Sequence Variations in Low Copy Repeats on 22q11.2

描述（由申请人提供）：低拷贝重复（lcr，也称为片段重复）约占人类基因组的5%，已知介导与智力迟钝障碍相关的染色体重排。我们项目的长期目标是使用22q11.2区域作为模型来评估lcr赋予基因组不稳定性的作用。两个240 kb低拷贝重复序列LCR22-2和LCR22-4之间的非等位基因同源重组（NAHR）事件，相隔3 Mb，导致几种基因组疾病，包括velo-cardio-facial/DiGeorge综合征（VCFS/DGS），发生在1/4,000活产婴儿中。基于初步数据，我们的假设是，两个lcr22之间存在共享多态性序列（SPSs）集群，代表了增强的历史基因转换事件的间隔。这些区域可能是负责NAHR事件的重组热点。这些集群两侧的序列对于一个LCR或另一个LCR来说是唯一的，并且会被旁系序列变体（psv）标记。基于PSV的标记可用于对VCFS/DGS家族进行分型，以缩小lcr22中的缺失端点，从而确定未来重排的热点。虽然22号染色体的两个等位基因有两个LCR22s的完整序列，这使我们能够产生这个假设，但不足以对其进行验证。为了验证我们的假设，我们建议从现有BAC文库中额外的22号染色体正常等位基因中生成LCR22-2和-4的完整序列，大约有4.4 Mb的序列。在Specific Aim 1中，我们将通过筛选来自不同文库的含有BAC克隆的过滤器，获得末端序列并使用PCR标记分型，生成基于BAC的物理图谱，从而表征LCR22-2和LCR22-4的模块化结构域组织。两个LCR22s的结构和序列变化尚未确定。在Specific Aim 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、缺失或反转以及核苷酸多态性的变异图。在Specific Aim 3中，我们将验证lcr22中存在不同历史基因转换区域的假设，并将定义一组位于这些区域两侧的PSV标记。PSV标记将在未来用于对250名典型3mb缺失的VCFS/DGS患者及其正常父母进行分型，以确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互惠重复疾病。此外，lcr22的详细地图将作为利用下一代方法的未来计划的理想模板。

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.