GHRand IGF2Rgenes may contribute to normal variations in craniofacial dimensions: Insights from an admixed population

Introduction

This study aimed to determine whether single nucleotide polymorphisms in the growth hormone receptor ( GHR ) and insulin-like growth factor 2 receptor ( IGF2R ) genes are associated with different craniofacial phenotypes.

Methods

A total of 596 orthodontic and 98 orthognathic patients from 4 cities in Brazil were included for analyses. Angular and linear cephalometric measurements were obtained, and phenotype characterizations were performed. Genomic DNA was collected from buccal cells and single nucleotide polymorphisms in GHR (rs2910875, rs2973015, rs1509460) and IGF2R (rs2277071, rs6909681, rs6920141) were genotyped by polymerase chain reactions using TaqMan assay. Genotype–phenotype associations were assessed in the total sample (statistical significance was set at P <8.333 × 10 −3 ) and by a meta-analytic approach implemented to calculate the single effect size measurement for the different cohorts.

Results

Rare homozygotes for the GHR rs2973015 showed increased measurements for the lower anterior facial height (ANS-Me) and mandibular sagittal lengths (Co-Gn and Go-Pg). In contrast, common homozygotes for the IGF2R rs6920141 presented reduced measurements for these dimensions (ANS-Me and Go-Pg). Furthermore, the less common homozygotes for IGF2R rs2277071 had reduced maxillary sagittal length (Ptm′-A′). The meta-analytical approach replicated the associations of rs2973015 with ANS-Me, rs2277071 with Ptm′-A′, and rs6920141 with Go-Pg.

Conclusions

Our results provide further evidence that GHR contributes to the determination of mandibular morphology. In addition, we report that IGF2R is a possible gene associated with variations in craniofacial dimensions. Applying meta-analytical approaches to genetic variation data originating from likely underpowered samples may provide additional insight regarding genotype and/or phenotype associations.

Highlights

  • Growth hormone receptor ( GHR ) and insulin-like growth factor 2 receptor ( IGF2R ) participate in the physiology of skeletal growth and development.

  • Rare homozygotes for GHR rs2973015 showed increased ANS-Me, Co-Gn, and Go-Pg measures.

  • Common homozygotes for IGF2R rs6920141 presented reduced ANS-Me and Go-Pg measures.

  • Less common homozygotes for IGF2R rs2277071 had reduced Ptm′-A′ measures.

  • Some of the associations did not remain significant after meta-analysis.

Various proteins and their encoding genes have been implicated in fetal, neonatal, and postnatal development. Among them, the growth hormone (GH), insulin-like growth factors 1 and 2 (IGF1 and IGF2), and their associated receptors (GH receptor [GHR], insulin-like growth factors 1 receptor [IGF1R], and insulin-like growth factors 2 [IGF2R]) have been recognized for playing an important role in the physiology of skeletal growth and development. GH and insulin-like growth factors (IGFs) must bind to their receptors to perform their functions and trigger specific signaling pathways. Alterations in these receptors could cause phenotypic variations such as idiopathic short stature or underdevelopment of facial bones. The presence of GHR and IGF1R in the mandibular condyle , suggests they can affect mandibular morphology and thus craniofacial skeleton. Previous studies have reported associations between single nucleotide polymorphisms (SNPs) in or flanking GHR and different craniofacial phenotypes, suggesting that this gene might be involved in normal variations of craniofacial morphology.

IGF2R works differently, binding to IGF2 and, although with lower affinity, to insulin and IGF1. IGFs promote cartilage and bone development. When IGF2 binds to the IGF2R, instead of initiating a signaling response to perform its functions, IGF2R causes uptake of IGF2, transporting it to the lysosomes for further degradation. Thus, this receptor has been recognized as a controller of extracellular IGF levels and regulator of the growth-promoting function of these growth factors during development. , A previous study mapped susceptibility loci for mandibular prognathism to chromosomes 1p36, 6q25, and 1913.2, suggesting that these chromosomal loci are linked to this mandibular phenotype. Then, considering that IGF2R is located in the 6q25.3 region, we hypothesized that this gene is involved in determining the morphology of craniofacial structures.

Although some aspects of the craniofacial growth have been extensively studied, the molecular regulation of postnatal development is still largely elusive. Our study aimed to assess whether genetic variants in GHR and IGF2R are associated with specific craniofacial phenotypes. Therefore, we tested 3 SNPs in GHR (rs2910875, rs2973015, rs1509460) that were previously suggested of having a possible role in the etiology of the Class III phenotype. , In addition, we genotyped 3 SNPs (rs2277071, rs6909681, rs6920141) flanking D6S305, which is near to IGF2R , because this microsatellite marker was linked to mandibular prognathism.

Material and methods

This cross-sectional multicenter study was approved by the Research Ethics Committees of the University Hospital Antônio Pedro from the Fluminense Federal University (33791314.3.0000.5243), School of Dentistry of Ribeirão Preto from the University of São Paulo (50765715.3.0000.5419), Positivo University (80846317.8.0000.0093), and the University of Pittsburgh (no. 12080056). Written informed consent was obtained from all participants or their legal guardians.

