Clinical Chemistry 45:8
1162–1167 (1999)
Molecular Diagnostics
and Genetics
Detection of Mitochondrial DNA Mutations
by Temporal Temperature Gradient
Gel Electrophoresis
Tian-Jian Chen,1,2 Richard G. Boles,2 and Lee-Jun C. Wong1,2*
Background: A unique requirement for the molecular
diagnosis of mitochondrial DNA (mtDNA) disorders is
the ability to detect heteroplasmic mtDNA mutations
and to distinguish them from homoplasmic sequence
variations before further testing (e.g., sequencing) is
performed. We evaluated the potential utility of temporal
temperature gradient gel electrophoresis (TTGE) for these
purposes in patients with suspected mtDNA mutations.
Methods: DNA samples were selected from patients
with known mtDNA mutations and patients suspected
of mtDNA disorders without detectable mutations by
routine analysis. Six regions of mtDNA were PCR
amplified and analyzed by TTGE. Electrophoresis was
carried out at 145 V with a constant temperature increment of 1.2 °C/h. Mutations were identified by direct
sequencing of the PCR products and confirmed by
PCR/allele-specific oligonucleotide or PCR/restriction
fragment length polymorphism analysis.
Results: In the experiments using patient samples containing various amounts of mutant mtDNA, TTGE detected as little as 4% mutant heteroplasmy and identified heteroplasmy in the presence of a homoplasmic
polymorphism. In 109 specimens with 15 different
known mutations, TTGE detected the presence of all
mutations and distinguished heteroplasmic mutations
from homoplasmic polymorphisms. When 11% of the
mtDNA genome was analyzed by TTGE in 104 patients
with clinically suspected mitochondrial disorders, 7
cases of heteroplasmy ('7%) were detected.
1
Institute for Molecular and Human Genetics, Georgetown University
Medical Center, Washington, DC 20007.
2
Division of Medical Genetics, Children’s Hospital Los Angeles, and
Department of Pediatrics, University of Southern California, School of Medicine, Los Angeles, CA 90027.
*Address correspondence to this author at: Institute for Molecular and
Human Genetics, 3800 Reservoir Rd. NW, Suite 4000, Georgetown University
Medical Center, Washington, DC 20007. Fax 202-784-1770; e-mail
[email protected].
Received February 11, 1999; accepted April 14, 1999.
Conclusions: TTGE distinguishes heteroplasmic mutation from homoplasmic polymorphisms and appears to
be a sensitive tool for detection of sequence variations
and heteroplasmy in patients suspected of having
mtDNA disorders.
© 1999 American Association for Clinical Chemistry
Mitochondrial genetics is non-Mendelian in many aspects. Mitochondrial DNA (mtDNA)3 is maternally inherited. Each cell contains hundreds to thousands of mitochondria. Mutant and wild-type mtDNA may coexist in
the same cell to various degrees, a phenomenon known as
heteroplasmy (1 ). The mitochondrial disorders represent
a group of heterogeneous diseases that are usually manifested in tissues with high energy demands, such as
nerve, muscle, endocrine gland, and kidney, and can be
caused by mutations in either nuclear DNA or mtDNA
(1 ). In the case with mtDNA mutations, clinical expression of disease depends on the percentage of mutant
mtDNA in various tissues. Confirmatory diagnosis of
a mtDNA disorder is achieved by mutational analysis
of mtDNA. In the past decade, .50 mutations in mtDNA
have been discovered (1, 2 ). In our recent studies, analysis
of 44 known mutations detected disease-causing heteroplasmic mutations in only 5.9% of 2000 patients referred
for mtDNA analysis (3 ), suggesting that clinical heterogeneity complicates the diagnosis and that the majority of
the pathogenic mutations may not have been identified.
These unidentified mutations may occur in either nuclear
DNA or mtDNA.
