Epigenetics in Legg–Calvé–Perthes disease: A study of global DNA methylation

Abstract

Objective

To examine the global methylation status of DNA in blood cells of children with Legg-Calvé-Perthes disease (LCPD), since the aetiopathogenesis of LCPD remains unclear, and many factors closely associated with DNA methylation may be linked to the occurrence of LCPD.

Methods

Children with LCPD and age-, sex- and body mass index-matched controls were evaluated. Methylation levels of the long interspersed nuclear element 1 (LINE-1), a biomarker of global DNA methylation, were quantified by methylation-specific polymerase chain reaction.

Results

Of 82 children with LCPD (68 male/14 female) and 120 matched controls (98 male/22 female), methylation of the LINE-1 promoter was significantly lower in patients with LCPD compared with controls. Subgroup analyses showed that methylation of the LINE-1 promoter was significantly lower in male patients with LCPD compared with male controls. No significant between-group differences were observed in female participants.

Conclusions

Reduced global DNA methylation may be associated with increased risk of LCPD in male children. Further research is required to understand whether detection of global DNA methylation may provide a basis for clinical diagnosis and early intervention of LCPD.

Introduction

Legg–Calvé–Perthes disease (LCPD) is a juvenile idiopathic osteonecrosis of the femoral head that affects children between the ages of 4 and 8 years, and its prevalence ranges between 0.2 and 19.1 per 100 000 population in various global regions. ¹ Since the initial reports of this condition ∼100 years ago,^2–4 a number of studies have been published regarding the aetiology, epidemiology, natural history, radiographic classifications, treatments and outcomes of LCPD.^5–13

Despite an increase in knowledge, LCPD remains one of the most controversial conditions in paediatric orthopaedics, and many aspects of the disease remain unclear. The causative gene for LCPD remains unidentified, although associations with various factors have been reported. Environment may play a key role in LCPD development, and many factors (including inflammation, vascular occlusion, thrombophilia, maternal smoking and passive smoke exposure) may be linked to LCPD occurrence.^14–16 Factors such as those described have been shown to be associated with epigenetic modifications.^17–19

Epigenetics is the study of gene function and heritable changes such as DNA methylation or chromatin conformational changes not caused by alternations in DNA sequence. DNA methylation, which is the principal mechanism of epigenetics, has been reported to play a role in cancer development,^20,21 ageing ²² and complex chronic diseases. ²³ Long interspersed nuclear element 1 (LINE-1) retrotransposons are a nonlong terminal repeat (LTR) class of retrotransposons that comprise ∼18% of the human genome. Because of their high frequency in the genome, levels of LINE-1 methylation serve as useful surrogate markers of global DNA methylation.^24,25

The aim of the present study was to investigate the role of epigenetics in LCPD. Thus, a preliminary epigenetic study of DNA methylation in LCPD (through analyses of LINE-1 methylation) was conducted, comparing the percentage of global methylation between patients with LCPD and matched controls.

Patients and methods

Study population and design

The present observational study included sequentially enrolled patients with LCPD, aged <18 years, admitted to the Department of Paediatric Orthopaedics, Nanjing Children’s Hospital Affiliated to Nanjing Medical University, Nanjing, China between January 2008 and December 2012. Criteria for inclusion in the LCPD group included patient’s medical history, and results of standard physical and imaging examinations undertaken to confirm LCPD. Features used to confirm LCPD included hip, knee, or groin pain, exacerbated by hip/leg movement, particularly internal hip rotation; reduced range of motion, particularly in abduction and internal rotation;, presence of a limp; X-radiographic examinations of the hip that suggested and/or verified the diagnosis (usually by demonstrating a flattened, and later fragmented, femoral head); bone scan or magnetic resonance imaging in cases where X-radiographic findings were inconclusive.

Patients with hypothyroidism, epiphyseal dysplasia, sickle-cell anaemia, metabolic bone disease or a history of using steroid compounds were excluded from the study. Patients with femoral head necrosis induced by femoral neck fracture or developmental dysplasia of the hip were also excluded. Control-group patients were enrolled from children hospitalized for trauma in the Department of Paediatric Orthopaedics, Nanjing Children’s Hospital Affiliated to Nanjing Medical University, during the same period as the LCPD patients. Control patients with any history of familial or genetic disease, or previous relevant medical history (such as hypothyroidism, epiphyseal dysplasia, sickle-cell anemia, metabolic bone disease or a history of using steroid compounds) were excluded. Body mass index (BMI; weight in  kg/height in m²) was recorded for each participant.

