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Introduction

Legg-Calvé-Perthes disease (LCPD) is a noninflammatory, aseptic, self-limiting, idiopathic, avascular necrosis of the femoral head, occurring in childhood, typically between the ages of 4 and 10 years and causing permanent femoral head deformity and premature osteoarthritis (1–3). The disease was first described by three independent authors in the year 1910: Perthes in Germany (4), Legg in the United States (5) and Calvé in France (6). The incidence of LCPD varies between 5.1 and 16.9 per 100,000 in various regions of the world (2,7). The etiology of LCPD remains unclear, which makes it impossible to create prevention strategies or to identify individuals at risk. A number of possible causes have been proposed, including trauma, an inflammatory process, vascular occlusion, hemodynamic alterations, endothelial injury, thrombophilia and insulin-like growth factor-1 pathway abnormality (2,8,9).

Nitric oxide (NO) is a multifunctional biomolecule synthesized by three isoforms of the nitric oxide synthase (NOS) enzyme, namely endothelial NOS (eNOS, also known as NOS III), inducible NOS (iNOS or NOS II) and neuronal NOS (nNOS or NOS I). NO participates in numerous physiological processes, including angiogenesis, thrombosis, coagulation and fibrinolysis. The eNOS gene is located on the long arm of chromosome 7 (7q35–36), has 26 exons and 25 introns, and encodes a protein of 1,203 amino acids (10,11). The most examined and functionally associated polymorphisms are G894T in exon 7 and 27-bp variable number tandem repeat (VNTR) polymorphism in intron 4, respectively (12,13). A number of studies have observed associations between genetic polymorphisms in the eNOS gene and cardiovascular diseases, including coronary artery disease, chronic heart failure, hypertension, atherosclerosis, stroke, renal diseases and avascular necrosis of the femoral head, mainly in adult patients (14–20). Numerous studies have indicated that disruption of the blood supply to the femoral head is a key pathogenic event resulting in bone necrosis (2,21,22). Thrombophilia and/or decreased fibrinolysis are possible mechanisms that have been investigated as potential causes of LCPD (23–25). It may be speculated that sequence variations in the eNOS gene could influence nitric oxide production, and thereby affect the progression of LCPD. To the best of our knowledge, no study has yet investigated the association between eNOS polymorphism and the pathogenesis of LCPD.

Therefore, in the present study, the aim was to examine a relevance of the 27-bp VNTR polymorphism in intron 4 and G894T polymorphism in exon 7 of the eNOS gene by conducting an analysis of 80 LCPD Chinese patients and 100 healthy controls.

Materials and methods

Study subjects

A total of 80 patients with LCPD (70 boys and 10 girls, aged between 4 and 10 years) were consecutively enrolled in this study at the Department of Orthopedics of the First Affiliated Hospital of Guangxi Medical University (Nanning, China) from October 2012 to September 2014. LCPD was diagnosed on the basis of radiography and clinical presentation. Magnetic resonance imaging (MRI) was used as a supplementary technique to identify the changes of the femoral head in the early stage of LCPD. None of the patients with LCPD had historical or clinical evidence of vascular diseases, such as atherosclerosis, arterial thrombosis, coronary artery disease, hypertension, renal diseases, ischemic heart diseases, cerebrovascular diseases, or coexisting disorders that may be associated with eNOS polymorphism. In addition, 100 healthy age- and gender-matched children were included as controls, none of whom had LCPD or clinical symptoms suggesting LCPD. This study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. Written informed consent was obtained from all participants or their parents.