Methods for the detection of point mutations as well as
small insertions and deletions in clinical diagnostics have
been reviewed recently (4 ). For unknown mutations, the
gold standard is direct DNA sequencing, although it is
not practical to routinely sequence the entire 16.6-kb
3
Nonstandard abbreviations: mtDNA, mitochondrial DNA; DGGE, denaturing gradient gel electrophoresis; TGGE, temperature gradient gel electrophoresis; TTGE, temporal temperature gradient gel electrophoresis; HA,
heteroduplex analysis; and EMC, enzyme mismatch cleavage.
1162
Clinical Chemistry 45, No. 8, 1999
1163
Fig. 1. TTGE mutation detection system.
In the TTGE mutation detection system, the denaturing environment is formed by a constant concentration of urea in the polyacrylamide gel and a temporal temperature
gradient (0.5–3 °C/h). During electrophoresis, double-stranded DNA will become partially denatured. When DNA denaturation begins, its electrophoretic mobility
decreases. Because of different melting behaviors, mutant (Mu) and wild-type (Wt) molecules are separated on the gel as they begin to denature at different
temperature.
mtDNA in all patients in which a mtDNA mutation is
suspected. Commonly used screening methods include
denaturing gradient gel electrophoresis (DGGE) (5 ), temperature gradient gel electrophoresis (TGGE) (6 ), singlestranded conformation polymorphism, heteroduplex
analysis (HA), chemical mismatch cleavage, enzyme mismatch cleavage (EMC), the protein truncation test, mismatch-binding protein, and cleavase fragment length
polymorphism. The drawbacks of these methods include
low sensitivity (single-stranded conformation polymorphism, HA), difficulty in casting gels (DGGE), synthesis
of GC-clamped primers (DGGE, TGGE), limitation of
detection to heterozygous or heteroplasmic mutations
(HA, chemical mismatch cleavage, EMC), high background
(chemical mismatch cleavage, EMC), lack of experience
(cleavase fragment length polymorphism, EMC, mismatchbinding protein), and preferential elimination of unstable
mutant transcripts (protein truncation test) (4).
mtDNA is highly polymorphic, and these single nucleotide polymorphisms are usually homoplasmic. Thus, a
unique requirement for the molecular diagnosis of
mtDNA disorders is the ability to detect heteroplasmic
mtDNA mutations and to distinguish them from homoplasmic sequence variations. Temporal temperature
gradient gel electrophoresis (TTGE) was first introduced
by Yoshino et al. (7 ) in 1991. It is based on the sequencespecific melting behavior of wild-type and mutant DNA
in a temporal temperature gradient that increases gradually in a linear fashion over the length of the electrophoresis (Fig. 1). TTGE differs from TGGE, which has been
reported several times, in that TGGE has a fixed temperature gradient from top to bottom of the gel (6 ). In TTGE,
the temperature at any location of the entire gel is the
same at any given time but changes with respect to time
(temporal temperature). Thus, it is easier to modulate the
temperature over time and provide a wider separation
range that increases sensitivity. TTGE, a modified highthroughput form (parallel form) of DGGE, is much more
robust and has a broader separation range than DGGE.
Thus, several fragments with different melting behaviors
can be analyzed on the same TTGE gel. TTGE does not
require the preparation of a chemical denaturant gradient
gel and can be performed without a GC clamp.
Recently, TTGE has been shown to be a powerful tool
for the detection of novel nuclear DNA mutations (8 –10 ).
In the study reported here, we first evaluated the sensitivity and specificity of TTGE in the analysis of previously
identified mtDNA homoplasmic and heteroplasmic mutations. We then used TTGE to search for unknown
mtDNA mutations. Our results demonstrate that heteroplasmic and homoplasmic mtDNA mutations can easily
and effectively be detected and distinguished from one
another by TTGE. Further testing and sequence confirmation of the mutations can be performed only on the
1164
Chen et al.: mtDNA Analyzed by TTGE
heteroplasmic cases, greatly reducing the time and effort
spent on the investigation of potentially benign polymorphic homoplasmic mutations.
Materials and Methods
Patients suspected of mtDNA disorders were referred to the
molecular genetics laboratory at Children’s Hospital Los
Angeles for mutational analysis according to protocol 90117, approved by the Committee on Clinical Investigations.
