Thalassemia is the most common monogenic inherited disease worldwide, affecting individuals originating from many countries to various extents. As the disease requires long-term care, prevention of the homozygous state presents a substantial global disease burden. The comprehensively preventive programs involve carrier detections, molecular diagnostics, genetic counseling, and prenatal diagnosis. Invasive prenatal diagnosis refers to obtaining fetal material by chorionic villus sampling (CVS) at the first trimester, and by amniocentesis or cordocentesis at the second trimester. Molecular diagnosis, which includes multiple techniques that are aimed at the detection of mutations in the α- or β-globin genes, facilitates prenatal diagnosis and definitive diagnosis of the fetus. These are valuable procedures for couples at risk, so that they can be offered options to have healthy offspring. According to local practices and legislation, genetic counseling should accompany the invasive diagnostic procedures, DNA testing, and disclosure of the results. The most critical issue in any type of prenatal molecular testing is maternal cell contamination (MCC), especially when a fetus is found to inherit a particular mutation from the mother. The best practice is to perform MCC studies on all prenatal samples. The recent successful studies of fetal DNA in maternal plasma may allow future prenatal testing that is non-invasive for the fetus and result in significant reduction of invasive diagnostic procedures.
Highlights
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It is necessary for all couples undergoing prenatal diagnosis to be counseled by a qualified counselor.
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Fetal blood analysis is now considered only in a relative late gestation when α-thalassemia hydrops fetalis has already been identified by ultrasound.
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For prenatal testing of thalassemia, the DNA diagnosis is always based on the findings whether the fetus has inherited the disease-causing alleles identified in both parents.
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Test to rule out maternal contamination or sample exchange is mandatory in molecular prenatal diagnosis.
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Newer techniques using cell-free DNA are being developed rapidly, and would result in significant reduction of invasive CVS and amniocentesis procedures
Introduction
Thalassemias are among the commonest autosomal recessive disorders worldwide and occur at high frequencies mainly in Mediterranean populations and in Africa, the Middle East, Central Asia, India, Southern China and the Far East . Because of population migration, currently, thalassemias are also common in many immigration countries worldwide . Thalassemias are caused by reduction or absent production of one or more of the globin chains that form the hemoglobin tetramers. According to the type of globin chain involved, two main types, the α- and β-thalassemias can be distinguished. The clinical types of thalassemias that are targets of prevention are β-thalassemia major resulting from homozygosity for β-thalassemia and hemoglobin Bart’s fetal hydrops syndrome caused by deletion or dysfunction of all four α-globin genes. Well-organized control programs based upon public awareness and education, carrier screening, and counseling as well as prenatal diagnosis have reduced dramatically the numbers of affected newborns .
Prenatal diagnosis is an integral component of a community control program for thalassemia. Invasive prenatal diagnosis can be performed from the first trimester by chorionic villus sampling (CVS) to the second trimester by amniocentesis or cordocentesis. DNA analysis can be performed using cells obtained from the chorionic villi, amniotic fluid, or fetal blood. It is important to note that the real value of DNA testing for the thalassemias is in prenatal testing. Postnatally, many types of thalassemias can be diagnosed with different hematological parameters combined with hemoglobin analysis and family history. This article emphasizes how the invasive procedures and the laboratory molecular techniques are facilitating the prevention of this ancient disorder.
Pre-procedural counseling
It is necessary for all couples undergoing prenatal diagnosis to be counseled by a qualified counselor. No woman should be subjected to an invasive procedure unless she has been thoroughly counseled. Irrespective of the couples’ previous experience, counseling should be offered for each at-risk pregnancy. The counseling begins with collecting the patient’s family history, ethnic background, past genetic, obstetrical, medical, and surgical history, and the indication for diagnostic fetal testing, and the personal values, cultural learning, and needs of the woman and her family should be evaluated .
Pre-procedural counseling requires an absolute understanding by patients of the level of genetic testing or diagnosis that is offered or requested. Patients welcome a clear explanation, in a way appropriate to their education, literacy, and language skills, of the thalassemia-screening results and the risk to the offspring that would lead them to consider prenatal diagnostic testing, so that they can provide informed consent. Once the criteria for offering prenatal invasive testing for an at-risk pregnancy have been met, counseling should include a description of the most appropriate procedure for the recommended or required prenatal genetic testing according to the gestation age and the risk related to the procedure. The evidence-based rates for spontaneous pregnancy loss and procedure-related pregnancy loss may be used during counseling . However, the importance of proper counseling cannot be over-emphasized. Ideally, counseling should be non-directive, enabling the couples to understand the probabilities, limitations, and potential consequences of the options. The final decision rests with the couple at risk.
