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Letters to the Editor |
1 Department of Chemical Pathology
2 Li Ka Shing Institute of Health Sciences
3 Centre for Emerging Infectious Diseases
4 Department of Obstetrics, and Gynaecology, The Chinese University of Hong Kong, Hong Kong SAR, China
aAddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38063, 1/F Clinical Sciences Building, Prince of Wales Hospital, 30–32 Ngan Shing Str., Shatin, New Territories, Hong Kong Special Administrative Region, China. Fax +852-2194-6171; e-mail loym{at}cuhk.edu.hk.
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 ß-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 the optimized SABER protocol (see the online Data Supplement). The fetal-specific allele was positively detected in 36 of the 41 SABER analyses in which the fetal genotype was informative (Table 1
). When applied as a panel, the assays were able to detect the presence of the paternally inherited, fetal-specific allele in at least one of the informative SNPs in all of the cases (Table 1
). There was no false-positive detection.
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In summary, we have developed a panel of 9 SNPs to serve as positive controls for circulating fetal DNA. Additional SNPs could be added to increase the informative rate of the panel. Alternatively, placental epigenetic signatures could also serve such a purpose (5). Because the latter approach is not polymorphism based, it negates the need to determine the fetomaternal genotypes, but the epigenetic approach relies on bisulfite modification, which enhances the technical complexity of the analysis. The availability of both genetic and epigenetic fetal DNA controls would allow individual laboratories to choose the technology platform(s) that would be most easily implemented for their expertise.
Acknowledgments
This work was supported by a Hong Kong Research Grants Council Earmarked Research Grant CUHK4395/03M. We thank SEQUENOM, Inc. for loaning the MassARRAY Compact system.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. K. Tong, R. W.K. Chiu, T. Y. Leung, C. Ding, T. K. Lau, T. N. Leung, and Y.M. D. Lo Detection of Restriction Enzyme Digested Target DNA by PCR Amplification Using a Stem-Loop Primer: Application to the Detection of Hypomethylated Fetal DNA in Maternal Plasma Clin. Chem., November 1, 2007; 53(11): 1906 - 1914. [Abstract] [Full Text] [PDF]
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