Molecular Dissection of the 22q11.2 Deletion Syndrome

22q11.2缺失综合征(22q11DS)是最常见的微缺失障碍。它与以下内容关联 显著的表型和神经精神病理学，两者差异很大。在大多数的 受影响的个体，由于四个大分子介导的异常重组而重新发生缺失 22q11中22号染色体特有的低拷贝重复序列(LCRA、-B、-C和-D)。它们的大小和存在的无数 序列几乎相同的片段将染色体特异的LCRs作为非等位基因的底物 同源重组(Nahr)。Nahr的结果是许多基因组疾病，包括 22q11DS。由于LCR22的结构特点，很难对其进行可靠的定位和测序 特点。目前，还不存在该区域的准确参考序列。此外，LCR22还 顽固的短读测序使它们的多态水平和变异性普遍存在 人口是未知的。然而，使用生物纳米基因组公司的IRYS技术对该区域进行光学测绘可以克服 这一困难。事实上，我们初步的生物纳米光学测绘数据表明，复制的 LCRA和LCRD内的160KB模块，包括副本数量和方向差异。此外，一个常见的 利用FISH技术对LCRC到LCRD区域的倒位多态性进行了鉴定。倒置 似乎极大地增加了删除的风险。值得注意的是，我们的初步数据表明这种多态 在非裔美国人(AAs)中没有倒置，这最终解释了AAs在我们的印章中的相对赤字- 基于22q11DS队列。在这项拟议的研究中，我们将使用创新的生物纳米光学测绘技术 为了确定这些22q11.2多态在普通人群中的频率，并确定 它们在促进Nahr导致删除方面发挥了作用。为了调查这些观察的基础 我们建议确定LCRC-D倒位在几个不同人群(CEU，非洲， 来自1000基因组计划的非洲裔美国人受试者；当地的非洲裔美国人和22q11DS三人组)。我们 还将详细分析这些人群中与22q11DS相关的LCR22包含区域，以 确定它们的结构和变异。将定义反转多态的断点，并使用 将开发一种将其作为NaHr危险因素进行筛查的聚合酶链式反应方法。最后，我们建议分析和 由生物纳米光学图谱引导的典型3MB 22q11.2缺失的断裂点的聚合酶链式反应 22q11DS三重奏中的先行者。通过利用长的单分子光学材料提供的更高的灵敏度 与10X基因组全基因组序列和分子梳理相结合的纳米通道阵列上的映射， 这一建议将阐明LCR22及其周围先前未绘制的结构和变异 地区。这里生成的数据和地图将提供对许多其他难以映射和排序的数据和地图的访问 基因组区域。

The 22q11.2 deletion syndrome (22q11DS) is the most common microdeletion disorder. It is associated with significant phenotypic and neuropsychiatric pathology, both of which are widely variable. In the majority of affected individuals, the deletion occurs de novo as a result of aberrant recombination mediated by four large chromosome 22-specific low copy repeats (LCRA, -B, -C and -D) in 22q11. Their size and the presence of numerous segments with near-identical sequence render chromosome specific LCRs as substrates for non- allelic homologous recombination (NAHR). The results of NAHR are numerous genomic disorders, including the 22q11DS. LCR22s are extremely difficult to reliably map and sequence because of their structural characteristics. Currently, an accurate reference sequence for the region does not exist. Also, LCR22s are recalcitrant to short read sequencing such that the level of their polymorphism and variability in the general population is unknown. However, optical mapping of the region with BioNano Genomics’ Irys technology overcomes this difficulty. In fact, our preliminary BioNano optical mapping data suggests a complex organization of a duplicated 160kb module within LCRA and LCRD that includes copy number and orientation differences. Further, a common inversion polymorphism in the LCRC to LCRD region has been identified with the aid of fiber- FISH. The inversion appears to dramatically increase the risk for deletion. Remarkably, our preliminary data suggests this polymorphic inversion is absent in African Americans (AAs), which would finally explain the relative deficit of AAs in our CHOP- based 22q11DS cohort. In this proposed study, we will use the innovative BioNano optical mapping technology to determine the frequency of these 22q11.2 polymorphisms in the general population and determine the role they play in facilitating NAHR leading to the deletion. In order to investigate the basis of these observations we propose to determine the prevalence of the LCRC-D inversion in several different populations (CEU, African, and African American subjects from the 1000 Genomes Project; local African Americans and 22q11DS trios). We will also analyze in detail the LCR22-containing regions associated with 22q11DS in these same populations to determine their structure and variation. The breakpoints for the inversion polymorphism will be defined and a PCR assay to screen for it as a risk factor for NAHR will be developed. Finally, we propose to analyze and PCR amplify the breakpoint of the typical 3Mb 22q11.2 deletion guided by BioNano optical maps derived from probands in the 22q11DS trios. By leveraging the increased sensitivity afforded by long single molecule optical mapping on nanochannel arrays coupled with 10X Genomics whole- genome sequence and molecular combing, this proposal will elucidate the previously unmapped structure and variation of the LCR22s and surrounding regions. The data and maps generated herein will provide access to many other difficult to map and sequence genomic regions.