Revised 4/5/02

Prenatal Diagnosis of Sickle Cell Disease


Despite recent advances in the management of sickle cell disease (SCD) through improved care, 1,2 re-induction of fetal hemoglobin synthesis,3,4 and bone marrow transplantation,5the condition nonetheless frequently causes major morbidity and early death.6,7 In addition, SCD has an enormous impact on the public health systems in the countries where the betaS gene occurs in high frequency, reflecting the significant investment required for patient care.
Table 1. Prenatal Diagnostic Techniques for
             Hemoglobinopathies
Sample Source Fetal Risk Analysis Current Status
Fetal Blood 1-2% Hemoglobin Separation Obsolete
Amniotic Cells >0.5% DNA Clinically Available
Trophoblastic Cells >0.05% DNA Clinically Available
Fetal Cells in Maternal Circulation 0% DNA Investigational
Consequently, prevention of the disease through carrier identification, genetic counseling, and prenatal diagnosis remain the only realistic approach to diminish the impact of the disease and allow better use of available resources for the existing patient populations.8-10 In this context, prenatal diagnosis offers a choice to couples at risk of having children with SCD. Certainly, the solution is far from ideal, and entails numerous moral, legal, technical, and financial issues. However, until gene therapy or other approaches can cure or significantly improve the clinical impact of this condition, prenatal diagnosis is the sole option available to many couples who wrestle with the issue of having children affected by SCD.

Approach to the Population and Acceptance of the Procedure

Indications for prenatal diagnosis of SCD include a potential outcome of homozygous SCD (one betaS gene on each chromosome) or compound heterozygous states with the betaS gene on one chromosome and beta-globin chain defects such as HbC, HbD, and the severe beta-thalassemias on the other. The latter group comprises the beta0 thalassemia genes (mainly the beta0 CD39 and IVS1-nt1 mutations) which completely abolish beta-globin chain synthesis, as well as the beta+ IVS1-nt 110 genotype which severely curtails beta-globin production.11

Couples at risk for these conditions may request prenatal diagnosis if there is:

  1. retrospective identification of the betaS trait in couples who have an affected child,
  2. retrospective identification of the carrier state in parents as a result of the diagnosis of an affected newborn at birth by examination of cord or neonatal blood,12-16
  3. prospective examination after information sent by mass media or school,
  4. prospective voluntary examination in the context of population screening,17-19 and
  5. prospective mandatory blood examination in the Army, school, or other organization.
The feasibility of population screening depends mainly on the economic resources of the country, the sensitivity and education level of the population group at risk, and the availability of appropriate laboratories. Population screening is not an issue in many heavily afflicted parts of the world because of the early death of homozygous babies.21 The issue emerges, however, where improved care allows children to survive the initial difficult years of life. Then the parents are likely to seek carrier testing before birth of another child.22 When the incidence of the betaS gene is relatively high, universal premarital or antenatal population screening is justified (e.g. Cuba and Guadeloupe).23,24 Unfortunately, logistical problems often make implementation difficult in regions where it is most needed, such as Africa, Brazil and India. Inadequate speciality services for sickle cell disease can hinder selective premarital or antenatal screening when parents from groups with a high incidence of the betaS gene immigrate to countries where the variant is rare (e.g. France, Germany, and the United Kingdom)15,25-28 In addition, selective examination of a group of people who commonly carry a harmful gene can create perceptions of discrimination.20 In the U.S., most states require neonatal testing by cord blood or heel prick.12-16

When at-risk populations harbor genes for both the betaS and beta-thalassemia in high frequency, universal prospective carrier identification in adults is the approach of choice (e.g. Greece, Southern Italy, Arab nations, and India).17,18 Other factors that influence the compliance of prospective parents with carrier identification efforts include religious, political, and societal views of the subjects, as well as the availability of prenatal diagnosis. In fact, the latter factor played the major role in the implementation of carrier identification programs published to date.29-31

Counselling

When both partners carry the betaS gene, genetic counseling can offer the following alternatives: (a) abstenance from childbearing, (b) assumption of the risks, (c) pregnancy with the option of prenatal diagnosis, and (d) in recent years - selection of a non-betaS embryo ex vivo that is implanted into the mother's uterus. The importance of proper counseling cannot be overemphasized. Ideally, counselling should be non-directive, enabling the partners to understand the probabilities, limitations, and potential consequences of the options. The final decision rests with the couple at risk.