All study participants or their legal proxies provided a written informed consent (guardians of the children provided informed consent) and the study was approved by Nanjing Children’s Hospital Ethics Committee.

Blood sample collection and DNA extraction

Peripheral blood samples (5 ml per patient) were collected using standard methods into anticoagulant tubes containing 1.5 mg/ml ethylenediaminetetra-acetic acid (EDTA). Blood samples were frozen within 30 min of collection and stored at −80℃ until use. Total DNA was extracted using a Multisource Genomic DNA Miniprep Kit (Axygen, Union City, CA, USA) according to the manufacturer’s protocol. Briefly, blood samples were defrosted at room temperature and red blood cells were lysed, followed by lysis of white blood cells and their nuclei. Cellular proteins were then removed by a salt-precipitation step, leaving the high molecular weight genomic DNA in solution. Finally, genomic DNA was concentrated and desalted by isopropanol precipitation, and dissolved in TE buffer (10 mM Tris–HCl, pH 7.4, 1 mM EDTA).

DNA modification and quantitative methylation-specific real-time PCR

Genomic DNA (1 mg per sample) was modified with sodium bisulphite using the CpGenome® DNA Modification Kit (Millipore, Billerica, MA, USA) and additional reagents (3 M NaOH pellets, β-mercaptoethanol, 70% EtOH, and 90% EtOH [Sigma-Aldrich, St Louis, MO, USA]) according to the manufacturer's instructions. The resultant bisulphite-treated genomic DNA was immediately subjected to quantitative methylation-specific real-time polymerase chain reaction (PCR) to quantify the level of LINE-1 methylation, which was used as a surrogate marker of global DNA methylation. DNA input was normalized by amplifying a region of β-actin that excluded CpG dinucleotides.

The following primer sequences were used: LINE-1 forward primer, LINE-1 M-F 5′-GTCGAATAGGAATAGTTTCGG-3′; LINE-1 reverse primer, LINE-1 M-R 5′-ACTCCCTAACCCCTTACGCT-3′; β- actin forward primer, Actin-F 5′-TGGTGATGGAGGAGGTTTAGTAAG-3′; β-actin reverse primer, Actin-R 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. Each 20 -µl reaction mix contained 10 µl 2 × LightCycler® 480 SYBR Green I Master (Roche, Basel, Switzerland), 10 ng modified DNA, 0.4 µl forward primer (10 pM/µl) and 0.4 µl reverse primer (10 pM/µl). Real-time PCR was performed using a LightCycler® 480 (Roche) with the following cycling conditions: 95℃ for 10 min followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ for 1 min, annealing at 62℃ for 10 s, then polymerization at 72℃ for 20 s. A melting curve was created by cooling the products at 50℃ for 30 s and then heating to 80℃ at a rate of 0.1℃/s, while simultaneously measuring the fluorescence. Data were analysed via the comparative threshold cycle (Ct) method. ΔCt values were first calculated using the formula: ΔCt = Ct _LINE-1−Ct _Actin; then the mean level of LINE-1 methylation status (2^–ΔCT) in all samples was determined. To exclude the impact of sex on DNA methylation levels, male patients with LCPD were compared with male controls, and female patients with LCPD were compared with female controls.

Statistical analyses

Data were presented as mean ± SD. Student’s t-test was used to examine the differences in proportions between groups. The difference of sex ratio between two groups was compared using χ²-test. All statistical analyses were carried out using Stata® software, version 9.0 (StataCorp LP, College Station, TX, USA) and SPSS® software, version 17.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data were analysed using unpaired two-sample Student’s t-test and P < 0.05 was considered to be statistically significant.

Results

A total of 82 patients with LCPD (68 male, 14 female), with a mean age of 7.7 years (range, 3–14 years) were included. Of these patients with LCPD, 43 were left-hip cases, 35 were right-hip cases, and four were bilateral hip cases who were all male. A total of 120 controls (98 male, 22 female) were included (10 per year of age, ranging between 3 and 14 years) with a mean age of 7.9 years. kg/height in m². No significant differences were observed between the two groups in terms of age, BMI or sex ratio (P = 0.434, P = 0.635 and P = 0.854, respectively).

DNA methylation status

Methylation levels of the LINE-1 promoter were significantly lower in children with LCPD compared with controls (Figure 1A)). Methylation levels of the LINE-1 promoter were significantly lower in male patients with LCPD compared with age-matched male controls (Figure 1B). Removal of the four male bilateral LCPD cases and repeat of the statistical analyses produced similar results: methylation levels of the LINE-1 promoter were significantly lower in male patients with unilateral LCPD compared with male controls (P < 0.05). No significant difference was observed between the female patient and control groups (Figure 1(C)).

