|Year : 2012 | Volume
| Issue : 3 | Page : 147-155
Rapid detection of multiple β-globin gene mutations by a real-time polymerase chain reaction in β-thalassemia carriers
Salwa M. Youssef1, Mohsen S. El Alfy2, Amany A. Osman1, Dina A. Khattab1, Mervat A. El Feky1, Marwa E. Hussein1
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||27-Feb-2012|
|Date of Acceptance||22-Mar-2012|
|Date of Web Publication||21-Jun-2014|
Mervat A. El Feky
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo
Source of Support: None, Conflict of Interest: None
β-Thalassemia is a heterogeneous disorder caused by mutations that reduce or abolish the synthesis of the β-globin chain. The clinical severity of thalassemia major makes it a priority genetic disease for prevention programs involving population screening of heterozygotes and an optional prenatal diagnosis for carrier couples.
Aim of the study
This study aimed to determine the most common β-globin gene mutations in Egypt using a real-time PCR and fluorescently labeled hybridization probes specific for each mutation and to assess the feasibility of introducing this technique in an overall thalassemia prevention program.
Participants and methods
The study was carried out on 45 individuals: 37 β-thalassemia carriers [including five amniotic fluid (AF) samples], seven β-thalassemia major cases (including two AF samples), and one normal AF sample. The most common β-thalassemia mutations were characterized by real-time PCR with fluorescently labeled hybridization probes specific for IVSI-110, IVSI-1, IVSI-6, codon 37, and codon 39 in 28/37 (75.7%) carriers.
The most common mutation encountered was IVSI-110 (46%), followed by IVSI-1 (16.2%) and then IVSI-6 (13.5%). Codon 37 and codon 39 were not characterized in any sample. The genotype of the uncharacterized carriers was determined using a less sensitive method (reverse hybridization technique) and a relatively less common set of mutation was characterized as follows: IVSII-1(10.8%), codon 5 (5.4%), IVSII-745 (5.4%), and IVSI-116 (2.7%). The overall number of alleles detected using both techniques was calculated to be 51. The real-time PCR alone, with its assigned probes, detected 38/51(74.5%). Thirteen mutations (13/51=25.5%) remained uncharacterized by this technique (because of the unavailability of the corresponding probes). However, the reverse hybridization technique detected 48/51 alleles (94.1%). However, comparison between both techniques in terms of the shared mutations showed that the real-time PCR detected 38/38 (100%) of these mutations, whereas the reverse hybridization technique detected only 36/38 (94.7%).
Real-time PCR is a very rapid and accurate method for the detection of the β-thalassemia mutation, which may be valuable in cases for which a rapid decision has to be taken. Impediments to prenatal diagnosis as encountered in this study were attributed to refusal of termination of pregnancy by the family for religious/reasons, abortion following amniocentesis, and failure to determine the correct genotype of the AF analyzed.
Keywords: amniocentesis, carriers, gene mutations, polymerase chain reaction, β-thalassemia
|How to cite this article:|
Youssef SM, El Alfy MS, Osman AA, Khattab DA, El Feky MA, Hussein ME. Rapid detection of multiple β-globin gene mutations by a real-time polymerase chain reaction in β-thalassemia carriers. Egypt J Haematol 2012;37:147-55
|How to cite this URL:|
Youssef SM, El Alfy MS, Osman AA, Khattab DA, El Feky MA, Hussein ME. Rapid detection of multiple β-globin gene mutations by a real-time polymerase chain reaction in β-thalassemia carriers. Egypt J Haematol [serial online] 2012 [cited 2020 Oct 19];37:147-55. Available from: http://www.ehj.eg.net/text.asp?2012/37/3/147/128293
| Introduction|| |
β-Thalassemias are heterogeneous hereditary anemias characterized by a reduced output of β-globin chains 1. The subsequent absence or decrease in hemoglobin production results in microcytosis with varying degrees of anemia 2. They are the most common monogenic disorder worldwide that occurs frequently in Africa, the Mediterranean, Middle East, India, and Asia 3. The disease is most prevalent in the temperate regions of the world, where it is a major health problem 1.
