Download Failures in Mitochondrial tRNA and tRNA Metabolism

Failures in Mitochondrial tRNAMet and tRNAGln
Metabolism Caused by the Novel 4401A>G Mutation Are
Involved in Essential Hypertension in a Han Chinese Family
Ronghua Li, Yuqi Liu, Zongbin Li, Li Yang, Shiwen Wang, Min-Xin Guan
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Abstract—We report here on the clinical, genetic, and molecular characterization of 1 Han Chinese family with maternally
transmitted hypertension. Three of 7 matrilineal relatives in this 4-generation family exhibited the variable degree of
essential hypertension at the age at onset, ranging from 35 to 60 years old. Sequence analysis of the complete
mitochondrial DNA in this pedigree identified the novel homoplasmic 4401A⬎G mutation localizing at the spacer
immediately to the 5⬘ end of tRNAMet and tRNAGln genes and 39 other variants belonging to the Asian haplogroup C.
The 4401A⬎G mutation was absent in 242 Han Chinese controls. Approximately 30% reductions in the steady-state
levels of tRNAMet and tRNAGln were observed in 2 lymphoblastoid cell lines carrying the 4401A⬎G mutation compared
with 2 control cell lines lacking this mutation. Failures in mitochondrial metabolism are apparently a primary contributor to
the reduced rate of mitochondrial translation and reductions in the rate of overall respiratory capacity, malate/glutamatepromoted respiration, succinate/glycerol-3-phosphate–promoted respiration, or N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine/
ascorbate-promoted respiration in lymphoblastoid cell lines carrying the 4401A⬎G mutation. The homoplasmic form, mild
biochemical defect, late onset, and incomplete penetrance of hypertension in this family suggest that the 4401A⬎G mutation
itself is insufficient to produce a clinical phenotype. Thus, the other modifier factors, eg, nuclear modifier genes and
environmental and personal factors, may also contribute to the development of hypertension in these subjects carrying this
mutation. These data suggest that mitochondrial dysfunctions, caused by the 4401A⬎G mutation, are involved in the
development of hypertension in this Chinese pedigree. (Hypertension. 2009;54:329-337.)
Key Words: hypertension 䡲 mitochondria 䡲 mutation 䡲 tRNA metabolism 䡲 maternal inheritance 䡲 risk factor
䡲 Chinese
tRNAIle gene.12–14 Most recently, the 4291T⬎C mutation in
tRNAIle gene has been associated with a cluster of metabolic
defects, including essential hypertension, hypercholesterolemia,
and hypomagnesemia in a large family.15
With an effort to understand a role of the mitochondrial
genome in the pathogenesis of cardiovascular disease in the
Chinese population, we have initiated a systematic and
extended mutational screening of mtDNA in a large cohort of
hypertension subjects in the Geriatric Cardiology Clinic at the
Chinese People’s Liberation Army General Hospital.16 –18 In
the present study, we performed the clinical, genetic, and
molecular characterizations of another Han Chinese family
with maternally transmitted hypertension. Three (2 men/1
woman) of 7 matrilineal relatives in this 4-generation family
exhibited the variable severity and age at onset in hypertension. Mutational analysis of the mitochondrial genome has
identified the novel 4401A⬎G mutation in this Chinese
family. This novel 4401A⬎G mutation is localized at the
C
ardiovascular disease is the leading cause of death in
America and the world. In particular, hypertension
affects ⬇1 billion individuals worldwide and 130 million in
China.1 The etiology of cardiovascular disease is not well
understood because of the multifactorial causes. Cardiovascular disease can be caused by a single gene or multifactorial
conditions, resulting from interactions between environment
and inherited risk factors. Of hereditary factors, the maternal
transmissions of cardiovascular disease have been implicated
in some pedigrees, suggesting that the mutation(s) in mitochondrial DNA (mtDNA) is one of the molecular bases for
this disorder.2– 6 Recently, several mtDNA point mutations
have been identified to be associated with cardiovascular
disease. These mutations included the 1555A⬎G mutation in
the 12S ribosomal RNA (rRNA) gene,7 the 3260A⬎G and
3303C⬎T mutations in the tRNALeu(UUR) gene,8,9 the
8348A⬎G and 8363G⬎A mutations in the tRNALys gene,10,11
and the 4295A⬎G, 4300A⬎G, and 4317A⬎G mutations in the
Received January 14, 2009; first decision February 9, 2009; revision accepted May 29, 2009.
From the Division of Human Genetics (R.L., L.Y., M.-X.G.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; Institute of Geriatric
Cardiology (Y.L., Z.L., S.W.), Chinese People’s Liberation Army General Hospital, Beijing, China; Department of Pediatrics (M.-X.G.), University of
Cincinnati College of Medicine, Cincinnati, Ohio.
