LAUSR

Cloning of blood group genes and subsequent elucidation of the molecular backgrounds to blood group polymorphisms have made it possible to predict blood group phenotypes from the DNA of a patient or donor, when a suitable red cell sample is not available [1]. One important application of this technology is determination of the blood group of a fetus without need for the difficult and risky procedure of obtaining fetal red blood cells. Fetal DNA can be obtained from amniocytes or chorionic villi, but the procedures for obtaining these materials are expensive, invasive, and present a significant risk to the fetus. The best source of fetal DNA is cell-free fetal DNA derived from placental trophoblasts and present in the plasma of pregnant women [2,3]. The main advantage of this source of fetal DNA is that it can be obtained from a maternal blood sample; the main disadvantage is that the fetal DNA cannot be separated from the maternal DNA. Between 10 and 20 weeks of gestation, 10–15% of the cell-free DNA isolated from the mother’s plasma is of fetal origin, with a range of 3–30%. After 21 weeks the fetal fraction increases by about 1% per week [4,5]. Hemolytic disease of the fetus and newborn (HDFN) occurs when IgG antibody present in a pregnant woman and directed at a fetal blood group antigen inherited from the father is transferred across the placenta, facilitating destruction of fetal red cells or their precursors and causing fetal or neonatal anemia and bilirubinemia. The most common cause of HDFN is antibody to the D (RH1) antigen of the Rh blood group system. Rh polymorphisms are predominantly controlled by the Rh genes, RHD and RHCE. About 15% of Caucasians are D-negative and, in almost all cases, this phenotype results from homozygosity for a deletion of the whole RHD gene. In African populations D-negative is less common, at around 5%. Although the most common cause of D-negative is homozygosity for an RHD deletion, inactive RHD genes, such as RHDΨ (RHD*08N.01) and RHD-CE-D hybrid genes complicate the situation in Africans. In eastern Asia, D-negative is rare [1]. Determination of fetal D type in pregnant women with anti-D demonstrates whether the fetus is at risk from HDFN. If the fetus is D-negative, then there is no risk and further intervention is not required; if D-positive, then the pregnancy can be monitored for a fetus at risk. Although fetal D typing in pregnant women with a significant level of anti-D is carried out as a routine service in some countries, where it may be considered the standard of care, it has not been universally adopted [3]. Fetal D typing involves testing for the presence of selected regions of RHD in DNA isolated from the maternal plasma. Pregnant women with anti-D usually lack RHD. Consequently, if any RHD sequence is detected it must be of fetal origin and the fetus can be assumed to be D-positive, whereas if no RHD is detected, the fetus is assumed to be D-negative. Detection of carefully selected RHD regions prevents false results in D-negative fetuses with uncommon inactive RHD genes. Use of quantitative PCR analysis reveals the presence of an inactive RHD gene in the D-negative mother, avoiding a false-positive result. A concern about this relatively simple method of testing for fetal genes in DNA samples that contain substantially more maternal DNA than fetal DNA arises from the absence of a positive control for a negative result. Failure to detect RHD in a D-positive fetus could result from too little fetal DNA being present or from a testing fault. Incorporation of reactions to a housekeeping gene, such as CCR5 or GAPDH, demonstrates that amplification has occurred, but will amplify fragments of both fetal and maternal DNA and so provides no control for the presence of adequate fetal DNA. At least three methods have been applied to provide a control for detection of fetal DNA in D-negative fetuses, though none is totally reliable and all add to the expense of the testing [3]. One is to include a test for a Y-linked gene, such as SRY, though this will only give a positive result if the fetus is male and introduces the ethical issue of an unrequested sex determination. Another method is to test for a variety of different polymorphisms in an attempt to detect a sequence present on fetal DNA, but not in the maternal DNA. A third involves epigenetics. RASSF1A is a tumour-suppressor gene in which the promoter is