The subjects of the present study were 40 patients with cataracts and 30 normal persons who visited the Department of Ophthalmology. The contents of the survey and examination were explained to all of the subjects, and written consent was obtained. The questionnaire assessed gender, age, and life habit variables such as smoking status, drinking status, and daily life, in addition to survey items about personal history. The medical examinations included measurements of height, weight, blood pressure, fasting plasma glucose, and total cholesterol and genetic tests for ABL1 and TDRD7. For the genetic and clinical examinations involving survey, we obtained approval from the institutional review board of Chosun University Hospital. Both groups belonged to the same ethnicity. Thirty healthy, age- and sex-matched subjects were also included for the controls. Blood for testing was obtained before noon the next day after instructing the subjects to keep their stomachs empty for 12 hours or longer after eating dinner. For serum cholesterol, plasma glucose and gene analysis, peripheral blood (4-5 mL) was taken into vacuum tubes treated with EDTA (ethylene diamine tetraacetic acid), and transported on ice from the time it was obtained until centrifugation. The blood that arrived at the test room was then centrifuged for 10 minutes at 3,000 rpm at 4℃ prior to testing. Total cholesterol and plasma glucose were measured using a commercial analytical system (Architect ci16200 Analyser; Abbott Laboratories, Abbott Park, IL, USA).

Total RNA was extracted from each venous blood sample with the EasyRed RNA extraction kit (Intronbio, Seoul, Korea) in accordance with the manufacturer's protocol. The cDNA synthesis was conducted using a Superscript VILO cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. For quantification of the TDRD7 transcript relative to the ABL1 control gene, we used the primers specific for TDRD7 (forward: 5'-ACGAGTAGAGATCACAAATG-3'; reverse: 5'-TCTGCTAAACAGCACTTAAT-3') and ABL1 (forward 5'-CTGCTAGAGAAGGACTACC-3'; reverse: 5'-TCGTCTGAGATACTGGATTC-3'). Real-time polymerase chain reaction (PCR) reactions with the above primers were conducted in a total volume of 20 µL using Phusion Flash (Thermo Scientific, Wilmington, DE, USA) with EvaGreen (Biotium, Hayward, CA, USA) in accordance with the manufacturer's recommendations. Thermocycling was carried out using a CFX96 real-time PCR detection system (Bio-rad, Hercules, CA, USA) with the following conditions: 1 cycle of 98℃ for 10 seconds, 40 cycles of 98℃ for 1 second, and 55℃ for 15 seconds. The amplification curves of 2 genes, TDRD7 and ABL1 mRNA, are shown in Fig. 1.

Amplification of an endogenous control may be conducted to standardize the amount of TDRD7. For the quantification of gene expression, we employed ABL1 mRNA as the endogenous control. The relative expression levels of TDRD7 mRNA, normalized to ABL1 mRNA, were calculated using the 2^-ΔΔCt value. In order to apply the 2^-ΔΔCt method, the results of real-time reverse transcription-PCRs were represented as cycle threshold (Ct) values. The Ct value was defined as the cycle at which a sample crosses a threshold that is significantly above the background fluorescence and within the exponential phase of the amplification. The average of three Ct measurements was calculated for both the TDRD7 and ABL1 mRNA. ΔCt was determined as the mean of the triplicate Ct values for the TDRD7 genes minus the mean of the triplicate Ct values for the ABL1 gene. The ΔΔCt represented the difference between the two groups for a given target gene, namely ΔΔCt = ΔCt (cataract group) - ΔCt (the mean of normal group). The ^x-fold higher expression of a given target gene in the cataract group compared to the normal group was calculated as 2^-ΔΔCt. All assays were executed in triplicate.

