Non-invasive antenatal RHD typing

Transfusion clinique et biologique 13 (2006) 53–57 http://france.elsevier.com/direct/TRACLI/

Original article

Non-invasive antenatal RHD typing Le génotypage RHD prénatal non invasif C.E. Van der Schoot *, A. Ait Soussan, J. Koelewijn, G. Bonsel L.G.C. Paget-Christiaens, M. de Haas Department of experimental immunohematology, Sanquin Research, 125, Plesmanlaan, 1066 CX Amsterdam, the Netherlands Disponible sur internet le 29 mars 2006

Abstract The existence of cell free fetal DNA, derived from apoptotic syncytiotrophoblast, in the maternal circulation has opened new possibilities of non-invasive prenatal diagnosis. Although still some technical problems exists, especially the lack of a generic positive control on the presence of fetal DNA and the aspecific amplification of background maternal DNA, non-invasive prenatal RHD typing has been successfully introduced in several laboratories, especially in Europe. The diagnostic accuracy reaches > 99%. In the Netherlands PCR guided administration of antenatal anti-D prophylaxis is cost-effective and nearby. In this review the main characteristics and applications of cell free fetal DNA are discussed, with an emphasis on prenatal RHD genotyping. © 2006 Elsevier SAS. Tous droits réservés. Keywords: Rhesus; RhD; Genotyping; antenatal diagnosis; Fetal DNA; Maternal plasma Mots clés : Rhésus ; RhD ; Génotyping ; Diagnostic prénatal non invasif ; ADN fœtal ; Plasma maternel

1. Introduction Since the introduction of immune prophylaxis to prevent Rhesus (Rh)D allo-immunization, the risk of anti-RhD allo-immunization has been markedly reduced. Once such an alloantibody is detected in maternal blood, and the laboratory parameters such as titer or tests on the biological activity are indicative for possible hemolysis, it is important to know the phenotype of the fetus [1,2]. Before the molecular basis of the Rh-antigens was known, the phenotype had to be determined by serological testing of fetal red cells, which could be obtained by cordocentesis or by chorionic villus sampling [3]. The elucidation of the molecular basis of the blood group systems allowed the development of polymerase chain reaction (PCR)-based assays for blood group typing. These assays can be performed with fetal DNA obtained via invasive means, such as amniocentesis or chorionic villus sampling. These procedures still carry a risk for the fetus, wit h a pregnancy loss rate of up to 0.3%. In addition, the procedures themselves * Corresponding

author. Tel.: 31 20 5123377; fax: 31 20 5123474. Adresse e-mail : [email protected] (C.E. Van der Schoot).

1246-7820/$ - see front matter © 2006 Elsevier SAS. Tous droits réservés. doi:10.1016/j.tracli.2006.02.021

might further sensitize the mother against fetal red cell antigens. More recently, it has been demonstrated that other sources for fetal DNA exist. Both fetal cells and cell free fetal DNA have been found in the maternal circulation during pregnancy. Moreover, the availability of non- invasive diagnostic assays for prenatal RHD typing makes it possible to restrict antenatal prophylaxis to only those women at risk for immunization. In the Caucasian population 40% of the D-negative women will receive unnecessary administration of antenatal anti-D as they are carrying a D-negative child. Administration of this human blood product carries a small but real risk of associated bloodborne infection. Furthermore, worldwide supplies of RhD immunoglobulin are limited. 2. Fetal cells in maternal circulation Already in 1893 Schmorl described the deportation of trophoblast sprouts into the pulmonary circulation of a pregnant woman [4]. At present, it is widely accepted that fetal cells are present in maternal blood (reviewed by Bianchi) [5]. Lo et al. were the first to describe that fetal sex can be determined by Ychromosome- specific PCR in cellular DNA isolated from

