Real‐Time Quantitative PCR Measurement of Circulatory Rhodopsin mRNA in Healthy Subjects and Patients with Diabetic Retinopathy

Abstract

Diabetic retinopathy is the commonest complication of diabetes and is the biggest single cause of registered blindness in the UK. No biochemical tests exist to determine the precise state and rate of change of the eyes in the diabetic patient. In the present study, using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we measured mRNA encoding the retina-specific pigment protein rhodopsin (RHO) in the peripheral blood of healthy individuals (n = 20) and diabetic patients (n = 46) with and without retinopathy. Beta-actin mRNA was also assayed and results are expressed as a ratio of RHO to beta-actin mRNA. Peripheral blood was taken by venipucture directly into PAXgene Blood RNA collection tubes and RNA extracted by use of the PAXgene Blood RNA extraction kit, as per the manufacturer's (Qiagen) instructions. Diabetic patients were divided into three groups defined by the severity of retinopathy as assessed by fundoscopy: A, diabetic without retinopathy; B, background retinopathy; and C, preproliferative retinopathy. Medians of the ratios between groups were compared. RHO mRNA was successfully detected and quantified in peripheral blood in all healthy and diabetic groups, with levels shown to be significantly higher in diabetic patients than in healthy controls (2.54 x 10(-5) vs. 1.29 x 10(-5); P = 0.002). Significant differences in RHO mRNA levels were also seen between healthy control subjects and diabetic groups A (2.52 x 10(-5); P = 0.022), B (1.98 x 10(-5); P = 0.028), and C (5.08 x 10(-5); P = 0.002). The results suggest that there is an increase in circulatory RHO mRNA with the severity of diabetic retinopathy.

Association of Circulating Markers With Outcome Parameters in the Bevacizumab and Ranibizumab in Diabetic Macular Edema Trial

“…It is generally assumed that retinoschisin and rhodopsin are retinaspecific proteins that are locally produced. 9,12,13,[18][19][20] This assumption is supported by our finding of higher mRNA levels of retinoschisin in donor retina than in plasma of DME patients. Limitations of this study include the fact that patients had been treated with either bevacizumab or ranibizumab, and our results may have been influenced by that particular anti-VEGF agent.…”

“…Previous reports indicated that circulating retina-specific mRNA, including retinoschisin, rhodopsin, and RPE65 mRNA, may be useful in assessing the progression of DR. 9,10,12,13 We found that mRNA levels of retinoschisin and rhodopsin are associated with changes in central subfield thickness for up to 3 months after initiation of anti-VEGF treatment. Although not all their functions are known, rhodopsin and retinoschisin may play an active role in maintaining retinal integrity.…”

“…Further research is needed to establish the relationship between these plasma mRNA levels, corresponding protein levels, and the pathogenesis of DME. Several previous reports have indicated that retina-specific transcripts in the blood, including retinoschisin and rhodopsin, may be promising candidate biomarkers in DR. 9,10,[12][13][14] The identification of retina-specific transcripts in the blood may have implications for a better understanding of disease progression and possibly stratification of patients that may allow the development of more effective treatment strategies in DME, for example, identifying patients most prone to worsening or whose response to therapy is the most significant. Because little is known about biomarkers in DME and because there are no robust methods to determine which patients with DME are good responders and which are nonresponders to anti-VEGF therapy, this study may contribute to the development of more effective treatment strategies of these patients.…”

Detecting cell-free circulating hTERT mRNA in the plasma may identify a subset of nonsmall cell lung cancer patients

“…This approach is based on the detection of genetic/epigenetic changes shared by primary tumors [1,2,11,37,54,59] or on the quantitative assays of cell-free total circulating DNA [57,58]. More recent and exciting, however, is the discovery that many different types of cell-free circulating mRNA molecules may be detected in the bloodstream of patients with a variety of malignancies such as melanomas [10,20,29,45], follicular lymphomas [9], and breast [3,15,28,45,55], large-bowel [9,35,56,72], liver [38,39,44], esophagus [10], nasopharynx [36], thyroid [34,45], prostate [5], and lung [12,28] carcinomas, in hepatic cirrhosis and chronic hepatitis [38,39], in patients with trauma [51] or diabetic retinopathy [18,19], or in women during pregnancy for either fetal [43,50,62,66,67] or maternal [42] mRNA. The occurrence of detectable cell-free and stable RNA in the blood of such patients [63] challenges the lo...…”

Genetics of diabetic retinopathy

“…An interesting recent study relating genetic factors to retinopathy used real-time quantitative reverse transcriptase-polymerase-chain reaction to measure plasma levels of mRNA from the retina-specific rhodopsin (RHO) gene in healthy subjects and diabetic patients with and without retinopathy [26]. Compared with levels in healthy controls, circulating RHO mRNA was significantly higher even in diabetic patients without retinopathy, but tended to be progressively higher in groups with increasingly severe retinopathy, from no disease to background retinopathy to preproliferative retinopathy.…”