Mutational Analysis of Peroxisome Biogenesis Disorders

描述（由申请人提供）：过氧化物酶体生物发生障碍（PBDs）是一种先天性代谢错误，具有相当大的临床、生化和遗传异质性。合理设计有效治疗这些疾病的方法需要精确识别每个PBD患者的致病分子缺陷。然而，PBD患者的诊断是耗时的，劳动密集型的，并且高度专业化，因为它需要建立成纤维细胞培养并对每个被测试的个体进行生化脂质分析。我们的目标是快速有效地确定pbd的遗传基础，以提高识别和开发对每位患者最有效的治疗方法的可能性。我们假设高通量突变检测和基因表达分析可以快速有效地诊断pbd的分子基础。为了验证我们的假设，我们将从Zellweger综合征-新生儿肾上腺白质营养不良-婴儿Refsum病（ZS-NALDIRD） PBDs谱患者的基因组DNA中识别致病突变，并分析其在已知突变患者培养成纤维细胞中的活性。在本提案中，我们将开发新的基于寡核苷酸微阵列的诊断方法，以快速有效地从患者血液样本中筛选5个基因（PEX1, PEX6, PEX10， PEX12和PEX26）的突变，这5个基因占ZS-NALD-IRD病例的90%以上（Specific Aim 1）。我们将通过对已知这些基因突变的ZS-NALD-IRD患者进行盲法突变分析来确定这些检测的特异性和敏感性。在这一验证步骤之后，我们将使用这些微阵列分析来发现导致100名ZS-NALD-IRD患者临床表型的遗传缺陷。此外，我们将进行功能分析，以确定错义突变的功能意义，以便更好地将患者基因型与临床表型联系起来（Specific Aim 2）。首先，我们将根据这些氨基酸在其他物种中的保守性和一般人群中的等位基因频率来优先分析错义变化。随后，我们将构建慢病毒载体，将野生型和突变的PEX基因转导到由于过氧化物酶体组装缺陷而缺乏功能性过氧化物酶体的PEX1、PEX6、PEX10、PEX12和PEX26零细胞系中。我们将通过筛选适当转导的零细胞系中过氧化物酶体的外观来确定含有错义变化的PEX基因相对于其野生型对应物的功能活性。总的来说，这些突变检测（Specific Aim 1）和功能检测（Specific Aim 2）将为ZS-NALD-IRD谱内患者的精确分子诊断和表征提供宝贵的资源。除了提供合理设计治疗所需的遗传信息外，这些高通量基因检测将允许对这些个体的临床结果进行更准确的预测，并促进未来怀孕的产前诊断。

DESCRIPTION (provided by applicant): Peroxisome biogenesis disorders (PBDs) are inborn errors of metabolism with considerable clinical, biochemical, and genetic heterogeneity. The rational design of effective treatments for these disorders requires the precise identification of the causative molecular defects in each PBD patient. However, the diagnosis of PBD patients is time consuming, labor intensive, and highly specialized since it requires establishing fibroblast cultures and performing biochemical lipid analyses for every individual tested. Our goal is to rapidly and efficiently determine the genetic basis for PBDs in order to improve the likelihood of identifying and developing therapies that are most effective for each patient. We hypothesize that high-throughput mutation detection and gene expression assays can rapidly and efficiently diagnose the molecular basis for PBDs. To test our hypothesis, we will identify causative mutations in genomic DNA from patients within the Zellweger syndrome-neonatal adrenoleukodystrophy-infantile Refsum disease (ZS-NALDIRD) spectrum of PBDs and analyze their activity in cultured fibroblasts from patients with known mutations. In this proposal, we will develop novel oligonucleotide microarray-based diagnostics to rapidly and efficiently screen genomic DNA from patient blood samples for mutations in five genes (PEX1, PEX6, PEX10, PEX12, and PEX26) which account for over 90% of ZS-NALD-IRD cases (Specific Aim 1). We will determine the specificity and sensitivity of these assays by blinded mutational analyses of ZS-NALD-IRD patients with known mutations in these genes. After this validation step, we will use these microarray assays to uncover the genetic defects responsible for clinical phenotypes in one hundred ZS-NALD-IRD patients. Furthermore, we will conduct functional assays to determine the functional significance of missense mutations in order to better correlate patient genotype with clinical phenotypes (Specific Aim 2). First, we will prioritize missense changes for analysis based on the conservation of these amino acids in other species and allele frequencies in the general population. Afterwards, we will construct lentiviral vectors that can transduce wild type and mutated PEX genes into PEX1, PEX6, PEX10, PEX12, and PEX26 null cell lines that lack functional peroxisomes due to defects in peroxisome assembly. We will determine the functional activity of PEX genes containing missense changes relative to their wild type counterparts by screening for the appearance of peroxisomes in the appropriately transduced null cell lines. Overall, these mutation detection (Specific Aim 1) and functional (Specific Aim 2) assays will provide valuable resources for the precise molecular diagnosis and characterization of patients within the ZS-NALD-IRD spectrum. In addition to providing the genetic information needed for the rational design of therapies, these high-throughput genetic tests will allow for more accurate predictions of clinical outcomes for these individuals and facilitate prenatal diagnosis in future pregnancies.