Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 47: 1641-1648, 2001;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohkubo, K.
Right arrow Articles by Ono, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohkubo, K.
Right arrow Articles by Ono, J.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Endocrinology and Metabolism
(Clinical Chemistry. 2001;47:1641-1648.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Mitochondrial Gene Mutations in the tRNALeu(UUR) Region and Diabetes: Prevalence and Clinical Phenotypes in Japan

Kumiko Ohkubo1a, Akemi Yamano2, Mariko Nagashima1, Yumiko Mori1, Keizo Anzai1, Yuko Akehi1, Riku Nomiyama2, Takashi Asano2, Akinori Urae3 and Junko Ono1

1 Department of Laboratory Medicine, Fukuoka University School of Medicine, 7-45-1, Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan

2 The First Department of Internal Medicine, Fukuoka University School of Medicine, 7-45-1, Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan.

3 Kyushu Clinical Pharmacology Research Clinic, 2-13-16, Jigyo, Chuo-ku, Fukuoka, 810-0065, Japan.

aAuthor for correspondence. Fax 81-92-873-1050; e-mail kokubo{at}cis.fukuoka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Mitochondrial gene mutations play a role in the development of diabetes mellitus. We have assessed the frequency of the A3243G and other mitochondrial mutations in Japan and in the relationship to clinical features of diabetes.

Methods: DNA was obtained from peripheral leukocytes of 240 patients with diabetes mellitus (39 with type 1; 188 with type 2; 13 with gestational diabetes) and 125 control subjects. We used PCR-restriction fragment length polymorphism analysis (ApaI) for A3243G and PCR-single-strand conformation polymorphism analysis to determine the mutations in the mitochondrial gene including nucleotide position 3243.

Results: The A3243G mutation was found in seven patients, and an inverse relationship was observed between the degree of heteroplasmy and the age at onset of diabetes. A3156G, G3357A, C3375A, and T3394C were detected in addition. Those who shared the same mutation showed similar clinical characteristics, thus representing a putative clinical subtype. The patients with A3156G had a sudden onset of hyperglycemia and showed a rapid progression to an insulin-dependent state with positive anti-glutamic acid decarboxylase antibody. Those with T3394C showed a mild defect in glucose-stimulated insulin secretion, and hyperglycemia appeared after adding such factors as aging or obesity.

Conclusions: The identification of mitochondrial gene mutations allows preclinical diagnosis of diabetes and prediction of the age at onset by evaluating the degree of heteroplasmy in cases with A3243G. Mutation detection may also be important for patient management and identification of affected family members.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Mitochondria are organelles that play an important role in the energy production of a cell, and they also have extranuclear genes, which are transmitted maternally. Especially in pancreatic ß cells, mitochondria are responsible for glucose-induced insulin secretion because the exocytosis of secretory granules is triggered by changes in the intracellular ATP and ADP concentrations and subsequent increase in the ATP/ADP ratio as a result of oxidative phosphorylation in the mitochondria caused by glucose metabolism (1). Mutations in mitochondrial genes may lead to disorders in ATP production, and thereby impair the glucose-induced insulin secretion.

The mitochondrial DNA is a ringed, double-stranded DNA, measuring 16 569 bp in overall length, and exists in the mitochondrial matrix. A cell contains 103–104 molecules of mitochondrial DNA. The mitochondrial DNA encodes 13 subunits of the mitochondrial respiratory chain, 22 tRNAs, and 2 rRNAs. The mutations of mitochondrial DNA constitute one of the main causes of the diseases that involve abnormalities in the brain and muscular tissues. An impaired glucose metabolism is also a symptom that is often seen in patients with mutations of mitochondrial DNA. Among these diseases, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)1 is known to be a syndrome that frequently accompanies diabetes mellitus. The screening of the A-to-G mutation at nucleotide position (np) 3243, found in 1992 in patients with MELAS (2), has been applied recently among diabetic patients. As a result, a maternally transmitted clinical subtype of diabetes mellitus, called diabetes-deafness syndrome (OMIM 520000), has been revealed. Different mutations based on the substitution of one base pair have been found in this region of mitochondrial DNA (3)(4)(5), thus showing it to be a hot spot area for mutations. Patients with the A3243G mutation have been reported to comprise 1–3% of all diabetic patients in Japan (6)(7)(8). The clinical characteristics of the patients with the A3243G mutation include impaired insulin secretion, sensorineural deafness, and maternal inheritance of the disease. Once impaired insulin secretion becomes overt, most cases progressively develop a disease state of insulin dependence. Therefore, the early or preclinical diagnosis of this disease by detection of such a mutation might help to prevent or delay the onset of diabetes, while also allowing for the selection of optimal treatment, thereby possibly avoiding diabetic complications altogether.