R.L., Y.L., and Z.L. contributed equally to this work.
Correspondence to Min-Xin Guan, Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH
45229-3039. E-mail [email protected]; or Shiwen Wang, Institute of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China.
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.109.129270
329
330
Hypertension
August 2009
systolic blood pressure of ⱖ140 mm Hg and/or a diastolic blood
pressure of ⱖ90 mm Hg.
I
1
2
Mutational Analysis of Mitochondrial Genome
II
1
2
3
4
3
4
III
2
1
4
5
6
7
8
9
IV
1
2
3
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Figure 1. The Chinese pedigree with hypertension. Affected
individuals are indicated by filled symbols. Arrowhead denotes
proband.
junction between the tRNAMet at the heavy strand and
tRNAGln at the light strand.19,20 Thus, it is hypothesized that
the 4401A⬎G mutation affects the processing of precursors
in these mitochondrial tRNAs. Functional significance of the
4401A⬎G mutation was evaluated by examining for the
steady-state levels of mitochondrial tRNAMet, tRNAGln, and
other tRNAs, including tRNALys, tRNAGly, and tRNASer(UCN), using lymphoblastoid cell lines derived from 2 affected
matrilineal relatives carrying the 4401A⬎G mutation and
from 2 married-in-control individuals lacking the mtDNA
mutation. These cell lines were further assessed for the effects
of the 4401A⬎G mutation on the rate of mitochondrial
protein synthesis, the endogenous respiration, and substratedependent respiration.
Materials and Methods
Subjects
As a part of genetic screening program for hypertension, a Han
Chinese family (Figure 1) was ascertained at the Institute of Geriatric
Cardiology of the Chinese People’s Liberation Army General Hospital. Informed consent, blood samples, and clinical evaluations were
obtained from all of the participating family members, under
protocols approved by ethic committee of the Chinese People’s
Liberation Army General Hospital and the Cincinnati Children’s
Hospital Medical Center Institute Review Board. Members of this
family were interviewed and evaluated to identify both personal or
medical histories of hypertension and other clinical abnormalities.
The 242 control DNA samples were obtained from a panel of
unaffected Han Chinese individuals from the same area.
Genomic DNA was isolated from whole blood of participants using
Puregene DNA Isolation kits (Gentra Systems). The entire mitochondrial genome of the proband II-1 was PCR amplified in 24 overlapping fragments by use of sets of the light-strand and the heavy-strand
oligonucleotide primers, as described elsewhere.23 Each fragment
was purified and subsequently analyzed by direct sequencing in an
ABI 3700 automated DNA sequencer using the Big Dye Terminator
Cycle sequencing reaction kit. The resultant sequence data were
compared with the revised Cambridge reference sequence (GenBank
accession No. NC㛭001807).24
For the quantification of the 4401A⬎G mutation, the first PCR
segments (903 bp) were amplified using genomic DNA as a template
and oligodeoxynucleotides corresponding with mtDNA at positions
3777 to 4679 to rule out the coamplification of possible nuclear
pseudogenes.25 Then, the second PCR product (225 bp) was amplified using the first PCR fragment as a template, and oligodeoxynucleotides corresponding with mtDNA at positions 4243 to 4467 and
subsequently digested with the restriction enzyme BfaI as the
4401A⬎G mutation creates the site for this restriction enzyme.
Equal amounts of various digested samples were then analyzed by
electrophoresis through 7% polyacrylamide gel. The proportions of
digested and undigested PCR products were determined by the
Image-Quant program after ethidium bromide staining to determine
whether 4401A⬎G mutation is in the homoplasmy in these subjects.
The allele frequency of the 4401A⬎G variant was determined by
PCR amplification using the genomic DNA derived from 242 Han
Chinese controls and subsequent restriction enzyme analysis of PCR
products, as described above.
