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中的重要组成部分¹，但目前我们才刚刚厘清其在人类孟德尔遗传病中的参与本质与覆盖范围。本研究证实，人类2号染色体上一段未注释的lncRNA位点的缺失，会引发严重的先天性肢体畸形。通过外显子测序（exome sequencing）与阵列比较基因组杂交（array CGH），我们在罹患复杂肢体畸形的患者体内，检测到位于Engrailed-1（EN1）基因上游300 kb处的纯合性27~63 kb缺失，该畸形表现为肢节缩短、并指、腹侧指甲异常伴背侧指（趾）重复畸形。在小鼠中重构人类的该缺失片段后，肢体特异性En1表达完全丧失，并出现双背侧肢体表型，完美重现了人类的畸形症状。对发育中小鼠肢体的全基因组分析显示，该缺失区域内存在一段包含4个外显子的长链非编码转录本，我们将其命名为Maenli（意为“肢体中En1的主激活因子”，英文全称Master activator of En1 in the limb）。对Maenli位点的功能解剖实验证实，肢体特异性的En1表达依赖于Maenli的转录，其缺失会引发双背侧肢体表型。在双杂合小鼠中同时对En1与Maenli进行单等位基因失活，并未挽救肢体畸形表型，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的转录过程，而非Maenli RNA本身。综上，肢体中的Maenli表达以顺式作用方式促进En1的转录激活；其缺失会引发先天性肢体畸形，这属于En1相关完整表型谱中的一个亚型。本研究通过外显子测序与阵列比较基因组杂交分析患者样本，借助CRISPR实验探究潜在致病变异的功能效应，利用全基因组分析鉴定出新的lncRNA位点，并通过功能研究阐明了敲除该lncRNA位点所产生的生物学效应。