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Keywords:

  • α-globin;
  • α-thalassemia;
  • molecular diagnosis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

Adult hemoglobin is a heterotetramer composed of two α-globin chains and two β-globin chains (α2β2), each of which contains a heme molecule capable of binding oxygen and facilitating oxygen transport. The α-globin chains are expressed from duplicated genes within a tandem gene cluster located on chromosome region 16p13.3. High-level expression of the α-globin genes commences early in fetal development and continues throughout life. The α-thalassemia syndromes are among the most single-gene disorders, resulting from decreased synthesis of α-globin chains or synthesis of functionally abnormal α-globin chains. These disorders are most common in South East Asia, but also occur in many other populations. The most common cause of α-thalassemia is gene deletions, of which more than seventy have been reported. In addition, a small but significant proportion of cases involve point mutations of the α-globin genes. Ideally, the diagnostic strategy should include allele-specific assays for commonly occurring deletions, as well as methods for detection of rare or novel deletions and point mutations. Here we provide an overview of the diagnostic methods available and our experience using these assays in a reference laboratory serving a heterogeneous at-risk population.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

The α-thalassemia syndromes are autosomal recessive hereditary anemias caused by deficient synthesis of normal α-globin components of fetal hemoglobin (Hb F, α2γ2) and adult hemoglobin (Hb A, α2β2), the predominant hemoglobins responsible for oxygen transport from early fetal development through to adulthood. The World Health Organization (WHO) has estimated that more than 20% of the world population is a carrier of some form of α-thalassemia [1] and as many as 14 700 births annually are affected by either Hb H disease or Hb Bart's hydrops fetalis syndrome [2]. These disorders are most common in regions of the world where falciparum malaria is or has been endemic, with the highest carrier rates in the Mediterranean region, sub-Saharan Africa, the Middle East, southern Asia, southern China, and South East Asia [3]. As a consequence of population migrations from these regions, these disorders now have a truly global distribution.

Hemoglobin genetics

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

The genes encoding the α-like chains of human hemoglobins are located on chromosome 16p13.3, whereas the β-like chains are encoded by a gene cluster on chromosome 11p15.5. High-level, tissue-specific expression is controlled by cis-acting enhancer sequences located upstream of each gene cluster, the locus control region (LCR) for the β-globin gene cluster, and HS-40 for the α-globin gene cluster [4].

The α-globin gene cluster contains several genes and pseudogenes that are tandemly arranged in the following order: HBZ-HBZP1-HBM-HBAP1-HBA2-HBA1-HBQ1 (Figure 1a). The HBZ gene encodes the ζ-globin chains, which are expressed early in embryogenesis and comprise the α-like chains of embryonic hemoglobins Gower I (ζ2ε2) and Portland I (ζ2γ2). Expression of the HBZ gene is silenced early in fetal development (6–7 weeks of gestation) and the HBA2 and HBA1 genes are activated and expressed at high levels thereafter, comprising the α-like chains of fetal (Hb F, α2γ2) and adult (Hb A, α2β2; Hb A2, α2δ2) hemoglobins [5]. Given that α-chains are components of the predominant hemoglobins responsible for oxygen transport from early fetal development to adulthood (Hb F and Hb A, respectively), mutations affecting the HBA2 and HBA1 genes can result in severe and potentially lethal disorders.