DNA extraction and genotyping

For all subjects, peripheral blood samples were collected into 2 ml EDTA tubes. Genomic DNA was extracted from whole blood using a commercial kit (DP318-03; Tiangen Biotech Co., Ltd., Beijing, China), in accordance with the manufacturer's protocol. The amplification of the eNOS 27-bp VNTR polymorphism of intron 4 was performed by polymerase chain reaction (PCR) using the following primers: Forward 5′-AGGCCCTATGGTAGTGCCTTT-3′ and reverse 5′-TCTCTTAGTGCTGTGGTCAC-3′. The PCR reactions were performed in a total volume of 25 µl, which contained containing 500 µM each dNTP, 20 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM MgCl2, 0.1 units Taq polymerase (Tiangen Biotech Co., Ltd.), 10 µM each primer and 100 ng genomic DNA. The reaction mixture was heated to 94°C (3 min), followed by 30 cycles of 94°C (30 sec), 58°C (30 sec), and 72°C (1 min), and then a final extension at 72°C (5 min). The PCR products were analyzed by electrophoresis in 2% agarose gels at 80 V for 40 min. A band size of 393 bp indicated a polymorphic genotype (aa, 4 repeats). A 420-bp fragment represented the wild-type genotype (bb, 5 repeats). Two fragments at 393 bp and 420 bp indicated a heterozygous genotype ab (Fig. 1). The PCR products were sequenced using an ABI3730XL genetic analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) (Fig. 2). The eNOS G894T polymorphism in exon 7 was determined by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The forward primer was 5′-AAGGCAGGAGACAGTGGATGG-A-3′ and the reverse primer was 5′-CCCAGTCAATCCCTTTGGTGCTCA-3′. PCR was conducted to amplify 0.1 µg genomic DNA. The 25 µl reaction mixture contained 500 µM each dNTP, 20 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM MgCl2, 0.1 units Taq polymerase, and 10 µM sense and antisense primers. PCR was performed by denaturation at 94°C for 3 min, followed by 30 cycles (30 sec at 94°C, 30 sec at 65°C and 60 sec at 72°C) and a final extension at 72°C for 5 min. This PCR amplification yielded a 248-bp fragment. Following amplification, PCR products were digested for 6 h using 2 units of BanII restriction enzyme (Takara Biotechnology Co., Ltd., Dalian, China) at 37°C. The final products of eNOS G894T polymorphism including GG, GT and TT genotypes had band sizes of 163 bp/85 bp, 248 bp/163 bp/85 bp and 248 bp, respectively. The digestion products were separated by electrophoresis on GoldView I-stained (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) 2.5% agarose gel (Fig. 3) with DNA marker I (Tiangen Biotech Co., Ltd.). The eNOS G894T polymorphisms were also sequenced using the ABI3730XL genetic analyzer (Fig. 4).

Figure 1. Agarose gel electrophoresis result of eNOS 27-bp VNTR polymorphism. Fragments containing 420 bp (eNOS b allele) and 393 bp (eNOS a allele) can be visualized. Lanes 1, 2, 4, 6, 7, 8 and 10 show the bb genotype; lanes 3, 5 and 9 show the ab genotype; lane M: DNA marker. The rare aa genotype was not found in patients with LCPD and healthy controls. eNOS, endothelial nitric oxide synthase; VNTR, variable number tandem repeat.

Figure 2. Sequence diagrams of the PCR amplification products of the eNOS 27-bp VNTR polymorphism. Sequence analysis of (A) bb genotype and (B) ab genotype. PCR, polymerase chain reaction; eNOS, endothelial nitric oxide synthase; VNTR, variable number tandem repeat.

Figure 3. Agarose gel electrophoresis result of eNOS G894T polymorphism provided by PCR-RFLP. The PCR product was digested with BanII restriction enzyme and visualized on a 2.5% agarose gel. Lanes 1, 2, 4, 5, 9, 10 show wild type GG genotype (163 and 85 bp); lanes 3,7,8 show heterozygous GT (248, 163 and 85 bp); and lane 6: homozygous TT (248 bp). Lane M: DNA marker. eNOS, endothelial nitric oxide synthase; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.

Figure 4. Sequence diagrams of the PCR amplification products of the eNOS G894T polymorphism. Sequence analysis of tbe (A) TT genotype. (B) GT heterozygous genotype and (C) GG genotype. PCR, polymerase chain reaction; eNOS, endothelial nitric oxide synthase.

