Journal of Perinatology (2015), 1–5 © 2015 Nature America, Inc. All rights reserved 0743-8346/15 www.nature.com/jp
ORIGINAL ARTICLE
Genetic association of AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia1, YD Apalasamy1, SZ Suki1, SZ Omar2 and Z Mohamed1 OBJECTIVE: Preterm birth (PTB) is a multifactorial complication in which genetic and environmental factors contribute to the phenotype. The AKAP10 protein encoded by AKAP10 gene has a vital role in the maintenance of myometrial quiescence and pregnancy. This study aimed to investigate whether polymorphisms in the AKAP10 gene are associated with the risk of PTB. STUDY DESIGN: A total of 664 women (132 preterm and 532 term) with spontaneous singleton deliveries were genotyped for AKAP10 polymorphisms (rs119672, rs203462 and rs169412) using Sequenom MassARRAY platform. RESULT: A significant association was observed between the CC and AC genotypes of AKAP10 rs169412 with reduced risk of PTB (CC: adjusted odds ratio (OR) 2.95, 95% confidence interval (CI): 1.23–7.09, P = 0.016. AC: adjusted OR 3.46, 95% CI: 1.38–8.68, P = 0.008), respectively. Following stratification by ethnicity, a significant association was observed between the AC and CC genotypes of rs169412 and term birth in the Malay ethnic subgroup. (CC: OR 2.9, 95% CI: 1.01–8.59, P = 0.041. AC: OR 3.14, 95% CI: 1.04–9.54, P = 0.043). A significant association was also observed between the CT genotypes of AKAP10 rs119672 with reduced risk of PTB deliveries (CT: OR 3.2, 95% CI: 1.06–9.76 P = 0.007, TT: OR 2.8, 0.98–8.34, P = .0.015) Alternatively, there was no association between AKAP10 rs169412 and rs119672 polymorphisms with PTB in the Indians and Chinese ethnic groups. CONCLUSION: This study indicates a significant association between the AKAP10 polymorphisms and reduced risk of PTB in the Malays. This demonstrates the potential role of AKAP10 polymorphisms in preterm complications. Journal of Perinatology advance online publication, 25 June 2015; doi:10.1038/jp.2015.68
INTRODUCTION Preterm birth (PTB) is a major cause of neonatal mortality and morbidity globally with a rising percentage each year.1 Every year, about 15 million babies are delivered prematurely and ~ 1.1 million lives are lost owing to PTB complications.1 Although neonatal care has improved tremendously over the years, most of the preterm neonates who survive still suffer many short- and long-term health problems including bronchopulmonary dysplasia, hypotension, patent ductus arteriosus, intracerebral hemorrhage, necrotizing enterocolitis, anemia and infant jaundice.1,2 PTB is a pregnancy complication involving multifactorial confounders including genetics and environmental factors.3 Early diagnosis of high-risk women for PTB either before pregnancy or early in the first few weeks of pregnancy would help clinicians to monitor and provide treatment when necessary for these groups of women. Maternal genetic factors may contribute up to ⩾ 20%4 in occurrence of PTB. The genes involved in biological pathways that keep the uterus relaxed or cause contraction, have important roles in child labor and birth.4–6 So far, many genes have been examined for their roles in PTB including genes related to immune system, androgenic β-receptor and others.7 One important pathway of PTB is the A kinase pathway involving phosphorylation and relaxation of myometrial smooth muscles by protein kinase A (PKA).8–10 PKA acts at very discrete sites in the cell thus colocalization or restriction of PKA to its specific substrate increases its action. AKAP10 is one among many AKAPs that have been identified to bind and maintain the PKA enzyme at its site of action.11,12 This is achieved by the binding of AKAP10 to PKA resulting in the formation of the AKAP10–PKA complex.6 The
formation of this complex enhances the action of PKA as it is restricted at the site of action or substrate. Genetic polymorphism in human AKAP10 gene has been shown to increase the formation of the AKAP10–PKA complex thereby enhancing compartmentalization and phosphorylation of myometrial smooth muscle thus preventing muscle contraction and maintaining myometrial quiescence.5,6,11,12 The activation of PKA by cyclic adenosine monophosphate and compartmentalization of PKA by AKAP10 is crucial for the role of PKA in smooth muscle relaxation.13,14 Function of AKAP10–PKA complex in the uterus inhibits the initiation of labor.15 The objective of this study was to investigate whether there is an association between maternal genotype of AKAP10 gene polymorphisms (rs119672, rs203462 and rs169412) and risk of PTB.
MATERIALS AND METHODS Study subjects A total of 664 women (132 preterm and 532 term) with spontaneous singleton delivery were prospectively enrolled in this study from 2011 to 2013. Ethnicities of the subjects were validated by confirmations of no mixed marriages for at least three generations. During data collection, all participants self-reported their ethnicity as Malay, Chinese or Indians with confirmation of no mixed marriage of at least three generations. The participants were classified into two groups; the preterm group consisted of mothers who delivered their baby between 24 and 36 weeks, whereas the controls group consisted of mothers with uncomplicated pregnancies who delivered their baby between 38 and 41 weeks. The prospective case–control study was approved by the University of Malaya Medical
1 Pharmacogenomics Laboratory, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia and 2Department of Obstetrics and Gynecology, University of Malaya, Kuala Lumpur, Malaysia. Correspondence: Professor Z Mohamed, Pharmacogenomics Laboratory, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Selangor 50603, Malaysia. E-mail: [email protected] Received 3 January 2015; revised 19 March 2015; accepted 14 April 2015
AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia et al
2 Centre (UMMC) Ethics Committee and written informed consent was obtained from all participants. The physical well being of the participants was assessed by trained gynecologists. The pregnancy age was calculated from the first day of the last menstrual cycle or obtained from ultrasound results of the patients. Eligibility for participation was based on the following inclusion criteria: healthy mothers between the ages of 18 and 35 years with normal competent cervix and uterus, without metabolic or autoimmune disease, conceived preterm but with normal and healthy fetus. Exclusion criteria included maternal age of o18 years or 435 years, abnormal fetus and still birth, mothers with history of drug abuse, mothers who are smokers, mothers pregnant with twins, birth delivery by induction due to fetal distress or placenta abruption or preeclampsia or hypertension or any known medical problem and mothers who were not able to sign informed consents were also excluded from the studies. The demographic details and information of the mothers including their lifestyle, nutrition, medical conditions and family history were collected.