The prevalence of β-thalassemias is increasing, and this is especially of concern in developing countries, as it increases the burden of the healthcare delivery system 4; therefore, the need to implement a thalassemia prevention program in these countries is paramount 5. In the last decades, several programs, aimed at controlling the birth rate of thalassemia newborns by screening and prenatal diagnosis of populations with a high risk of β-thalassemia, have been implemented successfully 1. As screening will continue to be the cornerstone of the strategies aimed at β-thalassemia control, attempts to develop effective and economic techniques for thalassemia screening have become very important, especially in countries that have populations with a high percentage of such diseases 6. Cascade testing is more practical than general population screening in a country with limited health facilities where consanguineous marriages are practiced. In addition, the need of extensive testing within families with index cases is important to identify the carriers of beta-thalassemia in order to reduce disease occurrence through awareness and genetic counseling 7.
The various clinical phenotypes in β-thalassemias have led to the study of genetic factors that could modify the manifestations of these diseases 8. Although more than 180 causative mutations have been reported for β-thalassemia, the spectrum of mutations and their frequencies in most populations consist of a limited number of common mutations and a slightly large number of rare mutations 9. It was found that in countries bordering the Mediterranean basin, the major mutations of β-globin are IVSI-110, IVSI-6, IVSI-1, codon 39, and codon 37. Studies on the Egyptian population have confirmed the heterogenous nature of thalassemia in Egypt and its synchronous pattern with that of the Mediterraneans 10.
The human β-globin gene has been genotyped using several methods including a PCR, followed by restriction digestion 11 and denaturating gradient gel electrophoresis 12. These methods require several hours and sometimes days for diagnosis 13. Real-time PCR is introduced as a novel technique, which is reported to be sensitive, accurate, and applicable for many genotype interactions 14. It is especially suitable for prenatal diagnosis as it is very rapid 15.
Aim of study
The current study aims to detect the most common β-globin gene mutations in Egypt among heterozygous β-thalassemia carriers by a recent technique using real-time PCR and fluorescently labeled hybridization probes specific for each mutation in an attempt to estimate the incidence of each mutation and determine the feasibility of introducing this technique in an overall thalassemia prevention program.
| Participants and methods|| |
This study was carried out on 45 individuals [37 peripheral blood (PB) samples and eight amniotic fluid (AF) samples]. These included 37 carriers (32 β-thalassemia carriers who are relatives of β-thalassemia children attending the outpatient clinic of Pediatric Hospital, Ain Shams University Hospitals, and five AF samples proved to be of carrier fetuses) and also seven β-thalassemia major individuals (five β-thalassemia major children who were offspring of the studied carriers included as positive controls and two AF samples proved to be of thalassemia major fetuses) together with one AF sample, which was of a normal fetus.
All carriers and β-thalassemia major individuals were subjected to the following.
- Complete history taking and a thorough clinical examination with a focus on the degree of consanguinity and number of thalassemic children (for carriers).
- Laboratory investigations, which included the following:
- Complete blood count using cell counter CELL-DYN 1800, Abott Diagnostics (ABOTT; ABOTT Park, Illinois, USA).
- Examination of PB films stained with Leishman stain.
- Manual reticulocytic count of brilliant-cresyl blue-stained blood films.
- Hemoglobin electrophoresis using Genio Electrophoresis – InterLab, Abott Diagnostics.
- Identification of β-globin gene mutations based on PCR and reverse hybridization using the β-globin strip assay, VienaLab Diagnostics GmbH (Gaudenzdorfer Gurtel, Vienna, Austria).
- Detection of the major gene mutations (IVSI-1, IVSI-110, IVSI-6, codon 37, and codon 39) of β-thalassemia by real-time PCR Light-Cycler (Roche Molecular Biochemicals; Sandhofer Strasse, Mannheim, Germany).
(1) PB samples (37 samples: 32 carriers and five β-thalassemia major children).
(i) Two microliters of PB samples were obtained on potassium EDTA (K2-EDTA) in vacutainers (final concentration of 1.5 mg/ml) for complete blood count, reticulocytic count, and hemoglobin electrophoresis.
(ii) Two microliters of PB samples were obtained on sterile EDTA for DNA extraction, amplification, and detection of mutations using a reverse hybridization technique and real-time PCR.
(2) AF samples (eight samples).
Ten to 15 ml of AF samples were collected through ultrasound-guided amniocentesis at 13th–15th weeks of gestation in special sterile tubes. Five AF samples were taken from the pregnant females included in the carriers group, whereas three AF samples were obtained from the genetics lab of the Holding Company for Biological Products and Vaccines (Vacsera, Cairo, Egypt) (with no available clinical or laboratory data).AF samples were used for DNA extraction, amplification, and mutation detection using the reverse hybridization technique and real-time PCR.