Acceptance of prenatal diagnosis for SCD differs from that reported for thalassemia. Influences include the mode and time of carrier identification; the age, education, religion, and national background of the prospective parents; the experience of having cared for or lost a child with SCD; the timing and effectiveness of genetic counseling; the attitude of the community; and the available medical resources. Reliable obstetric services are paramount. However, the key factor to increasing acceptance of prenatal diagnosis has been the early timing of the procedure. Women who were tested in the second trimester had difficulty with pregnancy termination in the 20th week or later. Earlier fetal diagnosis and selective abortion have become simple, reliable, and safe and have contributed to the wide acceptance of prenatal diagnosis of SCD.

Another decisive factor is the experience of having a child with SCD, which often convinces couples at risk to seek prenatal diagnosis. In the UK, prenatal diagnosis was offered to a group of unselected women attending the obstetric clinic of a hospital in London after detection of the trait. The option was also presented to another group of women at the clinic who had an affected child. The unselected women were not sufficiently motivated to present for testing in a timely fashion, to bring their partners for testing, and were less likely to proceed to prenatal diagnosis (22%). In contrast, almost all women in the second group had their partners tested and requested prenatal diagnosis if their partners bore the betaS gene.32 In Guadeloupe, where most betaS carriers are identified after examination of the cord blood, 64 out of 144 mothers at risk for having betaS/betaS children (44%) elected to have prenatal diagnosis. Of these, only 16 out of 27 given unfavorable news (59%) proceeded to termination of pregnancy. The respective percentages for 41 women at risk for the betaS/betaC combination were 34% and 60%.22 In Nigeria, where identification of carriers is limited to women who have had one or more affected children (retrospective screening), almost all 263 women at risk requested prenatal diagnosis. However, only 63% of those given an unfavorable diagnosis chose to terminate pregnancy, due most often to religious convictions or fear of the obstetrical procedure.32 Greece has a national program to identify carriers of inherited hemoglobin disorders at school age or before marriage. Women screened in this program are twice as likely to have a child with betaS/beta-thalassemia as the betaS/betaS combination. Each year nearly all 60-80 women at risk for SCD seek prenatal diagnosis, and undergo selective abortion in the case of unfavorable results.18,33

In Cuba, a national program offers carrier identification to all pregnant women seen in antenatal clinics, and enrolls about 300 couples at risk annually (one third for the betaS/betaS and two thirds for the betaS/betaC combination). Out of 343 evaluated couples tested during the years 1984 to 1989, 75 (22%) had split marriages (7 related to genetic risk). Of the remaining 268 stable couples, 52 (19%) proceeded to pregnancy, while the rest decided to have no more children, (a) because they already had the number of children they wanted, (b) because of the fear of giving birth to an affected child, or (c) because of the fear of the obstetrical procedure. Of the women proceeding to pregnancy, 46 sought prenatal diagnosis (12 with an affected child), while 6 others (all with low education levels) declined this offer.23

Termination of pregnancy when the fetal test results are unfavorable depends on the personal beliefs of the involved couples as well as the timing of the test. In a large U.S. study, only half of the women with a positive fetal diagnosis elected to terminate pregnancy when this was offered at the 17-20th. The number was much higher when the diagnosis was provided earlier (10th week) through improved diagnostic techniques.19 The degree of involvement of the prospective father also played a role in the decision. Furthermore, appropriate counselors whose origin and language are similar to those of the couple at risk and the views of the community with regard to racial discrimination and stigmatization powerfully influence decisions about pregnancy termination.

