ABSTRACT

Objective:

To evaluate the allele and genotype frequencies of the P561T and C422F polymorphic sites of the growth hormone receptor (GHR) gene and the relationship between mandibular prognathism (MP) and these two single-nucleotide polymorphisms (SNPs).

Materials and Methods:

A total of 99 subjects with severe skeletal Class III MP who planned to undergo orthognathic surgery and 99 subjects with Class I occlusion were examined in this study to evaluate the relationship between MP and two SNPs in exon 10 of the GHR gene. GHR was chosen as a candidate gene because growth hormone plays an important role in cartilage growth. A blood sample was used to extract genomic DNA, and the polymerase chain reaction–restriction fragment length polymorphism method was used to determine genotypes of P561T and C422F. The Minitab 14.0 packet program was used to perform statistical analysis.

Results:

Allele frequencies of the C422F and P561T variants were determined. Because of the low allele frequency of the control group, statistical analysis could not be performed to test the difference between MP and control groups. Therefore, the data were combined to determine the association between the P561T polymorphism and craniofacial measurements. Effective mandibular length (condylion-gnathion) and lower face height (anterior nasal spina-menton) were associated with the P561T variant.

Conclusion:

This finding supports that the GHR might be a candidate gene for mandibular morphogenesis in this population.

INTRODUCTION

Skeletal Class III malocclusion may be related to excessive mandibular growth (OMIM #176700), inefficient maxillary growth, or a combination of both, which is one of the most severe maxillofacial skeletal deformities in orthodontics.1 The prevalence of mandibular prognathism (MP) varies among different populations. It is reported that the prevalence of MP is the highest in East Asian populations (approximately 15%–23%), moderate in Sub-Saharan Africans (3%–8%), and lowest in white populations (0.48%–4%).2,3 Sarı et al.4 and Sayin and Turkkahraman5 reported the incidence of Class III malocclusion in Turkish population to be 10.2% and 12%, respectively. However, these data include not only mandibular prognathic patients but also patients with maxillary retrusion.

Although it has been mentioned in a number of previous studies that various environmental factors have contributed to the development of Class III malocclusion, MP has a significant genetic component.610 Craniofacial growth in patients who show more pronounced characteristics of mandibular development is difficult to predict.11 

Growth hormone (GH) is a peptide hormone produced in the anterior pituitary gland that plays a major role in regulating the growth and development of the craniofacial complex.12 GH must bind to its specific cell surface receptor (growth hormone receptor [GHR]) to commence these processes and activate various intracellular signaling pathways.13 GHRs are present in regions with cartilage growth, particularly the mandibular condyle.14 

The human growth hormone receptor (hGHR) gene is encoded by a single gene on chromosome 5p13.1-p12, is about 87 kilobases (kb) long, and consists of 10 exons, nine of which are coding.15 Exon 2 encodes the signal peptide, exons 3–7 the extracellular domain, exon 8 the transmembrane domain, and exon 9 and part of exon 10 the intracellular domain.16 To date, 88 different GHR gene mutations have been recorded in the Human Gene Mutation Database.17 These gene mutations were found to be related to idiopathic short stature,18 Laron syndrome (GH insensitivity syndrome), severe growth retardation, and undetectable serum GH-binding protein.19 

Previous research indicated possible effects of GHR gene mutations on MP, and these studies were usually carried out in Asian populations because of the high incidence (approximately 15%–23%).3 According to the results of the first report about GHR gene variants and MP, a relationship has been reported between mandibular ramus length and a heterozygous missense polymorphism of P561T.20 It has been reported that adult Chinese Han individuals with a genomic polymorphism I526L of the GHR gene have a greater mandibular ramus length.21 In another study, 33 children with mandibular protrusion and 27 normal children were evaluated, and the authors remarked that the P561T heterozygous polymorphism may have an effect on mandibular growth during early childhood.22 Tomayasu et al.23 examined the association between five single-nucleotide polymorphisms (SNPs) in exon 10 of GHR and MP in 167 normal Japanese adult subjects and in a multiethnic population of Han Chinese, African Americans, European Americans, and Hispanics to evaluate the effects of ethnic differences.