Clinical and radiographic records from Brazilian subjects were assessed. Orthodontic patients from private practice and graduate orthodontic clinics in 3 cities (Rio de Janeiro [mean age, 24.7 ± 10.7 years; 124 male, 210 female], Manaus [mean age, 26.5 ± 11.0 years; 51 male, 79 female], and Ribeirão Preto [mean age, 14.9 ± 7.0 years; 65 male, 68 female]) located in different regions of Brazil were selected. In addition, a sample of patients presenting severe skeletal sagittal and/or vertical disharmonies, which required orthognathic surgery, were recruited from another center in the city of Curitiba [mean age, 29.3 ± 9.1 years; 33 male, 65 female] to test if the studied genetic variants are also involved in more severe phenotypes. Sample characteristics, location setting, and ethnic composition of each city were already described in a previous study. Patients who received previous orthodontic treatment, who had concomitant systemic medical conditions, craniofacial congenital or syndromic anomalies, a substantial number of teeth missing, or suffered facial trauma were excluded.

Digital cephalometric tracings of preorthodontic or preorthognathic lateral cephalograms, with the mandible in centric occlusion positioning, were performed by previously trained orthodontists using Dolphin 3D Imaging software (version 8.0; Dolphin Imaging and Management Solutions, Chatsworth, Calif). The Steiner’s ANB and Ricketts’ NBa-PtGn angles were measured to determine sagittal (skeletal malocclusion) and vertical (facial type) skeletal jaw relations. Subjects were classified as Class I (0°-4°), Class II (>4°), or Class III (<0°) and as mesofacial (87°-93°), dolichofacial (<87°), or brachyfacial (>93°) according to the ANB and NBa-PtGn angles, respectively. In addition, angular and linear measurements were obtained in both sagittal and vertical dimensions ( Fig 1 ; Table I ). Linear measurements were only assessed on radiographs with a ruler included in the image that enabled the corresponding size calibration in the Dolphin 3D Imaging software.

Fig 1
Reference points and lines used to perform analyses. Angular measurements (°): NBa-PtGn, SN-GoGn, y-axis, SNA, SNB, and ANB. Linear measurements (mm): Co-Go, S-Go, ANS-Me, N-Me, S-N, Co-Gn, Go-Pg, and Ptm′-A′.

Table I
Craniofacial phenotypes and cephalometric measurements assessed
Variables Rio de Janeiro Manaus Ribeirão Preto Curitiba Total
Skeletal malocclusion, n (%) n = 334 n = 130 n = 132 n = 98 n = 596
Class I 128 (38.3) 68 (52.3) 73 (55.3) 21 (21.4) 269 (45.1)
Class II 173 (51.8) 50 (38.5) 42 (31.8) 19 (19.4) 265 (44.5)
Class III 33 (9.9) 12 (9.2) 17 (12.9) 58 (59.2) 62 (10.4)
Facial type, n (%) n = 334 n = 130 n = 132 n = 98 n = 596
Mesofacial 155 (46.4) 68 (52.3) 64 (48.5) 24 (24.5) 287 (48.2)
Dolichofacial 94 (28.1) 33 (25.4) 53 (40.2) 15 (15.3) 180 (30.2)
Brachyfacial 85 (25.4) 29 (22.3) 15 (11.4) 59 (60.2) 129 (21.6)
Angular measurements (°), mean (SD) n = 334 n = 130 n = 132 n = 98 n = 596
NBa-PtGn 89.9 (5.5) 89.8 (4.6) 87.9 (4.6) 94.2 (7.2) 89.4 (5.1)
SN-GoGn 31.5 (7.0) 30.5 (5.0) 30.2 (6.4) 30.6 (8.0) 31.0 (6.5)
y-axis 60.3 (5.0) 59.1 (3.8) 59.7 (4.7) 59.2 (6.1) 59.9 (4.7)
SNA 84.7 (4.7) 85.3 (4.0) 83.1 (4.9) 81.9 (4.7) 84.5 (4.6)
SNB 80.5 (4.8) 82.0 (4.2) 80.3 (5.1) 82.4 (6.4) 80.7 (4.7)
ANB 4.3 (3.5) 3.4 (2.5) 2.9 (2.7) −0.5 (5.4) 3.8 (3.2)
Linear measurements (mm), mean (SD) n = 228 n = 115 n = 132 n = 475
Co-Go 54.3 (6.9) 54.5 (5.4) 57.1 (7.5) 55.2 (6.9)
S-Go 74.9 (8.4) 72.4 (6.4) 73.5 (9.3) 73.9 (8.3)
ANS-Me 63.3 (7.4) 64.2 (5.9) 60.0 (7.6) 62.6 (7.3)
N-Me 113.1 (10.9) 113.1 (7.7) 109.6 (10.3) 112.1 (10.1)
S-N 66.4 (6.1) 66.3 (3.3) 67.3 (5.8) 66.6 (5.5)
Co-Gn 113.1 (11.0) 116.8 (7.7) 114.2 (11.2) 114.3 (10.4)
Go-Pg 71.6 (7.6) 75.7 (5.4) 67.6 (7.7) 71.5 (7.7)
Ptm′-A′ 50.6 (5.3) 48.1 (3.1) 49.8 (4.7) 49.8 (4.7)

Note. Linear measurements were not assessed in the Curitiba sample because the size of the radiographs could not be calibrated. The total sample includes the following cities: Rio de Janeiro, Manaus, and Ribeirão Preto.
SD, standard deviation.

The SNPs were chosen on the basis of their minor allele frequency >10%. Those selected in GHR (rs2910875, rs1509460, and rs2973015) are located in loci previously reported as candidate regions for mandible-related phenotypes. , , , , , SNPs in IGF2R (rs2277071, rs6909681, and rs6920141) were selected by their functional implications or location according to the SNP database ( www.ncbi.nlm.nih.gov/SNP ). The characteristics of the studied SNPs are shown in Table II .

