Definition of chromosomal abnormalities by next generation sequencing

描述(申请人提供)：在历史上，染色体重排是通过传统的染色体组型进行研究的，随后是艰难的定位克隆计划。最近，利用荧光原位杂交、阵列比较基因组杂交和SNP基因分型以及反向聚合酶链式反应和定量聚合酶链式反应等分子方法的分子细胞遗传学特征，使得对染色体异常的评估变得越来越准确，结果是一些可能的平衡重排被发现包括缺失、重复、插入或倒置。我将测试一种新的方法：有针对性的基因组捕获，然后进行下一代测序，以快速而准确地确定一组临床上有趣的病例中的分子异常。这些病例是在约翰·霍普金斯遗传医学研究所的遗传学诊所进行跟踪的。3例已知有易位的复杂畸形综合征患者：第一例(L982181家系)分离与颅面畸形相关的三代明显平衡的t(2；3)(p15；q12)易位；第二例(患者JHU2010)有t(2；6)(q22；p12.3)易位和染色体6p上RUNX2区域的断裂点，与锁骨颅骨发育不良相关(OMIM 119600)；第三例(患者L08-2709)有t(5；17)(q23.2；Q24)易位，在染色体17q上SOX9上游区域有一个断裂点，与无脐带性脐带发育不良(OMIM 114290)的临床表型相关。此外，我将研究Valle实验室收集的三个10q21-q23区域缺失的病例，这是他们在该区域识别精神分裂症易感基因的持续兴趣的一部分。它们包括我的系列中的第四位患者(JHU2020)，他的10q23.1缺失与多发性异常和发育延迟有关；第五和第六位患者涉及两个重叠缺失的家庭(JHU2000和JHU88293)，这两个家系都在10q21.1上使PCDH15失活，并与精神分裂症有关。对这些复杂重排的描述将有助于更好地了解其临床表型的分子基础。在例5和例6中，PCDH15作为精神疾病的候选基因的研究很有意义，因为它是这两个家族中唯一缺失的注释基因(JHU2000和JHU88293)。PCDH15的隐性截断突变导致Usher1F型(OMIM 602083)，其特征是先天性听力损失、前庭功能障碍和色素性视网膜病变。有趣的是，一份早期报告《基因座异质性预评估》显示，近20%的亚瑟综合征患者会发展成精神病[4]。 与公共卫生相关：新的方法表明，染色体异常比以前认识到的更频繁(例如CNV)；而且通常比以前通过检查认识到的更复杂，根据核型明显平衡的易位通常在边缘有相关的缺失或复制。我对利用染色体异常来识别与人类遗传病有关的基因很感兴趣，并一直在使用FISH、SNP基因分型、qRT-PCR、反向PCR、长程PCR和其他方法来定义易位和缺失的确切断点，但有时它们不允许在1个碱基对的分辨率下识别断点。我将测试一种新的方法：有针对性的基因组捕获，然后对捕获的材料进行下一代测序(100bp配对末端读取)，以快速和准确地确定在约翰霍普金斯大学麦库西克-纳桑斯遗传医学研究所(IGM)遗传学诊所跟踪的一组临床有趣病例中的分子异常。

DESCRIPTION (provided by applicant): Historically, chromosomal rearrangements have been investigated by conventional karyotype followed by arduous positional-cloning projects. More recently, molecular cytogenetic characterizations using fluorescence in situ hybridization, array comparative genomic hybridization, and SNP genotyping together with molecular methods such as inverse PCR and quantitative PCR have allowed an increasingly more precise evaluation of chromosome abnormalities with the result that some presumably balanced rearrangements have been found to include deletions, duplications, insertions or inversions. I will test a new approach: targeted genomic capture followed by next generation sequencing to rapidly and precisely define the molecular abnormalities in a set of clinically interesting cases followed in the Genetics clinics of the Johns Hopkins Institute of Genetic Medicine. 3 cases with complex dysmorphic syndromes already known to have translocations: the first (family L982181) is segregating an apparently balanced t(2;3)(p15;q12) translocation over 3 generations associated with craniofacial abnormalities; the second (patient JHU2010) has a t(2;6)(q22;p12.3) translocation and a breakpoint in the region of RUNX2 on chromosome 6p, associated with cleidocranial dysplasia (OMIM 119600); and the third (patient L08-2709) has a t(5;17)(q23.2;q24) translocation, with a breakpoint in the region upstream of the SOX9 on chromosome 17q, associated with the clinical phenotype of acampomelic campomelic dysplasia (OMIM 114290). Additionally, I will study three cases with deletions in the 10q21-q23 region that have been collected by the Valle lab as part of their ongoing interest in identification of schizophrenia susceptibility genes in this region. They include the fourth patient in my series (JHU2020), who has a 10q23.1 deletion associated with multiple anomalies and development delay; the fifth and sixth involve 2 families (JHU2000 and JHU88293) with overlapping deletions both of which inactivate PCDH15 on 10q21.1 and are associated with schizophrenia. The delineation of these complex rearrangements will lead to a better understanding of the molecular bases of their clinical phenotypes. In cases 5 and 6, the study of PCDH15 as a candidate gene for psychiatric disease is of interest because it is the only annotated gene that is deleted in both families (JHU2000 and JHU88293). Recessive truncating mutations in PCDH15 cause Usher type 1F (OMIM 602083) characterized by congenital hearing loss, vestibular dysfunction and pigmentary retinopathy. Interestingly, an early report, pre-appreciation of locus heterogeneity, suggested nearly 20% of patients with Usher syndrome develop psychosis [4]. PUBLIC HEALTH RELEVANCE: New methods have shown that chromosomal aberrations are more frequent than previously realized (e.g. CNV); and are often more complex than previously appreciated by examination, apparently balanced translocations by karyotype often have associated deletion or duplications at the margins. I am interested in taking advantage of chromosomal aberrations to identify genes responsible for human genetic disease and have been using methods such as FISH, SNP genotyping, qRT-PCR, inverse PCR, long range PCR, and others to define the exactly breakpoints of translocations and deletions but sometimes they do not allow the identification of breakpoints at the resolution of 1 base pair. I will test a new approach: targeted genomic capture followed by next generation sequencing (100 bp paired end reads) of the captured material to rapidly and precisely define the molecular abnormalities in a set of clinically interesting cases followed in the Genetics clinics of the Johns Hopkins McKusick-Nathans Institute of Genetic Medicine (IGM).