β-Globin strip assay
Principle of the assay
The β-globin strip assay is based on the reverse hybridization principle, and includes three successive steps: DNA is isolated from samples by a rapid and convenient procedure and then β-globin gene sequences are in-vitro amplified and biotin-labeled in a single (multiplex) amplification reaction. Finally, the amplification products are selectively hybridized to a test strip, which contains oligonucleotide probes (wild type and mutant specific) immobilized as parallel lines. Bound biotinylated sequences are detected using streptavidin-alkaline phosphatase and color substrates.
The assay covers 22 of the most frequent β-globin mutations:
–87 [C→G], –30 [T→A], codon 5 [–CT], hemoglobin C, hemoglobin S, codon 6 [–A], codon 8 [–AA], codon 8/9 [+G], codon 22 [7 bp del], codon 30 [G→C], IVS I-1 [G→A], IVS I-2 [T→A], IVS I-5 [G→C], IVS I-6 [T→C], IVS I-110 [G→A], IVSI-116 [T→G], IVS I-25 [25 bp del], codon 36/37 [–T], codon 39 [C→T], codon 44 [–C], IVS II-1 [G→A], and IVS II-745 [C→G].
Real-time polymerase chain reaction
For the design of primers and probes, we used the Light Cycler probe design software (Roche Molecular Biochemicals) [Table 1]. The method included real-time PCR amplification of a β-globin gene fragment of 609 bp, using the GLO-F and GLO-R primers together with a pair of hybridization probes. One of the probes, the anchor probe, was located in the vicinity of the mutation and was labeled in its 5º end with LC Red 640 or LC Red 705 and phosphorylated at the 3º end to prevent extension of the probes during PCR. An adjacent sensor probe was placed over the mutation two to five nucleotides apart from the anchor and was labeled with fluorescein at its 3º end.
|Table 1: Primers and probes used for genotyping of the β-thalassemia alleles|
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SPSS statistical software package (version 15.2, Echosoft Corp., USA, 2006) was used for data analysis.
| Results|| |
Distribution of the β-thalassemia mutations characterized
The results of mutation characterization by both methods and the detailed distribution among the patients are listed in [Table 2] and [Figure 1] [Figure 2], [Figure 3], [Figure 4]. The fluorescently labeled hybridization probes of the real-time PCR method were specific for the detection of IVSI-110, IVSI-1, IVSI-6, codon 37, and codon 39, whereas those of the reverse hybridization technique were used to detect a broader panel of mutations among the population studied.
|Table 2: Mutations detected by real-time polymerase chain reaction and the reverse hybridization technique among carriers and cases|
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|Figure 2: Real-time PCR result showing mutant [(a) heterozygous and (b) homozygous] IVSI-110.|
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|Figure 3: Real-time PCR showing mutant [(a) heterozygous and (b) homozygous] IVSI-1.|
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|Figure 4: Test strip results (reverse hybridization technique): from left to right=codon 5 homozygous, IVSI-6/IVSI-110 compound heterozygous and IVSI-110/IVSII-1 compound heterozygous.|
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Among the carriers group (N=37: 32 carriers and five AF samples proved to be carrier fetuses), the real-time PCR technique (with its five specific probes) characterized β-thalassemia mutations in 28/37 carriers (75.7%). The most common mutation encountered among carriers was IVSI-110, which was detected in 17/37 of carriers (46%), followed by IVSI-1 (6/37=16.2%) and then IVSI-6, which was detected in 5/37 of carriers (13.5%). Codon 37 and codon 39 were not detected in any of the carriers studied [Figure 1].
The genotype of the nine carriers that remained uncharacterized by real-time PCR (because of the unavailability of the corresponding probes) was determined by the reverse hybridization technique and was shown to be as follows: the IVSII-1 mutation was detected in four carriers (4/37=10.8%), codon 5 in 2/37=5.4%, IVSII-745 in 2/37=5.4%, and IVSI-116 in one carrier (1/37=2.7%) [Table 2]. The 28 carriers who were characterized by real-time PCR were also detected using the reverse hybridization technique and showed the same frequency as those of real-time PCR, except for one carrier (genotype IVSI-1), who was detected by real-time PCR and not by the reverse hybridization technique.
