Common miR-590 Variant rs6971711 Present Only in African Americans Reduces miR-590 Biogenesis

MicroRNAs (miRNAs) are recognized as important regulators of cardiac development, hypertrophy and fibrosis. Recent studies have demonstrated that genetic variations which cause alterations in miRNA:target interactions can lead to disease. We hypothesized that genetic variations in miRNAs that regulate cardiac hypertrophy/fibrosis might be involved in generation of the cardiac phenotype in patients diagnosed with hypertrophic cardiomyopathy (HCM). To investigate this question, we Sanger sequenced 18 miRNA genes previously implicated in myocyte hypertrophy/fibrosis and apoptosis, using genomic DNA isolated from the leukocytes of 199 HCM patients. We identified a single nucleotide polymorphism (rs6971711, C57T SNP) at the 17th position of mature miR-590-3p (= 57th position of pre-miR-590) that is common in individuals of African ancestry. SNP frequency was higher in African American HCM patients (n = 55) than ethnically-matched controls (n = 100), but the difference was not statistically significant (8.2% vs. 6.5%; p = 0.5). Using a cell culture system, we discovered that presence of this SNP resulted in markedly lower levels of mature miR-590-5p (39 ± 16%, p<0.003) and miR-590-3p (20 ± 2%, p<0.003), when compared with wild-type (WT) miR-590, without affecting levels of pri-miR-590 and pre-miR-590. Consistent with this finding, the SNP resulted in reduced target suppression when compared to WT miR-590 (71% suppression by WT vs 60% suppression by SNP, p<0.03). Since miR-590 can regulate TGF-β, Activin A and Akt signaling, SNP-induced reduction in miR-590 biogenesis could influence cardiac phenotype by de-repression of these signaling pathways. Since the SNP is only present in African Americans, population studies in this patient population would be valuable to investigate effects of this SNP on myocyte function and cardiac physiology.


Introduction
MicroRNAs (miRNA) play important regulatory roles in cardiac development and pathology via post-transcriptional gene silencing [1][2][3]. Base-pairing between the highly conserved, 5 0 proximal seed region (residues 2-8) of miRNA and the 3 0 -UTR (un-translated region) of target mRNA is important for miRNA: mRNA binding and target gene silencing [4,5]. Additionally, sequences outside the seed region can also impact target suppression [6][7][8]. Recent studies have shown that genetic variations in miRNA genes can predispose to disease [9][10][11][12][13]. However, it is unknown whether variants in miRNAs can affect the cardiac phenotype in cardiomyopathies, such as hypertrophic cardiomyopathy (HCM), which is the most common cardiac genetic disease and cause of sudden death in young individuals.
HCM is characterized pathologically by myocyte hypertrophy, disarray, fibrosis [14] and is caused by sarcomeric protein mutations in~60% of patients; genetic causes are unknown iñ 40% of patients. Inheritance is autosomal dominant with variable penetrance and phenotypic heterogeneity. The genetic mechanisms underlying variability in penetrance and expression in HCM are not well defined [15][16][17]. We hypothesized that genetic variations in miRNAs that regulate cardiac hypertrophy/fibrosis might be involved in generation of the cardiac phenotype in HCM patients. We used a literature search combined with online bioinformatics tools to identify miRNAs (n = 18) that have been implicated in the pathologic features of HCM, namely, myocyte hypertrophy, cardiac fibrosis and apoptosis. Using Sanger sequence analysis of genomic DNA obtained from peripheral blood in HCM patients, we identified 11 variants in 9 of the 18 miRNAs that we investigated. We focused on the C57T single nucleotide polymorphism (SNP, rs6971711) in the miR-590 gene for two reasons. First, the C57T SNP in miR-590 is a common SNP that is only seen in African Americans; minor allele frequency of the miR-590 SNP was slightly higher in African American HCM patients when compared to ethnicallymatched controls. Second, miR-590 has been reported to be an important regulator of signaling pathways involved in cardiac fibrosis/ventricular remodeling [18][19][20], embryonic stem cell proliferation [21], cardiac differentiation [22], metabolism [23][24][25], cardiac regeneration [26,27] and atrial fibrosis/fibrillation [23]. However, it is unknown whether the miR-590 C57T SNP affects levels of miR-590-5p or miR-590-3p. In order to address this question, we used an in vitro cell culture system to investigate functional effects of this SNP. We discovered that the C57T SNP markedly reduced levels of mature miR-590-5p and miR-590-3p (when compared with the wild-type sequence) without affecting levels of pri-miR-590 and pre-miR-590. Consistent with this finding, the C57T SNP also reduced target suppression by miR-590, which suggests that presence of the miR-590 SNP could influence cell function. Since miR-590 can regulate TGF-β, Activin A and Akt signaling, SNP-induced reduction in miR-590 biogenesis could modify cardiac phenotype by de-repression of these signaling pathways.

