microRNAs and inherited retinal dystrophies.

Inherited retinal dystrophies (IRD) are a heterogeneous group of blinding diseases that affect more than 200,000 Americans and millions worldwide (1). Identification of genes that are responsible for IRD when defective is of great importance to the basic understanding as well as development of efficient gene diagnosis and treatment. Thus far, 272 genetic loci are linked to various forms of IRD; among these, 232 protein-coding genes have been identified to cause IRD when defective (RetNet, https://sph.uth. edu/Retnet, last updated April 29, 2015). microRNAs (miRNAs) are endogenous, small, noncoding, regulatory RNAs (2). Since their discovery in 1993 (3, 4), miRNAs have proven to play important roles in the fine-tuning of gene expression at posttranscriptional levels (2). In spite of their importance in gene-expression regulation, few mutations of individual miRNAs have been found to cause Mendelian inherited diseases in humans, raising doubts about their importance in normal development and functions, and whether mutations in a miRNA can have significant functional impact to cause Mendelian inherited diseases, including IRD. Recently, a point mutation in the seed sequence of miR-184 was reported to cause familial keratoconus with cataract (5) and an anterior-segment dysplasia syndrome with endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning (6), suggesting that miR-184 is required for anterior-segment development and function, and proving that mutations in an individual miRNA can cause inherited ocular disease in humans. In mice, Lumayag et al. demonstrated that inactivation of the miR-183/96/ 182 cluster gene causes syndromic IRD with multisensory defects (7), suggesting that defects in miRNA genes can cause IRD in mammals. However, until now, no mutations in miRNAs have been found to cause IRD in humans. In PNAS, Conte et al. report a dominant mutation in miR-204 that causes IRD associated with ocular coloboma in a large five-generation family (8). This paper is the first report to show that a point mutation in an individual miRNA results in significant functional consequence and causes IRD in humans, and the third miRNA in which mutations cause Mendelian inherited disorders in humans, after miR-96, which causes nonsyndromic hearing loss (9, 10), and miR-184 (5, 6). Conte et al’s report (8) provides a new molecular mechanism of IRD and underscores the importance of miRNA in IRD. miR-204 resides in intron 8 of the transient receptor potential (TRP) channel gene, TRPM3, on human chromosome (chr) 9q21.12. Through a classic parametric linkage analysis, Conte et al. zoomed in on four regions segregating with the phenotypes, including 9q21 (8). Through exon sequencing, extensive bioinformatic studies to eliminate nondisease-causing variants using multiple publically available databases, including the Exon Variant Server of the National Heart, Lung and Blood Institute Exome Sequencing Project, dbSNP, and the 1000 genomes project, and segregation studies, Conte et al. found that a point mutation in miR-204, n.37C > T, cosegregates with the disease but is not present in all control databases and 21 sporadic cases of retinal dystrophy with microphthalmia anophthalmia and coloboma (MAC), 457 patients with isolated MAC, and 672 patients with autosomal dominant IRD, suggesting that n.37C > T is the disease-causing mutation in this family (8). Publically available control databases have become invaluable sources for evaluating the nature of a mutation and excluding nondisease-causing genomic variants. The Conte et al. report exemplifies an effective approach for identification of disease-causing mutations in a postgenomic era. In the family studied by Conte et al. (8), all affected individuals had progressive retinal dystrophy, vision loss, and coloboma; multiple affected individuals underwent bilateral cataract surgery at early ages, suggesting lens defects; and the proband also showed iridolenticular adhesions and scattered retinal pigmented epithelia (RPE) mottling. Mutation in miR-204 as the cause of the disease is consistent with the observation that miR204 is preferentially expressed in multiple ocular tissues, including the retina, RPE, ciliary body, and lens (11–13). In addition, previous in vitro and in vivo studies in medaka fish suggested its important roles in the development and function of the retina (13, 14), lens (13–15), and RPE (12). Knockdown of miR-204 in fish resulted in microphthalmia, abnormal lens formation, altered dorso-ventral (D-V) patterning of the retina, and coloboma (13). Mutations in miRNAs either disrupt their biogenesis and, therefore, expression levels of the mature miRNAs, and/or change their downstream target genes (5, 6, 9, 10). The seed sequence of a miRNA is the major determinant of its downstream target genes and, therefore, functions of the miRNA (16). The mutation identified in this family, n.37C > T, is located at the fourth residue of the seed sequence of miR-204. Conte et al. demonstrated that this mutation has no significant impact on the secondary structure of pre-miR-204 and biogenesis of miR-204 (8). However, target prediction analysis revealed a loss of 442 predicted targets of wild-type miR-204 (wt-miR-204) and a gain of 994 new predicted target genes of the mutant miR-204 (mut-miR-204) (Fig. 1) (8), in which genes involved in eye development, synaptic function, cell adhesion, axon guidance, D-V axis formation, and cellular response to stress are significantly enriched. Through overexpression of wt-miR-204 and mut-miR-204 in a human RPE cell line and gene-expression profiling, Conte et al. showed that n.37C > T mutation resulted in Fig. 1. n.37C > T mutation in miR-204 significantly changed its predicted target genes. Venn diagram summarizing the number of overlapping and unique predicted target genes of wt-miR-204 and mut-miR-204.