image

Figure 1. Molecular diagnostic strategies for detection of deletions and point mutations of the α-globin gene cluster. (a) α-Globin gene cluster showing the locations of the gene cluster on the distal short arm region of chromosome 16. The gene cluster is highlighted, as are ten of the most common α-thalassemia deletions detected in the Ontario population (>5 cases each). The first eight deletions inactivate both α-globin genes (α0-thalassemia), whereas the 3.7 and 4.2 inactivate one gene (α+-thalassemia). The 3.7 deletion results from homologous recombination between the HBA2 and HBA1 genes, resulting in a functional fusion gene consisting of the 5′ region of the HBA2 gene and the 3′ region of the HBA1 gene. (b) Detection of common gene deletions using gap-PCR. The first panel shows gap-PCR analysis for detection of the SEA, FIL THAI, MED-I, and 20.5 deletions. Positive samples could be heterozygous or homozygous. The remaining panels show gap-PCR assays for genotyping samples that are heterozygous or homozygous for the SEA, 3.7, and 4.2 deletions. (c) Detection of point mutations by resequencing of the α2-globin gene. The first panel is a patient hemizygous for a novel missense mutation (Hb Halifax, HBA2:c.371C>A). The second and third panels correspond to patients with Hb H disease who are hemizygous for two different initiation codon mutations.

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α-thalassemia syndromes

α-Globin gene mutations resulting in decreased expression of α-globin chains are associated with a group of disorders collectively known as the α-thalassemia syndromes. The hallmark feature of these syndromes is microcytic hypochromic anemia, the severity of which is directly proportional to the degree of imbalance between α- and β-chain expression.

Individuals with three functional α-globin genes are referred to as having ‘silent trait’ because they often have normal erythrocyte indices. In contrast, individuals with only two functional α-globin genes exhibit varying degrees of microcytosis and hypochromia, generally without significant anemia. This condition, known as α-thalassemia trait, represents the carrier state for the clinically significant α-thalassemia syndromes, Hb H disease and Hb Bart's hydrops fetalis syndrome.

Hb H disease is a clinically heterogeneous syndrome marked by moderate-to-severe hemolytic anemia [6]. Although most patients never require transfusions, some will have anemia necessitating occasional or even regular transfusions. The most common cause of Hb H disease is the loss of three α-globin genes through deletion (-α/--). Other causes include the loss of two genes through deletion and one through point mutation of the HBA2 gene (αTα/--) or the HBA1 (ααT/--) gene or homozygosity for particularly severe HBA2 point mutations (αTα/αTα).

The most severe form of α-thalassemia, Hb Bart's hydrops fetalis syndrome, is caused by the deletion of all four α-globin genes (--/--) [7]. This results in profound fetal anemia and hypoxia which, without intervention, usually results in fetal death late in pregnancy or shortly after birth. In cases where the diagnosis is made early in pregnancy, the pregnancy can be managed using in utero transfusions and early delivery, followed by regular transfusions and iron chelation therapy [8].

Mutation spectrum

There is a broad spectrum of mutations affecting expression of the α-globin genes, including rare deletions that remove the cis-acting HS-40 sequences and leave the α-globin genes intact, deletions that remove one (α+) or both (α0) of the α-globin genes, and point mutations of the HBA2 or HBA1 genes. To date, more than five hundred different α-globin gene cluster mutations have been documented [9, 10]. Approximately one-third of the mutations are loss-of-function thalassemic mutations, including 10 single-gene deletions, 60 deletions that inactivate both genes, and more than 80 loss-of-function point mutations of the HBA2 or HBA1 genes [3, 10]. There is a bias toward detection of point mutations involving the HBA2 gene because it is expressed at a higher level than the HBA1 gene, and carriers are more likely to have a thalassemic phenotype. The largest class of mutations are missense mutations that change the amino acid composition of the α-globin chain and can give rise to hemoglobin variants with altered properties. Fortunately, most α-chain Hb variants are functionally normal or have minimally altered function or stability.