Overall workflow of the studies
The overall workflow is illustrated in Fig 1 and in the Supporting Information section. Briefly, we used literature mining to select 18 miRNAs that have been implicated in cardiac hypertrophy, fibrosis and/or apoptosis by previous experimental studies. Sanger sequencing was initially performed in 199 patients with a clinical diagnosis of HCM. Eleven of the 199 patients (5.5%) were African American and 5 of the 11 African American HCM patients carried the miR-590 C57T SNP, rs6971711 (heterozygous). Targeted genotyping for the miR-590 C57T SNP, rs6971711 was performed in a second cohort of African American HCM patients (n = 44) and controls (n = 100) to increase sample size and examine possible significance of this SNP in HCM. Functional effects of the miR-590 C57T SNP were examined using a cell culture model (human embryonic kidney cells/HEK293T).
Please see Supporting Information section for detailed methods.  Patients enrolled in the HCM Registry at Johns Hopkins and the NIH HCM registry were retrospectively studied if they fulfilled the standard diagnostic criteria for HCM [36], namely, left ventricular hypertrophy (septal thickness>1.5cm) in the absence of other causes such as hypertension and valvular disease. African American individuals without heart disease were used as controls. DNA was isolated from buffy coats obtained from peripheral venous blood in most subjects. HCM patients' medical records were reviewed to obtain clinical information, including family history of HCM and results of imaging studies.  Table). The miRNA region was amplified using HotStar Taq DNA Polymerase (Qiagen). Excess primers and dNTPs were eliminated by Exo/SAP digestion. Purified PCR products were sequenced unidirectionally using either forward or reverse M13 universal primers and the DNA sequencing kit with Big Dye Cycle Sequencing Ready Reaction Kit version 3.1 (Applied Biosystems), according to the manufacturer's recommendations. Sequencing reaction products were separated using POP7 in a 50 cm 48 capillary array on an ABI3730 DNA Sequencer (Applied Biosystems). Sequencher 4.6 (GeneCodes) was used to align individual sequences to wild type reference sequence and identify sequence variations. All sequences were evaluated by two independent editors following criteria for identifying non-reference sequence changes validated by the Johns Hopkins DNA Diagnostic Laboratory. If a variant was detected in a single direction, then the opposite direction was sequenced for confirmation. Mfold was used to predict potential alteration of miRNA secondary structure. The Exome Aggregation Consortium (ExAC) database (http://exac.broadinstitute.org/) was used to obtain population-based allele and genotype frequency for the miR-590 C57T SNP rs6971711.

Plasmid construction
The miR-590 gene (pri-miR-590) was cloned from human DNA using DNA primers shown in S6 Table, using the XhoI and KpnI sites, into the GV268 vector (Geneche). The C57T SNP was introduced using the QuikChangeII XL Site-Directed Mutagenesis Kit (Stratagene). To construct dual luciferase reporters, psiCheck2 (Promega) was digested with XhoI and NotI, and the dsDNA oligos listed in S6 Table were inserted.

RNA preparation
Since endogenous expression of miR-590 is very low in HEK293T cells [37,38], we selected this cell line for analysis of miR-590 biogenesis following exogenous introduction of plasmids.