We developed a rapid diagnostic system using PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analysis for screening mutations in the tRNALeu(UUR) region of mitochondrial DNA. We determined the detection limit of our method, which was essential for applying this system as a screening test because mutant DNA and wild-type DNA could coexist in a single cell known as heteroplasmy. A total of 240 diabetic patients and 125 controls were examined by use of this screening method.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
patients and controls
Peripheral blood was obtained from 240 unrelated Japanese with diabetes mellitus (188 with type 2; 39 with type 1; 13 with gestational diabetes). The mean age was 52.8 ± 16.2 years. Individual patients were considered to be affected if they were being treated for type 2 diabetes or if the results of a standard 75-g oral glucose tolerance test indicated diabetes mellitus. Type 1 diabetes had been clinically diagnosed at the onset based on WHO criteria (9). The nondiabetic control group consisted of 125 unrelated Japanese (mean age, 22.9 ± 2.7 years) who showed fasting blood glucose of <=7 mmol/L and body mass index (BMI) of <=24 kg/m2. Informed consent, according to the medical ethics criteria of Fukuoka University, was obtained from all participants.

materials
A restriction enzyme, ApaI, was purchased from Takara. AmpliTaq DNA polymerase was from Roche Molecular Systems. SYBR Green I was obtained from FMC Bioproducts, and PhastGels, PhastGel Buffer Strips Native, and PhastGel DNA silver staining reagent sets were from Amersham Pharmacia Biotech. Plasmid vector pT7Blue T was from Novagen. SepaGeneTM was from Sanko Junyaku. Other reagents were of analytical grade.

preparation of dna and pcr amplification
Total DNA was extracted from peripheral leukocytes with SepaGene according to the manufacturer’s instructions. The fragments of mitochondrial DNA encompassing np 3243 were amplified by PCR with AmpliTaq DNA polymerase. PCR was carried out in a total volume of 100 µL containing 100 ng of extracted DNA, 200 µM each dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 1.0 U of Taq polymerase. The forward primers were 5'-CGTTTGTTCAACGATTAAAG-3' (MT1) and 5'-AGGACAAGAGAAATAAGGCC-3' (MT3), and the reverse primers were 5'-AGCGAAGGGTTGTAGTAGCC-3' (MT2) and 5'-CACGTTGGGGCCTTTGCGTA-3' (MT4). The DNA was initially denatured at 94 °C for 5 min and subjected to 30 PCR cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The PCR products were electrophoresed on 4% agarose gel and stained with ethidium bromide.

rflp analysis
The 422-bp fragment amplified by the primers MT1 and MT2 was digested by ApaI to identify any A-to-G mutation at np 3243 and electrophoresed on 4% agarose gels. After electrophoresis, the gels were stained with ethidium bromide or SYBR Green I. Fluoroscopic scans and analyses were performed with ChemiImager (Alpha Innotech).

sscp analysis
After PCR with the primers MT3 and MT4, the mixture containing the 294-bp amplified fragment was diluted twofold with a solution containing 950 mL/L formamide, 20 mmol/L EDTA, 0.5 g/L xylene cyanol, and 0.5 mL/L bromphenol blue. The sample was heated to 95 °C for 5 min, and after cooling on ice, 1 µL was separated on 20% polyacrylamide gels with PhastGel native buffer strips (0.25 mol/L Tris, 0.88 mol/L L-alanine, pH 8.8) on the PhastSystemTM (Amersham Pharmacia Biotech) (10). After electrophoresis, the gel was fixed and stained with a PhastGel DNA silver staining reagent set.

dna sequencing
Mutations detected by PCR-RFLP (ApaI) and/or PCR-SSCP were confirmed by direct DNA sequencing of PCR products or DNA sequencing after cloning into plasmid vectors. The sequencing reactions were carried out by use of the BigDye terminator cycle sequencing method (Perkin-Elmer), and reaction products were analyzed on an ABI-Prism 377 automated sequencer (Perkin-Elmer).

preparation for calibrators of the pcr products encompassing the mutation of a3243g
To determine the detection limit of the A3243G mutation, we constructed the mixture of PCR products with known proportions of amplified DNA that contained both the A3243G mutation and no mutation. The PCR products amplified with DNA from a patient with the heteroplasmic A3243G mutation, using primers MT1 and MT2, were subcloned into a plasmid vector, pT7Blue T. After cloning of the plasmid vector inserted with the mitochondrial DNA containing A3243G or no mutation, the inserted DNA was amplified with the primers MT1 and MT2 by PCR. The products containing only the A3243G mutation or no mutation were extracted with chloroform, precipitated with ethanol, resolubilized in distilled water, and quantified by measurement of the absorbance at 260 nm. The products from DNA containing the A3243G mutation or no mutation were diluted to a concentration of 150 ng/µL with distilled water and mixed at ratios of 0:100, 1:99, 2:98, 5:95, 10:90, 20:80, 40:60, 50:50, and 100:0 by volume. Each 150 ng of the mixed DNA was digested with 10 U of ApaI in 10 µL of the reaction mixture for 1 h at 37 °C. The digested DNA was electrophoresed on a 4% agarose gel and stained with ethidium bromide or SYBR Green I. Fluoroscopic scans and analyses were performed with ChemiImager. For PCR-SSCP, PCR products with or without mutation, amplified with the primers MT3 and MT4, were mixed as above and electrophoresed by use of the PhastSystem as described above.

