Prenatal Diagnosis of Sickle
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.
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.
|Table 1. Prenatal Diagnostic Techniques
|Fetal Cells in Maternal Circulation
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
Couples at risk for these conditions may request prenatal diagnosis
if there is:
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
retrospective identification of the betaS trait in couples who
have an affected child,
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
prospective examination after information sent by mass media or school,
prospective voluntary examination in the context of population screening,17-19
prospective mandatory blood examination in the Army, school, or other organization.
When at-risk populations harbor genes for both the betaS
in high frequency, universal prospective carrier identification in adults
is the approach of choice (e.g. Greece, Southern Italy, Arab nations, and
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
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.
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
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
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
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
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
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:
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
II cleavage. Precautions should be taken to avoid sample mix-up in
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
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
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
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
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
Reid CD, Charache S, Lubin B, et al. Management
and Therapy of Sickle Cell Disease. 3rd edition. NIH Publication
96-2117. Rockville MD. National Institutes of Health; National Heart, Lung
and Blood Institute. PHS; US Dept of Health and Human Services.
Lee A, Thomas P, Cupidore L, et al. Improved
survival of homozygous sickle cell disease: lessons from a cohort study.
Brit Med J 1995; 311:1600-1602.
Charache S, Terrin MN, Moore RD, et al. Effect
of hydroxyurea on the frequency of painfulcrises in sickle cell anemia;
Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia.
N Engl J Med 1995; 18:1317-1322.
Atweh GF, Sutton M, Nassif I, et al. Sustained
induction of fetal hemoglobin by pulse butyrate therapy in sickle cell
disease. Blood 1999;93:1790-1797.
Sullivan KM, Walters MC, Patience M, et al.
Collaborative study of marrow transplantation for sickle cell disease:
aspects specific for transplantation of hemoglobin disorders. Bone Marrow
Transplantation 1996; 19:102-105.
Bunn HF. Pathogenesis and treatment of sickle
cell disease. N Engl J Med. 1997;337:762-769.
Gilman PA, McFarlane JM, Huisman THJ. Natural
History of sickle cell anemia: re-evaluation of a 15 year cord blood testing.
Pediat Res 1976; 10:452-456.
Angastiniotis M, Modell B. Global Epidemiology
of Hemoglobin Disorders. Ann NY Acad Sci 1998; 850:251-269.
WHO Working Group. Community Control of hereditary
anaemias. Bull WHO 1983; 61:6369.
Modell B (1998) ApoGI Project (Accessible
Publishing of Scientific Information; Information Materials for Haemoglobin
Disorders. Department of Primary Care and Population Sciences; University
Voskaridou E, Kollia P, Loukopoulos D. Sickle
Cell Thalassemia in Greece. Identification and contribution of the interacting
b-thalassemia genes. Ann NY Acad Sci 1990; 612:508-509.
Garrick MD, Denbure P, Guthrie R. Sickle cell
anemia and other hemoglobinopathies. Procedures and strategy for screening
employing spots of blood on filter paper as specimens. N Engl J Med 1973;
Grover R, Shahidi S, Fisher B et al. Current
sickle cell screening program for newborns in New York City. Am J Pub Health
Harris MS, Eckman JR. Appoaches to screening.
Georgia's experience with newborn screening; 1981 to 1985. Pediatrics 1989;
83(suppl 5): 857-858.
Zeuner D, Ades AE, Karnon J et al. Antenatal
and neonatal haemoglobinopathy screening in the UK: review and economic
analysis. Health Technot Assess 1999; 3:1:186.
Nagel R. Neonatal screening. Management and
Therapy of Sickle Cell Disease. 3rd edition. NIH Publication
96-2117, Rockville MD. National Institutes of Health; National Heart, Lung
and Blood Institute. PHS; US Dept of Health and Human Services.
Cao A, Rosatelli MC, Leoni GB et al. Antenatal
diagnosis of b-thalassemia in Sardinia. Ann NY Acad Sci 1990; 612:215-225.
Loukopoulos D, Hadji A, Papadakis M, et al.
Prenatal diagnosis of thalassemia and of the sickle cell syndromes in Greece.
Ann NY Acad Sci 1990; 612:226-236.
Wang X, Seaman C, Paik M, et al. Experience
with 500 prenatal diagnosis for sickle cell disease: diseases: the effect
of gestational age on affected Pregnancy outcome. Prenat Diagn 1994, 14:851-857.
Beutler E, Boggs DR, Heller P, et al. Hazards
of indiscriminate screening for sickling. N Engl J Med 1971; 285: 1485-1486.