It is mandatory that blood samples should be obtained from both parents to confirm the carrier state of a thalassemia mutation before invasive procedures, and as a source of control DNA for the prenatal molecular analysis . This should be repeated with every prenatal diagnosis that a couple undergoes. Another issue is that prenatal diagnosis laboratories may or may not be associated with a cytogenetics laboratory. Although the indication for the invasive testing is thalassemia, karyotype analysis should be recommended. The options can be cell culture or rapid molecular karyotyping . However, thalassemia is an autosomal monogenic disease. Couples with the α- or β-thalassemia trait carry a 25% chance of having a fetus with either homozygous α-thalassemia or β-thalassemia major, respectively. This means that in one out of four, karyotyping would be performed in an affected fetus with no benefit for the couple. This issue should also be explained to the patients .
Pre-procedural counseling
It is necessary for all couples undergoing prenatal diagnosis to be counseled by a qualified counselor. No woman should be subjected to an invasive procedure unless she has been thoroughly counseled. Irrespective of the couples’ previous experience, counseling should be offered for each at-risk pregnancy. The counseling begins with collecting the patient’s family history, ethnic background, past genetic, obstetrical, medical, and surgical history, and the indication for diagnostic fetal testing, and the personal values, cultural learning, and needs of the woman and her family should be evaluated .
Pre-procedural counseling requires an absolute understanding by patients of the level of genetic testing or diagnosis that is offered or requested. Patients welcome a clear explanation, in a way appropriate to their education, literacy, and language skills, of the thalassemia-screening results and the risk to the offspring that would lead them to consider prenatal diagnostic testing, so that they can provide informed consent. Once the criteria for offering prenatal invasive testing for an at-risk pregnancy have been met, counseling should include a description of the most appropriate procedure for the recommended or required prenatal genetic testing according to the gestation age and the risk related to the procedure. The evidence-based rates for spontaneous pregnancy loss and procedure-related pregnancy loss may be used during counseling . However, the importance of proper counseling cannot be over-emphasized. Ideally, counseling should be non-directive, enabling the couples to understand the probabilities, limitations, and potential consequences of the options. The final decision rests with the couple at risk.
It is mandatory that blood samples should be obtained from both parents to confirm the carrier state of a thalassemia mutation before invasive procedures, and as a source of control DNA for the prenatal molecular analysis . This should be repeated with every prenatal diagnosis that a couple undergoes. Another issue is that prenatal diagnosis laboratories may or may not be associated with a cytogenetics laboratory. Although the indication for the invasive testing is thalassemia, karyotype analysis should be recommended. The options can be cell culture or rapid molecular karyotyping . However, thalassemia is an autosomal monogenic disease. Couples with the α- or β-thalassemia trait carry a 25% chance of having a fetus with either homozygous α-thalassemia or β-thalassemia major, respectively. This means that in one out of four, karyotyping would be performed in an affected fetus with no benefit for the couple. This issue should also be explained to the patients .
Fetal sampling
There are three procedures for fetal sampling, namely chorionic villus sampling (CVS), amniocentesis, and fetal blood sampling. The risk of miscarriage is remote if the invasive procedure is performed by an experienced hand. Prenatal diagnosis of thalassemia should preferably be carried out by CVS in the first trimester of pregnancy (10–12 weeks).
Chorionic villus sampling. CVS utilizes either a catheter or needle to biopsy placental tissue derived from the same fertilized egg as the fetus. Typically, CVS is performed at 10–14 weeks’ gestation. Although the procedure was initially developed as a transcervical technique, both transcervical and transabdominal approaches have now become available. The upper limit for transcervical sampling has been suggested as 12 weeks, while transabdominal procedure is usually undertaken at a gestational age of 10 weeks or greater. In most cases, operator or patient choice will determine the sampling route, but the choice of the route is usually decided on a case-by-case basis depending on the placental site. Anterior and fundal placentas are usually easily accessed transabdominally, while lower, posterior located placentas are more accessible transcervically. Both transabdominal and transcervical CVS have similar accuracy, but transcervical CVS is generally considered to be associated with a higher risk of abortion .
A 2–mm CVS sample tissue is adequate for PCR-based assays and a similar amount for establishing a backup culture. However, in a CVS tissue specimen,, both the fetal and maternal types are cultured when the chorionic villi are not well separated from the maternal decidua. Thus, CVS cultures present the highest potential for maternal cell contamination . Therefore, culture should be avoided as long as the sample is of adequate size. It is of primordial importance to isolate the villi from any potentially contaminating maternal tissue before DNA extraction and analysis. The risk of maternal contamination should be minimized by careful microscopic dissection to separate the villi from deciduas.
Amniocentesis. Amniocentesis is usually performed at 15–18 weeks of gestation. The major disadvantage of this procedure is that results of the prenatal diagnosis are not available until 17 to 20 weeks gestational age. If a genetic abnormality is identified in the fetus and the patient chooses termination of pregnancy, late abortion carries a greater emotional and physical risk to the woman than a first trimester abortion .