Although a large number of α-thalassemia mutations have been documented, most are quite rare, having been reported in one or few families. As with many genetic disorders, the mutation spectrum for each population group is dominated by a relatively small number of founder mutations that comprise the majority of mutant alleles in carriers and affected individuals [3, 10]. For example, in southern Chinese populations, the most common α-thalassemia alleles are the South East Asian α0-thalassemia deletion (SEA), the 3.7 kb and 4.2 kb α+-thalassemia deletions (3.7, 4.2), and the Hb Constant Spring (HBA2:c.427T>C) and Hb Quong Sze (HBA2:c.377T>C) point mutations. In contrast, the mutation spectrum is very different for Middle Eastern populations, dominated by three different α0-thalassemia deletions (MED-I, MED-II, 20.5), two α+-thalassemia deletions (3.7, 4.2), and two point mutations (HBA2c.95+2_95+6del and HBA2:c.*94A>G). In other population groups, such as African Blacks and South Asians, α+-thalassemia deletions (3.7, 4.2) are far more common than α0-thalassemia deletions. Lastly, it should be noted that α-thalassemia can also occur in populations generally not considered to be at high risk for hemoglobinopathies, as illustrated by a founder effect of the British α0-thalassemia deletion (BRIT) in Newfoundlanders [11].

Laboratory screening tests for α-thalassemia

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

Complete blood count (CBC)

Unlike most autosomal recessive disorders for which healthy carriers are phenotypically silent, carriers of α-thalassemia can be easily detected using routine hematological tests that are inexpensive and widely available. The most important screening tool for thalassemia carriers is the CBC done using automated cell counters. Adult carriers of α-thalassemia have mild microcytosis and hypochromia, with normal or slightly reduced hemoglobin levels [12]. For thalassemia screening, most laboratories employ cutoffs of mean corpuscular volume (MCV) <80 fL and mean corpuscular hemoglobin (MCH) <27 pg, values which are 2 SD below the normal values of 90 fL and 30 pg. Adult carriers of single-gene α+-thalassemia deletions have the mildest phenotype, with MCV of 81.2 ± 6.9 fL and MCH of 26.2 ± 2.3 pg [12]. Approximately one-half of all carriers of α+-thalassemia deletions have MCV and MCH values that fall within the normal ranges, hence the term ‘silent trait’. Carriers of loss-of-function point mutations of the HBA2 gene have more pronounced microcytosis (75.5 ± 4.7 fL) and hypochromia (24.8 ± 1.7 pg), as do homozygotes for α+-thalassemia deletions (MCV 71.6 ± 4.1 fL, MCH 22.9 ± 1.3 pg) and heterozygotes for α0-thalassemia deletions (MCV 69.1 ± 4.4 fL, MCH 21.7 ± 1.7 pg) [12].

It is important to appreciate that microcytosis and hypochromia are not specific for α-thalassemia trait. Similar indices can be found with carriers of β-thalassemia and with common nonthalassemic conditions such as iron deficiency anemia (IDA). Other parameters of the CBC may be useful for differentiating between thalassemia trait and IDA. These include the erythrocyte count that is elevated for thalassemia trait and decreased for IDA and the red cell distribution width (RDW) that often is elevated with IDA.

Hemoglobin analysis

Quantitative hemoglobin analysis is generally performed using high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE), with the BioRad VARIANT II and Sebia CAPILLARYS™ 2 platforms being the most widely used [13]. Both systems provide accurate quantitation of the Hb A2 levels, an important marker for distinguishing between α-thalassemia trait and β-thalassemia trait. The level of Hb A2 is normal or slightly decreased for α-thalassemia trait, whereas increased Hb A2 levels (>3.5%) are strongly indicative of β-thalassemia trait. Because both α- and β-thalassemia are common in many populations, it is not unusual to find individuals who are double heterozygotes for α- and β-thalassemia. In such instances, the level of Hb A2 remains elevated, potentially masking the α-thalassemia. For this reason, it is important to screen β-thalassemia carriers for concomitant α-thalassemia, particularly if their partner has α-thalassemia trait.

High-performance liquid chromatography and CE analyses are also important screening tools for Hb H disease in newborns and adults. In newborns, the presence of elevated levels of Hb Bart's (γ4) is indicative of α-thalassemia trait or Hb H disease. In adults, the presence of Hb H (β4) is indicative of Hb H disease.

