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Technical Briefs |
Departments of1 Chemical Pathology and2 Diabetes and Endocrinology, St. Thomas’ Hospital, London, UK
aaddress correspondence to this author at: Department of Chemical Pathology, 5th Floor, North Wing, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, United Kingdom; fax 44-020-2928-4226, e-mail r.swaminathan{at}kcl.ac.uk
Diabetes mellitus is a major health problem throughout the world, and retinopathy is a serious and common complication of diabetes (1). Most individuals with diabetes will eventually develop diabetic retinopathy (DR). In the United States, up to 24 000 patients with diabetes become legally blind each year, and DR is the leading cause of blindness in adults 20–74 years of age (2). The United Kingdom Prospective Diabetes Study (UKPDS) showed that 39% of men and 35% of women had some degree of retinopathy at the time of diagnosis (3). With the incidence of diabetes mellitus increasing in many countries, the prevalence of DR as a major cause of blindness or visual impairment is also likely to increase. Factors influencing the development of retinopathy include type and duration of diabetes, degree of glycemic control, blood pressure, and serum lipids (3)(4)(5). Of these, duration of diabetes is the major factor related to the incidence of retinopathy.
Clinical trials such as the Diabetic Retinopathy Study (DRS) (6) and the Early Treatment Diabetic Retinopathy Study (ETDRS) (7) have shown that effective laser photocoagulation could reduce the loss of vision by 90%. To be effective, regular screening for DR is required. Current recommendations are for annual screening for DR (8). Although recent studies suggest that in the absence of risk factors such as insulin use or duration of diabetes >20 years, screening every 2 or 3 years is adequate in type 2 diabetes (9). Despite these guidelines, 30–50% of patients with diabetes still do not get any eye examinations (10). Screening for DR includes visual acuity assessment as well as fundal photographs through dilated pupils. This involves cooperation between various healthcare professionals, such as diabetologists, optometrists, general practitioners, and photographic screening clinics. Patient eye status is classified according to a scale for the number of abnormalities observed (11), but this type of approach is subjective, and reliable identification requires expert training for accurate assessment. Such regular screening is not inexpensive, and it has been estimated that the cost is approximately $220 per visit (12). Diabetic nephropathy, another complication of diabetes, can be predicted by a simple and inexpensive test, microalbuminuria. There is as yet no reliable, independent, quantifiable, and nonsubjective test for DR.
During the past few years, tissue-specific nucleic acids have been identified in peripheral blood, and measurement of circulating nucleic acids offers enormous potential for prognostic and diagnostic purposes (13). It has been suggested that the concentration of plasma nucleic acids (DNA and RNA) reflects the degree of cell death. Increased amounts of circulating nucleic acids have been demonstrated in pathologic processes, including lung cancer (14), thyroid cancer (15), stroke (16), and trauma (17). Measurement of tissue-specific genetic markers in peripheral circulation is thought to give an early indication of the degree of tissue-specific damage. We postulated that measurement of retina-specific mRNA may be useful as a quantifiable marker for the assessment of eye status. The aim of this study was twofold: (a) to determine whether rhodopsin mRNA is detectable in the circulation of healthy individuals and diabetic patients by real-time quantitative PCR (18); and (b) whether this retina-specific mRNA marker is altered in DR.
Patients with diabetes were recruited from the Diabetes Clinic at St. Thomas’ Hospital, London. The protocol for this study was approved by the St. Thomas’ Hospital Local Research Ethics Committee. The eye status of these individuals was determined by independent examination of fundoscopic photographs. All diabetic patients were assigned to one of four groups: group A, diabetic patients without retinopathy (diabetic controls; n = 8); group B, diabetic patients with background retinopathy (n = 12); group C, diabetic patients with preproliferative retinopathy (n = 22); and group D, diabetic patients with proliferative retinopathy (n = 4). Blood samples were also collected from healthy volunteers (n = 20) with no known disease. Informed consent was obtained from each participant before blood collection. Peripheral venous blood (2.5 mL) was collected directly into PAXgeneTM Blood RNA tubes specifically designed for the collection and stabilization of RNA from whole blood (Qiagen).
Whole-blood RNA was extracted by use of the PAXgene Blood RNA Kit. This method includes treatment with DNase I to prevent contamination with genomic DNA. From this RNA, cDNA was synthesized by reverse transcription using SUPERSCRIPT IITM reverse transcriptase according to the manufacturer’s instructions (Invitrogen Life Sciences). The cDNA generated was stored at –80 °C until required for quantification of rhodopsin and ß-actin cDNA. ß-Actin was used to confirm amplification. Separately, samples were also subjected to the above procedure without SUPERSCRIPT II, which was replaced with water (negative control). cDNA was amplified in the ABI 7000 Sequence Detection System (PE Applied Biosystems), and the PCR products were detected by use of sequence-specific oligonucleotide probes and intron-spanning specific primers. ß-Actin cDNA was amplified with use of the Pre-Developed Assay Reagents TaqMan® assay (PE Applied Biosystems). For the rhodopsin (GenBank accession no. U49742) TaqMan assay, 5 µL of cDNA sample was mixed with 900 nM forward (5'-CCGGCTGGTCCAGGTACAT-3') and reverse primer (5'-TTGTTGACCTCCGGCTTGAG-3'), 250 nM probe (5'-FAM-CTGCAGTGCTCGTGTGGAATCGACTACT-TAMRA-3', where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine), and X2 TaqMan Universal Master Mix (25 µL) in a final reaction volume of 50 µL. All assays were done in triplicate. Calibration curves were prepared from serial dilutions of cDNA (Clontech) obtained from healthy human retina. A water blank was also incorporated in each run for the respective assays. Both assays were run simultaneously in 96-well optical reaction plates. PCR amplification included an initial phase of 2 min at 50 °C, followed by 10 min at 95 °C and 40 cycles of 15 s at 95 °C and 1 min at 60 °C.
