|Year : 2022 | Volume
| Issue : 2 | Page : 131-135
Parental transmission effects of the PAX7 Polymorphisms among non-syndromic cleft lip palate: A case-parent trio study
Mahamad Irfanulla Khan1, CS Prashanth2, Mohammed S Mustak3, Sheikh Nizamuddin4, Avinash Tejasvi5
1 Department of Orthodontics and Dentofacial Orthopedics, The Oxford Dental College, Bangalore, Karnataka, India
2 Department of Orthodontics and Dentofacial Orthopedics, DAPM R.V Dental College, Bangalore, Karnataka, India
3 Department of Applied Zoology, Mangalore University, Mangalore, Karnataka, India
4 Department of Urology, Medical Center-University of Freiburg, Germany, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany
5 Department of Oral Medicine and Radiology, Kamineni Institute of Dental Sciences, Narketpally, Telangana, India
|Date of Submission||16-Feb-2022|
|Date of Decision||13-May-2022|
|Date of Acceptance||18-May-2022|
|Date of Web Publication||22-Jun-2022|
Mahamad Irfanulla Khan
Department of Orthodontics and Dentofacial Orthopedics, The Oxford Dental College, Bangalore - 560 068, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Orofacial clefts (OFCs) are the most common congenital deformities in the human face, with a prevalence of around 1:800 to 1:1000 among the Indian population. Aim: To assess the Paired box 7 (PAX7) gene polymorphisms in the etiology of non-syndromic cleft lip and cleft palate (NSCL/P) in the Indian population using a case-parent trio design and to contemplate parent-of-origin effects. Materials and Methods: The study comprised forty case-parent trios of NSCL/P from the Indian population. Genomic deoxyribonucleic acid (DNA) was isolated from the cases and their parents. The polymorphisms rs6659735, rs553934, rs624761, rs609959, and rs4075768 of the PAX7 gene were genotyped using Agena Bio MassARRAY analysis. The transmission disequilibrium test (TDT) was performed using the PLINK software and the pairwise linkage disequilibrium analysis by the Haploview software. Results: The genotyping of the polymorphisms rs6659735, rs553934, rs624761, rs609959, and rs4075768 was done. We found a significant association of the rs6659735 (P-value = 0.03, Odds ratio = 4.5) with NSCL/P in the allelic frequencies of the Indian case-parent trios. In addition, the parent-of-origin effects were observed as the allelic TDT analysis showed a significant paternal transmission of the rs6659735 (P-value = 0.04), whereas the rs553934 (P-value = 0.04) showed an excess maternal transmission. Conclusions: The parent-of-origin effects were observed as the two polymorphisms demonstrated a significant parental transmission. The rs6659735 showed a significant paternal transmission, whereas the rs553934 showed an excess maternal transmission, suggesting that the PAX7 gene may influence the risk of NSCL/P in the Indian population.
Keywords: Case-parent trios, genotyping, orofacial clefts, paired box 7, parent-of-origin
|How to cite this article:|
Khan MI, Prashanth C S, Mustak MS, Nizamuddin S, Tejasvi A. Parental transmission effects of the PAX7 Polymorphisms among non-syndromic cleft lip palate: A case-parent trio study. J Indian Acad Oral Med Radiol 2022;34:131-5
|How to cite this URL:|
Khan MI, Prashanth C S, Mustak MS, Nizamuddin S, Tejasvi A. Parental transmission effects of the PAX7 Polymorphisms among non-syndromic cleft lip palate: A case-parent trio study. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Jul 1];34:131-5. Available from: https://www.jiaomr.in/text.asp?2022/34/2/131/347929
| Introduction|| |
Orofacial clefts (OFCs) are the most common congenital craniofacial malformations in humans, with a prevalence of around 1:800 to 1:1000 among the Indian population. The etiology is highly complex and heterogeneous, comprising genetic and environmental factors., Infants born with these congenital deformities may have difficulty in speech, hearing, feeding, and other psychosocial problems.
Several studies suggest that about 70% of the Cleft lip with or without cleft palate (CL/P) cases are non-syndromic and occur as isolated cases, whereas 30% are syndromic, which are associated with some other developmental anomalies., Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common form of OFC, but the birth prevalence rates vary in different populations and racial groups.
The Paired box 7 (PAX7) gene (OMIM 167410) belongs to the paired box (PAX) family and is located at 1p36.13. It plays a vital role in the development of neural crest cells.
