MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1,2,3,4,5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA–target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs.
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Data generated during this study are available in the Gene Expression Omnibus under accession number GSE98309. The human variant is deposited in the ClinVar database (SCV000586692.1). Human genome data from the individuals participating in the study is protected by Swedish law (2006:351), and raw Sanger sequencing data within the area of interest is available upon request. All other data will be made available upon request to the corresponding author.
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We thank patients and their family members for their participation in the study, M. Mannstadt, K. Leuter, M. Wein, H. Kronenberg, H. Jueppner, G. Björk, and A. Merker for advice and chemicals, J. Lundin for assistance with the human comparative genomic hybridization array databases, and Science for Life Laboratory, the National Genomics Infrastructure (NGI), Sweden, SNIC through UPPMAX under project b2014231 for providing assistance in massive parallel DNA sequencing and computational infrastructure. We also thank Center for Skeletal Research Core (NIH P30 AR066261) for access to histological analysis equipment; Harvard GMF for generation of knock-in mice; and DFCI and MGH Sequencing Cores for assistance with RNA and Sanger sequencing; the Robert A. Swanson (1969) Biotechnology Center at the Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology for technical support, specifically S. Levine and the staff of the BioMicro Center/KI Genomic Core Facility and G. Paradis, M. Jennings, and M. Saturno-Condon of the Flow Cytometry Core Facility. This project was supported by grants provided by the Stockholm County Council (ALF projects 20150143 and 20130315 to G. Grigelioniene. and A.N.) and by the National Institutes of Health (National Institute of Arthritis and Musculoskeletal and Skin Diseases grant R01-AR056645 to T.K., National Institute of General Medical Sciences grant R01-GM034277 and National Cancer Institute grant R01-CA133404 to P.A.S., and National Cancer Institute grant P30-CA14051 to the Koch Institute Core Facility). G.Grigelioniene was supported by a grant of the Sabbatical Leave Programme of the European Society for Paediatric Endocrinology through an educational grant from Eli Lilly International Corporation, travel grants from Fernström Foundation, Karolinska Institutet and Swedish Society of Medicine, scholarship from Stiftelsen Samariten, Stockholm, Sweden, Research Funds from Promobilia and Frimurare Barnhuset Stockholm, and project grant from Swedish Research Council 2018-03046. H.I.S. is supported by the Uehara Memorial Foundation Research Fellowship and the Osamu Hayaishi Memorial Scholarship for Study Abroad.