Newborn screening for PKU and BH4 responsiveness

描述（由申请人提供）：1958年，Robert Warner博士与Robert古特里博士接触，寻求帮助开发一种更好的方法来测量新生儿血液中的苯丙氨酸。在这种相互作用下，细菌抑制试验诞生了，并开始了基于人群的新生儿苯丙酮尿症（PKU）筛查。早期识别受影响的新生儿避免了不可逆的脑损伤使用苯丙氨酸限制饮食。虽然BIA已被串联质谱法取代作为测量苯丙氨酸的手段，但PKU仍然是通过前瞻性鉴定无症状患者有效治疗的疾病的范例。苯丙氨酸羟化酶（PAH）缺陷导致苯丙氨酸不能转化为酪氨酸。通过饮食方式治疗PKU在很大程度上保持不变，直到几个小组确定了一个使用6 R-四氢生物蝶呤（BH 4）（PAH酶的强制性辅因子）可治疗的患者子集，而无需苯丙氨酸限制饮食。虽然BH 4响应性患者倾向于轻度PKU和高苯丙氨酸贫血;经典PKU患者已被表征为BH 4响应性。PAH基因的分析正在成为确定BH 4反应的一个重要方面。PAH基因的综合分析可以使用普遍收集的新生儿筛查干血卡作为DNA来源进行。使用干血卡和新兴的高分辨率熔解曲线分析技术，PAH的综合分析可以在异常新生儿筛查结果的1.5天内轻松完成。当生理Phe/BH 4负荷试验结果完成时，基因型数据将在手。结合生理学分析和遗传学分析将导致BH 4应答性PKU患者的有效鉴定。导致BH 4反应性疾病的PAH突变的编目正在进行中，因此开发一种灵敏、快速和具有成本效益的方法来分析PAH基因将对临床医生和研究人员有用。本文提出了使用高分辨率熔解曲线来开发一种简化和流线型的方法来评估PAH基因中对mRNA加工至关重要的编码序列和内含子区域。高分辨率解链分析是快速的，灵敏度至少等于DNA序列分析，并超过其他序列前扫描技术。高分辨率熔解曲线分析将在鉴定BH 4应答性PKU患者中发挥作用。

DESCRIPTION (provided by applicant): In 1958, Dr Robert Guthrie was approached by Dr Robert Warner seeking aid to develop a better means to measure phenylalanine in the blood of newborns. Of this interaction was born the bacterial inhibition assay and the beginning of population-based newborn screening for phenylketonuria (PKU). Early identification of affected newborns avoided irreversible brain damage using a phenylalanine-restricted diet. While the BIA has been replaced by tandem mass spectrometry as the means to measure phenylalanine, PKU remains the paradigm for a disorder effectively treated by prospective identification of asymptomatic patients. PKU results from defects in phenylalanine hydroxylase (PAH) causing an inability to convert phenylalanine to tyrosine. Treating PKU by dietary means remained largely unchanged until several groups identified a sub- set of patients treatable using 6R-tetrahydrobiopterin (BH4), the obligatory co-factor of the PAH enzyme, without the phenylalanine restricted diet. While BH4-responsive patients are skewed to mild PKU and hyperphenyla'ianemia; classic PKU patients have been characterized as BH4 responsive. Analysis of the PAH gene is becoming an important aspect to determining BH4 response. Comprehensive analysis of the PAH gene may be performed using the universally collected newborn screening dried blood card as a source of DNA. Using the dried blood card and the emerging technology of high-resolution melt profiling, comprehensive analysis of PAH may be easily completed within 1.5 days of abnormal newborn screening results. Genotypic data will be in hand when results of the physiological Phe/ BH4 loading test are complete. Combining physiological analysis and genetic analysis will lead to effective identification of BH4 responsive PKU patients. Cataloging PAH mutations resulting in BH4 responsive disease is underway thus developing a sensitive, rapidly, and cost effective means to analyze the PAH gene will have utility to clinicians and researchers. Herein is proposed the use of high resolution melt profiling to develop a simplified and streamlined means of assessing the coding sequence and intronic regions critical to mRNA processing in the PAH gene. High resolution melt profiling is rapid with sensitivity at least equal to DNA sequence analysis and in excess of other pre-sequence scanning technologies. High resolution melt profiling will play a role to identify BH4 responsive PKU patients.