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BMC Cancer
BioMed Central
Open Access
Research article
Control region mutations and the 'common deletion' are frequent
in the mitochondrial DNA of patients with esophageal squamous
cell carcinoma
Christian C Abnet*1, Konrad Huppi1, Ana Carrera2, David Armistead2,
Keith McKenney2, Nan Hu1, Ze-Zong Tang3, Philip R Taylor1 and
Sanford M Dawsey1
Address: 1Cancer Prevention Studies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA, 2Clearant, Inc.,
401 Professional Drive, Gaithersburg, MD 20897, USA and 3Shanxi Cancer Hospital, Taiyuan, Shanxi Province, 030013, People's Republic of
China
Email: Christian C Abnet* - [email protected]; Konrad Huppi - [email protected]; Ana Carrera - [email protected];
David Armistead - [email protected]; Keith McKenney - [email protected]; Nan Hu - [email protected]; ZeZong Tang - [email protected]; Philip R Taylor - [email protected]; Sanford M Dawsey - [email protected]
* Corresponding author
Published: 01 July 2004
BMC Cancer 2004, 4:30
doi:10.1186/1471-2407-4-30
Received: 06 November 2003
Accepted: 01 July 2004
This article is available from: http://www.biomedcentral.com/1471-2407/4/30
© 2004 Abnet et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
Abstract
Background: North central China has some of the highest rates of esophageal squamous cell
carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA)
accumulates more mutations than nuclear DNA and because of its high abundance has been
proposed as a early detection device for subjects with cancer at various sites. We wished to
examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area
of China.
Methods: We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and
blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23
healthy persons. We completely sequenced the control region (D-Loop) from each of these
samples and used a PCR assay to assess the presence of the 4977 bp common deletion.
Results: Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55%) tumor samples had
mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and
the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged
from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp
'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined,
whereas no evidence of the common deletion was found in corresponding peripheral blood
samples.
Conclusions: Control region mutations were insufficiently common to warrant attempts to
develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly
prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential
utility of the common deletion in an early detection system will be pursued in further studies.
Page 1 of 8
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BMC Cancer 2004, 4:30
Background
The population of north central China is at very high risk
for ESCC with age standardized incidence rates > 125/100
000 per year [1]. Cumulative mortality attributed to
esophageal cancer is approximately 20% for women and
25% for men. The cause of these extraordinary rates
remains unknown, but previous studies suggest that age,
family history [2,3], selenium deficiency [4], and tooth
loss [5] are associated with higher risk of esophageal cancer in this population. Tobacco and alcohol use, the leading risk factors for ESCC in Western countries, have only a
minor role in this population [6].
Typically, there are 100–1000 mitochondria per cell and
each mitochondrion carries 1–10 copies of the mitochondrial genome. Thus there are 100–10,000 times as many
mtDNA genomes as there are nuclear genomes per cell.
The mitochondrion can repair DNA damage through base
excision repair but lacks nucleotide excision repair [7].
Mitochondrial DNA is not protected by histones and the
energy generating capacity of the mitochondrion produces high levels of potentially damaging reactive oxygen.
Therefore, the higher abundance of mtDNA, the reduced
DNA protection, and the limited DNA repair capacity
make mtDNA a potentially useful sensor for cellular DNA
damage and marker for development of cancer whether
these mutations are implicated in the disease process or
not.
Mitochondrial DNA from solid tumors or hematologic
malignancies often carries acquired alterations [8]. The
detection of mutated mtDNA in body fluids [9] and fine
needle aspirates [10] suggests that these changes could
serve as disease markers. Somatic mtDNA mutations have
been found in colorectal, head and neck, esophageal, gastric, bladder, ovarian, and breast cancers among others.
Many of the detected changes occur within the non-coding control region (CR; also known as the D-loop) of the
mitochondrial genome. A study of ovarian cancer found
that 60% of tumors had at least one mtDNA mutation,
with 33% of the mutations in the CR [11]. In one recent
breast cancer study, 74% of tumor samples had at least
one acquired mutation and 81% of the mutations identified were within the CR, demonstrating that this region of
the mitochondrial genome is much more susceptible to
mutation than the coding region [12].
In addition to alterations in the CR, several studies have
examined the 4977 bp 'common deletion' of the mitochondrial genome in cancer and in degenerative diseases.