The overall allele frequency of the detected mutations was determined among the individuals studied using both techniques; a total number of alleles (51) were calculated, with 32 heterozygous carriers, five AF heterozygous carriers, five thalassemia children (10 alleles), and two AF samples that proved to be β-thalassemia major (four alleles). IVSI-110 was the most common (22/51=43.1%), followed by IVSI-1 (10/51=19.6%); IVSI-6 and IVSII-1 were each detected in 6/51 alleles (11.8%). Codon 5, IVSII-745, and IVSI-116 were less common and accounted for (4/51=7.8%), (2/51=3.9%), and (1/51=1.9%), respectively. A summary of the number and percent of the resultant 51 alleles is presented in [Table 3].
|Table 3: Distribution of the overall allele frequency of β-thalassemia mutations among the groups studied|
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The genotype of one (AF) sample (1/45=2.2%) was not determined by either of the two techniques used, although the AF sample was withdrawn and analyzed twice (i.e. failed twice). The parents of this case had the genotypes of IVSI-110 and IVSII-1; therefore, the correct genotype of this AF sample was subsequently predicted from the genotype of the parents and the thalassemia major phenotype after delivery and was designated as compound heterozygous for both the parents’ genotypes (IVSI-110/IVSII-1). The real-time PCR method could detect the IVSI-110 allele of this case and not the other IVSII-1 allele (as the corresponding probe was not available). The reverse hybridization technique could not detect either of the two alleles.
The real-time PCR alone, with its assigned probes, detected 38 alleles of the estimated 51 alleles of all the studied individuals (38/51=74.5%). Thirteen mutations (13/51=25.5%) remained uncharacterized by this technique (because of the unavailability of the corresponding probes) [Table 3].
However, the reverse hybridization technique detected 48/51 alleles (94.1%) [Table 2] and [Table 4]. However, comparison between the reverse hybridization technique and real-time PCR in terms of the shared mutation between them (IVSI-110, IVSI-1, IVSI-6, codon 37, and codon 39) showed that the real-time PCR detected 38/38 (100%) of these mutations, whereas the reverse hybridization technique detected only 36/38 (94.7%) of these shared mutations [Table 4].
|Table 4: Comparison between real-time polymerase chain reaction and reverse hybridization technique in terms of the shared mutations between them|
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The most common four genotypes among carriers (IVSI-110, IVSI-1, IVSI-6, and IVSII-1) were compared together and comparison indicated no significant difference between these genotypes in hematological data (data not shown). However, when multiple comparisons were performed between each two of these four genotypes, a significantly lower hemoglobin level was found in heterozygotes of IVSI-110 and IVSI-1 when compared with IVSI-6 and lower mean corpuscular hemoglobin (MCH) of IVSI-1 and IVSII-1 compared with IVSI-6. Also, significantly higher red cell distribution width values were found in IVSI-1 compared with IVSI-6. The results of these comparisons are presented in [Table 5].
|Table 5: Multiple comparisons between each two of the four most common genotypes among carriers in terms of hematological data|
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| Discussion|| |
β-Thalassemia is a heterogenous disorder caused by mutations that reduce or abolish the synthesis of the β-globin chain 16. Recently, it has been reported that 1.5% of the world’s population are β-thalassemia carriers, that is, at least 80 to 90 million individuals, with an estimated 60 000 new carriers born each year. More than 200 β-thalassemia mutations have now been characterized worldwide; however, relatively small number of common β-thalassemia mutations can be found for each high-risk population 16. For example, in the Mediterranean region, where β-thalassemia is endemic, four mutations (namely IVSI-110, IVSI-6, IVSI-1, and IVSII-1) account for more than 75% of all cases 17.
The advancements in molecular genetics and the completion of the human genome project have raised the possibility of a genetic diagnosis. A prerequisite for prenatal diagnosis involves obtaining fetal material promptly and safely. In addition, the parental mutations have to be characterized before analysis of the fetal sample. The majority of methods used currently for the genetic analysis of prenatal samples and for a prenatal diagnosis are based on PCR (e.g. ASO, ARMS, RFLP, and direct DNA sequencing). Recently, the application of real-time PCR has offered a means for the use of rapid and potentially high-throughput assays without compromising accuracy, making it an ideal approach for genotyping prenatal and fetal samples for a prenatal diagnosis 18.
β-Thalassemia is a major health problem in Egypt. It has been estimated that 1000 children out of the 1.5 million live births are born annually with thalassemia major. In multicenter studies, the carrier rate has been reported to be in the range of 9–10% of the Egyptian population 19. Therefore, the current study was carried out with the aim of detecting the most common β-globin gene mutations in Egypt among heterozygous β-thalassemia carriers by a recent technique using real-time PCR and fluorescently labeled hybridization probes specific for each mutation in an attempt to estimate the incidence of each mutation and determine the practicability of introducing this technique in an overall thalassemia prevention program.
