Gene expression changes in Dhcr7 knockout brain, lung and liver at E18.5 compared to wild-type embryos

The genetic defect underlying the human Smith-Lemli-Opitz dysmorphological disorder is loss-of-function mutations affecting the cholesterol synthesis enzyme dehydrocholesterol delta7 reductase, DHCR7. Dhcr7 knockout mice recapitulate the biochemical characteristics, but all knockout pups die within 14h of birth. Tissues of knockout mice accumulate the precursor sterol, 7-dehydrocholesterol, and show reduced levels of cholesterol (J. Clin. Invest. (2001) 108: 905-915). We compared the global gene expression changes in lung, liver and brain from knockout mice to those seen from organs harvested from same-pregnancy wild-type embryos, harvested before birth (E18.5). Since the P0 knockout pups die, we expected that there would be significant changes in gene expression between knockout and wild-type organs, and further comparing the altered genes in common to brain, lung and liver would point to a common mechanistic pathway where disruption of normal sterol synthesis in all cells leads to pathophysiology. Remarkably, these data show that the global gene expression between knockout and wild-type organs is hardly altered, despite the complete loss of cholesterol synthesis. There are so few genes that are altered, and furthermore, those that are altered are very limited in sharing similarities in all three tissues. This suggests that disorganized gene expression is not the cause of the early neonatal lethality. Dhcr7+/- females were mated with Dhcr+/- males, and timed pregnancy obtained. At E18.5, pregnant dams were sacrificed, and embryos harvested, organs removed and RNA extracted, and all embryos were genotyped. Matching wild-type and knockout embryos were taken from each pregnancy, and two females yielded all genotypes used herein.人类史密斯-莱姆利-奥皮茨畸形障碍（Smith-Lemli-Opitz dysmorphological disorder）的潜在遗传缺陷为影响胆固醇合成酶7-脱氢胆固醇Δ7还原酶（DHCR7）的功能丧失突变。Dhcr7基因敲除小鼠可重现该疾病的生化特征，但所有敲除幼崽均在出生后14小时内死亡。该敲除小鼠的组织会积累前体甾醇7-脱氢胆固醇，并表现出胆固醇水平降低的表型（《临床研究杂志》(2001) 108: 905-915）。本研究将Dhcr7基因敲除小鼠肺、肝、脑的全局基因表达变化，与同胎野生型胚胎在产前（胚胎发育第18.5天，E18.5）采集的对应器官的基因表达谱进行了对比。鉴于出生后0天（P0）的敲除幼崽会发生死亡，我们预期敲除与野生型器官间会存在显著的基因表达差异；进一步比对脑、肺、肝中共有的差异表达基因，有望揭示一条共同的分子机制通路，即所有细胞中正常甾醇合成紊乱如何引发病理生理变化。值得注意的是，尽管胆固醇合成完全丧失，敲除与野生型器官间的全局基因表达几乎未发生改变。差异表达基因的数量极少，且在三种组织中仅存在极少量共通的相似性。这表明基因表达紊乱并非早期新生小鼠致死的原因。本研究将Dhcr7杂合子（Dhcr7+/-）雌性小鼠与Dhcr7杂合子雄性小鼠交配，获得精准计时的孕鼠。在胚胎发育第18.5天（E18.5）时处死孕母鼠，采集胚胎并分离器官、提取总RNA，随后对所有胚胎进行基因分型。从每胎中选取匹配的野生型和敲除胚胎，本研究使用的全部基因型样本均来自两只杂合母鼠。