This somatic mutation appears to accumulate with age, in
tumors, and in tissue under other forms of stress, such as
liver cirrhosis [13]. A study of gastric cancer demonstrated
that 26/32 (81%) of gastric tumors harbored either a CR
http://www.biomedcentral.com/1471-2407/4/30
alteration or the common deletion in tumor tissue
mtDNA [14].
A single case-series has examined mitochondrial DNA
alterations in ESCC [15]. This study was conducted in
Japan, a population at moderate risk for ESCC. The
authors reported that only 2/37 (5%) of ESCC tumors
harbored CR mutations. In contrast, a recent analysis
restricted to the two hypervariable regions of the D-loop
found that 13/38 (34%) of ESCC tumors in a Japanese
series had acquired mutations [16]. A study of esophageal
adenocarcinoma in Germany found 8/20 (40%) had CR
alterations in the tumor or tumor-associated Barrett's epithelium [17]. Esophageal adenocarcinoma has a distinct
etiology and is primarily thought to result from reflux of
stomach or duodenal contents into the esophagus [18].
No published studies have examined the common deletion in esophageal cancer tissue.
Methods
To determine the frequency of CR alterations and the
4977 bp common deletion in ESCC, we examined mtDNAs of tumor and adjacent endoscopically normal
esophageal tissues and blood from 21 ESCC cases accrued
at Shanxi Cancer Hospital in Taiyuan, Shanxi Province,
People's Republic of China in 1995 and 1996, using direct
DNA sequencing and PCR. For cases, samples of tumor
and normal-appearing squamous esophageal tissue were
snap-frozen in liquid nitrogen immediately after removal
of the esophagus. Frozen tissue was ground to a powder
and total DNA (nuclear and mitochondrial) was extracted
using standard methods. Also, representative pieces of
tumor tissue were fixed in formalin, embedded in paraffin, sectioned, and stained with H&E for confirmation of
diagnosis. Subjects with tumors without significant
inflammation and sections that contained mostly tumor
cells were selected to reduce contamination from inflammatory cell and normal cell mtDNA. From both cases and
a healthy reference group of 23 healthy individuals, 10 ml
of venous blood was collected and total DNA was
extracted using standard methods. The Institutional
Review Boards of the Shanxi Cancer Hospital and the U.S.
National Cancer Institute approved this study.
The mitochondrial control region from each sample was
amplified by PCR (forward primer 5'-CTAATACACCAGTCTTGTAAACC-3'; reverse primer 5'-GGTGATGTGAGCCCGTCTAAAC-3') and sequenced with BigDye
terminator chemistry (Applied Biosystems) and the ABI
PRISM 377 genetic sequencer (Applera Corporation).
Eight primers, four forward and four reverse (all primer
sequences can be obtained from the corresponding
author), were used to sequence the relatively short stretch
to ensure high quality factors. The first set of four primers
used spanned the CR. The second set of four primers were
Page 2 of 8
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BMC Cancer 2004, 4:30
used to span the homopolymer region, which tends to be
longer in Asian individuals and tends to cause more
sequencing errors. DNA sequences were assembled using
the STADEN Gap4 program.
To identify point mutations, insertions, and deletions in
the CR of the tumor tissues, we compared each of the case
subjects CR sequence from tumor DNA to their sequence
from blood DNA. We ensured accurate comparisons by
completing short tandem repeat analysis (STR) on all
samples using ABIAmpFlSTR Profiler Plus PCR STR multiplex assay on an ABI 310 genetic analyzer (Applera Corporation). Profiles were determined using Genescan 3.1.2
and Genotyper 2.5.
To assess the presence of the common deletion, we
employed a modification of the method of Maximo [19].
Two pairs of forward and reverse PCR primers were
designed such that one (Mitin2) produced a 271 bp
amplicon from intact mitochondrial DNA in the region of
cytochrome oxidase subunit III (Mitin2F 9500–9520;
Mitin2R 9761–9742) and the other (Mitout2) produced a
5,190 bp amplicon spanning the region between ATPase
8 and NADH Dehydrogenase Subunit 5 in wild type
mtDNA (Mitout2F 8370–8392; Mitout2R 13560–
13539). The Mitin2 amplification product, which should
be present in all samples, was used as an amplification
control. If the 4977 bp common deletion was present, the
Mitout2 primers were positioned such that a 213 bp fragment was generated from the fusion product. Standard
PCR conditions were used (55°C annealing) following
the manufacturer's conditions (Supermix, Invitrogen).