Statistical analysis Statistical analysis of subjects was performed using SPSS version 18.0 (IBM Corp., Chicago, IL, USA). Data were presented as percentage or mean ± s.d. Categorical and continuous variables were compared between preterm and term using Pearson’s χ2-test. Hardy–Weinberg equilibrium (HWE) was checked for the SNPs prior to genetic analysis using a goodness of fit χ2-test. The test for associations of the AKAP10 rs119672 and AKAP10 rs169412 with PTB was performed using logistic regression. Multivariate logistic regression models were adjusted for history of PTB, gestational diabetes and miscarriage. Confounding factors such as smoking, substance abuse, mothers who were themselves born preterm, hypertension and alcohol consumption were not included for statistical analysis in this paper because all subjects were free from these factors. Construction of linkage disequilibrium (LD) blocks and haplotype analyses were performed using Haploview 4.2 software (Broad Institute of MIT and Harvard, USA). Two AKAP10 SNPs were included in the analysis therefore α was 0.025 after Benferroni correction. The probability of the haplotype distributions were compared between term and preterm group by calculating the permutation P-values generated using a χ2 statistics with one degree of freedom. The power of the study was calculated using Quanto software (University of Southern California, USA).
AKAP10 genotyping A total of 2 ml of maternal venous blood samples were collected in both term and preterm subjects. The genomic DNA was extracted from whole blood using the GeneAll Exgene DNA purification kit (GeneAll Biotechnology, Korea). The rs119672, rs203462 and rs169412 single-nucleotide polymorphisms (SNPs) of A-kinase-anchoring protein 10 gene were genotyped using the Sequenom MassARRAY platform. SNPs with call rate of 490% were considered for statistical analysis. For the three PGR SNPs genotyped, two (PGR rs660149 and rs471767) with call rates of 99% were considered for statistical analysis, whereas the PGR rs10895068 SNP with a call rate of 81% was excluded from statistical analysis.
Table 1.
Demographic features of 532 term and 132 preterm subjects
Characteristics Maternal age Maternal BMI (kg m − 2) Married subjects (%) Gestational Diabetes (%) History of PTB (%) Previous miscarriage (%) Low income (%) Average income (%) High income (%)
Term
Preterm
P-value
29.3 ± 3.7 24.5 ± 5.4 98 0 0 15 2 33 64
29.8 ± 3.0 24.3 ± 5.1 98 11 14 25 3 38 62
0.23 0.69 0.72 o0.001 o0.001 0.011 0.551
Abbreviations: BMI, body mass index; PTB, preterm birth. Data are expressed as mean ± s.d. for continuous data and as percentage for categorical data. P-values were obtained using Mann–Whitney U-test or independent t-test. P-values o 0.05 were considered significant.
Table 2.
RESULTS The demographic and clinical parameters of the subjects are shown in Tables 1 and 2. Past history of PTB, miscarriage and gestational diabetes were significantly different between the preterm and term groups. There were no significant differences in maternal age, BMI, marital status, income and between preterm and term groups (Tables 1 and 2). There was no deviation from Hardy–Weinberg equilibrium HWE for AKAP10 rs119672 and AKAP10 rs169412 SNPs in both preterm and term subjects (Table 3). Genetic modeling revealed a significant association between AKAP10 rs119672 variants with preterm and term deliveries (Table 4). The odds of the TT genotype occurring among women with term delivery were twice those among the preterm subjects (odds ratio (OR) 2.95, 95% confidence interval (CI) 1.23–7.06, P = 0.015; Table 4). The CT genotype of rs119672 was also significantly high in the term than preterm subjects. The odds of the CT genotype occurring among the term group were thrice those among the preterm subjects (OR 3.58, 95% CI 1.42–8.98, P = 0.007). For rs169412, the AC genotype was significantly high in the term than preterm subjects (OR 3.46, 95% CI 1.38–8.68, P = 0.008). There was also a significant difference in the CC genotype of rs169412 between preterm and term subjects (OR 2.95, 95% CI 1.23–7.09, P = 0.016). The results show a strong association between AKAP10 polymorphisms and term birth. After stratification according to ethnicity, the CT genotype of rs119672 and the AC or CC genotypes of the rs169412 polymorphisms were high in the Malay ethnic group (Table 4).