Although these findings support the idea that sequence variations in GHR may be associated with differences in mandibular morphology and that GH plays an important role in cartilage growth, there is no study in the literature examining the minor allele frequencies of the P561T and C422F SNPs of GHR in a Turkish population. The aim of this study was to evaluate the allele and genotype frequencies of P561T and C422F polymorphic sites of GHR and the relationship between MP and these two SNPs in this population.

MATERIALS AND METHODS

Subjects

A total of 101 mandibular prognathic patients with severe skeletal Class III malocclusion who planned to undergo or had undergone orthognathic surgery and 99 patients with normal occlusion at the Orthodontics Department of Selcuk University Faculty of Dentistry participated in this study. Mandibular prognathic patients were chosen according to these inclusion criteria:

  • Patients older than 16 years who have completed their growth and development

  • Patients with severe Class III malocclusion with MP

  • Patients with no congenital anomalies such as cleft lip and palate, hereditary diseases, or endocrine problems

  • ANB angle and Wits value less than 0°

Two subjects were excluded from the study because of the poor quality of the lateral cephalograms. The control group consisting of 99 patients was chosen according to these inclusion criteria:

  • Patients older than 16 years who have Class I occlusion with orthognathic profile

  • Patients with no congenital anomalies, hereditary diseases, or endocrine problems

  • ANB angle 2°–4° and Wits value of 0–2

All of the MP subjects and Class I subjects who participated in this study are unrelated and Turkish. All patients participated in a protocol approved by the Ethical Committee of Selcuk University, Faculty of Medicine, and all participating subjects gave written informed consent before blood samples were taken. Lateral cephalometric radiographs and blood samples were obtained from 198 subjects. Demographic characteristics of all participants are shown in Table 1.

Table 1.

Demographic Characteristics of All Subjects

Demographic Characteristics of All Subjects
Demographic Characteristics of All Subjects

Genotyping of P561T and C422F Codons

Whole blood was collected in a sterile tube with K3-EDTA and stored at −20°C for later examination. Genomic DNA was extracted from whole blood samples using a standard phenol/chloroform method.24 The partial sequence of exon 10 of the GHR gene, which encodes the cytoplasmic domain of GHR, was amplified by polymerase chain reaction (PCR). The PCR amplification was performed in a 30-µL PCR volume containing 100 ng template DNA, 0.750 units Taq polimeraz (Pittsburgh, PA, USA) 1× Mg++ free PCR buffer (Fermentas), 200 µM dNTP (Fermentas), 1.5 mM MgCl++, 10 pMol of each primers (5′-GGGAAGCAGATCTCTTATGC-3′ and 5′-TATAGTCTGGGACAGGCATCT-3′). A touchdown-PCR profile was carried out on the BioRad MyCycler-Thermal Cycler (BioRad Laboratories Inc, Hercules, Calif). After agarose gel electrophoresis, the 10-µL PCR-amplified DNA was digested with Eco147I (StuI) and Cac8I, which digests the GHR gene at codon 561 and 422, respectively. Digested PCR products were electrophoresed on 1.5% gels and visualized after ethidium bromide staining and allele genotypes of 561 and 422 codons were determined.

Craniofacial Measurements

Lateral cephalometric radiographs were used to classify subjects into two groups and to determine the maxillary, mandibular, and cranial lengths. Cephalometric radiographs of 198 subjects were traced and analyzed with Quick Ceph Studio (Quick Ceph Systems, San Diego, Calif) by the same researcher. Twenty randomly selected radiographs were retraced and remeasured after 2 weeks, and method error was calculated according to Dahlberg's formula.25 Angular and linear measurements used in our study are shown in Figure 1.

Figure 1.

Linear and angular measurements. 1. Wits appraisal. 2. Ramus height (articulare-gonion; Ar-Go). 3. Corpus length (gonion-pogonion′; Go-Pog′). 4. Mandibular length (gonion-menton; Go-Me). 5. Effective length of mandible (condylion-pogonion; Co-Pog). 6. Effective midface length (condylion-A point; Co-A). 7. Maxillomandibular difference. 8. Maxillary length (A point′-pterygomaxillary point′; A′-Ptm′). 9. Sella-nasion (S-N). 10. Sella-basion (S-Ba). 11. Sella-articulare (S-Ar). 12. Lower face height (anterior nasal spina-menton; ANS-Me). 13. SNA angle. 14. SNB angle. 15. ANB angle.