High-risk polymorphisms rs6659735, rs553934, rs624761, rs609959, and rs4075768 of the PAX7 gene are involved in the etiology of NSCL/P and have been evaluated in different populations worldwide. However, the current literature review reveals no reported case-parent trio studies among the Indian population to evaluate the possible association between the PAX7 gene and the risk of NSCL/P. Hence, we selected these high-risk polymorphisms from the literature (previous genetic studies) to test for the possible association between these polymorphisms of the PAX7 gene with NSCL/P in the Indian population considering the parent-of-origin (PoO) effects.
| Material and Methods|| |
Study participants and ethics statement
A total of 40 NSCL/P case-parent trios (120 subjects) were selected from the cleft lip and palate clinic of the Mallige medical center, Bangalore. The craniofacial specialist examined each case to exclude the syndromic forms of cleft lip with or without cleft palate. The research was approved by the Institutional Review Board of the D. A. Pandu Memorial (DAPM) RV Dental College, Bangalore (IRB No. 230/Vol-2/2017, dated:10.06.2017). The research protocol was performed following the declaration of Helsinki's ethical principles for medical research involving human subjects. The phenotypic features and the samples included were Cleft lip and palate (CLP: 21 subjects), Cleft lip (CL: 13 subjects), and Cleft palate (CP: 6 subjects) only.
Sample size estimation
The sample size was estimated using G* Power software (Universitat Dusseldorf, Germany) with a statistical power of 80% and a significance level of 0.05. Therefore, it was determined to include 40 case-parent trios (120 subjects).
In this study, polymorphisms rs6659735, rs553934, rs624761, rs609959, and rs4075768 of the PAX7 gene, were selected from the literature (previous genetic studies) and the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/projects/SNP).
DNA isolation and genotyping
After obtaining the written informed consent from the cases and parents, the general information, including age, gender, ethnicity, and congenital disabilities, were recorded. 3 ml of venous blood was taken in the Ethylene Diamine Tetra Acetic acid (EDTA) coated vials from each participant for the genotyping.
Genomic deoxyribonucleic acid (DNA) was extracted from the blood lymphocytes of each sample using a commercial DNA extraction kit (QIAamp DNA Mini kit, Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions and was stored at -70°C. After that, all the polymorphisms were genotyped using the Agena Bio MassARRAY analyzer (Agena Bioscience, Inc., San Diego, USA), which works based on the Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Finally, the genomic data obtained was sent for statistical analysis.
PLINK software (v1.09; http://pngu.mgh.harvard.edu/purcell/plink/) was used for the statistical analysis of the SNP assay data obtained from the MassArray system Hardy-Weinberg equilibrium (HWE) was assessed for all polymorphisms. We performed the allelic transmission disequilibrium test (TDT) to assess the transmitted and non-transmitted target alleles. The parent-of-origin effects were analyzed using the same PLINK software. The odds ratio (OR) and 95% confidence intervals (CIs) were calculated, and a P value <0.05 was set as statistically significant.
Haplotype identification and haplotype-TDT were performed using the Haploview tool. The pairwise linkage disequilibrium (LD) [Figure 1] was assessed for all the polymorphisms using Haploview software.
|Figure 1: The Linkage disequilibrium blocks for the PAX7 haplotype analysis|
Click here to view
| Results|| |
All the polymorphisms rs6659735, rs553934, rs624761, rs609959, and rs4075768 had a call rate of 100% and followed the Hardy-Weinberg equilibrium test. [Table 1] summarizes the results of TDT analysis and family-based association of PAX7 with NSCL/P. We found a significant association of the rs6659735 (P-value = 0.03, odds ratio = 4.5) with NSC/LP in the allelic frequencies of the Indian case-parent trios.
Parent-of-origin effects were evaluated using the TDT; the two polymorphisms of the PAX7 gene demonstrated a significant parental transmission. The rs6659735 (A allele, P = 0.04) showed a paternal transmission, whereas rs553934 (G allele, P = 0.04) showed an excess of maternal transmission [Table 2].
The LD plot of the five polymorphisms analyzed is presented in [Figure 1]. A haplotype block was constructed in this region by the D′ (D′ = standardized coefficient of LD) and r2 (pairwise correlation coefficient) values. In LD block 1, the haplotypes showed a significant association and transmission (P = 0.03) among the Indian case-parent trios.
| Discussion|| |
The development of NSCL/P is complex and heterogeneous, involving various genetic and environmental risk factors. Despite the complex etiology and pathogenesis of NSCL/P, several genetic factors have been identified by genome-wide association (GWA) studies, linkage studies, and whole-exome sequencing, providing a much more efficient way to detect susceptibility genes and loci causing NSCL/P. The PAX7 gene is located at 1p36.13, which encodes specific DNA binding transcription factors, and in humans, it is involved in neural crest development.