Demographic characteristics of subjects by ethnicity
Characteristics
Malays (N = 479)
Indians (N = 93)
Chinese (N = 93)
Term (N = 388) Preterm (N = 91) P-value Term (N = 74) Preterm (N = 19) P-value Term (N = 70) Preterm (N = 22) P-value Maternal age Maternal BMI (kg m − 2) Married subjects (%) Gestational Diabetes (%) History of PTB (%) Previous miscarriage (%) Low income (%) Average income (%) High income (%)
29.2 ± 5.6 24.3 ± 5.6 100 0 0 14 2.8 29 68
29.8 ± 4.1 24.6 ± 5.4 99 8.0 14 24 3.0 35 61
0.249 0.267 0.974 o 0.001 o 0.001 0.017 0.212
30.0 ± 3.7 25.6 ± 5.7 98 0 0 19 3 55 41
30.3 ± 3.7 24.2 ± 5.5 99 10 15 26 5 31 63
0.938 0.550 0.961 0.004 o0.001 0.511 0.161
25.9 ± 3.6 23 ± 5.5 100 0 0 17 1 27 72
29.4 ± 3.5 24.8 ± 4.5 99 20 12 28 0 36 64
0.946 0.329 0.920 o0.001 0.005 0.288 0.593
Abbreviations: BMI, body mass index; PTB, preterm birth. Data are expressed as mean ± s.d. for continuous data and as percentage for categorical data. P-values were obtained using Mann–Whitney U-test or independent t-test. P-values o0.05 were considered significant.
Journal of Perinatology (2015), 1 – 5
© 2015 Nature America, Inc.
AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia et al
3 Table 3.
Genotype distribution of AKAP10 SNPs Genotype frequencies N(%)
Ethnicity
HWE- P
Preterm vs term
Preterm/term
IL1R2, rs119672 C/T All ethnicity Malay Indians Chinese
CC 0.08 0.07 0.09 0.05
vs vs vs vs
0.03 0.02 0.07 0.01
CT 0.30 0.31 0.33 0.27
vs vs vs vs
0.34 0.35 0.38 0.24
TT 0.61 0.63 0.58 0.68
vs vs vs vs
0.63 0.63 0.55 0.74
0.134/0.532 0.253/0.251 0.188/0.901 0.295/0.533
IL1R2 rs169412A/C All ethnicity Malay Indians Chinese
AA 0.08 0.06 0.07 0.05
vs vs vs vs
0.03 0.02 0.06 0.01
AC 0.31 0.31 0.32 0.32
vs vs vs vs
0.34 0.34 0.44 0.25
CC 0.60 0.62 0.47 0.64
vs vs vs vs
0.63 0.64 0.49 0.74
0.179/0.532 0.431/0.295 0.640/0.342 0.702/0.481
Abbreviation: SNPs, single-nucleotide polymorphisms. X2P: Chi square P-value, HWE-P:Hardy Weinberg equilibrium P-value. Po0.05 was considered significant.
Table 4.
Logistic regression analysis for AKAP10 genotype association with preterm birth in overall ethnicity, Malay Indians and Chinese ethnic
groups SNPs
Genotypes
Overall ethnicity (N = 664) OR (95% CI)
rs119672 rs169412
CC CT TT AA AC CC
Reference 3.58 (1.42–8.98) 2.95 (1.23–7.06) Reference 3.46 (1.38–8.68) 2.95 (1.23–7.09)
P-value 0.007 0.015 0.008 0.016
Malays (N = 479) OR (95% CI) Reference 3.2 (1.06–9.76) 2.8 (0.98–8.34) Reference 3.14 (1.04–9.54) 2.94 (1.0–8.59)
Indians (N = 93)
Chinese (N = 92)
P-value
OR (95% CI)
P-value
OR (95% CI)
P-value
0.039 0.055
0.27 (0.55–1.30) 0.27 (0.06–1.22)
0.103 0.091
Reference 0.35 (0.02–6.57) 0.29 (0.02–4.89)
0.485 0.389
0.043 0.041
0.23 (0.05–1.13) 0.31(0.07–1.41)
0.071 0.130
0.41 (0.02–7.55) 0.28 (0.02–4.67)
0.550 0.275
Abbreviations: CI, confidence interval; OR, odds ratio. P-values were adjusted for GDM, previous PTB and previous miscarriage.
Figure 1.
LD block of AKAP10 SNP (rs119672, rs169412) in the overall ethnicity, Malays, Chinese and Indians.
Carriers of one or two risks allele (CT/TT-rs119672, AC/CCrs169412) were associated with reduced risk of PTB. No significant association was seen in the Indian and Chinese ethnic group between AKAP10 SNPs and PTB. Figure 1 shows the LD pattern of the AKAP10 gene. The LD between AKAP10 rs119672 and AKAP10 rs169412 in the overall ethnicity was high (D′ = 0.88). High LD was also seen in the Malays (D′ = 0.87), Chinese (D′ = 0.91) and Indians (D′ = 0.97) ethnic group. Table 5 shows the overall population haplotype block frequency. The haplotype TC was seen frequently compared with CA, TA and CC haplotypes. The frequency of CA haplotypes was higher in the preterm group compared with the term group. However, none of the haplotypes were significantly associated with PTB. © 2015 Nature America, Inc.
Table 5.
Haplotype association analysis of AKAP10 gene variants
Haplotype Block 1 TC CA TA CC
Frequency All
Preterm
Term
χ2
P-value
0.773 0.188 0.02 0.019
0.748 0.225 0.016 0.012
0.779 0.179 0.021 0.02
1.207 2.896 0.338 0.831
0.2719 0.0888 0.5611 0.3621
Block 1 involves SNP rs119678 and rs169412. P-value corrected for 1000 permutation test.
