Independent Origin of c.57 C > T Mutation in MIR184 Associated with Inherited Corneal and Lens Abnormalities

MicroRNAs (miRNAs) are small non-coding RNAs that suppress post-transcriptional gene expression by base pairing with their target messenger RNAs (mRNAs) and inducing either translational repression or mRNA degradation of their targets.1 In contrast to the protein coding genes, germline sequence variants in mature miRNAs are extremely rare possibly due to the extreme conservation and importance of mature miRNAs as well as to the really small size of these molecules (18–25 base pairs). Known variants were limited to the polymorphism in MIR125a2 and the mutation in MIR96 causing autosomal dominant progressive hearing loss,3^,4 until a recurring heterozygous mutation c.57 C > T in the seed region of MIR184 [MIM 613146] was repeatedly identified in the Northern Irish family with keratoconus and cataracts,5 a family with EDICT (endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning) syndrome,6 and most recently by our group in a five generation family with cataracts and varying corneal abnormalities including severe keratoconus and non-ectatic corneal thinning from Galicia, Spain.7

The maternal line of the Galician family (Figure 1) has originated in the city of La Estrada in an autonomous community of Galicia in Northwest Spain. The paternal side of the family includes Eastern European (Russian/Ukranian Jewish, proband’s grandfather) and German (proband’s grandmother) roots. We performed targeted testing of the two rare polymorphisms located in close proximity to the c.57 C > T mutation previously described in a Northern Irish family5 (Table 1, Supplementary Methods – online only) in the founding member of the Galician family (Figure 1, individual 101). Neither of the two rare alleles identified in the Northern Irish family was present in the tested individual. Then we performed genetic ancestry testing of this family by genotyping the proband, his mother and maternal grandmother (all carrying c.57 C > T mutation) using HumanOmni 2.5 BeadChip and comparing SNP data with reference population genotypes using principal component analysis (PCA, Supplementary Methods, Supplementary Figure 1 – online only). PCA with subjects from different continents identified all members of the family as having European ancestry. Further investigation revealed the proband’s mother and maternal grandmother having Iberian ancestry originating either in Spain or in Portugal; whereas the proband’s ancestry was found to be admixed between Iberian and Ashkenazi Jewish populations thus confirming their ancestry as identified by family history. Based on the absence of adjacent rare polymorphisms described in the Northern Irish family and by in-depth ancestral testing which conclusively showed lack of Irish genetic roots in this family we conclude that the deleterious c.57 C > T MIR184 mutation arose independently in the Galician and Northern Irish families and thus represents a first observation of the recurrent germline mutation in the microRNA gene leading to the genetic disease described up to date.

FIGURE 1. Pedigree structure of the Galician family. Genotyped individuals are identified by numbers.

TABLE 1. Results of testing of rare variants identified in the Northern Irish family in the Galician family.

Gene	Genbank Accession	Sequence variant	Northern Irish family	Galician Family
DNAJA4	NM_001130183	g.2099_2100 delAT	Present	Absent
IREB2	NM_004136	c.*2043G > T	Present	Absent
MIR184	NR_029705	r.57c > u	Present	Present

We further hypothesized that genomic variants in the gene targets of MIR184 may act as nuclear modifier(s) leading to intra- and interfamily variability of age of onset and clinical symptoms. To identify SNPs which could potentially modify phenotype severity in the members of the Galician family we used MiRNASNP2.0 database to identify SNPs with the potential to influence structure and function of MIR184.8 We found that out of the total of 513 SNPs annotated in the database, eight SNPs, one located in MIR184 genomic region and seven in the 3′ UTR regions of nuclear genes, were polymorphic in one or more of the three tested family members (Supplementary Table 1 – online only). The Proband (individual 301) affected with a more severe clinical presentation of the ocular symptoms was found to be a homozygote for the minor (potentially detrimental) allele for SNPs located in genes: IYD, C1orf158, and WSCD2. In addition, we performed analysis of predicted gene targets of MIR184 using MiRecords which combines the results of 11 established miRNA target prediction programs9 and compared the results of such predictions between eight genes identified as candidate genes for keratoconus in recent studies10–16 and seven genes known to be validated targets of MIR1845^,17–20 (Supplementary Table 2 – online only). We found that on average candidate genes were predicted to be MIR184 targets by two programs, whereas validated genes were predicted by an average of 2.4 programs (Mann-Whitney p value = 0.4) implying that genes important for corneal disease such as keratoconus may be regulated by MIR184 and may contain phenotype modifying polymorphisms.

Further analysis of genes expressed in the ocular tissues and potentially regulated by MIR184 will bring new understanding of microRNA regulation in health and disease and could suggest novel treatment options making therapeutic use of miRNA in the genetic eye diseases a real prospect.

Supplementary Material Available Online

Supplementary Tables 1 and 2

Supplementary Figure 1

Supplementary Methods

Acknowledgments

We thank all family members for their participation.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by the National Eye Institute [grant NEI 09052], the Skirball Foundation for Molecular Ophthalmology, and the Eye Defects Research Foundation Inc.