Introduction
Mandibular retrognathism is a type of malocclusion that refers to an abnormal posterior position of the mandible as a result of a developmental abnormality. From the literature, it is evident that the mandibular growth pattern is determined by the intramembranous ossification of the mandibular body and endochondral ossification of the condyle. Matrilin-1 is a cartilage extracellular matrix protein, and matrilin-1 gene ( MATN1 ) polymorphisms have been found to be involved in dental malocclusions of humans. In this study, we aimed to examine the association between MATN1 polymorphisms and the risk of mandibular retrognathism, in a case-control study with a South Indian population.
Methods
Eighty-one patients with mandibular retrognathism (SNB, <78°) and 71 controls having an orthognathic mandible (SNB, 80° ± 2°) were recruited. In both the patient and control groups, subjects with an orthognathic maxilla (SNA, 82° ± 2°) were included. Three single nucleotide polymorphisms of the MATN1 gene (rs1149048, rs1149042, and rs1065755) were genotyped using polymerase chain reaction-restriction fragment length polymorphism. The statistical association analysis was performed using the chi-square test. Pair-wise linkage disequilibrium was computed, and haplotypes were compared between subjects and controls. Nonparametric tests were used to compare cephalometric measurements between groups.
Results
No polymorphic site deviated from Hardy-Weinberg equilibrium in the controls. The rs1149042 genotypes and alleles were found to be associated with reduced risk of mandibular retrognathism. Furthermore, rs1149042 genotypes were associated with mandibular measurements (SNB and ANB). There was no strong and consistent linkage disequilibrium linkage disequilibrium across two different single nucleotide polymorphisms and haplotypes were not associated with mandibular retrognathism.
Conclusions
The results of our study suggest an association between the MATN1 gene polymorphisms and mandibular retrognathism.
Highlights
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The matrilins are a family of noncollagenous extracellular matrix proteins.
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We aimed to evaluate MATN1 gene polymorphisms as a risk factor for mandibular retrognathism.
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Polymorphisms of MATN1 gene (rs1149048, rs114904, and rs1065755) were analyzed for 81 subjects and 71 controls.
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The rs1149042 genotypes and alleles were associated with reduced risk of mandibular retrognathism.
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The rs1149048 genotypes showed significant differences in mandibular corpus length.
Malocclusion broadly refers to any irregular arrangement and contact of the maxillary teeth with the mandibular teeth. Malocclusions can vary, which could be viewed characteristically in different parts of the world, among and in different ethnic groups. The incidence of Angle Class II Division 1 malocclusion in a South Indian population is 6.8% to 15.5%. Patients with a Class II Division 1 malocclusion often exhibit a significant retrognathic mandible. Mandibular retrognathism is 1 congenital or acquired anomaly of the skeletal jaw-cranial base relationship. Analysis of lateral and frontal roentgenographic cephalograms of Class II malocclusion patients and their parents showed high correlation coefficients between parents and their offspring, indicating a strong familial tendency in the development of a Class II malocclusion. Assessment of skeletal and dental patterns showed that the skeletal measurements are more correlated with Class II Division 1 malocclusion than are the dental measurements. The incidence of Class II malocclusion in a particular population warrants a basis for planning preventive and interceptive orthodontics to address the retrusive mandible. The relative influence of genetics and environmental factors in the etiology of malocclusion has been a matter of debate in the orthodontic literature. Genes responsible for regulation of bone and cartilage development can be divided into those encoding bone-matrix and cartilage-matrix proteins and those regulating cellular or other gene activities.