Journal of Perinatology (2015), 1 – 5
AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia et al
4
DISCUSSION In this study, we showed an association between AKAP10 variants and genetic association with term birth. AKAP10 rs119672 and rs169412 were both shown to confer reduced risk of PTB. To the best of our knowledge, this is the first study that investigates the AKAP10 polymorphisms (rs119672 and rs169412) in mothers with PTB. In this study, the genotypes of AKAP10 rs119672 and rs169412 were significantly associated with term birth in overall subjects. Mothers with TT or CT genotypes of rs119672 and CC or AC genotypes of rs169412 delivered more at normal pregnancy duration after 37 weeks of gestation. After stratification by ethnicity, the same trend was observed in the Malay ethnic group. The CT genotype of 119672 and AC genotype of 169412 had higher frequency in mothers who delivered at normal pregnancy duration after 37 weeks of gestation. On the other hand, no significant association was seen in the Indian and Chinese ethnic group. However, AKAP10 genotype frequencies varied among the Malays, Chinese and Indians and our results showed variability in LD patterns of AKAP10 SNPs among the three ethnic groups.16 Previous study has shown that the racial disparity in the occurrence of PTB among Caucasians and African American women is due to the differences their genetic makeup.17 Malaysia has a multiethnic population with three major ethnic groups and we suggest that the difference in the occurrence of PTB among women in this population is owing to differences in the genetic makeup specific to each ethnicity. The AKAP10 variants confer reduced risk of PTB in the Malays, which may indicate that the AKAP10 polymorphisms are prognostic markers among the Malays. We cautiously consider our sample to be adequately powered to detect the genetic associations. This study had a power of 80% and 87% for rs119672 and rs169412, respectively. This study demonstrates the potential role of AKAP10 polymorphisms in PTB complication and therapeutic target for development of new diagnostic and treatment strategies. The role of PKA/AKP10 complex in pregnancy, especially in maintenance of myometrial smooth muscle relaxation, has been shown in animal studies.9 The AKAPs are crucial in targeting PKA to its site of action for PKA phosphorylation of myometrial smooth muscles. The active form of PKA in the cell can be distracted from phosphorylating smooth muscles by protein kinase inhibitors. Protein kinase inhibitors act as pseudosubstrates to the active form of protein kinases, by binding to the active form of the kinase enzyme, thereby preventing phosphorylation. Verli and his colleagues suggested that a combination of selective phosphodiesterases inhibitors and β-adrenergic agonist that will increase the levels of cAMP should be considered for treatment of preterm contractions. The current drug treatment for preterm labor is with the use of the β2 agonists such as salbutamol or terbutaline.18–20 but theoretically, it may be possible to also use a substance that increases PKA targeting to the substrate site or a phosphodiesterase inhibitor.4,20,21 Genetic variants in the anchoring enzyme (AKAP10) might result in a conformational change that increase AKAP10 binding to PKA leading to increased formation of AKAP10–PKA complex. This will result in increased compartmentalization allowing for enhanced binding of cyclic AMP to PKA. Studies have shown that binding and activation of PKA by cAMP increases phosphorylation of myometrial smooth muscles and thus maintains pregnancy.4,22 CONCLUSION In conclusion, our results demonstrated a significant association between the AKAP10 rs119672 and rs169412 with term birth. This study suggests the role of AKAP10 gene variants in PTB Journal of Perinatology (2015), 1 – 5
complication. Prior information on the genetic composition of women may help in the identification and management of high-risk mothers. Future studies leading to the discovery of new drugs that can be used to enhance the relaxation of myometrial smooth muscles till the end of gestation period might result in prevention of PTB globally. Discovery of new biomarkers that can be used for early prediction of high-risk women will also help to diminish PTB. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank the patients and staffs of University of Malaya Medical Centre (UMMC) for their participation in this study. This study was funded by the University of Malaya research grant PG118-2012B and HIR MOHE E000025-20001.
REFERENCES 1 Vogel J, Lee A, Souza J. Maternal morbidity and preterm birth in 22 low- and middle-income countries: a secondary analysis of the WHO Global Survey dataset. BMC Pregnancy Childbirth 2014; 14: 56. 2 Ward RM, Beachy JC. Neonatal complications following preterm birth. BJOG 2001; 20: 8–16. 3 Zhang Y-P, Liu X-H, Gao S-H, Wang J-M, Gu Y-S, Zhang J-Y et al. Risk factors for preterm birth in five maternal and Child Health Hospitals in Beijing. PLoS One 2012; 7: e52780. 4 Verli J, Klukovits A, Kormanyos Z, Hajagos-Toth J, Ducza E, Seres AB et al. Uterusrelaxing effect of beta2-agonists in combination with phosphodiesterase inhibitors: studies on pregnant rat in vivo and on pregnant human myometrium in vitro. J Obstet Gynaecol Res 2013; 39: 31–39. 5 Price SA, Bernal AL. Uterine quiescence: the role of cyclic AMP. Exp Physiol 2001; 86: 265–272. 6 MacDougall MW, Europe-Finner GN, Robson SC. Human myometrial quiescence and activation during gestation and parturition involve dramatic changes in expression and activity of particulate type II (RII alpha) protein kinase A holoenzyme. J Clin Endocrinol Metab 2003; 88: 2194–2205. 7 Loniewska B, Clark JS, Kaczmarczyk M, Adler G, Binczak-Kuleta A, Kordek A et al. Possible counter effect in newborns of 1936 A4G (I646V) polymorphism in the AKAP10 gene encoding A-kinase-anchoring protein 10. J Perinatol 2012; 32: 230–234. 8 Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain. Proc Natl Acad Sci USA 1997; 94: 11184–11189. 9 Ku CY, Sanborn BM. Progesterone prevents the pregnancy-related decline in protein kinase A association with rat myometrial plasma membrane and A-kinase anchoring protein. Biol Reprod 2002; 67: 605–609. 10 Kim SH, Serezani CH, Okunishi K, Zaslona Z, Aronoff DM. Distinct protein kinase A anchoring proteins direct prostaglandin E2 modulation of Toll-like receptor signaling in alveolar macrophages. J Biol Chem 2011; 286: 8875–8883. 11 Edwards AS, Scott JD. A-kinase anchoring proteins: protein kinase A and beyond. Curr Opin Cell Biol 2000; 12: 217–221. 12 Amieux PS, McKnight GS. The essential role of RI alpha in the maintenance of regulated PKA activity. Ann NY Acad Sci 2002; 968: 75–95. 13 Kammerer S, Burns-Hamuro LL, Ma Y, Hamon SC, Canaves JM, Shi MM et al.. Amino acid variant in the kinase binding domain of dual-specific A kinaseanchoring protein 2: a disease susceptibility polymorphism. Proc Natl Acad Sci USA 2003; 100: 4066–4071. 14 Tingley WG, Pawlikowska L, Zaroff JG, Kim T, Nguyen T, Young SG et al. Gene-trapped mouse embryonic stem cell-derived cardiac myocytes and human genetics implicate AKAP10 in heart rhythm regulation. Proc Natl Acad Sci USA 2007; 104: 8461–8466. 15 Mehats C, Tanguy G, Paris B, Robert B, Pernin N. Pregnancy Induces a Modulation of the cAMP Phosphodiesterase 4-Conformers Ratio in Human Myometrium: Consequences for the Utero-Relaxant Effect of PDE4-Selective Inhibitors. J Pharmacol Exp Ther 2000; 292: 817–823. 16 Teo YY, Sim X, Ong RT, Tan AK, Chen J, Tantoso E et al. Singapore Genome Variation Project: a haplotype map of three Southeast Asian populations. Genome Res 2009; 19: 2154–2162.