Table II
Characteristics of the SNPs studied
Gene Locus Reference sequence Type of alteration Base change (context sequence) Global MAF
GHR 5p12 rs2910875 utr variant 3 prime ATG[A /G]CTA 0.4557/2282
GHR 5p12 rs2973015 intron variant TTT[A/G ]CTG 0.4493/2250
GHR 5p13.1 rs1509460 intron variant CAG[G /T]ACT 0.4407/2207
IGF2R 6q25.3 rs2277071 intron variant ATA[A /G]CAT 0.2718/1361
IGF2R 6q25.3 rs6909681 intron variant AAC[A/T ]GTC 0.4287/2147
IGF2R 6q25.3 rs6920141 intron variant TGT[C /T]GAT 0.4629/2318

MAF , minor allele frequency.

Lower frequency allele.

Genomic DNA was extracted and purified from buccal mucosa cells from saliva as previously reported. Genotyping was blindly performed by polymerase chain reactions (PCR) using end-point analysis and TaqMan assay on a real-time PCR system (Applied Biosystems Prism QuantStudio 6 Flex PCR System, Thermo Fisher Scientific, Foster City, Calif) according to an established protocol.

Statistical analysis

Statistical analyses were performed using 2-tailed tests (α = 0.05) on GraphPad Prism (GraphPad Software, San Diego, Calif) and Epi Info (Version 3.5.2.; Centers for Disease Control and Prevention, Atlanta, Ga). Chi-square (with Yate correction for continuity, when necessary) or Fisher exact tests were performed to evaluate the association between genotype and allele frequencies for each SNP and the skeletal malocclusion and facial type. Analysis of variance or Kruskal-Wallis tests were applied according to the data distribution (assessed by the Kolmogorov-Smirnov and Shapiro-Wilk tests) to compare the means or medians of cephalometric measurements according to genotypes, respectively. The threshold for statistical significance after Bonferroni correction for multiple testing was P <8.333 × 10 −3 (0.05/6.00 SNPs).

Chi-square tests were used to confirm the Hardy-Weinberg equilibrium for each SNP in each of the samples assessed, considering only the subjects with the phenotypes of reference (Class I malocclusion and mesofacial-type). Statistical power for the total sample was estimated using the Genetic Power Calculator tool assuming a marker allele frequency of 0.45, a prevalence of the phenotype of 0.1, the high-risk allele frequency of 0.3, D′ of 1.0, genotype relative risk for heterozygotes of 2.0, and genotype relative risk for homozygotes of 4.0. The power for the comparison of Class I vs Class III malocclusion (which had the smallest number of subjects) under an alpha of 0.05 was 80%. Changing the high-risk allele frequency for less than 0.3 or the effect sizes of heterozygotes and homozygotes for less than 2.0 or 4.0, respectively, will cause a substantial decline in the statistical power. All other comparisons such as Class I with Class II or mesofacial with dolichofacial and/or brachyfacial should have better power than the comparison of Class I with Class III.

Analyses were performed in the total sample, and with a concern that combining different geographic cohorts of Brazilians may be affected by population substructure, we tested a fixed-effects meta-analysis model to calculate the single effect size measurement for the different cohorts. This approach was performed only for the results that showed a significant association in the total sample using RevMan (version 5.1; Nordic Cochrane Centre, Copenhagen, Denmark). Heterogeneity was assessed by the I 2 Index. The genotypes were independently compared and also were pooled to perform analyses in dominant and recessive models. The analysis was done with and without the sample from Curitiba because this cohort included subjects with phenotypes of more severe craniofacial variations.

Results

The distribution of genotypes followed Hardy-Weinberg equilibrium (data not shown). There were no associations between the skeletal malocclusion or facial type and the genotype or allele frequencies for any SNP assessed in GHR and IGF2R in the total sample. Associations were detected when specific cephalometric measurements were assessed ( P <8.333 × 10 −3 ) ( Table III ). Rare homozygotes for the GHR rs2973015 marker showed increased measures for the lower anterior facial height (ANS-Me) and mandibular sagittal lengths (Co-Gn and Go-Pg). In contrast, common homozygotes for the IGF2R rs6920141 presented reduced measures for these dimensions (ANS-Me and Go-Pg). In addition, the less common homozygotes for the IGF2R rs2277071 had reduced maxillary length (Ptm′-A′).

Table III
Genotype–phenotype associations unveiled in the total sample
SNPs Genotypes P value
GHR rs2973015 AA AG GG
n 72 145 119
ANS-Me (mm), median (Q1-Q3) 65.2 (60.8-70.1) 63.3 (59.3-67.1) 61.4 (58-65.6) 0.006
Co-Gn (mm), median (Q1-Q3) 116.5 (113.3-121.7) 113.4 (108.3-119.5) 113.6 (106.2-117.8) 0.003
Go-Pg (mm), median (Q1-Q3) 75.1 (71.3-78.7) 72.7 (69.1-77.2) 71.2 (66.8-75.2) <0.001
IGF2R rs2277071 AA AG GG
n 45 161 258
Ptm′-A′ (mm), median (Q1-Q3) 47.4 (45.8-50.2) 49.4 (47.1-52.5) 49.4 (47.2-51.8) 0.003
IGF2R rs6920141 CC CT TT
n 69 179 200
ANS-Me (mm), median (Q1-Q3) 62.1 (58.4-66.5) 63.6 (58.6-67.4) 61.1 (56.8-65.3) 0.007
Go-Pg (mm), median (Q1-Q3) 73.1 (69.3-78.3) 72.4 (68.3-76.8) 69.1 (64.8-74.5) <0.001

Note. The total sample includes the following cities: Rio de Janeiro, Manaus and Ribeirão Preto. Ribeirão Preto sample was not included on analysis of the SNP rs2973015 because the genotyping presented low call rate.