The matrilins are a family of noncollagenous extracellular matrix proteins. Matrilin-1 is secreted primarily by chondrocytes and has a role in the assembly of cartilage extracellular matrix. It is a noncollagenous protein that is secreted by chondrocytes in the maturation area of the growth plate. The matrilin family at present has 4 members (matrilin-1, matrilin-2, matrilin-3, and matrilin-4). The matrilin genes may be strictly and differently regulated, and their expressions may serve as markers for cellular differentiation. Matrilin-1 is a constituent of collagen-dependent and collagen-independent fibrils and is associated with cartilage proteoglycans. It is expressed mainly in cartilaginous tissues in the growth plate of developing long bones. Matrilin-1 plays a decisive role in endochondral bone formation. Chondrocytes in the temporomandibular joint condyle can also express matrilin-1. The MATN1 gene is mapped to chromosome 1p35. The gene spans over 12 kilobase (kb) and contains 8 exons coding for 496 amino acids, including a 22-residue signal peptide. Matrilin-1 gene is known to contribute to the genetic susceptibility of mandibular prognathism. However, the association of matrilin-1 gene with mandibular retrognathism in the Indian population has not been studied so far. Thus, the aim of this study was to evaluate the association between matrilin-1 gene polymorphisms and the risk of mandibular retrognathism.
Material and methods
The study group consisted of 152 persons, including 71 controls and 81 subjects with mandibular retrognathism. All subjects were recruited from the Department of Orthodontics and Dentofacial Orthopaedics, Faculty of Dental Sciences, Sri Ramachandra University in Chennai, India. The study was approved by the institutional ethics committee and followed the Helsinki guidelines on ethics for human research. Written informed consent was obtained from all adult subjects. Parents or legal guardians provided written consent on behalf of minors. From each study subject, lateral cephalograms were obtained. Tracings of the lateral cephalograms were done manually on acetate matte tracing paper. All tracings were done by 2 independent researchers (P.B.B., A.B.C.). Assessment of interrater reliability to demonstrate consistency among measurements taken by the 2 observers was used. The interrater reliability was assessed by computing intraclass correlations, which incorporate the magnitude of the disagreement and marginal distributions of each observer in which the agreement expected by chance was determined by the marginal frequencies. The intraclass correlation coefficient and marginal distributions of variables documented in the Supplementary Table and the Supplementary Figure , respectively, demonstrate the reliability of the measurements. Patients having a retrognathic mandible (SNB, <78°) were selected as subjects, and those with an orthognathic mandible (SNB, 80° ± 2°) were selected as controls. Furthermore, both subjects and controls had an orthognathic maxilla (SNA, 82° ± 2°). Patients with a retrognathic or prognathic maxilla (SNA, <80° and >84°), diagnosed syndromes, and those under orthodontic treatment were excluded from the study. Hence, the subjects in our study were a uniquely selected subset, with the maxilla orthognathic and the mandible retrognathic based on the analysis of Steiner. Power analysis showed that the sample sizes used in this study were large enough to detect a significant odds ratio of 0.32, with power of 71% and an alpha of 5%, and an exposure difference of 0.155 between subjects and controls. An increased sample size of 100 subjects or 88 controls would increase the power of the study to 80%. Since the power in our sample was smaller than that usually obtained, an additional 20 mandibular retrognathism subjects and 20 controls would be important to achieve sufficient statistical power.
A 3-mL peripheral blood sample was collected from all subjects. DNA was obtained from the blood samples using a standard procedure. Genotyping of the 3 important MATN1 polymorphisms (rs1149048, rs1149042, and rs1065755) was performed by using polymerase chain reaction-restriction fragment length polymorphism. Departure of genotype frequencies from the Hardy-Weinberg equilibrium was assessed using the chi-square test with 1 degree of freedom. To test the associations between MATN1 gene polymorphisms and mandibular retrognathism, the chi-square test was performed. Odds ratios and 95% confidence intervals were calculated using the STATCALC feature of EpiInfo software (version 7; Centers for Disease Control and Prevention, Atlanta, Ga). Haplotypes and pair-wise linkage disequilibrium measures (D’ and r 2 ) were estimated using the Haploview program (version 3.12; Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, MA) that uses an accelerated expectation–maximization algorithm to construct haplotypes. Because the cephalometric measures did not follow the assumptions of normal distribution of the data, nonparametric tests were adopted. For the analysis of discrete cephalometric measures, the nonparametric Kruskal-Wallis test was used to compare mean values between the groups, and the Mann-Whitney U test was used to compare their mean values. All analyses were performed with statistical software (version 16.0; SPSS, Chicago, Ill) for Windows; P ≤0.05 (2-tailed) was considered statistically significant.