The most common mutations encountered in this study are in agreement with other similar surveys conducted in Egypt. Although El-Gawhary et al. 20 reported the most common mutations to be IVSI-110 (23.3%), followed by IVSI-6 (15%), El-Gawhary et al. 21 reported that IVSI-6 constituted 36.3% of the alleles studied, IVSI-110 (25.8%) and IVSI-1 (19%). The two studies used direct sequence analysis for mutation characterization and the relatively high frequency of IVSI-6 in both studies was attributed to the high number of β-thalassemia intermedia patients in their series (as aiming at IVSI-6 mutation is commonly associated with an intermediate phenotype). Hussein et al. 19, however, used PCR amplification, followed by restriction enzyme analysis to identify these common Egyptian β-thalassemia mutations. The most common mutations found in their results were IVSI-110 (31.4%), IVSI-1 (17.6%), IVSI-6 (17.6%), and IVSII-1 (5.9%). In addition, Abdel Messih et al. 22 used PCR (β-globin strip assay MED), on the basis of the reverse hybridization principle, and found that the most common mutation was IVSI-6 (27%, 10/37 cases), followed by the IVSI-110 mutation and the IVSI-1 mutation (21.6%, 8/37 cases for each), followed by the IVSII-745 mutation (13.5%, 5/37 cases).
In the current study, the rare frame-shift mutation of codon 5 (which was detected by aiming at reverse hybridization technique) accounted for 7.8% of the cases (4/51 cases). The four alleles were all members of the same family (two heterozygous parents and one homozygous child who was presented with a thalassemia major phenotype). This allele is quite rare and can be found in Greeks, Macedonians, and Turks. However, several studies have reported its occurrence in Egypt; Hussein et al. 23 reported a frequency of 3.6%, whereas Weatherall and Clegg 24 and Abdel Messih et al. 23 reported a frequency of 1.7 and 5.4%, respectively. The higher calculated frequency of this mutation in the current study is obviously related to the deliberate inclusion of all related family member alleles in the calculation of its frequency among the overall alleles. Another less frequent mutation that was identified in two cases (3.9%) was that of the second intervening sequence, position 745 (IVSII-745). The very uncommon mutation IVSI-116 was found in only one carrier case (<2%). These two mutations were detected using the reverse hybridization technique and the higher percentage for both may be because of the small number of cases in this study.
Real-time PCR alone, with its assigned probes, detected 38 alleles out of the estimated 51 alleles of all the studied patients (38/51=74.5% of mutant alleles). Thirteen mutations (13/51=25.5%) remained uncharacterized by this technique (because of the limited number of probes available). Similarly, Waye et al. 25 and Omar et al. 26 could not characterize 11 and 27%, respectively, of the mutant alleles examined in their series despite using different techniques such as reverse dot-blot hybridization, restricted fragment length polymorphism, and ARMS techniques. However, El-Gawhary et al. 21 found that all the alleles examined could be identified only using direct DNA sequence analysis.
However, the reverse hybridization technique detected 48/51 of alleles (94.1%). However, comparison between the reverse hybridization technique and real-time PCR in terms of the shared mutation between them (IVSI-110, IVSI-1, IVSI-6, codon 37, and codon 39) showed that real-time PCR detected 38/38 (100%) of these mutations, whereas the reverse hybridization technique detected only 36/38 (94.7%) of these shared mutations.
One of these two undetected alleles by the reverse hybridization technique was a PB sample of a carrier and real-time PCR determined its genotype as IVSI-1 heterozygous. The other undetected allele was IVSI-110 in an AF sample of a compound heterozygous IVSI-110/IVSII-1 fetus. Real-time PCR detected only the IVSI-110 allele (by the available probe), but the reverse hybridization technique failed to detect both alleles.
When we compared the four most common genotypes in terms of their hematological data, significantly lower hemoglobin and MCH levels were found in patients with genotypes IVSI-110 and IVSI-1 than patients with IVSI-6. Also, the red cell distribution width was shown to be significantly higher in heterozygotes of IVSI-1 when compared with IVSI-6 heterozygotes. This shows that the IVSI-6 mutation may be presented with a better clinical phenotype. The homozygous state of this particular mutation is usually characterized by mild clinical phenotypes that is thalassemia intermedia 27; also, Galenello et al. 28 reported that heterozygotes of IVSI-6 generally have higher mean corpuscular volume and MCH when compared with other β-thalassemia mutations. In addition, their HbA2 levels are usually mildly elevated (3.4–4%).
