Personal Practice

Indian Pediatrics 1999;36: 1119-1125

Carrier Screening and Prenatal Diagnosis of b-Thalassemia

From the Genetic Unit, Department of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India.
Reprint requests: Dr. P.S.N. Menon, Additional Professor, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi 110 029, India.

Beta-thalassemia is the most common single gene disorder in our country. The incidence of thalassemia is very high, with over 30 million people carrying the defective gene. Carrier frequency varies from 3 to 17% in different populations(1). Over 9000 thalassemic children are born every year and the treatment is very expensive(2). Hence the burden induced on the society is tremendous. The most effective approach to reduce the burden of the society and reduce the disease incidence is implementation of a carrier screening program offering genetic counselling, prenatal diagnosis and selective termination of affected fetuses.

Though there is a definite need for carrier screening in our country, it is hard to draw a consensus regarding the time of screening. Due to lack of education and public awareness about the disease, even carrier status for a disease can become a stigma. Various options available are: (i) Screening of school going children; (ii) Screening of high risk communities; (iii)  Premarital screening; (iv) Extended family screening - screening of relatives if there is a thalassemic child in a faimly; and (v) Routine antenatal screening in early pregnancy ideally between 10-12 weeks. The first three options are logistically not feasible in our country due to social and economic reasons. Extended family screening is acceptable and is being practised to a certain extent, but with such a program we are likely to miss many carriers.

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. We are at present offering this facility to the mothers registering at the antenatal clinic of AIIMS under an ICMR funded project and find it quite feasible and useful.

Carrier Screening Methods

There are various methods available for carrier screening. The definitive test for b thalassemia carrier status is HbA2 level of �3.5 per cent. There are some rapid screening tests for mass screening. These are described below.

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(3) and some on MCV or MCH alone(4,5). In our own study on 1286 antenatal women (unpublished data from ongoing ICMR project) sensitivity and specificity of MCV (<77 fl) and MCH (<27 pg) for determination of carrier status was 98% and 92% respectively. When either MCV or MCH was considered for carrier status, sensitivity and specificity were 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(6) 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(7-11). 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 (Fig. 1).

Fig. 1. NESTROFT stand showing positive and negative samples in different tubes. Tubes from L to R: Tubes 1-3 and 6-8 are positive samples where black line is not visible through the solution, Tubes 4-5 and 9-10 negative samples where black line is visible through the solution.

It has a sensitivity ranging from 94 to 99 per cent (8,9,12). In a recent survey done by us (results unpublished) on antenatal women (n=1048), sensitivity specificity, positive predictive and negative predictive values of NESTROFT were 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. HbA₂ levels can be measured by various methods such as microcolumn chromatography(13), high performance liquid chromatography (HPLC)(14) and capillary isoelectrofocu-sing(15). 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. In a survey conducted by us on antenatal women (n=1286), we found that 16% were positive by preliminary screening using NESTROFT and red cell indices. However, only 4.5% were confirmed as carriers by HbA₂ estimation.

4. DNA Mutation Analysis

Once the carrier status of the couple is confirmed, the next step is to prevent the birth of thalassemic child by offering prenatal diagnosis and selective abortion of the fetuses affected with thalassemia. 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(16), reverse dot blot(17), and restriction endonuclease allele recognition(18). 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(19) 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(20). Studies conducted in India have identified about 28 mutations in Indian population. Of these five to six mutations are found to be common(21-24). 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(25-27). 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(24). Overall common mutations account for 90-93.6 per cent(23,24).

Still 5-10 per cent needs further investiga-tions 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(28). DGGE is based on the resolution of DNA fragments differing by single nucleotide substitution(29). Both the methods could be used for detection of rare mutations. This can be followed by sequencing using automated sequencers which are available now. We are using ARMS technique for character-isation of mutations in our laboratory. Using this technique we are able to detect five common mutations, namely, IVS 1-5, IVS 1-1, 619 bp del, Fr41-42 and Fr8-9 (Fig. 2) in 90-95% of the subject and 12 rare mutations in 1-2% of the subjects. The families where mutations were not characterized could be helped by doing linkage studies.

Fig. 2. Gel photograph showing 5 common mutations.

Lanes L to R: Lane_1 IVS 1-5, Lane_2 IVIS 1-1, Lane 3_Fr 41-42, Lane_4 Fr 8-9, Lane 5_619 bp Del, Lane_6 Negative Control, Lane 7_Molecular weight marker.

Prenatal Diagnosis

Until the development of DNA techno-logies, prenatal diagnosis was carried out by estimating the rate of globin chain synthesis in fetal blood samples(30) and it is still a method of choice where DNA mutations are unidenti-fied in parents, or when the couples report for the first time with advanced pregnancy(31). In this procedure fetal blood sample is collected  after 18 weeks of gestation and globin chains are separated on CM cellulose column. Ratio of b/a chains is studied.

Fetal DNA analysis was adopted subsequently by many centers. Fetal DNA analysis could be done by extracting DNA from amniotic fluid after 15 weeks of gestation and chorionic villus samples (CVS) between 10-12 weeks or later gestation(32). CVS is preferable as the result of prenatal diagnosis are available early in pregnancy. Usual reporting time is about one week. The experience of our unit on prenatal diagnosis has been published recently(33). We are doing more than 100 prenatal diagnostic tests every year in our laboratory with very gratifying results. We are routinely screening women attending the antenatal OPD for thalassemia carrier status if they report early in pregnancy. We prefer to take cases in first trimester but may accept samples upto 20 weeks. If the lady is found to be NESTROFT and red cell indices positive, HbA₂ is done to confirm the carrier status. If her HbA₂ is �3.5. per cent, husband's carrier status is tested. If both partners are carriers we study their DNA for 5 common and 12 rare mutations. Prenatal diagnosis is offered if mutations are identified. In case the mutations are not identified linkage studies using restriction fragment length polymorphisms (RFLP) or globin chain synthesis by cord blood sampling are other options used for prenatal diagnosis.

Apart from women screened in the antenatal OPD, we get known thalassemic carriers (parents having affected children). These couples are directly screened for mutations in the b-thalessemia gene and offered prenatal diagnosis. Fig. 3 shows the approach we are following for carrier screening and prenatal diagnosis at our hospital.

Fig. 3. Flow chart for carrier detection and prenatal diagnosis of thalassemia.

With the availability of a battery of cost effective preliminary screening tests and advent of DNA based diagnostic technologies, it will  be possible, in the years to come, to reduce the burden of the disease on the society and in turn reduce its frequency in the population at large.

Acknowledgement

We are thankful to the Indian Council of Medical Research for providing partial financial assistance.

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