Wakeling Erin N, Nahhas Fatimah A, Feldman Gerald L
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan; Molecular Genetics Laboratory, Detroit Medical Center University Laboratories, Detroit, Michigan.
Molecular Genetics Laboratory, Detroit Medical Center University Laboratories, Detroit, Michigan; Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan.
J Mol Diagn. 2014 Nov;16(6):689-96. doi: 10.1016/j.jmoldx.2014.06.006.
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.
三重引物PCR检测已成为脆性X综合征检测的首选方法。使用一种市售检测方法，我们在13161份临床样本的0.5%中检测到了可重复的额外峰。本研究的目的是确定这些额外峰的原因；确定这些峰是否代表检测特异性假象、潜在的染色体非整倍体或体细胞镶嵌性；并确定它们的临床相关性。通过实验室开发的PCR以及对部分样本进行FMR1 5'UTR测序或Southern印迹法，证实了额外等位基因的存在。实验室开发的方法在64份样本中的57份中检测到了额外等位基因。因此，我们在所有样本的约0.4%中证实了一个额外峰，通常丰度较低。在这些样本中，5份来自男性，52份来自杂合或纯合女性。6名患者可能存在X染色体非整倍体。在82.3%的样本中，额外等位基因的重复次数少于主要等位基因。通过FMR1三重引物PCR检测到的额外等位基因不是检测特异性假象，可能是由于X染色体非整倍体或体细胞重复不稳定性。额外的正常等位基因可能对脆性X综合征携带者或患病状态没有临床意义。核型正常个体中的额外等位基因可能代表FMR1体细胞变异。