On combining the two methods, real-time PCR (with high sensitivity) and the conventional method of the reverse hybridization technique (with a broader panel of mutations) in the genetic analysis of this study samples, the mutations were characterized successfully in all the samples (PB or AF) apart from the AF sample, in which the reverse hybridization technique failed to detect its genotype twice, whereas real-time PCR showed its genotype to be IVSI-110 heterozygous. The correct genotype of this sample was subsequently predicted from the thalassemia phenotype after delivery and the genetic analysis of the parents and was designated as compound heterozygous for the parental mutations (IVSI-110/IVSII-1).
Assessment of real-time PCR with fluorescently labeled hybridization probes in the current study showed that the newly established method was able to detect 100% of the mutations assigned to its included probes. Moreover, this method showed 100% reliability in differentiating di(normal) from mutant alleles and also homozygous from heterozygous mutations (by melting curve analysis). In agreement with the current finding, Gaafar et al. 29 reported that the greater sensitivity of the results obtained using the newly established method when compared with conventional methods and the lack of false-positive results in nonthalassemics clearly confirm the superiority of the real-time method for the detection of the mutations studied. In addition, the real-time method has the advantage of being considerably less time consuming than the conventional methods.
In a study carried out by Pornprasert et al. 30, it was concluded that the real-time PCR method for the detection of mutations was useful for screening couples at risk before prenatal diagnosis. It is also suitable for prenatal diagnosis as it is a very rapid and highly sensitive method. They added that the method is cost-effective and high throughput. Thus, it is more convenient for screening a large number of samples. In addition, in 2004, Vrettou et al. 14 standardized and validated a protocol involving the analysis of β-globin genotypes with real-time PCR for a preimplantation genetic diagnosis. They concluded that the protocol showed 100% concordance with conventional DNA sequencing methods and, overall, it was found to be sensitive, accurate, reliable, rapid, and applicable for many genetic interactions with internal monitoring of contamination, thus fulfilling all requirements for clinical applications.
In this study, the conventional reverse hybridization technique (VienaLab Diagnostics GmbH) that could screen for 22 mutations detected only 48 out of the 51 alleles examined and when it was compared with real-time PCR, its diagnostic sensitivity was found to be 94.7%. The same commercial kit was used by Keser et al. 31 for screening β-globin gene mutations, where it was followed by automated DNA sequencing in all the samples analyzed. Also, Rahim et al. 32 used the reverse hybridization kit to initially screen for the 22 common mutations and only the negative samples were further analyzed by DNA sequencing of the entire β-globin gene. However, the diagnostic sensitivity of the kit was not determined in either of these two series.
In a study carried out by Tamhankar et al. 33 for the prenatal diagnosis of β-thalassemia in India, all affected pregnancies were medically terminated by the respective couples as they realized that thalassemia is a public health problem and could produce immense mental and economic pressure on them. However, in the Egyptian study carried out by El-Gawhary et al. 20, although they were able to make a definitive diagnosis in all fetuses investigated, only a few couples opted for an abortion. They attributed the refusal of termination of pregnancy in the majority of carriers who were found to have a positive fetus to several factors: cultural and religious beliefs that consider abortion as a criminal act, higher levels of illiteracy, and lack of confidence in the predictive value of these new techniques. Their results highlighted the importance of proper health education and the need for implementing large-scale proper counseling programs, as well as the importance of premarital screening and prevention of carrier marriage rather than prenatal diagnosis, in developing countries with limited resources for healthcare before implementing such expensive techniques on a wider scale.
Moreover, countries such as Pakistan, Iran, Saudi Arabia, and Lebanon, which initially carried out only premarital screening to disallow a marriage in which both partners were carriers, have now reinterpreted religion to include prenatal diagnosis and abortion up to a particular gestational age 34–36.
Characterization of mutation in carriers is a prerequisite when making a prenatal diagnosis for couples at risk of having an affected child and frequently needs to be carried out as quickly as possible, especially when the couple presents after pregnancy is confirmed 29. For rapid DNA analysis, real-time PCR has become an important tool in clinical diagnostics. The proposed method is most useful when applied for a prenatal diagnosis. Once the parental mutations are known, the method is very fast and reliable. Compared with other methods, this is beneficial for the couple and the pregnancy. Furthermore, real-time PCR facilitates simultaneous detection of mutation through the flexible design of detection probes 15.
