The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 3 992-994
Copyright © 1998 by The Endocrine Society
Mitochondrial Deoxyribonucleic Acid 3256C-T Mutation in a Japanese Family with Noninsulin-Dependent Diabetes Mellitus1
Masashi Hirai,
Susumu Suzuki,
Masatoshi Onoda,
Yoshinori Hinokio,
Aki Hirai,
Masataka Ohtomo,
Masaki Chiba,
Shigeru Kasuga,
Satoshi Hirai,
Yoshinori Satoh,
Hiroaki Akai,
Shigeaki Miyabayashi and
Takayoshi Toyota
Third Department of Internal Medicine (M.H., S.S., M.O., Y.H.,
A.H., M.O., M.C., S.K., S.H., Y.S., H.A., T.T.) and Department of
Pediatrics (S.M.), Tohoku University School of Medicine, Sendai,
Japan
Address all correspondence and requests for reprints to: Susumu Suzuki, M.D., Third Department of Internal Medicine, Tohoku University School of Medicine, 1–1 Seiryo-machi, Aoba-ku, Sendai 980, Japan.
 |
Abstract
|
|---|
Accumulating reports indicate a relationship between mitochondrial DNA
mutation and impaired glucose-induced insulin secretion leading to a
subtype of noninsulin-dependent diabetes mellitus. DNA from a 45-yr-old
Japanese woman with noninsulin-dependent diabetes mellitus and muscle
atrophy was isolated and studied for mitochondrial DNA mutations. We
identified a mitochondrial DNA C-T heteroplasmic mutation at nucleotide
position 3256. The mutation was located in the transfer ribonucleic
acidLeu in a region conserved in evolution. Eight other
members of her family were examined for the mutation. Six of them had
the same mutation together with noninsulin-dependent diabetes mellitus,
and one teenage boy had the mutation and impaired glucose tolerance.
The other family member who did not have this mutation had normal
glucose tolerance. The enzyme activity of the mitochondrial oxidative
phosphorylation pathway in the muscle of the proband was measured. The
enzyme activity was decreased in the proband, especially in complex I.
This mutation might be responsible for the abnormal glucose metabolism.
|
Introduction
|
|---|
NONINSULIN-DEPENDENT diabetes
mellitus (NIDDM) is characterized by disturbances in insulin action and
insulin secretion (1), and heredity plays a significant role in the
development of the disease (2). Mitochondrial oxidative phosphorylation
plays an important role in glucose-stimulated insulin secretion in
pancreatic ß-cells. Thus, mitochondrial DNA (mtDNA) is a candidate
gene for NIDDM. Recently, there have been many reports suggesting that
several mtDNA mutations cause a subtype of diabetes mellitus (3, 4, 5, 6, 7, 8). In
this paper, we report a mtDNA 3256 C-T mutation in a Japanese family
with NIDDM. This mutation might be responsible for their abnormal
glucose metabolism.
|
Subjects and Methods
|
|---|
Patients
The proband’s pedigree is shown in Fig. 1
. The proband was a 54-yr-old Japanese
woman who was 147 cm tall and weighed 41 kg. Her birth and early life
had been unremarkable until age 44 yr, when she was diagnosed with
diabetes. She controlled it with diet and oral hyperglycemic agents
until the age of 46 yr, when she required insulin injection. Her
insulin secretory capacity was considered low based on urinary C
peptide immunoreactivity response excretion (10.4 µg/day) and a low
plasma C peptide immunoreactivity response 6 min after iv
administration of 1 mg glucagon (0.8 ng/mL). Her hemoglobin
A1c was 8.8%, and she occasionally had hypoglycemia
attacks. Glutamic acid decarboxylase antibody was negative. She began
to have hearing disturbances at the age of 45 yr. She was diagnosed as
having proliferative retinopathy and received photocoagulation therapy
at the age of 46 yr. She had had progressive muscle weakness and
atrophy, mainly in the proximal muscles of the lower extremities since
54 yr of age.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Pedigree of the family with diabetes
mellitus and mtDNA 3256 C-T mutation. 3256 C-T mutation and wild type
are represented by MT and WT, respectively; untested individuals by
question marks; and the proband by an arrow. The age of
the patient at the time of study is shown in
parentheses.
|
|
DNA sequencing
DNA was prepared from the proband’s muscle. The 193-bp fragment
including the transfer ribonucleic acidLeu
(tRNALeu) region was amplified with PCR (forward primer,
5'-AGGACAAGAGAAATAAGGCC-3'; reverse primer,
5'-CACGTTGGGGCCTTTGCGTA-3'), subcloned into a TA cloning vector
(Invitrogen, San Diego, CA), and sequenced by an ALF DNA sequencer
(Pharmacia, Uppsala, Sweden).
