COMPREHENSIVE CYP21 GENOTYPING

男性化先天性肾上腺增生症（CAH）描述了一组涉及从胆固醇到皮质醇途径的类固醇生成疾病。95%的CAH源于CYP21基因的改变。盐消耗形式的CAH在新生儿时期可能是致命的，而非经典形式的CAH在生命后期会引起健康问题。CAH的筛查包括测量17-羟孕酮。在筛查CAH时，17-OHP的临界值过高，导致许多可治疗的CAH无法被检测到。如果降低临界值并进行CYP21突变的分子测定，这些CAH病例将被识别。分子分析消除假阳性，同时确定受影响的个体。荧光共振能量转移（FRET）探针的快速循环PCR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8 bp del 706-713加上缺失/重复事件。使用肽质量特征基因分型（PSMG）进行全面的CYP21分析。PSMG包括扩增产物的表达，通过MALDI-TOF对表达肽进行质量测定，以及对质量数据进行计算反褶积以确定遗传变化。CYP21外显子8是模型系统。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CYP21剂量和突变分析允许改进CAH筛选程序。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学家。对于内分泌学家来说，CYP21基因分型将有很大的市场，因为他们看到的患者通常是谜一般的轻度17-OHP升高，以及在一般人群中常见的推定迟发性非典型性CAH。

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.