Figure 1.

Linear and angular measurements. 1. Wits appraisal. 2. Ramus height (articulare-gonion; Ar-Go). 3. Corpus length (gonion-pogonion′; Go-Pog′). 4. Mandibular length (gonion-menton; Go-Me). 5. Effective length of mandible (condylion-pogonion; Co-Pog). 6. Effective midface length (condylion-A point; Co-A). 7. Maxillomandibular difference. 8. Maxillary length (A point′-pterygomaxillary point′; A′-Ptm′). 9. Sella-nasion (S-N). 10. Sella-basion (S-Ba). 11. Sella-articulare (S-Ar). 12. Lower face height (anterior nasal spina-menton; ANS-Me). 13. SNA angle. 14. SNB angle. 15. ANB angle.

Statistical Analysis

The experiment, with the current sample size (99 MP, 99 control), was established to detect the differences with 95% chance at the usual level of statistical significance (alpha  =  .05) for the ANB angle to differentiate groups. The Minitab 14.0 (Minitab Inc, State College, Penn) packet program was used to perform all statistical analyses. Allele and genotype frequencies of P561T and C422F polymorphic sites and Hardy-Weinberg equilibrium were calculated using the GenAlEx6 (Peakall and Smouse 2006) program. The Mann-Whitney U-test was performed to analyze the association between polymorphisms and craniofacial measurements.

RESULTS

A comparison of the lateral cephalometric measurements between the MP and the control groups is shown in Table 2. If the data showed normal distribution, a two-sample t-test was used to analyze the difference between two groups; otherwise, the Mann- Whitney U-test was performed. Although there was no difference between the two groups in maxillary measurements, all mandibular measurements were statistically significant between the two groups, indicating Class III malocclusion with MP.

Table 2.

Comparison of Lateral Cephaometric Measurements between MP and Control Groups

Comparison of Lateral Cephaometric Measurements between MP and Control Groups
Comparison of Lateral Cephaometric Measurements between MP and Control Groups

There was no subject with the C422F and P561T homozygous polymorphism. Two of the MP subjects and one of the control subjects had the C422F heterozygous polymorphism. In 561T locus, five MP subjects and one of the control subjects were heterozygous. Allele and genotype frequencies are shown in Figure 2. C422F and P561T genotypes were also confirmed by forward and reverse sequencing, indicating that PCR restriction fragment length polymorphism analyses were in concordance.

Figure 2.

Allele and genotype frequencies of P561T and C422F regions.

Figure 2.

Allele and genotype frequencies of P561T and C422F regions.

The haplotype distribution of both polymorphic sites was evaluated with the Hardy-Weinberg equilibrium and found to be stable (P > .05; Table 3). Because of the lack of heterozygous genotype at both the C422F and P561T codons in the control group, statistical analysis could not be performed to evaluate the difference between the two groups. Therefore, the data were integrated to define the effect of P561T SNP on craniofacial growth.

Table 3.

Hardy-Weinberg Equilibrium

Hardy-Weinberg Equilibrium
Hardy-Weinberg Equilibrium

According to the Mann-Whitney U statistical analysis, two craniofacial measurements including effective mandibular length (condylion-gnathion [Co-Gn]) and lower face height (anterior nasal spina-menton [ANS-Me]) were found to be associated with the P561T variant (P < .05; Tables 4 and 5).

Table 4.

Association Between P561T Mutation and Mandibular and Maxillary Measurements

Association Between P561T Mutation and Mandibular and Maxillary Measurements
Association Between P561T Mutation and Mandibular and Maxillary Measurements
Table 5.

