Prenatal diagnostic testing involves testing the fetus before birth to determine whether the fetus has certain abnormalities, including certain hereditary or spontaneous genetic disorders. Kan and Dozy carried out the first prenatal DNA diagnosis in 1978 for sickle cell anemia, and since then a lot has changed in the scenario of prenatal diagnosis in maternal anemia. Beta thalassemia is one of the conditions in which prenatal diagnostic testing is gaining popularity in India.
There are various forms of Thalassemia in the mother.
α Thalassemia is characterised by deletion or inactivation of α globin alleles. As a result the α globin chains’ production is affected. Deletion of all 4 alleles results in Hb Bart syndrome, deletion of 3 alleles results in Hb H disease. When 2 alleles are inactive results in α0 Thalassemia and when only one allele is inactive, the condition is called α1 Thalassemia. There are two clinically significant forms: Hb Bart syndrome and Hb H disease. Hb Bart syndrome is more severe and is usually lethal while Hb H disease is mild. α0 Thalassemia is clinically seen as α Thalassemia trait and α1 Thalassemia is clinically seen as α Thalassemia silent carrier.
Genetic counseling. Alpha-thalassemia is usually inherited in an autosomal recessive manner.
Hb Bart Syndrome:- At conception, each sib of an individual with Hb Bart syndrome has a 25% chance of having Hb Bart syndrome, a 50% chance of having αº-thalassemia (α-thalassemia trait), and a 25% chance of being unaffected and not a carrier. Once an at-risk sib is known to be unaffected, the risk of his/her having αº-thalassemia (α-thalassemia trait) is 2/3.
HB H Disease:-At conception, each sib of an individual with HbH disease has a 25% chance of having HbH disease, a 25% chance of having αº-thalassemia (α-thalassemia trait), a 25% chance of having α+-thalassemia (α-thalassemia silent carrier), and a 25% chance of being unaffected and not a carrier. Once an at-risk sib is known to be unaffected, the risk of his/her having either αº-thalassemia (α-thalassemia trait) or α+-thalassemia (α-thalassemia silent carrier) is 2/3. Each child of an individual with HbH disease inherits the mutation for either αº-thalassemia or α+-thalassemia and is thus an obligate heterozygote.
Family members, members of ethnic groups at risk, and gamete donors should be considered for carrier testing. Couples who are members of at-risk populations for αº-thalassemia carrier status can be identified prior to pregnancy to avoid conceiving a fetus with Hb Bart syndrome. Prenatal testing may be carried out for couples who are at high risk of having a fetus with Hb Bart syndrome or for a pregnancy in which one parent is a known αº-thalassemia carrier and it is unknown whether the other parent has the mutation.
β Thalassemia is characterised by inactivation or deletion of β globin alleles. This results in variable dysfunction of βglobin chain production.
Clinically, β Thal major is characterised by severe anemia with dependence on blood transfusion; β Thal Intermiadia is characterised by milder forms of anemia and β Thal minor are carriers of the trait.
Genetic counseling. The β-thalassemias are inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Heterozygotes may be slightly anemic but are clinically asymptomatic. Carriers are often referred to as having thalassemia minor (or β-thalassemia minor). Carrier testing for individuals at risk (including family members, gamete donors, and members of at-risk populations) is possible. Prenatal testing for pregnancies at increased risk is possible if the disease-causing mutations in the family are known.
Common Indian mutations β Thalassemia are:-IVS-1-5 (G→C), IVS-1-1 (G→T), codons 8/9 (+G), codons 41/42(-CTTT) and 619 base pair deletion.
Population screening conducted in past years in Indian population2 has helped to recognize certain communities like Sindhis, Gujaratis, Bengalis, Punjabis and Muslims with high prevalence of β-Thalassemia.
The population to screen includes those groups who have high incidence of β Thalassemia, parents of a previous affected sib , a pregnancy with known β Thal carrier parent or family history of the disease.
The first step is to screen the mother.The most feasible option in our view is to test the mothers antenatally in early pregnancy preferably in the first trimester. The parents are often receptive and would usually agree to get any tests done for the well being of their baby. If the mother is found to be a carrier, her husband can be tested for carrier status and if he is also a carrier, prenatal diagnosis can be offered after proper genetic counselling.
1. Red Cell Indices
Red cell indices obtained through standard electronic cell counters provide valuable tool for preliminary screening of thalassemic traits. Thalassemic traits in general have reduced mean corpuscular volume (MCV) and reduced mean corpuscular hemoglobin (MCH) with normal mean corpuscular hemoglobin concentration (MCHC). Specific cut off points for each index varies from laboratory to laboratory. Some laboratories concentrate on both reduced MCV and MCH and some on MCV or MCH alone. When either MCV or MCH was considered for carrier status, sensitivity and specificity is less. Hence, selection of both MCV (<77 fl) and MCH (<27 pg) is ideally suited for further rapid confirmation of carrier status. Low MCV or MCH sometimes poses a problem by giving false positive results due to iron deficiency anemia or other nonthalassemic microcytosis. Various formulae like Bessman index, Shine and Lal index, England index, Mentzler index are used as good indicators to differentiate between thalassemic and nonthalassemic microcytosis.
2. Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT)
All laboratories do not have the facility of electronic cell counters to measure red cell indices. In such cases NESTROFT, a rapid, simple and cost effective screening test could be used. The principle of NESTROFT is based on the limit of hypotonicity which the red cell can withstand. In this procedure 2 ml of 0.36% buffered saline is taken in a test tube, 20ml of whole blood is added to it, and is allowed to stand at room temperature. After 20 minutes reading is taken on a NESTROFT stand on which a thin black line is marked. If the line is visible through the solution, the test is considered as negative and if line is not visible it is considered as positive. Positive test is due to the reduced osmotic fragility of red cells.
It has a sensitivity ranging from 94 to 99 per cent Th sensitivity specificity, positive predictive and negative predictive values of NESTROFT were reported to be 91%, 95%, 55% and 99% respectively. A lower positive predictivity suggested false positive results probably due to associated iron deficiency which requires confirmation by estimation of HbA2 levels. Based on high negative predictive values, the use of NESTROFT has been recommended for mass screening due to its low cost and simplicity. Though NESTROFT is a simple and rapid test, combination of NESTROFT and red cell indices increases the sensitivity and negative predictive value to almost 100 per cent.
3. Hemoglobin A2 Estimation
Raised hemoglobin A2 level is the gold standard for diagnosis of thalassemic trait. Subjects found to be positive in preliminary screening test by red cell indices and NESTROFT are confirmed for thalassemic carrier status by HbA2 measurement. Subjects with HbA2 levels of 3.5 per cent and above are considered to have thalassemic trait. However, precautions have to be taken when HbA2 levels fall between 3.3 and 3.8 per cent. In such cases it is recommended to repeat the assay to rule out a technical error while performing the assay.
Once the mother is diagnosed to be having thalassemic trait, father is investigated on similar lines. If both husband and wife are diagnosed to be carrier, the next step is to prevent the birth of thalassemic child by offering prenatal diagnosis and selective abortion of the fetuses affected with thalassemia.
DNA Mutation Analysis in parents
To offer prenatal diagnosis to the couples it is essential to characterize the DNA mutations of the parents. There are various methods available to study DNA mutations such as allele specific oligonucleotide (ASO) screening, reverse dot blot and restriction endonuclease allele recognition. ASO method detects point mutations, nucleotide insertion or deletion in genomic DNA. In this method ASO probes of 18-20 mer sequence are used. DNA is denatured and dot blotted on to a nylon membrane and then hybridized to different probes. In reverse dot blot probes are attached to the membrane and DNA hybridizes with dot corresponding to the mutation. A recent method is amplifica- tion refractory mutation system (ARMS) technique in which specific primers against normal and mutant sequences are used.
More than 150 mutations causing b- thalassemia have been reported from different parts of the world. Studies conducted in India have identified about 28 mutations in Indian population. Of these five to six mutations are found to be common. These include IVS 1-5, 619 bp del, IVS 1-1, frameshift 8-9, 41-42, and codon 15. The type of mutation varies in different ethnic groups with a particular type of mutation being more common in a specific ethnic group. The frequency of mutations in carriers originated from different states in India varies from region to region with the predominant mutation in most of the states being IVS 1-5. In migrants from West Pakistan the predominant mutation is 619 bp del. Overall common mutations account for 90-93.6 per cent.
Still 5-10 per cent needs further investigations which could be characterized using techniques such as single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). SSCP is based on the mobility shift in a neutral polyacrylamide gel due to conformational change caused by substitution of a base in a single stranded DNA fragment. DGGE is based on the resolution of DNA fragments differing by single nucleotide substitution. Both the methods could be used for detection of rare mutations. This can be followed by sequencing using automated sequencers which are available now. The families where mutations were not characterized could be helped by doing linkage studies.
Armed with the DNA diagnosis of the parents, one now moves to prenatal testing.
Fetal DNA analysis could be done by extracting DNA from amniotic fluid after 15 weeks of gestation or chorionic villus samples (CVS) between 10-12 weeks or later gestation. CVS is preferable as the result of prenatal diagnosis are available early in pregnancy. Usual reporting time is about one week. Since its inception in the 1980s, CVS has
gained popularity as a means of rapid prenatal diagnosis in early pregnancy. Its advantages over a second trimester procedure like amniocentesis include reduced emotional and physical stress in couples at risk, a less obvious pregnancy and therefore more privacy.
Termination of pregnancy, if indicated, can be carried out at a safer time in such cases. Old, et al., were the first to report successful diagnosis of beta thalassemia using CVS.