Figure 1.

Methylation of long interspersed nuclear element 1 (LINE-1) promoter in children aged ≤14 years with Legg-Calvé-Perthes disease (LCPD) compared with controls. Data presented as mean ± SD. (A) Levels of LINE-1 promoter methylation were significantly lower in children with LCPD (1.0 ± 0.1475) compared with controls (1.3899 ± 0.2182, P < 0.05; Student’s t-test); (B) Levels of LINE-1 promoter methylation were significantly lower in male patients with LCPD (1.0 ± 0.1567) compared with male controls (1.4439 ± 0.2098, P < 0.05; Student’s t-test); (C) Levels of LINE-1 promoter methylation were not significantly different between female patients with LCPD (1.0 ± 0.4134) and female controls (0.8467 ± 0.3928, P > 0.05; Student’s t-test)

Discussion

The results of the present study suggest that genomic DNA methylation may be significantly altered in male children with LCPD. To the best of the author’s knowledge, this is the first study comparing LINE-1 methylation levels in peripheral blood cells of patients with LCPD and control subjects.

The aetiology and pathophysiology of LCPD remain unclear. To date, no gene has been conclusively reported to be associated with LCPD; however, epigenetics may contribute to its pathogenesis. DNA methylation is an important aspect of epigenetics as it can influence gene expression, including genes closely related to the growth and development of cartilage, such as sex determining region Y-box 9 (SOX9), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2), among others.^26–28 Literature reports regarding candidate genes for methylation in LCPD are limited, therefore, global DNA methylation analysis was considered to be an appropriate first step in the present study to examine the epigenetics of genes associated with the disease. Peripheral blood is an easily accessible clinical sample, so methylation levels in genomic DNA extracted from the peripheral blood of patients with LCPD were analysed. Levels of LINE-1 methylation were found to be significantly lower in male patients with LCPD compared with male controls.

Based on the present results, the authors suggest that changes in genome-wide methylation may affect gene expression during the process of bone growth and development, influencing normal growth and development of the femoral head and leading to femoral head necrosis. Growth, development and diseases of the bones and joints have been shown to be closely associated with DNA methylation.^29–31 Although the aetiology remains unclear, smoke exposure, hormonal disorders, obesity and coagulopathy appear to be closely associated with the occurrence and/or development of LCPD,^14–16,32 and the relationship between these factors and DNA methylation is of substantial research interest.^16,18,19 It may be reasonable to suggest, therefore, that these factors could alter DNA methylation levels, leading to femoral head necrosis.

Sex is an important factor affecting global genome methylation, thus, in the present study, DNA methylation levels between patients with LCPD and age- and sex-matched controls were compared in female and male subgroup analyses. LINE-1 promoter methylation levels were significantly lower in male patients with LCPD compared with male controls, while no significant differences were observed between female subgroups. This could be due to the smaller sample sizes of the female subgroups.

Children with bilateral LCPD may have an underlying genetic predisposition to LCPD. The presence of multiple epiphyseal dysplasia (caused by mutations in at least five different genes) or a collagen type II mutation was not checked in the present study,^33,34 however, removal of the four male bilateral LCPD cases in the present study, and repeat of the statistical analyses did not alter the statistical results.

The present preliminary study explored the relationship between epigenetics and LCPD, and the positive results may form the basis for further research into the role of epigenetics in the development of LCPD. Although 10 control cases were included for each year of age in order to reduce the influence of age on methylation levels, the sample size in the present study was relatively small. Further studies with larger sample sizes are required to support the present data, and to enable analyses of data stratified by age. Future studies should also consider that epigenetic modifications such as methylation of CpG islands is likely to occur in tissue-specific regions such as femoral head cartilage, joint capsules, acetabulum cartilage and blood vessels. In addition, future studies should examine specific candidate genes/regions implicated by other evidence (e.g., gene sequencing studies), expression and methylation changes in major candidate genes, and the detection of methylation at specific sites.

In summary, the present preliminary study found significant differences in the global methylation of peripheral blood DNA between patients with LCPD and matched controls, suggesting a need to further investigate methylation in key tissues and specific target genes or regions. Aberrant DNA methylation patterns may serve as epigenetic biomarkers for early detection of LCPD, and understanding the epigenetic mechanisms involved in LCPD may provide novel avenues for diagnosis and treatment of this disease.