Mitochondrial tRNA Analysis
Lymphoblastoid cell lines were immortalized by transformation with the
Epstein-Barr virus, as described elsewhere.26 Cell lines derived from 1
proband II-1 and her son III-3 carrying the 4401A⬎G mutation and 2
Chinese married-in controls (II-2 and III-4) lacking this mutation were
grown in RPMI 1640 (Invitrogen), supplemented with 10% FBS. Total
mitochondrial RNA were obtained using a TOTALLY RNA kit
(Ambion) from mitochondria isolated from lymphoblastoid cell lines
(⬇4.0⫻108 cells), as described previously.27 Two micrograms of total
mitochondrial RNA were electrophoresed through a 10% polyacrylamide/7 mol/L urea gel in Tris-borate-EDTA buffer (after heating the
sample at 65°C for 10 minutes) and then electroblotted onto a positively
charged nylon membrane (Roche) for the hybridization analysis with
oligodeoxynucleotide probes. For the detection of tRNAMet, tRNAGln,
tRNALys, tRNAGly, and tRNASer(UCN), the following nonradioactive
digoxigenin (DIG)-labeled oligodeoxynucleotides specific for each
RNA were used: 5⬘-TAGTACGGGAAGGGTATAACC-3⬘ (tRNAMet);
5⬘-CTAGGACTATGAGAATCGAA-3⬘ (tRNAGln); 5⬘-TCACTGTA
AAGAGGTGTTGG-3⬘ (tRNALys); 5⬘-TACTCTTTTTTGAATGTT
GTC-3⬘ (tRNAGly); and 5⬘-CAAGCCAACCCCATGGCCTC-3⬘
(tRNASer(UCN)).24 DIG-labeled oligodeoxynucleotides were generated
by using a DIG Oligonucleotide Tailing Kit (Roche). The hybridization
was carried out as detailed elsewhere.28 Quantification of density in each
band was made as detailed previously.28 –30
Measurements of Blood Pressure
Analysis of Mitochondrial Protein Synthesis
Members of this Chinese family underwent a physical examination,
laboratory assessment of cardiovascular disease risk factors, and
routine electrocardiography. A physician measured the systolic and
diastolic blood pressures of subjects using a mercury column
sphygmomanometer and a standard protocol. The first and the fifth
Korotkoff sounds were taken as indicative of systolic and diastolic
blood pressures, respectively. The average of 3 such systolic and
diastolic blood pressure readings was taken as the examination blood
pressure. Hypertension was defined according to the recommendation of the Sixth Joint National Committee on the Detection,
Evaluation, and Treatment of High Blood Pressure.21 and the World
Health Organization-International Society of Hypertension22 as a
Pulse labeling of the cell lines for 30 minutes with [35S]methionine[35S]cysteine in methionine-free DMEM in the presence of emetine,
electrophoretic analysis of the translation products, and quantification of
radioactivity in the whole-electrophoretic patterns or in individual
well-resolved bands was carried out as detailed previously.31
O2 Consumption Measurements
Rates of O2 consumption in intact cells were determined with a YSI
5300 oxygraph (Yellow Springs Instruments) on samples of 1⫻107
cells in 1.5 mL of special DMEM glucose lacking glucose and
supplemented with 10% dialyzed FBS.32 Polarographic analysis of
digitonin-permeabilized cells using different respiratory substrates
Li et al
Hypertension-Associated Mitochondrial DNA Mutation
Table 1. Summary of Clinical Data for Some Members in 1
Han Chinese Family
Subjects
Sex
Age of
Test, y
Age of
Onset, y
Systolic Pressure,
mm Hg
Diastolic Pressure,
mm Hg
331
Table 2. MtDNA Variants in 1 Han Chinese Subject (II-1)
With Hypertension
Gene
Replacement
Conservation
(H/B/M/X)*
Previously
Reported†
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II-1
F
65
60
180
110
73
A to G
Yes
II-2
M
68
NA
125
75
249
DelA
Yes
II-3
M
63
NA
130
75
310
T to CTC
Yes
III-1
M
40
NA
115
82
489
T to C
Yes
III-2
F
38
NA
100
70
16145
G to A
Yes
III-3
M
41
36
140
110
16223
C to T
Yes
III-4
F
39
NA
118
78
16298
T to C
Yes
III-5
M
37
35
140
95
16519
T to C
III-6
F
34
NA
126
76
750
A to G
A/A/G/A
Yes
IV-1
M
20
NA
122
70
1438
A to G
A/A/A/G
Yes
IV-2
M
18
NA
116
74
16S rRNA
2706
A to G
A/G/A/A
Yes
IV-3
F
16
NA
112
72
ND1
3552
T to A
NC2
4401
A to G
ND2
4715
A to G
M indicates male; F, female; NA, not applicable.
D-Loop
Position
12S rRNA
and inhibitors to test the activity of the various respiratory complexes
was carried out as detailed previously.33
Results
CO1
Clinical Presentation
The proband (II-1) began suffering from hypertension at the
age of 60 years. She came to the Geriatric Cardiology Clinic
of the Chinese People’s Liberation Army General Hospital
for further clinical evaluations at the age of 65 years. Her
blood pressure was 180/110 mm Hg. Physical examination,
laboratory assessment of cardiovascular disease risk factors,
and routine electrocardiography showed no other clinical
abnormalities, including diabetes mellitus, vision and hearing
impairments, or renal and neurological disorders. Therefore,
she exhibited a typical essential hypertension. The family
originated from Beijing in northern China, and the majority of
family members live in the same area. As shown in Figure 1,
this familial history is consistent with a maternal inheritance.