Amniotic cells can be used for molecular analysis directly after centrifugation of the amniotic fluid. The DNA yield is generally lower than from CVS samples, but provided that 10 ml of fluid is obtained, it is usually sufficient for analysis with PCR-based methods. Direct analysis from uncultured amniotic fluid should be carried out with caution as the fetal cells can be contaminated with maternal cells. This is best avoided by careful inspection for the presence of blood in the aspirated amniotic fluid and by discarding the first 1–2 ml of the sample withdrawn, which will contain maternal skin fibroblasts. It is important to ensure that only the second or subsequent draws are sent to the laboratory. Amniotic cell culture can greatly increase the amounts of fetal DNA, but unlike culturing chorionic villi, it minimizes the risk of maternal contamination since maternal lymphocytes do not grow well. The drawback of amniotic cell culture is however the delay in reporting time
Fetal blood sampling. Currently, fetal blood analysis is seldom arranged for prenatal testing of thalassemia as early screening and accurate and less expensive PCR methods enable earlier and more reliable prenatal diagnosis in the first half of pregnancy. In some centers where PCR-based techniques are not available, quantity of cord HbA levels is still used as an approach for prenatal diagnosis of severe β-thalassemia. Cordocentesis can be used to obtain fetal blood, usually after 18 weeks. The Hb A levels in the second trimester cord blood of fetuses with β-thalassemia major are ranged from 0% to 0.5%, and these were distinguishable from heterozygous fetuses where the Hb A levels were >1.3% in different studies by HPLC or Hb electrophoresis . For the diagnosis of β-thalassemia, globin chain synthesis in fetal blood is no longer used by most centers as it is technically more demanding than the current DNA diagnosis. Fetal blood sampling is associated with a higher rate of miscarriage, and the results are available much later in pregnancy.
Fetal blood sampling may be more useful in women at-risk of α-thalassemia hydrops fetalis . In these cases, a quick diagnosis, available in a few minutes, may be obtained on the fetal blood using HPLC, as Hb F will be absent if the fetus is affected . However, clinical decision-making for pregnancies at-risk for hemoglobin Bart’s hydrops fetalis has been changing at many centers. Ultrasound now plays a central role in the early detection of homozygous α-thalassemia. In fetuses with homozygous α-thalassemia, anemia occurs very early, and thus, signs of fetal cardiomegaly and placentomegaly can be detected on ultrasound from the second trimester onward . Under skilled hands, detection of these signs by ultrasound has been shown to be reliable at as early as 11–13 weeks of gestation in predicting this condition in the fetus . Therefore, fetal blood analysis is now considered only in relatively late gestation when α-thalassemia hydrops fetalis has already been identified by ultrasound .
Inherent problems with current prenatal diagnosis, coupled with benefits of earlier reassurance or termination, would make an alternative earlier method very valuable. Although CVS can be undertaken earlier, concern over the risks of miscarriage and limb defects has restricted the use of CVS to 10 weeks and beyond. Early amniocentesis has been associated with pulmonary hypoplasia. During the first trimester of pregnancy, the coelomic cavity surrounds both the fetus and the amniotic cavity, reaching the maximum volume at 7–9 weeks, and then subsequently disappearing at around 13 weeks. This makes possible the selective aspiration of coelomic fluid from as early as five weeks of gestation . However, celiocentesis for prenatal diagnosis of thalassemia is not common, and only practiced at a single center with accurate genotyping results . Definite conclusions on both the short- and long-term safety of the technique can only be drawn after the study of a much larger number of cases.
Molecular prenatal diagnosis
For prenatal testing of thalassemia, the DNA diagnosis is always based on the findings of whether the fetus has inherited the disease-causing alleles identified in both the parents. The oldest DNA methods for thalassemia diagnosis were the restriction endonuclease technology and Southern blotting, which are obsolete and seldom used in current practice. Today PCR-based methods, which are capable of detecting most defects in the wide spectrum of mutations described on the globin genes, are the main techniques for genetic analysis and prenatal diagnosis ( Table 1 ). The molecular genetics laboratory should choose the technique(s) best suited to their own infrastructure, expertise, and target population.