Real-time qPCR
Reverse-transcription was performed using oligo-dT primer and SuperScript II reverse transcriptase (Invitrogen). Real-time quantitative PCR was performed using Luminaris HiGreen qPCR master mix (Life Technologies) in a CFX96 system (Biorad). The sequences of the oligo primers used are listed in S6 Table. Northern blot Northern blot was performed as described previously [39]: 35 μg total RNA was denatured in formamide loading buffer (98% v/v formamide, 0.1% w/v bromophenol blue, 0.1% w/v xylene cyanol, and 10mM EDTA) at 95C for 5 min and was resolved on a 0.4 mm thick, 15% denaturing polyacrylamide 7 M urea sequencing gel in 0.5×TBE (Tris-Borate-EDTA) buffer. After electrophoresis, RNA was transferred at 20 V for 1 hr to a Hybond-N+ membrane (GE healthcare) in 0.5×TBE buffer using a semi-dry transfer system (Transblot SD, Bio-Rad). The RNA was UV cross-linked (HL2000, UVP) to the membrane and pre-hybridized in Church buffer for at least 60 min at 37C. DNA oligo nucleotide probes (S6 Table) were 5 0 32 P-radiolabeled with γ-32 P-ATP and T4 polynucleotide kinase (NEB). After labeling, non-incorporated nucleotides were removed using a Sephadex G-25 spin column (GE healthcare). The probes were added to the Church buffer and hybridized for at least 6h at 37°C. The miR-590-3p-WT and miR-590-3p-SNP probes were hybridized at 32 and 25C, respectively, because of their lower Tm values. Membranes were washed three times for 10 min in 2×SSC containing 0.05% (w/v) SDS, subsequently exposed to Storage Phosphor Screens (GE healthcare), and analyzed using FLA-9500 (GE healthcare). Probes were stripped from the membranes in boiling 0.1% SDS solution. The membranes were re-probed with the next probe.
Luciferase assay S2 cell dual luciferase reporter assays using the psiCheck2 vector (Promega, Madison, WI, USA) were performed as described previously [39]. HEK293T cells (1x10 5 ) were co-transfected with 20 ng of the psiCheck2 luciferase reporter plasmids and 100 ng of the pri-miR-590 plasmids (WT, SNP, empty vector), using Dharmafect Duo (GE healthcare), 24h after seeding. The media was replaced 24h after transfection. Firefly (Photinus pyralis) luciferase and Renilla luciferase activities were measured using the Dual-Glo luciferase assay system (Promega) 48h after transfection. Firefly luciferase served as the internal control.
U test was used to test significance between groups depending on their distributions. Chisquare test was used for categorical variables. A p-value<0.05 was considered statistically significant. All statistical analyses were performed using the SPSS statistical package (v.13.0).

HCM cohort
A total of 243 patients (mean age 46 ± 16 years; 149 men) were studied. The demographic and clinical features of the entire study population (n = 243) and the cohort of African American HCM patients (n = 55) are described in Table 1.

Identification of miRNA variants in HCM patients
Sanger sequencing of the 18 miRNA genes identified by literature search and bioinformatics analysis revealed 11 variants in 9/18 miRNAs in 89/199 HCM patients ( Table 2 and Supporting Information section). Only one variant, C57T (rs6971711), was located in a mature miRNA: miR-590-3p. The allele frequency of miR-590 C57T was higher in the African American HCM cohort (8.2%, 9 of 110 alleles) compared to the control group (6.5%, 13 of 200 alleles), but the difference was not statistically significant (p = 0.5) ( Table 3). Since allele frequency can vary depending on the population, we also assessed frequency of this allele in European and African populations in the Exome Aggregation Consortium (ExAC) database for comparison. Frequency of miR-590 C57T in individuals with African ancestry in the ExAC database (7.0%, 728 of 10340 alleles) was slightly lower than that seen in our African American HCM cohort (8.2%), but again the difference was not statistically significant (p = 0.5). Notably, this SNP was not seen in whites in our HCM cohort or in the European (Finnish) population in the ExAc database; minor allele frequency in the European (non-Finnish) population was very low (0.0003%, 22 of 66296 alleles) (S4 Table).