None of the offspring of affected fathers had hypertension.
Two male matrilineal relatives exhibited hypertension as the
sole clinical symptom, whereas other members of this family
had normal blood pressure. As shown in Table 1, subject III-3
experienced the hypertension (blood pressure was 140/
110 mm Hg) at the age of 36 years, whereas his brother (III-5)
had hypertension (blood pressure was 140/95 mm Hg) at the
age of 35 years. There is no evidence that any member of this
family had any other known cause to account for hypertension. Comprehensive family medical histories of these individuals showed no other clinical abnormalities, including
diabetes mellitus, vision and hearing impairments, or renal
and neurological disorders.
Mitochondrial DNA Analysis
The maternal transmission of hypertension in this family
suggested the mitochondrial involvement and led us to
analyze the mitochondrial genome of matrilineal relatives.
For this purpose, the DNA fragments spanning the entire
mtDNA of the proband II-1 were PCR amplified, and each
fragment was purified and subsequently analyzed by direct
ATP6
CO3
ND3
ND4
4769
A to G
5262
G to A (Ala to Thr)
Yes
Yes
A/A/A/A
No
Yes
Yes
A/M/I/F
Yes
5993
C to T
No
6338
A to G
Yes
6386
C to T
Yes
7028
C to T
Yes
7196
C to A
Yes
8584
G to A (Ala to Thr)
A/V/V/I
Yes
8701
A to G (Thr to Ala)
T/S/L/Q
Yes
8860
A to G (Thr to Ala)
T/A/A/T
Yes
9540
T to C
Yes
9545
A to G
Yes
10398
A to G (Thr to Ala)
10400
C to T
T/T/T/A
Yes
Yes
10873
T to C
11447
G to A (Val to
Met)
11719
G to A
Yes
11914
G to A
Yes
12705
C to T
Yes
13263
A to G
ND6
14318
T to C (Asn to
Ser)
Cytb
14783
T to C
Yes
15043
G to A
Yes
15301
G to A
Yes
15326
A to G (Thr to Ala)
15487
T to C
ND5
Yes
V/V/I/V
Yes
Yes
N/N/D/S
T/M/I/I
Yes
Yes
Yes
*Conservation of amino acid for polypeptides or nucleotide for rRNAs in
human (H), bovine (B), mouse (M), and X laevis (X).
†See http//www.mitomap.org and http://www.genpat.uu.se/mtDB/.
sequence. As shown in Table 2, the comparison of the
resultant sequences with the Cambridge consensus sequence
identified a number of nucleoside changes, belonging to the
Eastern Asian haplogroup C.34 Of these nucleoside changes,
August 2009
Mitochondrial tRNA Analysis
To examine whether the 4401A⬎G mutation affects the processing of the precursors in the tRNAMet and tRNAGln, the
steady-state levels of the tRNAMet and tRNAGln were determined by isolating total mitochondrial RNA from cell lines
derived from 2 affected individuals (II-1 and III-3) carrying the
4401A⬎G mutation and 2 married-in controls (II-2 and III-4)
lacking this mutation in this Chinese family, separating them by
a 10% polyacrylamide/7 mol/L urea gel, electroblotting, and
hybridizing with a nonradioactive DIG-labeled oligodeoxynucleotide probe specific for tRNAMet and tRNAGln. After stripping the blots, the DIG-labeled oligodeoxynucleotide probes,
A
4401
III-2
ACCCCATCCT AAAGTAAGGTCAG
III-1
ACCCCATCCT AAAGTAAGGTCAG
4401
G
4400 4402
4329
A
T
tRNA Gl n
RNase P a
C
c
c
G 4329
-- U UA C U UU A
A U
G C
4401 C
G C
A U
T G
UU A
G
G G A U A C UC
U A G U GU G
G
A
A U G GG C
UU
G
G
G G GC A C
G
G
U
G CAG
A U
G C
A U
A UA
U
G
U
UU G
4469
tRNA Met
RNase P a
c
c
4469
A
-- U CC U A A A U
G C
U A
4401 G
A U
A G
G C
G C C
U
C
A
U UC C C A
A U C GA
U
G U U GG
A
UU A
U
U A A G CU
A
A
U AAA
C G
G C
G C
G C
C
C
C
A
CA U
t RNA Met
t RNAGln
IV-3
III-5
III-4
III-3
III-2
C
Un-digested
B
III-1
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there were 8 polymorphisms in the D-loop region, 2 variants
in the 12S rRNA gene, 1 variant in the 16S rRNA gene, 1
novel 4401A⬎G mutation in the spacer between tRNAMet
and tRNAGln genes, 20 silent mutations (1 novel and 19
known), and 8 missense mutations in protein-encoding
genes.35 These missense mutations are 5262G⬎A (264A⬎T)
in the ND2 gene, 8584G⬎A (20A⬎T), 8701A⬎G (59T⬎A),
and 8860A⬎G (112T⬎A) in the ATP6 gene, 10398A⬎G
(114T⬎A) in the ND3 gene, 11447G⬎A (230V⬎M) in the
ND4 gene, 14318T⬎C (119N⬎S) in the ND6 gene, and
15326A⬎G (194T⬎A) in the Cytb gene. These variants in
rRNAs and polypeptides were further evaluated by phylogenetic analysis of these variants and sequences from other
organisms, including mouse,36 bovine,37and Xenopus laevis.38
None of variants in the polypeptides and rRNAs were highly
evolutionarily conserved and implicated to have significantly
functional consequence.