Obstetric Aspects

Fetal Blood

Initially, prenatal diagnosis of SCD was performed on a small amount of fetal blood aspirated from the placenta, umbilical cord, or even fetal heart, either blindly, with ultrasound guidance, or through a fetoscope at the 20th week of pregnancy.34-38 The procedure was followed by blood analysis described below, but was associated with 1-2% fetal loss plus other complications. Also, the mid-trimester diagnosis of an affected fetus often led to a painful abortion. The complexity of this approach prevented its large scale implementation in countries such as the U.S. and Britain. The largest series of cases is from Greece where 170 prenatal tests on fetal blood, mostly at risk for betaS/beta-thalassemia (26% affected), were performed successfully from 1977 to 1985.18

Fetal DNA

The introduction of molecular DNA techniques that allow accurate fetal diagnosis in the first trimester and termination of pregnancy, if indicated, not later than the 12th week of gestation changed the picture dramatically.

Amniotic Cells

Use of amniotic cells was the easiest approach to DNA analysis, even though they provide reliable results only after the 17th week of pregnancy. Aspiration of amniotic fluid with a long needle is relatively painless and safe. About 20 ml of fluid contain enough amniotic cells for extraction of up to 20ug of DNA, which is more than enough to identify the defect by PCR. The amniotic cells also can be expanded by in vitro culture if needed. The risk of fetal loss is lower than 0.5%.17,18

Trophoblastic cells

Trophoblasts derive from the trophoectoderm, which is the outer wall of the blastocyst before implantation. From the 2nd week of pregnancy, this structure develops villi which grow across the outer surface of the chorion. By the 8-10th week, the villi form the placenta at the implantation site and gradually disappear on the opposite side until the process is complete by the 14-15th week. These villi, which will be lost anyway, can be biopsied with no apparent harm to the ongoing pregnancy. Chorionic villi sampling (CVS) can be performed transcervically or transabdominally.41 Transcervical aspiration can be carried out blindly, under direct visualization, or with ultrasound monitoring. The risk of infection is greater with this approach, however. Ultrasound monitoring has made transabdominal CVS as effective and less risky. The procedure requires no anesthesia since it usually produces only mild pain. Prenatal diagnosis by CVS has a risk-rate for fetal loss or malformations of around 5 per 10,000 cases.42,43

Fetal Cells in the Maternal Circulation

This still experimental approach aims to spare mothers at risk from an obstetric procedure. Fetal cells present in the maternal circulation can be tagged with appropriate antibodies and collected by either fluorescence-activated or magnetic sorting. The nuclei are microdissected, and the DNA is extracted for analysis. The technique has produced prenatal diagnosis in two pregnancies at risk for sickle cell disease and thalassemia.39,40

Laboratory Aspects

Fetal Blood

Isoelectic focusing (IEF), high performance liquid chromatography (HPLC) or variations of these techniques can detect sickle hemoglobin in fetal blood.44-47 When a sample contains solely fetal blood, the heterozygous condition reveals a major fraction of HbF (80-90%) and equal amounts of HbA and HbS that are easily identified. In contrast, homozygous normal and homozygous HbS fetuses will display either HbA or HbS only, respectively. When a mixture of placental and maternal blood is present, the latter red cells (and reticulocytes) can be eliminated by selective hemolysis.48 The remaining cells can be used for globin biosynthetic studies. Today, this approach has been replaced by DNA techniques, except in the rare case when the thalassemic component cannot be identified at the molecular level.

Chorionic villi or amniotic cell DNA

DNA extraction. Maternal decidua contamination of trophoblastic tissue must be avoided categorically. Failure to do so can produce catastrophic errors.

Identification of the BS mutation at the DNA level. Early wrok used a Hpa I restriction fragment length polymorphism (RFLP) to distinguish betaA-globin and betaS-globin genes.49-51 The current method of choice employs the polymerase chain reaction (PCR) to amplify the mutated region (CCT GAG GAG to CCT GTG GAG, codons 5-7) for diagnosis. There are two main approaches:

