Effect of a Novel Mutation in
5′-Regulatory Region of Aldose Reductase Gene
on Its Expression

XIE Ping, LI Qing-Jie, LIU Mei-Lian,
HUANG Jian-Jun, ZENG Wei-Min, CHEN Shu-Hua, SONG Hui-Ping*

(
Department of Biochemistry, Xiangya School of Medicine, Central South University, Changsha
410078, China )

Abstract        To study
the genetic variation in 5′-regulatory region of aldose reductase (AR) gene that might
influence expression and the relationship between variations and diabetic
complication (DC), PCR-single stranded conformational polymorphism (SSCP) was
used to screen the 5′-regulatory region of AR gene in Chinese patients with type 2
diabetes mellitus. A novel mutation, C－167→A
substitution which created a new CCAAT box was found only in two diabetic
patients. These two patients have no retinopathy, and the AR activity of their
erythrocytes was within low range in patients without DC. The DNA segments of
AR wild type and mutant were subcloned into pCAT reporter vector, and CAT
assays were performed to assess promoter activity. The interaction between the
DNA segments and nuclear proteins was determined by using competitive gel
electrophoretic mobility shift assay (EMSA). The transcriptional activity of
mutant (5.7%±2.9%) was lower than that of wild type (15.7%±4.1%) (P<0.01), and the mobility shift of mutant was also slower than that of wild type. The results indicated the mutation C－167→A in AR gene
might prevent or delay the development of DC by repressing the expression of AR
gene.

Key
words     aldose
reductase; gene; mutation; diabetic complication

Long-standing hyperglycemia has
been indicated as the most important cause of diabetic microvascular
complications[1]. On the other hand, some genetic factors have been proposed to
be responsible for the development and progression of diabetic complications
(DC)[2], but the definitive genes have not been identified yet. Aldose
reductase (AR; EC 1.1.1.21) is the first enzyme of the polyol pathway and
catalyzes the reduction of D-glucose to D-sorbitol. The excessive accumulation
of sorbitol in cells is believed to contribute to the etiology of various
DC[3], and abnormally high level of AR activity plays a pivotal role in this
pathogenesis[4].

The level of AR is mainly decided by the
expression of AR gene which is mostly regulated by the 5′-untranslate region (5′-UTR) of the gene. Recently, a
polymorphism C－106→T in the promoter region of AR gene
was found to be strongly negatively associated with diabetic retinopathy in
Australian type 1 diabetes mellitus adolescent patients[5]. In addition, two
polymorphisms C－106→T and C－12→G in the 5′region of AR gene had been identified to be positively associated
with diabetic retinopathy in Chinese type 2 diabetes mellitus patients[6].

In this study, a
novel mutation C－167→A in the promoter region of AR gene
was found in two Chinese diabetic subjects. These two patients suffered from
hyperglycemia for a relatively long time, but they did not appear diabetic
retinopathy and other complications. This experiment exploited the effect of
this mutation C－167→A on the AR transcription and the
relationship between the mutation and DC.

1    Materials and Methods

1.1   Subjects

321 Chinese
volunteers were totally recruited from Xiangya Hospital and the Second Affiliated
Hospital of Xiangya School of Medicine. Among them, 198 patients were
clinically diagnosed to have type 2 diabetes mellitus (105 with retinopathy and
93 without retinopathy), other 123 were control subjects. The patients have
been in diabetes for 8.1－14.2 years. Informed consent was obtained from each participant.

1.2   Assay of activity of AR in
erythrocytes

The activity of
aldose reductase was determined by fluorometric method[7]. 20 μL erythrocytic hemolysate was
incubated with 50 mmol/L potassium phosphate (pH 6.0), 0.4 mmol/L lithium
sulfate, 5 mmol/L 2-mercaptoethanol, 0.1 mmol/L NADPH and 10 mmol/L
glyceraldehyde at 37 ℃ for 5 min in a total volume of 0.2 mL. The enzymatic reaction was
stopped by addition of 0.2 mL 10% TCA, and the solution was mixed thoroughly to
precipitate proteins and destroy the remaining NADPH, and 0.3 mL supernatant
was obtained. 1.5 mL 6 mol/L NaOH containing 10 mmol/L imidazole was added to
the supernatant, and the mixture was detected by F-4000 spectro-fluorometer (Hitachi,
Japan) at excitation
wavelength of 360 nm and emission wavelength of 460 nm. A standard solution
containing 0.1－10 nmol NADP+
was treated similarly and used as a control. The activity of aldose reductase
was represented as u/g Hb.