MiR-590 C57T SNP rs6971711 changes G-C pair to G-U pair
The miR-590 gene is located within intron 5 of the eukaryotic translation initiation factor 4H (eIF4H) gene in 7q11.23. SNP rs6971711 is located at the 57th position of pre-miR-590 (the 17th position of miR-590-3p). Therefore, it is referred to as miR-590 C57T SNP (and C57U SNP when we refer to pre-miR-590 RNA molecule). Alignment of available pre-miR-590 sequences revealed that the miR-590 C57T SNP rs6971711 is highly conserved in mammals (Fig 2A). Nucleotide C57 is located in the stem of pri-miR-590 and pre-miR-590, and forms a G-C base pair with nucleotide G3 (Fig 2B), which is also highly conserved among mammals (Fig 2A). The C57T SNP changes this conserved G-C base pair to a G-U wobble pair. This change could potentially affect miR-590 maturation, including pri-miR-590 processing by Drosha, pre-miR-590 processing by Dicer, and/or miR-590 duplex stability. MiR-590 C57T SNP rs6971711 reduces abundance of miR-590-5p and miR-590-3p First, we examined whether presence of the C57T SNP in miR-590 affects abundance of miR-590 by quantifying miR-590 production in HEK293T cells. We chose HEK293T because these cells do not express endogenous miR-590, based on high-throughput sequencing data [37,38].
doi:10.1371/journal.pone.0156065.g003 expression to 29 ± 3% of the empty vector control (Fig 5A). In contrast, the pri-miR-590-SNP plasmid suppressed Rr luciferase expression to 40 ± 6% of the empty vector control. The difference between the WT and SNP was significant (p-value < 0.03). Similarly, co-transfection of near perfect match target sites reporter plasmid with the pri-miR-590-WT plasmid suppressed Rr luciferase expression to 62 ± 5% of the empty vector control, while the pri-miR-590-SNP plasmid co-transfection suppressed Rr luciferase expression to 75 ± 2% of the empty vector ( Fig 5B). The difference between the WT and SNP was significant (p-value < 0.02). The control, no target site reporter was not suppressed by pri-miR-590-WT or pri-miR-590-SNP plasmids (Fig 5C). We concluded that the miR-590 C57T SNP reduces miR-590-5p target suppression, which is consistent with our results that this SNP reduces abundance of miR-590-5p (Fig 3).