Products were examined following 35 cycles of PCR
amplification by electrophoresis in 1.0% low melt agarose
gels. The identities of the amplified products were confirmed through cloning the PCR products, sequencing the
inserts, and comparing them to the appropriate mtDNA
sequence.
Results and Discussion
We compared each of the CR sequences from blood of the
healthy individuals and cases to the Revised Cambridge
Reference Sequence (12) and found a total of 285 variants
(polymorphisms) for the healthy group (median = 12 variants per subject, range 9–17) and 243 variants for the
case group (median = 11, range 7–18). The site and frequency of each novel variant or variant with a frequency ≥
5 is reported in Table 1. The number of variants was not
different between the two groups (Wilcoxon rank-sum
test, P = 0.33). We aligned all CR sequences from the
healthy individuals and determined a consensus sequence
for the group of 23 individuals. Next, we compared each
of the case CR sequences to this healthy consensus CR
sequence. Because mtDNA sequences show distinct
ethno-geographic patterns, we expected and found fewer
http://www.biomedcentral.com/1471-2407/4/30
variants in the comparison of our case sequences to this
local control group (median = 9, range 6–15) than in the
comparison to the Cambridge sequence. Analysis of this
limited data set using a phylogenetic tree (AlignX module,
Vector NTI Suite 7.0, Informax, Bethesda, MD) produced
no distinct groupings of cases separate from the healthy
subjects (data not shown).
To examine acquired mutations we compared the blood
sequence from each individual to the tumor and adjacent
normal CR sequences (Table 2). We found that 7/21
(33%) subjects had at least one acquired mutation. The
95% confidence interval around this proportion of 33%
was 17%–55% (logit transform). Of the 7 subjects with
alterations, 3 showed a single change while 4 (19% of the
total) showed greater than one acquired mutation. The
total numbers of acquired mutations for these four samples were 2, 2, 8, and 16. The 16 changes in a single subject represent a mutation frequency of 1.4% of the total
sequenced bases. The 33% rate of mutation that we found
is comparable to some of the findings for other solid
tumors [9,10], but is higher than a previous report for gastric cancer [20]. or one of two reports for ESCC [15]. In
contrast, a recent analysis restricted to the two hypervariable regions of the D-loop found that 13/38 (34%) ESCC
tumors in a Japanese series had acquired mutations [16].
We detected a clustering of acquired changes in two areas
of the CR (Figure 1A). First, 4 of the 7 subjects with alterations in their tumors had a change in the homopolymeric stretch of Cs that are assigned to position 309. This
region accounts for 22% of detected mutations across a
broad spectrum of tumors [21]. The homopolymer region
was also a site with a large number of variants in the blood
samples with 76% of ESCC cases and 96% of healthy individuals in our study having a polymorphism at this site.
Second, hypervariable segment one (HVS 1) was a
broader region that contained a concentration of alterations. HVS1, has previously been identified as a 'hotspot'
for both germline and somatic mutations [22], but the
functional significance of mutations in this region
remains unknown.
For each of the cancer cases we also sequenced the CR in
mtDNA isolated from adjacent normal squamous esophageal tissue. In most cases the normal esophageal tissue
sequence matched that of the blood (Table 2) and in no
case did we find sequence alterations that were restricted
to the adjacent normal tissue. Of the seven subjects with
CR alterations in their tumors, 3 had an adjacent normal
tissue CR sequence identical to blood and 4 shared some
of the mtDNA changes seen in the adjacent tumor. In
total, 11/31 (36%) of the CR alterations found in the
tumor samples were also present in the adjacent normal
tissue.
Page 3 of 8
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BMC Cancer 2004, 4:30
http://www.biomedcentral.com/1471-2407/4/30
Table 1: Novel and high frequency mtDNA polymorphisms detected in Shanxi, China.