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maternal blood samples [6]. Meanwhile, many groups have shown that also the fetal RhD status can be determined with a similar method [7–11]. However, the reported accuracy of these tests was low. This may be due to the scarcity of fetal cells (about 1.2 cell/ml maternal blood) [12]. Moreover, it has been shown that the presence of fetal cells can persist post partum. Bianchi et al. reported the persistence of male CD34 + progenitor cells in maternal blood for as long as 27 years after delivery [13]. It is evident that these cells from previous pregnancies can lead to falsepositive fetal blood group typing during a new pregnancy. Presently, most investigators are studying terminally differentiated cell types, such as trophoblast sprouts and nucleated erythroid cells, which are probably less persistent. The advantage of trophoblast cells is that specific monoclonal antibodies can be used for their identification [14]. However, in normal pregnancies it is extremely difficult to detect trophoblast cells [15]. Many investigators have successfully isolated nucleated red blood cells (NRBCs). Because not all NRBCs are of fetal origin [16], markers against fetal and especially embryonic hemoglobin can be used to identify fetal cells [17,18]. But also with this approach the success rate is low. Moreover, it is not clear how many of the positive results have been obtained in blood drawn from women after an invasive diagnostic procedure, a procedure, which has been shown to increase the number of circulating fetal cells [19]. Since the NRBCs express blood- group antigens, their mRNA can be used for RTPCR. Unlike DNA, RNA is present in multiple copies in the cell, increasing the sensitivity of the tests. Al-Mufti et al. and Cunningham et al. enriched fetal erythroblasts from the peripheral circulation in RhD-negative pregnant women, and performed RT-PCRs [20,21]. Their assays were 63% and 75% accurate, respectively at predicting fetal RhD status. These results were better than PCRs on genomic DNA isolated from these cells. The discordant data were due to falsenegatives in the majority of cases, probably because no fetal cells were isolated. A theoretical explanation for this low sensitivity might be AB0 incompatibility between mother and child, causing rapid clearance of fetal NRBCs. To our knowledge, it has not been investigated yet whether the high rate of false-negative results obtained with NRBCs is due to this immunological clearance. This might in particular be problematic in pregnancies in which anti-Rh or anti-Kell antibodies are present in the serum of the mother, whereas especially in these pregnancies there is need of fetal genotyping. In summary, despite the fact that many investigators have shown that it is theoretically possible to isolate fetal cellular DNA from maternal blood, none of the applied methodologies for cell enrichment have resulted in tests that meet the accuracy needed for clinical usage. Furthermore, the persistence of fetal cells from previous pregnancies renders this approach susceptible to false-positive results. 3. Cell free fetal DNA in maternal plasma All above described investigations have focused on complete and intact fetal cells in the maternal circulation. Prompted

by reports about large quantities of tumor DNA in plasma from cancer patients [22,23], Lo et al. shifted their investigation towards the a-cellular fraction of the blood. In 1997 the Lo-group showed that Y-chromosomal sequences could be amplified from DNA isolated from the plasma from pregnant women carrying male fetuses [24]. Fetal DNA can be detected in maternal plasma already at the 5th week of gestation, [25] the concentration of fetal DNA increases with gestational age. A sharp increase is detected during the last 8 weeks of pregnancy. Lo et al. reported that on average 25 fetal genome equivalents/ ml maternal plasma were detected in the first trimester, increasing to 100 genome equivalents in the third trimester [26]. Slightly higher copy numbers (82 genome equivalents/ml) in the first trimester were detected by the Swiss group [27]. We measured on average 89 genome equivalents/ml (range 15703) in 160 plasma samples of RhD-negative women carrying an RhD-positive child at 30th week of gestation [28]. Fetal DNA represents a substantial proportion of the total DNA in maternal plasma, contributing ~3.4% (range 0.4-12%) and 6.2% (range 2.3-11.5%) of total plasma DNA in the first and third trimester of pregnancy, respectively [26]. However, the relative concentration of fetal DNA in total maternal plasma DNA is difficult to determine because the blood-processing protocols influence especially the total maternal DNA concentration [27,29]. The excess of maternal DNA in plasma severely hampers the use of cell free fetal DNA for screening on chromosomal aneuploidies. Methods to decrease the amount of maternal by direct fixation of the maternal blood in paraformaldehyde has been reported [30], but were not found to be irreproducible [31]. Moreover, not all the maternal DNA is derived from blood cells, as we have shown in the plasma of patients after allogeneic stem cell transplantation. In these patients about 25% of the cell free DNA is not of donor origin (unpublished observation). Most likely this DNA is derived fom apoptotic syncitiorophoblasts. The human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. But also in mice, in which the placenta is bichorial, cell free fetal DNA is present. Recently a mouse model has been established to study the mechanism involved in fetomaternal trafficking [32]. It has also been demonstrated that microvesicles containing RNA and DNA are shed into the maternal circulation. These microvesicles contain placental specific RNA sequences, proving their origin. However only a small proportion (<1%) of fetal DNA is present in these vesicles [33]. It is very rapidly cleared from the circulation, the T1/2 being only 15 minutes [26]. Therefore, false positivity due to the persistence of fetal cells from previous pregnancies is excluded [34]. Recently, Chan et al. analyzed plasma DNA samples from 21 pregnant women, who were carrying male fetuses, for the size distribution of DNA fragments encoding the SRY gene [35]. They reported that the median relative concentration of the SRY gene determined with primers producing amplicons longer than 313 bps was <1%. They also suggest that most of the circulating fetal DNA molecules are in the range of 145-