statistical analysis
The statistical analysis was performed with the Stat View-J 4.02 program (Abacus Concepts). Fisher exact test or the {chi}2 test was used for statistical analyses to compare the frequencies of mutations between the diabetics and controls. A regression analysis was performed to determine the correlation between the percentage of heteroplasmy and the age at onset of diabetes in the patients with A3243G.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
detection limit for a3243g mutation
The PCR product amplified by the primers MT1 and MT2 was 422 bp in length. After digestion with ApaI, it was separated into 210 bp or 212 bp if it had a substitution of A to G at np 3243 to constitute the recognition site for ApaI. When electrophoresed on a 4% agarose gel, both the 210 bp and 212 bp overlapped and appeared as a single band, thus leading to an improvement in the sensitivity of the PCR-RFLP analysis.

After analysis of the calibrators, which consisted of DNA fragments containing A3243G and no mutation in the various known ratios, it turned out that PCR-RFLP could detect the A3243G fragment at a percentage as low as 1.0% when stained with ethidium bromide and 0.2% with SYBR Green I (Fig. 1 ). PCR-SSCP could detect 5.0% fragments of A3243G (Fig. 2 ). The detection limit of RFLP was lower than that of SSCP; however, the detection rates of the A3243G mutation among patients were the same between these two methods because all of the A3243G mutations showed heteroplasmy of >5%.



View larger version (62K):
[in this window]
[in a new window]
  		Figure 1. Detection limit of the A3243G mutation by PCR-RFLP (ApaI).

PCR products containing various percentages of A3243G mutation indicated above each lane were digested with ApaI for 1 h at 37 °C. After electrophoresis on a 4% agarose gel, they were stained with ethidium bromide (lanes 1–9) or SYBR Green I (lanes 10–14). The arrow indicates the two fragments overlapping after digestion with ApaI. MW, molecular weight.



View larger version (65K):
[in this window]
[in a new window]
  		Figure 2. Detection limit for the A3243G mutation by PCR-SSCP.

PCR products containing various percentages of A3243G mutation indicated above each lane were electrophoresed on a 20% polyacrylamide gel and stained with silver. The arrow accompanying an asterisk shows the additional band resulting from the PCR products having A3243G.

screening of the mutations
Of 240 diabetic patients examined, five kinds of one-point mutations including A3243G were detected by PCR-SSCP (Figs. 3 and 4 ). They consisted of A3156G, A3243G, G3357A, C3375A, and T3394C mutations. Only A3243G is heteroplasmic, whereas the others are homoplasmic mutations.



View larger version (52K):
[in this window]
[in a new window]
  		Figure 3. PCR-RFLP and PCR-SSCP of the A3243G mutation.

PCR products were analyzed by PCR-RFLP (ApaI) and PCR-SSCP. Controls are represented in lanes 1 (PCR-RFLP), 5, and 6 (PCR-SSCP). The A3243G mutation is indicated in lanes 2 and 7 (case 1 in Table 2Up ), lanes 3 and 8 (case 2), and lanes 4 and 9 (case 3).



View larger version (86K):
[in this window]
[in a new window]
  		Figure 4. PCR-SSCP of mitochondrial gene mutations other than A3243G.

PCR products were electrophoresed as described in Subjects and Methods. Lanes 1 and 6 are controls. Four kinds of mutations are shown: A3156G in lane 2, G3357A in lane 3, C3375A in lane 4, and T3394C in lane 5.

A3243G, A3156G, and G3357A were found only in diabetic patients, whereas C3375A and T3394C were also detected in the controls. No statistically significant differences in the frequencies of mutations were observed between the diabetic patients and controls (Table 1 ), although the frequencies of the A3243G mutation tended to be higher in the diabetic patients than in the controls (P = 0.057).


View this table:
[in this window]
[in a new window]
  		Table 1. Prevalence of mitochondrial gene mutations in diabetics and controls.

The A3243G mutation was found in seven patients (one male and six females). The ages of the patients diagnosed to have A3243G mutations were 29–62 years. All patients showed hearing loss and a low height. The mean height of the six female patients was 146.3 ± 2.9 cm, and one male patient was 155 cm in height, all belonging to the lower 2.5 percentile of Japanese. Their clinical profiles are summarized in Table 2 . The percentage of A3243G mutations were widely distributed, from 7.4% to 38.9%. The degree of heteroplasmy for A3243G was significantly correlated with the age at onset of hyperglycemia (P <=0.05; Fig. 5 ).


