Download Expression of the Mitochondrial ATPase6 Gene and Tfam in Down

Mol. Cells, Vol. 15, No. 2, pp. 181-185
M olecules
and
Cells
KSMCB 2003
Expression of the Mitochondrial ATPase6 Gene and Tfam in
Down Syndrome
Sook Hwan Lee1, Suman Lee1, Hye Sun Jun1, Hye Jin Jeong1, Won Tae Cha2, Yong Sun Cho1,
Jung Hwan Kim1, Seung Yup Ku*, and Kwang Yul Cha1
Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul 110-744, Korea;
1
Department of Obstetrics and Gynecology, Human Genetics Laboratory, CHA Infertility Medical Center, College of Medicine,
Pochon CHA University, CHA General Hospital, Seoul 135-081, Korea;
2
Duke University, Durham, USA.
(Received September13, 2002; Accepted November 11, 2002)
We investigated the expression of the mitochondrial
ATPase6 gene whose product is active in oxidative
phosphorylation (OXPHOS), and compared it to the
expression of Tfam, an important regulator of the
transcription and replication of mtDNA. Our aim was
to examine a possible relation between mitochondrial
gene expression and Down syndrome. The expression
of ATPase6 and Tfam was analyzed by RT-PCR amplification of the mRNA in cultured amniocytes from
Down syndrome and normal fetuses. The band intensities obtained were normalized against those of HPRT.
The Down syndrome fetuses were found to have lower
ATPase6 and Tfam expression than the normal fetuses.
This finding suggests that mitochondrial dysfunction
resulting from decreased ATPase6 and Tfam expression during meiotic oocyte maturation of oocytes
might affect ATP generation and cause the nondisjunctional error. Hence this study suggests that mitochondrial dysfunction may be associated with the developmental mechanism of Down syndrome.
Keywords: ATPase6; Down Syndrome; Mitochondria;
Tfam.
Introduction
Down syndrome is the most common autosomal trisomy
among live births. In more than 95% of the cases studied,
Down syndrome is caused by nondisjunction at meiotic
division. Most cases are maternally inherited since 91.5%
Demo
* To whom correspondence should be addressed.
Tel: 82-2-760-1971; Fax: 82-2-762-3599
E-mail: [email protected]
of the extra chromosomes are of maternal origin (Nicolaidis et al., 1998). It has therefore been suggested that
the risk of Down syndrome is closely linked to maternal
age. In the past the association of Down syndrome with
age was explained by relaxation of selection against
trisomic fetuses in older women, but recently there have
been suggestions that it is due to meiotic error caused by
the aging of oocytes. However, the exact mechanism has
not yet been determined. The incidence of Down syndrome increases dramatically in children born to women
35 years of age and over, but 75% of Down syndrome
children are born to mothers under the age of 35 (Jorde et
al., 1999).
Mitochondrial DNA consists of a 16.5-kb doublestranded circular molecule, which is self-replicating and
maternally inherited. Mitochondria provide the ATP for
all energy-requiring cellular activities through the oxidative phosphorylation (OXPHOS) pathways (Zeviani et al.,
1997). Deletion, mutation or replication abnormalities of
mitochondrial DNA (mtDNA) due to genetic abnormalities, hypoxia, oxidative stress or old age can cause mitochondrial dysfunction. In fact, deletions in mtDNA occur
frequently in the oocytes of women of advanced reproductive age (Keefe et al., 1995). Mitochondrial dysfunction in the oocytes and early embryo can influence ATP
generation, which in turn can cause aberrant chromosomal
segregation or developmental arrest (Van Blerkom et al.,
1998).
On the basis of these reports we hypothesized that when
there is mitochondrial dysfunction during oocyte meiotic
maturation, aneuploidies such as Down syndrome may
occur because insufficient energy is available for spindle
assembly. In this study, we investigated the involvement
of the mitochondrial ATPase6 gene in OXPHOS, and
compared it with the expression of Tfam, an important
182
Mt ATPase6 Gene and Tfam Expression in Down Syndrome
regulator of the transcription and replication of mtDNA,
in Down syndrome and normal fetuses.
Table 1. Oligonucleotide primer sequence and PCR products size.
Materials and Methods
ATPase6 (MIT3)
Patients Amniocytes were obtained from 20 patients with 21
trisomy, and 21 patients with normal karyotype, from January
1998 to June 1999 in the Human Genetics Laboratory of CHA
General Hospital. The average gestational week at the time of
amniocentesis of the Down syndrome and normal fetuses was
19+2 weeks and 18+2 weeks respectively. The average ages of the
mother was 33.9 years and 33.8 years, respectively, and 11 of
the mothers in each group were below 35. All of the patients
gave informed consent and the study was approved by the Institutional Review Board of our hospital.