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201 bp. This finding is in line with studies in cancer patients in which it has also been shown that the lengths of plasma DNA molecules are very short and are in multiples of nucleosomal DNA [36]. This size difference between fetal and maternal DNA (which is in mainly >300 bp) might be used in future to enrich fetal DNA for the development of new genotyping assays [37]. Furthermore, the size of fetal DNA fragments should be taken into account when developing fetal genotyping assays. For example, we recently experienced that a RHD genotyping assay that was able to discriminate between a normal RHD gene and an RHD pseudogene on genomic DNA was not working on cell free fetal DNA (Tax et al. submitted for publication). 4. PCR based assays for non-invasive prenatal genotyping Since the discovery of cell free fetal DNA numerous potential applications have been reported. Apart from blood group typing and fetal sexing, most of them have been concentrated on genetic single gene disorders, especially on paternal inherited dominant traits such as achondroplasia, Duchenne’s myodystrophia, adrenogenital syndrome etc. (reviewed in ref. [38]) In a large network of excellence on non-invasive prenatal diagnosis (SAFE, see www.safenoe.org) European investigators collaborate on the development of new technologies and applications. In a workshop organized to evaluate the performance of the different technical platforms for DNA isolations it has been shown that the different DNA isolation methods were highly reproducible between the different SAFE partners. Interestingly, one isolation method which was originally developed for viral DNA isolation was clearly superior (QIAamp DSP Mini-elute virus kit of Qiagen) and showed on average a two fold higher recovery than the other methods. (Legler et al. manuscript in preparation) There are still two major issues that affect the interpretation of PCR results: ● false negativity. The recognition of false negative cases, which are mainly due to either a lack of fetal DNA in the maternal sample due to early gestation, or methods that are not sensitive enough to detect low amounts of fetal DNA. Strategies to confirm that fetal DNA is indeed present in the maternal sample include amplification of the SRY sequence (demonstration of Y DNA confirms that fetal DNA is present if the fetus is male). If the SRY amplification is negative, the fetus is presumed to be female, and a panel of highly polymorphic markers can be used to search for differences between fetal and maternal DNA. For that purpose we have developed allele-specific RQ- PCRs for bi-allelic insertion/deletion polymorphisms. When 10 different primer -probe combinations are applied, the mean observed number of informative polymorphisms in motherchild combinations in 20 pairs tested so far is 2.3 [39]. The recent demonstration that the tumor suppressor gene maspin is hypermethylated in maternal blood and hypomethylated in placenta holds promise as a universal way to confirm that fetal (placental) DNA is present in the sample [40]. However, to date