Nucleotide Position
Base Change
Healthy Reference Samples (n = 23)
ESCC Case Samples (n = 21)
73
150
152
179
195
249
263
309
310–311
315
320
358
368
489
523–524
548
574
16155
16163
16172
16183
16223
16289
16298
16362
16519
16524
A-G
C-T
T-C
T-C*
T-C
A del
A-G
C-CC/CCC
TC del*
C-CC
C-T*
A-C*
A-G*
T-C
AC del*
C-T*
A-G*
A-T*
A-C*
T-C
A-C
C-T
A-C*
T-C
T-C
T-C
A-T*
21
10
6
1
5
4
21
16/6
1
19
2
2
0
12
6
0
0
1
1
2
6
19
0
4
9
12
0
21
4
5
0
2
5
21
12/4
0
21
0
0
1
14
7
1
1
0
0
6
5
13
1
5
7
9
1
Novel polymorphisms (denoted by *) were those not reported in the Mitomap database as of 6/01/2003. High frequency was defined as
polymorphisms present in ≥ 5 samples in either group. Nucleotide position was defined by the Cambridge reference sequence (GenBank NC
001807).
We found 8 instances of heteroplasmy when comparing
blood and tumor CR sequences. A typical example of heteroplasmy is presented in Figure 1B (from case 21 at position 16 164). In total, we saw 4 positions where
heteroplasmy was present in the tumor but not in the
blood and 4 positions where heteroplasmy was present in
the blood but not in the tumor. Case 18 showed a heteroplasmy in the normal adjacent tissue that was intermediate between the normal blood sequence and the tumor
sequence. Heteroplasmy of mitochondrial DNA mutations is typical, and several models have been developed
to explain this phenomenon [23,24].
Using a PCR assay, we investigated the presence or
absence of the 4977 bp common deletion in each of the
case DNA samples (Table 2). To control for the ability to
PCR amplify mtDNA, a primer set (Mitin2) that amplified
a 271 bp product within the common deletion region
(cytochrome oxidase III) was used as an amplification
control (Figure 1C). The common deletion was considered present if the Mitout2 primers produced a 213 bp
fusion product of the ATPase 8 and NADH subunit 5
genes. The common deletion was not detected in any of
the case blood samples. In contrast, the common deletion
was found in 19/20 (95%, 95% CI = 72%–99%) of the
adjacent normal samples and in 17/19 (89%, 95% CI =
66%–97%) of the tumor samples examined. Since we
were unsuccessful in amplifying Mitin2 in three samples,
we could not assess the presence of the common deletion
in those samples.
The 4977 bp common deletion has been reported in a
wide range of tumors, stressed tissues, and even normal
appearing tissue. Certain diseases of aging, such as ocular
myopathy, are commonly associated with an increase in
mtDNA mutations and these mutations are often found to
be clustered to regions such as the common deletion. A
detailed study of thyroid tumors demonstrated that 43/43
(100%) of Hürthle cell adenomas and carcinomas carried
the common deletion, while 0–33% of the adjacent
parenchyma carried the same change [25]. In our study we
found that 89% of tumors and 95% of the adjacent normal tissue carried the deletion. Our attempt to select
tumors with few normal cells does not preclude the possiPage 4 of 8
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BMC Cancer 2004, 4:30
http://www.biomedcentral.com/1471-2407/4/30
A
HVS1
HVS2
16024
16569/0
309
576
Mitochondrial DNA Control Region
B
19
T
AN
Ca
se
19
Ca
se
3
Ca
se
3
Ca
se
T
AN
Blood
T
2
Ca
se
Ca
se
2
T
18
18
AN
Ca
se
Ca
se
10
0
bp
la
dd
er
C
Tumor
AN
Adjacent Normal
Mitin
Mitout
Figure 1
A, Location of mtDNA CR mutations in 21 esophageal cancer cases
A, Location of mtDNA CR mutations in 21 esophageal cancer cases. 7/21 cases demonstrated CR mutations and a vertical bar
indicates position within the region. In total, 31 mutations are indicated. Numbers below the bar refer to the standard mtDNA
genome position numbering system. B, DNA sequencing electropherograms showing a heteroplasmy present at position
16164 in the blood DNA from case 21. Although a preponderance of G was found, a clear minority of the sequence was A as
seen in the tumor DNA. C, 1% agarose gel from the Mitin2/ Mitout2 assay for the 4977 bp common deletion. AN refers to
adjacent normal tissue and T to tumor tissue. A very faint band was present in the tumor sample from case 19.