Association Between P561T Mutation and Craniofacial Measurements

Association Between P561T Mutation and Craniofacial Measurements
Association Between P561T Mutation and Craniofacial Measurements

DISCUSSION

Class III malocclusion with MP has attracted the attention of many researchers because it is frequently encountered in the orthodontic clinic and relatively difficult to treat. MP is important to the understanding of the molecular pathogenesis of skeletal Class III malocclusion. In particular, etiological factors of MP described in the literature59 and genetic susceptibilities on mandible growth have been defined in recent studies.3,2023,2731 Although none of the genomewide studies found a significant marker on chromosome 5 for MP, we analyzed two SNPs (C422F and P561T) located on exon 10 of the GHR in Turkish subjects. We chose GHR as a candidate gene because GH plays an important role in cartilage growth and GHRs were identified in the mandibular condyle.31 These polymorphisms can cause variance in the signal transduction by affecting the expression of IGF-1, which can affect endochondral bone growth.

The first polymorphism of hGHR was defined in a study consisting of 76 patients with Laron syndrome, and heterozygous polymorphisms were associated with this syndrome.32 Initially, allele and genotype frequencies of P561T and C422F polymorphic sites of the GHR gene in the Turkish population were determined in the present study. No difference in the frequency of the C422F and P561T variants was found between the two groups. The lack of heterozygous polymorphism at the C422F region in both the MP and control group showed that this polymorphism was not associated with MP in this population. Positive correlations were observed between body height and all mandibular measurements, but it was the highest in effective mandibular length. In this study, an association was also determined between the P561T polymorphism and effective mandibular length and lower face height. Even though a previous study23 of 167 Japanese adults reported an association between the C422F locus and mandibular ramus height, no such association was observed in this study. Findings of a study of 95 Chinese adult subjects did, however, agree with our findings at the C422F locus.21 Zhou et al.21 reported an association between the I526L polymorphism of the GHR and mandibular ramus height. The findings from both Turkish and Chinese populations did not show any relationship between mandibular length and C422F polymorphism, which is different from findings in the Japanese population. This discrepancy may be related to the ethnic differences of the groups.

According to the first study examining the association between MP and the single polymorphism of GHR, the P561T heterozygous polymorphism and ramus height were found to be associated.20 Sasaki et al.22 reported differences in all cranial measurements except maxillary length between heterozygous subjects and subjects without polymorphism. Another study of 167 Japanese subjects reported an association between the P561T variant and mandibular ramus height.23 Unlike those results, we found that the P561T variant was associated with effective mandibular length and lower face height in the Turkish population. Previous studies showed that ethnicity is a risk factor for MP.2,3 According to these results, it can be thought that GHR can affect not only the longitudinal development but also the horizontal development of the mandible. Therefore, statistically significant results can be obtained in ramus height if the sample size is increased. From these data, it can be concluded that there is a relationship between the GHR polymorphism and mandibular growth.

In this study, subjects with the genotype CA had a longer mandible and a higher lower face than those with the genotype CC, whereas cranial measurements of heterozygous subjects tend to be lower in other studies.20,22,23 

Although a number of etiological factors have been found to contribute to mandibular morphology, genetic factors play a significant role.33 Bailey34 found that 30 genes can affect the morphogenesis of the mandible in mice. Different sites of GHR and new candidate genes must be examined to explain the relationship between different phenotypes and genetic variants as well as to understand the effects of genetic factors in the multifactorial etiology of MP.

The advantage of determining candidate genes for MP is to predict mandibular length in growing patients by using DNA from a simple blood sample. This genotyping could also be done in a less invasive manner from buccal cells collected in saliva, but the quality and quantity of DNA were inefficient in this method. We planned an association study to investigate the relationship between GHR and MP, but a linkage study will be helpful to understand the effect of this gene and its promoter region on MP.

In the past decade, interest in growth factors and other agents that may control cartilage proliferation and differentiation has increased. In the near future, this knowledge could be used for growth regulation in patients with MP and might be useful for orthodontic diagnosis and orthopedic treatment of the mandible.

CONCLUSIONS

  • C422F and P561T heterozygous polymorphisms of the GHR gene did not justify the difference between the MP group and control group in this population.

  • Subjects with the CA genotype of P561T have a greater effective mandibular length (Co-Gn) and lower face height (ANS-Me) than those with genotype CC.

  • This finding supports that the GHR might be a candidate gene for mandibular morphogenesis.