Oligonucleotide hybridization
This technique involves using a synthetic, labelled oligonucleotide (about 20 kilobases long) that binds specifically to the CCT GTG GAG sequence under stringent conditions, but not to the sequence CCT GAG GAG. The reaction is carried out on a membrane using a drop of PCR product. The binding is shown by probes tagged with radioactive isotopes or enzymatic chromogens. The literature contains several variation of this approach.52-54 Cross-checks with control probes, e.g. the betaA sequence, are required.
Analysis of restriction endonulcease site
In the past, identification of the mutation by the absence of a restriction endonuclease site was carried out by the Southern blot technique on extracted DNA. The lost cleavage site produces a larger RFLP fragment.55-56 Now the technique is used on the PCR amplification product, with the enzyme Mst II, which cuts the normal CCT-GAG-GAG sequence in two DNA fragments, 228 and 202 bp long. The enzyme cannot cleave the mutant sequence, CCT-GTG-GAG, and yields a single 430 bp DNA fragment.57 Other enzymes that recognize the CCTGTGGAAG mutation are Dde I and Bsu 361 (an isoschizomer of Mst II).58,59
Misdiagnosis can occur for many reasons. The most serious is inadequate isolation of the fetal sample from contaminating maternal tissues, which can lead to misinterpretation,particularly with PCR amplified DNA.60 To avoid this problem, most laboratories use a small amount of DNA (less than 3-4 micrograms) and limit the number of PCR cycles to minimize undesirable co-amplification of traces of maternal DNA. DNA polymorphisms in the trophoblastic sample should be compared to those of the father and mother, to exclude maternal contamination. Technical failures can be reduced by using two techniques for each sample, e.g. both allele-specific hybridization and Mst II cleavage. Precautions should be taken to avoid sample mix-up in the laboratory.

Pre-conception and Pre-implantation Technology

The discovery that PCR could detect the beta gene sequence in a single cell was published in 1990.61 Since then, increasing numbers of laboratories have applied this technology for preimplantation diagnosis of inherited monogenic diseases. The procedure is still complicated, costly, and perhaps risky. Moreover, coordination of various disciplines is required.62 Preimplantation selection is feasible, however, and can be offered to couples who cannot use conventional prenatal diagnosis because selective abortion is prohibited, or who already have had several abortions after diagnoses of affected fetuses. The technique can also be offered to couples for whom multiple attempts at pregnancy are risky.

Pre-conception diagnosis is carried out on the DNA of the first polar body, which is extruded upon maturation of oocytes. The oocytes are obtained after ovarian stimulation, and the polar bodies, which contain identical DNA, are aspirated into micropipettes for analysis. The procedure has been shown to not affect fertilization of the oocytes or viability of the resultant embryos.62 A search for the defect is carried out by PCR methods, and one or more polymorphic markers usually are checked as precautions against errors. Finally, non-affected oocytes are fertilized and transferred into the uterus of the prospective mother to start pregnancy. The procedure already has been carried out on over 28 couples at risk for various single-gene disorders, that included thalassemia and sickle cell disease, and has produced 191 mutation-free oocytes. Transfer of the latter into mothers resulted in 20 successful pregnancies and birth of 19 healthy infants.64,65

Post-conception diagnosis of genetic diseases can be carried out on the second polar body, which is extruded after fertilization. Sequential study of both polar bodies is strongly recommended, because it increases the accuracy of the results when the first polar body reveals an heterozygous state.66 Then the unaffected, fertilized oocyte(s) is transferred into the uterus of the prospective mother for continuation of the pregnancy.

Alternatively, pre-implantation diagnosis can be made by biopsy of the early blastomere that has few cells. The technique is not associated with harm to the fetus.67 In a major study, nested PCR was used to detect the abnormal gene, along with two linked polymorphic markers (a short tandem repeat (STR) 5' to the beta-globin gene and HUMTH01) in order to avoid misdiagnosis due to preferential amplification or allele drop out. Three non-linked STRs were used to detect potential contamination, and identify the right embryos after selection. An overall experience was reported for 23 couples who had 37 fertilization cycles and 96 fetal transfers prior to pregnancy. The pregnancies had come to completion in 7 couples by the date of publication.68

Conclusions

Prenatal diagnosis is considered to be the only solution to prevent sickle cell disease all over the world. The procedure has been simplified by both obstetric and laboratory techniques, and can be carried out with safety in many countries, early enough to allow elective termination of pregnancy.

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