1.3   Determination of hemoglobin
concentration

Hemoglobin
concentration was determined by cyanmethemoglobin method[8].

1.4   PCR-SSCP and DNA sequencing

Total DNA was
extracted from leucocyte pellets[9]. 
0.2 μg DNA was
used as PCR template. The primers were designed for the amplification of the 5′-regulatory region (－609 to +40 bp) as three overlapping
regions according to published sequences of the human AR gene (Table 1). The
PCR amplification were performed as following: an initial incubation at 95 ℃ for 3 min; 32 cycles: 95 ℃ for 50 s, 65 ℃ for fragment 1, 58 ℃ for fragment 2 or 62 ℃ for fragment 3 for 45 s, 72 ℃ for 1 min; a final extension at 72
℃ for 5 min.

Table 1   Primers
for three overlapping fragments of 5′-regulatory
region of AR

The products of
PCR were then subjected to SSCP analysis[10]. Electrophoresis was performed on
10% nondenaturing polyacrylamide gels (Arc:Bis=49:1) with (or without) 10%
glycerol at a constant voltage of 100V/300V for an appropriate duration at 25 ℃. After electrophoresis, the DNA
bands were visualized by silver staining. All SSCP variants were submitted for
DNA sequence analysis on an ABI PRISMTM 377 DNA Sequencer.

1.5   Plasmid construction

The plasmid CAT
enhancer vector without promoter was used for the plasmid construction. The
SSCP variant and a wild-type DNA fragment were amplified from genomic DNA by
PCR using a pair of oligonucleotide primers and Pfu DNA polymerase. The
proximal primer 5′-atggctgcagcgctccccagacccccgc-ccagt-3′ with a PstI site and the distal
primer 5′-cgcta-aagctttcgctttcccaccagatacagc-3′ with a HindIII restriction site
were constructed. After purification, PCR products and pCAT enhancer vectors
were digested with HindIII and PstI, and linked by T4 DNA ligase at 16 ℃ for 4 h. The recombinant was
introduced into E. coli DH5α by using TSS buffer (10% PEG 8000, 5% DMSO and 20
mmol/L MgSO4)[11].

For the samples
belonging to heterozygotes, two recombinants will be generated from cloning. In
order to select the DNA constructs with wild type and variant regulatory
regions, several colonies were harvested at random from each LB plate with
ampicillin and cultured in LB media with ampicillin overnight separately. DNA
constructs were extracted by the alkaline lysis method, and purified by the
polyethylene glycol (PEG) precipitation method[12]. The recombinants with
promoters of the wild-type or variations were identified by HindIII/PstI
digestion, SSCP and DNA sequence analysis. The M13 reverse primer was used for
sequence primer.

1.6   Transient transfection and CAT
assays

In a 60-mm
tissue culture dish, seeded 5－6×10⁵
HeLa cells in 2 mL of RPMI 1640 supplemented with 10% bovine fetal serum were
incubated at 37 ℃ and 5% CO2
until the cells were 40%－60% confluent. These cells were cotransfected with 2 μg of pCAT enhancer vector with
promoters of the wild-type or mutant and 2 μg of pSV-β-galactosidase by using 5 μL of cationic liposome in serum-free medium. After incubation for 4
h at 37 ℃, the medium
containing liposome-DNA complex was removed and incubated in normal medium with
10% bovine fetal serum for another 48 h. Then, the cells were harvested after
washing with phosphate-buffered saline and adding 400 μL of lysis buffer (Promega). The
pCAT control vector controlled by the SV40 promoter with enhancer and the basic
pCAT were served as positive control and negative control respectively.

CAT assays were
performed using the CAT enzyme assay system (Promega) and the liquid
scintillation counting method. The cpm measured represented the CAT activity.
β-Galactosidase activity was measured using the β-Gal enzyme assay system
(Promega) and presented in milliunit.

