Objective: Because prenatal testing of fetal RhD status by amniocentesis carries small yet finite risks to the fetus and mother, this study sought to determine whether fetal DNA in maternal serum could be used to detect fetal RhD status by polymerase chain reaction (PCR).
Methods: A retrospective analysis was made of frozen serum specimens from 20 sensitized RhD-negative pregnant women (ranging from 15.0 to 36.0 weeks' gestation) who were confirmed by serology at birth to have been carrying RhD-positive fetuses. Eleven serum specimens from RhD-negative individuals served as controls. DNA was isolated from serum and used in two PCR-based methods to detect a 99 base pair (bp) DNA fragment specific for the RhD gene and a 113 bp fragment specific for the RhCE gene as control.
Results: Overall, in 14 (70%) of 20 RhD-positive fetuses the 99 base pair RhD-specific PCR product was detected. There was no false positive detection among the 11 control serum specimens.
Conclusion: The results illustrate the ability to detect fetal RhD sequences in maternal serum of sensitized women. Moreover, the findings demonstrate that fetal single-gene disorders can be detected prenatally by using DNA isolated only from maternal serum.
3. Cherif-Zahar B, Bloy C, Le Van Kim C, et al. Molecular cloning and protein structure of a human Rh polypeptide. Proc Natl Acad Sci USA1990;87:6243-6247.
4.
4. Colin Y, Cherif-Zahar B, Le Van Kim C, Raynal V, van Huffel V, Carton JP. Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood1991;78:2747-2752.
5.
5. Le Van Kim C, Mouro I, Cherif-Zahar B, et al. Molecular cloning and primary structure of the human blood group RhD polypeptide. Proc Natl Acad Sci USA1992;89:10925-10929.
6.
6. Bennett PR, Kim CL, Colin Y, et al. Prenatal determination of fetal RhD type by DNA amplification following chorion villus biopsy or amniocentesis. N Engl J Med1993;329:607-610.
7.
7. Fisk NM, Bennett P, Warwick RM, et al. Clinical utility of fetal RhD typing in alloimmunized pregnancies by means of polymerase chain reaction on amniocytes or chorionic villi. Am J Obstet Gynecol1994;171:50-54.
8.
8. Lighten AD, Overton TG, Warwick RM, Sepulveda W, Fisk NM, Bennett PR, Accuracy of prenatal determination of RhD type status by polymerase chain reaction with amniotic cells. Am J Obstet Gynecol1995;173:1182-1185.
9.
9. Sagot P, Bonneville F, Bignon JD, Cesbron A, Boog G, Muller JY. Management of platelet and RhD maternal immunizations by PCR phenotyping after early amniocentesis. Fetal Diagn Ther1995;10:373-380.
10.
10. Rossiter JP, Blakemore KJ, Kicler TS, et al. The use of polymerase chain reaction to determine fetal RhD status. Am J Obstet Gynecol1994;171:1047-1051.
11.
11. Van den Veyver IB, Subramanian SB, Hudson KM, Werch J, Moise KJ, Hughes MR. Prenatal diagnosis of the RhD fetal blood type on amniotic fluid by polymerase chain reaction. Obstet Gynecol1996;87:419-422.
12.
12. Spence WC, Maddalena A, Demers DB, Bick DP. Molecular Analysis of the RhD genotype in fetuses at risk for RhD hemolytic disease. Obstet Gynecol1995;85:296-298.
13.
13. Queenan JT, Tomai TP, Ural SH, King JC, Deviation in the amniotic fluid optical density at a wavelength of 450 nm in Rh-immunized pregnancies from 14 to 40 weeks' gestation: A proposal for clinical management. Am J Obstet Gynecol1993;168:1370-1376.
14.
14. Lo YMD, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet1997;350:485-487.
15.
15. Lo YMD, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: IMplications for noninvasive prenatal diagnosis. Am J Hum Genet1998;62:768-775.
16.
16. Findlay I, Quirke P. Fluorescent polymerase chain reaction Part I. A new method allowing genetic diagnosis and DNA fingerprinting of single cells. Hum Reprod Update1996;2:137-152.
17.
17. Hattori M, Yoshioka K, Sakaki Y. Highly sensitive fluorescent DNA sequencing and its application for detection and mass-screening of point mutations. Electrophoresis1992;13:560-565.
18.
18. Aubin JT, Le Van Kim C, Mouro I, et al. Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification. Br J Haematol1997;98:356-364.
19.
19. Carton JP. Defining the Rh blood group antigens: biochemistry and molecular genetics. Blood Rev1994;8:199-212.
20.
20. Kemp TJ, Poulter M, Carritt B. A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D phenotype. Am J Hum Genet1996;59:1066-1073.
21.
21. Matassi G, Cherif-Zahar B, Mouro I, Cartron JP. Characterization of the recombination hot spot involved in the genomic rearrangement leading to the hybrid D-CE-D gene in the D(VI) phenotype. Am J Hum Genet1997;60:808-817.
22.
22. Avent ND, Jones JW, Liu W, et al. Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein. Br J Haematol1997;97:366-371.