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5 17 Dolan SM. Genetic and environmental contributions to racial disparities in preterm birth. Mt Sinai J Med 2010; 77: 160–165. 18 Engelhardt S, Zieger W, Kassubek J, Michel MC, Lohse MJ. Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium. J Clin Endocrinol Metab 1997; 82: 1235–1242. 19 Kosasa TS, Nakayama RT, Hale RW, Rinzler GS, Freitas CA. Ritodrine and terbutaline compared for the treatment of preterm labor. Acta Obstet Gynecol Scanda 1985; 64: 421–426.
© 2015 Nature America, Inc.
20 Phupong V, Charakorn C, Charoenvidhya D. Oral salbutamol for treatment of preterm labor. J Med Assoc Thai 2004; 87: 1012–1016. 21 Klukovits A, Verli J, Falkay G, Gaspar R. Improving the relaxing effect of terbutaline with phosphodiesterase inhibitors: studies on pregnant rat uteri in vitro. Life Sci 2010; 87: 733–737. 22 Soloff MS, Jeng YJ, Izban MG, Sinha M, Luxon BA, England SK. Effects of progesterone treatment on expression of genes involved in uterine quiescence. Reprod Sci 2011; 18: 781–797.
Journal of Perinatology (2015), 1 – 5
ORIGINAL ARTICLE
Genetic association of AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia1, YD Apalasamy1, SZ Suki1, SZ Omar2 and Z Mohamed1 OBJECTIVE: Preterm birth (PTB) is a multifactorial complication in which genetic and environmental factors contribute to the phenotype. The AKAP10 protein encoded by AKAP10 gene has a vital role in the maintenance of myometrial quiescence and pregnancy. This study aimed to investigate whether polymorphisms in the AKAP10 gene are associated with the risk of PTB. STUDY DESIGN: A total of 664 women (132 preterm and 532 term) with spontaneous singleton deliveries were genotyped for AKAP10 polymorphisms (rs119672, rs203462 and rs169412) using Sequenom MassARRAY platform. RESULT: A significant association was observed between the CC and AC genotypes of AKAP10 rs169412 with reduced risk of PTB (CC: adjusted odds ratio (OR) 2.95, 95% confidence interval (CI): 1.23–7.09, P = 0.016. AC: adjusted OR 3.46, 95% CI: 1.38–8.68, P = 0.008), respectively. Following stratification by ethnicity, a significant association was observed between the AC and CC genotypes of rs169412 and term birth in the Malay ethnic subgroup. (CC: OR 2.9, 95% CI: 1.01–8.59, P = 0.041. AC: OR 3.14, 95% CI: 1.04–9.54, P = 0.043). A significant association was also observed between the CT genotypes of AKAP10 rs119672 with reduced risk of PTB deliveries (CT: OR 3.2, 95% CI: 1.06–9.76 P = 0.007, TT: OR 2.8, 0.98–8.34, P = .0.015) Alternatively, there was no association between AKAP10 rs169412 and rs119672 polymorphisms with PTB in the Indians and Chinese ethnic groups. CONCLUSION: This study indicates a significant association between the AKAP10 polymorphisms and reduced risk of PTB in the Malays. This demonstrates the potential role of AKAP10 polymorphisms in preterm complications. Journal of Perinatology advance online publication, 25 June 2015; doi:10.1038/jp.2015.68
INTRODUCTION Preterm birth (PTB) is a major cause of neonatal mortality and morbidity globally with a rising percentage each year.1 Every year, about 15 million babies are delivered prematurely and ~ 1.1 million lives are lost owing to PTB complications.1 Although neonatal care has improved tremendously over the years, most of the preterm neonates who survive still suffer many short- and long-term health problems including bronchopulmonary dysplasia, hypotension, patent ductus arteriosus, intracerebral hemorrhage, necrotizing enterocolitis, anemia and infant jaundice.1,2 PTB is a pregnancy complication involving multifactorial confounders including genetics and environmental factors.3 Early diagnosis of high-risk women for PTB either before pregnancy or early in the first few weeks of pregnancy would help clinicians to monitor and provide treatment when necessary for these groups of women. Maternal genetic factors may contribute up to ⩾ 20%4 in occurrence of PTB. The genes involved in biological pathways that keep the uterus relaxed or cause contraction, have important roles in child labor and birth.4–6 So far, many genes have been examined for their roles in PTB including genes related to immune system, androgenic β-receptor and others.7 One important pathway of PTB is the A kinase pathway involving phosphorylation and relaxation of myometrial smooth muscles by protein kinase A (PKA).8–10 PKA acts at very discrete sites in the cell thus colocalization or restriction of PKA to its specific substrate increases its action. AKAP10 is one among many AKAPs that have been identified to bind and maintain the PKA enzyme at its site of action.11,12 This is achieved by the binding of AKAP10 to PKA resulting in the formation of the AKAP10–PKA complex.6 The
formation of this complex enhances the action of PKA as it is restricted at the site of action or substrate. Genetic polymorphism in human AKAP10 gene has been shown to increase the formation of the AKAP10–PKA complex thereby enhancing compartmentalization and phosphorylation of myometrial smooth muscle thus preventing muscle contraction and maintaining myometrial quiescence.5,6,11,12 The activation of PKA by cyclic adenosine monophosphate and compartmentalization of PKA by AKAP10 is crucial for the role of PKA in smooth muscle relaxation.13,14 Function of AKAP10–PKA complex in the uterus inhibits the initiation of labor.15 The objective of this study was to investigate whether there is an association between maternal genotype of AKAP10 gene polymorphisms (rs119672, rs203462 and rs169412) and risk of PTB.