| Method | Advantages | Disadvantages |
|---|---|---|
| Gap-PCR | Simple, rapid, and inexpensive | Need control DNA to validate test |
| Can be multiplexed | Limited to diagnosis of deletions with known DNA breakpoint sequences | |
| Susceptible to allele drop-out | ||
| MLPA | Can detect any targeted copy number variation | Automated sequencer required for fragment analysis |
| DNA quality may be critical | ||
| RDB | Simple, rapid, reliable, and inexpensive | Need good technical expertise to set up and validate RDB |
| Simultaneous detection for many mutations | ||
| Usually no radioactivity | ||
| ARMS-PCR | Simple, rapid, and inexpensive | Primers can degrade, provides non-specific signal |
| Stringent PCR conditions paramount for accuracy |
Gap-polymerase chain reaction
Gap-PCR is a simple technique to identify known large gene deletions. It is based upon the inability of PCR primers complementary to DNA sequences that are too far apart to direct amplification unless a deletion brings them closer together ( Figure 1 ). PCR primer pairs are designed to flank a known deletion, generating a unique amplicon that will be smaller in the mutant sequence compared with the wild type. The presence or absence of a PCR product is detected by electrophoresis. This technique is the standard method for diagnosis of the common α + -thalassemia (-α 3.7 and -α 4.2 ) and α 0 -thalassemia (– 20.5 /αα, — Med /αα, — SEA /αα, — FIL /αα, and — THAI /αα) . It is also used to detect the δ/β globin gene crossover responsible for Hb Lepore and the large deletions responsible for hereditary persistence of fetal hemoglobin (HPFH) . For a homozygous α 0 -thalassemia fetus, the Gap-PCR result presents only the deletion allele with no normal allele. The normal allele is detected by amplifying across one of the deletion breakpoints, using a third primer complementary to part of the deleted sequence near to the flanking normal DNA sequence.
Multiplex ligation-dependent probe amplification
If one parent has a rare α 0 -thalassaemia mutation, it cannot be diagnosed by gap-PCR because its breakpoint sequence was unknown. Instead, this kind of deletion mutation can be diagnosed by the multiplex ligation-dependent probe amplification assay (MLPA) . This approach is very useful as it permits the diagnosis of any α-thalassemia deletion (known and unknown) in a single test. The technique consists of two probe oligonucleotides hybridizing to immediately adjacent target sequences. Only when the two probe oligonucleotides are both hybridized to their adjacent targets can they be ligated during the ligation reaction. All ligated probes have identical sequences at their 5′ and 3′ ends, permitting simultaneous and quantitative amplification in a PCR containing only one primer pair. Each probe yields an amplification product of unique size. The amplification products are separated using capillary electrophoresis. Probe oligonucleotides that are not ligated only contain one primer sequence. As a consequence, they cannot be amplified exponentially and will not generate a signal. The removal of unbound probes is therefore unnecessary in MLPA and makes the MLPA method easy to perform. Fragments are analyzed by specific programs. Peak heights are compared with control sample and ratios are calculated. For a homozygous α 0 -thalassemia fetus, the MLPA result presents zero copy of α-globin gene.
Amplification refractory mutation system (ARMS)
Several PCR techniques for detecting β-thalassemia mutations have been developed based on the principle of primer-specific amplification. The most widely used method is known as the ARMS . It is simple and reliable for the detection of single nucleotide polymorphisms (SNPs). ARMS is performed by using a pair of PCR primers that are specific to either the mutant or the wild-type sequences. The polymorphic nucleotides should be present at the 3′ end of the primers to enhance the specificity of the reaction. Extension would only occur if the 3′ end of the allele-specific primer is bound to the complementary target sequences. Polymerases with no 3′ endonuclease activities are required to prevent correction of terminal mismatch. The genotype of SNPs is determined by analysis of PCR products with gel electrophoresis. A positive PCR amplification with the wild-type–specific primers indicates a homozygous wild-type genotype. Furthermore, a positive PCR with both wild-type and mutant primers indicates heterozygous and a positive PCR with mutant primers indicates a homozygous mutant genotype. The internal control PCR fragment must be always present, indicating that the reaction is working well. The technique has been established for prenatal diagnosis in countries such as India and Pakistan, because of its rapid and inexpensive features . For prenatal diagnosis, if the parents have different mutated alleles, then two separate reactions for each mutation are required, or if the parents have the same mutated allele, an ARMS reaction complementary to the normal sequence and an ARMS reaction complementary to the mutated allele are required for genotyping the fetal DNA.
Reverse dot-blot
Unlike the dot blot analysis, the reverse dot blot provides the immobilization of oligonucleotide probes on a membrane rather than DNA samples. The basis of this method is to detect which of a variety of known mutations exists in the target DNA by determining whether mutant or normal membrane-fixed probes capture PCR-amplified and labeled target DNA ( Figure 2 ). Primers were developed for simultaneous amplification of two β-globin gene fragments (duplex PCR) that encompass all the known β-thalassemia mutations of a specific population . The number of mutations screened in one hybridization is limited by the reaction conditions required to distinguish numerous allelic pairs. Because normal and mutant probes for each mutation are dotted side by side on the membrane strip, it is very easy to distinguish the heterozygous or homozygous state for a specific mutation. Although it is somewhat time-consuming compared to the conventional gap-PCR, RDB is virtually fool proof and robust enough to correctly detect the specific mutations. This method is especially popular in China .
