| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters to the Editor |
Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume, Japan
a Address correspondence to this author at: Department of Forensic Medicine and Human Genetics, Kurume University School of Medicine, Kurume 830-0011, Japan, e-mail ykoda{at}med.kurume-u.ac.jp
To the Editor:
Anhaptoglobinemic patients have been reported to experience severe anaphylactic reactions to transfusions due to the production of antihaptoglobin (anti-HP) antibodies(1)(2). Anhaptoglobinemia in patients homozygous for HPdel, which is a deletion of an approximately 28-kb segment of chromosome 16 extending from the promoter region of the HP (haptoglobin) gene to exon 5 of HPR (haptoglobin-related protein), has been adequately characterized only recently(1). Use of a simple duplex PCR method has detected the HPdel allele in East and Southeast Asian populations at frequencies of 1%–3% but this allele has not been detected in African, West and South Asian, and European populations(1)(3)(4). Thus, diagnosing HPdel homozygosity prior to blood transfusion or the infusion of blood components into individuals from East and Southeast Asian populations is effective for preventing anaphylactoid shock due to anti-HP antibodies. We have developed a simple method that uses a 5' nuclease real-time PCR assay (TaqMan; Applied Biosystems) to detect the HPdel allele without having to isolate genomic DNA. The ethics committee of Kurume University School of Medicine approved this study protocol.
To distinguish alleles, we performed real-time PCR assays that detect the 2 regions that encompass the HPdel breakpoint and the 5' region of HP exon 1, which is deleted in HPdel. The 20-µL PCR reaction contained 200 µmol/L deoxynucleoside triphosphates, 1 µL of template (diluted blood or genomic DNA), 0.5 U of Ex TaqHS with its buffer (Takara), and the following primers and TaqMan probes (see Fig. 1
legend for sequences): Hp5'-F and -R primers (450 nmol/L); Hp5'–TaqMan probe (125 nmol/L) for detecting the 5' region of HP; Hpdel-F and -R primers (900 nmol/L); and Hpdel–TaqMan probe (250 nmol/L) for detecting HPdel. The PCR temperature profile was 95 °C for 30 s, followed by 50 cycles of denaturing at 95 °C for 5 s and annealing and extension at 60 °C for 30 s. All oligonucleotides were synthesized by Biosearch Technologies. We monitored amplification progress by monitoring the fluorescence at the end of each cycle with an Mx3000P instrument (Stratagene) with excitation and emission wavelengths of 492 and 516 nm (FAM), and 585 and 610 nm (CAL Fluor Red 610).
|
With genomic DNA (5 ng/µL) as a template, we used dual-color scatter plots to distinguish individuals previously determined to have the HP/HP, HP/HPdel, and HPdel/HPdel genotypes. Samples with the HP/HP genotype had little FAM fluorescence and plotted along the x-axis, HPdel/HPdel samples had little CAL Fluor Red 610 fluorescence and plotted along the y-axis, and HP/HPdel samples were located between the homozygote samples in the plot (Fig. 1
). To the TaqMan real-time PCR mixture, we directly added 1 µL of samples diluted 100-fold with PCR-grade water (previously frozen samples of buffy coat from 47 Indonesians from Surabaya or a freshly drawn blood sample from 1 Japanese individual from Fukuoka). Blood was collected in EDTA-containing tubes (Indonesian and Japanese samples) and in a heparin-containing tube (the Japanese sample). The results from 2 independent experiments showed no discrepancies. In addition, the results obtained with the present TaqMan real-time PCR method were fully concordant with those obtained with a previously described PCR method for the same individuals (i.e., 46 HP/HP individuals and 2 Indonesians with HP/HPdel; Fig. 1
)(1). We previously had collected blood samples from 105 Indonesian individuals from Surabaya and had not found the HPdel allele in 58 of these individuals(3); however, in the present study we did find 2 HPdel alleles among the remaining 47 individuals in this population sample. Thus, the HPdel allele is also present in Southeast Asian populations, Thais(4), and Indonesians (this study) at frequencies similar to those of East Asians.
A previous study demonstrated that TaqMan real-time PCR methods can distinguish alleles from samples of dried whole blood(5). In the present study, we instead diluted samples of whole blood 100-fold to decrease the concentrations of substances that could inhibit the PCR reaction or quench the fluorescence signal. This procedure is simple, but a proper assessment of its reliability requires further evaluation with many more samples. Compared with the conventional PCR approach, the present TaqMan real-time PCR assay is simple and rapid (<1.5 h). Moreover, this automated method is cost-effective and amenable to high throughput, eliminates preparation of genomic DNA and post-PCR handling, and prevents contamination via PCR product carryover. Thus, application of this method for routine clinical diagnosis prior to blood transfusions or the infusion of human blood products may prevent anaphylactoid shock caused by anti-HP antibodies. This method may also be useful for detecting HPdel/HPdel homozygotes among donors of blood samples used for preparing blood products, such as albumin for anhaptoglobinemic recipients, and for large-scale screening of HPdel in various populations.
Acknowledgments
Grant/Funding Support: This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan.
Financial Disclosures: None declared.
Acknowledgments: We thank Katherine Ono for editing the English in this manuscript.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  M. Soejima and Y. Koda TaqMan-Based Real-Time PCR for Genotyping Common Polymorphisms of Haptoglobin (HP1 and HP2) Clin. Chem., November 1, 2008; 54(11): 1908 - 1913. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
