Definition of chromosomal abnormalities by next generation sequencing

通过下一代测序定义染色体异常

• 批准号: 8063822
• 负责人: Nara Sobreira
• 金额: $ 3.84万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063822
• 关键词: 10q21; 10q21.1; 17q; Base Pairing; Candidate Disease Gene; Chromosomal Rearrangement; Chromosome abnormality; Chromosomes; Cleidocranial Dysplasia; Clinic; Complex; Craniofacial Abnormalities; Cytogenetics; DNA Sequence Rearrangement; Detection; Development; Developmental Delay Disorders; Disease; Equilibrium; Evaluation; Family; Fluorescent in Situ Hybridization; Functional disorder; Generations; Genes; Genetic; Genetic Medicine; Genomics; Hereditary Disease; Heterogeneity; Human; Human Genetics; Hybridization Array; Institutes; Inverse Polymerase Chain Reaction; Karyotype; Lead; Mental disorders; Methods; Molecular; Molecular Abnormality; Molecular Cytogenetics; Mutation; NRG3 gene; Online Mendelian Inheritance In Man; Patients; Phenotype; Predisposition; Psychotic Disorders; Reading; Reporting; Resolution; Resources; Retinitis Pigmentosa; SNP genotyping; Schizophrenia; Series; Susceptibility Gene; Syndrome; Testing; Translocation Breakpoint; Usher Syndrome; base; campomelic dysplasia; clinical phenotype; comparative genomic hybridization; hearing impairment; interest; malformation; next generation; novel strategies; positional cloning; proband

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).

描述（由申请人提供）：历史上，染色体重排已通过常规核型研究，然后进行艰苦的定位克隆项目。最近，使用荧光原位杂交、阵列比较基因组杂交和SNP基因分型以及分子方法如反向PCR和定量PCR的分子细胞遗传学表征已经允许越来越精确地评估染色体异常，结果发现一些可能平衡的重排包括缺失、重复、插入或倒位。 我将测试一种新的方法：靶向基因组捕获，然后进行下一代测序，以快速准确地确定约翰霍普金斯遗传医学研究所遗传学诊所的一组临床有趣病例中的分子异常。3例已知存在易位的复杂畸形综合征：第一例（家族L982181）是分离一个明显平衡的t（2;3）（p15;q12）易位超过3代与颅面畸形;第二个（患者JHU 2010）具有t（2;6）染色体6p上RUNX 2区域的（q22;p12.3）易位和断裂点与锁骨颅骨发育不良相关第三个（患者L08-2709）具有t（5;17）（q23.2;q24）易位，在染色体17 q上的SOX 9上游区域中具有断点，与缺肢性缺肢发育不良的临床表型相关（OMIM 114290）。此外，我将研究3例10 q21-q23区域缺失的病例，这些病例是由瓦莱实验室收集的，作为他们对鉴定该区域精神分裂症易感基因的持续兴趣的一部分。他们包括我的系列中的第四名患者（JHU 2020），他有一个10q23.1缺失，与多个异常和发育迟缓相关;第五和第六涉及两个家族（JHU 2000和JHU 88293），两个家族都有重叠缺失，这两个家族都在10q21.1上缺失PCDH 15，与精神分裂症相关。 这些复杂的重排的描绘将导致更好地了解其临床表型的分子基础。在病例5和6中，将PCDH 15作为精神疾病的候选基因的研究是令人感兴趣的，因为它是在两个家族（JHU 2000和JHU 88293）中缺失的唯一注释基因。PCDH 15中的隐性截短突变导致Usher 1F型（OMIM 602083），其特征在于先天性听力损失、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，基因座异质性的预先评估，表明近20%的Usher综合征患者发展为精神病[4]。 公共卫生相关性：新的方法已经表明，染色体畸变比以前认识到的更频繁（例如CNV）;并且通常比以前通过检查认识到的更复杂，核型的明显平衡易位通常在边缘有相关的缺失或重复。我感兴趣的是利用染色体畸变来鉴定导致人类遗传疾病的基因，并一直在使用FISH、SNP基因分型、qRT-PCR、反向PCR、长距离PCR等方法来确定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鉴定断点。我将测试一种新的方法：靶向基因组捕获，然后对捕获的材料进行下一代测序（100 bp配对末端读取），以快速准确地确定约翰霍普金斯麦库西克-纳森遗传医学研究所（IGM）遗传学诊所中一组临床有趣病例中的分子异常。