MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis

doi:10.1073/pnas.0403962101

MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis

^aBioinformatics Program and Center for Advanced Biotechnology, Boston University, Boston, MA 02215; Departments of ^eChemical Pathology and ^gObstetrics and Gynaecology, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; ^hDivision of Haematology, Department of Pathology, University of Hong Kong, Hong Kong; ⁱFaculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; ^jDivision of Genetics, Department of Paediatrics, KK Women's and Children's Hospital, Singapore 229899; ^kDepartment of Medical Genetics, First Military Medical University, Guangzhou 510515, China; ^lDivision of Medical Genetics, Guangzhou Municipal Maternity and Child Healthcare Hospital, Guangzhou 510180, China; ^mDepartment of Molecular Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; ⁿDepartments of Medicine and Pathology, Boston University School of Medicine, Boston, MA 02118; and ^oSequenom, Inc., San Diego, CA 92121

Contributed by Charles R. Cantor, June 3, 2004

Abstract

The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian β-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the β-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical β-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation.

Footnotes

↵ r To whom correspondence should be addressed at: Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Room 38063, 1/F Clinical Sciences Building, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR. E-mail: loym{at}cuhk.edu.hk.
↵ b C.D. and R.W.K.C. contributed equally to this work.
↵ c C.D., R.W.K.C., C.R.C., and Y.M.D.L. have filed a patent on the technology described in this article.
↵ d C.D. is a shareholder of Sequenom, Inc.
↵ f Y.M.D.L. and R.W.K.C. have filed patents on fetal nucleic acid analysis from maternal plasma.
↵ p C.R.C. is the chief scientific officer of Sequenom, Inc.
↵ q Aspects of fetal nucleic acid analysis in maternal plasma have been licensed to BTG and Plasmagene Limited, for which Y.M.D.L. is a consultant and shareholder.
Abbreviations: SABER, single allele base extension reaction; SNP, single-nucleotide polymorphism.