Statistical analysis

Continuous data are presented as the mean ± standard deviation. Categorical data are expressed in percentages. The allelic frequencies were calculated by the chromosome counting method. The genotype frequencies for eNOS polymorphism among patients and healthy controls were assessed for Hardy-Weinberg equilibrium (HWE) using the Pearson's Chi-square method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using relevant 2×2 contingency tables. The data was analyzed using the SPSS statistical software package (version 16.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

Results

General characteristics of the study subjects

In this study, 80 LCPD patients and 100 controls were included in the final analysis. LCPD was more common in boys than in girls. The general characteristics of the study population are summarized in Table I. There were more boys than girls in both the patient group (87.5% boys) and the gender-matched pediatric control group (85% boys). There were no significant differences between the groups regarding age and gender.

Table I. General characteristics of the study subjects.

Table I.

General characteristics of the study subjects.

Variable	LCPD (n=80)	Healthy control (n=100)	P-value
Gender, n (%)
Male	70 (87.5)	85 (85.0)	>0.05
Female	10 (12.5)	15 (15.0)	>0.05
Age, yearsa	7.3±2.0	6.8±2.1	>0.05
Site of LCPD, n (%)
Right	38 (47.5)
Left	30 (37.5)
Bilateral	12 (15.0)
Surgery
Yes	49
No	31

a Mean ± standard deviation. LCPD, Legg-Calvé-Perthes disease.

eNOS gene polymorphisms in the case and control groups

The genotypic and allelic frequencies of the two eNOS gene polymorphisms in the case and control groups are shown in Table II. The genotype frequencies of the analyzed polymorphism were in HWE. The genotypic and allelic frequencies of the two eNOS gene polymorphisms differed between the control and patient groups. Representative electrophoresis results and sequence diagrams for the eNOS 27-bp VNTR polymorphism are shown in Figs. 1 and 2. The LCPD case group showed a higher frequency of the ab genotype than the healthy control group (27.5 vs. 14%; OR, 2.33; 95% CI, 1.10–4.92; P=0.024). The frequency of the a allele was significantly higher in the LCPD case group than in the healthy controls (13.8 vs. 7%; OR, 2.12; 95% CI, 1.05–4.29; P=0.034). Representative electrophoresis results and sequence diagrams for the eNOS G894T polymorphism are shown in Figs. 3 and 4 The heterozygous genotype GT was observed in 35% of the patients (28/80) and 17% (17/100) of the controls (OR, 2.67; 95% CI, 1.33–5.36; P=0.005). The frequency of allele T in the LCPD case group (20%) was greater than that of the healthy control group (10.5%), and the difference in frequency distribution between alleles was significant (OR, 2.13; 95% CI, 1.18–3.87; P=0.011).

Table II. Comparison of genotypic distributions and allele frequencies of the two eNOS gene polymorphisms between the LCPD and healthy control groups.

Table II.

Comparison of genotypic distributions and allele frequencies of the two eNOS gene polymorphisms between the LCPD and healthy control groups.

				LCPD vs. healthy control
Polymorphism	Genotype/Allele	LCPD (n=80)	Healthy control (n=100)	OR	95% CI	P-value
eNOS 27 bp VNTR, n (%)	bb	58 (72.5)	86 (86.0)	1
	ab	22 (27.5)	14 (14.0)	2.33	1.10–4.92	0.024
	aa	0 (0)	0 (0)
	b	138 (86.2)	186 (93.0)	1
	a	22 (13.8)	14 (7.0)	2.12	1.05–4.29	0.034
eNOS G894T, n (%)	GG	50 (62.5)	81 (81.0)	1
	GT	28 (35.0)	17 (17.0)	2.67	1.33–5.36	0.005
	TT	2 (2.5)	2 (2.0)	1.62	0.22–11.87	0.632
	G	128 (80.0)	179 (89.5)	1
	T	32 (20.0)	21 (10.5)	2.13	1.18–3.87	0.011

[i] eNOS, endothelial nitric oxide synthase; VNTR, variable number tandem repeat; LCPD, Legg-Calvé-Perthes disease.