However, the novel A to G transition at the position 4401
(4401A⬎G) mutation, as shown in Figure 2, lies at the
junction of tRNAMet at the H-strand and tRNAGln at the
L-strand.19,20 Here the 5⬘ end of the flanking sequence is
4401A/AGTAAG in the tRNAMet gene, whereas the 5⬘ end of
the flanking sequence is 4401T/TGAGAT in the tRNAGln
gene.39 In fact, the processing of mitochondrial tRNAs
requires the precise endonucleolytic cleavage at both 3⬘ and
5⬘ ends catalyzed by RNase P and 3⬘ endonuclease.20,40,41
Thus, the 4401A⬎G mutation may affect the reaction efficiency of the RNase P involved in tRNAMet and tRNAGln 5⬘
end metabolism. The 4401A⬎G mutation was further assessed by phylogenetic analysis of this variant and sequences
from mouse, bovine, and X laevis, as well as other 13
primates including Gorilla gorilla, Pan paniscus, Pan troglodytes, Pongo pygmaeus, Pongo abelii, Hylobates lar, Macaca mulatta, Macaca sylvanus, Papio hamadryas, Cebus
albifrons, Tarsius bancanus, Nycticebus coucang, and Lemur
catta (Genbank). In fact, the adenine at the 4401 position is
extraordinarily conserved among these species. To determine
whether the 4401A⬎G mutation is present in homoplasmy,
the fragments spanning the tRNAMet and tRNAGln genes were
PCR amplified and subsequently digested with BfaI, because
the 4401A⬎G mutation creates the site for this restriction
enzyme. As shown in Figure 2C, there was no detectable
wild-type DNA in 4 matrilineal relatives, indicating that the
4401A⬎G mutation was present in homoplasmy in these
matrilineal relatives. In addition, this mutation was absent in
242 Han Chinese controls.
II-2
Hypertension
II-1
332
225 bp
159 bp
66 bp
Figure 2. Identification and qualification of the 4401A⬎G mutation in
the junction between mitochondrial tRNAMet and tRNAGln genes. A,
Partial sequence chromatograms of tRNAMet and tRNAGln genes from
an affected individual (III-2) and a married-in control (III-1). An arrow
indicates the location of the base changes at position 4401. B, A
schema of location of 4401A⬎G in the precursors of tRNAMet and
tRNAGln genes. Cloverleaf structures of human mitochondrial tRNAMet
and tRNAGln are derived from Florentz et al.39 Processing sites in the
mitochondrial tRNAMet and tRNAGln precursors were determined for
RNase P. 40 Arrow indicates the position of the 4401A⬎G mutation.
C, Quantification of the mtDNA 4401A⬎G mutation in 8 members of
the Chinese family. PCR products around the 4401A⬎G mutation
were digested with BfaI and analyzed by electrophoresis in a 7%
polyacrymide gel stained with ethidium bromide. Patients and control
individuals are indicated.
Li et al
Hypertension-Associated Mitochondrial DNA Mutation
333
tRNAGln in the mutant cells were significantly reduced
relative to the controls. In particular, the average levels of
tRNAMet in the mutant cell lines derived from II-1 and
III-3 ranged from ⬇71% of controls after normalization to
tRNAGly, ⬇67% of controls after normalization to tRNALys, to
⬇70% of controls after normalization to tRNASer(UCN). Similarly, the average levels of tRNAGln in the mutant cell lines
derived from II-1 and III-3 ranged from ⬇75% of controls
after normalization to tRNAGly, ⬇71% of controls after
normalization to tRNALys, to ⬇70% of controls after normalization to tRNASer(UCN).
