MicroRNA Expression in Red Blood Cells from Patients with PNH.

Abstract Although recent advances in microarray technology and analytic tools have led to an explosion of knowledge in many human diseases, the application of these tools to erythrocyte diseases has been limited by the long-held belief that mature erythrocytes lack most RNA expression. Recently, we have found that although mature erythrocytes lack most large-sized RNA, they possess abundant and diverse microRNAs (miRNAs), a class of small-sized RNAs with important regulatory functions [2]. While their functional role in mature erythrocytes remains to be investigated, these miRNAs are likely to play important regulatory roles during earlier stages of erythropoeisis. The discovery of these expressed miRNAs also allows us to use genomic tools and advanced bioinformatics to elucidate the biological role of miRNAs in the erythrocyte, particularly their contribution to phenotypic variations in erythrocyte disorders. As an initial step toward this aim, we obtained reticulocyte-free erythrocyte RNAs from seven HbAA individuals, sixteen HbSS individuals (homozygous sickle cell anemia) and six individuals with paroxysmal nocturnal hemoglobinuria (PNH). We analyzed these samples with microRNA microarrays and compared their gene expression of mature erythrocytes. The most prominent feature is the robust separation of HbAA and HbSS samples into two distinct groups with unsupervised analysis with hierarchical clustering. Interestingly, the new 7 PNH samples were split into two branches, one branch (four samples) was arranged together with normal individuals (thus termed “normal-like” group) while the other branch (three samples) was arranged with sickle cell individuals (thus termed “HbSS-like”). The four patients with the “normal-like” gene expression all exhibited a low level of hemolysis when compared with the other three patients with the “HbSS-like” gene expression. Three of the PNH patients with “normal-like” gene expression were receiving Eculizumab, a humanized antibody blocking complement C5 protein expected to reduce hemolysis and transfusion requirement in patients with PNH. The fourth patient, thought not receiving eculizumab treatment, has very mild hemolysis clinically. In contrast, the two PNH patients with “HbSS-like” gene expression pattern exhibit significant hemolysis. It also suggests that one important component of dysregulated gene expression seen in HbSS erythrocytes may reflect hemolysis, a clinical feature shared by all HbSS patients and a subset of PNH patients. Supervised analysis of erythrocyte gene expression with SAM also allowed the identification of 256 miRNAs associated with one of the three groups (HbAA, PNH or HbSS) of erythrocytes with a SAM analysis. The PNH-specific genes included miR-17 and miR-130a. The HbSS-specific genes included miR-223 and miR-451. And the normal-specific genes included miR-320 and let-7 family. These selected microRNAs may be valuable as diagnostic tools and reveal distinct pathophysiological processes in these diseases. Taken together, the genomic analysis of erythrocyte microRNA expression may provide relevant biomarkers and important insights into the diseased phenotypes of erythrocytes in human anemia disorders.