COMPREHENSIVE CYP21 GENOTYPING

病毒性先天性肾上腺增生症(CAH)描述了一组类固醇合成障碍，涉及从胆固醇到皮质醇的途径。95%的慢性肝炎是由细胞色素P21基因的改变引起的。盐耗型CAH在新生儿期可能是致命的，而非经典性CAH会在晚年导致健康问题。筛查CAH需要测定17-羟孕酮。在CAH的筛查中，17-OHP的截止水平如此之高，以至于许多可治疗的CAH都没有被检测到。如果降低截止水平并进行CYP21突变的分子检测，这些CAH病例就会被识别出来。分子分析消除了假阳性，同时识别了受影响的个体。快速循环聚合酶链式反应结合荧光共振能量转移(FRET)探针分析是突变和基因剂量分析的一种创新方法。快速循环PCR和FRET分析将用于检测CYP21突变I172N、I2和8bpdel 706-713加上缺失/复制事件。使用多肽质量签名基因分型(PSMG)进行全面的CYP21分析。PSMG包括扩增产物的表达，MALDI-TOF对表达多肽的大量测定，以及大量数据的计算去卷积来确定遗传变化。以CYP21外显子8为模型系统。突变、基因剂量和PSMG分析的模型系统将证明CYP21基因分型服务的可行性。建议的商业应用：快速的CyP21剂量和突变分析允许改进CAH筛查计划。这项服务将在内部使用，并提供给其他筛查项目、儿科医生和儿科内分泌科医生。对于内分泌学家来说，CYP21基因分型将发现一个很大的市场，内分泌学家看到的患者通常患有神秘的轻度17-OHP升高和推测的迟发性非经典型CAH，这些在普通人群中很常见。

Virilizing congenital adrenal hyperplasia (CAH) describes a group of disorders of steroidogenesis involving the pathway from cholesterol to cortisol. Ninety-five percent of CAH results from alteration of the CYP21 gene. The salt wasting form of CAH may be fatal in the neonatal period while non-classical forms cause health problems late in life. Screening for CAH involves measuring 17-hydroxyprogesterone. In screening for CAH, cut-off levels for 17-OHP are held so high that many treatable forms of CAH are not detected. These cases of CAH would be identified if cut-off levels were lowered and a molecular assay for CYP21 mutations was performed. Molecular analysis eliminates false positives while identifying affected individuals. Rapid cycle PCR with analysis of fluorescence resonance energy transfer (FRET) probes is an innovative approach to mutation and gene dosage analysis. Rapid cycle PCR and FRET analysis will be used to detect CYP21 mutations I172N, I2, and 8 bp del 706-713 plus deletion/duplication events. Comprehensive CYP21 analysis is performed using Peptide Mass-Signature Genotyping (PSMG). PSMG involves expression of amplification products, mass determination of expression peptides by MALDI-TOF, and computational deconvolution of mass data to determine genetic changes. CYP21 exon 8 is the model system. The model systems for mutational, gene dosage, and PSMG analysis will demonstrate feasibility of a CYP21 genotyping service. PROPOSED COMMERCIAL APPLICATIONS: Rapid CYP21 dosage and mutation analysis allows for an improved CAH screening program. This service will be used in-house and offered to other screening programs, pediatricians, and pediatric endocrinologists. CYP21 genotyping will find a large market to endocrinologists seeing patients with the often enigmatic mild 17-OHP elevations and putative late-onset non-classical forms of CAH which are common in the general population.