Mitochondrial Protein Synthesis Defect
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Figure 3. Northern blot analysis of mitochondrial tRNA. A, Equal
amounts (2 ␮g) of total mitochondrial RNA from various cell lines
were electrophoresed through a denaturing polyacrylamide gel,
electroblotted, and hybridized with DIG-labeled oligonucleotide
probes specific for the tRNAMet. The blots were then stripped and
rehybridized with DIG-labeled tRNAGln, tRNAGly, tRNALys, and
tRNASer(UCN), respectively. B, Quantification of mitochondrial tRNA
levels. Average relative tRNAMet and tRNAGln content per cell, normalized to the average content per cell of tRNAGly, tRNALys, or
tRNASer(UCN) in 2 control cell lines and in 2 mutant cell lines. The
values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were
based on 3 independent determinations of tRNAMet and tRNAGln
content in each cell line and 3 determinations of the content of
each reference RNA marker in each cell line. Error bars indicate
2 standard error of the means (SEMs).
including tRNAGly and tRNALys as representatives of the whole
H-strand transcription unit and tRNASer(UCN) derived from the
L-strand transcription unit,19,20 were hybridized with the same
blots for normalization purposes.
As shown in Figure 3A, the amounts of tRNAMet and
tRNAGln in mutant cells were markedly decreased as compared with those in control cells. For comparison, the average
levels of tRNAMet and tRNAGln in various control or mutant
cell lines were normalized to the average levels in the same
cell line for the tRNAGly, tRNALys, and tRNASer(UCN), respectively. As shown in Figure 3B, the levels of tRNAMet and
To examine whether a defect in mitochondrial translation
occurred in lymphoblastoid cell lines carrying the 4401A⬎G
mutation, cells derived from 2 affected individuals (II-1 and
III-3) carrying the 4401A⬎G mutation and 2 married-in
controls (II-2 and III-4) lacking this mutation in this Chinese
family were labeled for 30 minutes with [35S]methionine[35S]cysteine in methionine-free regular DMEM in the presence of 100 ␮g/mL of emetine to inhibit cytosolic protein
synthesis.31 Figure 4A shows typical electrophoretic patterns
of the mitochondrial translation products of the mutant and
control cell lines. Patterns of the mtDNA-encoded polypeptides of the cells carrying the 4401A⬎G mutation were
qualitatively identical in terms of electrophoretic mobility of
the various polypeptides to those of the control cells and of
143B.TK⫺ cells. However, the cell lines carrying the
4401A⬎G mutation showed a clear tendency toward a
decrease in the total rate of labeling of the mitochondrial
translation products relative to those of control cell lines.
Figure 4B shows a quantification of the results of a large
number of labeling experiments and electrophoretic runs,
which were carried out by the Image-Quant program of
appropriate exposures of the fluorograms and normalization
to data obtained for the 143B.TK⫺ sample. In fact, the overall
rates of labeling of the mitochondrial translation products in
the cell lines derived from 2 affected individuals (II-1 and
III-3) carrying the 4401A⬎G mutation were decreased 31.7%
and 20.8%, with an average of 26.0% relative to the mean
value measured in the control cell lines.
Respiration Defects in the Cell Lines
The endogenous respiration rates of cell lines derived from 2
affected individuals (II-1 and III-3) carrying the 4401A⬎G
mutation and 2 married-in controls (II-2 and III-4) lacking
this mutation in this Chinese family were measured by
determining the O2 consumption rate in intact cells, as
described previously.32 As shown in Figure 5A, the rate of
total O2 consumption in the lymphoblastoid cell lines derived
from 2 affected individuals (II-1 and III-3) ranged between
⬃74.9% and 80.6%, with an average reduction of ⬃77.8%
relative to the mean value measured in the control cell lines.
To investigate which of the enzyme complexes of the
respiratory chain was affected in the mutant cell lines, O2
consumption measurements were carried out on digitoninpermeabilized cells using different substrates and inhibitors.33
As shown in Figure 5B, in the cell lines derived from 2
affected individuals, the rate of malate/glutamate-driven res-
334
Hypertension
August 2009
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Figure 4. Mitochondrial translation assay. A, Electrophoretic patterns of the mitochondrial translation products of lymphoblastoid cell
lines and of 143B.TK⫺ cells labeled for 30 minutes with [35S]methionine in the presence of 100 ␮g/mL of emetine. Samples containing
equal amounts of protein (30 ␮g) were run in SDS/polyacrylamide gradient gels. COI, COII, and COIII indicate subunits I, II, and III of
cytochrome c oxidase; ND1, ND2, ND3, ND4, ND4L, ND5, and ND6, subunits 1, 2, 3, 4, 4L, 5, and 6 of the respiratory chain reduced
nicotinamide-adenine dinucleotide dehydrogenase; A6 and A8, subunits 6 and 8 of the H⫹-ATPase; and CYTb, apocytochrome b. B,
Quantification of the rates of labeling of the mitochondrial translation products, after a 30-minute [35S]methionine pulse, in lymphoblastoid cell lines. The rates of mitochondrial protein labeling, determined in detailed elsewhere,31 were expressed as percentages of the
value for 143B.TK⫺ in each gel, with error bars representing 2 SEMs. A total of 3 independent labeling experiments and 3 electrophoretic analyses of each labeled preparation were carried out on lymphoblastoid cell lines. The vertical arrows refer to 2 SEMs.