DNA was extracted from peripheral blood lymphocytes according to published procedures and stored at
4 °C (11 ). The presence of large deletions and 12 common
point mutations were studied by standard Southern analysis and the PCR/allele-specific oligonucleotide method
(3, 12 ). The percentage of heteroplasmic mutations was
determined by the PCR/restriction fragment length polymorphism method as described previously (13 ). One
hundred nine samples with 15 different known mutations
and various percentages of heteroplasmy were chosen for
the evaluation of potential utility of TTGE method.
From .2000 patients who tested negative for the
routine analysis to exclude the presence of the most
common heteroplasmic mutations—A3243G, A8344G,
G8363A, T8993C, and T8993G (3, 14 )—104 patients were
selected for TTGE analysis. The selection criteria were
based on strong indications of mitochondrial disorders
from other studies, such as clinical presentations, biochemical assays, histochemistry of muscle biopsies, and
pedigree analysis.
Regions of mtDNA were PCR amplified, followed by
TTGE analysis. Each region was named based on the
tRNAs located within it, although the majority of each
region consists of protein-coding genes. The primers used
in PCR amplification and the temperature range of the
TTGE analysis are listed in Table 1. Each 100-mL PCR
reaction mixture contained 13 Promega PCR buffer (50
mmol/L KCl, 10 mmol/L Tris-HCl, pH 9.0, 1 mL/L
Triton X-100), 1.5 mmol/L MgCl2, 0.2 mmol/L each
dNTP, 0.5 mmol/L each primer, 1 U of Taq DNA polymerase (Promega), and 100 ng of genomic DNA. The
reaction mixture was denatured at 94 °C for 4 min,
followed by 30 cycles of 30 s of denaturation at 94 °C, 45 s
of reannealing at 55 °C, and 45 s of extension at 72 °C. The
PCR reaction was completed by a final extension cycle at
72 °C for 4 min. PCR products were denatured at 95 °C for
30 s and slowly cooled to 45 °C for a period of 45 min at
a rate of 1.1 °C/min. The reannealed homoduplexes and
heteroduplexes were kept at 4 °C until being loaded onto
the gel. TTGE was performed on a Bio-Rad D-Code
apparatus. Two back-to-back 20 cm 3 20 cm 3 1 mm 6%
polyacrylamide (acrylamide:bis ratio, 37.5:1, by weight)
gels were prepared in 1.253 Tris-acetate-EDTA buffer
containing 6 mol/L urea. Denatured and reannealed PCR
product (5 mL) was loaded onto the gel. The electrophoresis was carried out at 145 V for 6 –7 h at a constant
temperature increment of ;1.2 °C/h as shown in Table 1.
The temperature range was determined by computer
simulation (MacMelt software; Bio-Rad Laboratories). The
gels were stained in 2 mg/L ethidium bromide for 5 min
and imaged with a digital CCD gel documentation system. Confirmation of the nucleotide alteration was performed by direct DNA sequencing of the PCR product,
using a dye terminator cycle sequencing kit (PerkinElmer) and an ABI 373A or 377 (Applied Biosystems)
automated sequencer. To detect low-percentage mutant
mtDNA by sequencing, the homoduplex mutant or the
heteroduplex bands were excised from the TTGE gel and
PCR amplified before sequence analysis. Once the mutation was identified by sequencing, a second method such
as PCR/allele-specific oligonucleotide dot blot or PCR/
restriction fragment length polymorphism analysis was
used to confirm the status of homoplasmy or heteroplasmy (13, 14 ).
Results
The ability of TTGE to detect known heteroplasmy was
demonstrated by the analysis of different percentages of
the mutant A3243G heteroplasmy. As shown in Fig. 2,
wild-type mtDNA (lanes 1 and 11) consistently produces
a sharp single band, whereas homoplasmic sequence
Table 1. Summary of analyzed mtDNA fragments and TTGE conditions.
Nucleotide
positions
Fragment
size, bp
L
3116–3758
643
IQM
4013–4508
496
WANCY
5451–6016
566
K
7804–8380
577
ATPase8/6
8334–9199
866
11688–12360
673
Regions
HSL
a
F, forward; R, reverse.