The threshold for statistical significance after Bonferroni correction was P <8.333 × 10 −3 .

The meta-analytical approach replicated the associations of rs2973015 with ANS-Me, rs2277071 with Ptm′-A′, and rs6920141 with Go-Pg. Subjects with the AA genotype for GHR rs2973015 showed higher ANS-Me measurements than subjects carrying the GG genotype ( P = 0.008, I 2 = 49%; Fig 2 ). In contrast, subjects with the AA genotype in IGF2R rs2277071 presented reduced measures for Ptm′-A′; this effect was stronger when both A alleles were present (recessive model for AA vs AG + GG ; P = 0.03, I 2 = 29%; Fig 3 ). Furthermore, subjects carrying at least 1 C allele for IGF2R rs6920141 had higher Go-Pg measurements (dominant model for CC + CT vs TT ; P = 0.02, I 2 = 48%; Fig 4 ). No other association remained significant when the single effect size measurement for the different cohorts was calculated in meta-analytic approaches ( Supplementary Figs 1-3 ).

Fig 2
Forest plots showing the effect size on ANS-Me (mm) measure according to the genotype presented for the GHR rs2973015. SD, standard deviation; CI, confidence interval.

Fig 3
Forest plots showing the effect size on Ptm′-A′ (mm) measure according to the genotype presented for the IGF2R rs2277071. SD, standard deviation; CI, confidence interval.

Fig 4
Forest plots showing the effect size on Go-Pg (mm) measure according to the genotype presented for the IGF2R rs6020141. SD, standard deviation; CI, confidence interval.

The Curitiba sample showed significant differences in angular measurements of both jaws (SNA and SNB) for rs1509460 in GHR ( P <8.333 × 10 −3 ). Complete results are presented in the Supplementary Data ( Supplementary Tables I-V ).

Discussion

The roles of GH, IGFs, and their receptors have been well described and recognized for participating in the physiology of skeletal growth and development. The expression of some of them in the mandibular condyle, , , where cartilage-mediated growth occurs, suggests they can influence the mandibular morphology. Previous studies have suggested an association of missense mutations in GHR with the mandibular ramus height. , , , Our results showed that this gene could also affect the sagittal dimensions of the mandibular body.

Although P561T in exon 10 of GHR has already been associated with horizontal and longitudinal variations in the morphology of the mandible, , , this is the first study reporting association between rs2973015 and this dimension. Our findings indicated that this SNP could also affect lower anterior facial height. Interestingly, a previous study identified, by a principal component analysis, the involvement of this marker in the genetic background of horizontal and vertical maxillomandibular discrepancies in Brazilians. Because subjects carrying the AA genotype in the total sample presented significantly greater measures for Co-Gn and Go-Pg in the present study, we suggest that this SNP could be considered as a prognostic indicator of mandibular prognathism. It is important to mention that the genetic contribution of this SNP could vary on different populations; the present findings differ from other previously reported findings that found no association between this SNP and mandibular prognathism in a sample from the United States. In addition, the associations unveiled in the present study between this genetic variant and the measurements Co-Gn and Go-Pg were not replicated when the total sample was divided according to the region. These negative findings, however, are likely a direct result of a lack of statistical power because the cohorts contributing to these analyses had reduced sample sizes.

An interesting finding is that GHR could also be associated with the severity of skeletal variations. Subjects carrying the GG genotype for rs1509460 showed significantly higher angular measures for both jaws. This result from the cohort that had patients who underwent orthognathic surgery (therefore, from patients with more severe craniofacial deformities) could suggest a regulatory role of this genetic variant on the size and not as a contributor to a specific craniofacial characteristic.

Our results suggesting a role of IGF2R in variations of the craniofacial morphology may be explained by the regulatory function of the extracellular IGF levels of the receptor that this gene codes. IGF2R is a transmembrane receptor that transports IGF2 to lysosomes. IGF2R can remove IGF2 from circulation and extracellular medium and thus regulate the growth-promoting function of this growth factor. Increased systemic IGF2 levels could cause embryonic overgrowth and perinatal lethality ; in contrast, overexpression of IGF2R in a tissue-specific manner has been shown to cause a local reduction in organ size. It is important to mention that IGF2R not only binds to IGF2 but also to IGF1, although with less affinity. This means that alterations in IGF2R could also modify IGF1 levels and consequently their effect on target tissues. A previous study showed that Igf1 null mutant mice exhibited decreased craniofacial size and prominent changes in the facial and cranial areas. In humans, a previous case-control study of Chinese subjects found no association between IGF1 and mandibular prognathism. However, mandibular prognathism was defined on the basis of the facial features, presence of crossbite, and ANB angle (under −2.0°), and there could be a contribution of other structures besides the mandible for this phenotype.

Considering that IGFs are GH mediators and are believed to also stimulate growth independently by promoting cartilage and bone development, the association of rs6920141 with the Go-Pg measure may be due to the following: (1) SNPs in IGF2R that may alter the role of this receptor modifying the IGF levels exerting its function on the condyle and (2) the condylar growth that contributes to vertical height gains but also to the sagittal length of the mandible. In addition, a sagittal linear measurement for the maxilla (Ptm′-A′) showed a difference depending on the genotype for rs2277071. We believe that IGF has a role in the development of the maxilla because in the embryonic period, its presence in the maxillary prominences has been detected. Furthermore, although the maxilla has an intramembranous ossification, this bone is enlarged and displaced, to some extent, by the influence of the structures of the skull base that have endochondral ossification. Therefore, it appears that IGF2R may influence both the maxillary and mandibular morphology.

