Mantripragada Kiran Kumar, Tapia-Páez Isabel, Blennow Elisabeth, Nilsson Peter, Wedell Anna, Dumanski Jan P
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital, SE-751 85 Uppsala, Sweden.
Int J Mol Med. 2004 Feb;13(2):273-9.
Deletions and duplications of genomic segments commonly cause developmental disorders. The resolution and efficiency in diagnosing such gene-dosage alterations can be drastically increased using microarray-based comparative genomic hybridization (array-CGH). However, array-CGH currently relies on spotting genomic clones as targets, which confers severe limitations to the approach including resolution of analysis and reliable gene-dosage assessment of regions with high content of redundant sequences. To improve the methodology for analysis, we compared the use of genomic clones, repeat-free pools of amplified genomic DNA and cDNAs (single and pooled) as targets on the array. For this purpose, we chose q11.2 locus on chromosome 22 as a testing ground. Microdeletions at 22q11 cause birth defects collectively described as the DiGeorge/velocardiofacial syndrome. The majority of patients present 3 Mb typical deletions. Here, we report the construction of a gene-dosage array, covering 6 Mb of 22q11 and including the typically deleted region. We hybridized DNA from six DiGeorge syndrome patients to the array, and show that as little as 11.5 kb non-redundant, repeat-free PCR-generated sequence can be used for reliable detection of hemizygous deletions. By extrapolation, this would allow analysis of the genome with an average resolution of 25 kb. In the case of cDNAs our results indicate that 3.5 kb sequence is necessary for accurate identification of haploid/diploid dosage alterations. Thus, for regions rich in redundant sequences and repeats, such as 22q11, a specifically tailored array-CGH approach is good for gene copy number profiling.
基因组片段的缺失和重复通常会导致发育障碍。使用基于微阵列的比较基因组杂交技术（array-CGH）可大幅提高诊断此类基因剂量改变的分辨率和效率。然而，目前的array-CGH依赖于将基因组克隆点样作为靶标，这给该方法带来了严重限制，包括分析分辨率以及对冗余序列含量高的区域进行可靠基因剂量评估。为改进分析方法，我们比较了将基因组克隆、无重复的扩增基因组DNA池以及cDNA（单个和混合）作为阵列靶标的情况。为此，我们选择22号染色体上的q11.2位点作为测试对象。22q11微缺失会导致统称为DiGeorge/心面综合征的出生缺陷。大多数患者存在3 Mb的典型缺失。在此，我们报告构建了一个基因剂量阵列，覆盖22q11的6 Mb区域，包括典型缺失区域。我们将6例DiGeorge综合征患者的DNA与该阵列杂交，结果表明，低至11.5 kb的非冗余、无重复PCR生成序列可用于可靠检测半合子缺失。由此推断，这将允许以平均25 kb的分辨率分析基因组。对于cDNA，我们的结果表明，3.5 kb序列对于准确鉴定单倍体/二倍体剂量改变是必要的。因此，对于富含冗余序列和重复序列的区域，如22q11，一种专门定制的array-CGH方法有利于基因拷贝数分析。