Isolation of RNA mRNA from cultured amniocytes of the 20
Down syndrome and 21 normal cases was carried out with Trizol LS Reagent (Invitrogen, USA) which is prepared by the acid
phenol-guanidium isothiocyanate-chloroform method (Chomzyncski and Sacci, 1987). The cells were collected directly into
a culture flask by adding 0.5 ml of Trizol LS Reagent. 0.1 ml of
chloroform (Sigma, USA) was added to the lysed cells. The
tubes were vigorously shaken by hand for 15 seconds and incubated at room temperature for 10 min. The samples were then
centrifuged at 12,000 × g for 15 min at 4oC, the aqueous phase
transferred to a clean tube and 0.5 ml isopropanol (Sigma)
added to precipitate the RNA. The samples were further incubated
at room temperature for 10 min and centrifuged at 12,000 × g for
10 min at 4oC. Following this the RNA pellet was washed by
adding 75% ethanol, vortexed and centrifuged at 7,500 × g for 5
min at 4oC. The RNA was redissolved in 0.1% DEPC-water.
cDNA synthesis and PCR amplification of cDNATwo sets of
oligonucleotide primers for regions of the mitochondrial ATPase6 gene and one set for the Tfam gene region were designed
from published DNA sequences (Table 1). RT-PCR was performed using the SuperScript Preamplification System for First
Strand cDNA Synthesis (Invitrogen) with slight modifications.
Any DNA contaminating the mRNA was removed by treating
with 2 units of DNase I at room temperature for 15 min followed by addition of 25 mM EDTA and incubation at 65oC for
15 min.
The RNA that was added to 1 µl of oligo-dT primer had been
reverse transcribed in 20 µl aliquots of reaction mixture consisting of 0.5 mM dNTPs, 2.5 mM MgCl2, 0.01 M DTT, 10× PCR
buffer, and 200 units of reverse transcriptase. The reaction was
carried out for 50 min at 42oC, followed by heating at 70oC for
15 min. It was then incubated at 37oC for 20 min before treatment with 2 units of RNase H. Hypoxanthine-guanine phosphoribosyltransferase (HPRT), a uniformly expressed housekeeping
gene, was amplified along with the target genes to correct for
inter-sample variability. One aliquot of each reverse transcription product (20 µl) was amplified with the ATPase6 gene, Tfam,
Gene
Size of PCR
products (bp)
Sense
Antisense
283
Sequence of oligonucleotide
5′-CCATACACAACACTAAAGGACG-3′
5′-CGAAAGCCTATAATCACTGTGC-3′
ATPase6 (MIT6)
Sense
Antisense
293
5′-GAGGCCACAACTACCTCCTCG-3′
5′-CCTACTCATGCACCTAATTGGA-3′
Tfam
Sense
Antisense
226
5′-CCGAGGTGGTTTTCATCTGT-3′
5′-TATATACCTGCCACTCCGCC-3′
HPRT
Sense
Antisense
226
5′-GCCGGCTCCGTTATGGCG-3′
5′-AGCCCCCCTTGAGCACACAGA -3′
and HPRT primers with 2.5 units of Taq DNA polymerase
(Promega, Madison, WI), MgCl2 (1.5 mM), and dNTP (1 mM)
in the following amplification sequence: 94oC for 1 min, 55oC
for 1 min for the mitochondrial ATPase6 gene, or 60oC for 1
min for Tfam and 67oC for 2 min for HPRT; then at 72oC for 1
min (30 cycles), followed by a final extension at 72oC for 3 min.
Each PCR product was run through an agarose gel (2%) and
visualized by ethidium bromide staining. The relative abundance of the PCR products was determined using an Imaging
Densitometer (Model GS-700; Bio-Rad Hercules, USA), and the
results expressed as the ratio of the 2 regions of the mitochondrial ATPase6 gene and Tfam divided by the HPRT gene.
Results
Due to the similar age and gestational period of the mothers in the groups investigated (see above), we could exclude any influence of age on the frequency of mitochondrial deletions. Our analysis showed that the agarose gel
bands were of lower intensity in the case of the Down
syndrome fetuses compared to the normal fetuses (Figs. 1
and 2). The band size of the 2 regions of the ATPase6
gene and the Tfam region were as follows: MIT3 (283 bp),
MIT6 (299 bp) and Tfam (261 bp). The mRNA of each
sample was not quantified, and the variability between
samples was corrected by means of the internal control
using the housekeeping gene, HPRT (226 bp). The band
intensity of the RT-PCR products was quantitatively assayed using an imaging densitometer and the level of expression calculated as the ratio of MIT3, MIT6, and Tfam
to HPRT.
The Down syndrome fetuses showed decreased ATPase6 gene and Tfam expression compared to the normal
Sook Hwan Lee et al.
Table 2. Densitometric analysis of RT-PCR in Down syndrome
fetus (n = 20) and normal fetus (n = 21).