Results for rhodopsin mRNA were expressed as the ratio to total blood ß-actin mRNA. Statistical analysis was performed with SPSS. Differences in rhodopsin mRNA between groups were analyzed by the Kruskal–Wallis test followed by a post hoc test where appropriate. A P value <0.05 was considered statistically significant.
The slopes of eight consecutive calibration curves showed good reproducibility for both target and housekeeping genes (CVs of 5.1% for rhodopsin and 4.8% for ß-actin). Rhodopsin mRNA was detected in the peripheral blood of all healthy and diabetic individuals in this study. The rhodopsin mRNA concentration in patients with diabetes (median ratio of rhodopsin mRNA to total blood ß-actin RNA, 2.54 x 10–5; n = 46) was significantly higher than that in the healthy individuals (median ratio, 1.29 x 10–5; P = 0.002). We observed a significant difference in rhodopsin mRNA between groups (P = 0.008). Diabetic patients without retinopathy (group A) had significantly (P = 0.022) higher median mRNA concentrations than did healthy individuals. We also observed a significant difference in mRNA between healthy individuals and DR groups B (P = 0.028) and C (P = 0.002), respectively (Fig. 1
). The rhodopsin mRNA concentration in diabetic patients with proliferative retinopathy (group D), however, was not different from the concentration in healthy individuals. When we compared groups A, B, and C, we observed a tendency for the mRNA concentration to increase with increasing severity of retinopathy (P = 0.041 for trend).
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To the best of our knowledge, this is the first study to use real-time PCR to quantify rhodopsin mRNA in peripheral blood for assessment of DR retinopathy. The results showed that rhodopsin mRNA was present in quantifiable amounts in both healthy individuals and patients with and without DR. The presence of RNases in the circulation meant that the sample had to be treated at the time of collection to prevent degradation of RNA. We therefore chose to collect whole blood by the PAXgene method, which serves the twofold purpose of lysing all cells and stabilizing RNA from degradation, thereby maximizing mRNA yield. However, this method does not distinguish between the possible sources of this RNA, i.e., retinal cells, blood cells, or other cells. It is also possible that the rhodopsin mRNA detected here may be attributable to the presence of "illegitimate transcription", i.e., a basal amount of transcription of tissue-specific genes outside of the tissues in which they are typically active. Although this phenomenon is known to occur for certain genes, the illegitimate transcripts are thought to be of very low abundance (19), and this may account, in part, for the detection of rhodopsin mRNA in healthy individuals.
With the exception of the group of patients with proliferative retinopathy, there was a trend for the relative amounts of rhodopsin mRNA detected to increase with severity of retinopathy. This upward trend may be related to the increasingly severe damage and widespread retinal pathology associated with the progressive stages of DR. Although the precise mechanisms are unclear, it is plausible that (a) release from dead or dying retinal cells as a result of ischemia, (b) possible up-regulation of rhodopsin transcription, or (c) controlled secretion of rhodopsin mRNA may be the underlying reasons for the increase in rhodopsin mRNA observed in peripheral blood. Indeed, other studies have implicated apoptosis, necrosis, and active secretion as possible mechanisms for the presence of nucleic acids (in particular, DNA) in the peripheral circulation (20). In the case of the proliferative retinopathy group, in whom peripheral blood rhodopsin mRNA concentrations appeared to be the lowest, it may be argued that so much damage has occurred to this point on the disease scale that retinal cells are exhausted, either in a metabolic capacity or in total number. In this preliminary study, we examined only a marker specific for the rods because these are more abundant than cones (ratio of 20:1). Investigation of other retina-specific markers may help to confirm these preliminary findings. Further research into the origin of circulating RNA could help to reveal the exact nature of the significant differences seen in this study and, possibly, the mechanisms involved. Longitudinal and large-scale studies are required to determine the diagnostic and prognostic value of this measurement.
We conclude that rhodopsin mRNA in peripheral blood, measured by real-time quantitative PCR, is increased in DR and may be a useful marker of this condition.
Acknowledgments
We appreciate the cooperation of the Molecular Working Group at St. Thomas’ Hospital for use of facilities in the Molecular Diagnostics Laboratory. Our thanks to Julie Clayton for assistance in manuscript preparation.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Rani, M. Clynes, and L. O'Driscoll Detection of Amplifiable mRNA Extracellular to Insulin-Producing Cells: Potential for Predicting Beta Cell Mass and Function Clin. Chem., November 1, 2007; 53(11): 1936 - 1944. [Abstract] [Full Text] [PDF]
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E. Orlandi, A. Butt, D. Goldsmith, and R. Swaminathan Factors Affecting Circulating mRNA for Nephrin Clin. Chem., October 1, 2005; 51(10): 1982 - 1983. [Full Text] [PDF]
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