Additional tests

Additional tests, such as measurement of serum ferritin and zinc protoporphyrin (ZPP) levels, can aid in differentiating between α-thalassemia and IDA. Reduced serum ferritin is an indicator of iron deficiency, whereas increased ZPP levels is an indicator of IDA.

Another useful screening test for α-thalassemia is brilliant cresyl blue (BCB) staining for Hb H inclusion bodies. For this test, the blood film is stained with BCB and the appearance of a small percentage of erythrocytes containing Hb H inclusion bodies is indicative of α0-thalassemia trait. As a screening test for carriers of α0-thalassemia deletions, this test has 70% sensitivity and 90% sensitivity. The presence of numerous Hb H–positive cells per field is highly indicative of Hb H disease.

Molecular diagnostic testing

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

Almost forty years have passed since the first molecular test was used to diagnose human genetic disease, specifically the application of comparative genomic hybridization to detect α0-thalassemia deletions [14]. Since that time, there have been many advances in molecular diagnostic approaches to gene deletions as well as point mutations. Currently, the most commonly methods for diagnosis of α-thalassemia include: (i) deletion-specific gap-PCR for detection of the common α0- and α+-thalassemia deletions, (ii) multiplex ligation-dependent probe amplification (MLPA) and microarray analysis for detection of common, rare or private gene deletions, (iii) allele-specific assays for detection of common point mutations, and (iv) α-globin gene resequencing for detection of common, rare, and private point mutations.

Deletion-specific gap-PCR

Gap-PCR is the most commonly used strategy for detection of α-thalassemia deletions with known end-points. This approach utilizes primers that are located near the 5′ and 3′ breakpoints to amplify a small fragment that spans the deletion junction. For normal alleles, the primers are situated too far apart to allow efficient amplification.

Gap-PCR strategies have been developed for numerous deletions of the α-globin gene cluster and can be used individually or in multiplex panels. One of the most popular multiplex assay covers seven deletions, specifically the SEA, FIL, MED-I, THAI, 20.5, 3.7, and 4.2 deletions [15-18]. In many laboratories, particularly those serving heterogeneous populations, this has become the standard panel for α-thalassemia screening. In our laboratory, we employ a simple multiplex gap-PCR assay to screen for five common α0-thalassemia deletions (SEA, FIL, THAI, MED-I, 20.5) (Figure 1b). We also have genotyping assays for the several of the most commonly encountered deletions (SEA, 3.7, 4.2) (Figure 1b). For samples that test negative for the seven common α-thalassemia deletions, we have additional single- and multiplex gap-PCR assays for other recurrent deletions (e.g., BRIT, SA, MED-II) (Figure 1a).

MLPA and microarrays

Multiplex ligation-dependent probe amplification is a technique designed for the detection of gene deletions, duplications, and rearrangements [19]. MRC-Holland has developed a commercial MLPA assay specifically for detection of α-globin gene deletions and rearrangements (Salsa® MLPA® probemix P140-B4 HBA). The assay employs 24 probes spaced throughout the distal short arm of chromosome 16, with a high density of probes within the α-globin gene cluster. This approach has proven to be very useful for initial detection and characterization of rare and private deletions [20, 21]. MLPA is recommended as a supplementary screening test for patients with indices highly suggestive of α-thalassemia, yet negative for the common deletions or point mutations. In our experience, this is required for only a small minority (<1%) of all cases.

Chromosome microarray analysis (CMA) has rapidly become a routine test for detection of copy number variants (CNVs) throughout the genome [22]. CMA is capable of detecting CNVs as small as several kilobases, including rearrangements of the α-globin genes. As such, α-thalassemia is a common incidental finding of microarray testing performed for other indications (e.g., developmental delay).

Allele-specific assays

A host of allele-specific assays have been developed for detection of the most common point mutations of the α-globin genes. Popular methods include reverse dot-blot hybridization, amplification refractory mutation systems (ARMS), restriction endonuclease cleavage, pyrosequencing, and quantitative real-time PCR. These methods are ideally suited for homogeneous populations with small numbers of common mutations, as opposed to heterogeneous populations. A further limitation is the inability to detect rare or private mutations.