View this table:
[in this window]
[in a new window]
  		Table 2. Clinical characterization of the diabetic patients having the A3243G mutation.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. Relationship between the degree of heteroplasmy and the age at onset of diabetes in the patients with A3243G.

The degree of heteroplasmy (percentage) was significantly associated with the age at onset of diabetes (y = 35.4 - 0.5x; r = -0.63; P <=0.05).

An A-to-G mutation at np 3156 is located in the 3' portion of 16S ribosomal RNA. The patient carrying this mutation (case 1 in Table 3 ) showed the clinical features of type 1 diabetes. The same mutation was shared with her mother and younger sister, and her mother abruptly developed hyperglycemia with a high titer of anti-glutamic acid decarboxylase (GAD) antibody, fell into an insulin-dependent state, and was diagnosed as having type 1 diabetes mellitus 4 years after the detection of the A3156G mutation. The patient’s younger sister, who is 43 years of age, currently shows an impaired glucose tolerance.


View this table:
[in this window]
[in a new window]
  		Table 3. Clinical characterization of the diabetic patients with mitochondrial gene mutations other than A3243G.

G3357A, C3375A, and T3394C mutations are located in the coding region of ND1, the gene that encodes NADH dehydrogenase subunit 1. G3357A and T3394C are missense mutations (Met17Ileu and Tyr30His, respectively), and C3375A is a silent mutation. The patient with G3357A (case 2) had type 1 diabetes and was also positive for the anti-GAD antibody. The C3375A mutation was detected in two patients with type 2 diabetes. The T3394C mutation was found in six patients, all of whom had type 2 diabetes. Four patients (cases 5, 6, 7, and 8) were diagnosed with hyperglycemia between the ages of 51 and 63 years for the first time. They have all maintained relatively good insulin secretion and thus have only undergone diet therapy or drug therapy with hypoglycemic agents. The remaining two patients (cases 9 and 10) became diabetic in their youth and have been treated with insulin. These two patients also showed a relatively high BMI, and their clinical features are summarized in Table 3Up .


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Mitochondrial DNA is predominantly inherited maternally. Less than 0.1% of the mitochondrial DNA is contributed by a sperm. Each cell contains hundreds of mitochondria and thousands of copies of the mitochondrial DNA genome. Therefore, cells can harbor a mixture of mutant and wild-type mitochondrial DNAs, a phenomenon called heteroplasmy. Over multiple cell divisions, the proportion drifts toward either predominantly mutant or wild-type mitochondrial DNAs, leading to homoplasmy. The percentage of mitochondrial DNA with mutations varies from tissue to tissue, and is observed to be the highest in the affected tissues (7)(11)(12). In the case of diabetic patients with the A3243G mutation, which was responsible for a deterioration in insulin secretion, pancreatic ß cells showed the highest percentage of heteroplasmy (13), and therefore ß cells were considered the most suitable tissue for the examination. However, obtaining ß cells by a biopsy of the pancreas is a hazardous and difficult procedure. The peripheral blood leukocytes are relatively easy to obtain and, therefore, are used most frequently to screen for gene mutations, although the observed percentages of heteroplasmy might be lower than those in the affected tissue (14)(15). In our diagnostic system, DNA is extracted from peripheral blood leukocytes, and then PCR is performed, followed by SSCP or RFLP. It takes 4.5–5.0 h to complete all processes, which makes it possible to diagnose mitochondrial DNA mutations the same day the sample is taken. The lower limit of detection for A3243G heteroplasmy by PCR-RFLP is 1.0% with ethidium bromide staining and 0.2% with SYBR Green I staining, whereas in PCR-SSCP, the lower limit of detection is 5.0% in our system. By the use of ligation-mediated PCR method, which can detect 0.01% heteroplasmy, only diabetic patients, and not controls, have been reported to show >0.01% heteroplasmy (16). As a result, the possibility of overlooking mutated mitochondrial DNAs if their number is <=0.2% cannot be ruled out. However, ligation-mediated PCR takes a much longer time than PCR-RFLP or PCR-SSCP, and thus is not suitable for routine screening methods.