Several linkage analyses, genome-wide association studies, and whole-exome sequencing have been widely used to explain the etiopathogenesis of NSCL/P. Although GWA studies have reported multiple genes or loci involved in the etiology of NSCL/P,,, only 20% of the genetic heritability of these congenital disabilities can be explained.
PAX7 is confirmed by genome-wide association (GWA) studies and meta-analysis in the etiology of NSCL/P. Therefore, the identification of this PAX7 gene in our population would clarify the etiology and pathogenesis involved in CL/P. Meanwhile, most GWA investigations were largely focused on the European population. Other ethnic populations are usually small, making it difficult to identify the specific genes involved in the development of NSCL/P.
Parent-of-origin effects have been widely considered alternative mechanisms to study the genetic etiology of NSCL/P. It is essential to contemplate the parent-of-origin effects when studying congenital disabilities such as cleft lip and palate because maternal genotype controls the in-utero environment of the developing fetus., When the mother is exposed to any specific environmental factor, an incompatibility between the maternal and fetal genomes may disrupt the normal embryogenesis. Each gene may contribute to the development of NSCL/P. Excess maternal transmission effects in the development of NSCL/P have also been reported for the Methylenetetrahydrofolate reductase (MTHFR), Cystathionine Beta-Synthase (CBS), and Transforming Growth Factor Beta 3 (TGFB3) genes.,
The family studies for the Indian population reported that several genes such as CRISPLD2, RUNX2, SOX1-OT, MAPK4, CTIF, MYO5B, SMAD7, LOXHD1, and SKA1 are not associated with NSCLP in Indian multiplex families.,, We conducted a family-based case-parent trio study as they are very limited in the Indian population. The trio studies are another method to conventional case-control studies in which the cleft children and their parents (father and mother) are genotyped. The advantages of these studies include the robust sample selection, testing maternal versus paternal effects, and being unbiased as the affected cleft child and the parents share the same genetic ancestry.
But A et al. recruited cleft lip and palate patients from Iowa, Japan, Mongolia, and the Philippines and showed a significant association of rs6695765 with NSCL/P in all the cleft groups combined in the Iowa population. In a case-control study by Guo Q et al. on non-syndromic orofacial clefts from northern China, the rs6695765 showed no significance.
A study by Sull JW et al., comprised of Taiwanese, Singaporean, Korean, and Maryland case-parent trios, revealed an excess maternal transmission of the polymorphism rs553934, whereas the rs624761 and rs609959 showed no significant association with NSCL/P.
In the European case-parent trios, when they tested for gene-gene (G × G) interaction, the polymorphism rs4075768 showed no significance.
Few genetic studies reported an association between PAX7 polymorphisms and risk of NSCL/P, whereas few studies showed no association. These differences and inconsistencies in results may be due to geographic origin, ethnicity, and the susceptibility of the loci associated with the complex etiology of NSCL/P.
Our case-parent trio study showed that the polymorphisms rs6659735 and rs553934 of PAX7 genes are associated with NSCL/P in our population may provide new insight into the previous GWA studies. The evidence of excess maternal (rs553934) and paternal transmission (rs6659735) suggest the genomic imprinting or the genotype effects of the parents.
| Conclusion|| |
The current study revealed that in the Indian case-parent trios, the polymorphism rs6659735 is associated with NSCL/P. The parent-of-origin effects were observed as the two polymorphisms demonstrated a significant parental transmission. The rs6659735 showed a significant paternal transmission, whereas the rs553934 showed an excess maternal transmission suggesting that the PAX7 gene may influence the risk of NSCL/P in the Indian population.
Further research is needed to study the role of these polymorphisms with a larger sample size for the Indian population and establish the impact of the PAX7 gene in the etiology of NSCL/P.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their clinical information to be reported in the journal. The patients understand that their names and initials will not be published, and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Schutte BC, Murray JC. The many faces and factors of orofacial clefts. Hum Mol Genet 1999;8:1853-9.
Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: Understanding genetic and environmental influences. Nat Rev Genet 2011;12:167-78.
Rahimov F, Jugessur A, Murray JC. Genetics of non-syndromic orofacial clefts. Cleft Palate Craniofac J 2012;49:73-91.
Spritz RA. The genetics and epigenetics of Orofacial clefts. Curr Opin Pediatr 2001;13:556-60.
Murthy J. Management of cleft lip and palate in adults. Indian J Plast Surg 2009;42(Suppl): S116-22.
Wong FK, Hagg U. An update on the etiology of orofacial clefts. Hong Kong Med J 2004;10:331-6.