MATERIALS AND METHODS Study subjects A total of 664 women (132 preterm and 532 term) with spontaneous singleton delivery were prospectively enrolled in this study from 2011 to 2013. Ethnicities of the subjects were validated by confirmations of no mixed marriages for at least three generations. During data collection, all participants self-reported their ethnicity as Malay, Chinese or Indians with confirmation of no mixed marriage of at least three generations. The participants were classified into two groups; the preterm group consisted of mothers who delivered their baby between 24 and 36 weeks, whereas the controls group consisted of mothers with uncomplicated pregnancies who delivered their baby between 38 and 41 weeks. The prospective case–control study was approved by the University of Malaya Medical
1 Pharmacogenomics Laboratory, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia and 2Department of Obstetrics and Gynecology, University of Malaya, Kuala Lumpur, Malaysia. Correspondence: Professor Z Mohamed, Pharmacogenomics Laboratory, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Selangor 50603, Malaysia. E-mail: [email protected] Received 3 January 2015; revised 19 March 2015; accepted 14 April 2015
AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia et al
2 Centre (UMMC) Ethics Committee and written informed consent was obtained from all participants. The physical well being of the participants was assessed by trained gynecologists. The pregnancy age was calculated from the first day of the last menstrual cycle or obtained from ultrasound results of the patients. Eligibility for participation was based on the following inclusion criteria: healthy mothers between the ages of 18 and 35 years with normal competent cervix and uterus, without metabolic or autoimmune disease, conceived preterm but with normal and healthy fetus. Exclusion criteria included maternal age of o18 years or 435 years, abnormal fetus and still birth, mothers with history of drug abuse, mothers who are smokers, mothers pregnant with twins, birth delivery by induction due to fetal distress or placenta abruption or preeclampsia or hypertension or any known medical problem and mothers who were not able to sign informed consents were also excluded from the studies. The demographic details and information of the mothers including their lifestyle, nutrition, medical conditions and family history were collected.
Statistical analysis Statistical analysis of subjects was performed using SPSS version 18.0 (IBM Corp., Chicago, IL, USA). Data were presented as percentage or mean ± s.d. Categorical and continuous variables were compared between preterm and term using Pearson’s χ2-test. Hardy–Weinberg equilibrium (HWE) was checked for the SNPs prior to genetic analysis using a goodness of fit χ2-test. The test for associations of the AKAP10 rs119672 and AKAP10 rs169412 with PTB was performed using logistic regression. Multivariate logistic regression models were adjusted for history of PTB, gestational diabetes and miscarriage. Confounding factors such as smoking, substance abuse, mothers who were themselves born preterm, hypertension and alcohol consumption were not included for statistical analysis in this paper because all subjects were free from these factors. Construction of linkage disequilibrium (LD) blocks and haplotype analyses were performed using Haploview 4.2 software (Broad Institute of MIT and Harvard, USA). Two AKAP10 SNPs were included in the analysis therefore α was 0.025 after Benferroni correction. The probability of the haplotype distributions were compared between term and preterm group by calculating the permutation P-values generated using a χ2 statistics with one degree of freedom. The power of the study was calculated using Quanto software (University of Southern California, USA).
AKAP10 genotyping A total of 2 ml of maternal venous blood samples were collected in both term and preterm subjects. The genomic DNA was extracted from whole blood using the GeneAll Exgene DNA purification kit (GeneAll Biotechnology, Korea). The rs119672, rs203462 and rs169412 single-nucleotide polymorphisms (SNPs) of A-kinase-anchoring protein 10 gene were genotyped using the Sequenom MassARRAY platform. SNPs with call rate of 490% were considered for statistical analysis. For the three PGR SNPs genotyped, two (PGR rs660149 and rs471767) with call rates of 99% were considered for statistical analysis, whereas the PGR rs10895068 SNP with a call rate of 81% was excluded from statistical analysis.
Table 1.
Demographic features of 532 term and 132 preterm subjects
Characteristics Maternal age Maternal BMI (kg m − 2) Married subjects (%) Gestational Diabetes (%) History of PTB (%) Previous miscarriage (%) Low income (%) Average income (%) High income (%)
Term
Preterm
P-value
29.3 ± 3.7 24.5 ± 5.4 98 0 0 15 2 33 64
29.8 ± 3.0 24.3 ± 5.1 98 11 14 25 3 38 62
0.23 0.69 0.72 o0.001 o0.001 0.011 0.551
Abbreviations: BMI, body mass index; PTB, preterm birth. Data are expressed as mean ± s.d. for continuous data and as percentage for categorical data. P-values were obtained using Mann–Whitney U-test or independent t-test. P-values o 0.05 were considered significant.
