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Mitochondrial Gene Transfer Ribonucleic Acid (tRNA)^Leu(UUR) 3243 and tRNA^Lys 8344 Mutations and Diabetes Mellitus in Korea

Division of Endocrinology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea

Address all correspondence and requests for reprints to: Dr. Hyun Chul Lee, Division of Endocrinology, Department of Internal Medicine, Yonsei University College of Medicine, 134 Shinchondong, Sudaemungu, Seoul 120–752, Korea.

	Abstract

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The high prevalence of diabetic patients with a mutation in the mitochondrial gene (the 3243 and 8344 bp mutations) has been identified in Japan. To determine the prevalence of diabetic patients with those mutations in Korea, we randomly screened selected 503 clinical diabetic patients regardless of their diabetes types. We found only 1 patient with the mitochondrial DNA mutation at position 3243 (percent mutation, 32%), and the mitochondrial DNA mutation at position 8344 was not found in any of the patients. The affected subject was a 22-yr-old man with a history of myoclonic epilepsy and mild sensorineural hearing loss, a 1-yr duration of diabetes mellitus, and a low level C peptide response to oral glucose. Because of the low frequency of these mutations in the Korean diabetic population, we concluded that these particular mutations of mitochondrial DNA may not be a common contributor to diabetes mellitus in Korea.

	Introduction

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IT IS NOW well established that genetic factors play an important role in the development of diabetes mellitus, but extensive international research has not been able to locate any genetic lesions. Only rare cases of diabetes mellitus have been reported to be associated with mutations in the insulin gene, the insulin receptor gene (1, 2), the glucokinase gene (4, 5, 6), the glucagon receptor gene (7), the insulin receptor substrate-1 gene (8), the fatty acid-binding protein-2 gene (9), and the ß₃-adrenergic receptor gene (10). In recent years, however, an increasing number of publications have shown that certain deletions (11, 12), insertions, or point mutations (13) of mitochondrial DNA (mtDNA) are associated with diabetes mellitus, although mtDNA mutations are rare.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode [A to G transition mutation in the mitochondrial transfer RNA^Leu (tRNA^Leu) gene at position 3243] (14, 15), myoclonic epilepsy with ragged red fiber (A to G transition mutation in the mitochondrial tRNA^Lys gene at position 8344) (16), and other mitochondrial cytopathies are associated with diabetes mellitus. The above observations suggest that alterations of mitochondrial DNA (mtDNA) may to some degree contribute to the development of diabetes mellitus. The tRNA^Leu(UUR) 3243 and tRNA^Lys 8344 mutations in mtDNA have been studied in other countries (17); however, the prevalence of mtDNA mutation has not been surveyed in the Korean population with diabetes mellitus. Therefore, we examined the prevalence of tRNA^Leu(UUR) 3243 and tRNA^Lys 8344 mutations in mtDNA in the Korean population with diabetes mellitus.

	Subjects and Methods

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We examined 503 randomly selected Korean patients with diabetes mellitus without prior information about the type of diabetes, age, or other clinical data. All patients were recruited from Severance Hospital. All subjects fulfilled the diagnostic criteria of the WHO. All subjects gave written informed consent, and the study was approved by the ethics committee of Yonsei University College of Medicine.

mtDNA was extracted from the mitochondria of peripheral blood lymphocytes. PCR was performed for 35 cycles on a thermal cycler: denaturation at 95 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 1 min. The forward and reverse primers for tRNA^Leu(UUR)3243 were 5'-AGGACAAGAGAAATAAGGCC-3' and 5'-CACGTTGGGGCCTTTGCGTA-3', respectively; the forward and reverse primers for tRNA^Lys 8344 were 5'-CTACCCCCTCTAGAGCCCAC-3' and 5'-TAGTATTTAGTTGGGGCATTTCACTGTAAAGCCGTGTTGG-3', respectively. PCRs for two pairs of primers were performed separately. PCR products (resultant 294- and 225-bp fragments) were digested with endonucleases. mtDNA mutated at the 3243 position (GAGCCCGGGCCC) could be cleaved by ApaI, and two fragments of 179 and 115 bp were produced; mtDNA mutated at 8344 position (GCCAACA CCTCGCCNNNNNGGC) could be cleaved by BglI, and two fragments of 187 and 38 bp were produced. The digested PCR products were electrophoresed in 3% agarose gels.

The degree of heteroplamy was determined by densitometric analysis with the CSC Chemiluminescence Detection Module (Raytest Isotopemßgeräte, Straubenhardt, Germany), and PCR-digested sample produced by the mutation at a ratio of less than 3.2% could be detected.

	Results

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The characteristics of our subjects are shown in Table 1. They consisted of 45 patients with insulin-dependent diabetes mellitus (IDDM), 435 patients with noninsulin-dependent diabetes mellitus (NIDDM), 5 patients with gestational diabetes mellitus, and 18 patients with postrenal transplant diabetes mellitus. Among the 503 diabetic patients, the mutation in tRNA^Leu(UUR) 3243 was detected in only 1 subject (Fig. 1), and the relative proportion of mutant and wild-type mtDNA was about 32:68 (percent mutation, 32%), whereas the mutation in tRNA^Lys 8344 could not be found in any of the patients. We also examined the mtDNA of his family members.

View larger version (103K): [in this window] [in a new window]   Figure 1. Among the 503 diabetic patients, only 1 subject had the tRNALeu(UUR) 3243 mutation in mtDNA (lane 1). Lanes 2, 3, and 4 indicate his family members. His mother and brother also showed the tRNALeu(UUR) 3243 mutation, but his father did not.