Huang L, Jia Z, Shi Y, Du Q, Shi J, Wang Z, et al
. Genetic factors define CPO and CLO subtypes of the non-syndromic orofacial cleft. PLoS Genet 2019;15:e1008357.
Basch ML, Bronner-Fraser M, Garcia-Castro MI. Specification of the neural crest occurs during gastrulation and requires Pax7. Nature 2006;441:218-22.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al
. PLINK: A tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559-75.
Spielman RS, Mc Ginnis RE, Ewens WJ. Transmission test for linkage disequilibrium: The insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 1993;52:506-16.
Mansouri A, Stoykova A, Torres M, Gruss P. Dysgenesis of cephalic neural crest derivatives in Pax7 mutant mice. Development 1996;122:831-38.
Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, et al
. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet 2010;42:525-9.
Sun Y, Huang Y, Yin A, Pan Y, Wang Y, Wang C, et al
. Genome-wide association study identifies a new susceptibility locus for cleft lip with or without a cleft palate. Nat Commun 2015;6:6414.
Yu Y, Zuo X, He M, Gao J, Fu Y, Qin C, et al
. Genome-wide analyses of non-syndromic cleft lip with palate identify 14 novel loci and genetic heterogeneity. Nat Commun 2017;8:14364. doi: 10.1038/ncomms14364.
Leslie EJ, Carlson JC, Shaffer JR, Buxó CJ, Castilla EE, Christensen K, et al
. Association studies of low-frequency coding variants in non-syndromic cleft lip with or without cleft palate. Am J Med Genet A 2017;173:1531-8.
Weinberg CR, Umbach DM. A hybrid design for studying genetic influences on the risk of diseases with onset early in life. Am J Hum Genet 2005;77:627-36.
Wilkins JF, Haig D. What good is genomic imprinting: The function of parent-specific gene expression. Nat Rev Genet 2003;4:359-68.
Ludwig KU, Mangold E, Herms S, Nowak S, Reutter H, Paul A, et al
. Genome-wide meta-analyses of non-syndromic cleft lip with or without cleft palate identify six new risk loci. Nat Genet 2012;44:968-71.
Van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocké MC, Zielhuis GA, Goorhuis-Brouwer SM, et al
. Does the interaction between maternal folate intake and the methylenetetrahydrofolate reductase polymorphisms affect the risk of cleft lip with or without cleft palate? Am J Epidemiol 2003;157:583-91.
Reutter H, Birnbaum S, Mende M, Lauster C, Schmidt G, Henschke H, et al
. TGFB3 displays parent-of-origin effects among central Europeans with nonsyndromic cleft lip and palate. J Hum Genet 2008;53:656-61.
Neela PK, Reddy SG, Husain A, Mohan V. CRISPLD2 Gene polymorphisms with non-syndromic cleft lip and palate in Indian population. Global Med Genet 2020;7:22-5.
Neela PK, Gosla SR, Husain A, Mohan V, Thumoju S, Bv R. Association of MAPK4 and SOX1-OT gene polymorphisms with cleft lip palate in multiplex families: A genetic study. J Dent Res Dent Clin Dent Prospect 2020;14:93-6.
Neela PK, Gosla SR, Husain A, Mohan V, Thumoju S, BV R. Association of single nucleotide polymorphisms on locus 18q21.1 in the etiology of non-syndromic cleft lip palate (NSCLP) in Indian multiplex families. Global Med Genet 2021;8:24-31.
Khan MI, Prashanth CS. Case-parent trio studies in cleft lip and palate. Global Med Genet 2020;7:75-9.
Butali A, Suzuki S, Cooper ME, Mansilla AM, Cuenco K, Leslie EJ, et al
. Replication of genome wide association identified candidate genes confirm the role of common and rare variants in PAX7 and VAX1 in the etiology of non-syndromic CL (P). Am J Med Genet A 2013;161A: 965-72.
Guo Q, Li D, Meng X, Liu T, Shi J, Hao Y, et al
. Association between PAX7 and NTN1 gene polymorphisms and nonsyndromicorofacial clefts in a northern Chinese population. Medicine (Baltimore) 2017;96:e6724.
Sull JW, Liang KY, Hetmanski JB, Fallin MD, Ingersoll RG, Park J, et al
. Maternal transmission effects of the PAX genes among cleft case-parent trios from four populations. Eur J Hum Genet 2009;17:831-9.
Xiao Y, Taub MA, Ruczinski I, Begum F, Hetmanski JB, Schwender H, et al
. Evidence for SNP-SNP interaction identified through targeted sequencing of cleft case-parent trios. Genet Epidemiol 2017;41:244-50.
[Table 1], [Table 2]