Mass spectrometric detection of an SNP panel as an internal positive control for fetal DNA analysis in maternal plasma.

To the Editor: Applications of fetal DNA detection in maternal plasma have been reported for the prenatal assessment of fetal RhD status, sex-linked disorders, and -thalassemia. Because fetal DNA constitutes only 3% to 6% of the total DNA in maternal plasma (1 ), fetal sequences may occasionally go undetected because of low fetal DNA concentrations or fetal DNA loss during sample processing. Such false-negative results may lead to misinterpretation of the fetal genotype and consequently, false diagnoses. Thus the incorporation of analytical controls to confirm the presence of fetal DNA is recommended. Other investigators have developed a panel of insertion-deletion polymorphisms to serve this purpose (2). Single nucleotide polymorphisms (SNPs), however, are the most abundant class of polymorphisms in the human genome. We have recently developed a mass spectrometry based protocol, the single allele base extension reaction (SABER), that allows the sensitive and specific detection of fetal SNPs in maternal plasma (3). We applied SABER to develop an SNP panel to serve as an internal positive control for circulating fetal DNA detection. Nine SNPs [see the Data Supplement that accompanies the online version of this letter at http://www. clinchem.org/content/vol53/issue1], each with a minor allele frequency of 35%, were selected from the RealSNPTM Assay Database (www. realsnp.com, Sequenom). For each SNP locus, primer extension assays based on the standard Homogenous MassEXTEND (hME) and the SABER principles were developed (3 ). The former was used to determine the fetal and maternal genotypes and the latter was for the detection of the fetal-specific SNP alleles from maternal plasma (3 ). The assay designs are summarized in the online Data Supplement. The extension products of all assays were then resolved and detected by the MassARRAYTM Analyzer Compact Mass Spectrometer (Bruker), a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) system (4 ). Forty-one women in the first trimester of pregnancy (gestational age range: 11–13 weeks) were recruited from the Prince of Wales Hospital, Hong Kong, with informed consent and institutional ethical approval. Peripheral blood (12 mL) was collected into EDTA tubes before chorionic villus sampling (CVS) and was processed to fractionate the plasma and buffy coat (3 ). DNA was extracted (4 ) and the maternal and fetal genotypes at each SNP were determined by the hME assays using maternal buffy coat and CVS tissues, respectively. A case would be considered informative if the mother was homozygous while the fetus was heterozygous for at least 1 SNP. This protocol was used because the paternally inherited fetal allele is most readily distinguishable from other maternal DNA molecules in maternal plasma because it is not possessed by the mother. If the SNP panel was adopted for clinical use, however, the fetal genotype would not be known at the time of analysis. Hence, SNP loci that would be applicable for any given case would be those for which the mother’s genotype was homozygous. For each of the applicable SNP loci, the SABER assay targeting the SNP allele not possessed by the mother would then be used for fetal DNA detection from maternal plasma. The positive detection of a unique SNP allele in maternal plasma but not the buffy coat fraction would suggest the presence of circulating fetal DNA. In our use of this scheme, the number of SNPs applicable to any 1 of the 41 fetomaternal pairs was 1 to 7. The number of applicable SNPs for which the fetus was heterozygous, i.e., informative, was 0 to 5. Overall, 90% of the cases were informative for at least 1 SNP from the panel. Maternal plasma fractions from half of the informative cases were used to optimize the SABER assays. Fetal allele detection from maternal plasma was then conducted blindly on the remaining 18 informative cases with