183
Table 3. Densitometric analysis of RT-PCR in Down syndrome
fetus (n = 11) and normal fetus with < 35 age (n = 11).
Gene
Down syndrome (A.U.)
Normal (A.U.)
Gene
Down syndrome (A.U.)
Normal (A.U.)
MIT3
MIT6
Tfam
HPRT
1.90 ± 0.89
2.33 ± 1.79
0.55 ± 0.29
1
2.69 ± 2.25*
3.51 ± 3.60*
1.10 ± 1.02*
1
MIT3
MIT6
Tfam
HPRT
1.86 ± 1.00
2.19 ± 1.65
0.50 ± 0.27
1
2.94 ± 2.78*
4.04 ± 4.87*
0.86 ± 0.35*
1
All values are mean ± SD.
A.U. = arbitrary unit.
* P < 0.01, Down syndrome vs. Normal fetus.
The age range of Down syndrome group = 27−33 (mean: 30.18 ±
1.90).
The age range of control group = 27−34 (mean: 30.45 ± 2.10).
Total ATPase6 and Tfam mRNA expression was calculated against
the level of HPRT mRNA.
A. U.
All values are mean ± SD.
A.U. = arbitrary unit.
* P < 0.01, Down syndrome vs. Normal fetus.
The age range of Down syndrome group = 27−40 (mean: 33.9 ± 4.7).
The age range of control group= 27−40 (means: 33.8 ± 4.1).
Total ATPase6 and Tfam mRNA expression was calculated against
the level of HPRT mRNA.
Fig. 1. Expression of ATPase6, Tfam and HPRT mRNA in amniocytes of Down syndrome fetuses analyzed by RT-PCR. Lane
M, 100 bp DNA ladder; lane 1, MIT3; lane 2, MIT6; lane 3,
Tfam; lane 4, no-RT control; lane 5, HPRT.
MIT3
MIT6
Tfam
Fig. 3. Densitometric analysis of ATPase6 and Tfam mRNA
expression in amniocytes of Down syndrome and normal fetuses
(U, down; T, normal; A.U., arbitrary units).
Discussion
Fig. 2. Expression of ATPase6, Tfam and HPRT mRNA in amniocytes of normal fetuses analyzed by RT-PCR. Lane M, 100
bp DNA ladder; lane 1, MIT3; lane 2, MIT6; lane 3, Tfam; lane
4, no-RT control; lane 5, HPRT.
fetuses (Table 2 and Fig. 3). Mitochondrial gene expression in the Down syndrome fetuses was also lower than in
the normal fetuses in the age group below 35 years of age
(Table 3 and Fig. 4).
In this study we have shown that decreased ATPase6 gene
expression during oocyte meiotic maturation may reduce
the capacity for oxidative phosphorylation and influence
ATP generation, leading to chromosomal nondisjunction.
Furthermore, we have shown that decreased Tfam expression may cause nondisjunction because the required energy for spindle assembly has not been supplied. Therefore, our research suggests that mitochondrial dysfunction
due to various extrinsic or intrinsic influences can induce
aneuploidies such as Down syndrome.
Since mitochondrial DNA (mtDNA) can only be inherited through the maternal line, we hypothesized that mitochondrial gene expression measured in amniocytes, a fetal
tissue, may reflect mitochondrial gene expression during
meiotic division in oocytes.
Human mtDNA is a double-stranded, circular molecule
of 16,569 bp containing 37 genes coding for two rRNAs,
22 tRNAs and 13 polypeptides. The polypeptides are all
subunits of enzyme complexes of the OXPHOS. ATPase6
184
Mt ATPase6 Gene and Tfam Expression in Down Syndrome
MIT3
MIT6
Tfam
Fig. 4. Densitometric analysis of ATPase6 and Tfam mRNA
expression in amniocytes of Down syndrome and normal fetuses
(mothers < 35 years old) (U, down; T, normal; A.U., arbitrary
units).
and ATPase8 are subunits of complex V of the ATP synthase. The energy liberated in redox reactions is partially
stored as a transmembrane proton gradient generated by
active extrusion of protons from the inner mitochondrial
compartment, and ultimately utilized by complex V to
phosphorylate ADP to ATP. The entire process is known
as the OXPHOS, which supplies most of the ATP in the
cell (Taanman et al., 1999; Zeviani et al., 1997). Therefore, decreased ATPase6 gene expression coculd influence the generation of ATP (Lee et al., 2000).
Tfam is an important regulator of both transcription and
replication of mtDNA (Antoshechkin et al., 1997; Dairaghi
et al., 1995; Ghivizzani et al., 1994; Larsson et al., 1994;
Poulton et al., 1994). Moreover, it also regulates mtDNA
copy number. Heterozygous knockout mice have reduced
mtDNA copy number while in homozygous knockout
embryos mtDNA is depleted and OXPHOS eradicated
(Larsson et al., 1998). The low mtDNA copy number observed in the sperm cells may be directly due to decreased
expression of Tfam preventing paternal transmission of
mtDNA (Larsson et al., 1997). Thus, decreased Tfam expression in Down syndrome may reduce the capacity of
OXPHOS.