piration, which depends on the activities of reduced
nicotinamide-adenine dinucleotide:ubiquinone oxidoreductase (complex I), ubiquinol-cytochrome c reductase (complex
III), and cytochrome c oxidase (complex IV), but usually
reflects the rate-limiting activity of complex I,33 was very
significantly decreased, relative to the average rate in the
control cell lines, by 77% to 80% (⬃78% on average).
Similarly, the rate of succinate/glycerol-3-phosphate– driven
respiration, which depends on the activities of complexes III
and IV but usually reflects the rate-limiting activity of
complex III, was significantly affected in the mutant cell
lines, relative to the average rate in the control cell lines, by
76% to 81% (⬃78% on average). Furthermore, the rate of
N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine/ascorbate-driven
respiration, which reflects the activity of complex IV, exhibited a 78% to 82% reduction in complex IV activity (⬃80%
on average) in the mutant cell lines relative to the average rate
in the control cell lines.
Discussion
In the present study, we performed the clinical, genetic, and
molecular characterization of a Han Chinese family with
essential hypertension. The hypertension as a sole clinical
phenotype was only present in all of the matrilineal relatives
of this 4-generation pedigree. Clinical and genetic evaluations
revealed the variable severity and age at onset in hypertension
among 3 of 7 matrilineal relatives in this Chinese family. In
particular, the age at onset in hypertension was 60, 36, and 35
years in 3 affected matrilineal relatives, with an average age
of 44 years. The maternal transmission of hypertension in this
family suggested that the mtDNA mutation(s) is 1 of the
molecular bases for this disorder. Mutational analysis of the
mitochondrial genome in this family identified 40 variants
belonging to the Eastern Asian haplogroup C.34 Of these, 39
variants appeared to be polymorphisms, because these variants were not highly evolutionarily conserved and implicated
to have significantly functional consequence. However, the
homoplasmic A-to-G transition at position 4401 lies in the
spacer immediately to the 5⬘ end of the tRNAMet and tRNAGln
genes.19,24 Furthermore, the adenine at the 4401 position of
the mitochondrial genomes is highly conserved among various primates. This mutation is present only in matrilineal
relatives of this family in the homoplasmic form but not in the
242 Han Chinese controls, indicating that this mutation may
be involved in the pathogenesis of hypertension.
In fact, 22 human mitochondrial tRNAs are interspersed
among the other functional mitochondrial RNAs (2 rRNAs
and 11 mRNAs encoding 13 polypeptide subunits of the
oxidative phosphorylation complexes) on long precursor
transcripts.19 Of these, 8 tRNAs, including tRNAGln and
tRNASer(UCN), are synthesized from the polycistronic precursors of the L-strand, whereas the other 14 tRNAs, eg,
tRNAMet, tRNALys, and tRNAGly, are transcribed from the
precursors of the H-strand transcripts.19,42 The processing of
Li et al
Total O2 consumption
(mol/min/cell)
A
Hypertension-Associated Mitochondrial DNA Mutation
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
O2 consumptin (succ+G-3-p)
(mol/min/cell)
0.0
2.5
2.0
1.5
1.0
0.5
0.0
O2 consumption (asc/TIMPD)
(mol/min/cell)
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
O2 consumption (glu+mal)
(mol/min/cell)
B
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
II-2
III-4
II-1
III-3
Figure 5. Respiration assays. A, Average rates of endogenous O2
consumption per cell measured in different cell lines are shown,
with error bars representing 2 SEMs. A total of 4 determinations
were made on each of lymphoblastoid cell lines. B, Polarographic
analysis of O2 consumption in digitonin-permeabilized cells of the
various cell lines using different substrates and inhibitors. The
activities of the various components of the respiratory chain were
investigated by measuring on ⬃1⫻107 digitonin-permeabilized
cells the respiration dependent on malate/glutamate, on succinate/
glycerol-3-phosphate, and on N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine/ascorbate. A total of 4 determinations were made on each
of the lymphoblastoid cell lines. Graph details and symbols are
explained in the legend to Figure 3. mal/glu indicates malate/
glutamate-dependent respiration; succ/G-3-P, succinate/glycerol3-phosphate– dependent respiration; and asc/TMPD, N,N,N⬘,N⬘tetramethyl-p-phenylenediamine/ascorbate-dependent respiration.
