COMPREHENSIVE CYP21 GENOTYPING

男性化先天性肾上腺皮质增生（CAH）是一组涉及胆固醇向皮质醇代谢途径的类固醇生成障碍。95%的CAH是由CYP 21基因改变引起的。CAH的盐耗形式在新生儿期可能是致命的，而非经典形式在生命后期引起健康问题。CAH的筛查包括测量17-羟孕酮。在CAH的筛查中，17-OHP的截止水平太高，以至于许多可治疗的CAH形式无法检测到。如果降低临界水平并进行CYP 21突变的分子测定，则可以识别这些CAH病例。分子分析消除了假阳性，同时确定受影响的个人。荧光共振能量转移（FRET）探针分析的快速循环PCR是突变和基因剂量分析的创新方法。快速循环PCR和FRET分析将用于检测CYP 21突变I172 N、I2和8 bp del 706-713加缺失/重复事件。使用肽质量特征基因分型（PSMG）进行全面的CYP 21分析。PSMG涉及扩增产物的表达、通过MALDI-TOF的表达肽的质量测定以及质量数据的计算解卷积以确定遗传变化。CYP 21外显子8是模型系统。用于突变、基因剂量和PSMG分析的模型系统将证明CYP 21基因分型服务的可行性。拟议的商业应用：快速CYP 21剂量和突变分析允许改进CAH筛查程序。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。CYP 21基因分型将为内分泌学家发现一个巨大的市场，这些内分泌学家看到的患者通常具有神秘的轻度17-OHP升高和推定的晚发型非经典形式的CAH，这些CAH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.