Discussion

The precise pathogenetic basis of LCPD is currently unknown. Repeated interruptions of the vascular blood supply to the femoral head, which results in ischemic necrosis, appears to be a key pathogenic event causing pathological and subsequent structural changes to the growing femoral head (2,21,22). However, at present, the exact pathogenetic basis of the reductions in blood flow to the femoral head vasculature in patients with LCPD is unclear. LCPD is considered to be a multifactorial disease, in which genetic and environmental factors play a role (2). Since genetic background is a determinant of LCPD, extensive genomics research is required to identify polymorphic loci and elucidate the pathogenesis. eNOS gene polymorphisms have been investigated as potential risk factors in numerous vascular diseases, as aforementioned (14–20). To the best of our knowledge, no studies have investigated the role of eNOS gene polymorphism in the risk of LCPD. In the present study, the association between eNOS gene polymorphism and the risk of LCPD was analyzed in children from the Guangxi province of China, and it was found that the frequency of an a allele and ab genotype in intron 4 and allele 894T, GT genotype in exon 7 were significantly higher in patients with LCPD than in healthy controls.

NO, synthesized by eNOS, has various roles in the vasculature, which include the regulation of vascular tone and blood pressure via vasodilation (26). NO also been suggested to be involved in the modulation of angiogenesis (27). In a previous study, it was observed that the development of angiogenesis in response to limb ischemia was severely reduced in mice lacking the eNOS gene (28). Another study demonstrated that the presence of a 27-bp VNTR polymorphism in intron 4 and G894T polymorphism in exon 7 can cause changes in eNOS expression and enzymatic activity; the NO levels in the plasma of subjects with the a allele of eNOS in intron 4 were significantly lower than those without the a allele (29). The G894T polymorphism causes a structural change in the eNOS protein that is associated with impaired eNOS activity (30). Therefore, allele a and G894T gene polymorphisms can lead to low expression levels and activity of eNOS. The present study also reveals that the 27-bp VNTR polymorphism in intron 4 and G894T polymorphism in exon 7 may be risk factors for LCPD, and indicates that NO produced by constitutively expressed eNOS may play a protective role in the pathogenesis of LCPD. The eNOS enzyme is constitutively expressed in vascular endothelial cells. Endothelial cells are important in the pathogenesis of diseases that involve thrombosis and/or inflammation (31). Changes in hemodynamics (22), vascular thrombotic tendency due to hypercoagulability (32), and hypofibrinolysis (9,33,34) have all been suggested to be significant factors in the pathogenesis of LCPD. Intron 4 and G894T polymorphisms of eNOS have been shown to decrease NO levels in human plasma (35,36). As a result, polymorphisms of the eNOS gene and resultant reductions in the quantity of synthesized NO are likely to lead to different types of vascular diseases, as previously mentioned; these diseases may share some common risks through inheritance or exposure. Polymorphisms of the eNOS gene have been indicated to be risk factors for avascular necrosis of the femoral head (20), an adult disease that has a similar pathogenesis to LCPD. The finding in the present study that eNOS polymorphism is associated with the pathogenesis of LCPD is, therefore, not remarkable.

The present study has certain limitations: As all of the samples are from the same hospital, potential selection bias cannot be fully excluded. Also, in consideration of the low incidence of LCPD, the sample size was small, and the rare aa genotype was not found in the subjects investigated.

In summary, to the best of our knowledge, this is the first report investigating an association between eNOS gene polymorphism and the risk of LCPD. The results support the role of eNOS in the pathogenesis of LCPD. However, as the sample size of this study was relatively small, additional studies with larger cohorts are required in order to confirm the observed association.

Glossary

Abbreviations

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References