335
precursors in mitochondrial tRNAs requires the precise endonucleolytic cleavage at both 5⬘ and 3⬘ ends. Extra nucleotides at their 5⬘ termini are removed by RNase P, whereas the
excision of tRNAs from primary polycistronic mitochondrial
transcripts at their 3⬘ end is catalyzed by the 3⬘ endonuclease.19,43 Thus, it is anticipated that the A-to-G transition at
position 4401 in the H-strand may lead to defective tRNAMet
5⬘ end processing in the H-strand transcripts, and the T-to-C
transition at position 4401 may cause the reduced efficiency
of the tRNAGln precursor 5⬘ end cleavage in the L-strand
transcripts. There is increasing evidence showing that the 5⬘
and 3⬘ end processing defects arising from pathogenic mitochondrial tRNA mutations could contribute to clinical abnormalities. The deafness-associated 7445T⬎C mutation in the
precursor of the tRNASer(UCN) gene and the cardiomyopathies-associated 4269A⬎G and 4295A⬎G mutations in the
tRNAIle gene altered 3⬘ end processing efficiency of corresponding tRNAs.41,44 Similarly, the mitochondrial encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS)associated 3243A⬎G and 3271T⬎C mutations and
mitochondrial myopathy-associated 3302A⬎G mutation in the
tRNALeu(UUR) led to the tRNA 5⬘ end processing defects.45,46
Alternatively, a taurine modification deficiency at the anticodon
wobble position of tRNALeu(UUR) carrying the 3243A⬎G or
3271T⬎C mutation is involved in the decreased translation of
ND6 with a high content of the UUG codon.47
In the current study, compared with a control cell lacking
the mutation, a ⬇30% reduction in the levels of tRNAMet and
tRNAGln were observed in cells carrying the 4401A⬎G
mutation. The lower levels of tRNAMet and tRNAGln in cells
carrying the 4401A⬎G mutation most probably result from a
defect in the 5⬘ end processing of tRNAMet and tRNAGln
precursors. As a result, a shortage of the tRNAMet and
tRNAGln leads to the reduced rate of mitochondrial protein
synthesis. These defects appear to be responsible for the
reduced activities of the mitochondrial respiration chain.
Subsequently, these defects lead to the reduction of ATP
production and an increase of reactive oxygen species production. These mitochondrial dysfunctions likely contribute
to the development of hypertension.48 –50 However, the levels
of total tRNAMet and tRNAGln in mutant cells are above a
proposed threshold, which is 30% of the control level of
tRNA, to support a normal rate of mitochondrial translation.20,28 Thus, the homoplasmic form, mild mitochondrial
dysfunctions, late onset, and incomplete penetrance of hypertension in this family carrying the 4401A⬎G mutation
indicated that the 4401A⬎G mutation itself is insufficient to
produce a clinical phenotype, as in the cases of hypertensionassociated tRNA Met 4435A⬎G mutation, 18 deafnessassociated 12S rRNA 1555A⬎G mutation,51,52 and Leber’s
hereditary optic neuropathy–associated ND4 11778G⬎A mutation.53 The other modifier factors, eg, nuclear modifier
genes, environmental factors, and personal lifestyles, also
contribute to the development of hypertension in these
subjects carrying the 4401A⬎G mutation. Therefore, the
4401A⬎G mutation, acting as an inherited risk factor, is
involved in the development of hypertension in this Chinese
family.
336
Hypertension
August 2009
Perspectives
The genetic and biochemical evidence indicate that the
mtDNA 4401A⬎G mutation is involved in essential hypertension. The tissue specificity of this pathogenic mtDNA
mutation is likely attributed to tissue-specific RNA processing or the involvement of nuclear modifier genes. The
4401A⬎G mutation should be added to the list of inherited
risk factors for future molecular diagnosis for hypertension.
Thus, our finding will provide new insights into the molecular
mechanism, management, and treatment of maternally inherited hypertension. Future research should further explore the
emerging link among hypertension, mitochondrial dysfunction, and their causative-effect relationship.
14.
15.
16.
17.
18.
Sources of Funding
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This work was supported by National Institutes of Health grants
RO1DC05230 and RO1DC07696 from the National Institute on
Deafness and Other Communication Disorders (to M-X.G.) and
National Key Basic Research and Development Project 973 Fund
2007CB07403 (to S.W.).
19.
20.
Disclosures
21.
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Failures in Mitochondrial tRNAMet and tRNAGln Metabolism Caused by the Novel
4401A>G Mutation Are Involved in Essential Hypertension in a Han Chinese Family
Ronghua Li, Yuqi Liu, Zongbin Li, Li Yang, Shiwen Wang and Min-Xin Guan
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Hypertension. 2009;54:329-337; originally published online June 22, 2009;
doi: 10.1161/HYPERTENSIONAHA.109.129270
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