In the family member search, the patient’s mother and brother were heteroplasmic for this mutation, but his father was not. His elderly brother died during the perinatal period of an unknown illness. Neither his parents nor his younger brother had any evidence of neurological symptoms or diabetes mellitus, and their oral glucose tolerance test results were within normal limits. His grandmother died at the age of 60 yr without any known specific disease. One of his aunts has mild hearing loss, but none of his other relatives have had any known neurological symptoms or diabetes mellitus. As the mtDNA mutation at position 3243 was also detected in his mother and brother, but not in his father, maternal inheritance is suggested. However, the blood samples of other relatives, aunts, uncles, and cousins were not available.

	Discussion

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Redrawn et al. (13) described two patients with the mutation of mtDNA who had poor insulin secretory responses to glucose. Kadowaki et al. (18) and others (19) demonstrated a significant reduction of maximal insulin secretory capacities and early secretion response of insulin to glucose administration in diabetic patients with the tRNA^Leu(UUR) 3243 mutation. The patients with the mtDNA mutation have exhibited a lower frequency of obesity in the past and needed insulin more often than the diabetic patients without the mutation. Oka et al. (20) reported that the tRNA^Leu(UUR) 3243 mutation is missing in patients who were initially diagnosed with NIDDM and then progressed to insulin dependency within a few years. They speculated that the tRNA^Leu(UUR) 3243 mutation may cause gradual ß-cell destruction, but no direct correlation was found between the percentage of mutation in tRNA^Lys 8344 in peripheral leukocyte and ß-cell secretory function (16). Whereas Van den Ouweland et al. (21) reported that insulin secretion was apparently normal in affected patients, suggesting the pathogenic importance of peripheral insulin resistance, Alcolado et al. (22) and others (23, 24) have shown that in the diabetes with mtDNA mutations, the sensing pathway may be intact, and the prevailing hyperglycemia results in increased concentrations of insulin precursors typical of those seen in patients with NIDDM and impaired glucose tolerance. Metabolic dysfunction of the muscle may be involved in insulin resistance, because muscle tissue is an important target for insulin. Glucose intolerance in patients with the mtDNA mutation may be due not only to a deficiency in insulin secretion, but also to insulin resistance in peripheral tissues. Therefore, mitochondrial genes are plausible candidates as the cause of both IDDM and NIDDM.

In our study we detected only one patient with the tRNA^Leu(UUR) 3243 mutation and found no patient with the tRNA^Lys 8344 mutation in mtDNA in 503 diabetic patients. However, about 0.9% of diabetic patients have the tRNA^Leu(UUR) 3243 mutation, and a slightly lower percentage of diabetic patients have the tRNA^Lys8344 mutation in Japan (17). This percentage is slightly higher than that of mutations in the insulin gene and the insulin receptor gene. Due to the above and other facts, Kadowaki et al. (18) suggested that this mitochondrial mutation should be considered a cause of slowly progressive IDDM or insulin-deficient NIDDM.

Based on our patient’s clinical findings, his type of diabetes is an unusual form and can be classified as the so-called slowly progressive IDDM. However, we regard it as an insulin-requiring NIDDM diagnosed at young age. Our patient did not have the cardinal features of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode syndrome. The patient’s history suggests that his phenotype can be classified as diabetes mellitus associated with myoclonic epilepsy with ragged red fiber syndrome, which has previously (25) been demonstrated as a pathogenic mutation at the mtDNA 8344 position. However, the tRNA^Lys 8344 mutation was not found in his pedigree, although the patient with the tRNA^Leu(UUR) 3243 mutation has a variety of clinical phenotypes, as demonstrated. It is not clear that his mother and brother do not have diabetes mellitus or neuromuscular symptoms despite the presence of the mtDNA mutation. It is possible that even in the absence of obvious functional symptomatology, subclinical defects may be occasionally observed with detailed study. More importantly, it is possible that its phenotypic variability is due to differential expression of the mutant genotype at different times with different thresholds. Therefore, close observation might provide an opportunity to learn more about the onset and natural course of diabetes with the tRNA^Leu(UUR) 3243 mutation.

Otabe et al. (17) reported that 0.9% of diabetic patients have the 3243 bp mutation of mtDNA in Japan, whereas we found only 1 patient with that mtDNA mutation among 503 Koreans with diabetes mellitus. The reason for the different results may be in the selection of patients, the difference in races, the use of different methods, and the degree of heteroplasmy. The degree of heteroplasmy reported differs in various tissues and in various subjects (15, 16, 26). Larsson et al. (25) reported that >92% of mtDNA with the tRNA^Lys 8344 mutation in muscle is needed to cause respiratory chain dysfunction detected by biochemical methods, and the levels of mutated mtDNA in lymphocytes were 12.0 to 27.6% of those in muscle. Therefore, measurements in blood cells might not precisely reflect the situation in the affected tissue. The relative proportions of mutant and wild-type mtDNA can be determined by densitometric analysis or radiolabeled nucleotide and autoradiograms. We used densitometric analysis and found 32% of mtDNA to have the tRNA^Leu(UUR) 3243 mutation in the patient.

In this screening of 503 Korean diabetic patients, we identified only 1 subject and his family members with the mtDNA mutation at position 3243. At present, a precise statement cannot be made on the frequency of this mutation and its association with diabetes mellitus, but a lower frequency of this mutation in the Korean diabetic population was found than in that in other countries. Therefore, we conclude that the particular mutation of mtDNA may not be a common contributor to diabetes mellitus in Korea. The further study of mtDNA mutation with muscle tissue and/or pancreas tissue may give us more information about the pathogenesis of diabetes mellitus and the relation of diabetes mellitus to mitochondrial myopathy.

Received March 22, 1996.

Revised August 29, 1996.

Accepted October 11, 1996.

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