Newborn screening for PKU and BH4 responsiveness

描述（由申请人提供）：1958年，Robert Warner博士找到Robert Guthrie博士寻求帮助，以开发一种更好的方法来测量新生儿血液中的苯丙氨酸。这种相互作用产生了细菌抑制试验和基于人群的新生儿苯丙酮尿症（PKU）筛查的开始。通过限制苯丙氨酸的饮食，早期识别受影响的新生儿可以避免不可逆的脑损伤。虽然BIA已被串联质谱法取代，成为测量苯丙氨酸的手段，但PKU仍然是通过前瞻性识别无症状患者有效治疗疾病的范例。PKU是由苯丙氨酸羟化酶（PAH）缺陷引起的，它不能将苯丙氨酸转化为酪氨酸。通过饮食治疗PKU的方法基本保持不变，直到几个小组确定了一组可以使用6r -四氢生物蝶呤（BH4）治疗的患者，这是多环芳烃酶的强制性辅助因子，而不需要苯丙氨酸限制饮食。而bh4反应性患者倾向于轻度PKU和高苯贫血；典型的PKU患者具有BH4反应性。多环芳烃基因的分析正成为确定BH4反应的一个重要方面。使用普遍收集的新生儿筛查干血卡作为DNA来源，可以进行多环芳烃基因的综合分析。利用干血卡和新兴的高分辨率熔融剖面技术，在新生儿异常筛查结果出现后1.5天内即可轻松完成对多环芳烃的全面分析。当生理Phe/ BH4负荷测试结果完成后，基因型数据将到手。将生理分析与遗传学分析相结合，可有效鉴别BH4反应性PKU患者。对导致BH4反应性疾病的多环芳烃突变进行编目正在进行中，因此开发一种敏感、快速、经济有效的方法来分析多环芳烃基因，将对临床医生和研究人员有实用价值。本文建议使用高分辨率熔体分析来开发一种简化和流线的方法来评估PAH基因中mRNA加工的关键编码序列和内含子区域。高分辨率熔体分析速度快，灵敏度至少与DNA序列分析相当，超过其他序列前扫描技术。高分辨率熔体分析将在确定BH4反应性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 While the BIA has been replaced by tandem mass spectrometry as the means to measure phenylalanine, PKU remains the paradigm for a disorder effectively treated by prospective identification of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.