INSERM UMRS 937, Pierre and Marie Curie University (UPMC, Paris 6) and Medical School, Paris, France.
PLoS One. 2011;6(9):e23956. doi: 10.1371/journal.pone.0023956. Epub 2011 Sep 1.
The hypothesis of dosage compensation of genes of the X chromosome, supported by previous microarray studies, was recently challenged by RNA-sequencing data. It was suggested that microarray studies were biased toward an over-estimation of X-linked expression levels as a consequence of the filtering of genes below the detection threshold of microarrays.
METHODOLOGY/PRINCIPAL FINDINGS: To investigate this hypothesis, we used microarray expression data from circulating monocytes in 1,467 individuals. In total, 25,349 and 1,156 probes were unambiguously assigned to autosomes and the X chromosome, respectively. Globally, there was a clear shift of X-linked expressions toward lower levels than autosomes. We compared the ratio of expression levels of X-linked to autosomal transcripts (X∶AA) using two different filtering methods: 1. gene expressions were filtered out using a detection threshold irrespective of gene chromosomal location (the standard method in microarrays); 2. equal proportions of genes were filtered out separately on the X and on autosomes. For a wide range of filtering proportions, the X∶AA ratio estimated with the first method was not significantly different from 1, the value expected if dosage compensation was achieved, whereas it was significantly lower than 1 with the second method, leading to the rejection of the hypothesis of dosage compensation. We further showed in simulated data that the choice of the most appropriate method was dependent on biological assumptions regarding the proportion of actively expressed genes on the X chromosome comparative to the autosomes and the extent of dosage compensation.
CONCLUSION/SIGNIFICANCE: This study shows that the method used for filtering out lowly expressed genes in microarrays may have a major impact according to the hypothesis investigated. The hypothesis of dosage compensation of X-linked genes cannot be firmly accepted or rejected using microarray-based data.
X 染色体基因的剂量补偿假说得到了先前微阵列研究的支持，但最近的 RNA 测序数据对其提出了挑战。有人认为，由于微阵列对基因的过滤低于检测阈值，微阵列研究可能存在高估 X 连锁表达水平的偏差。
方法/主要发现：为了研究这个假说，我们使用了来自 1467 个人的循环单核细胞的微阵列表达数据。总共，25349 个和 1156 个探针分别明确地分配给常染色体和 X 染色体。总体而言，X 连锁表达明显向比常染色体更低的水平转移。我们使用两种不同的过滤方法比较了 X 连锁与常染色体转录物表达水平的比值（X∶AA）：1. 基因表达使用检测阈值过滤，而不考虑基因染色体位置（微阵列中的标准方法）；2. 分别在 X 染色体和常染色体上以相等的比例过滤出基因。对于广泛的过滤比例，使用第一种方法估计的 X∶AA 比值与 1 没有显著差异，1 是达到剂量补偿的预期值，而使用第二种方法，它明显低于 1，导致剂量补偿假说被拒绝。我们进一步在模拟数据中表明，选择最合适的方法取决于关于 X 染色体上活性表达基因的比例与常染色体的比例以及剂量补偿程度的生物学假设。
结论/意义：这项研究表明，在微阵列中用于过滤低表达基因的方法可能会根据所研究的假说产生重大影响。使用基于微阵列的数据不能坚定地接受或拒绝 X 连锁基因的剂量补偿假说。