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PCR methods based on differential methylation are not sensitive enough for routine application; ● false positivity as stated above only 3-6% of the cell free DNA in plas ma is from fetal origin. The development of robust assays for the discrimination of single nucleotide polymorphisms (SNPs) such as almost all blood group antigens is hampered by aspecific amplification from the background maternal allele. PNA clamping might solve this problem [41]. Our own preliminary results indeed indicate that the addition of a PNA probe specific for the k-allele prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal K-allele in the presence of excess of maternal k-allele. Promising results are also coming from a method based on single base extension and MassARRAY (Sequenom). Recently Ding et al. applied this technology successfully for the routine demonstration of beta-thalassemia [42]. 5. Non-invasive RHD genotyping Non- invasive RHD genotyping is relatively easy, as aspecific amplification of maternal DNA is less likely because in most cases, D-negative mothers simply miss the RHD gene. Already in 1998 Lo et al. [43]. and our group [44] demonstrated almost simultaneously that the RHD sequence could be reliably amplified from the plasma of pregnant women with high sensitivity and specificity. Because of variant RHD genes it is wise to apply multiplex PCR assays in which several regions of the RHD gene are amplified. In this way false negativity can be circumvented. False positivity due to the existence of silent alleles cannot completely be prevented, but the inclusion of an RHD-specific PCR that is negative on the RHD- pseudogene will greatly decrease its incidence. In the meantime many groups have developed robust PCR assays in which several RHD specific nucleotides are present in primers and probes. Indeed, RHD DNA detection has reached close to 100% accuracy. Several studies appeared during the last years on non-invasive prenatal RHD genotyping in relatively large patient series (see Table 1 and ref [45]). The International Blood Group Reference Laboratory (IBGRL) in Bristol, United Kingdom described their experiences with 359 cases, in which in only 12 they were unable to report a result, but no false results were encountered [46]. Similar large-scale reports have been published reflecting multi-year experience in France. In a pre-clinical study Costa et al. also demonstrated that fetal RhD genotype could be achieved efficiently, even during the first trimester Table 1 Diagnostic accuracy of prenatal RHD genotyping on cell free fetal DNA Faas et al. 1998 [43] Lo et al. 1998 [44] Finning et al. 2004 [46] Rouillac et al. 2004 [49] Van der Schoot et al. 2004 [50] Gautier et al. 2005 [48] Total

Number tested 31 57 359 851 1257 283

Number correct 31 55 347 842 1249 283 97.4%

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[47]. The test was then systematically introduced for all RhD negative pregnant women who chose to undergo amniocentesis. During the last two years, Gautier et al. studied 283 RhD negative pregnant women, and detected 170 RhD positive fetuses and 102 RhD negative fetuses, with no false positive or negative results [48]. In 11 cases no fetal or neonatal follow-up was available. Rouillac- Le Sciellour et al. [49] described their experiences with plasma obtained from 851 pregnant women. Ninety-eight per cent (193/197) of women with a non-invasive diagnosis of an RHD negative fetus had confirmed results; 4/ 197 were false negatives due to low fetal DNA concentrations, but were correctly genotyped on a repeat sample. Ninetynine per cent (649/654) of women were accurately diagnosed as carrying an RhD positive fetus. Three false positive cases were due to the presence of RHD variants or a presumed “vanishing” triplet in a case of assisted reproduction; two cases could not be confirmed due to lack of follow -up. In the Netherlands 2,359 plasma samples from D-negative pregnant women have been tested for the presence of RHD sequences at 30 weeks of gestation using an automated approach [50]. Fifteen of these serologically Dnegative women were found to carry a variant RHD gene (6 of them a RHD-pseudogene) and could not be evaluated for prenatal RHD typing in the current assay. In 1,257 cases the PCR results could be compared with the results of cord blood serology. The diagnostic accuracy was 99.4% (1249/1257). Three false negative results and 5 false positive results were obtained. For this latter study we developed a fully automated assay for fetal RHD genotyping using cell-free fetal DNA from maternal plasma. 1 ml of maternal plasma is automatically presented (Tecan) to a DNA isolation-robot (Roche). The DNA -eluate is tested (after automatically pipetting) in triplicate in a real-time quantitative RHD exon7- PCR based on Taqman chemistry. This automated approach lowers the assay costs below the price of anti-D immunoglobulin. Indeed, an economic evaluation showed that the implementation of RHD- PCR in the Dutch situation is cost-effective. It is therefore expected that in the Netherlands this screening will soon be introduced to restrict the antenatal anti-D immunoprophylaxis to women carrying RhD-positive fetuses. 6. Conclusion The accuracy of non-invasive prenatal RHD genotyping is satisfactory high (> 99%), which makes invasive RHD prenatal genotyping almost obsolete. For screening purposes the absence of a positive control is not needed, and depending on the local situation PCR-guided administration of antenatal anti-D is cost-effective. In case of prenatal RHD genotyping in an allo-antiD immunized woman the lack of a generic positive control on the presence of fetal DNA might in some cases cumbersome, but it is to be expected that this problem will be solved in the next coming years. References [1]

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