Decreased Levels of DNA Methylation in the PCDHA Gene Cluster as a Risk Factor for Early-Onset High Myopia in Young Children

Abstract

Purpose: High myopia (HM), an eye disorder with at least -6.0 diopters refractive error, has a complex etiology with environmental, genetic, and likely epigenetic factors involved. To complement the DNA methylation assessment in children with HM, we analyzed genes that had significantly lower DNA methylation levels.

Methods: The DNA methylation pattern was studied based on the genome-wide methylation data of 18 Polish children with HM paired with 18 controls. Genes overlapping CG dinucleotides with decreased methylation level in HM cases were assessed by enrichment analyses. From those, genes with CG dinucleotides in promoter regions were further evaluated based on exome sequencing (ES) data of 16 patients with HM from unrelated Polish families, Sanger sequencing data of the studied children, and the RNA sequencing data of human retinal ARPE-19 cells.

Results: The CG dinucleotide with the most decreased methylation level in cases was identified in a promoter region of PCDHA10 that overlaps intronic regions of PCDHA1-9 of the PCDHA gene cluster in myopia 5q31 locus. Also, two single nucleotide variants, rs200661444, detected in our ES, and rs246073, previously found as associated with a refractive error in a genome-wide association study, were revealed within this gene cluster. Additionally, genes previously linked to ocular phenotypes, myopia-related traits, or loci, including ADAM20, ZFAND6, ETS1, ABHD13, SBSPON, SORBS2, LMOD3, ATXN1, and FARP2, were found to have decreased methylation.

Conclusions: Alterations in the methylation pattern of specific CG dinucleotides may be associated with early-onset HM, so this could be used to develop noninvasive biomarkers of HM in children and adolescents.

Figure 1.

Detailed workflow of DNA methylation analyses in children with HM and controls. The research steps marked in white have been already published. Boxes colored in gray indicate the steps performed in the current study.

Figure 2.

Comparisons of methylation levels of selected CG dinucleotides between cases with HM and controls. Presented are the highest-ranked CG dinucleotides, with at least a 20% methylation difference between HM cases and controls and location in gene promoter regions. Standard deviation is included and asterisk (*) stands for the statistically significant difference in methylation level (FDR-corrected P < 0.01 × 10⁻¹⁶)

Figure 3.

Genes of PCDHA cluster overlapping cg27494055 with decreased methylation level in HM children. The PCDHA gene cluster is tandemly localized on chromosome 5q31 and it consists of 15 genes and one pseudogene. The 13 upstream genes and the pseudogene have highly similar sequence, while a subfamily C contains two more (C1 and C2) distantly related coding sequences. The CG dinucleotide is localized in TSS1500 of PCDHA10 gene and in introns of PCDHA1–9. SNVs rs200661444 in exon 1 of PCDHA10, rs246073, and rs1581364290 colocalized with CG dinucleotide were indicated.

Figure 4.

Pedigree of the HM-78 family showing the results of segregation analyses of rs1581364290, rs200661444, and rs246073 in the PCDHA10 gene. Individuals with HM are indicated by black-filled symbols and control individuals by the open symbols, whereas symbol with M represents individual (21 years old) with myopia (OD: –4.0 D, axial length (AL) 24.11 mm, OS: –4.5 D, AL 24.82 mm). Individuals assessed in the segregation analysis are numbered under their symbols in the pedigree. Exome sequencing was applied for individuals with the underlined numbers. Members HM-78-07, HM-78-10, HM-78-11, and HM-78-08 carried a 16,794-bp deletion, starting in exon 1 of PCDHA8 and ending in intron 1 of PCDHA10 (marked by “del”).

Figure 5.

HM/myopia loci at selected chromosomes, with indicated positions of CG dinucleotides with at least a 15% decreased methylation level in children with HM versus controls. (A) CG dinucleotides at chromosome 5. Known myopia loci, 5p15.33–p15.2 (MYP16), 5p15.1–p13.3 (MYP19), and 5q31 (MYP25), are marked by a red frame and enlarged. cg27494055 in the PCDHA10 gene is indicated in red. (B) CG dinucleotides at chromosome 7. Two previously identified HM loci (7p22.1–7p21.1, 7p12.3–7p11.2) and the HM locus 7p15 (MYP17) are marked by a red frame and enlarged. (C) CG dinucleotides at chromosome 12. Previously identified HM loci 12p12.3–12p12.1 and 12q21–q23 (MYP3) and myopia locus 12q13 (MYP24) are marked by a red frame and enlarged.