Some of the associations found in the combined sample did not remain significant when the single effect size measurement for the different cohorts was calculated in a meta-analysis. It has been reported that, as a whole, the ancestry contribution of Brazilians is 62% European, 21% African, and 17% Amerindian; however, the genetic admixture varies greatly depending on the region evaluated. For example, although the European contribution in the Southern region is higher with 77%, the Northern region has the largest contribution of Amerindian ancestry with 32%. The subjects included in our analyses belonged to cities located in different regions of the country; Rio de Janeiro and Ribeirão Preto are in the Southeast, Manaus in the North, and Curitiba in the Southern region of Brazil. Therefore, undetected population substructure is possible, and implementing approaches to minimize this effect is warranted.

Conclusions

In summary, our results provide additional evidence that GHR contributes to the determination of mandibular morphology. In addition, we report that IGF2R is a possible gene associated with variations in craniofacial dimensions.

Supplementary data

Supplementary Fig 1
Forest plots showing the effect size on Co-Gn (mm) measure according to the genotype presented for the GHR rs2973015. SD, standard deviation; CI, confidence interval.

Supplementary Fig 2
Forest plots showing the effect size on Go-Pg (mm) measure according to the genotype presented for the GHR rs2973015. SD, standard deviation; CI, confidence interval.

Supplementary Fig 3
Forest plots showing the effect size on ANS-Me (mm) measure according to the genotype presented for the IGF2R rs6020141. SD, standard deviation; CI, confidence interval.

Supplementary Table I
Genotype and allele frequencies according to the skeletal malocclusion for the SNPs assessed in GHR
SNP Class Genotypes Alleles
GHR rs2910875 AA AG GG P value A G P value
RJ Class I 25 (20.7) 51 (42.1) 45 (37.2) Reference 101 (41.7) 141 (58.3) Reference
Class II 25 (14.8) 90 (53.3) 54 (32) 0.1526 140 (41.4) 198 (58.6) >0.9999
Class III 7 (25.9) 11 (40.7) 9 (33.3) 0.8270 25 (46.3) 29 (53.7) 0.5466
M Class I 18 (26.9) 28 (41.8) 21 (31.3) Reference 64 (47.8) 70 (52.2) Reference
Class II 11 (24.4) 20 (44.4) 14 (31.1) 0.9465 42 (46.7) 48 (53.3) 0.8922
Class III 1 (9.1) 7 (63.6) 3 (27.3) 0.3198 9 (40.9) 13 (59.1) 0.6473
RP Class I 5 (7.9) 36 (57.1) 22 (34.9) Reference 46 (36.5) 80 (63.5) Reference
Class II 7 (18.9) 19 (51.4) 11 (29.7) 0.2618 33 (44.6) 41 (55.4) 0.2953
Class III 4 (26.7) 6 (40.0) 5 (33.3) 0.1136 14 (46.7) 16 (53.3) 0.3064
RJ+M+RP Class I 48 (19.1) 115 (45.8) 88 (35.1) Reference 211 (42.0) 291 (58.0) Reference
Class II 43 (17.1) 129 (51.4) 79 (31.5) 0.4584 215 (42.8) 287 (57.2) 0.8481
Class III 12 (22.6) 24 (45.3) 17 (32.1) 0.8228 48 (45.3) 58 (54.7) 0.5892
C Class I 3 (14.3) 8 (38.1) 10 (47.6) Reference 14 (33.3) 28 (66.7) Reference
Class II 7 (36.8) 5 (26.3) 7 (36.8) 0.2554 19 (50.0) 19 (50.0) 0.1734
Class III 9 (15.8) 23 (40.4) 25 (43.9) 0.956 41 (36.0) 73 (64.0) 0.8510
RJ+M+RP+C Class I 51 (18.8) 123 (45.2) 98 (36.0) Reference 225 (41.4) 319 (58.6) Reference
Class II 50 (18.5) 134 (49.6) 86 (31.9) 0.5337 234 (43.3) 306 (56.7) 0.5388
Class III 21 (19.1) 47 (42.7) 42 (38.2) 0.8989 89 (40.5) 131 (59.5) 0.8710

GHR rs2973015 AA AG GG P value A G P value
RJ Class I 13 (10.2) 56 (44.1) 58 (45.7) Reference 82 (32.3) 172 (67.7) Reference
Class II 18 (10.5) 80 (46.8) 73 (42.7) 0.8737 116 (33.9) 226 (66.1) 0.7252
Class III 1 (3.1) 16 (50.0) 15 (46.9) 0.4339 18 (28.1) 46 (71.9) 0.5510
M Class I 30 (44.8) 24 (35.8) 13 (19.4) Reference 84 (62.7) 50 (37.3) Reference
Class II 22 (46.8) 20 (42.6) 5 (10.6) 0.4296 64 (68.1) 30 (31.9) 0.4811
Class III 5 (41.7) 7 (58.3) 0 (0.0) 0.1596 17 (70.8) 7 (29.2) 0.4973
RJ+M Class I 43 (22.2) 80 (41.2) 71 (36.6) Reference 166 (42.8) 222 (57.2) Reference
Class II 40 (18.3) 100 (45.9) 78 (35.8) 0.5299 180 (41.3) 256 (58.7) 0.6720
Class III 6 (13.6) 23 (52.3) 15 (34.1) 0.3104 35 (39.8) 53 (60.2) 0.6342
C Class I 8 (38.1) 11 (52.4) 2 (9.5) Reference 27 (64.3) 15 (35.7) Reference
Class II 6 (31.6) 6 (31.6) 7 (36.8) 0.1081 18 (47.4) 20 (52.6) 0.1761
Class III 28 (48.3) 19 (32.8) 11 (19.0) 0.2528 75 (64.7) 41 (35.3) >0.9999
RJ+M+C Class I 51 (23.7) 91 (42.3) 73 (34.0) Reference 193 (44.9) 237 (55.1) Reference
Class II 46 (19.4) 106 (44.7) 85 (35.9) 0.5370 198 (41.8) 276 (58.2) 0.3477
Class III 34 (33.3) 42 (41.2) 26 (25.5) 0.1359 110 (53.9) 94 (46.1) 0.0339

