CFTR GENOTYPING BY PEPTIDE MASS-SIGNATURE GENOTYPING

囊性纤维化（CF）是最常见的、致死性的常染色体隐性遗传病。每2500个新生儿中就有1人感染，携带者频率为25分之一。CF是由囊性纤维化膜传导调节基因（CFTR）突变引起的。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——免疫反应性胰蛋白酶原、Delta F508分析和汗液氯化物检测——被证明不如以前认为的可靠。CFTR基因分型在CF诊断中的作用越来越大。CFTR基因分型服务价格昂贵，通常只检测不到10%的已知突变。Peptide MassSignature基因分型是一种廉价、高通量的CFTR基因分型方法。PSMG分析需要1。包括外显子和内含子/外显子边界的基因组DNA扩增；带表位标签的帧内扩增子的克隆；3 .克隆片段的表达；4 .通过表位标签进行亲和纯化；MALDI-TOF分析多肽质量；计算反卷积的肽质量数据，以确定基因突变。MALDI-TOF的灵敏度很高，可以进行多路并行分析。以外显子10和11为模型系统，验证PMSG进行CFTR基因分型的可行性。建议的商业应用：基本上所有CF中心都对其患者进行CFTR分析。使用PMSG提供全面的CFTR基因分型。现有服务成本的1/3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服务扩展到基因型分析是有价值工具的其他疾病。

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.