DNA Methylation at Birth Predicts Intellectual Functioning and Autism Features in Children with Fragile X Syndrome

Abstract

Fragile X syndrome (FXS) is a leading single-gene cause of intellectual disability (ID) with autism features. This study analysed diagnostic and prognostic utility of the Fragile X-Related Epigenetic Element 2 DNA methylation (FREE2m) assessed by Methylation Specific-Quantitative Melt Analysis and the EpiTYPER system, in retrospectively retrieved newborn blood spots (NBS) and newly created dried blood spots (DBS) from 65 children with FXS (~2-17 years). A further 168 NBS from infants from the general population were used to establish control reference ranges, in both sexes. FREE2m analysis showed sensitivity and specificity approaching 100%. In FXS males, NBS FREE2m strongly correlated with intellectual functioning and autism features, however associations were not as strong for FXS females. Fragile X mental retardation 1 gene (FMR1) mRNA levels in blood were correlated with FREE2m in both NBS and DBS, for both sexes. In females, DNAm was significantly increased at birth with a decrease in childhood. The findings support the use of FREE2m analysis in newborns for screening, diagnostic and prognostic testing in FXS.

Keywords: DNA methylation (DNAm); autism spectrum disorder (ASD); fragile X mental retardation 1 gene (FMR1 gene); fragile X syndrome (FXS); intellectual disability (ID); newborn screening.

Figure 1

Methods and comparisons flowchart. (A) Sixty five Australian children with fragile X syndrome (FXS) aged 1.71 to 16.93 years old: (35.4% female) were recruited into the study and provided consent to: (B) undergo neuropsychological assessments (Autism Diagnostic Observation Schedule – Second Edition (ADOS-2) and Intelligence Quotient (IQ)); (C) provide blood to create dried blood spot (DBS) for Fragile X-Related Epigenetic Element 2 methylation (FREE2m) analyses and for isolation of peripheral blood mononuclear cells (PBMCs) for fragile X mental retardation 1 gene (FMR1) expression analyses; and (D) for retrieval of newborn blood spots (NBS) collected at birth for FREE2m analyses. The differences in numbers analysed for outcomes from (A) to (D) reflect differences due to proportion of participants that: (1) did not provide blood at recruitment, but did provide consent for NBS retrieval, where NBS samples could be located and retrieved; (2) did provide blood at recruitment and consent for NBS retrieval, where NBS samples could not be located and retrieved; (3) did not complete neuropsychological assessments and/or did not obtain valid scores. FMR1 mRNA levels in PBMCs were analysed using the reverse transcription real-time PCR relative standard curve method [30]. FREE2m of NBS and DBS samples was analysed using Methylation Specific Quantitative Melt Analysis (MS-QMA) and the EpiTYPER system [19]. While both methods target the same locus consisting of 12 CpG sites, the EpiTYPER system is unable to analyse methylation of CpG’s 3, 4 and 5 as the cluster of fragments is too big in size (Daltons) to be captured by the mass-spectrum utilised by this system, as previously described [19,22]. For the remaining CpG sites the EpiTYPER system is able to provide CpG site-specific methylation. MS-QMA analysis provides a single aggregate measure of methylation across 11 out of 12 CpG sites (all but CpG 1).

Figure 2

FREE2 aggregate methylation ratio (MR) determined using: (A) MS-QMA (CpG 2-12) in retrieved NBS samples stratified by gender and allelic group (males: 89 control (CN) newborns, and 6 PM and 37 FXS participants aged 1.89 to 16.93 years old at time of retrieval; females: 95 CN newborns, and 10 PM and 21 FXS participants aged 0.54 to 18.27 years old at time of retrieval); (B) FREE2 aggregate methylation output ratio (MOR) determined using the EpiTYPER system (mean CpG’s 1, 2, 6/7, 8/9 and 10-12 MOR) in the same cohort (males: 79 CN, and 10 PM and 35 FXS participants; females: 74 CN newborns, and 10 PM and 19 FXS participants. CN = control; F = female; FREE2 = fragile X-related epigenetic element 2; FXS: fragile X syndrome (FM and PM/FM); M = male; MS-QMA = methylation specific-quantitative melt analysis; PM = premutation. Circles represent males, triangles represent females, hollow symbols represent PM/FM mosaic individuals, and a hollow square represents the normal size (NS)/PM/FM mosaic individual (here NS represents alleles <44 CGGs). *** Significant difference between FXS males and PM and CN males (p < 0.001); ### significant difference between FXS females and PM and CN females (p <0.001); +++ significant difference between PM and CN females (p = 0.016). Dotted lines represent the optimal threshold for differentiating FXS participants from other groups, determined for males by the maximum control value and for females by the receiver operating characteristic curve. Significant p values are in bold.

Figure 3

FREE2m assessed using MS-QMA and the EpiTYPER system in NBS samples from FXS males. FREE2 aggregate methylation ratio (MR) determined using MS-QMA (CpG 2-12) correlation with (A) corrected full scale IQ (cFSIQ) and (B) Autism Diagnostic Observation Schedule-Second Edition overall calibrated severity scores (ADOS-2 CSS); FREE2 aggregate methylation output ratio (MOR) determined using the EpiTYPER system correlation with (C) cFSIQ and (D) ADOS-2 CSS. FREE2 = fragile X-related epigenetic element 2; FXS: fragile X syndrome (FM only and PM/FM mosaic); MS-QMA = methylation specific-quantitative melt analysis. Note: Circles represent males, triangles represent females, hollow symbols represent PM/FM mosaic individuals, and a hollow square represents the normal size (NS)/PM/FM mosaic individual (here NS represents alleles <44 CGGs). Significant p values are in bold.

Figure 4

Correlations in FXS males between FREE2m levels of individual CpG sites in NBS samples using the EpiTYPER system, and cFSIQ and ADOS-2 CSS results determined at time of recruitment. (A) Organisation of the Xq27.3 sequence encompassing specific FREE2 CpG sites (GenBank L29074 L38501) targeted by the EpiTYPER system. Correlations between FREE2m levels and: (B) cFSIQ in 23 FXS males aged 3.32 to 16.93 years old; and (C) ADOS-2 CSS in 31 FXS males aged 1.89 to 16.93 years old. Note: Hollow circles represent PM/FM mosaic cases and a hollow square represents the normal size (NS)/PM/FM male. ADOS-2 = Autism Diagnostic Observation Schedule-Second Edition; CSS = Calibrated Severity Score; FREE2 = Fragile X-Related Epigenetic Element 2; MS-QMA = methylation specific-quantitative melt analysis. Significant p values are highlighted in bold.