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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...

Background: PFA0210c is an exported Plasmodium falciparum protein.Results: PFA0210c is a phospholipid transfer protein with similarities to proteins of the family of START domain-containing proteins and can transfer various phospholipids between membranes in vitro.Conclusion: PFA0210c and its orthologs are phospholipid transfer proteins with a broad specificity.Significance: Identification of the function of conserved Plasmodium proteins allows deeper insight into the basis of pathogenesis.

StAR-related lipid transfer (START) domains are phospholipid- or sterol-binding modules that are present in many proteins. START domain-containing proteins (START proteins) play important functions in eukaryotic cells, including the redistribution of phospholipids to subcellular compartments and delivering sterols to the mitochondrion for steroid synthesis. How the activity of the START domain is regulated remains unknown for most of these proteins. The Plasmodium falciparum START protein PFA0210c (PF3D7_0104200) is a broad-spectrum phospholipid transfer protein that is conserved in all sequenced Plasmodium species and is most closely related to the mammalian START proteins STARD2 and STARD7. PFA0210c is unusual in that it contains a signal sequence and a PEXEL export motif that together mediate transfer of the protein from the parasite to the host erythrocyte. The protein also contains a C-terminal extension, which is very uncommon among mammalian START proteins. Whereas the biochemical properties of PFA0210c have been characterized, the function of the protein remains unknown. Here, we provide evidence that the unusual C-terminal extension negatively regulates phospholipid transfer activity. Furthermore, we use the genetically tractable Plasmodium knowlesi model and recently developed genetic technology in P. falciparum to show that the protein is essential for growth of the parasite during the clinically relevant asexual blood stage life cycle. Finally, we show that the regulation of phospholipid transfer by PFA0210c is required in vivo, and we identify a potential second regulatory domain. These findings provide insight into a novel mechanism of regulation of phospholipid transfer in vivo and may have important implications for the interaction of the malaria parasite with its host cell.

Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2′FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2′FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2′FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2′FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2′FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup. IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2′FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

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Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.