Resequencing

Given the small size of the HBA2 and HBA1 genes (~1.0 kb), resequencing is an attractive alternative to allele-specific assays for point mutations. Although the coding sequences of the HBA2 and HBA1 are identical, sequence differences in the 5′ and 3′ regions can be used to selectively amplify the individual genes prior to resequencing [23]. This approach has the advantage of being able to detect all known pathogenic mutations, as well as mutations that have not been reported in the literature or mutation databases (Figure 1c).

The ontario experience

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

Our laboratory has served as a hemoglobinopathy reference laboratory for the province of Ontario, Canada (population 13 million), since 1989. Ontario has the highest ethnic diversity of any province in Canada, with one in every four individuals belonging to populations at high risk for hemoglobinopathies. Of particular relevance, there are more than 1 million individuals from regions of the world in which the gene frequency of α0-thalassemia is highest (southern China, South East Asia).

To date, the laboratory has conducted more than 18 400 investigations of patients referred because the erythrocyte indices and hemoglobin profiles were suggestive of an α-thalassemia syndrome. Forty percent were negative for the common α-globin gene deletions, in agreement with a previous study demonstrating that half of all patients referred for α-thalassemia investigations had other causes for microcytic anemia, such as IDA [24].

The hematological indices for the deletion-negative patients favor a diagnosis of IDA rather than α-thalassemia trait. For example, the adult carriers of α0-thalassemia deletions had higher erythrocyte counts compared to the deletion-negative patients (females 5.30 ± 0.54 × 1012/L vs. 4.54 ± 0.51 × 1012/L, males 6.10 ± 0.71 × 1012/L vs. 5.21 ± 0.51 × 1012/L). Moreover, 89% of the adult carriers of α0-thalassemia deletions were positive for Hb H inclusions bodies, compared to only 2% of the deletion-negative patients. Overall, these results indicate that the deletion-negative samples most likely are true negatives for α0-thalassemia. Nevertheless, only a portion of our deletion-negative samples (<20%) were subjected to α-globin gene resequencing, leaving open the possibility that some of these patients may be carriers of point mutations.

Sixty percent of our patients investigated for α-thalassemia were positive for α-globin gene mutations. The breakdown of genotypes was as follows: 37.86% heterozygous α+-thalassemia trait (-α/αα, αTα/αα, ααT/αα), 17.66% homozygous α+-thalassemia trait (-α/-α, -α/αTα, -α/ααT, αTα/αTα), 33.35% heterozygous α0-thalassemia trait (--/αα), 6.45% Hb H disease (-α/--, αTα/--, ααT/--, αTα/αTα), and 0.91% Hb Bart's hydrops fetalis syndrome (--/--). In addition, 3.77% of cases were positive for missense mutations resulting in nonthalassemic α-chain Hb variants (αVα/αα, -α/αVα, αTα/αVα).

Table 1 summarizes the spectrum of mutations detected in our α-thalassemia investigations. More than twelve different α0-thalassemia deletions were detected, with the SEA and FIL deletions accounting for the vast majority (>95%). Multiplex gap-PCR screening for the SEA, FIL, THAI, MED-I, and 20.5 deletions detected 98.5% of the deletional α0-thalassemia mutations in our patients.