A past history of ophthalmologic disease such as ocular trauma and cataract surgery was checked, as well as whether the subjects had systemic disease such as hypertension, diabetes, or cardiovascular disease, along with family history. In addition to uncorrected visual acuity, corrected visual acuity was examined in the case of subjects that wore corrective lenses, and the best corrected visual acuity was measured after checking the refractive state via a manifest refraction test. The state of the lens was classified by Lens Opacities Classification System (LOCS) III through an examination using a slit lamp microscope after mydriasis [12]. If, in one eye or two eyes, there was nuclear opacity of second stage or higher in LOCS III classification, cortical opacity of second stage or higher, or posterior subcapsular opacity of first stage or higher, those subjects were classified as cataract patients, and were otherwise categorized as control group members. Cataract patients were classified into nuclear, cortical, posterior subcapsular, or mixed types. Cases of pseudophakic eye after cataract surgery were excluded from the classifications.

Data analysis was conducted using SPSS ver. 17.0 (Chicago, IL, USA) and the actual numbers, percentage, average, and standard deviation were obtained as descriptive statistics in order to study the general characteristics of the patient group and control group. The comparison of the general characteristics of the patient group and the control group was conducted via t test and χ² test, and, in comparison of the genetic values of the patient group and the control group, cases in which the p-value was found to be below 0.05 by an independent sample t test were determined to be statistically significant. In order to compare the TDRD7 gene values of different types of cataracts, one-way ANOVA was used; to compare the TDRD7 gene values of normal eyes of different age objects, correlation analysis was conducted. Cases in which the p-value was below 0.05 were determined to be statistically significant.

The ABL1 and TDRD7 values of the control group were 26.95 ± 0.91 and 29.68 ± 1.07, respectively, and those of the cataract patients were 26.17 ± 1.25 and 29.11 ± 1.34, respectively. ΔCt was determined as the mean of the triplicate Ct values for the TDRD7 genes minus the mean of the triplicated Ct values for the ABL1 gene. ΔΔCt represented the difference between the two groups for a TDRD7 gene, more precisely ΔΔCt = ΔCt (cataract group) - ΔCt (normal group). The mean and standard deviations for all ΔCt and ΔΔCt values (cataract versus normal group) are provided in Table 2. The actual expression level of a TDRD7 gene was analyzed using the 2^-ΔΔCt value. The results, including standard deviations, are listed in Table 2. As the result of adjusting the TDRD7 value in reference to ABL1 mRNA, the TDRD7 expression level of the cataract patients was found to be significantly lower than that of the control group, with a value of 1.05 ± 0.34, whereas that of the control group was 1.52 ± 0.63 (p = 0.048) (Table 2). To allow for a better overview and to show the broad spectrum of expression rates, all 2^-ΔΔCt values (with standard deviations) are illustrated within a single figure (Fig. 2).

The results of our comparisons of the genetic values of different types of cataracts demonstrated that the TDRD7 expression levels of the cortical type and mixed type were statistically significantly lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017) (Table 3). To allow for a better overview and to demonstrate the wide spectrum of expression rates, all 2^-ΔΔCt values (with standard deviations) are illustrated within a single figure (Fig. 3). The results of measuring the TDRD7 gene expression levels of the normal eyes of different age objects demonstrated that there was no statistically significant correlation between the two variables (p = 0.785) (Fig. 4).

Lachke et al. [11] suggested that RNA-containing granules can regulate the subcellular localization of mRNA as well as the processing which plays an important role in the transparency of the lens. Cytoplasmic RNA granules play a role in determining whether mRNAs undergo degradation, stabilization, or intracellular localization. The structure of the lens in vertebrates requires a very high concentration of specialized cytoplasmic proteins: the crystalline, a specialized intermediate filament cytoskeleton to stabilize the structure of the lens [15]; cellular membranes with high concentrations of both the lens-specific water channel, aquaporin 0; and lens-preferred connexins, which allow the lens to transport nutrients and waste products despite its lack of blood vessels [16]. A number of the genes encoding for these proteins are abundantly expressed in the lens, resulting in a highly biased transcriptome [17]. Lachke et al. [11] have reported the identification of Tudor domain-containing 7 protein, or TDRD7, as an RNA granule component with a highly enriched and conserved pattern of developmental expression in the ocular lens. They suggested that human TDRD7 mutations result in cataract formation via the misregulation of specific, developmentally critical lens transcripts.

