Sequence Variations in Low Copy Repeats on 22q11.2

描述（由申请人提供）：低拷贝重复序列（LCR;也称为节段性重复）约占人类基因组的5%，并且已知介导与精神发育迟滞疾病相关的染色体重排。我们计划的长期目标是以22q11.2区域为模型，评估LCR赋予基因组不稳定性的作用。两个240 kb低拷贝重复序列（称为LCR 22 -2和LCR 22 -4，相距3 Mb）之间的非等位基因同源重组（NAHR）事件导致几种基因组疾病，包括1/4，000活产婴儿中发生的腭心面/DiGeorge综合征（VCFS/DGS）。我们的假设，根据初步数据，是有集群的共享多态性序列（SPS）之间的两个LCR 22，代表增强的历史基因转换事件的间隔。这些区域可能作为重组热点负责NAHR事件。这些簇侧翼的序列对于一个LCR或另一个LCR是独特的，并且将由旁系同源序列变体（PSV）标记。基于PSV的标记可用于对VCFS/DGS家族进行分型，以缩小LCR 22中的缺失端点，从而鉴定将来重排的热点。为了验证我们的假设，我们建议从现有BAC文库中的22号染色体的额外正常等位基因产生LCR 22 -2和-4的完成序列，大约4.4 Mb的序列。在具体目标1中，我们将通过筛选含有来自不同文库的BAC克隆的过滤器生成基于BAC的物理图谱，获得末端序列并用PCR标记分型，来表征LCR 22 -2和LCR 22 -4的模块化结构域组织。两个LCR 22内的结构和序列变异尚未确定。在特定目标2中，将对包含5个等位基因的BAC克隆的最小平铺路径进行测序，并生成包含所有插入、缺失或倒位以及核苷酸多态性的变异图谱。在特定目标3中，我们将检验LCR 22内存在历史基因转换的不同区域的假设，并将定义这些区域侧翼的一组PSV标记。PSV标记物将在未来用于对我们的250名具有典型的3 Mb缺失的VCFS/DGS患者及其正常父母进行分型，以鉴定基因组不稳定性的特征。在序列水平上理解22q11.2重排的基础可以作为其他新发现的基因组疾病的模型，例如22q11.2和基因组其他地方的相互复制障碍。此外，LCR 22的详细地图将作为未来利用下一代方法的计划的理想模板。 公共卫生相关性：染色体22q11.2区域与多种发育障碍相关。我们建议通过DNA序列分析来确定22q11.2上的不稳定区域。这将使我们能够理解为什么染色体可以在减数分裂过程中重新排列，导致DNA拷贝数的丢失或增加，从而导致出生缺陷。

DESCRIPTION (provided by applicant): Low copy repeats (LCRs; also known as segmental duplications) constitute roughly 5% of the human genome and are known to mediate chromosome rearrangements associated with mental retardation disorders. The long-term goal of our program is to assess the roles in which LCRs confer genome instability using the 22q11.2 region as a model. Non-allelic homologous recombination (NAHR) events between two, 240 kb low copy repeats, termed LCR22-2 and LCR22-4, mapping 3 Mb apart, lead to several genomic disorders including velo-cardio-facial/DiGeorge syndrome (VCFS/DGS), occurring in 1/4,000 live births. Our hypothesis, based upon preliminary data, is that there are clusters of shared polymorphic sequences (SPSs) between the two LCR22s, representing intervals of enhanced historic gene conversion events. Such regions might serve as recombination hotspots responsible for NAHR events. Sequences flanking these clusters would be unique to one LCR or the other and would be marked by paralogous sequence variants (PSVs). PSV based markers could be used to then type VCFS/DGS families to narrow deletion endpoints in the LCR22s to identify hotspots for rearrangements in the future. Although the complete sequence of the two LCR22s is available for two alleles of chromosomes 22 and it allowed us to generate this hypothesis, it is insufficient to test it. To test our hypothesis, we propose to generate the finished sequence of LCR22-2 and -4 from additional normal alleles of chromosome 22 from existing BAC libraries, roughly 4.4 Mb of sequence. In Specific Aim 1, we will characterize the modular domain organization of LCR22-2 and LCR22-4, by generating BAC based physical maps by screening filters containing BAC clones from different libraries, obtaining end sequence and typing with PCR markers. Structural and sequence variation within the two LCR22s has not been defined. In Specific Aim 2, the minimal tiling path of BAC clones encompassing five alleles will be sequenced and a variation map containing all the insertions, deletions or inversions as well as nucleotide polymorphisms, will be generated. In Specific Aim 3, we will test the hypothesis that there are varied regions of historic gene conversion within the LCR22s and will define a set of PSV markers flanking these regions. The PSV markers will be used in the future to type our cohort of 250 VCFS/DGS patients with the typical 3 Mb deletion and their normal parents to identify signatures of genomic instability. Understanding the basis for 22q11.2 rearrangements on the sequence level can serve as a model for other newly discovered genomic disorders, such as the reciprocal duplication disorder on 22q11.2 and elsewhere in the genome. In addition, the detailed maps of the LCR22s will serve as an ideal template for future programs utilizing next generation approaches. PUBLIC HEALTH RELEVANCE: The chromosome 22q11.2 region is associated with multiple developmental disorders. We propose to define unstable regions on 22q11.2 by DNA sequence analysis. This will enable us to understand why chromosomes can rearrange during meiosis resulting in loss or gains in DNA copy number causing birth defects.