Small ncRNA Expression-Profiling of Blood from Hemophilia A Patients Identifies miR-1246 as a Potential Regulator of Factor 8 Gene

Abstract

Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). Genetic mutations in the gene encoding FVIII (F8) have been extensively studied. Over a thousand different mutations have been reported in the F8 gene. These span a diverse range of mutation types, namely, missense, splice-site, deletions of single and multiple exons, inversions, etc. There is nonetheless evidence that other molecular mechanisms, in addition to mutations in the gene encoding the FVIII protein, may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-à-vis the role of nc-RNAs in the regulation of F8 expression. These hypotheses have not been exhaustively tested in this study as they require carefully curated clinical samples.

Fig 1. Hierarchical clustering and principal component analyses of significantly differentially expressed ncRNAs from ANOVA analysis.

(A) Unsupervised hierarchical clustering analysis of 100 significantly differentially expressed ncRNAs between all hemophilia A patients (yellow and orange bars) and controls (red bar) shows the distinct ncRNA expression pattern of the two group (p-value < 0.01). Each row represents a subject, whereas each column is a ncRNA probe. Blue color in the heat map indicates down-regulation, whereas red indicates up-regulation. (B) Principal component analysis of samples on the basis of the 100 significantly differentially expressed ncRNAs identified by the ANOVA analysis described above. Yellow spheres represent HA samples, whereas turquoise spheres represent unaffected controls.

Fig 2. Principal component analysis of significantly differentially expressed ncRNAs from 3-class ANOVA analysis and 3-class SAM analysis.

(A) 3-class ANOVA analysis. Principal component analysis of samples based on 88 significantly differentially expressed ncRNAs from 3-class ANOVA analysis (p-value < 0.01) reduces the dimentionality of the data and shows the clear separation between the hemophilia A patients with inhibitor development (red), the hemophilia A patients without inhibitor development (yellow), and the controls (turquoise). (B) 3-class SAM analysis. Principal component analysis of samples based on 11 significantly differentially expressed ncRNA probes from 3-class SAM analysis (FDR < 5%) reveals the distinct separation between the hemophilia A patients with inhibitor development (red), the hemophilia A patients without inhibitor development (yellow), and the controls (turquoise).

Fig 3. Differentially expressed ncRNA probes identified by 3-class ANOVA and SAM analyses.

(A) Significant ncRNA probes were identified using 3-class ANOVA analysis with p-value < 0.01 and 3-class SAM analysis with FDR < 5%. A total of 4 ncRNAs (hsa-miR-1246, hsa-miR-4521, HBII-13, and SNORD121B) were found to overlap between the two statistical analyses. (B) Hsa-miR-1246 (red dot) is significantly down-regulated in HA patients with inhibitor relative to patients without inhibitor.

Fig 4. Sequence alignment of hsa-miR-1246 and F8 mRNA.

Fig 5. Fold change in hsa-miR 1246 and F8 mRNA levels in HA patients compared to normal donors.

(A) The fold change in has-miR 1246 was determined by RT-PCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a significant increase in the has-miR 1246 levels in samples from HA patients compared to controls. (B) The fold change in F8 mRNA was determined by RT-PCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a significant decrease in the F8 mRNA levles in samples from HA patients compared to controls.