We recommend the use of real-time PCR for the rapid characterization of mutations among Egyptian β-thalassemia mutation carrier couples at risk of having a thalassemic child. Once the parental mutations are identified, a prenatal diagnosis can be made easily for the detection of this specific mutation(s), thus minimizing both the time and the cost for prenatal diagnosis. The reliability of the real-time PCR technique is an advantage when making an informed decision on the fate of the investigated pregnancy.
| References|| |
|1.||Cao A, Moi P, Galanello R. Recent advances in β-thalassemias. Pediat Rep. 2011;3:e17 |
|2.||Jones AK, Poon A. Evaluation of a single tube multiplex polymerase chain reaction screen for detection of common alpha thalassemia genotypes in a clinical laboratory. Am J Clin Pathol. 2002;118:18–24 |
|3.||Thein S. Genetic modifiers of β-thalassemia. Haematologica. 2005;90:649–660 |
|4.||Sarnaik S. Thalassemia and related hemoglobinopathies. Indian J Pediatr. 2005;72:319–324 |
|5.||Al-Allawi NA, Jubrael JM, Hughson M. Molecular characterization of beta-thalassemia in the Dohuk region of Iraq. Hemoglobin. 2006;30:479–486 |
|6.||Sachdev R, Dam A, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: report of 2600 cases. Indian J Pathol Microbio. 2010:53–57 |
|7.||Baig SM, Din MA, Hassan H, Azhar A, Baig JM, Aslam M, et al. Prevention of beta-thalassemia in a large Pakistani family through cascade testing. Community Genet. 2008;11:68–70 |
|8.||Fernandes AC, Shimmoto MM, Furuzawa GK, Vicari P, Fiqueiredo MS. Molecular analysis of β- thalassemia patients: first identification of mutation HBB: c.93.2 A>G and HBB: c.114 G>A in Brazil. Hemoglobin. 2011;35:358–366 |
|9.||Hardison RC, Chui DH, Giardine B, Riemer C, Patrinos G, et al. Hb Var: a relational data base of human hemoglobin variants and thalassemia mutations at the globin gene server. Hum Mutat. 2002;19:225–233 |
|10.||Moreno I, Bolufer P, Perez M, Barrangan E, Miguel A. Rapid detection of the major Mediterranean β-thalassemia mutations by real-time polymerase chain reaction using fluorophore-labeled hybridization probes. Br J Hematol. 2002:554–557 |
|11.||Zhang YH, McCabe LL, Wilborn M, Therrell BL Jr, McCabe ER. Application of molecular genetics in public health: improved follow-up in a neonatal hemoglobinopathy screening program. Biochem Med Metab Biol. 1994:27–35 |
|12.||Girodon E, Ghanem N, Vidaud M, Riou J, Martin J, Galacteros F, Goossens M. Rapid molecular characterization of mutations leading to unstable hemoglobin beta-chain variants. Ann Hematol. 1992;65:188–192 |
|13.||Herrmann MG, Dobrowolski SF, Wittwer CT. Rapid beta-globin genotyping by multiplexing probe melting temperature and color. Clin Chem. 2000;46:425–428 |
|14.||Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C, Kanavakis E. Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat. 2004;23:513–521 |
|15.||Vrettou C, Traeger-Synodinos J, Tzetis M, Malamis G, Kanavakis E. Rapid screening of multiple beta-globin gene mutations by real-time PCR on the Light-Cycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes. Clin Chem. 2003;49:769–776 |
|16.||Li D, Liao C, Li J, Huang Y, Xie X, Wei J, Wu S. Prenatal diagnosis of β-thalassemia by reverse dot-blot hybridization in southern China. Hemoglobin. 2006;30:365–370 |
|17.||Naja R, Kaspar H, Shbalko H, Chakar N, Makhoul N, Zalloua P. Accurate and rapid prenatal diagnosis of the most frequent East Mediterranean β-thalassemia mutations. Am J Hematol. 2004;75:220–224 |
|18.||Traeger-Synodinos J, Vrettou C, Kanavakis EHahn S, Jackson L. Rapid detection of fetal Mendelian disorders. Methods in Molecular Biology. 2008 Totowa, NJ Humana Press:133–145 |