The official name of the TDRD7 gene is "tudor domain containing 7"; it is also referred to by other names such as CATC4, KIAA1529, PCTAIRE2BP, RP11-508D10.1, and TRAP. It is located on the long(q) arm of chromosome 9 at position 22.33. More accurately, it is located from base pair 100,174,301 to base pair 100,258,406 on chromosome 9. TDRD7 is a member of a large family of Tudor domain-containing proteins that mutually interact with methylated arginine residues on other proteins. Many members of the TDRD family, including TDRD7, are found in RNA granules-cytoplasmic complexes of protein and RNA that regulate gene expression by influencing RNA degradation, stabilization, and the intracellular localization of RNA [18]. Although TDRD7 transcripts and proteins are found in a variety of cells, especially of the male germ line, TDRD7 expression is very high in lens fiber cells and may control the production of extremely high amounts of structural proteins of the lens. TDRD7 is a Tudor domain RNA binding protein which is expressed in lens fiber cells, and is required for the posttranscriptional control of mRNAs that are essential for normal lens development and RNA granule function. Lachke et al. [11] demonstrated that TDRD7 is found in a granular distribution in lens fiber cells and colocalizes with concentrations of RNA found in the cytoplasm of these cells. They demonstrated that human organogenesis defects can result from the perturbation of a distinct, tissue-specific RNA granule component that regulates the posttranscriptional steps of developmentally critical mRNAs. Simple RNA distribution within lens fibers would presumably be inefficient because of the relatively long distance between the lens fiber cell nucleus and the fiber cell tips, as well as the high concentration of proteins in the fiber cell cytoplasm [19]. Accordingly, when we look into the matter retrospectively, the requirement for a lens-preferred RNA transport mechanism may not be entirely surprising. Most notably, the lens epithelial cells express mRNAs for lens structural proteins that are not translated into proteins until the onset of fiber cell differentiation, which is also when the onset of TDRD7 expression occurs [20]. Additionally, another possible role for TDRD7-containing RNA granules would be to regulate the translation of fiber cell structural proteins. Lachke et al. [11] determined that reduced TDRD7 expression in a lens epithelial cell line induced statistically significant changes in the expression levels of 6% of the genes expressed in those cells, including some crystallins. Overall, Lachke et al. [11] demonstrated that the vertebrate lens contains several classes of RNA granules and that TDRD7-containing granules are critically important for the normal functioning of the lens. These findings open up a new field of investigation that may ultimately help to elucidate both the physiological and biochemical functions that regulate RNA granules in the lens.

When we look into different risk factors of cataract, there are some risk factors which clearly affect specific types of cataracts. For example, smoking increases the prevalence of nuclear cataracts, and steroids are related to the occurrence of posterior subcapsular cataract [5]. The results of comparing the gene values of different types of cataracts in this study demonstrated that the TDRD7 expression ratios of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular type; this implies that, while many senile cataracts are of nuclear type, many cataracts caused by genetic loss-of-function mutations may be of the cortical type.

Although blood cholesterol is not known as a risk factor for cataracts, a case was recently reported wherein posterior subcapsular cataract occurred in two eyes of a lathosterolosis patient showing a defect of cholesterol biosynthesis [21]; Klein et al. [22] suggested that the ingestion of statin, which is widely used for the purpose of preventing cardiovascular disease by lowing the blood cholesterol value lowers the incidence rate of nuclear cataracts. In this study, although the total cholesterol value of cataract patients was higher than that of the control group, this was not statistically significant. The result of comparing the age and the TDRD7 gene expression ratios of the patients with normal eyes demonstrated that there is no statistically significant correlation between the two variables (Fig. 4).

Although this study is significant in that it is the first study regarding the relationship between the TDRD7 gene and cataracts using Koreans as subjects, further proactive studies with a greater number of subjects selected from a larger number of regions are needed to more clearly establish the relationship between the TDRD7 gene and cataracts.

In conclusion, as the TDRD7 gene expression levels of the cataract patients found in a test conducted with 70 subjects living in a local region in the present study were statistically significantly lower than those of the control group, we can assert that there is a causal relationship between the occurrence of human cataracts and the loss- of-function mutations of the TDRD7 gene.