Results
The mean values for both angular and linear cephalometric variables from the control and mandibular retrognathism groups are shown in Table I . The maxillary measurements were not significantly different between the control and subject groups, but all mandibular measurements were statistically significant between the groups. Comparison of the cephalometric analysis related to growth pattern between the subjects and controls showed that only the Frankfort horizontal to mandibular plane measurement had significant differences ( P = 0.010), with mean values of 26.3° in the controls and 28.4° in the subjects. The mandibular corpus length (Go-Pog) in the subject group was significantly smaller than in the control group ( P = 0.010).
| Cephalometric variable | Control | MR | P value ∗ |
|---|---|---|---|
| Age (Y) | 20 (11-30) | 19 (12-31) | 0.123 |
| Sex: Female (%) | 43 (60.6) | 40 (49.4) | |
| Male (%) | 28 (39.4) | 41 (50.6) | 0.112 |
| SNA(°) | 82 (80-84) | 82 (80-84) | 0.237 |
| SNB(°) | 79 (79-82) | 75 (68-78) | <0.001 |
| ANB (°) | 2 (1-4) | 6 (4-12) | <0.001 |
| Go-Gn to SN (°) | 31 (20-43) | 32 (22-75) | 0.064 |
| FH to MP (°) | 26 (12-38) | 28 (18-46) | 0.010 |
| Ramus height (mm) | 41 (33-58) | 42 (31-57) | 0.460 |
| Go-Pog (mm) | 68 (61-80) | 66 (56-81) | 0.010 |
| Gonial angle(°) | 128 (113-139) | 129 (114-141) | 0.353 |
∗ Mann-Whitney U test exact significance (1-tailed) P value.
All 3 single nucleotide polymorphisms were polymorphic in both the subject and control groups. The Hardy-Weinberg equation is a basic principle of population genetics, which states that the amount of genetic variation in a population will remain constant from generation to generation in the absence of evolutionary factors. Hardy-Weinberg equilibrium testing is an essential step in genetic association studies, since its deviation indicates the genotyping error, and selection and ascertainment biases, and it is essential to exclude such loci from further analysis. The genotypic frequency distributions of all polymorphisms did not deviate significantly from the Hardy-Weinberg equation in the control group ( Table II ). Results on the association between mandibular retrognathism and MATN1 gene polymorphisms are given in Table II . The rs1149048 single nucleotide polymorphism genotypes and alleles were not significantly different between the subjects and the controls ( Table II ). The rs1149042 minor allele frequency was slightly higher in the control group (14.8%) than in the subject group (5.6%). The genotype frequencies were significantly different between the subject and control groups for rs1149042 polymorphism ( P = 0.029). Furthermore, this polymorphism has shown a significant association with mandibular retrognathism in dominant ( P = 0.011) and allelic models ( P = 0.007). This statistical significance persisted in dominant ( P = 0.034) and allelic models (0.021) even after the Bonferroni correction ( Table II ). Odds ratios calculated under different models showed that the MATN1 rs1149042 single nucleotide polymorphism reduces the risk of developing mandibular retrognathism ( Table II ). The third polymorphism, rs1065755, did not show a significant association with mandibular retrognathism at the genotype or the allele level ( Table II ). Linkage disequilibrium is the nonrandom association of neighboring alleles. The extent of linkage disequilibrium is closely and inversely correlated with nucleotide diversity; hence, it varies along the chromosomes and also among populations. Linkage disequilibrium makes tightly linked variants strongly correlated, increasing the statistical power for an association with the disease. Lower r 2 values are recorded in the Figure , indicating that the pairwise linkage disequilibrium is not strong and significant between the pairs of loci. Comparison of haplotypes between the subjects and the controls did not show evidence for an association with mandibular retrognathism ( Table III ).
