SEARCH

SEARCH BY CITATION

Keywords:

  • α-thalassaemia;
  • α-globin gene triplication;
  • multiplex PCR;
  • GC-rich PCR;
  • genetic screening

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common α-thalassaemia deletions and one α-globin gene triplication. The new assay detects the α0 deletions – –SEA, – (α)20.5, – –MED, – –FIL and – –THAI in the first multiplex PCR, the second multiplex detects the –α3.7 deletion and αααanti3.7 variant, the third multiplex detects the –α4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where α-thalassaemias are prevalent.

The α-thalassaemias are the most common single-gene diseases in the world ( Weatherall, 1998). They are characterized by a reduction or complete absence of α-globin gene expression. Normal individuals have two α genes on each chromosome 16 (αα/αα). The loss of one (– α) or both (– –) of these cis-linked genes are the most common causes for α-thalassaemias. Patients with haemoglobin H disease (– –/– α) develop chronic haemolytic anaemia of variable severity, whereas fetuses with Hb Bart's hydrops fetalis (– –/– –) die either in utero or shortly after birth as a result of severe intrauterine anaemia. Although individuals with three functional α genes (– α/αα) are clinically and haematologically silent and carriers with α-thalassaemia trait (– α/– α or – –/αα) only result in very mild hypochromic microcytic anaemia, couples with these genotypes are at risk of having a hydrop baby or offspring with HbH disease ( Bernini & Harteveld, 1998). Originally, α-thalassaemias were only endemic in malarial regions of the tropics and subtropics, but recent global migration of people from these areas has resulted in the world-wide occurrence of these diseases, including in the UK.

Traditionally, molecular characterization of α-thalassaemias has been carried out using Southern blot analysis. The method is both labour intensive and expensive. Moreover, its success depends heavily on the quality of the genomic DNA under test and the quantity available. PCR-based assays, which are more rapid, less expensive and more sensitive, have been described previously ( Bowden et al, 1992 ; Dode et al, 1993 ; Baysal & Huisman, 1994; Oron-Jarni et al, 1998 ),but for various reasons have not found their way into routine use. As the majority of α-thalassaemias world-wide are mainly caused by a few common underlying deletions, for example – α3.7, – α4.2, – –SEA, – –MED, – (α)20.5, – –FIL and – –THAI, we therefore developed a universal PCR approach, based on three simple and reliable multiplex PCR assays, that allows rapid detection of these seven common deletions as well as the αααanti3.7 triplication, which could be useful in predicting the clinical phenotype of thalassaemia intermedia ( Traeger-Synodinos et al, 1996 ). In addition, we have overcome the inherent problem of PCR amplification of the GC-rich α-globin locus by utilizing betaine and dimethyl sulphoxide (DMSO), together with an automatic hot-start, in our PCR assays, hence making this approach highly robust, reproducible and likely to be suitable for routine diagnostic and screening use.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Genomic DNA was extracted from peripheral blood using standard methods. PCR was carried out in 25-μl reactions containing 0.75 mol/l betaine, 5% DMSO, 200 μmol/l dNTPs, 1.25 units of AmpliTaq Gold polymerase (Perkin-Elmer) and 100 ng genomic DNA, in 1× GeneAmp PCR buffer provided by the manufacturer. The concentrations of the primers used in each multiplex PCR assay, their locations and sequences in the α-globin gene cluster are shown in Table I. The relative amounts of primers and reagents were optimized with the use of AmpliTaq Gold polymerase. Some adjustment of the relative concentrations will be necessary for the successful amplification with other Taq polymerases. Primers were either newly designed or modified from published primers ( Bowden et al, 1992 ; Oron-Jarni et al, 1998 ). Amplification was performed with an initial heat activation step of 15 min at 95°C followed by 35 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 2 min 30 s, and then an extension step of 72°C for 10 min in a DNA Engine thermal cycler (MJ Research, USA). After amplification, 10 μl of product was electrophoresed in a 2% agarose gel, stained in ethidium bromide solution and visualized on a UV transilluminator.

Table 1.  . Sequence and location of the PCR primers. *Final concentrations of the primers used in the three multiplex PCR assays.†Z69706 and Z84721 are GenBank accession nos. The co-ordinates are in nucleotides.Thumbnail image of

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

The multiplex PCR assays were carried out on 52 DNA samples that had been previously genotyped by Southern blot analysis as part of either haemoglobinopathy screenings or anthropological surveys: αα/αα (n = 3); – –SEA/– –SEA (n = 2); – –SEA/αα (n = 20); – (α)20.5/– (α)20.5 (n = 1); – (α)20.5/αα (n = 1); – –MED/– (α)20.5 (n = 1); – –FIL/αα (n = 1); – –THAI/αα (n = 1); – α3.7/– α3.7 (n = 8); – α3.7/αα (n = 6); – α4.2/– α4.2 (n = 4); – α4.2/αα (n = 4); – α3.7/– α4.2 (n = 2); αααanti3.7/αα (n = 5); and αααanti3.7/αααanti4.2 (n = 1). The results of the assays agreed completely with those obtained by Southern blotting. Representative results are shown in Fig 1.

image

Figure 1. 9, – α3.7/– α4.2.

