Large-Scale Whole Genome Sequence Analysis of >22,000 Subjects Provides no Evidence of FMR1 Premutation Allele Involvement in Autism Spectrum Disorder

Abstract

Expansion of a CGG repeat in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene on the X chromosome is the cause of Fragile X Syndrome (FXS). The repeat length of unaffected individuals varies between 5-40 repeats, whereas >200 repeats are observed in cases of FXS. The intermediate range between 55-200 repeats is considered the premutation range and is observed in roughly 1:300 females and 1:900 males in the general population. With the availability of large-scale whole genome sequence (WGS) data and the development of computational tools to detect repeat expansions, we systematically examined the role of FMR1 premutation alleles in autism spectrum disorder (ASD) susceptibility, assess the prevalence, and consider the allelic stability between parents and offspring. We analyzed the WGS data of 22,053 subjects, including 32 FXS positive controls, 1359 population controls, and 5467 ASD families. We observed no FMR1 full mutation range repeats among the ASD parent-offspring families but identified 180 family members with premutation range alleles, which represents a higher prevalence compared to the independent WGS control sample and previous reports in the literature. A sex-specific analysis between probands and unaffected siblings did not reveal a significant increase in the burden of premutation alleles in either males or females with ASD. PCR validation, however, suggests an overestimation of the frequency of FMR1 premutation range alleles through computational analysis of WGS data. Overall, we show the utility of large-scale repeat expansion screening in WGS data and conclude that there is no apparent evidence of FMR1 premutation alleles contributing to ASD susceptibility.

Keywords: ExpansionHunter; FMR1; Fragile X Syndrome; autism spectrum disorder; repeat expansion.

Figure 1

Workflow of calculating the FMR1 repeat length of 22,350 samples using ExpansionHunter: 22,350 sequence alignment files (CRAM format) were downloaded from either the (1) European Genome Archive (EGA), (2) Australian Medical Reference Genome Bank (MRGB), (3) Simons Foundation Autism Research Initiative (SFARI), or (4) the Simons Foundation Powering Autism Research (SPARK) initiative. ExpansionHunter was able to calculate repeat length genotypes for 21,977 samples (98.4%). Four SFARI families with at least one member predicted to carry an expanded repeat were used for molecular validation by PCR. PCR showed overestimation in the repeat lengths calculated by ExpansionHunter, which was shown to be caused by the use of off-target reads in repeat length calculation. To reduce overestimating expanded repeat lengths, samples originally identified to have repeats exceeding the normal range were calculated using only the FMR1 reference region.

Figure 2

Validating repeat lengths calculated by ExpansionHunter. 118 samples identified to have expanded repeats at loci associated with eight different diseases were processed through ExpansionHunter. ExpansionHunter correctly identified all samples with expanded FMR1 repeat lengths, however, discrepancies were observed in the exact length of the repeats as the samples known to be carriers of full mutation range repeats were classified as being in the premutation range by ExpansionHunter.

Figure 3

Estimated repeat length of the FMR1 trinucleotide repeat in SFARI. For each subject the longer allele for the FMR1 genotype is displayed in (A) SFARI, (B) SPARK, and (C) Australia MRGB. Alleles have repeat lengths within the normal (blue), premutation (yellow), or full mutation (red) range. Repeat length estimates were observed to fall within distributions within the normal range (30 bp–120 bp) or around the start of the premutation range.