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Blood, 1 July 2001, Vol. 98, No. 1, pp. 250-251
CORRESPONDENCE
To the editor:
A rapid and reliable 7-deletion multiplex polymerase chain
reaction assay for  -thalassemia
The molecular genetics of the  -thalassemias has been
comprehensively reviewed.1 Of the numerous mutations that
have been described, deletions at the -globin gene locus account for
the vast majority of -thalassemia alleles.2 The most
widely occurring of these are the -3.7 and
-4.2 single -globin gene deletions, while double
-globin gene deletions in cis, such as the
- -SEA, - -FIL, and - -THAI
alleles are very common within Southeast Asia, and the
- -MED and -()20.5 double-gene deletions
occur more frequently in the Mediterranean area. Since the publication of the entire sequence of the human -globin
gene cluster,3 we and others have developed multiplex polymerase chain reaction (PCR) methods to diagnose different subsets
of -thalassemia deletional determinants.4,5 Generally, however, the high G+C nucleotide content and high degree of homology between the genes and pseudogenes at this locus have made it
technically challenging to develop a multiplex PCR assay capable of
detecting all 7 of the above mutations [-3.7,
-4.2, - -SEA, - -FIL,
- -MED, -()20.5, and - -THAI]
in a single reaction. We have now successfully developed an improved
single-tube multiplex PCR assay that can detect heterozygosity, homozygosity, and compound heterozygosity of these 7 -globin gene deletions. To achieve this, several aspects of our original multiplex PCR assay
were modified. These included the redesign of several primers,
inclusion of additional new primers, reoptimization of primer
concentrations, and use of a different, chemically modified automatic
hot-start DNA polymerase. Each 50-µL reaction contained 200 µM of
each dNTP, 1.5 mM MgCl2, 1 × Q-solution (Qiagen, Hilden, Germany), 2.5 U HotStarTaq DNA polymerase in supplied reaction buffer (Qiagen), 100-200 ng of genomic DNA, and 16 different primers at
various concentrations (Table 1).
Reactions were conducted in a T3 thermal cycler (Biometra,
Göttingen, Germany), with an initial 15-minute
denaturation at 96°C, followed by 30 cycles of 98°C denaturation
for 45 seconds, 60°C annealing for 90 seconds, and 72°C extension
for 135 seconds. A final 5-minute extension at 72°C completed the
reaction. Ten microliters of each amplified product was analyzed by
electrophoresis through a 1% agarose gel in 1 × Tris-Borate-EDTA
buffer at 10 volts/cm for an hour. The expected amplicon sizes
for each of the deletion junction fragments and the control
2 globin gene and LIS1 gene 3'
untranslated region (UTR) fragments are listed in Table 1. Because any
of the 7 deletions either partially or completely removes the
2 globin gene, its positive amplification
serves to indicate heterozygosity when a deletion allele is also
present. The LIS1 gene 3' UTR fragment serves as a separate
control for general amplification success. Multiplex PCR results from
representative DNA samples with various -thalassemia genotypes are
shown in Figure 1.
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| Figure 1.
Strategy and results of -thalassemia multiplex polymerase chain
reaction analysis.
(A) Schematic representation of the -globin gene cluster,
indicating extents of the 7 deletions and relative positions of the
primers (except for the control LIS1-F and LIS1-R primers, which are
located on a different chromosome). Locations of X, Y, and Z sequence
homology boxes and hypervariable regions (HVRs) are also shown. (B)
Multiplex PCR results from genomic DNA samples with various -globin
genotypes. M indicates Generuler 1kb DNA ladder (Fermentas, St
Leon-Rot, Germany).
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This simple assay has been validated on over two hundred DNA samples
from -thalassemia-1 and HbH disease individuals, and represents a
rapid and reliable method for detecting 7 of the most common mutations
of -thalassemia.
Arnold S.-C. Tan, Thuan C. Quah, Poh S. Low, and Samuel S. Chong
Correspondence: Samuel S. Chong, Department of Pediatrics,
National University of Singapore, Level 4, Main Building, National
University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore
Acknowledgments
Supported by Singapore grant NMRC/0365/1999 to S.S.C.
References
1.
Higgs DR.
Molecular mechanisms of -thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge United Kingdom: Cambridge University Press; 2001:405-430.
2.
Bernini LF.
Geographic distribution of -thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge United Kingdom: Cambridge University Press; 2001:878-894.
3.
Flint J, Thomas K, Micklem G, et al.
The relationship between chromosome structure and function at a human telomeric region.
Nature Genet
1997;15:252-257[CrossRef][Medline]
[Order article via Infotrieve].
4.
Chong SS, Boehm CD, Higgs DR, Cutting GR.
Single-tube multiplex-PCR screen for common deletional determinants of -thalassemia.
Blood.
2000;95:360-362[Abstract/Free Full Text].
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Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB.
Rapid detection of -thalassemia deletions and -globin gene triplication by multiplex PCRs.
Br J Haematol.
2000;108:295-299[CrossRef][Medline]
[Order article via Infotrieve].
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