of the subjects in the case group had isolated nonsyndromic clefts, without congenital malformations or major developmental disorders. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University, Chennai, India. As many of the children were younger than 15 years, consent was requested from their parents. A sample of 3 mL of blood was collected from all of the participants after obtaining informed consent. Genomic DNA of the above samples was isolated by standard protocols with phenol-chloroform extraction and ethanol precipitation (Sambrook and Russell, 2001). Genotyping for CBS c.844ins68 polymorphism was performed following a published polymerase chain reaction (PCR)–based method (Mostowska et al., 2010). Amplifications were performed in a 10-lL volume containing 23 Ampliqon master mix, 1 pmol of forward primer, 1 pmol of reverse primer, and 40 ng of genomic DNA. All PCR products were examined by agarose gel electrophoresis, and the genotypes were independently scored by two researchers to minimize error. Allele
frequencies were determined by direct counting of alleles at each locus. The genotype distribution for c.844ins68 in each group was evaluated for Hardy-Weinberg equilibrium (HWE) using the HWSIM program (Cubells et al., 1997). The association between CBS polymorphism and NSCLP was analyzed using the chi-square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The chi-square homogeneity test was employed to assess the minor allele frequency (MAF) difference between ethnic groups. All statistical analyses were performed with SPSS statistical software version 17.0 (SPSS Inc, Chicago, IL) for Windows. RESULTS Population-specific frequencies and counts of CBS c.844ins68 genotypes among different populations are shown in Figure 1. The CBS c.844ins68 allele is present in six of the seven populations analyzed, and allele frequencies range from 1.5% in Balija to 9.1% in Sugali populations. Genotype distributions follow the Hardy-Weinberg equation in all seven Indian populations studied. Figure 1 also depicts the distribution of CBS c.844ins68 genotypes, allele frequencies, and 95% confidence intervals from controls of various published studies around the world. Data showed significant differences in the frequency of the CBS c.844ins68 allele across the populations (P , .0001, df ¼ 34). The CBS c.844ins68 polymorphisms follow the HWE in the control group as well as in cases (Table 1). The c.844ins68 insertion of the CBS gene was found in 37 of 320 control subjects with MAF of 5.2%. In the patient group, 21 subjects had the c.844ins68 insertion with MAF of 3%. Homozygosity for the c.844ins68 insertion of the CBS gene was not observed in either the case or control groups. The CBS c.844ins68 showed a significant association with NSCLP at genotype (WW versus WI: OR ¼ 0.54, 95% CI ¼ 0.31 to 0.95, P ¼ .030) and allele level (W versus I: OR ¼ 0.56, 95% CI ¼ 0.32 to 0.96, P ¼ .033; Table 1). DISCUSSION An analysis of seven populations has shown significant difference in allele frequencies among populations, and none of the populations showed the homozygous condition for the c.844ins68 insertion allele. The c.844ins68 insertion allele occurs more frequently among Africans and Europeans than the other populations (Franco, Elion, et al., 1998b). The c.844ins68 insertion allele has been reported in the homozygous condition, with a very low frequency, from few European and African populations (Franco, Elion, et al., 1998b; Relton et al., 2004; Golimbet et al., 2009; Eussen et al., 2010; Galbiatti et al., 2010). The current study suggests that the CBS c.844ins68 insertion polymorphism is associated with cleft lip and palate in the Indian population. The presence of more insertions in controls than in the NSCLP group demon-
Murthy et al., CBS GENE POLYMORPHISMS AND OROFACIAL CLEFTS
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FIGURE 1 The frequency of CBS c.844ins68 polymorphism in the current study compared with the frequency in different ethnic groups in different studies. The points and whiskers in the forest plot represent MAF and the 95% confidence interval (CI), respectively. Whiskers overlap in the forest plot, indicating no significant difference in MAF between populations. Data from previous studies are denoted by the citations. N, sample size; W, wild; I, insertion; HWp, Hardy-Weinberg equilibrium P value.