Among the 240 patients investigated, we identified 7 patients who had the A3243G mutation. The prevalence of this mutation was 2.9%, and this finding correlates with reports that the prevalence was within a range of 1–3% in Japanese diabetic populations (6)(7)(8)(17). In patients with the A3243G mutation, impaired insulin secretion tends to become overt between 20 and 30 years of age. Once a patient becomes diabetic, the amount of secretable insulin progressively declines. Thus, all cases in whom we detected the A3243G mutation had to receive insulin therapy. Among the six female patients carrying the mutation, four had experienced an abnormal pregnancy, leading to either still birth or premature birth. In these cases, it appears that hyperglycemia might have occurred during pregnancy for the first time. Among the population of Japanese women attending a diabetic pregnancy clinic, it was reported (18) that the prevalence of the A3243G mutation was higher than that observed in randomly selected Japanese diabetic patients. Moreover, the prevalence of spontaneous abortions in patients with A3243G was also higher than in those without this mutation. Therefore, the diagnosis of the A3243G mutation before a hyperglycemic state develops seems to be very important in female patients not only to prevent hyperglycemia, but also to prevent abnormal pregnancy. The percentages of heteroplasmy for the A3243G mutation that were detected with amplified DNA from peripheral leukocytes were significantly associated with age at the onset of hyperglycemia (P <=0.05; Fig. 5Up ). Although various percentages of heteroplasmy were observed in the tissues and organs, heteroplasmy in leukocytes was thought to be associated with heteroplasmy in the pancreatic ß cells. These findings may make it possible to predict the onset of hyperglycemia for the siblings and descendants of the patients who were unaffected at the time of diagnosis, while also allowing for a better control of blood glucose and the prevention of diabetic complications.

Regarding the etiology of impaired glucose tolerance, the A3243G mitochondrial gene mutation is thought to be a cause of impaired, glucose-induced insulin secretion. The clonal cybrid cells between the cells lacking mitochondrial DNA and fibroblasts from a patient with the A3243G mutation showed poor respiration, an increased ratio of lactate to pyruvate, and marked defects in the mitochondrial morphology and respiratory chain complex I and IV activities (19). Janssen et al. (20) indicated that cells harboring patient-derived mitochondria with the A3242G mutation displayed a severe loss of respiration, whereas the rate of mitochondrial translation was not seriously affected despite the low degree of leucylation of tRNALeu (UUR). Chomyn et al. (21) observed that cell lines that were nearly homoplasmic for this mutation exhibited a strong (70–75%) reduction in the degree of aminoacylated tRNALeu(UUR), as well as a decrease in mitochondrial protein synthesis. They also revealed a reduced association of mRNA with ribosomes, possibly attributable to the presence of a tRNALeu(UUR) aminoacylation defect. The cultured ß-cell line MIN6 with knocked-out mtDNA ({rho}0 MIN6) showed a loss of mitochondrial transcription, translation, and respiration activity and a glucose-stimulated increase in insulin secretion (22). Similar results were also obtained for the mitochondrial DNA-deficient {rho}0 INS-1 cells established by Kennedy et al. (23).

In addition to the A3243G mutation, we detected four types of one-point mutations in this region of the mitochondrial gene. These mutations were A3156G, G3357A, C3375A, and T3394C. A3156G, G3357A, and C3375A were novel mutations. The np 3156 in the mitochondrial gene is located in the region close to the 3' end of 16S rRNA, whereas 3357, 3375, and 3394 are located in the coding region of NADH dehydrogenase subunit 1 (ND1). The two mutations, G3357A and T3394C, induce an exchange of amino acids. In the case of G3357A, methionine is substituted for isoleucine and, in T3394C, tyrosine for histidine. It has not yet been determined whether these mutations cause mitochondrial dysfunctions, which reduce insulin secretion in the affected patients. However, some groups of the patients with the same mutation tended to show very similar clinical manifestations, including a sudden onset of hyperglycemia and a rapid progression to an insulin-dependent state with positive anti-GAD antibody in the case of the A3156G mutation, and a mild defect in glucose-stimulated insulin secretion and the appearance of hyperglycemia with aging or obesity in the cases of the T3394C mutation. These diabetic states are suggested to result from the same etiologies related to the same mutations in the mitochondrial gene.

The A3156G mutation was detected in three subjects in one family. The proband (case 1 in Table 3Up ) and her mother became abruptly hyperglycemic and fell into an insulin-dependent state at 42 and 70 years of age, respectively, thus showing the typical clinical features of type 1 diabetes mellitus. The mother had anti-GAD antibody with a high titer at onset, whereas the proband was negative at the time of the investigation, 3 years after her own onset of the disease. Her younger sister had the same mutation and showed impaired glucose tolerance at the time of the investigation when the sister was 42 years of age. Although we could not complete the pedigree of the A3156G mutation because the extended family declined to participate in further studies, the examined subjects who demonstrated the A3156G mutation might represent a group of phenotypes with slowly progressive insulin-dependent diabetes mellitus (24)(25). Recent studies have shown that mitochondria are frequently involved in a variety of key events of apoptosis, including the release of such caspase activators as cytochrome c, changes in electron transport, a loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins (26)(27). The mitochondrial dysfunction, possibly resulting from the translational disorders produced by the mutated 16S rRNA with A3156G, could be associated with the apoptosis of ß cells in islets, which is considered one of the causes of type 1 diabetes mellitus (28)(29). Apoptotic features were observed in the muscle fibers of patients carrying a high percentage of single mtDNA deletions and tRNA point mutations including A3243G (30), but not in the islet ß cells of patients with A3243G (13)(31).