GHR rs1509460 GG GT TT P value G T P value
RJ Class I 30 (26.8) 58 (51.8) 24 (21.4) Reference 118 (52.7) 106 (47.3) Reference
Class II 52 (32.1) 73 (45.1) 37 (22.8) 0.5194 177 (54.6) 147 (45.4) 0.6639
Class III 5 (20.0) 17 (68.0) 3 (12.0) 0.3214 27 (54.0) 23 (46.0) 0.8770
M Class I 18 (27.3) 37 (56.1) 11 (16.7) Reference 73 (55.3) 59 (44.7) Reference
Class II 9 (19.6) 29 (63.0) 8 (17.4) 0.6376 47 (51.1) 45 (48.9) 0.5867
Class III 3 (25.0) 6 (50.0) 3 (25.0) 0.7866 12 (50.0) 12 (50.0) 0.6614
RP Class I 15 (20.5) 41 (56.2) 17 (23.3) Reference 71 (48.6) 75 (51.4) Reference
Class II 11 (26.8) 26 (63.4) 4 (9.8) 0.1920 48 (58.5) 34 (41.5) 0.1685
Class III 3 (17.6) 5 (29.4) 9 (52.9) 0.0453 11 (32.4) 23 (67.6) 0.1253
RJ+M+RP Class I 63 (25.1) 136 (54.2) 52 (20.7) Reference 262 (52.2) 240 (47.8) Reference
Class II 72 (28.9) 128 (51.4) 49 (19.7) 0.6313 272 (54.6) 226 (45.4) 0.4475
Class III 11 (20.4) 28 (51.9) 15 (27.8) 0.4819 50 (46.3) 58 (53.7) 0.2894
C Class I 1 (4.8) 14 (66.7) 6 (28.6) Reference 16 (38.1) 26 (61.9) Reference
Class II 0 (0.0) 17 (89.5) 2 (10.5) 0.2019 17 (44.7) 21 (55.3) 0.6506
Class III 5 (8.6) 46 (79.3) 7 (12.1) 0.2039 56 (48.3) 60 (51.7) 0.2823
RJ+M+RP+C Class I 64 (23.5) 150 (55.1) 58 (21.3) Reference 278 (51.1) 266 (48.9) Reference
Class II 72 (26.9) 145 (54.1) 51 (19.0) 0.6141 289 (53.9) 247 (46.1) 0.3613
Class III 16 (14.3) 74 (66.1) 22 (19.6) 0.0827 106 (47.3) 118 (52.7) 0.3825

Note. Values are n (%). The Ribeirão Preto sample was not included on analysis of the SNP rs2973015 because the genotyping presented low call rate.
RJ , Rio de Janeiro; M , Manaus; RP , Ribeirão Preto; C , Curitiba.

Statistically significant association (nominal level, P <0.05); Chi-square test conditions were not met.

Supplementary Table II
Genotype and allele frequencies according to the facial type for the SNPs assessed in GHR
SNP Facial type Genotypes Alleles
GHR rs2910875 AA AG GG P value A G P value
RJ Mesofacial 27 (18.4) 72 (49) 48 (32.7) Reference 126 (42.9) 168 (57.1) Reference
Dolichofacial 15 (16.7) 45 (50) 30 (33.3) 0.9465 75 (41.7) 105 (58.3) 0.8483
Brachyfacial 15 (18.8) 35 (43.8) 30 (37.5) 0.7189 65 (40.6) 95 (59.4) 0.6909
M Mesofacial 15 (22.7) 30 (45.5) 21 (31.8) Reference 60 (45.5) 72 (54.5) Reference
Dolichofacial 5 (16.1) 14 (45.2) 12 (38.7) 0.6907 24 (38.7) 38 (61.3) 0.4381
Brachyfacial 10 (38.5) 11 (42.3) 5 (19.2) 0.2478 31 (59.6) 21 (40.4) 0.1019
RP Mesofacial 10 (18.2) 24 (43.6) 21 (38.2) Reference 44 (40.0) 66 (60.0) Reference
Dolichofacial 5 (10.4) 31 (64.6) 12 (25.0) 0.1023 41 (42.7) 55 (57.3) 0.7768
Brachyfacial 1 (8.3) 6 (50.0) 5 (41.7) 0.7047 8 (33.3) 16 (66.7) 0.6467
RJ+M+RP Mesofacial 52 (19.4) 126 (47.0) 90 (33.6) Reference 230 (42.9) 306 (57.1) Reference
Dolichofacial 25 (14.8) 90 (53.3) 54 (32) 0.3413 140 (41.4) 198 (58.6) 0.6738
Brachyfacial 26 (22.0) 52 (44.1) 40 (33.9) 0.8025 104 (44.1) 132 (55.9) 0.8131
C Mesofacial 6 (25.0) 9 (37.5) 9 (37.5) Reference 21 (43.8) 27 (56.3) Reference
Dolichofacial 5 (33.3) 3 (20.0) 7 (46.7) 0.5117 13 (43.3) 17 (56.7) >0.9999
Brachyfacial 8 (13.8) 24 (41.4) 26 (44.8) 0.4653 40 (34.5) 76 (65.5) 0.2897
RJ+M+RP+C Mesofacial 58 (19.9) 135 (46.2) 99 (33.9) Reference 251 (43.0) 333 (57.0) Reference
Dolichofacial 30 (16.3) 93 (50.5) 61 (33.2) 0.5406 153 (41.6) 215 (58.4) 0.6866
Brachyfacial 34 (19.3) 76 (43.2) 66 (37.5) 0.7240 144 (40.9) 208 (59.1) 0.5395