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Table 2: mtDNA mutations detected in esophageal cancer cases in Shanxi, China.
Sample Age
Sex
Control Region Mutations
Nucleotide
Position
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
Case 10
Case 11
Case 12
Case 13
Case 14
Case 15
Case 16
Case 17
Case 18
56
64
47
55
54
59
58
53
66
50
55
62
49
59
65
59
50
65
F
M
F
M
F
M
M
M
M
F
M
F
M
F
M
M
M
F
Case 19
63
M
Case 20
46
F
Case 21
57
F
Cases (n = 21)
309
16148
309
309
16289
16293
16319
16185
16223
16256
16260
16266
16270
16298
16399
150
309
16067
16164
16171
16172
16182
16183
16184
16298
16362
16443
16470
16471
16473
16519
7/21 (33%)
Change Blood → Tumor
None
None
None
None
None
None
None
None
None
None
None
None
None
None
del C
C → T/c
del C
del C
C→A
A → G/a
G/a → A
C→T
C→T
T→C
C→T
C → G/c
T→C
T→C
G→A
T→C
del C
T/c → C
G/a → A
G/a → A
C→T
C→A
C→A
C→T
T → C/t
C→T
T→C
G→A
G→A
G→A
C→T
4977 bp 'Common' Deletion
Adjacent Normal
Sequence
C
C
C
C
A/c
A
A
T
T
C
T
C
C
C
A
T
C
T
G
G
C
C
C
C
T
C
T
G
G
G
C
Either Change
Adjacent
Normal
Tumor
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
ND
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
ND
Y
Y
Y
ND
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
ND
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
19/20 (95.0%)
17/19 (89.5%)
20/20 (100%)
Y = Yes, N = No, ND = No data, and X/x indicates heteroplasmy with the predominant base as a capital letter.
bility that the presence of the common deletion in tumor
samples was due to contamination from normal cells. The
high prevalence of the common deletion in normal tissue
is surprising and potentially useful as a risk marker, but
this remains to be determined. We did not have tissue
samples from persons with non-diseased esophagi for this
study and we cannot conclude that the presence of the
common deletion is limited to subjects with esophageal
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cancer. The cause of this ubiquitous deletion cannot be
assessed in our current study, but a possible link between
the mtDNA common deletion and DNA damage caused
by exogenous agents was investigated in a study of oral
cancer [26]. These investigators used a quantitative assay
to show that people who chew betel quid, which contains
multiple genotoxic substances, had an increased percentage of mtDNA carrying the common deletion in both
tumor and histologically normal oral tissue.
5.
Conclusions
6.
In summary, CR mutations were present in 33% of the
tumors we examined. Although we have not formally
tested the ability of CR mutations to serve as a cancer
marker, this rate does not appear to be sufficient to warrant attempts to develop a CR mutation assay as an early
detection method for ESCC. The incidence of the common deletion in the esophageal tissue of cancer patients
was very high (95%), making it a potential ESCC marker.
The absence of the common deletion in the peripheral
blood of the same patients suggests that exposure to carcinogens, aging, or both could be causing selectively
higher mutation in the esophagus. Further study of this
deletion in the esophageal tissue of healthy individuals
and those with squamous dysplasia, the precursor lesion
of ESCC, will help to determine whether it is directly associated with disease or is an age-related or exposure-related
phenomenon in the esophageal tissue of members of this
high risk population.
List of abbreviations
The abbreviations used are: bp; base pair; CI, confidence
interval; CR, control region; ESCC, esophageal squamous
cell carcinoma; HVS, hypervariable segment; mtDNA,
mitochondrial DNA.
2.
3.
4.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Competing interests
None declared.
17.
Author contributions
CCA designed and coordinated the study, tabulated and
analyzed the results, and drafted the manuscript. KH
designed and completed the common deletion assays and
drafted the manuscript. AC and DA completed the CR
sequencing and made the inter- and intra-subject comparisons. KM designed and supervised the CR sequencing
protocols and drafted the manuscript. NH and Z-ZT collected the samples, isolated the DNA, and participated in
drafting the manuscript. PRT and SMD oversaw study
design, completion, and interpretation and drafted the
manuscript.
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