title: Extra alleles in FMR1 triple-primed PCR: artifact, aneuploidy, or somatic mosaicism?
abstract: Triple-primed PCR assays have become the preferred fragile X syndrome testing method. Using a commercially available assay, we detected a reproducible extra peak(s) in 0.5% of 13,161 clinical samples. The objectives of this study were to determine the cause of these extra peaks; to identify whether these peaks represent an assay specific artifact, an underlying chromosome aneuploidy, or somatic mosaicism; and to ascertain their clinical relevance. The presence of an extra allele(s) was confirmed by a laboratory-developed PCR, with sequencing of the FMR1 5' UTR or Southern blot for some samples. The laboratory-developed procedure detected the extra allele(s) in 57 of 64 samples. Thus, we confirmed an extra peak, typically of lower abundance, in approximately 0.4% of all samples. Of these samples, 5 were from males and 52 were from heterozygous or homozygous females. Six patients likely had X chromosome aneuploidies. In 82.3% of samples, the extra allele had fewer repeats than the predominant allele(s). Additional alleles detected by FMR1 triple-primed PCR are not an assay-specific artifact and are likely due to X chromosome aneuploidies or somatic repeat instability. Additional normal alleles likely have no clinical significance for fragile X syndrome carrier or affected status. Extra alleles in individuals with normal karyotypes probably represent FMR1 somatic variation.
authors.affiliation: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan; Molecular Genetics Laboratory, Detroit Medical Center University Laboratories, Detroit, Michigan. Electronic address: ewakelin@dmc.org.
authors.forename: Erin N
authors.initials: EN
authors.lastname: Wakeling
authors.affiliation: Molecular Genetics Laboratory, Detroit Medical Center University Laboratories, Detroit, Michigan; Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan.
authors.forename: Fatimah A
authors.initials: FA
authors.lastname: Nahhas
authors.affiliation: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan; Molecular Genetics Laboratory, Detroit Medical Center University Laboratories, Detroit, Michigan; Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan; Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan.
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Feldman
mesh_terms: D000328:Adult; D000483:Alleles; D000782:Aneuploidy; D016477:Artifacts; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D006801:Humans; D008297:Male; D009030:Mosaicism; D016133:Polymerase Chain Reaction / Q000379:methods*; D055815:Young Adult
chemical_list: D017931:DNA Primers; D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human