Detection of Trisomy 21 by Quantitative Mass Spectrometric Analysis of Single-Nucleotide Polymorphisms

Abstract

tients with the p.R110X mutation, as was described recently (13 ). Although p.R110X is one of the few recurrent mutations of this gene, we were unable to find other clinical descriptions of female patients carrying this mutation, and the movement disorder in CLS, although common, is not well studied.No clear hot spot mutations were found, thus highlighting the functional relevance of both protein kinase domains. In patients negative for RSK2 mutations by DHPLC analysis, direct sequencing of the entire gene failed to detect any variations. Moreover, no false-positive results were reported, each abnormal chromatogram containing 1 mutation or polymorphism.The detection rate in our patients was 7 of 16 (44%), and despite the small number of patients, we believe that it represents a significant increase in the sensitivity of RSK2 mutational screening compared with the recent literature. Among patients who were negative on RSK2 mutation analysis, only 2 exhibited a typical CLS phenotype. As suggested previously (14 ), genetic heterogeneity or defects in regions not investigated by this assay can account for the failure in detecting RSK2 mutations in those patients.Including the time to set up the PCR reaction and to perform the DHPLC, analysis time was ϳ6 h to screen the entire coding region of the RSK2 gene, thus providing a high-throughput alternative to single-strand conformational polymorphism-based analysis for molecular prescreening in CLS patients.We gratefully acknowledge financial support from the Italian Ministry of Health. Down syndrome is the most common chromosomal abnormality of live-born babies. Currently, cytogenetic analysis of fetal cells such as full chromosome karyotyping (1 ) and fluorescence in situ hybridization (2 ) are the most widely used techniques for prenatal diagnosis of this chromosomal aneuploidy. Recently, however, alternative molecular strategies have been developed. Quantitative fluorescence PCR involves the detection of short tandem repeats on chromosome 21 and is by far the most thoroughly evaluated molecular technique. Additional reported molecular detection methodologies include realtime quantitative PCR (3, 4 ), multiplex probe ligation assays (5 ), and paralogous sequence quantification (6 ). Using melting curve analysis of single-nucleotide polymorphisms (SNPs), Pont-Kingdon et al. (7 ) have measured the relative allele copy number in fixed cells and amniocyte cell cultures of trisomy 21 samples. Compared with short tandem repeats, SNPs occur much more frequently in the human genome (8 ) and thus would potentially provide more information on chromosomal abnormalities. In the present study, we investigated the feasibility of measuring the allelic ratios of the chromosome-21 SNPs to identify trisomy 21 in various clinical samples. We detected and quantified the allelic ratios by PCR followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) 2358Technical Briefs

Fetal-Specific DNA Methylation Ratio Permits Noninvasive Prenatal Diagnosis of Trisomy 21

“…Additionally, our diagnostic approach is not affected by fetal gender or the presence of informative polymorphic sites in contrast to previous studies 18,[24][25][26][27] . This advantage is of high importance as the technology will be available population wide.…”

Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA

“…Genotyping was performed by the use of MassARRAY homogenous MassEXTEND assays (Sequenom) as previously described. 20 Genomic DNA obtained from the peripheral blood samples of the pregnant hemophilia carriers was used for hemophilia mutation detection. PCRs were performed for all exons covering coding regions, intron/exon boundaries, promoter, and 3Ј UTR.…”

Accurate Single-Nucleotide Polymorphism Allele Assignment in Trisomic or Duplicated Regions by Using a Single Base–Extension Assay with MALDI-TOF Mass Spectrometry

“…The iPLEX assay is a multiplexed singlebase extension assay that generates allele-specific products for detection by MALDI-TOF mass spectrometry. Although originally designed for SNP analysis, the assay has also been used for detecting trisomy (2)(3)(4), as well as high-throughput validation of CNVs (5,6 ). Therefore, the iPLEX assay has potential for integrated high-throughput joint analyses of SNPs and CNVs.…”