Table 2.
RESULTS The demographic and clinical parameters of the subjects are shown in Tables 1 and 2. Past history of PTB, miscarriage and gestational diabetes were significantly different between the preterm and term groups. There were no significant differences in maternal age, BMI, marital status, income and between preterm and term groups (Tables 1 and 2). There was no deviation from Hardy–Weinberg equilibrium HWE for AKAP10 rs119672 and AKAP10 rs169412 SNPs in both preterm and term subjects (Table 3). Genetic modeling revealed a significant association between AKAP10 rs119672 variants with preterm and term deliveries (Table 4). The odds of the TT genotype occurring among women with term delivery were twice those among the preterm subjects (odds ratio (OR) 2.95, 95% confidence interval (CI) 1.23–7.06, P = 0.015; Table 4). The CT genotype of rs119672 was also significantly high in the term than preterm subjects. The odds of the CT genotype occurring among the term group were thrice those among the preterm subjects (OR 3.58, 95% CI 1.42–8.98, P = 0.007). For rs169412, the AC genotype was significantly high in the term than preterm subjects (OR 3.46, 95% CI 1.38–8.68, P = 0.008). There was also a significant difference in the CC genotype of rs169412 between preterm and term subjects (OR 2.95, 95% CI 1.23–7.09, P = 0.016). The results show a strong association between AKAP10 polymorphisms and term birth. After stratification according to ethnicity, the CT genotype of rs119672 and the AC or CC genotypes of the rs169412 polymorphisms were high in the Malay ethnic group (Table 4).
Demographic characteristics of subjects by ethnicity
Characteristics
Malays (N = 479)
Indians (N = 93)
Chinese (N = 93)
Term (N = 388) Preterm (N = 91) P-value Term (N = 74) Preterm (N = 19) P-value Term (N = 70) Preterm (N = 22) P-value Maternal age Maternal BMI (kg m − 2) Married subjects (%) Gestational Diabetes (%) History of PTB (%) Previous miscarriage (%) Low income (%) Average income (%) High income (%)
29.2 ± 5.6 24.3 ± 5.6 100 0 0 14 2.8 29 68
29.8 ± 4.1 24.6 ± 5.4 99 8.0 14 24 3.0 35 61
0.249 0.267 0.974 o 0.001 o 0.001 0.017 0.212
30.0 ± 3.7 25.6 ± 5.7 98 0 0 19 3 55 41
30.3 ± 3.7 24.2 ± 5.5 99 10 15 26 5 31 63
0.938 0.550 0.961 0.004 o0.001 0.511 0.161
25.9 ± 3.6 23 ± 5.5 100 0 0 17 1 27 72
29.4 ± 3.5 24.8 ± 4.5 99 20 12 28 0 36 64
0.946 0.329 0.920 o0.001 0.005 0.288 0.593
Abbreviations: BMI, body mass index; PTB, preterm birth. Data are expressed as mean ± s.d. for continuous data and as percentage for categorical data. P-values were obtained using Mann–Whitney U-test or independent t-test. P-values o0.05 were considered significant.
Journal of Perinatology (2015), 1 – 5
© 2015 Nature America, Inc.
AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia et al
3 Table 3.
Genotype distribution of AKAP10 SNPs Genotype frequencies N(%)
Ethnicity
HWE- P
Preterm vs term
Preterm/term
IL1R2, rs119672 C/T All ethnicity Malay Indians Chinese
CC 0.08 0.07 0.09 0.05
vs vs vs vs
0.03 0.02 0.07 0.01
CT 0.30 0.31 0.33 0.27
vs vs vs vs
0.34 0.35 0.38 0.24
TT 0.61 0.63 0.58 0.68
vs vs vs vs
0.63 0.63 0.55 0.74
0.134/0.532 0.253/0.251 0.188/0.901 0.295/0.533
IL1R2 rs169412A/C All ethnicity Malay Indians Chinese
AA 0.08 0.06 0.07 0.05
vs vs vs vs
0.03 0.02 0.06 0.01
AC 0.31 0.31 0.32 0.32
vs vs vs vs
0.34 0.34 0.44 0.25
CC 0.60 0.62 0.47 0.64
vs vs vs vs
0.63 0.64 0.49 0.74
0.179/0.532 0.431/0.295 0.640/0.342 0.702/0.481
Abbreviation: SNPs, single-nucleotide polymorphisms. X2P: Chi square P-value, HWE-P:Hardy Weinberg equilibrium P-value. Po0.05 was considered significant.
Table 4.
Logistic regression analysis for AKAP10 genotype association with preterm birth in overall ethnicity, Malay Indians and Chinese ethnic
groups SNPs
Genotypes
Overall ethnicity (N = 664) OR (95% CI)
rs119672 rs169412
CC CT TT AA AC CC
Reference 3.58 (1.42–8.98) 2.95 (1.23–7.06) Reference 3.46 (1.38–8.68) 2.95 (1.23–7.09)
P-value 0.007 0.015 0.008 0.016
Malays (N = 479) OR (95% CI) Reference 3.2 (1.06–9.76) 2.8 (0.98–8.34) Reference 3.14 (1.04–9.54) 2.94 (1.0–8.59)
Indians (N = 93)
Chinese (N = 92)
P-value
OR (95% CI)
P-value
OR (95% CI)
P-value
0.039 0.055
0.27 (0.55–1.30) 0.27 (0.06–1.22)
0.103 0.091
Reference 0.35 (0.02–6.57) 0.29 (0.02–4.89)
0.485 0.389
0.043 0.041
0.23 (0.05–1.13) 0.31(0.07–1.41)
0.071 0.130
0.41 (0.02–7.55) 0.28 (0.02–4.67)
0.550 0.275
Abbreviations: CI, confidence interval; OR, odds ratio. P-values were adjusted for GDM, previous PTB and previous miscarriage.
