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Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks 1 , but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) 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 En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease. There has been enormous progress in exploring disease variants in the human genome. Yet, the interpretation of variants in the non-coding genome remains a challenge owing to the myriad mechanisms by which they can potentially cause disease. Besides disrupting cis-regulatory elements, non-coding variants may interfere with the function of non-coding transcripts. Indeed, a substantial fraction of the human genome is transcribed into RNA, although most transcripts lack protein-coding potential and are referred to as non-coding transcripts 2. Characterization of a small number of these RNA molecules has revealed that they may have roles as regulators of gene expression through diverse modes of action 3. However, the identification of functional non-coding transcript loci remains challenging. Thus, annotating non-coding transcript loci and unravelling their function will substantially improve our knowledge about gene regulation and the identification and interpretation of non-coding genetic variants with respect to disease pathogenesis. Non-coding deletions cause limb malformations We identified 27-63-kb non-coding deletions of chromosome 2 in three unrelated individuals (patients 1-3) with a type of limb malformation that, to our knowledge, remains undescribed. Affected individuals had a severe shortening and deformation of the legs and feet, 3/4 syndactyly of the hands, as well as the presence of nails on the palmar side of fingers IV and V (Fig. 1a, Extended Data Fig. 1a, b, Supplementary Note 1). Radiographs showed normal femora but severely shortened tibiae, triangular fibulae and malformed or absent bones in the feet (Fig. 1a, Extended Data Fig. 1a, Supplementary Note 1). Exome s...

Significance The relationship between long-range Polycomb-associated chromatin contacts and the linear propagation of histone H3 lysine 27 trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) is not well-characterized. Here, we nominate a role for developmental loci as genomic architectural elements that enable long-range spreading of H3K27me3. Polycomb-associated loops are disrupted upon loss of PRC2 binding and deletion of loop anchors results in alterations of H3K27me3 deposition and ectopic gene expression. These results suggest that Polycomb-mediated genome architecture is important for gene repression during embryonic development.