Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [scRNA-seq]

Long non-coding RNAs (lncRNAs) can be important components in gene regulatory networks1, but we are only beginning to understand the nature and extent of their involvement in human Mendelian disease. Here we show that deletions of an unannotated lncRNA locus on human chromosome 2 cause a severe congenital limb malformation. Using exome sequencing and array CGH, we identified homozygous 27-63 kb deletions located 300 kb upstream of the engrailed-1 (EN1) gene in patients with a complex limb malformation, featuring mesomelic shortening, syndactyly, and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of limb-specific En1 expression and a double dorsal limb phenotype, recapitulating the human malformation. Genome-wide analysis in the developing mouse limb revealed the presence of a 4-exon long non-coding transcript within the deleted region, which we named Maenli (for Master activator of En1 in the limb). Functional dissection of the Maenli locus showed that limb-specific En1 expression depends on transcription of Maenli and its loss resulted in the double dorsal limb phenotype. Concomitant monoallelic inactivation of En1 and Maenli in double heterozygous mice did not rescue the limb phenotype, indicating that En1 activation in the limb requires the cis-acting regulatory element Maenli. Moreover, our results strongly suggest that En1 activation is dependent on Maenli transcription, but not on the Maenli RNA itself. Thus, Maenli expression in the limb acts in cis to promote En1 transcriptional activation; its loss results in congenital malformation of the limbs, a subset of the full En1 associated phenotype. Together, our findings provide evidence that mutations involving lncRNAs loci can result in human Mendelian disease. We used exome sequencing and array CGH to analyze patients' samples, CRISPR experiments to investigate the effect of the potential pathogenic variants, genome wide analysis to identify a new lncRNA locus, and functional studies to characterize the effect of the knockout lncRNA locus.

长链非编码RNA（long non-coding RNAs，lncRNAs）可作为基因调控网络的重要组成部分¹，但目前我们仅刚起步了解其在人类孟德尔遗传病中的作用本质与参与程度。本研究证实，人类2号染色体上一处未注释的lncRNA位点的缺失可引发严重的先天性肢体畸形。研究人员通过外显子组测序与阵列比较基因组杂交（array comparative genomic hybridization，array CGH），在患有复杂肢体畸形的患者中，鉴定出位于engrailed-1（EN1）基因上游300 kb处的纯合性27~63 kb缺失，该畸形表现为肢中段缩短、并指（趾）畸形以及掌侧甲异常（伴背侧重复指/趾畸形）。在小鼠体内重现该人类缺失片段后，研究人员观察到肢体特异性En1表达完全丧失，并出现双背侧肢体表型，完美复刻了人类的肢体畸形。对发育中小鼠肢体的全基因组分析显示，缺失区域内存在一段含4个外显子的长链非编码转录本，研究人员将其命名为Maenli（即“肢体中En1的主激活因子”，Master activator of En1 in the limb）。对Maenli位点的功能解析实验表明，肢体特异性En1的表达依赖于Maenli的转录，其缺失会导致双背侧肢体表型。在双杂合子小鼠中同时对En1和Maenli进行单等位基因失活，并未挽救肢体表型，这表明肢体中的En1激活需要顺式作用调控元件Maenli。此外，本研究结果强烈提示，En1的激活依赖于Maenli的转录，而非Maenli RNA本身。因此，肢体中的Maenli表达通过顺式作用促进En1的转录激活；其缺失会导致先天性肢体畸形，这是完整En1相关表型的一个亚型。综上，本研究的发现为涉及lncRNA位点的突变可导致人类孟德尔遗传病提供了实证依据。本研究通过外显子组测序与阵列比较基因组杂交分析患者样本，利用CRISPR实验探究潜在致病性变异的影响，通过全基因组分析鉴定新的lncRNA位点，并借助功能研究表征敲除该lncRNA位点后的效应。