PCR primers
F:a CCTCCCTGTACGAAAGGAC
R: AGTAGAATGATGGCTAGGGTGAC
F: CCCTCACCACTACAATCTT
R: GATGGTAGAGTAGATGACG
F: ACACTCATCGCCCTTACCA
R: CGAATAAGGAGGCTTAGAG
F: AGTCCTCATCGCCCTCCC
R: ATTTAGTTGGGGCATTTCACTGTA
F: GATTAAGAGAACCAACACCTCTTTACGGT
R: TGTCGTGCAGGTAGAGGCTT
F: CCGGCGCAGTCATTCTCA
R: GGTTATAGTAGTGTGCATG
Temperature range for
TTGE, °C
56–64
54–58
54–60
54–60
53–62
53–61
Clinical Chemistry 45, No. 8, 1999
1165
Fig. 2. Detection of the heteroplasmic A3243G
mutation.
PCR products of 643 bp (nucleotides 3116 –3758) containing the tRNALeu (UUR) and parts of the 16S rRNA and ND1
regions were analyzed. Lanes 1 and 11, wild type; lane 2,
homoplasmic T3197C; lane 3, homoplasmic T3197C with
47% heteroplasmy for A3243G; lanes 4 –10, different proportions of A3243G mutant DNA. The numbers under each
lane indicate the percentages of mutant A3243G mtDNA.
variations produce a shifted band (Fig. 2, lane 2). Under
optimal conditions, a single heteroplasmic mutation produces four bands representing the wild-type and mutant
homoduplex bands (Fig. 2, lower two bands of lanes 3–10)
and two wild-type/mutant heteroduplex bands (Fig. 2,
upper two bands of lanes 3–10). As can be seen in Fig. 2,
the intensities of the two heteroduplex and the mutant
homoduplex bands increase as the percentage of the
mutant A3243G mtDNA increases from 4% to 62% (Fig. 2,
lanes 4 –10). Mutant heteroplasmy as low as 4% was
detected. The detection of the heteroplasmic A3243G
mutation in the presence of a homoplasmic T3197C background is also illustrated, where the wild-type homoduplex and all other bands are down-shifted (Fig. 2, lane 3).
Fig. 3 shows the banding shift of homoplasmic single
nucleotide substitutions in two different regions of
mtDNA. These results clearly display the power of TTGE
to detect and distinguish homoplasmic and heteroplasmic
mutations as well as to identify heteroplasmy in the
presence of a homoplasmic polymorphism.
To further establish the utility of TTGE, 109 samples
with 15 different known mutations in 6 different regions
of mtDNA were analyzed. The percentage of mutant
mtDNA in the samples with heteroplasmy varied from
4% to 95%. The results summarized in Table 2A demonstrate that every one of these known mutations was
detected and correctly identified as either homoplasmic or
heteroplasmic. Each mutation showed a distinct pattern
on TTGE that is reproducible on repeat analysis.
TTGE was also applied to detect unknown mutations
in a patient population as described in Materials and
Methods. In each of these 104 cases, a mitochondrial
disorder was suspected to various degrees by the referring clinician. All samples had tested negative for large
deletions and common point mutations. Three regions,
the L, K, and WANCY regions, representing ;11% of the
entire mitochondrial genome were screened by TTGE.
Table 2B summarizes the results. Homoplasmic sequence
variations (shifted single band) was detected in 11 patients. Seven samples repetitively showed multiple bands,
suggesting the presence of heteroplasmy.
Outside the hypervariable D-loop region, heteroplasmy is believed to be rare and is more likely to be
associated with disease. Thus, heteroplasmy should occur
at a higher frequency in a patient population than in the
general population. This hypothesis was tested by the
analysis of DNA samples from 50 individuals with phenylketonuria, a disorder unrelated to mtDNA mutations.
