CFTR GENOTYPING BY PEPTIDE MASS-SIGNATURE GENOTYPING

通过肽质量特征基因分型进行 CFTR 基因分型

• 批准号: 6294880
• 负责人: EDWIN W NAYLOR
• 金额: $ 9.79万
• 依托单位: PEDIATRIX SCREENING, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6294880
• 关键词: affinity chromatography; chloride channels; genetic screening; genotype; mass spectrometry; molecular cloning; nucleic acid amplification techniques; peptides; protein purification; technology /technique development

Cystic Fibrosis (CF) is the most common, lethal, autosomal recessive disorder. It affects 1 in 2500 births and has a carrier frequency of 1 in 25. CF results from mutations in the cystic fibrosis membrane conductance regulator gene (CFTR). The current paradigm of CF screening and diagnosis is being challenged because the traditional battery of assays - immune reactive trypsinogen, Delta F508 analysis, and sweat chloride testing - are proving to be less reliable than previously thought. CFTR genotyping is playing an increasing role in CF diagnosis. CFTR genotyping services are expensive and typically assay for fewer than 10% of known mutations. Peptide MassSignature Genotyping is an inexpensive, high throughput method for CFTR gentyping. PSMG analysis entails 1. Amplification of genomic DNA encompassing the exons and intron/exon boundaries, 2. Cloning the amplicons in-frame with an epitope tag, 3. Expression of the cloned fragment, 4. Affinity purification through the epitope tag, 5. Analysis of peptide mass by MALDI-TOF, and 6. Computational deconvolution of peptide mass data to determine genetic mutations. The exquisite sensitivity of MALDI-TOF allows for multiplex and parallel analysis. The feasibility of CFTR genotyping by PMSG will be demonstrated using exons 10 and 11 as model systems. PROPOSED COMMERCIAL APPLICATIONS: Essentially all CF centers have CFTR analysis performed on their patients. Offering comprehensive CFTR genotyping using PMSG at approx. 1/3 the cost of services currently available has significant commercial potential. Establishing a CFTR genotyping service will set a precedent to expand PMSG services to other disorders where genotype analysis is a valuable tool.

囊性纤维化是最常见的致死性常染色体隐性遗传病。它影响2500名新生儿中的1名，携带者频率为25名中有1名。囊性纤维性纤维膜电导调节基因(CFTR)突变所致。目前的CF筛查和诊断模式正受到挑战，因为传统的检测方法-免疫反应性胰酶原、Delta F508分析和汗液氯化物检测-被证明不如之前认为的可靠。CFTR基因分型在CF诊断中发挥着越来越重要的作用。Cftr基因分型服务很昂贵，通常只检测不到10%的已知突变。多肽大量签名基因分型是一种廉价、高通量的CFTR分型方法。PSMG分析包括：1.扩增包含外显子和内含子/外显子边界的基因组DNA；2.用表位标签克隆框架内的扩增片段；3.克隆片段的表达；4.通过表位标签进行亲和纯化；5.用MALDI-TOF分析多肽质量；6.计算多肽质量数据以确定基因突变。MALDI-TOF的精致灵敏度允许多路分析和并行分析。本研究将以外显子10和外显子11为模型系统，验证PMSG基因分型方法的可行性。建议的商业应用：基本上所有的CF中心都对他们的患者进行了CFTR分析。使用PMSG提供全面的CFTR基因分型，大约。1/3的现有服务成本具有巨大的商业潜力。建立CFTR基因分型服务将开创先例，将PMSG服务扩展到其他以基因分析为宝贵工具的疾病。

项目成果

Detection of cystic fibrosis mutations by peptide mass signature genotyping.

通过肽质量特征基因分型检测囊性纤维化突变。

• DOI: 10.1373/49.8.1318
• 发表时间: 2003
• 期刊: Clinical chemistry
• 影响因子: 9.3
• 作者: Malehorn,DavidE;Telmer,CherylA;McEwen,SherriB;An,Jiyan;Kinsey,AshleyD;Retchless,AdamC;Mason,Christopher;Vieta,WilliamM;Jarvik,JonathanW
• 通讯作者: Jarvik,JonathanW