Discussion
MiRNAs are important regulators of multiple genes via post-transcriptional gene silencing and of physiologic as well as pathologic processes such as cardiac development, hypertrophy, fibrosis, proliferation, apoptosis, and cancer. Previous studies have revealed that occurrence of SNPs or variants in human pre-miRNA sequences is relatively rare:~10% of human pre-miR-NAs have documented SNPs and <1% of human miRNAs have documented SNPs in the seed region, which is crucial for target gene regulation [45]. Base-pairing between the highly conserved, 5 0 proximal seed region (residues 2-8) of miRNA and the 3' UTR of target mRNA is important for miRNA:mRNA binding and silencing [4,5]. Additionally, sequences outside the miRNA seed region can also impact target silencing efficiency and/or the spectra of targeted transcripts [6][7][8]. Our study revealed a SNP in the 17th position of mature miR-590-3p. Experimental studies indicated a 'loss of function effect' of the miR-590 C57T SNP (rs6971711) on mature miR-590 abundance, which is most likely due to SNP-induced change in the G-C Watson-Click base pair within the pri-and pre-miR-590 stem and miR-590 duplex into a G-U wobble base pair. It is possible that this change alters geometry of the RNA helix and thus affects recognition of the pri-and/or pre-miR-590 stem and miR-590 duplex by enzymes or RNA-binding proteins [46][47][48], while the stems of most of pri-miRNAs and pre-miRNAs, including pri-miR-590 and pre-miR-590 ( Fig 2B) contain G-U wobble base pairs, mismatched base pairs, and/or bulged nucleotides. Where in the miR-590 biogenesis step, does the C57T SNP exert its effect? Considering that only mature miR-590-5p and -3p are reduced, but pri-miR-590 and pre-miR-590 levels are unaffected, it appears that the SNP negatively affects miR-590 maturation downstream of pre-miR-590 production by Drosha in nucleus. The possibilities include the following: C57T SNP may reduce (i) transport of pre-miR-590 from nucleus to  [44] were analyzed. The dark gray bars and light gray bars represent IPSC-CMs with and without Endothelin-1 treatment, respectively. Mean ± SD (n = 3). Endothelin-1 (ET-1) treatment is commonly used to stimulate cardiac myocyte hypertrophy [44,49,59].
doi:10.1371/journal.pone.0156065.g006 miR-590 SNP rs6971711 Reduces miR-590 Biogenesis cytoplasm by Exportin-5, (ii) processing of pre-miR-590 into miR-590 duplex by Dicer, (iii) loading of miR-590 duplex to Argonaute and/or (iv) stability of the miR-590 duplex. We performed an in vitro pre-miR-590 processing assay using recombinant Dicer protein to test possibility (ii) and observed no effect of the SNP on pre-miR-590 processing. Future work is needed to test if this is the case in cells and in vivo. We also tested possibility (iv) using an in vitro system, and found that the miR-590 C57T SNP did not affect stability of the miR-590 duplex in HEK293T cell lysate; the WT and SNP miR-590 duplex were degraded at similar rates by cellular RNases present in the HEK293T cell lysate (S2 Fig). Further studies are required to identify the specific step(s) in the miR-590 biogenesis pathway that is negatively affected by the C57T SNP.
Mutations or SNPs in miRNAs can cause disease via two main mechanisms: First, variations in miRNA coding regions, especially the seed region, can act as causal mutations in inherited disease. For example, a mutation in the seed region of miR-96 was segregated with human hearing loss in a large family and this was further reproduced using animal models [10]. MiRNAs may also serve as modifier genes [50]. SNPs (or variants) in miRNA genes have been demonstrated to influence miRNA expression, processing and/or maturation thereby affecting downstream gene targeting [6]. A recent study demonstrated that miR-499 is up-regulated in cardiac hypertrophy and cardiomyopathy [51] and a variant located outside the seed region (miR-499-5p c17) conveyed a favorable impact on the cardiac phenotype when compared to wild-type miR-499-5p by altering the target gene profile [8]. Interestingly, the location of the SNP rs6971711 in mature miR-590-3p (detected in our study) and the miR-499-5p variant are identical; both SNPs are located at the 17th nucleotide position of the respective mature miRNA. Since the miR-590 C57T SNP was observed in 6.5% of controls, it is unlikely to be a causal gene in cardiomyopathies. But it could be a modifier of the cardiac phenotype in heart disease including HCM. SNP-induced reduction of miR-590 levels could lead to de-repression of TGFβRII (target of miR-590-5p [23]) and Acvr2a (target of miR-590-5p/3p), receptors involved in TGF-β and Activin A signaling, respectively, which in turn could influence cardiac hypertrophy and fibrosis (cardiac remodeling) and thus, clinical outcomes in the setting of cardiomyopathies and following myocardial infarction [18].

Clinical implications
African-Americans with heart disease have higher cardiovascular mortality rates [52], disproportionately higher rates of heart failure [53][54][55] and higher mortality [56] in the setting of heart failure, when compared to whites. However, the mechanisms underlying differences in outcomes between African Americans and whites is unknown. Since the C57T SNP in miR-590 is common in African-Americans and can regulate TGF-β signaling, an important regulator of cardiac fibrosis/ventricular remodeling [57,58], presence of the miR-590 C57T SNP could promote cardiac fibrosis by de-repression of TGF-β signaling.

Limitations
The small number of African American HCM patients in our study precluded assessment of the relationship between the common miR-590 C57T SNP and cardiac phenotype. This result is not unexpected because common genetic polymorphisms are known to have small effects on disease phenotype. Future basic and epidemiologic studies in large numbers of African Americans are needed to investigate effect of the miR-590 C57T SNP on myocyte physiology and cardiac fibrosis/function.