A case of type 1 diabetes with the G3357A mutation (case 2) showed a high titer of anti-GAD antibody and was complicated with Down syndrome. Her mother showed impaired glucose tolerance by a 75-g oral glucose tolerance test.

The C3375A mutation was detected in two patients with type 2 diabetes. They showed a slight increase of blood glucose, and diet therapy has been introduced, thus leading to good control of the glucose concentration. The proband, an 8-year-old girl with the C3375A mutation (case 4) had an early onset of type 2 diabetes, whereas her mother showed normal glucose tolerance with a low insulin response after the 75-g oral glucose tolerance test. Her elder brother and sister also showed impaired glucose tolerance in their early teens.

The T3394C mutation has previously been reported to be implicated in Leber hereditary optic neuropathy (32)(33) and also in patients with type 2 (non-insulin-dependent) diabetes mellitus in the United Kingdom (34) and in Japan (35). All patients with the T3394C mutation who were detected by our screening process also showed the clinical features of type 2 diabetes. Among the four cases whose onset of disease started at more than 50 years of age, three showed a normal BMI and a mild disturbance in glucose tolerance and were treated with either dietary treatment or with acarbose or glibenclamide (cases 5, 7, and 8). On the other hand, patients who demonstrated diabetes before 40 years of age (cases 9 and 10) and had BMIs of 27.2 and 28.0, respectively, were placed on insulin therapy. In the case of this T3394C mutation, the disorder of insulin secretion might become overt when combined with such factors as aging or obesity. The prevalence of this mutation was 2.5% among diabetic patients and 3.2% among the controls according to our screening, whereas the prevalence was 4.9% among diabetic patients and 1.3% among the controls in another study on Japanese patients with non-insulin-dependent diabetes mellitus (35). Because the controls in our study were younger than the patients and were not age-matched to the patient group, there is a possibility that the control group might have contained some individuals who might become diabetic in middle age by aging or obesity. In addition, it is also possible that this T3394C mutation is a modifier locus for one or many other genes.

As a result of this screening method, we found both novel and known mutations of the mitochondrial gene and determined the potential clinical subtypes based on the site of the mitochondrial gene mutations. Our findings regarding mitochondrial gene mutations should provide valuable information related to the preclinical diagnosis of diabetes mellitus, the treatment and follow-up of such diabetic patients, and the identification of their relatives with maternally transmitted mitochondrial gene mutations.


   Acknowledgments
 
This work was supported in part by funds (No. 986006) from the Central Research Institute of Fukuoka University.