Fleming AF. 1989. The presentation, management
and prevention of crisis in sickle cell disease in Africa. Blood Rev 3:
Akinyanju OO, Disu RF, Akinde JA, et al. Initiation
of prenatal diagnosis of sickle cell disease in Africa. Prenat Diagn 1999;
Granda H, Gispert S, Dorticos A, et al. Cuban
Programme for prevention of sickle cell disease. Lancet 1991; 1:152-153.
Alexandre L, Keklard L, Romana M, et al. Efficiancy
of prenatal counseling for sickle cell disease in Guadeloupe. Genet Couns
Cronin EK, Normand C, Henthorn JS, et al.
Organisation and cost-effectiveness of antenatal haemopglobinopathy screening
and follow up in a community-based programme. Br J Obst Gynecol 2000; 107:486-491.
De Montalembert M, Guilloud-Bataille M, Ducros
A, et al. Implication of prenatal diagnosis of sickle cell disease. Genet
Couns 1996; 7:9-15.
Dickerhoff R, Kulozik AE, Kohne E. Monitoring
pregnant women from risk countries with sickle cell desease and thalassemia.
Clinical aspects, screening and prenatal diagnosis. Geburtshilfe Frauenheilkd
Lena-Russo D, Erny N, Serradimigni F, et al.
Genetic hemoglobin diseases. Prevention at centers for family planning
and education of maternal-child protection in Marseille. Presse Med 1996;
25:151-153 (in French).
Modell BM, Ward RHT, Fairweather DVI. Effect
of introducing antenatal diagnosis on reproductive behaviour of families
at risk for thalassemia major. Br med J 1980; 1:1347-1348.
Dorticos-Balea A, Martin-Luiz M, Hechevarria-Fernandez
P, et al. Reproductive behavior of couples at risk for sickle cell disease
in Cuba. A follow-up study. Prenat Diagn 1997; 17:737-742.
Greengross P, Hickman M, Gill M, et al. Outcomes
of universal antenatal Screening for hemoglobinopathies. J Med Screen 1999;
Durosinmi MA, Odebiyi AI, Adediran IA, et
al. Acceptability of prenatal diagnosis of sickle cell anemia (SCA) by
female patients and parents of SCA patients in Nigeria. Soc Sci Med 1995;
Loutradi A. Report of the activity of the
Center of Thalassemia. Unpublished statistics. Athens, 2000.
Hobbins JC, Mahoney MJ. In utero diagnosis
of hemoblobinopathies. Technique for obtaining fetal blood. N Engl J Med
Kan YW, Valenti C, Carnazza V, et al. Fetal
in utero. Lancet 1974; 1:79-80.
Rodeck CH, Campbell S. Sampling pure fetal
blood by fetoscopy in the second trimester of pregnancy. Br Med J 1978;
Fairweather DVI, Ward RHT, Modell B. Obstetric
aspects of midtrimester fetal blood sampling by needling or fetoscopy.
Br J Obstetr Gynaecol 1980; 3:271-7.
Daffos F, Capella-Pavlovsky M, Forrestier
F. Fetal blood sampling via the umbilical cord using a needle guided by
ultrasound. Prenat Diagn 1983; 3:271-7.
Bianchi DW, Flint AF, Pizzimenti MF, et al.
Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc
Nat Acad Sci USA 1990; 87:3279-3283.
Cheung MC, Goldberg JD, Kan YW. Prenatal diagnosis
of sickle cell anemia and thalassemia by analysis of fetal cells in maternal
Antsaklis A. First trimester prenatal diagnosis
by chorionic villi sampling. In: Prenatal diagnosis of thalassemia and
the hemoglobinopathies. D. Loukopoulos ed., CRC Press 1988. Boca Raton
Fl, pp. 132-143.
Hogge WA, Schonberg SA, Golbus MS. Chorionic
villis sampling: experience of the first 1000 cases. Am J Obstetr Gynecol
Brambati B. Lanzani A, Oldrini A. Transdominal
chorionic villus sampling; clinical experience of 1159 cases. Pren Diagn
Blouquit Y, Beuzard Y, Varnavides L, et al.
Antenatal diagnosis of haemoglobinopathies by Biorex chromatography of
haemoglobins. Bri J Haematol 1982; 50:7-15.
Dubart A, Goossens M, Beuzard Y, at al. Prenatal
diagnosis of hemoglobinopathies: comparison of the results obtained by
isoelectric focusing of homoglobins and by chromatography of radioactive
globin chains. Blood 1980; 56:1092-9.