|19.||Hussein G, Fawzy M, El-Serafi T, Ismail E, El-Metwally D, Saber M, et al. Rapid detection of β-thalassemia alleles in Egypt using naturally or amplified created restriction sites and direct sequencing: a step in disease control. Hemoglobin. 2007;31:49–62 |
|20.||El-Gawhary S, Sahar M, Rashad W, Mosaad M, Abdalla M, Ezzat G, et al. Prenatal diagnosis of β-thalassemia in Egypt: implementing accurate high tech methods did not reflect much on the outcome. Pediatr Hematol Oncol. 2008;25:541–548 |
|21.||El-Gawhary S, El-Shafie S, Niazi M, Aziz M, El-Beshlawy A. Study of β-thalassemia mutations using the polymerase chain reaction-amplification refractory mutation system and direct sequencing techniques in a group of Egyptian thalassemia patients. Hemoglobin. 2007;31:63–69 |
|22.||Abdel Messih IY, Mokhtar GM, Youssef SR, Elmogy MT, Mohamoud HM, Pessar SA. Evaluation of HPLC and molecular genetic confirmatory testing as a starting point of national screening program for β-thalassemia carriers in Egypt. 2011 Cairo, Egypt Thesis, Clinical Pathology Department; Ain Shams University:140 |
|23.||Hussein I, Temtamy S, El-Beshlawy A, Dearon C, Shalaby Z, Vassilopoulos G, Kazazian H. Molecular characterization of β-thalassemia in Egyptians. Hum Mutat. 1993;2:48–52 |
|24.||Weatherall D, Clegg J The Thalassaemia Syndromes: Distribution and population genetics of thalassemias. 20014th ed. Oxford Blackwell Science:248 |
|25.||Waye J, Bory S, Eng B, Patterson M, Chui D, Badr El-Din O, et al. Spectrum of β-thalassema mutations in Egypt. Hemoglobin. 1999;23:255–261 |
|26.||Omar A, Abdel Karim E, Gendy WE, Marzouk I, Wagdy M. Molecular basis of beta-thalassemia in Alexandria. Egypt J Immunol. 2005;12:15–24 |
|27.||Weatherall DStamatoyannopoulos G, Majerus P, Perlmutter R, Varmus H. The thalassemia. The Molecular Basis of Blood Diseases. 2001 6th ed. McGraw Hill Medical W.B. Saunders Company:183 |
|28.||Galanello R, Eleftheriou A, Traeger-Synodinos J, Old J, Petrou M, Angastiniotis M. Beta thalassemia mutations data tables: frequency and distribution, Annex II. In: Prevention of Thalassemias and other Hemoglobin Disorders. 2003;Vol. 1 Thalassemia International Federation Publications:156 |
|29.||Gaafar T, El-Beshlawy A, Aziz M, Abdelrazik H. Rapid screening of β-globin gene mutations by real-time PCR in Egyptian thalassemia children. Afr J Health Sci. 2006;13:3–4 |
|30.||Pornprasert S, Sukunthamala K, Sacome J, Phusua A, Saetung R, Sanguansermsri T, Leechanachai P. Analysis of Real-time SYBR polymerase chain reaction cycle threshold for diagnosis of the thalassemia-1 Southeast Asian type deletion: application to carrier screening and prenatal diagnosis of Hb Bart’s hydrops fetalis. Hemoglobin. 2008;32:393–402 |
|31.||Keser I, Manguoglu E, Kayisli O, Yesilipek A, Luleci G. Combination of Hb Knossos (cod 27 G-T) and IVSII-745 (C-G) in a Turkish patient with beta thalassemia major. Genet Test. 2007;11:228–230 |
|32.||Rahim F, Keikhaei B, Aberumand M. Prenatal diagnosis (PND) of β-thalassemia in Khuzestan province, Iran. J Clin Diag Res. 2007;6:454–459 |
|33.||Tamhankar P, Agarwal S, Arya V, Kumar R, Gupta U, Agarwal S. Prevention of homozygous beta thalassemia by premarital screening and prenatal diagnosis in India. Prenat Diagn. 2008;29:83–88 |
|34.||Akhlaghpoor S. Chorionic villus sampling for beta thalassemia: the first report of experience in Iran. Prenat Diagn. 2006;26:1131–1136 |
|35.||Inati A, Zeinith N, Isma’eel H, Koussa S, Gharzuddine W, Taher A. β-thalassemia: the Lebanese experience. Clin Lab Haematol. 2006:217–227 |
|36.||Naseem S, Ahmed S, Vahidy F. Impediments to prenatal diagnosis for beta thalassemia: experiences from Pakistan. Prenat Diagn. 2008;28:1116–1118 |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]