Genotyping assays for the most common mutations associated with abacavir hypersen

描述(由申请人提供)：我们建议开发一种邻近患者的检测方法，以获得与阿巴卡韦-严重皮肤不良反应(SCAR)相关的2个SNP；即，HLAB*5701和Hsp70-Hom M493T等位基因(又名。Rs2227956CT)。核酸模板将从使用Trimgen‘s BuccalQuick“(马里兰州斯帕克斯)的面颊拭子中获得的细胞中分离。我们将使用我们专有的解旋酶依赖扩增(HDA)技术开发两种基因分型方法。HDA产品将通过安装在扩张器容器中的侧向流动装置进行检测，以防止实验室受到污染。在第一阶段，我们将实施设计控制，并确保BioHelix获得国际标准化组织13485认证。我们还将利用这一技术平台开发上述SNP基因座的分型分析。最后，我们将与PGXL合作，使用PGXL研究人员收集的一组50个临床标本，相对于黄金标准(DNA测序)验证这些分析的性能。在第二阶段，我们将进行一项前瞻性的多点临床研究，旨在验证这些检测方法是否可以预测SJS和10名服用阿巴卡韦治疗艾滋病的患者的风险。与公共卫生相关：阿巴卡韦(ABC)是一种人类免疫缺陷病毒-1(HIV-1)核苷类似物逆转录酶抑制剂，在Trizivir中与拉米夫定(3TC)和齐多夫定(ZDV)共同配制；每天一片，没有食物限制，药物-药物相互作用有限，具有良好的耐药性。Trizivir与其他HIV药物联合使用，是美国卫生与公众服务部共识小组指南建议的，作为抗逆转录病毒初治患者的可能初始治疗。在Epzicom(注册商标)中，ABV也与拉米夫定联合使用。此外，由于其低成本的(www.who.int/hiv/pub/guidelines/adult/en/index.html).，ABC(以ZiAGEN(R)的名称作为单一药物出售)是发展中国家使用的一线疗法的一部分两个单核苷酸多态(SNP)与阿巴卡韦严重皮肤不良反应(SCAR)高度相关。人类白细胞抗原B*5701是高加索人ABC过敏症的已知遗传风险因素(Martin等人。2005)。Hsp70-Hom M493T等位基因(又名Rs2227956CT)与人类白细胞抗原-B*5701的结合与ABC超敏反应密切相关。尽管存在用于人类白细胞抗原配型的分析方法，但这些方法成本高昂，而且产生的信息比检测ABC超敏反应风险所需的信息更多。例如，靶向DNA测序用于高分辨率分型，而序列特异性引物(SSP)或基于序列特异性寡核苷酸探针(SSOP)的分析用于低分辨率HLA分型。SSP和SOPP分析通常对每个基因座使用20到100个不同的引物或探针。DNA测序仍然是一种复杂且昂贵的DNA诊断方法。我们建议开发的方法将成本更低，并且易于在患者附近的环境中实施。

DESCRIPTION (provided by applicant): We propose to develop a near patient assays to score 2 SNPs associated with abacavir-severe cutaneous adverse reactions (SCAR); i.e., HLA B*5701 and the Hsp70-Hom M493T allele (a.k.a. rs2227956CT). Nucleic acid templates will be isolated from cells obtained from cheek swabs with the Trimgen's BuccalQuick" (Sparks, MD). Two genotyping assays will be developed using our proprietary helicase dependent amplification (HDA). HDA products will be detected with a lateral flow device encased in an amplicon containment vessel that prevents contamination of the laboratory. In Phase I we will Implement design control, and secure ISO 13485 certification for BioHelix. We will also develop typing assays for the aforementioned SNP loci using this technology platform. Finally we will collaborate with PGXL to validate the performance of these assays relative to a gold standard (DNA sequencing) using a set of 50 clinical specimens collected by PGXL researchers. In Phase II, we would perform a prospective, multi-site clinical study aimed at validating the assays as a means of predicting the risk of SJS and TEN in patients being prescribed abacavir for the treatment of AIDS. PUBLIC HEALTH RELEVANCE: Abacavir (ABC), a human immunodeficiency virus-1 (HIV-1) nucleoside- analogue reverse transcriptase inhibitor, is coformulated with lamivudine (3TC ) and zidovudine (ZDV) in Trizivir; a single one a day pill with no food restrictions, limited drug-drug interactions, and a favorable resistance profile. Trizivir in combination with other HIV drugs, is recommended by the US Department of Health and Human Services Consensus Panel Guidelines as a possible initial treatment in antiretroviral-naive patients. ABV is also used in a combo with lamivudine in Epzicom(R). In addition, ABC (sold as a single drug under the name Ziagen(R)) is part of the first-line regimens used in developing countries because of its low cost (www.who.int/hiv/pub/guidelines/adult/en/index.html). Two single nucleotide polymorphisms (SNP) are highly association with abacavir-severe cutaneous adverse reactions (SCAR). HLA B*5701 is a known genetic risk factor for ABC hypersensitivity in Caucasians (Martin et al. 2005). The Hsp70-Hom M493T allele (a.k.a. rs2227956CT) in combination with HLA-B*5701 is strongly associated with ABC hypersensitivity. Although assays exist for HLA typing, these are costly, and yield more information that the strict necessary for detecting the risk of ABC hypersensitivity. For example, targeted DNA sequencing is used for high resolution typing, and sequence-specific primer (SSP), or sequence specific oligonucleotide probe (SSOP) based assays are used for low resolution HLA typing. SSP and SOPP assays typically 20 to 100 different primer sets or probes are used for each locus. DNA sequencing remains a complex and costly method for DNA diagnostics. The method we propose to develop would be lower cost and easy to implement in near patient settings.