Determination of mtDNA 3256 C-T mutation
DNA was prepared from muscle, hair roots, or buccal cells. The
C-T transition at nucleotide 3256 was detected by restriction
site-generating PCR restriction fragment length polymorphism. A
modified reverse primer for restriction site-generating PCR in which
the nucleotide at position 3258 was changed from an A to a G
(5'-CTGACTGTAAAGTTTTAAGTTTTGT-3', corresponding to position
3257–3281) was synthesized and used together with a forward primer
(5'-AAAGGACAAGAGAAATAAGGCC-3', corresponding to position 3128–3149) to
create a new AfaI restriction site when the mutated sequence
was amplified. The PCR conditions were as follows: 20 cycles of 1-min
denaturation at 94 C, 1-min annealing at 55 C, and 45-s extension at 72
C. It was confirmed that the reaction did not reach its plateau in this
condition. The fragment that originated from the mutated sequence was
digested by AfaI into 128- and 25-bp fragments, whereas the
fragment that originated from the wild type was not digested by
AfaI, yielding a 153-bp fragment. The digests were
electrophoresed through a 12% polyacrylamide gel, stained with
ethidium bromide, and visualized by UV, or silver staining was
performed. The proportion of mutant mtDNA and the total mtDNA were
calculated by reflectance densitometry on photographs of the gels.
Assay of enzyme activities of mitochondrial respiratory chain
The enzyme activities of respiratory chain, such as
NADH-cytochrome c reductase, succinate dehydrogenase,
succinate cytochrome c reductase, and cytochrome
c oxidase, were assayed using freshly purified mitochondria
from skeletal muscle biopsy specimens of the proband and controls, as
described by Miyabayashi et al. (9). Controls were normal
glucose-tolerant subjects with no neuromuscular disease.
|
Results
|
|---|
Twenty clones were sequenced, and it was revealed that nucleotide
position 3256 was C (wild type) in 10 clones and T (mutant) in the
other 10 clones. Figure 2 shows the
sequences encompassing position 3256 in normal and mutant DNA from the
proband. It was evident that the 3256 C-T mutation was a heteroplasmic
mutation. C at position 3256 pairs with a G in the D stem, and both
nucleotides were highly conserved throughout evolution (from
Saccharomyces cervisiae through human).
View larger version (65K):
[in this window]
[in a new window]
|
Figure 2. mtDNA sequence of the proband’s skeletal
muscle. Both wild-type (A) and mutated mtDNA (B) were sequenced in the
proband, suggesting that the mtDNA 3256 C-T mutation was a
heteroplasmic mutation.
|
|
The enzyme activity of the mitochondrial respiratory chain was
decreased by 79% in NADH-cytochrome c reductase in the
proband’s muscle (Table 1).
We analyzed mtDNA from the muscle of the proband (II-6); from the hair
roots and buccal cells of the proband’s sister (II-5) and niece
(III-1); from the blood of III-3, -4, -5, and -6; and from the hair
roots of IV-1 and -2. PCR restriction fragment length polymorphism
analysis showed that individual II-6 (proband), II-5, III-1, III-3,
III-4, III-5, III-6, and IV-2 had the same heteroplasmic mutation (data
not shown). Although densitometry of PCR products gives an indirect and
rather inaccurate estimate of the proportions of two populations of
mtDNA, it is the only method available that gives an idea of the degree
of heteroplasmic point mutations. The proportions of wild-type
vs. mutant DNA in the muscle of the proband and the mutant
DNA were considered to be approximately 50% according to densitometric
analysis. The mutant DNA band from the proband’s blood sample was very
faint, and it was difficult to quantify. Therefore, muscle had a much
higher level of mutant DNA than blood. We were not able to obtain the
proband’s hair or buccal cells. The mutant DNA was approximately 50%
in hair roots of individual II-5, and it was about 20% in buccal cells
in the same individual. III-1 also had higher levels of mutant DNA in
hair roots (45%) than in buccal cells (18%). Their blood showed very
faint bands. It is thought that muscle and hair roots have relatively
high levels of mutant DNA, buccal cells have somewhat less, and blood
cells have very low levels of mutant DNA. Based on 75-g oral glucose
tolerance tests, individuals II-5, III-1, III-3, III-4, III-5, and
III-6 had NIDDM, and IV-2 showed impaired glucose tolerance (IGT). The
mutation was not detected in IV-1 in this study, and he showed normal
glucose tolerance. Insulin levels during an oral glucose tolerance test
were lower in IV-2 than in IV-1. Insulinogenic indexes (increment of
immunoreactive insulin/increment of plasma glucose in the first 30 min
after 75 g glucose oral administration) were 1.14 and 0.21 pmol/mg
in IV-1 and IV-2, respectively. II-2 died from heart failure in her
thirties, II-3 and II-4 died suddenly from unknown causes in their
twenties, and III-6 has been mentally retarded since she suffered from
encephalitis at the age of 4 yr. Individuals II-5, III-1, III-3, III-4,
III-5, IV-1, and IV-2 did not have any neuromuscular findings during
the study. II-6 was the offspring of marriage between individuals I-2
and I-3. Note that the father of individual II-6 had previously been
married to individual I-1, who was the sister of individual I-3 and who
died of an unknown cause at a relatively young age (Fig. 1
).
