HIC2 is a novel dosage-dependent regulator of cardiac development within the distal 22q11 deletion syndrome region

Rationale: 22q11 deletion syndrome arises from recombination between low copy repeats on chromosome 22. Typical deletions result in hemizygosity for TBX1 associated with congenital cardiovascular disease. Deletions distal to the typically deleted region result in a similar cardiac phenotype but lack extra-cardiac features of the syndrome suggesting that a second haploinsufficient gene maps to this interval. Objective: The transcription factor HIC2 is lost in most distal deletions as well as a minority of typical deletions. We used mouse models to test the hypothesis that HIC2 hemizygosity causes congenital heart disease. Methods and Results: We created a genetrap mouse allele of Hic2. The genetrap reporter was expressed in the heart throughout the key stages of cardiac morphogenesis. Homozygosity for the genetrap allele was embryonic lethal before embryonic day E10.5 while the heterozygous condition exhibited a partially penetrant late lethality. One third of heterozygous embryos had a cardiac phenotype. Magnetic resonance imaging demonstrated a ventricular septal defect with overriding aorta. Conditional targeting indicated a requirement for Hic2 within the Nkx2.5+ and Mesp1+ cardiovascular progenitor lineages but not in the Wnt1+ neural crest or Mef2c+ second heart field lineages. Microarray analysis revealed increased expression of BMP10. Conclusions: Our results demonstrate a novel role for Hic2 in cardiac development. Hic2 is the first gene within the distal 22q11 interval to have a demonstrated haploinsufficient cardiac phenotype in mice. Together our data suggests HIC2 haploinsufficiency likely contributes to the cardiac defects seen in distal 22q11 deletion syndrome. The aim of this microarray experiment was to compare gene expression changes in the E13.5 mouse heart in a conditional knockout of Hic2 (Mesp1Cre/+; Hic2FL/FL) against control. All samples represent pools of isolated hearts from E13.5 mouse embryos. 3 replicates of each genotype were assayed. The genotypes are: 'WT' (Hic2FL/FL) 'KO' (Mesp1Cre/+; Hic2 FL/FL)研究背景：22q11缺失综合征（22q11 deletion syndrome）源于22号染色体上低拷贝重复序列之间的重组。典型缺失会导致TBX1基因处于半合子状态，进而引发先天性心血管疾病。位于典型缺失区域远端的缺失虽会产生相似的心脏表型，但缺乏该综合征的心脏外特征，这提示另有一个单倍剂量不足基因定位于此基因组区间。 研究目标：转录因子HIC2在大多数远端缺失病例以及少数典型缺失病例中发生丢失。我们借助小鼠模型验证了“HIC2半合子状态可导致先天性心脏病”这一假说。 方法与结果：我们构建了Hic2基因的基因陷阱（genetrap）小鼠等位基因。该基因陷阱报告基因在心脏形态发生的关键阶段全程于心脏组织中表达。纯合基因陷阱等位基因会在胚胎发育第10.5天（E10.5）前导致胚胎致死，而杂合状态则表现为外显率不完全的晚期致死现象。三分之一的杂合胚胎出现心脏表型。磁共振成像检测显示，该表型为室间隔缺损伴主动脉骑跨。条件性靶向实验表明，Hic2在Nkx2.5+和Mesp1+心血管祖细胞谱系中发挥必需作用，但在Wnt1+神经嵴细胞或Mef2c+第二心场谱系中并非必需。基因芯片（Microarray）分析显示，BMP10的表达水平显著上调。 研究结论：我们的研究结果揭示了Hic2在心脏发育中的全新功能。Hic2是22q11远端区间首个在小鼠模型中被证实具有单倍剂量不足心脏表型的基因。综合本研究数据表明，HIC2单倍剂量不足很可能是远端22q11缺失综合征患者心脏缺陷的致病诱因之一。本次基因芯片实验旨在对比Hic2条件性敲除小鼠（Mesp1Cre/+; Hic2FL/FL）与对照小鼠在胚胎发育第13.5天（E13.5）时的心脏基因表达变化。所有样本均为E13.5小鼠胚胎的分离心脏组织混合样本。每种基因型设置3次生物学重复。基因型分为："WT"（野生型，Hic2FL/FL）与"KO"（敲除型，Mesp1Cre/+; Hic2 FL/FL）