strates a protective effect of this allele. There is no consistent evidence that the CBS c.844ins68 polymorphism affects CBS function or homocysteine levels (Grobelny et al., 2011). Furthermore, the CBS enzyme assay conducted on individuals carrying different CBS c.844ins68 genotypes showed heterozygous insertion and homozygous insertion activities comparable with the wild-type genotype (Tsai, Garg, et al., 1996). A significant distortion in the transmission of the c.844ins68 allele in mothers of Italian cleft children led to an 18.7-fold increase in the risk of oral clefts due to genomic imprinting or the additive effect of fetal and maternal genotypes (Rubini et al., 2005). The presence of both CBS insertion and the MTHFR c.677T alleles in all c.844ins68 heterozygous cleft children suggested the interaction between the CBS c.844ins68 allele and MTHFR c.677T leading to an increase in the risk of NSCLP (Rubini et al., 2005). A reevaluation study conducted in Central Europeans failed to show such distortion in transmission of CBS c.844ins68 in mothers of the children with cleft or a co-occurrence of the c.844ins68 allele and MTHFR c.677T in children with cleft (Birnbaum et al., 2007). A Norwegian population-based study demonstrated that the offspring of mothers homozygous for CBS c.699C.T (Y233Y; rs234706) showed a reduced risk of cleft lip and palate, but this association was independent of folate status of mothers (Boyles et al., 2008). Although there is no convincing direct evidence for linkage disequilibrium (LD) between c.699C.T and c.844ins68, the presence of a small number of haplotypes carrying these alleles might be indicative of LD between these two variants (Urreizti et al., 2007). Furthermore, a pathway-wide association study in
Norway’s families with orofacial clefts revealed that the multiple tightly linked single-nucleotide polymorphisms of the CBS gene contributed to the incidence of cleft lip with or without cleft palate (Boyles et al., 2009). The CBS c.844ins68 insertion allele was distributed in equal proportion between cases and controls and did not show a significant association with orofacial clefts in Polish samples (Mostowska et al., 2010). Cysteine-rich secretory protein LCCL domain containing 1 and 2 genes (CRISPLD1 and CRISPLD2) plays a role in NSCLP through the interaction with the CBS gene, but this interaction is population specific. CBS interacts with CRISPLD1 in non-Hispanic and with CRISPLD2 in Hispanic populations (Chiquet et al., 2011). Furthermore, the interaction between the CBS gene and several other folate metabolism genes such as BHMT2, FOLR1, FOLR2, MTHFD1, MTHFR, MTRR, NOS3, SLC19A1, and TYMS is found to cause NSCLP pathogenesis (Blanton et al., 2011). A recent family-based association test using Italian cleft lip and palate families suggested that the intergenerational fetal-maternal interaction of CBS genoTABLE 1 Results of Association Tests With CBS c.844ins68 Polymorphisms in Cleft Lip and Palate Genotype WW WI II W* allele I** allele HWp§
Controls, n (%) NSCLP, n (%) 320 (89.6) 37 (10.4) 0 (0) 677 (94.8) 37 (5.2) .301
334 (94.1) 21 (5.9) 0 (0) 689 (97.0) 21 (3.0) .565
OR (95% CI) Reference 0.54 (0.31–0.95) — Reference 0.56 (0.32–0.96)
W*, wild; I**, insertion; HWp§, Hardy-Weinberg equilibrium P value.
P Value .030
.033
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types mediates the development of orofacial structures (Martinelli et al., 2011). In conclusion, our results support the involvement of the CBS c.844ins68 polymorphism in NSCLP. It is well known that the multiple risk factors contribute to NSCLP, and the effect of each factor may be pretty small in itself, but together they multiply into a much bigger interaction between them (Murthy and Bhaskar, 2009). Furthermore, limited available data on folate metabolism does not support their direct role in NSCLP pathogenesis. Hence, additional studies are warranted to identify the potential functional variants in the genes controlling homocysteine as contributors to the formation of oral clefts. Acknowledgments. L.V.K.S. Bhaskar acknowledges funding from the Indian Council of Medical Research (ICMR), Government of India (Project Ref. No. 56/15/2007-BMS).
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