PROJECT II: VARIANTS FROM COMPLEMENTARY GENOMIC TECHNOLOGIES WILL YIELD

在项目II(来自互补基因组技术的变体将识别候选致病CDH基因)中，Charies Lee博士正在完成一项广泛的平台比较，以评估源自阵列CGH平台和Affymetrix 6.0阵列平台的拷贝数变异(CNV)。 这项工作是李实验室、桑格协会和多伦多大学谢勒实验室合作完成的，结果正在准备发表。我们预计，这一分析将为各自平台未来对CNV的解释提供指导。我们将使用高分辨率的Agilent IM阵列CGH来研究我们的孤立和综合征型CDH患者，以确定新的候选基因座。将对父母/患者三人组进行分析，以确定未报告的CNV是从头开始还是遗传的。由于CDH等出生缺陷预计是多基因的，我们应该预料到遗传性CNV的组合将是强烈的促成因素，这些CNV在患者和对照组中的频率增加。 Affymetrix 6.0芯片将用于研究多个CDH家族，以确定连锁区块和杂合性丢失区域，当与整个外显子测序相结合时，可以揭示新的候选CDH基因。为这项研究创建的生物信息学算法CNV CONNECT将对CNV基因座中的所有基因和那些来自杂合性缺失区域的基因进行优先排序，以选择那些与其他已知对CDH有显著影响的基因相互作用的基因。 随着新兴测序平台的成本逐步降低，我们计划对50名患有系统性或复杂性CDH的患者进行完整的外周基因组测序。我们的假设是，共同基因座的变异会导致合并心脏和横隔膜缺陷的患者。我们还假设，类似地，有限数量甚至单个变异将导致具有CDH表型星座和其他显著先天异常的各种综合征CDH患者。我们预测，对这一特殊群体的患者进行筛查将增加揭示新的因果变异的可能性。 用于对更大的孤立性先天性横隔疝患者队列进行亚基因组测序。Lee和Donahoe实验室将创建患者特有的DNA文库。然后，Lee实验室将在捕获过滤器上串联Pober博士在项目I中选择的所有候选基因，并将患者DNA样本杂交。洗脱后，将使用下一代测序技术对150名单独的CDH患者的整个队列中的所有候选基因进行测序。 拥有Lee实验室的专业知识和Seidman实验室的咨询，以及他对Sanger和多伦多财团的广泛接触，增加了我们选择平台的有效性，并确保这些技术将得到适当的选择、使用和解释，以期影响患者护理的应用。

In PROJECT II (Variants from Complementary Genomic Technologies will Identify Candidate Causative CDH Genes) Dr. Charies Lee is completing an extensive platform comparison to evaluate copy number variations (CNV) derived from array CGH platforms, with those from the Affymetrix 6.0 array platform. This work has been done in collaboration between the Lee laboratory, the Sanger Consorslum, and the Sherer laboratory in the University of Toronto, and results are now being readied for publication. We anticipate that this analysis will provide guidance for future interpretations of CNVs from the respective platforms. We will use the high resolution Agilent IM array CGH to study our patients with isolated and syndromic CDH to identify new candidate loci. Parent/patient trios will be analyzed to determine whether an unreported CNV is de novo or inherited. Since birth defects such as CDH are anticipated to be polygenic, we should anticipate that combinations of inherited CNVs which have an increased frequency in patients vs. controls will be strong contributing factors. Affymetrix 6.0 chips will be used to study multiplex CDH families to identify blocks of linkage and for regions of loss of heterozygosity, which, when combined with whole exomic sequencing, can reveal new candidate CDH genes. A bioinformatic algorithm created for this study, CNV connect, will then prioritize all the genes in the CNV loci and those derived from regions loss of heterozygosity to select those that significantly interact with other genes know to contribute to CDH. As the cost of emerging sequencing platforms is progressively reduced, we are planning to undertake whole exomic sequencing on 50 patients with sydromic or complex CDH. Our hypothesis is that variant in common loci will be causative In patients with comorbidities of heart and diaphragm defects. We also hypothesize that similarly, a limited number or even a single variant will be responsible for patients with various syndromic CDH who have a phenotype constellation of CDH with other significant congenital anomalies. We predict that screening this special group's of patients will increase the probability of revealing novel causal variants. For subgenomic sequencing of a larger cohort of patients with isolated Congenital Diaphragmatic Hernia. The Lee and Donahoe laboratories will create the patient specific DNA libraries. The Lee laboratory will then concatamerize on the capture filter all the candidate genes selected by Dr. Pober in Project I and hybridize the patient DNA samples. After elution, next generation sequencing techniques will be employed to sequence all the candidate genes in the entire cohort of 150 patients with isolated CDH. Having the expertise of the Lee laboratory with the consultation of the Seidman laboratory and with his extensive outreach to the Sanger and the Toronto consortia gives added validity to our choice of platforms and the assurity that these technologies will be appropriately selected, used, and interpreted, with the hope of affecting an application to patient care.