Variant detection and variant analysis process for diagnosis of CH and MODY

描述（由申请人提供）：项目摘要/摘要：Correlagen开发和商业化基于DNA的单基因疾病诊断测试，如先天性高胰岛素血症（CH）和年轻人成熟型糖尿病（MODY），在内分泌学和免疫学领域。目前，序列变异检测是基于PCR扩增和基因编码区和内含子/外显子连接的全测序。第一个具体目标是扩展这种变异检测方法，以允许检测基因组缺失以及序列变异。最近的研究表明，基因组缺失以显著的频率引起疾病表型（Eichler，2006; McCarroll，2006;沃尔什，2006）。如果这些缺失跨越整个PCR扩增片段（扩增子），则不能通过基因组DNA的直接桑格DNA序列分析检测到，因为序列仍然是从常染色体基因的另一染色体拷贝获得的。因此，仅通过DNA测序进行的变体检测可能导致假阴性结果。这种局限性对于通常与基因中的大缺失相关的疾病的诊断尤其令人担忧，例如MODY 5（Bellanne-Chantelot，2005）。因此，将开发一种用于缺失检测的标准化定量PCR方案，该方案可以很容易地与Correlagen现有的全测序平台集成。第二个具体目标侧重于改进Correlagen当前的变体评分方法，以创建一个新的多因素评分方案。这种先进的评分方案将用于分析诊断测试期间CH和MODY基因中发现的变异。这些变异中的一些是新发现的，目前被归类为意义不明的变异，限制了它们的诊断价值。将分析各种现有的基于算法的方法来解释变异的意义，以准确地预测变异的已知功能和导致错误预测的模式的能力。将对来自普通人群的DNA样本进行患病率研究，以确定CH和MODY基因的常见多态性，并开发用于评估基因型/表型相关性强度的计算器。基于这些研究，将开发用于评估变体的新算法和遗传参数。与此同时，将开发一个信息技术平台，以跟踪评分协议演变过程中的评分变化。具体目标1和2中提出的研究所产生的改进的变异检测和变异评分方案将被纳入Correlagen目前的服务流程，以提高其遗传疾病诊断测试服务的临床效用。项目叙述/相关性：我们的努力将导致一种系统和可靠的变异检测和分析方法，可应用于Correlagen和我们技术的其他参考实验室许可证测试的其他基因中发现的变异。更广泛地说，这些方法也有可能成为学术界和工业界进行基因检测的标准方法。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Correlagen develops and commercializes DNA-based diagnostic testing for monogenic diseases, such as congenital hyperinsulinism (CH) and maturity-onset diabetes of the young (MODY), in the areas of endocrinology and immunology. Currently, sequence variant detection is based on PCR amplification and full sequencing of gene coding regions and intron/exon junctions. The first specific aim is to expand on this variant detection methodology to allow detection of genomic deletions in addition to sequence variation. Recent studies have shown that genomic deletions cause disease phenotypes at a significant frequency {Eichler, 2006; McCarroll, 2006; Walsh, 2006). Such deletions are not detectable by direct Sanger DNA sequence analysis of genomic DNA if they span an entire PCR-amplification fragment (amplicon) since sequence is still obtained from the other chromosome copy for autosomal genes. Thus, variant detection by DNA sequencing alone can potentially lead to a false negative result. This limitation is of particular concern for the diagnosis of diseases commonly associated with large deletions in a gene, such as MODY5 (Bellanne-Chantelot, 2005). Therefore, a standardized quantitative PCR protocol for deletion detection will be developed that can be easily integrated with Correlagen's existing full-sequencing platform. The second specific aim focuses on refining Correlagen's current variant scoring method to create a new, multi-factorial scoring protocol. This advanced scoring protocol will be used to analyze variants discovered in CH and MODY genes during diagnostic testing. A number of these variants are newly discovered and are currently classified as variants of unknown significance, limiting their diagnostic value. Various existing algorithmic-based methods for interpreting variant significance will be analyzed for their capacity to predict the known function of variants accurately and for patterns leading to false predictions. A prevalence study on DNA samples derived from the general population will be performed to identify common polymorphisms in CH and MODY genes, and calculator for assessing the strength of genotype/phenotype correlations will developed. Based on these studies, new algorithms and genetic parameters for evaluating variants will be developed. Concurrently, an information technology platform will be developed to track changes in scores as the scoring protocol evolves. The improved variant detection and variant scoring protocols resulting from the studies proposed in specific aims 1 and 2 will be incorporated into Correlagen's current service processes to enhance the clinical utility of its testing services for the diagnosis of genetic diseases. Project Narrative/Relevance: Our efforts will lead to a systematic and reliable method of variant detection and analysis that can be applied to variants found in other genes tested by Correlagen and by other reference laboratory licensees of our technology. More broadly, these methods also have the potential to become standard methodology for genetic testing performed in both academia and industry.