New Testing Approach in HLA Genotyping Helps Overcome Barriers in Effective Clinical Practice

Cheng, Suk Hang; Kwan, Patrick; Ng, Ho Keung; Ng, Margaret H.L.

doi:10.1373/clinchem.2009.127894

Cited by 25 publications

(25 citation statements)

References 11 publications

(8 reference statements)

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“…Recent studies have demonstrated that heat-treated blood samples are applicable for LAMP (LAMP-HB) as a template. 16,17 This time-and cost-saving method is now in common use for LAMP analysis. On the other hand, a blood sample diluted with 50 mmol/L NaOH (and heated) is used as a template for our real-time PCR for diagnosis of HP del and isothermal single nucleotide polymorphism genotyping.…”

Section: Resultsmentioning

confidence: 99%

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Soejima

Egashira

Kawano

et al. 2011

The Journal of Molecular Diagnostics

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Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin antibodies. Being homozygous for the haptoglobin gene deletion allele (HP del ) is the only known cause of congenital anhaptoglobinemia, and detection of HP del before transfusion is important to prevent anaphylactic shock. In this study, we developed a loop-mediated isothermal amplification (LAMP)-based screening for HP del . Optimal primer sets and temperature for LAMP were selected for HP del and the 5= region of the HP using genomic DNA as a template. Then, the effects of diluent and boiling on LAMP amplification were examined using whole blood as a template. Blood samples diluted 1:100 with 50 mmol/L NaOH without boiling gave optimal results as well as those diluted 1:2 with water followed by boiling. The results from 100 blood samples were fully concordant with those obtained by real-time PCR methods. Detection of the HP del allele by LAMP using alkaline-denatured blood samples is rapid, simple, accurate, and cost effective, and is readily applicable in various clinical settings because this method requires only basic instruments. In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests. The absence of a serum protein such as IgA or haptoglobin (Hp) is one of the factors that can lead to anaphylactic transfusion reactions due to production of serum antibodies against the absent protein after a transfusion. 1 At present, a homozygous deletion of the haptoglobin gene (HP del ) is the only known cause of anhaptoglobinemia.Human Hp has a genetic polymorphism of two codominant alleles, HP 1 and HP 2 , that give rise to the three common phenotypes, Hp1, Hp2-1, and Hp2. 2,3 The HP 2 allele appears to have occurred by a 1.7-kb intragenic duplication of exons 3 and 4 of the HP 1 allele ( Figure 1A). Anomalous inheritance of the Hp phenotypes was encountered during determinations of parentage, and HP del was identified by genetic analyses of one such family in Japan. 4 The HP del allele lacks an approximately 28-kb segment of chromosome 16 extending from the promoter region of the HP gene to intron 4 of the haptoglobinrelated gene (HPR) ( Figure 1A). 4,5 The HP del allele has been found only in East and Southeast Asian populations (Chinese, Korean, Japanese, Mongols, Thais, and Indonesians), not in African, West and South Asian, and European populations so far. [5][6][7][8][9] Detection of homozygosity for HP del before blood transfusion or blood component infusion is important to prevent severe side effects of transfusion because washed red blood cells and platelet concentrate do not cause transfusion-related anaphylactic reactions. 10 Recently, we established two real-time PCR methods for detection of HP del by use of a 5=-nuclease assay using dual-labeled (TaqMan; Applied Biosystems, Foster City, CA) probes and SYBR Green I (Invitrogen, Carlsbad, CA). 11,12 These methods are rapid, robust, and suitable for high-throughput analysis but req...

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Section: Resultsmentioning

confidence: 99%

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Soejima

Egashira

Kawano

et al. 2011

The Journal of Molecular Diagnostics

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“…10 To meet the increasing need for fast and easily performable genotyping assays, loop mediated isothermal amplification (LAMP) is a promising tool. [12][13][14] LAMP method has the advantages of reaction simplicity and amplification efficiency when compared with PCR technology. 15 Since the HBV genotypes B and C were the predominant strains in East Asia, we aimed to develop a LAMP method for genotyping and quantification of HBV genotypes B and C simultaneously in this study.…”

Section: Introductionmentioning

confidence: 99%

Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification

Cai

Lou

Cai

et al. 2011

Journal of Clinical Virology

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“…However, current HLA genotyping methods are hard to apply in a clinical setting [4]. For example, sequencebased typing (SBT) or PCR with sequence-specific oligonucleotide hybridization methods yield very precise and high resolution data, but these methods require the effort of many intensive steps and long turnaround times [5,6].…”

mentioning

confidence: 99%

“…As drug hypersensitivity is related to specific HLA allele, pharmacogenetic testing is required to detect one HLA allele specifically [1,4]. Based on this concept, real-time PCR using TaqMan or hybridization probe [7], and the loop-mediated isothermal amplification (LAMP) method [4] have been proposed for pharmacogenetic testing.…”

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confidence: 99%

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Development of new HLA-B*3505 genotyping method using Invader assay

Hosono

Chantarangsu

Kiyotani

et al. 2010

Pharmacogenetics and Genomics

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Several pharmacogenetic studies have revealed strong associations between specific human leukocyte antigen (HLA) alleles and the susceptibility to drug hypersensitivity. Recently, we reported HLA-B*3505 as a strong genetic biomarker for the nevirapine-induced skin rash in Thai population. Here, we developed a new HLA-B*3505 genotyping method by a combination of the Universal Invader assay and sequence-specific primer PCR. From the sequence alignment of 68 HLA-B alleles in the Thai population, we selected the two most discriminative SNPs (rs1140412 and rs4997052) as target SNP sites. When we carried out the assay using 324 Thai individuals, fluorescence intensities of HLA-B*3505-positive and HLA-B*3505-negative samples were apparently discriminated at the endpoint of the reaction. Our results were 100% concordant with those obtained by a sequence-based typing method. As our assay is simple and rapid, we believe our method will be a useful tool for pharmacogenetic testing of the nevirapine-induced skin rash.

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New Testing Approach in HLA Genotyping Helps Overcome Barriers in Effective Clinical Practice

Cited by 25 publications

References 11 publications

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

Development of a novel genotype-specific loop-mediated isothermal amplification technique for Hepatitis B virus genotypes B and C genotyping and quantification

Development of new HLA-B*3505 genotyping method using Invader assay

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