Molecular Dissection of the 22q11.2 Deletion Syndrome

22q11.2 缺失综合征 (22q11DS) 是最常见的微缺失疾病。它与 显着的表型和神经精神病理学，两者都有很大的差异。在大多数 在受影响的个体中，由于四个大的介导的异常重组，缺失从头发生。 22q11 中的 22 号染色体特异性低拷贝重复序列（LCRA、-B、-C 和 -D）。它们的规模和数量众多 具有几乎相同序列的片段使得染色体特异性LCR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。 LCR22 由于其结构原因，极难可靠地作图和测序。 特点。目前，该区域不存在准确的参考序列。此外，LCR22 是 难以接受短读长测序，因此其多态性和变异性水平一般 人口未知。然而，使用 BioNano Genomics 的 Irys 技术对该区域进行光学测绘克服了 这个困难。事实上，我们初步的 BioNano 光学测绘数据表明，重复的生物体具有复杂的组织结构。 LCRA 和 LCRD 内的 160kb 模块包括拷贝数和方向差异。此外，一个常见的 借助纤维 FISH 已鉴定出 LCRC 至 LCRD 区域的倒位多态性。反转 似乎大大增加了删除的风险。值得注意的是，我们的初步数据表明这种多态性 非裔美国人 (AA) 中不存在倒置，这最终可以解释我们的 CHOP 中 AA 的相对不足- 基于 22q11DS 队列。在这项拟议的研究中，我们将使用创新的 BioNano 光学测绘技术 确定一般人群中这些 22q11.2 多态性的频率并确定 它们在促进 NAHR 导致删除方面发挥的作用。为了调查这些观察的基础 我们建议确定 LCRC-D 倒置在几个不同人群（CEU、非洲、 以及来自千人基因组计划的非裔美国人受试者；当地非裔美国人和 22q11DS 三人组）。我们 还将详细分析这些相同人群中与 22q11DS 相关的含有 LCR22 的区域，以 确定它们的结构和变异。将定义反转多态性的断点并 将开发 PCR 检测方法，将其作为 NAHR 的危险因素进行筛查。最后，我们建议分析和 PCR 扩增典型 3Mb 22q11.2 缺失的断点，由源自 BioNano 的光学图引导 22q11DS 三人组中的先证者。通过利用长单分子光学提供的更高的灵敏度 纳米通道阵列上的绘图结合 10X Genomics 全基因组序列和分子梳理， 该提案将阐明之前未映射的 LCR22 及其周围的结构和变化 地区。本文生成的数据和图谱将提供对许多其他难以绘制和排序的数据和图谱的访问 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.