GHR rs2973015 AA AG GG P value A G P value
RJ Mesofacial 15 (9.8) 71 (46.4) 67 (43.8) Reference 101 (33.0) 205 (67.0) Reference
Dolichofacial 10 (10.8) 45 (48.4) 38 (40.9) 0.8958 65 (34.9) 121 (65.1) 0.6944
Brachyfacial 7 (8.3) 36 (42.9) 41 (48.8) 0.7483 50 (29.8) 118 (70.2) 0.5365
M Mesofacial 27 (41.5) 30 (46.2) 8 (12.3) Reference 84 (64.6) 46 (35.4) Reference
Dolichofacial 21 (65.6) 10 (31.3) 1 (3.1) 0.0602 52 (81.3) 12 (18.8) 0.0196
Brachyfacial 9 (31.0) 11 (37.9) 9 (31) 0.0916 29 (50.0) 29 (50.0) 0.0759
RJ+M Mesofacial 42 (19.3) 101 (46.3) 75 (34.4) Reference 185 (42.4) 251 (57.6) Reference
Dolichofacial 31 (24.8) 55 (44.0) 39 (31.2) 0.4771 117 (46.8) 133 (53.2) 0.2989
Brachyfacial 16 (14.2) 47 (41.6) 50 (44.2) 0.1836 79 (35.0) 147 (65.0) 0.0659
C Mesofacial 7 (29.2) 11 (45.8) 6 (25.0) Reference 25 (52.1) 23 (47.9) Reference
Dolichofacial 7 (46.7) 3 (20.0) 5 (33.3) 0.2554 17 (56.7) 13 (43.3) 0.8162
Brachyfacial 28 (47.5) 22 (37.3) 9 (15.3) 0.2780 78 (66.1) 40 (33.9) 0.1127
RJ+M+C Mesofacial 49 (20.2) 112 (46.3) 81 (33.5) Reference 210 (43.4) 274 (56.6) Reference
Dolichofacial 38 (27.1) 58 (41.4) 44 (31.4) 0.2962 134 (47.9) 146 (52.1) 0.2577
Brachyfacial 44 (25.6) 69 (40.1) 59 (34.3) 0.3383 157 (45.6) 187 (54.4) 0.5239

GHR rs1509460 GG GT TT P value G T P value
RJ Mesofacial 47 (33.8) 67 (48.2) 25 (18) Reference 161 (57.9) 117 (42.1) Reference
Dolichofacial 26 (29.9) 39 (44.8) 22 (25.3) 0.4148 91 (52.3) 83 (47.7) 0.2450
Brachyfacial 14 (19.2) 42 (57.5) 17 (23.3) 0.0801 70 (47.9) 76 (52.1) 0.0521
M Mesofacial 19 (29.7) 34 (53.1) 11 (17.2) Reference 72 (56.3) 56 (43.8) Reference
Dolichofacial 6 (19.4) 22 (71.0) 3 (9.7) 0.2503 34 (54.8) 28 (45.2) 0.8772
Brachyfacial 5 (17.2) 16 (55.2) 8 (27.6) 0.3214 26 (44.8) 32 (55.2) 0.1573
RP Mesofacial 13 (20.6) 34 (54) 16 (25.4) Reference 60 (47.6) 66 (52.4) Reference
Dolichofacial 13 (24.5) 30 (56.6) 10 (18.9) 0.6771 56 (52.8) 50 (47.2) 0.5100
Brachyfacial 3 (20.0) 8 (53.3) 4 (26.7) 0.9950 14 (46.7) 16 (53.3) >0.9999
RP+M+RP Mesofacial 79 (29.7) 135 (50.8) 52 (19.5) Reference 293 (55.1) 239 (44.9) Reference
Dolichofacial 45 (26.3) 91 (53.2) 35 (20.5) 0.7445 181 (52.9) 161 (47.1) 0.5779
Brachyfacial 22 (18.8) 66 (56.4) 29 (24.8) 0.0739 110 (47.0) 124 (53.0) 0.0414
C Mesofacial 0 (0.0) 20 (83.3) 4 (16.7) Reference 20 (41.7) 28 (58.3) Reference
Dolichofacial 0 (0.0) 13 (86.7) 2 (13.3) 0.9608 13 (43.3) 17 (56.7) >0.9999
Brachyfacial 6 (10.2) 44 (74.6) 9 (15.3) 0.2685 56 (47.5) 62 (52.5) 0.6066
RJ+M+RP+C Mesofacial 79 (27.2) 155 (53.4) 56 (19.3) Reference 313 (54.0) 267 (46.0) Reference
Dolichofacial 45 (24.2) 104 (55.9) 37 (19.9) 0.7594 194 (52.2) 178 (47.8) 0.5949
Brachyfacial 28 (15.9) 110 (62.5) 38 (21.6) 0.0184 166 (47.2) 186 (52.8) 0.0499

Note. Values are n (%). The Ribeirão Preto sample was not included on analysis of the SNP rs2973015 because the genotyping presented low call rate.
RJ , Rio de Janeiro; M , Manaus; RP , Ribeirão Preto; C , Curitiba.