Table 1. Mutations detected in 18 400 laboratory investigations for α-thalassemia syndromes
MutationTypeHGVS nomenclatureN%
South East Asian deletion (SEA)α0-thalNC_000016.9:g.215396_234699363526.25
Filipino deletion (FIL)α0-thalNC_000016.9:g.201640_232315del7465.39
THAI deletion (THAI)α0-thalNC_000016.9:g.199862_233311del310.22
Mediterranean-I deletion (MED-I)α0-thalNC_000016.9:g.212861_230268delins134800.58
20.5-kb deletion (20.5)α0-thalNC_000016.9:g.207337_227006del210.15
British deletion (BRIT)α0-thalNC_000016.9:205465_233537del360.26
South African deletion (SA)α0-thalNC_000016.9:209051_232787del70.05
Mediterranean-II deletion (MED-II)α0-thalNC_000016.9:197943_229150del70.05
Other α0 deletions (>4 types)α0-thal 190.14
3.7-kb rightward deletion (3.7)α+-thalMultiple deletion end-points770455.64
3.7-kb (Hb G-Philadelphia)α+-thalNM_000517.4(HBA2):c.207C>G150.11
4.2-kb leftward deletion (4.2)α+-thalMultiple deletion end-points5183.74
4.2 kb (Hb Q-Thailand)α+-thalNM_000558.3(HBA1):c.223G>C540.39
Other α+ deletions (2 types)α+-thal 40.03
α2, IVS-I (-TGAGG)α+-thalNM_000517.4(HBA2):c.95+2_95+6del2681.94
α2, Hb Constant Springα+-thalNM_000517.4(HBA2):c.427T>C1270.92
α2, Hb Quong Szeα+-thalNM_000517.4(HBA2):c.377T>C480.35
α2, polyA AATAAA[RIGHTWARDS ARROW]AATAAGα+-thalNM_000517.4(HBA2):c.*94A>G330.24
α2, polyA AATAAA[RIGHTWARDS ARROW]AATAα+-thalNM_000517.4(HBA2):c.*93_94del180.13
α2, initiation codon ATG[RIGHTWARDS ARROW]AGα+-thalNM_000517.4(HBA2):c.2del120.07
Other α2 point mutations (32 types)α+-thal 880.64
Other α1 point mutations (4 types)α+-thal 40.03
α2 and α1 Hb variants (87 types)Hb variant 3702.67
147 mutations   13 845 100.00

The 3.7-kb and 4.2-kb single-α-globin-gene deletions accounted for 92.5% of the α+-thalassemia alleles and almost 60% of all mutant alleles detected. Approximately 7.5% of all α+-thalassemia alleles were point mutations. There were 42 different point mutations, 38 involving the α2-globin gene and four involving the α1-globin gene. Again, a small number of common mutations accounted for the majority (84.6%) of the point mutations encountered in our population (Table 1).

Lastly, we detected 87 different missense mutations resulting in nonthalassemic α-chain Hb variants. Many of these variants were detected during newborn screening for sickle cell disease or Hb A1c monitoring for diabetes.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

The diagnostic strategy employed by our laboratory involves both hematological screening tests and simple DNA tests designed to detect common, rare, and private α-thalassemia mutations. The first line of investigation is hematology screening tests (CBC, quantitative Hb analysis, BCB staining) used to identify with individuals with α-thalassemia phenotypes. The second line of investigation is gap-PCR assays designed to detect the most common deletions found in thalassemic populations. We routinely screen for seven common deletions (SEA, FIL, THAI, MED, 20.5, 3.7, 4.2), but also have gap-PCR assays for several other recurrent deletions. For the rare cases involving deletions not covered by our gap-PCR assays, MLPA can be used to confirm the existence of a deletion and identify the approximate end-points. The third line of investigation is resequencing of the α-globin genes, which is applied to cases that are negative for deletions but require further investigation to exclude the possibility of α-thalassemia point mutations. Collectively, these tests have a sensitivity of detection approaching 100%.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References

We are indebted to the past and present staff of the Molecular Hematology and Genetics Laboratory, particularly Laurie Hellens, Betty-Ann Hohenadel, Lisa Nakamura, Margie Patterson, and Lynda Walker. Special thanks to Dr. David H.K. Chui, founder and former director of the laboratory.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Hemoglobin genetics
  5. Laboratory screening tests for α-thalassemia
  6. Molecular diagnostic testing
  7. The ontario experience
  8. Conclusion
  9. Acknowledgements
  10. References
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