ACKNOWLEDGMENTS

This study was produced from the PhD thesis of Sinem Bayram and supported by Selcuk University Research Projects (11202032).

REFERENCES

REFERENCES
1.
Proffit
WR
.
Contemporary Orthodontics
.
St Louis, Mo
:
Mosby-Year Book
;
1992
.
2.
El-Gheriani
AA
,
Maher
BS
,
El-Gheriani
AS
,
et al
.
Segregation analysis of mandibular prognathism in Libya
.
J Dent Res
.
2003
;
82
:
523
527
.
3.
Yamaguchi
T
,
Park
SB
,
Narita
A
,
Maki
K
,
Inoue
I
.
Genome-wide linkage analysis of mandibular prognathism in Korean and Japanese patients
.
J Dent Res
.
2005
;
84
:
255
259
.
4.
Sari
Z
,
Uysal
T
,
Karaman
AI
,
Basciftci
FA
,
Usumez
S
,
Demir
A
.
Orthodontic malocclusions and evaluation of treatment alternatives: an epidemiologic study
.
Turkish J Orthod
.
2003
;
16
:
119
126
.
5.
Sayin
MO
,
Turkkahraman
H
.
Malocclusion and crowding in an orthodontically referred Turkish population
.
Angle Orthod
.
2004
;
74
:
635
639
.
6.
Jena
AK
,
Duggal
R
,
Mathur
VP
,
Parkash
H
.
Class-III malocclusion: genetics or environment? A twins study
.
J Indian Soc Pedod Prev Dent
.
2005
;
23
:
27
30
.
7.
Angle
EH
.
Treatment of Malocclusion of the Teeth. 7th ed
.
Philadelphia, Pa
:
S.S. White Manufacturing Company
;
1907
.
8.
Downs
WG
.
Studies in the causes of dental anomalies
.
J Dent Res
.
1928
;
8
:
267
379
.
9.
Gold
JK
.
A new approach to the treatment of mandibular prognathism
.
Am J Orthod
.
1949
;
35
:
893
912
.
10.
Pascoe
J
,
Hayward
JR
,
Costich
ER
.
Mandibular prognathism, its etiology and a classification
.
J Oral Surg
.
1960
;
18
:
21
24
.
11.
Stiles
KA
,
Luke
JE
.
The inheritance of malocclusion due to mandibular prognathism
.
J Hered
.
1953
;
44
:
241
245
.
12.
Ramirez-Yanez
GO
,
Smid
JR
,
Young
WG
,
Waters
MJ
.
Influence of growth hormone on the craniofacial complex of transgenic mice
.
Eur J Orthod
.
2005
;
27
:
494
500
.
13.
Pilecka
I
,
Whatmore
A
,
van Huijsduijnen
RH
,
Destenaves
B
,
Clayton
P
.
Growth hormone signalling: sprouting links between pathways, human genetics and therapeutic options
.
Trends Endocrinol Metab
.
2007
;
18
:
12
18
.
14.
Simmons
KE
.
Growth hormone and craniofacial changes: preliminary data from studies in Turner's syndrome
.
Pediatrics
.
1999
;
104
:
1021
1024
.
15.
Leung
DW
,
Spencer
SA
,
Cachianes
G
,
et al
.
Growth hormone receptor and serum binding protein: purification, cloning and expression
.
Nature
.
1987
;
330
:
537
543
.
16.
Godowski
PJ
,
Leung
DW
,
Meacham
LR
,
et al
.
Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism
.
Proc Nat Acad Sci
.
1989
;
86
:
8083
8087
.
17.
Stenson
PD
,
Mort
M
,
Ball
EV
,
Shaw
K
,
Phillips
AD
,
Cooper
DN
,
Human Gene Mutation Database (HGMD): 2013 update
.
18.
Goddard
AD
,
Covello
R
,
Luoh
SM
,
et al
.
Mutations of the growth hormone receptor in children with idiopathic short stature. The Growth Hormone Insensitivity Study Group
.
N Engl J Med
.
1995
;
333
:
1093
1098
.
19.
Kaji
H
,
Nose
O
,
Tajiri
H
,
et al
.
Novel compound heterozygous mutations of growth hormone (GH) receptor gene in a patient with GH insensitivity syndrome
