Array-Based FMR1 Sequencing and Deletion Analysis in Patients with a Fragile X Syndrome–Like Phenotype

Background: Fragile X syndrome (FXS) is caused by loss of function mutations in the FMR1 gene. Trinucleotide CGG-repeat expansions, resulting in FMR1 gene silencing, are the most common mutations observed at this locus. Even though the repeat expansion mutation is a functional null mutation, few conventional mutations have been identified at this locus, largely due to the clinical laboratory focus on the repeat tract. Methodology/Principal Findings: To more thoroughly evaluate the frequency of conventional mutations in FXS-like patients, we used an array-based method to sequence FMR1 in 51 unrelated males exhibiting several features characteristic of FXS but with normal CGG-repeat tracts of FMR1. One patient was identified with a deletion in FMR1, but none of the patients were found to have other conventional mutations. Conclusions/Significance: These data suggest that missense mutations in FMR1 are not a common cause of the FXS phenotype in patients who have normal-length CGG-repeat tracts. However, screening for small deletions of FMR1 may be of clinically utility

Novel FMR1 sequence variants identified in FXS-like males.

<p>Novel FMR1 sequence variants identified in FXS-like males.</p

Detection of known polymorphisms in FMR1 by array resequencing.

<p>P-values reflect the result of Fisher's exact tests.</p

Phenotypic characteristics of FXS-like patients.

<p>Patients enrolled as FXS-like exhibited at least two of these characteristics.</p

Targeted resequencing of <i>FMR1</i>.

<p>The horizontal axis is formed by intronic sequence, and the numbered vertical spokes represent the 17 exons of <i>FMR1</i>. Coding exonic sequence is shown in blue, while noncoding exonic sequence is shown in white. The black region upstream of exon 1 is the minimal promoter of <i>FMR1</i>. The grey bars represent the four LR-PCR amplicons used for sequencing. The green boxes represent the <i>FMR1</i> regions sequenced with the custom resequencing array.</p

FMRP expression in control and fragile X tissues.

<p>Western blot of lymphoblastoid cell lysate from a healthy control, a fragile X patient, and a patient harboring a novel deletion in the 5′UTR of <i>FMR1</i>. The protein eIF4e was assessed as a loading control.</p