Figure 1.
LD block of AKAP10 SNP (rs119672, rs169412) in the overall ethnicity, Malays, Chinese and Indians.
Carriers of one or two risks allele (CT/TT-rs119672, AC/CCrs169412) were associated with reduced risk of PTB. No significant association was seen in the Indian and Chinese ethnic group between AKAP10 SNPs and PTB. Figure 1 shows the LD pattern of the AKAP10 gene. The LD between AKAP10 rs119672 and AKAP10 rs169412 in the overall ethnicity was high (D′ = 0.88). High LD was also seen in the Malays (D′ = 0.87), Chinese (D′ = 0.91) and Indians (D′ = 0.97) ethnic group. Table 5 shows the overall population haplotype block frequency. The haplotype TC was seen frequently compared with CA, TA and CC haplotypes. The frequency of CA haplotypes was higher in the preterm group compared with the term group. However, none of the haplotypes were significantly associated with PTB. © 2015 Nature America, Inc.
Table 5.
Haplotype association analysis of AKAP10 gene variants
Haplotype Block 1 TC CA TA CC
Frequency All
Preterm
Term
χ2
P-value
0.773 0.188 0.02 0.019
0.748 0.225 0.016 0.012
0.779 0.179 0.021 0.02
1.207 2.896 0.338 0.831
0.2719 0.0888 0.5611 0.3621
Block 1 involves SNP rs119678 and rs169412. P-value corrected for 1000 permutation test.
Journal of Perinatology (2015), 1 – 5
AKAP10 gene polymorphism with reduced risk of preterm birth IM Langmia et al
4
DISCUSSION In this study, we showed an association between AKAP10 variants and genetic association with term birth. AKAP10 rs119672 and rs169412 were both shown to confer reduced risk of PTB. To the best of our knowledge, this is the first study that investigates the AKAP10 polymorphisms (rs119672 and rs169412) in mothers with PTB. In this study, the genotypes of AKAP10 rs119672 and rs169412 were significantly associated with term birth in overall subjects. Mothers with TT or CT genotypes of rs119672 and CC or AC genotypes of rs169412 delivered more at normal pregnancy duration after 37 weeks of gestation. After stratification by ethnicity, the same trend was observed in the Malay ethnic group. The CT genotype of 119672 and AC genotype of 169412 had higher frequency in mothers who delivered at normal pregnancy duration after 37 weeks of gestation. On the other hand, no significant association was seen in the Indian and Chinese ethnic group. However, AKAP10 genotype frequencies varied among the Malays, Chinese and Indians and our results showed variability in LD patterns of AKAP10 SNPs among the three ethnic groups.16 Previous study has shown that the racial disparity in the occurrence of PTB among Caucasians and African American women is due to the differences their genetic makeup.17 Malaysia has a multiethnic population with three major ethnic groups and we suggest that the difference in the occurrence of PTB among women in this population is owing to differences in the genetic makeup specific to each ethnicity. The AKAP10 variants confer reduced risk of PTB in the Malays, which may indicate that the AKAP10 polymorphisms are prognostic markers among the Malays. We cautiously consider our sample to be adequately powered to detect the genetic associations. This study had a power of 80% and 87% for rs119672 and rs169412, respectively. This study demonstrates the potential role of AKAP10 polymorphisms in PTB complication and therapeutic target for development of new diagnostic and treatment strategies. The role of PKA/AKP10 complex in pregnancy, especially in maintenance of myometrial smooth muscle relaxation, has been shown in animal studies.9 The AKAPs are crucial in targeting PKA to its site of action for PKA phosphorylation of myometrial smooth muscles. The active form of PKA in the cell can be distracted from phosphorylating smooth muscles by protein kinase inhibitors. Protein kinase inhibitors act as pseudosubstrates to the active form of protein kinases, by binding to the active form of the kinase enzyme, thereby preventing phosphorylation. Verli and his colleagues suggested that a combination of selective phosphodiesterases inhibitors and β-adrenergic agonist that will increase the levels of cAMP should be considered for treatment of preterm contractions. The current drug treatment for preterm labor is with the use of the β2 agonists such as salbutamol or terbutaline.18–20 but theoretically, it may be possible to also use a substance that increases PKA targeting to the substrate site or a phosphodiesterase inhibitor.4,20,21 Genetic variants in the anchoring enzyme (AKAP10) might result in a conformational change that increase AKAP10 binding to PKA leading to increased formation of AKAP10–PKA complex. This will result in increased compartmentalization allowing for enhanced binding of cyclic AMP to PKA. Studies have shown that binding and activation of PKA by cAMP increases phosphorylation of myometrial smooth muscles and thus maintains pregnancy.4,22 CONCLUSION In conclusion, our results demonstrated a significant association between the AKAP10 rs119672 and rs169412 with term birth. This study suggests the role of AKAP10 gene variants in PTB Journal of Perinatology (2015), 1 – 5
complication. Prior information on the genetic composition of women may help in the identification and management of high-risk mothers. Future studies leading to the discovery of new drugs that can be used to enhance the relaxation of myometrial smooth muscles till the end of gestation period might result in prevention of PTB globally. Discovery of new biomarkers that can be used for early prediction of high-risk women will also help to diminish PTB. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank the patients and staffs of University of Malaya Medical Centre (UMMC) for their participation in this study. This study was funded by the University of Malaya research grant PG118-2012B and HIR MOHE E000025-20001.
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