1.7   Competitive gel electrophoretic
mobility shift assays

The probes were
prepared by digesting DNA constructs with HindIII/PstI. The obtained 282-bp DNA
fragment was purified and the 3’-end of DNA fragments was labeled with [α-³²P]dATP
using the Klenow fragment. The activity of the probe is over 2000 cpm/ng
protein. Nuclear extracts were prepared from HeLa cells[13]. HeLa nuclear
extract (5 μg) and labeled
probe (0.5 pmol) with/without unlabeled 50× wild type or mutant DNA segment were used in each reaction in a
volume of 20 μL. Reaction
mixtures were incubated at 25 ℃ for 30 min. DNA-protein complexes were resolved from free probes on
8% (W/V) polyacrylamide gels (Arg:Bis=29:1) in a 0.25×TBE buffer. Gels were dried, and
labeled complexes were visualized by autoradiography at –30 ℃.

1.8   Statistical
analysis

All values were
analyzed using one-way ANOVA and u-test by SPSS software. P<0.05 was accepted as a level of statistical significance.

2    Results

2.1   Identification of the polymorphisms
in the AR gene 5′-regulatory region

In PCR-SSCP analysis, there was only one
pattern for fragment 2 and 3, which means no variations existed in these two fragments.
While in the fragment 1, four different patterns of SSCP were totally detected,
and DNA sequencing results confirmed that their genotypes are WT/WT, WT/C－12→G, WT/C－106→T and WT/C－167→A, respectively. WT/C－12→G and WT/C－106→T had been reported in previous report[6], but WT/C－167→A was found for first time here (Fig.1). In 321 subjects, this
genotype WT/C－167→A was only found in two diabetic
patients without DC. The clinical data was also shown in Table 2. In addition,
clinical diagnosis defined that these two patients suffer from neither diabetic
nephropathy nor diabetic neuropathy. The stage of diabetic nephropathy was
defined based on the urinary albumin excretion rate (AER) in 24 h urine sample
collected twice or more, their AER<20 μg/min. The distal conduction velocity (finger-wrist) of the two
patients were 57 and 59 m/s separately,and their proximal conduction velocities
(wrist-elbow)  were 64 and 67 m/s
respectively. Their distal (wrist) sensory nerve action  amplitudes (SNAP) were 45 and 49 μV, and the proximal (elbow) SNAP
were 27 μV and 30 μV separately.2.2Activity of AR in
erythrocytes of subjects

The activity of aldose reductase in
erythrocytes of subjects is displayed in Table 2. The activity of AR in
patients without retinopathy was significantly lower than that of patients with
retinopathy, and the activity of AR of two patients with the mutation was
within the low range in patients without retinopathy.

Fig.1       SSCP
analysis of 5′-regulatory region in AR

The result was from fragment
1. 1, regulatory sequence with mutation C－167→A; 2, wild-type
regulatory sequence.

2.3   Relative transcription activities
of different AR gene regulatory regions

The relative
transcription activities were calculated as follows:

Relative
activity=[(S_cpm－B_cpm)/(S_β–G－B_β–G)]/[(C_cpm－B_cpm)/( C_β–G－B_β–G)]

Where, S_cpm
was cpm of sample; B_cpm was cpm of vacant cells; C_cpm was
cpm of positive control (pCAT control vector); S_β-G was β-Gal
activity of sample; B_β-G was β-Gal activity of vacant cells; C_β-G
was β-Gal activity of positive control. The relative transcription activity of
regulatory sequences for the wild type and C^–167→A were (15.7%±4.1%) and (5.7%±2.9%), respectively, as shown in
Fig.2 (P<0.05).

2.4   Competitive gel electrophoretic
mobility shift assays

The regulatory
region with mutation C－167→A had slower
shift mobility than the wild-type regulatory sequence, and two retarded bands
were completely competed by both DNA segments respectively (Fig.3).

Table 2   Clinic
feature and activity of AR of patients with type 2 diabetes mellitus

FPG,
fasting plasma glucose. NIDDM, non insulin dependent diabetes mellitus. *P<0.01 vs. control subject. ^#P<0.01 vs. Patients with retinopathy (u-test).

Fig.2       The
relative transcription activity of the wild-type and mutant in pCAT

Control, pCAT vector with a SV40 promoter; Wild type, DNA construct
with wild-type fragment; Mutant, DNA construct with the mutation C－167→A. Data are
represented as x±s, (n=5). *P<0.05 vs. wild type.