| Genotype | Mandibular retrognathism (%) | Control (%) | Odds ratio (95% CI) | P value | |
|---|---|---|---|---|---|
| rs1149048 | |||||
| Genotypic model | AA | 53 (65.4) | 47 (66.2) | Reference | 0.496 |
| AG | 18 (22.2) | 19 (26.8) | 0.84 (0.39-1.79) | ||
| GG | 10 (12.3) | 5 (7.0) | 1.77 (0.56-5.56) | ||
| Dominant model | AA | 53 (65.4) | 47 (66.2) | Reference | |
| AG+GG | 28 (34.6) | 24 (33.8) | 1.03 (0.53-2.03) | 0.920 | |
| Allelic model | A | 124 (76.5) | 113 (79.6) | Reference | |
| G | 38 (23.5) | 29 (20.4) | 1.19 (0.69-2.06) | 0.524 | |
| HWp | 0.535 | 0.664 | |||
| rs1149042 | |||||
| Genotypic model | TT | 73 (90.1) | 53 (74.6) | Reference | |
| GT | 7 (8.6) | 15 (21.1) | 0.34 (0.13-0.89) | 0.087 ∗ | |
| GG | 1 (1.2) | 3 (4.2) | 0.24 (0.02-2.39) | ||
| Dominant model | TT | 73 (90.1) | 53 (74.6) | Reference | |
| GT+GG | 8 (9.9) | 18 (25.4) | 0.32 (0.13-0.79) | 0.034 ∗ | |
| Allelic model | T | 153 (94.4) | 121 (85.2) | Reference | |
| G | 9 (5.6) | 21 (14.8) | 0.34 (0.15-0.77) | 0.021 ∗ | |
| HWp | 0.112 | 0.173 | |||
| rs1065755 | |||||
| Genotypic model | GG | 58 (71.6) | 51 (71.8) | Reference | |
| GA | 17 (21.0) | 18 (25.4) | 0.83 (0.39-1.77) | ||
| AA | 6 (7.4) | 2 (2.8) | 2.64 (0.51-13.65) | 0.401 | |
| Dominant model | GG | 58 (71.6) | 51 (71.8) | Reference | |
| GA+AA | 23 (28.4) | 20 (28.2) | 1.01 (0.498-2.05) | 0.975 | |
| Allelic model | G | 133 (82.1) | 120 (84.5) | Reference | |
| A | 29 (17.9) | 22 (15.5) | 1.19 (0.65-2.18) | 0.575 | |
| HWp | 0.010 | 0.789 | |||
| Haplotype ∗ | Control (%) | Mandibular retrognathism (%) | Odds ratio (95% CI) | P value |
|---|---|---|---|---|
| A-T-G | 43 (60.8) | 55 (68.1) | Reference | |
| A-G-G | 6 (8.5) | 2 (2.6) | 0.26 (0.05-1.35) | 0.089 |
| A-T-A | 7 (10.3) | 5 (5.8) | 0.56 (0.17-1.88) | 0.342 |
| G-G-G | 5 (6.9) | 2 (3.0) | 0.31 (0.06-1.69) | 0.157 |
| G-T-A | 4 (5.1) | 10 (12.1) | 1.95 (0.57-6.66) | 0.278 |
| G-T-G | 6 (8.5) | 7 (8.4) | 0.91 (0.29-2.91) | 0.876 |
Comparison of various cephalometric variables that determine the growth pattern among genotypes using the Kruskal-Wallis test showed that these variables are not different among the genotypes of the polymorphisms analyzed ( Table IV ). However, the mandibular measurement SNB showed significant differences among genotypes of rs1149042 ( P = 0.019).