Download figure to PowerPoint

The Thailand α0 deletion (– –THAI) breakpoint was characterized during the course of developing the α0-multiplex PCR assay (unpublished data). The 5′ breakpoint lies between 10724 and 10725 of GenBank accession no. Z84721, and the 3′ breakpoint lies between 1219 and 1220 of Z69706. A total of 33 452 nucleotides were found to be deleted. Our result confirmed that the breakpoint determined in a previous publication was actually that of the – –FIL instead of the – –THAI deletion, as reported previously ( Ko et al, 1998; Higgs et al, 1999 ; Ko & Li, 1999).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

The inherently problematic GC-rich α-globin locus has long been a major obstacle for developing any reliable and reproducible PCR assays for the detection of α-thalassaemias because the secondary structures formed in the target are often refractory to amplification. The introduction of the enhancing agents betaine and DMSO not only allowed disruption of the base pairing in the GC-rich region but also led to destablization of the secondary structure by making the G + C and A + T base pairs equally stable in the DNA duplex ( Henke et al, 1997 ). In addition, the built-in automatic hot-start of the AmpliTaq Gold polymerase further prevents any non-specific amplifications before the start of thermal cycling. This step is essential in population screening when large numbers of PCR reactions are required to be set up at ambient temperatures.

In this report, we have demonstrated that the multiplex PCR approach described here can reliably detect the homozygosity and heterozygosity of seven common α-thalassaemia deletions. Although only five α0-thalassaemia deletions are included in the α0-multiplex PCR assay at present, other population-specific deletions, such as – –CAL, should be easily incorporated into the assay if required. Moreover, when designing primers for the – α3.7 and – α4.2 multiplex PCRs, we have selected unique sequences in the non-homologous I, II and III regions for specific amplification instead of the rare unique sequences within the highly homologous X, Y and Z boxes ( Liebhaber et al, 1981 ; Michelson & Orkin, 1983; Hess et al, 1984 ), hence further ensuring the specificity of these assays.

Although it is straightforward to carry out all three multiplex assays on any individual sample, it may not be necessary to do all three assays if the ethnic origin and the haematological phenotypes of the sample are taken into account. In addition, when screening for the Mediterranean α0 alleles [– –MED and – (α)20.5] in a local population, the primers for the South-east Asian α0 alleles (– –SEA, – –FIL and – –THAI) can be omitted without loss of function of the assay (and vice versa).

Individuals with three functional α genes are carriers for α+-thalassaemia, a mild form in which only one α-globin gene is active. Their key haematological indices of mean cell haemoglobin (MCH) and mean cell volume (MCV) are only slightly reduced from normal values. Because the 3.7-kb deletion (– α3.7) and the 4.2-kb deletion (– α4.2) are the common causes of α+-thalassaemia, only the – α3.7 and – α4.2 multiplex PCRs are required for this diagnosis. Those individuals with an MCH of 27–25 pg need only be screened with the – α3.7 and the – α4.2 multiplex PCRs. Individuals with two functional α genes have clearly reduced MCH (below 25 pg) and MCV values. Such individuals may be homozygous for α+-thalassaemia (– α/– α) or are carriers for α0-thalassaemia (– –/αα), a severe form in which both α-globin genes are inactive. In such cases, all three multiplex PCRs are necessary for accurate diagnosis. Individuals with Hb H disease also require the three multiplex PCRs to determine the genotypes. However, prenatal diagnosis of Hb Bart's hydrops fetalis syndrome can be diagnosed effectively by the α0-multiplex PCR, which covers the five most common types of α0-thalassaemia gene deletions found world-wide.

The three multiplex reactions can thus be used singly or in any combination to suit the spectrum of the α-thalassaemia mutations expected from ethnic and haematological considerations.

In order to avoid tedious DNA purifications, we tested the feasibility of applying the assays to whole blood PCR ( Rees et al, 1996 ). Ten blind samples were tested with the α0-multiplex PCR assay; two – –SEA/αα and eight αα/αα were diagnosed (unpublished data). These results were subsequently confirmed by Southern blot analysis, suggesting that this method may be applicable to whole blood as well as to genomic DNA.

These universal multiplex PCR assays should considerably simplify carrier detection, population screening ( Lau et al, 1997 ) and molecular diagnosis for these common α-thalassaemia deletions in at-risk populations or individuals. These assays gave the correct genotype on testing 52 mixed DNA samples. However, this screening test was not carried out blind and laboratories adopting this approach should test the primers on their own samples with known genotypes before establishing the method as a reproducible diagnostic tool. In populations where β-thalassaemia with a normal HbA2 is common, a simple and reliable method for distinguishing heterozygous α0-thalassaemia from β-thalassaemia trait would also facilitate carrier screening and prenatal diagnosis for β-thalassaemia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We are grateful to Helena Ayyub and Helen Shaw for organizing DNA and blood samples; Eric Prieur for expert advice on using Power Point. We thank Professor Douglas Higgs and Dr Swee-Lay Thein for providing DNA samples. This work was supported by the Wellcome Trust, the Medical Research Council and a grant to J.M.O. from the International Atomic Energy Agency (Technical Contract no. 10511/RO).

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References