Dash S, Panourgias J, Karababa P, Loukopoulos
D. Prenatal diagnosis of thalassaemia by haemoglobin chromatography on
Biorex. Prenat Diagn 1984; 4:11-20.
Gianni Am, Polli E, Giglioni B, et al. Isoelectric
focusing of globin chains for antenatal diagnosis of b0thalassemia.
Hemoglobin 1981; 5:349-56.
Alter BP, Metzger JB, Yock PG, et al. Selective
hemolysis of adult red blood cells; an aid to prenatal diagnosis of hemoglobinopathies.
Blood 1979; 53:279-87.
Kan YW, Dozy AM. Polymorphism of DNA sequences
adjacent to the human B-globin structural gene: relationship to sickle
mutation. Proc Natl Acad Sci 1978; 75:5631-5.
Kan YW, Dozy Am. Antenatal diagnosis of sickle
cell anemia by DNA analysis of amniotic fluid cells. Lancet 1978; 2:910-2.
Kazazian HHJr, Phillips JA, Boehm CD, et al.
Prenatal diagnosis of B-thalassemia by amniocentisis; linkageanalysis of
multiple polymorphic restriction endonuclease sites. Blood 1980; 56:926-30.
Saikii RK, Walsh PS, Levenseon CH, et al.
Genetic analysis of amplifies DNA with immobilized sequence specific oligonucleotide
probes Proc Natl Acad Sci USA 1989; 17:2503-2516.
Newton CR, Graham A, Hepteinstall LE et al.
Analysis of any point mutation in DNA with immobilized sequence specific
olignucleotide probes Proc Natl Acad Sci USA 1989; 17:2503-2516.
Geever RF, Wilson LB, Nallaseth HHJr, Boehm
CD. Improved detection of the sickle cell mutation by DNA analysis: application
to prenatal diagnosis. N Engl J Med 1982; 307:32-6.
Chang JC, Kan YW. A sensitive new prenatal
test for sickle cell anemia. N Engl J Med 1982; 307:30-32.
Orkin SH, Little PFR, Kazazian HHjr, Boehm
CD. Improved detection of the sickle cell mutation by DNA analysis: application
to prenatal diagnosis. N Engl J Med 1982; 307:32-6.
Embury SH, Sharf SJ, Saiki RK, et al. Rapid
prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.
N Engl J Med 1987; 316:656-61.
Gurgey A, Becsac S, Mesci L, et al. Prenatal
diagnosis of sickle cell anemia using PCR and restriction enzyme Dde I.
Turkish J Pediatr 1993; 35:159-162.
Husian SM, Kalavathi P, Anandaraj MP. Analysis
of sickle cell gene using the polymerase chain reaction and restriction
enzyme Bsu 361. Indian J Med Res 1995; 101:273-276.
Cao A, Rosatelli C. Screening and prenatal
diagnosis of the haemoglobinopathies. Balliere's Clnics in Haematoology
Monk M, Holding C. Amplification of a B-haemoglobin
sequence in individual human oocytes and polar bodies. Lancet 1990; 985-088.
Kuliev A, Jackson L, Froster U, et al. Chorionic
villus sampling safety. Report of World Health Organization/EURO meeting
in association with the 7th international conference on early
prenatal diagnosis of genetic disease. Am J Obstet Gynecol 1996; 174:807-811.
Verlinsky Y, Retchitsky S, Verlinsky O, et
al. Pre-pregnancy testing for single cell disorders by polar body analysis.
Genet Test 1999; 3:185-190.
Xu K, Shi ZM, Veeck LL, et al. First unaffected
pregnancy using pre-implantation genetic diagnosis for sickle cell anemia.
JAMA 1999; 281:1701-1706.
Kuliev A, Retchitsky S, Verlindky O, et al.
Birth of healthy children after pre-implantation diagnosis of thalassemias.
J Assist Reprod Genet 1999; 16:207-211.
Verlinsky Y, Retchitsky S, Cieslak J, et al.
Pre-implantation diagnosis of single gene disorders by two-step oocyte
genetic analysis using first and second polar body. Biochem Mol Med 1997;
De Vos A, Van Steirteghem A. 2001. Aspects
of biopsy procedures prior to preimplantation genetic diagnosis. Prenat
Palmer GA, Traeger-Synodinos J, Davies S,
Tzetis M, Vrettou C, Mastrominas M, Kanavakis E. 2002. Pregnancies following
blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.
Hum Reprod 17:25-31.