   Footnotes
 
1 Nonstandard abbreviations: MELAS, mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; np, nucleotide position; PCR-RFLP, PCR-restriction fragment length polymorphism; PCR-SSCP, PCR-single-strand conformation polymorphism; BMI, body mass index; and GAD, glutamic acid decarboxylase.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Gerbitz K-D, Gempel K, Brdiczka D. Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit. Diabetes 1996;45:113-126.[Abstract]
  2. van den Ouweland JM, Lemkes HHJ, Ruitenbeek W, Snadkuijl LA, de Vijlder MF, Struyvenberg PA, et al. Mutation in mitochondrial tRNALeu(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nat Genet 1992;1:368-371.[ISI][Medline] [Order article via Infotrieve]
  3. Suzuki Y, Hosokawa K, Suzuki S, Shimada A, Hinokio Y, Asahina T, et al. Diabetes associated with a novel 3264 mitochondrial tRNALeu(UUR) mutation. Diabetes Care 1997;20:1138-1140.[Abstract]
  4. Tsukuda K, Suzuki Y, Kameoka K, Osawa N, Goto Y, Katagiri H, et al. Screening of patients with maternally transmitted diabetes for mitochondrial gene mutations in the tRNALeu(UUR) region. Diabet Med 1997;14:1032-1037.[ISI][Medline] [Order article via Infotrieve]
  5. Nakano S, Fukuda M, Hotta F, Ito T, Ishii T, Kitazawa M, et al. Mitochondrial DNA point mutation at nucleotide pair 3316 in a Japanese family with heterogeneous phenotypes of diabetes. Endocr J 1998;45:625-630.[ISI][Medline] [Order article via Infotrieve]
  6. Katagiri H, Asano T, Ishihara H, Inukai K, Anai M, Yamanouchi T, et al. Mitochondrial diabetes mellitus: prevalence and clinical characterization of diabetes due to mitochondrial tRNALeu(UUR) gene mutation in Japanese patients. Diabetologia 1994;37:504-510.[ISI][Medline] [Order article via Infotrieve]
  7. Tokunaga M, Mita S, Sakuta R, Nonaka I, Araki S. Increased mitochondrial DNA in blood vessels and ragged-red fibers in mitochondrial myopathy, enchephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Ann Neurol 1993;33:275-280.[ISI][Medline] [Order article via Infotrieve]
  8. Kadowaki T, Kadowaki H, Mori Y, Tobe K, Sakuta R, Suzuki Y, et al. A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. N Engl J Med 1994;330:962-968.[Abstract/Free Full Text]
  9. Alberti KGMM, Zimmet PZ, . for the WHO Consultation. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med 1998;15:539-553.[ISI][Medline] [Order article via Infotrieve]
  10. Dockhorn-Dworniczak B, Dworniczak B, Brommelkamp L, Bulles J, Horst J, Bocker WW. Non-isotopic detection of single strand conformation polymorphism (PCR-SSCP): a rapid and sensitive technique in diagnosis of phenylketonuria. Nucleic Acids Res 1991;19:2500.[Free Full Text]
  11. Moraes CT, Ciacci F, Bonilla E, Jansen C, Hirano M, Rao N, et al. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. J Clin Invest 1993;92:2906-2915.
  12. Smith ML, Hua XY, Marsden DL, Liu D, Kennaway NG, Ngo KY, et al. Diabetes and mitochondrial encephalomyophathy with lactic acidosis and stroke-like episodes (MELAS): radiolabeled polymerase chain reaction is necessary for accurate detection of low percentages of mutation. J Clin Endocrinol Metab 1997;82:2826-2831.[Abstract/Free Full Text]
  13. Kobayashi T, Nakanishi K, Nakase H, Kajio H, Okubo M, Murase T, et al. In situ characterization of islets in diabetes with a mitochondrial DNA mutation at nucleotide position 3243. Diabetes 1997;46:1567-1571.[Abstract]
  14. Chinnery PF, Zwijnenburg PJ, Walker M, Howell N, Taylaor RW, Lightowlers RN, et al. Nonrandom tissue distribution of mutant mtDNA. Am J Med Genet 1999;85:498-501.[ISI][Medline] [Order article via Infotrieve]
  15. Suzuki Y, Goto Y, Taniyama M, Nonaka I, Murakami N, Hosokawa K, et al. Muscle histopathology in diabetes mellitus associated with mitochondrial tRNALeu(UUR) mutation at position 3243. J Neurol Sci 1997;145:49-53.[ISI][Medline] [Order article via Infotrieve]
  16. Urata M, Wakiyama M, Iwase M, Yoneda M, Kinosita S, Hamasaki N, et al. New sensitive method for the detection of the A3243G mutation of human mitochondrial deoxyribonucleic acid in diabetes mellitus patients by ligation-mediated polymerase chain reaction. Clin Chem 1998;44:2088-2093.[Abstract/Free Full Text]
  17. Fukui M, Nakano K, Obayashi H, Kitagawa Y, Nakamura N, Mori H, et al. High prevalence of mitochondrial diabetes mellitus in Japanese patients with major risk factors. Metabolism 1997;46:793-795.[ISI][Medline] [Order article via Infotrieve]
  18. Yanagisawa K, Uchigata Y, Sanaka M, Sakura H, Minei S, Shimizu M, et al. Mutation in the mitochondrial tRNALeu at position 3243 and spontaneous abortions in Japanese women attending a clinic for diabetic pregnancies. Diabetologia 1995;38:809-815.[ISI][Medline] [Order article via Infotrieve]
  19. van den Ouweland JM, Maechler P, Wollheim CB, Attardi G, Maassen JA. Functional and morphological abnormalities of mitochondria harbouring the tRNALeu(UUR) mutation in mitochondria derived from patients with maternally inherited diabetes and deafness (MIDD) and progressive kidney disease. Diabetologia 1999;42:485-492.[ISI][Medline] [Order article via Infotrieve]