Since then few studies in the West and also from India have demonstrated the
high efficiency, safety and acceptability of this procedure prompting experts to suggest that first trimester CVS should be the gold standard for prenatal diagnosis. This message can be propagated through thalassemia clinics and thalassemia welfare societies who have a major role in the creation of awareness regarding the problem of thalassemia.
The various methods of analysis used are:-
Targeted mutation analysis. The β-thalassemias can be caused by more than 200 different HBB gene mutations ,however, the prevalent molecular defects are limited in each at-risk population. This phenomenon has greatly facilitated molecular genetic testing. Commonly occurring HBB mutations in a specific population(s) can be detected by a number of PCR-based procedures. The most commonly used methods are reverse dot blot analysis or primer specific amplification, with a set of probes or primers complementary to the most common mutations in the population from which the affected individual originated . Other methods based on real-time PCR or microarray technology because of their reproducibility, rapidity, and easy handling are potentially suitable for the routine clinical laboratory .
Sequence analysis detects mutations in the HBB coding region and associated flanking regions.Sensitivity is 99%. Mutations in the non-coding region and heterozygous deletions of an exon(s) or other gene region are not detected by this analysis.
Deletion/duplication analysis. Deletions of variable extent of the HBB gene or of the beta-globin gene cluster that result in β-thalassemia or in the complex β-thalassemias called γδβ-thalassemia and δβ-thalassemia are rare causes of β-thalassemia. Some HBB alleles with deletion mutations can be common in certain ethnic groups (e.g., the 619-bp deletion in Asian Indians; globin.cse.psu.edu).)
DNA diagnosis is usually done on chorionic villus samples at 10-12 weeks gestation in informative families(1,10-12). However, it has certain limitations. There are very few centers for molecular diagnosis and couple has to travel to established center for prenatal sampling and DNA analysis in majority of cases. Moreover, for DNA diagnosis mutations should be detected or the markers should be informative for linkage. Linkage requires sample from the affected hemophilia child, which is not available in all cases. When family comes late in pregnancy and common mutations are not identified in thalassemia, it is not possible to further characterize the unidentified mutation due to time constraints.
In such cases, the diagnosis is made by quantification of Hb A in fetal sample obtained by cordocentesis. Normal foetuses have low but demonstrable levels of Hb A in mid pregnancy. This is reduced in affected foetuses. Thus, this test can be used to diagnose foetuses with Thalassemia where advanced molecular studies are not available.
Serial Sonography for diagnosis of Thalassemia.
Abnormal MCA PSV is defined as value more than 1.5 MOM for the GA.It is important to emphasize that normal MCAPSV at 18 to 22 weeks reduces the probability that a fetus is affected by the disease and may obviate the need for invasive procedures to confirm normality, although follow-up sonographic examinations may be required. Despite the high accuracy of MCA-PSV, the diagnosis should be confirmed with definitive methods such as fetal blood analysis to exclude false-positive findings, although very infrequent, and hydrops fetalis secondary to other causes. The measurement of MCA-PSV may represent an alternative cost-effective method in identifying cases for referral or follow-up with serial sonography in their own hospitals.
2)Fetal CTR (Cardio thoracic ratio)-
A four chamber view of the heart is obtained. At this level, fetal transverse cardiac diameter (TCD) was measured. The same plane was used to measure transverse thoracic diameter (TTD). CTR was calculated as TCD/TTD. Cardiomegaly was defined as a CTR ≥0.5 at 12–17 weeks, ≥0.52 at 18–20 weeks and ≥0.59 at 25–30 weeks.
Because affected babies have hydrops and the first sign of hydrops is cardiomegaly. This ratio is used to diagnose thalassemia.
3)Placental Thickness (PT)
The thalassemic babies are associated with placentomegaly. Hence PT is used diagnose thalassemia. Placental thickness is measured at the centre of the placenta. A line is drawn tangential to the margin of placenta and the thickness is measured at right angle to this line. PT > 18 mm at 12-15 weeks is considered significant.
It has been suggested that echogenic bowel in fetuses with homozygous α-thalassemia-1 may be bowel wall edema as a result of anemia and hypoxia. The sequestration of fluid in the bowel wall may give it the echobright appearance.
It has been suggested that MCA PSV > 1.5 MOM, PT >18 mm and CTR >0.5 be used to screen foetuses at risk.
With this approach, invasive procedures could be performed selectively, fewer fetuses would be lost unnecessarily and medical expense could be reduced. Therefore, cordocentesis or amniocentesis should be reserved only for cases with positive sonographic markers, especially high MCA PSV. Although a small proportion of affected fetuses may be missed by this approach, it is possible that they could be detected on serial sonographic examinations because of later hydropic changes.This approach has been used in countries where legal termination of pregnancy is permitted even in advanced gestational age.
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Dr.Sameer Dikshit, MD, DGO, FCPS,FICOG
A fetal medicine consultant of vast practical experience. Trained at prestigious King’s College London.