Statistically significant association (nominal level, P <0.05).

Supplementary Table III
Genotype and allele frequencies according to the skeletal malocclusion for the SNPs assessed in IGF2R
SNP Class Genotypes Alleles
IGF2R rs2277071 AA AG GG P value A G P value
RJ Class I 6 (4.8) 50 (40.0) 69 (55.2) Reference 62 (24.8) 188 (75.2) Reference
Class II 6 (3.5) 69 (40.6) 95 (55.9) 0.8607 81 (23.8) 259 (76.2) 0.8459
Class III 2 (6.5) 8 (25.8) 21 (67.7) 0.3396 12 (19.4) 50 (80.6) 0.4086
M Class I 13 (19.1) 28 (41.2) 27 (39.7) Reference 54 (39.7) 82 (60.3) Reference
Class II 12 (24.5) 14 (28.6) 23 (46.9) 0.3697 38 (38.8) 60 (61.2) 0.8931
Class III 4 (33.3) 5 (41.7) 3 (25.0) 0.4584 13 (54.2) 11 (45.8) 0.2615
RP Class I 4 (5.7) 19 (27.1) 47 (67.1) Reference 27 (19.3) 113 (80.7) Reference
Class II 4 (9.8) 17 (41.5) 20 (48.8) 0.1596 25 (30.5) 57 (69.5) 0.0709
Class III 0 (0.0) 4 (25.0) 12 (75.0) 0.5916 4 (12.5) 28 (87.5) 0.4528
RJ+M+RP Class I 23 (8.7) 97 (36.9) 143 (54.4) Reference 143 (27.2) 383 (72.8) Reference
Class II 22 (8.5) 100 (38.5) 138 (53.1) 0.9324 144 (27.7) 376 (72.3) 0.8898
Class III 6 (10.2) 17 (28.8) 36 (61.0) 0.5016 29 (24.6) 89 (75.4) 0.6454
C Class I 2 (9.5) 9 (42.9) 10 (47.6) Reference 13 (31.0) 29 (69.0) Reference
Class II 1 (5.6) 11 (61.1) 6 (33.3) 0.5194 13 (36.1) 23 (63.9) 0.6797
Class III 3 (5.6) 20 (37.0) 31 (57.4) 0.6873 26 (24.1) 82 (75.9) 0.4115
RJ+M+RP+C Class I 25 (8.8) 106 (37.3) 153 (53.9) Reference 156 (27.5) 412 (72.5) Reference
Class II 23 (8.3) 111 (39.9) 144 (51.8) 0.8158 157 (28.2) 399 (71.8) 0.7903
Class III 9 (8.0) 37 (32.7) 67 (59.3) 0.6171 55 (24.3) 171 (75.7) 0.4230

IGF2R rs6909681 AA AT TT P value A T P value
RJ Class I 27 (21.1) 74 (57.8) 27 (21.1) Reference 128 (50.0) 128 (50.0) Reference
Class II 51 (29.5) 87 (50.3) 35 (20.2) 0.2466 189 (54.6) 157 (45.4) 0.2834
Class III 7 (21.2) 21 (63.6) 5 (15.2) 0.7334 35 (53.0) 31 (47.0) 0.6809
M Class I 17 (26.6) 27 (42.2) 20 (31.3) Reference 61 (47.7) 67 (52.3) Reference
Class II 13 (32.5) 18 (45.0) 9 (22.5) 0.6005 44 (55.0) 36 (45.0) 0.3213
Class III 5 (41.7) 5 (41.7) 2 (16.7) 0.4630 15 (62.5) 9 (37.5) 0.2659
RP Class I 22 (32.8) 32 (47.8) 13 (19.4) Reference 76 (56.7) 58 (43.3) Reference
Class II 17 (41.5) 15 (36.6) 9 (22.0) 0.5117 49 (59.8) 33 (40.2) 0.6731
Class III 5 (29.4) 9 (52.9) 3 (17.6) 0.9277 19 (55.9) 15 (44.1) >0.9999
RJ+M+RP Class I 66 (25.5) 133 (51.4) 60 (23.2) Reference 265 (51.2) 253 (48.8) Reference
Class II 81 (31.9) 120 (47.2) 53 (20.9) 0.2753 282 (55.5) 226 (44.5) 0.1689
Class III 17 (27.4) 35 (56.5) 10 (16.1) 0.4819 69 (55.6) 55 (44.4) 0.4235
C Class I 4 (22.2) 11 (61.1) 3 (16.7) Reference 19 (52.8) 17 (47.2) Reference
Class II 4 (21.1) 11 (57.9) 4 (21.1) 0.9418 19 (50.0) 19 (50.0) 0.8208
Class III 15 (27.8) 25 (46.3) 14 (25.9) 0.5379 55 (50.9) 53 (49.1) >0.9999
RJ+M+RP+C Class I 70 (25.3) 144 (52.0) 63 (22.7) Reference 284 (51.3) 270 (48.7) Reference
Class II 85 (31.1) 131 (48.0) 57 (20.9) 0.3108 301 (55.1) 245 (44.9) 0.2049
Class III 32 (27.6) 60 (51.7) 24 (20.7) 0.8497 124 (53.4) 108 (46.6) 0.5847
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Jan 9, 2021 | Posted by in Orthodontics | Comments Off on GHRand IGF2Rgenes may contribute to normal variations in craniofacial dimensions: Insights from an admixed population
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