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. 2008 Dec 16;105(50):19920-5.
doi: 10.1073/pnas.0810373105. Epub 2008 Dec 5.

Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma

Fiona M F Lun  1 , Nancy B Y Tsui, K C Allen Chan, Tak Y Leung, Tze K Lau, Pimlak Charoenkwan, Katherine C K Chow, Wyatt Y W Lo, Chanane Wanapirak, Torpong Sanguansermsri, Charles R Cantor, Rossa W K Chiu, Y M Dennis Lo
Affiliations

Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma

Fiona M F Lun et al. Proc Natl Acad Sci U S A. .

Abstract

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.

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Conflict of interest statement

Conflict of interest statement: F.M.F.L., N.B.Y.T., K.C.A.C., C.R.C., R.W.K.C., and Y.M.D.L. have filed patent applications on the detection of fetal nucleic acids in maternal plasma for noninvasive prenatal diagnosis. Part of this patent portfolio has been licensed to Sequenom. C.R.C. is chief scientific officer of and holds equities in Sequenom. Y.M.D.L is a consultant to and holds equities in Sequenom.

Figures

Fig. 1.
Fig. 1.
Schematic illustration of the principles of (A) digital RMD and (B) digital NASS. (A) When a pregnant woman and her fetus are both heterozygous for a gene mutation, the amounts of the mutant allele (M) and wild-type allele (N) would be in allelic balance in maternal plasma. When the fetus is homozygous for the wild-type or mutant allele, there would be an underrepresentation or overrepresentation of the mutant allele, respectively. Digital RMD determines if the mutant and wild-type alleles in maternal plasma are in allelic balance or imbalance. (B) The scheme of the ZFY/X digital NASS assay is shown. The assay can discriminate between ZFX, denoted by X, and ZFY, denoted by Y, DNA molecules. In addition, the assay can distinguish if the ZFX and ZFY DNA molecules are long or short. Digital PCR is performed using two forward primers and one reverse primer (arrows), or vice versa, that are oriented to produce a short amplicon overlapping with the long amplicon. When a single DNA molecule at least as long as that specified by the long amplicon is captured in the reaction well, both the long and short PCR products would be generated. When a single DNA molecule shorter than the span of the long amplicon is captured, only the short amplicon would be generated. The presence of the long and/or short amplicons can be detected by strategically located hybridization probes or extension primers. An extension primer, L (boxed), is designed to detect the presence of the long amplicon. An extension primer is located within the short amplicon and the extension products are used to discriminate the ZFX and ZFY alleles (shown as boxed X and boxed Y, respectively). The identities of the DNA molecules could be interpreted by counting the products present within each well. +, present; −, absent.
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