All 50 samples showed a single band in each of the
previous three different mtDNA regions (Table 2B), demonstrating the absence of heteroplasmy. Two samples did
show a shifted single band, a homoplasmic sequence
variation pattern, which is consistent with the known
high frequency of polymorphisms in mtDNA. However,
multiple banding patterns, which were detected in 7% of
the patients when 11% of the mtDNA regions were
analyzed, were not detected in any of the 50 phenylketonuria patients. These data suggest that heteroplasmic
Fig. 3. Detection of homoplasmic mtDNA variants.
PCR products from the L (643 bp) and IQM (496 bp) regions
were analyzed by TTGE according to the conditions described in Table 1. Homoplasmic mtDNA variants, G3315A
and G3460A (L region) and T4216C and T4336C (IQM
region) showed a single band shift on TTGE gel.
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Chen et al.: mtDNA Analyzed by TTGE
Table 2. Summary of mutational analysis by TTGE.
A. Patients with known mtDNA mutations tested by TTGE
Base substitution
T3197C
A3243G
A3243G 1
T3197C
G3315A
T3394C
G3460A
T4216C
A4317G
T4336C
A8344G
G8363A
T8993C
T8933G
G11778A
A12308G
T12311C
Total
Homoplasmy or
heteroplasmy
Polymorphism or mutation
(phenotype)
No. of patients
tested
Homoplasmy
Heteroplasmy
Homo1hetero
Polymorphism
Mutation (MELAS)b
Mutation, polymorphism
Homoplasmy
Homoplasmy
Homoplasmy
Homoplasmy
Homoplasmy
Homoplasmy
Heteroplasmy
Heteroplasmy
Heteroplasmy
Heteroplasmy
Homoplasmy
Homoplasmy
Homoplasmy
Polymorphism
Polymorphism
Mutation, (LHON)d
Mutation (LHON)
Disease associatione
Disease associatione
Mutation (MERRF)b
Mutation (cardiomyopathy)
Mutation (NARP)d
Mutation (NARP)
Mutation (LHON)
Polymorphism
Disease associatione
No. of patients with
positive results
10
30
1
10
30
1
5
4
1
10
1
7
12
6
10
1
1
9
1
109
5
4
1
10
1
7
12
6
10
1
1
9
1
109
Number of bands
detected
Singlea
Multiplec
Shifted, multiplec
Singlea
Singlea
Singlea
Singlea
Singlea
Singlea
Multiplec
Multiplec
Multiplec
Multiplec
Singlea
Singlea
Singlea
B. Patients with various phenotypes and unknown mutations detected by TTGE
R/O mitochondrial disorders
Phenylketonuria
Area amplified
Region
name
No. of patients
tested
No. of patients
shift banda
No. of patients
mult. bandc
No. of patients
tested
No. of patients
shift bandc
No. of patients
mult. bandc
3116–3758
5451–6016
7804–8380
Lf
WANCYg
Kh
104
104
104
5
3
3
1
3
3
50
50
50
2
0
0
0
0
0
Single band on gel shifted relative to control 5 homoplasmy sequence variation pattern.
MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; homo, homoplasmy; hetero, heteroplasmy; LHON, leber hereditary optic
neuropathy; MERRF, myoclonic epilepsy and ragged red fibers; NARP, neuropathy, ataxia, and retinitis pigmentosa; mult., multiple.
c
Multiple bands on gel 5 heteroplasmy pattern.
d
In LHON, homoplasmic mutations lead to adult-onset visual loss. NARP is a childhood-onset mitochondrial disorder.
e
A4317G associated with FICP (fatal infantile cardiomyopathy); T4336C associated with increased risk of Alzheimer/Parkinson disease; T12311C associated with
LIMM (lethal infantile mitochondria myopathy).
f
L region includes the transcription terminator, parts of 16S rRNA and ND1 genes, and t-RNA for Leu.
g
WANCY region includes part of ND2 gene, the light strand origin of replication, and t-RNAs for Trp, Ala, Asn, Cys, and Tyr.
h
K region includes part of COXII gene and t-RNA for Lys.
a
b
mtDNA mutations are rare in controls and, when present,
are likely disease causing.