  20. Janssen GMC, Maassen JA, van den Ouweland JM. The diabetes-associated 3243 mutation in the mitochondrial tRNALeu(UUR) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate. J Biol Chem 1999;274:29744-29748.[Abstract/Free Full Text]
  21. Chomyn A, Enriquez JA, Micol V, Fernandez-Silva P, Attardi G. The MELAS syndrome-associated human mitochondrial tRNALeu(UUR) mutation causes aminoacylation deficiency and concomitant reduced association of mRNA with ribosomes. J Biol Chem 2000;275:19198-19209.[Abstract/Free Full Text]
  22. Soejima A, Inoue K, Takai D, Kaneko M, Ishihara H, Oka Y, et al. Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic cell line, MIN6. J Biol Chem 1996;271:26194-26199.[Abstract/Free Full Text]
  23. Kennedy ED, Maechler P, Wollheim CB. Effects of depletion of mitochondrial DNA on metabolism-secretion coupling in INS-1 cells. Diabetes 1998;47:374-380.[Abstract]
  24. Kobayashi T, Itoh T, Kosaka K, Tsuji K. Time course of islet cell antibodies and ß-cell function in non-insulin-dependent stage of type I diabetes. Diabetes 1987;36:510-517.[Abstract]
  25. Kobayashi T.. Subtype of insulin-dependent diabetes mellitus (IDDM) in Japan: slowly progressive IDDM–the clinical characteristics and pathogenesis of the syndrome. Diabetes Res Clin Pract 1994;24((Suppl)):S95-S99.
  26. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309-1312.[Abstract/Free Full Text]
  27. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999;397:441-446.[Medline] [Order article via Infotrieve]
  28. Loweth AC, Williams GT, James RF, Scarpello JH, Morgan NG. Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1ß and Fas ligation. Diabetes 1998;47:727-732.[Abstract]
  29. Mauricio D, Mandrup-Poulsen T. Apoptosis and the pathogenesis of IDDM. Diabetes 1998;47:1537-1543.[Abstract]
  30. Mirabella M, Giovanni SD, Silvestri G, Tonali P, Servidei S. Apoptosis in mitochondrial encephalomyopathies with mitochondrial DNA mutations: a potential pathogenic mechanism. Brain 2000;123:93-104.[Abstract/Free Full Text]
  31. Otabe S, Yasuda K, Mori Y, Shimokawa K, Kadowaki H, Jimi A, et al. Molecular and histological evaluation of pancreata from patients with mitochondrial gene mutation associated with impaired insulin secretion. Biochem Biophys Res Commun 1999;259:149-156.[ISI][Medline] [Order article via Infotrieve]
  32. Johns DR, Neufeld MJ, Park RD. An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. Biochem Biophys Res Commun 1992;187:1551-1557.[ISI][Medline] [Order article via Infotrieve]
  33. Brown MD, Voljavec AS, Lott MT, MacDonald I, Wallace DC. Leber’s hereditary optic neuropathy: a model for mitochondrial neurogenerative disease. FASEB J 1992;6:2791-2799.[Abstract]
  34. Thomas AW, Edwards A, Sherratt EJ, Majid A, Gagg J, Alcolado JC. Molecular scanning of candidate mitochondrial tRNA genes in type2 (non-insulin dependent) diabetes mellitus. J Med Genet 1996;33:253-255.[Abstract]
  35. Hirai M, Suzuki S, Onoda M, Hinokio Y, Ai L, Hirai A, et al. Mitochondrial DNA 3394 mutation in the NADH dehydrogenase subunit 1 associated with non-insulin-dependent diabetes mellitus. Biochem Biophys Res Commun 1996;219:951-955.[ISI][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 J. Clin. Endocrinol. Metab.Home page
R. P. Sahu, A. Aggarwal, G. Zaidi, A. Shah, K. Modi, S. Kongara, S. Aggarwal, S. Talwar, S. Chu, V. Bhatia, et al.
Etiology of Early-Onset Type 2 Diabetes in Indians: Islet Autoimmunity and Mutations in Hepatocyte Nuclear Factor 1{alpha} and Mitochondrial Gene
J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2462 - 2467.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
Q.-P. Kong, H.-J. Bandelt, C. Sun, Y.-G. Yao, A. Salas, A. Achilli, C.-Y. Wang, L. Zhong, C.-L. Zhu, S.-F. Wu, et al.
Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations
Hum. Mol. Genet., July 1, 2006; 15(13): 2076 - 2086.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
R.-K. Bai and L.-J. C. Wong
Simultaneous Detection and Quantification of Mitochondrial DNA Deletion(s), Depletion, and Over-Replication in Patients with Mitochondrial Disease
J. Mol. Diagn., November 1, 2005; 7(5): 613 - 622.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
R.-K. Bai and L.-J. C. Wong
Detection and Quantification of Heteroplasmic Mutant Mitochondrial DNA by Real-Time Amplification Refractory Mutation System Quantitative PCR Analysis: A Single-Step Approach
Clin. Chem., June 1, 2004; 50(6): 996 - 1001.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
J. A. Maassen, Leen. M. 't Hart, E. van Essen, R. J. Heine, G. Nijpels, R. S. Jahangir Tafrechi, A. K. Raap, G. M.C. Janssen, and H. H.P.J. Lemkes
Mitochondrial Diabetes: Molecular Mechanisms and Clinical Presentation
Diabetes, February 1, 2004; 53(90001): S103 - 109.
[Abstract] [Full Text]

Home page
 J. Immunol.Home page
Y. Gu, C. Wang, C. M. Roifman, and A. Cohen
Role of MHC Class I in Immune Surveillance of Mitochondrial DNA Integrity
J. Immunol., April 1, 2003; 170(7): 3603 - 3607.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohkubo, K.
Right arrow Articles by Ono, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohkubo, K.
Right arrow Articles by Ono, J.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Endocrinology and Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS