Value of DNA methylation in predicting curve progression in patients with adolescent idiopathic scoliosis

Abstract

Background: There is considerable discordance in the curve progression of adolescent idiopathic scoliosis (AIS) patients between monozygotic (MZ) twins, indicating that nongenetic factors must be involved in the curve progression of AIS patients. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to curve progression in AIS patients.

Methods: The genome and methylome of peripheral monocytes were compared between MZ twins discordant for curve progression. Sets of differentially methylated sites were validated using the MassARRAY platform of Sequenome on additional samples.

Results: In the discovery study, we found evidence suggesting a lack of differences at the genome sequence level and the presence of epigenetic differences related to the curve progression of AIS patients. The top 4 differentially methylated CpG sites associated with curve severity were tested, and only site cg01374129 (CpG site located at chr8:122583383, Hg19) was confirmed in two replication cohorts. The methylation levels of site cg01374129 were significantly lower in the progression group than in the nonprogression group. Cox regression analysis demonstrated that hypo-methylation of site cg01374129 was an independent prognostic factor for curve severity. Site cg01374129 methylation as a marker achieved a sensitivity of 76.4% and a specificity of 85.6% in differentiating between samples from patients with and without curve progression (AUC = 0.827; 95% CI: 0.780 to 0.876).

Conclusion: Increased curvature is associated with decreased methylation at site cg01374129. Our results indicate that methylation of site cg01374129 may therefore serve as a promising biomarker in differing between patients with and without curve progression.

Keywords: Adolescent idiopathic scoliosis; Methylation; Predictor; Progression; Whole genome sequencing.

Fig. 1

Overview of the study design.

Fig. 2

Two pairs of monozygotic twins in the discovery study. (A and B) Posteroanterior and lateral views of individual Pair 1A show a main thoracic curve of 47° and thoracic kyphosis of 13°. (C and D) Posteroanterior and lateral views of individual Pair 1B show a main thoracic curve of 22° and thoracic kyphosis of 27°. (E) Pedigree and segregation pattern in the Pair 1 family. (F and G) Posteroanterior and lateral views of individual Pair 2A show a main thoracic curve of 50° and thoracic kyphosis of 10°. (H and I) Posteroanterior and lateral views of individual Pair 2B shows a main thoracic curve of 21° and thoracic kyphosis of 28°. (J) Pedigree and segregation pattern in the Pair 2 family. Solid symbols indicated affected individuals. Squares signify males and circles, females.

Fig. 3

Genome-wide methylation analyses in the discovery study. (A-B) The distribution of intra-pair differentiated methylation sites in various chromosomes. (C) Venn diagram showing the overlaps (middle) of intra-pair hypermethylated CpG sites. (D) Venn diagram showing the overlaps of intra-pair hypo-methylated CpG sites. (E) Plotting of the difference of beta values of the methylation sites in Pair 1A versus 1B (Pair 1A/1B) against that in Pair 2A versus 2B (Pair 2A/2B). Pearson correlation, r = 0.91 (P < .0001). Each symbol represents a methylation site. Pair 1A and 2A represent the individuals with large curves, and Pair 1B and 2B represent the individuals with small curves.

Fig. 4

Replication study in Cohort 1. (A) The methylation levels of site cg01374129 were significantly lower in the progression group (n = 50) than in the nonprogression group (n = 42). (B) Receiver operating characteristic (ROC) curves for methylation levels of site cg01374129 in Cohort 1. (C) Correlation of methylation status and Cobb angle of patients in Cohort 1. Dot plot showing correlation between the methylation biomarkers (site cg01374129) and Cobb angle. The r-value represents the square of Pearson correlation coefficient.

Fig. 5

Replication study in Cohort 2. (A) The methylation level of site cg01374129 was significantly lower in the progression group (n = 112) than in the nonprogression group (n = 164). (B) ROC curves for methylation levels of site cg01374129 in Cohort 2. (C) Kaplan-Meier curves of progression-free survival according to methylation levels of site cg01374129.

Fig. S1

Schematic diagram shows the CpG position of cg01374129 (chr8:122583383, A), cg00017851 (chr2:106351195, B), cg18441082 (chr5:16466057, C), and cg11421255 (chr15:60690974, D) in the human genome (HG19).

Fig. S2

Relationship confirmation from IBD estimates for twins in the discovery study. Estimates of the IBD coefficients, Z0 and Z1, are used to infer relatedness. Each point is for a pair of samples and the diagonal line is Z0 + Z1 = 1. Twin pairs are expected to occur at MZ area at the lower left corner of the diagonal (Z0 = Z1 = 0). Unrelated pairs are expected to occur at the lower right end of the diagonal.

Fig. S3

Methylation status of site cg01374129 stratified by sex in Cohort 1 (A) and Cohort 2 (B). Correlation of methylation status and Cobb angle in females (C) and males (D) in Cohort 1. Dot plot showing correlation between the methylation biomarkers (site cg01374129) and Cobb angle. The r-value represents the square of Pearson correlation coefficient.