More than 95% of cases of Down syndrome are caused
by nondisjunction due to abnormal chromosomal segregation during stage I or II of meiosis. Mitochondria play an
important role in meiosis of mammalian oocytes. In an
experiment with mouse oocytes, mitochondria were seen
to translocate to the perinuclear region during formation
of the first metaphase spindle and to disperse subsequently during the release of the first polar body. These
rearrangements of mitochondria are regarded as essential
for the specification of localized activities of ATP at the
meiotic spindle. In order to facilitate development, a large
supply of ATP is required (Van Blerkom et al., 1984,
1995; 1997; 1998).
According to Van Blerkom et al. (1998), mitochondrial
dysfunction due to a variety of intrinsic and extrinsic influences can profoundly influence the level of ATP generation in oocytes and early embryos, and this in turn may
result in aberrant chromosomal segregation or developmental arrest (Hsieh et al., 2001). Chromosomal movements during meiosis are directed by microtubule assemblies within the spindle. According to Battaglia et al.
(1996), the spindle exhibited abnormal tubulin placement
and one or more chromosomes were displaced from the
metaphase plate during the second meiotic division in
79% of oocytes in an older age group under investigation.
In contrast, only 17% of the oocytes from a younger age
group exhibited aneuploidy. This indicates that regulatory
mechanisms responsible for the assembly of the meiotic
spindle are significantly altered in older women, leading
to a higher prevalence of aneuploidy (Battaglia et al.,
1996). By inhibiting mitochondrial function in mice with
chloramphenicol, Beermann and Hansmann (1986) demonstrated that defective mitochondrial function interferes
with ordered chromosome segregation during the first
meiotic division (Beerman et al., 1986; 1988).
Mitochondrial dysfunction leading to oxidative damage
and apoptosis, hypoxia, and deletion or point mutations in
the mitochondrial genome, especially in oocytes of older
women, are adverse influences that may contribute to reduced mitochondrial function in the human female gamete. Linnane et al. (1989) speculated that the accumulation of mtDNA mutations and the resultant reduction in
gene expression can cause organ dysfunction.
The Down syndrome mothers in the present study were
between the ages of 27−40, and their average age was
33.9 (± 4.7). Within this group, 9 mothers were aged 35 or
above while 11 were below the age of 35 (the average age
was 30.18 ± 1.90). Although maternal age is strongly correlated with the risk of Down syndrome, it should be
pointed out that approximately 75% of Down syndrome
children are born to mothers under the age of 35. This is
because the great majority of children (> 90%) are born to
women under 35 (Jorde et al., 1999). Therefore, not only
maternal age but also the biological mechanisms underlying chromosomal nondisjunction need to be identified. In
this study, the average maternal age of the Down syndrome fetuses and normal fetuses was 33.9 (± 4.7) and
33.8 (± 4.1) respectively. Despite the similarity in average
maternal age, the Down syndrome fetuses exhibited decreased mitochondrial ATPase6 gene and Tfam expression
when compared to the normal fetus. Furthermore, this was
true even in the group below 35 years of age. This means
that not only advanced maternal age but also intrinsic or
extrinsic factors that can damage the mitochondrial genome in the oocytes can cause aneuploidy. Thus, although
advanced maternal age is the only well documented risk
factor for maternal meiotic nondisjunction, mitochondrial
dysfunction may be the underlying mechanism of this age
Sook Hwan Lee et al.
effect.
Not only Down syndrome, but other autosomal trisomies involving extra chromosomes of maternal origin,
such as trisomy 13 (88.1%), 15 (88.2%), 16 (100%), and
18 (91.5%) are closely related to advanced maternal age
(Nicolaidis et al., 1998). Therefore, the correlation between autosomal trisomies and the mitochondrial genome
should be further evaluated. Moreover, just as mitochondrial transfer between oocytes can increase pregnancy rate
by increasing mitochondrial function, so ooplasmic transfer may increase the possibility of a having a healthy baby
in an ensuing pregnancy in older women who harbor the
risk of Down syndrome. More research is required to
evaluate this possibility.
In conclusion, we have shown that mitochondrial dysfunction may be associated with the developmental
mechanism of Down syndrome. Mitochondrial dysfunction resulting from decreased ATPase6 gene and Tfam
expression during oocyte meiosis could affect ATP generation and cause nondisjunctional error.
Acknowledgments This study was supported by the Genome
Research Center for Reproductive Medicine and Infertility (01PJ10-PG6-01GN13-0002) from the Ministry of Health and Welfare, Korea.
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