|
Discussion
|
|---|
Recently, several groups, including ours, reported that mutations
and deletions of mtDNA were related to maternally inherited diabetes
mellitus (3, 4, 5, 6, 7, 8). In this study we have identified a mtDNA 3256 C-T
point mutation in a diabetic patient with hearing disturbances and
muscle atrophy. The mutation exists in the tRNALeu(UUR)
region and may interfere with protein synthesis and decrease
mitochondrial function. Eight other members of her family were examined
for the mutation; six of them had the same mutation and NIDDM, and one
teen-aged boy had the mutation and IGT. Individual IV-1 had no
detectable levels of mutant mtDNA and showed normal glucose tolerance
by oral glucose tolerance test. As heteroplasmic cells undergo mitosis
and meiosis, random segregation of mutant and normal mitochondria
occurs. Therefore, it is considered that wild-type mtDNA was dominantly
transmitted from III-1 to IV-1. However, we do not know whether there
is a significant level of the mutant mtDNA in the pancreatic ß-cells
in IV-1. The C at position 3256 pairs with a G in the D stem of the
tRNALeu, and both nucleotides have been highly conserved
throughout evolution (from S. cervisiae through human),
suggesting that this mutation is pathogenic. Moraes et al.
reported the same mutation in a patient with a neurological syndrome
resembling myoclonus epilepsy and ragged-red fibers as well as diabetes
mellitus (10). They showed impaired respiratory chain function and
decreased protein synthesis in mitochondria. Sato et al.
also reported this mutation in a patient with mitochondrial myopathy,
encephalopathy, lactic acidosis, and stroke-like episodes and showed
decreased mitochondrial enzyme activity (11). Consistent with their
findings, the enzyme activity of the mitochondrial respiratory chain in
the patient with the mtDNA 3256 C-T mutation was decreased, especially
in complex I. It is speculated that this mitochondrial 3256 mutation
contributed to impaired insulin secretion, which might lead to abnormal
glucose metabolism (NIDDM and IGT) in this family. Further studies are
needed to characterize this mutation in this family.
|
Acknowledgments
|
|---|
We thank Dr. Daisuke Goto (Asahi General Hospital) and Dr.
Ko-ichi Kikuta (Public Tsukidate Hospital) for their help with the
familial survey. We also thank Chitose Suzuki for excellent technical
assistance.
|
Footnotes
|
|---|
1 This work was supported in part by a Grant for Diabetes Research
from the Ministry of Health and Welfare and a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science, and
Culture of Japan. 
Received July 23, 1997.
Revised November 20, 1997.
Accepted December 1, 1997.
|
References
|
|---|
-
DeFronzo RA, Bonadonna RC, Ferrannini E. 1992 Pathogenesis of NIDDM: a balanced overview. Diabetes Care. 15:318–368.[Abstract]
-
Barnett AH, Eff C, Leslie RDG, et al. 1981 Diabetes in identical twins. Diabetologia. 20:87–93.[CrossRef][Medline]
-
van den Ouweland JMW, Lemkes HHPJ, Ruitenbeek W, et
al. 1992 Mutation in mitochondrial tRNALeu(UUR) gene
in a large pedigree with maternally transmitted type II diabetes
mellitus and deafness. Nat Genet. 1:368–371.[CrossRef][Medline]
-
Kadowaki T, Kadowaki H, Mori Y, et al. 1994 A
subtype of diabetes mellitus associated with a mutation of
mitochondrial DNA. N Engl J Med. 330:962–968.[Abstract/Free Full Text]
-
Suzuki S, Hinokio Y, Hirai S, et al. 1994 Diabetes
with mitochondrial gene tRNALYS mutation. Diabetes Care. 17:1428–1432.[Abstract]
-
Suzuki S, Hinokio Y, Hirai S, et al. 1994 Pancreatic beta-cell secretory defect associated with mitochondrial
point mutation of tRNALeu(UUR) gene. Diabetologia. 37:818–825.[Medline]
-
Hirai M, Suzuki S, Onoda M, et al. 1996 Mitochondrial DNA 3394 mutation in the NADH dehydrogenase subunit 1
associated with non-insulin-dependent diabetes mellitus. Biochem
Biophys Res Commun. 219:951–955.[CrossRef][Medline]
-
Nakagawa Y, Ikegami H, Yamato E, et al. 1995 A new
mitochondrial DNA mutation associated with non-insulin dependent
diabetes mellitus. Biochem Biophys Res Commun. 209:664–668.[CrossRef][Medline]
-
Miyabayashi S, Haginoya K, Hanamizu H, et al. 1980 Defective pattern of mitochondrial respiratory enzyme in mitochondrilal
myopathy. J Inher Metab Dis. 12:373–377.
-
Moraes CT, Ciacci F, Bonilla E, et al. 1993 Two
novel pathogenic mitochondrial DNA mutations affecting organelle number
and protein synthesis. Is the tRNALeu(UUR) gene an
etiologic hot spot? J Clin Invest. 92:2906–2915.
-
Sato W, Hayakawa K, Shoji Y, et al. 1994 A
mitochondrial tRNALeu(UUR) mutation at 3,256 associated
with mitochondrial myopathy, encephalopathy, lactic acidosis, and
stroke-like episodes (MELAS). Biochem Mol Biol Int. 33:1055–1061.[Medline]
Top
Abstract
Introduction