Fig.3       Competitive
gel mobility shift assay of wild type or mutant DNA fragment in regulatory
region of AR gene

1, 32P-labled wild-type DNA fragment only; 2, 32P-labled wild-type DNA
fragment interacting with nuclear extract; 3, 32P-labled wild-type DNA fragment
interacting with nuclear extract and (50×) unlabled wild-type DNA
fragment, showing complete competition; 4, 32P-labled wild-type DNA fragment
interacting with nuclear extract and (50×) unlabled mutant DNA
fragment, showing complete competition; 5, 32P-labled mutant DNA fragment
interacting with nuclear extract; 6, 32P-labled mutant DNA fragment interacting
with nuclear extract and (50×) unlabled wild-type DNA fragment, showing complete competition; 7,
32P-labled mutant DNA fragment interacting with nuclear extract and (50×) unlabled mutant
DNA fragment, showing complete competition.

3    Discussion

In this study, a
novel mutation, C－167→A, in the promoter region of human
AR gene was only identified in two diabetic patients who had suffered from
diabetes and sustained hyperglycemia for more than ten years. Clinic data
showed that no diabetic complications such as retinopathy, nephropathy and
neuropathy occurred in the two patients. We also found that their activity of
AR in erythrocytes was within the low range in patients without diabetic
retinopathy. AR is strongly associated with pathogenesis of DC, and it has been
shown that the activity of AR in erythrocytes is in positive relation with diabetic
retinopathy. The low activity of AR in erythrocytes may be one of the important
reasons that prevent the development and progression of diabetic complications
in the two patients.

One of the main
reasons of reducing AR activity is abnormally low level of this enzyme which is
decided by the expression of AR gene. Gene expression is controlled by the
level of transcription, which is usually regulated by the interaction between
cis-elements in untranslation region (UTR, including promoter, enhancer and silencer)
of genes and corresponding trans-factors[14]. Among them, the promoter region,
which RNA-polymerase combined with, is especially important for the
transcription. Therefore, variations in this region might interfere with
interaction between RNA polymerase and DNA segment, then influence gene
expression severely[15]. The results of reporter gene assays confirmed that the
transcription activity of the 5′-promoter region with mutation was significantly lower than that of
wild type during transient expression of the promoters in a CAT coding sequence
fusion system in transfected Hela cells. Obviously the mutation in the promoter
region reduced the level of transcription of AR gene, and possibly resulted in
low activity of AR in tissues of the two patients.

The promoter
region of AR gene contains a TATA (－25 to －28) box, a
CCAAT (－94 to －98) promoter element, three Sp1
protein binding consenses sequences and a GA-rich region (－186 to －146) upstream of the translation
start ATG codon[16]. Analyzed the sequence near the mutation site, it was found
that the segment in the region from －164 to －168 had changed from CCACT in wild-type to CCAAT in the mutant. The
CCAAT box is an important cis-element for transcription[17]. According to the
position where the box locates and trans-factors that combine with the box, the
transcription of genes can be regulated positively[18] or negatively[19]. The
CCAAT box preferentially locates in －80/－100 region in
TATA-containing promoters[18]. So the novel CCAAT box in the region (－164 to －168) might repress the
transcription of AR gene. Moreover, the new CCAAT box possibly interferes with
the interaction between the primary CCAAT box (－94 to －98) and its
trans-factor, and affects the transcription of AR gene.

The competitive gel electrophoretic
mobility shift assay showed that the shift mobility of the regulatory sequence
with mutation was slower than that of the wild-type regulatory sequence, and
retarded bands were completely competed by both DNA segment. These meant the
novel CCAAT box possibly combined the same protein with the primary CCAAT box,
but the novel box interfered with the normal interaction between the primary
CCAAT box and its corresponding trans-factor, thereby influenced the
transcription of AR gene.

Furthermore, this
novel CCAAT box just locates between the two CGGAAA/G motifs, which could
interact with GA-binding proteins and show promoter activity in the AG-rich
region of AR gene[16]. So, the interaction between the novel CCAAT box and its
corresponding trans-factor might hinder the two CGGAAA/G motifs from combining
with GA-binding proteins, and repress the transcription of AR gene.

Though the two
patients had suffered diabetic mellitus and sustained hyperglycemia for many
years, the mutation C^–167→A in 5′ regulatory
region of the AR gene represses the expression of gene, and prevent or delay
the development of diabetic complications probably.

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_______________________________________

Received: May 14, 2003Accepted: July
17, 2003

The work was supported by a grant from the
National Natural Science Foundation of China (No. 39670352)

*Corresponding author: Tel, 86-731-4805415;
e-mail, [email protected]

Updated at: 2003-10-09