Some of our TTGE results for the K and WANCY
regions in the patient population are shown in Fig. 4. Each
heteroplasmic mutation (Fig. 4, lanes 2– 6) demonstrates a
distinct TTGE pattern. Theoretically, a single heteroplasmic mutation in the DNA fragment to be analyzed would
produce four bands on TTGE. However, depending on
the melting behavior of the mutant DNA fragment, the
location of the mutation in the fragment, and the TTGE
conditions, it is possible to have three or two bands as
shown in Fig. 4. To identify the mutations, mutant bands
were excised from the TTGE gel, PCR amplified, and
sequenced. The samples in lanes 3– 6 were identified as
having the heteroplasmic mutations of T8300C, nt8042del
AT, C5499T, and A5951G, respectively. The mutations
were further confirmed by either PCR/allele-specific oli-
gonucleotide or PCR/restriction fragment length polymorphism analysis. Other heteroplasmic variations detected by TTGE are being confirmed by sequencing
analysis. The nt8042del AT mutation in the gene encoding
the cytochrome C oxidase II subunit produces a truncated
protein that is 68 amino acids shorter than normal. This
mutation is thus predicted to be detrimental. The quantification of the heteroplasmy and the biochemical and
clinical significance of these novel mutations will be
described elsewhere.
Discussion
In two recent reports (15, 16 ), DGGE was used as a rapid
and sensitive method for the exhaustive scanning of
mtDNA mutations. A narrow- or broad-range denaturant
gradient parallel to the direction of electrophoresis was
required (15 ). In addition, at least 15 pairs of GC-clamped
1167
Clinical Chemistry 45, No. 8, 1999
TTGE is suitable for the mutational screening of the entire
mitochondrial genome in large patient populations. The
existence of a cost-effective screening assay for mtDNA
mutations would have high utility considering the high
variability in the clinical presentation of these disorders.
As an example of the potential usefulness of such screening assay, in this study, 7 specimens with heteroplasmy
were identified from 104 patients by scanning only 11% of
the mitochondrial genome. Our data suggest that mtDNA
heteroplasmy may be more common than previously
reported. As a heteroplasmy screening assay, TTGE may
also be a suitable screening technique for other applications such as in evolutionary studies and forensics.
References
Fig. 4. Detection of unknown mutations in mtDNA.
PCR products from the K (lanes 1– 4) and WANCY (lanes 5–7) regions, 577 bp
and 566 bp in size, respectively, were analyzed by TTGE. Lanes 1 and 7, wild
type; lanes 2–5, various heteroplasmic mutations; lane 6, possible multiple
heteroplasmic mutations.
primers were synthesized, with most PCR fragments of
;200 –300 base pairs in size. In TTGE, which is based on
the principle of DGGE, the denaturant gradient is
achieved by temporal temperature gradient, thus eliminating the inconvenience of preparing the chemical denaturant gradient gel. We also improved the method by
increasing the fragment size up to 1 kb and eliminating the
use of GC clamps. Thus, TTGE is simple and more costeffective without sacrificing sensitivity. The separation range
of TTGE is flexible and can be easily regulated by adjusting
the temperature range (narrow or broad) and ramp.
Heteroplasmy is characteristic of pathogenic mtDNA
mutations. An apparent TTGE heteroplasmic pattern
may, however, be the result of blood transfusion or bone
marrow transplantation. Therefore, analysis of a second
blood specimen or a tissue specimen other than blood is
necessary to confirm the finding.
There are two major goals in molecular diagnosis of
mtDNA disorders. The first is to detect any sequence
variations at any position of the mtDNA genome. The
second is to determine whether the sequence variation is
homoplasmic or heteroplasmic. Our data demonstrate
that TTGE accomplishes both of these goals; it is both
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detected as close as 29 nucleotides from the end of a
fragment. At the present configuration, as many as 50
samples can be analyzed simultaneously for fragments up to
1 kb in length, and the entire procedure can be completed
within 1 working day.
By screening for the presence of heteroplasmy and
ignoring the frequent homoplasmic polymorphisms,
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