.
J Clin Endocrinol Metab
.
1997
;
82
:
3705
3709
.
20.
Yamaguchi
T
,
Maki
K
,
Shibasaki
Y
.
Growth hormone receptor gene variant and mandibular height in the normal Japanese population
.
Am J Orthod Dentofacial Orthop
.
2001
;
119
:
650
653
.
21.
Zhou
J
,
Lu
Y
,
Gao
XH
,
Chen
YC
,
et al
.
The growth hormone receptor gene is associated with mandibular height in a Chinese population
.
J Dent Res
.
2005
;
84
:
1052
1056
.
22.
Sasaki
Y
,
Satoh
K
,
Hayasaki
H
,
Fukumoto
S
,
Fujiwara
T
,
Nonaka
K
.
The P561T polymorphism of the growth hormone receptor gene has an inhibitory effect on mandibular growth in young children
.
Eur J Orthod
.
2009
;
31
:
536
541
.
23.
Tomoyasu
Y
,
Yamaguchi
T
,
Tajima
A
,
Nakajima
T
,
Inoue
I
,
Maki
K
.
Further evidence for an association between mandibular height and the growth hormone receptor gene in a Japanese population
.
Am J Orthod Dentofacial Orthop
.
2009
;
136
:
536
541
.
24.
Sambrook
J
,
Fritsch
EF
,
Maniatis
T
,
Molecular Clonning: A Laboratory Manual. 2nd ed. Vol 2
.
Cold-Spring Harbor, NY
:
Cold Spring Harbor Laboratory Press
;
1989
.
25.
Dahlberg
G
,
Statistical Methods for Medical and Biological Students
.
London, UK
:
Allen & Unwin Ltd.
1940
:
122
132
26.
Peakall
R
,
Smouse
PE
.
Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research
.
Molecular Ecology Notes
.
2006
;
6
:
288
295
.
27.
Frazier-Bowers
S
,
Rincon-Rodriguez
R
,
Zhou
J
,
Alexander
K
,
Lange
E
.
Evidence of linkage in a Hispanic cohort with a Class III dentofacial phenotype
.
J Dent Res
.
2009
;
88
:
56
60
.
28.
Jang
JY
,
Park
EK
,
Ryoo
HM
,
et al
.
Polymorphisms in the Matrilin-1 gene and risk of mandibular prognathism in Koreans
.
J Dent Res
.
2010
;
89
:
1203
1207
.
29.
Xue
F
,
Wong
R
,
Bakr
A
,
Rabie
M
.
Identification of SNP markers on 1p36 and association analysis of EPB41 with mandibular prognathism in a Chinese population
.
Arch Oral Biol
.
2010
;
55
:
867
872
.
30.
Cruz
RM
,
Hartsfield
JK
, Jr,
Falcão-Alencar
G
,
et al
.
Exclusion of Class III malocclusion candidate loci in Brazilian families
.
J Dent Res
.
2011
;
90
:
1202
1205
.
31.
Li
Q
,
Li
X
,
Zhang
F
,
Chen
F
.
The identification of a novel locus for mandibular prognathism in the Han Chinese population
.
J Dent Res
.
2011
;
90
:
53
57
.
32.
Lewinson
D
,
Bialik
GM
,
Hochberg
Z
.
Differential effects of hypothyroidism on the cartilage and the osteogenic process in the mandibular condyle: recovery by growth hormone and thyroxine
.
Endocrinology
.
1994
;
135
:
1504
1510
.
33.
Berg
M.A
,
Guevara-Aguirre
J
,
Rosenbloom
AL
,
Rosenfeld
RG
,
Francke
U
.
Mutation creating a new splice site in the growth hormone receptor genes of 37 Ecuadorean patients with Laron syndrome
.
Hum Mutat
.
1992
;
1
:
24
32
.
34.
Chang
HP
,
Tseng
YC
,
Chang
HF
.
Treatment of mandibular prognathism
.
J Formos Med Assoc
.
2006
;
105
:
781
790
.
35.
Bailey
DW
.
Genes that affect the shape of the murine mandible
.
J Hered
.
1985
;
76
:
107
114
.