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Articles |
1
Department of Laboratory Medicine, Landeskrankenanstalten Salzburg, Muellner Hauptstrasse 48, A-5020 Salzburg, Austria.
Departments of
2
Surgery and
3
Medicine,
Krankenhaus Hallein, A-5400 Hallein, Austria.
a Author for correspondence. Fax 011 43 662 4482 885; e-mail w.patsch{at}lkasbg.gv.at
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We measured allele-specific apoC-III expression in liver biopsies of five obese subjects, using restriction isotyping and a primer extension method, both based on the SstI polymorphism.
Results: The expression of mRNA by the S1 and S2 alleles was similar in two patients, whereas the mRNA encoded by the S2 allele was 14%, 26%, and 29% more abundant than the wild-type mRNA in the remaining three patients. Because other polymorphisms at the apoC-III gene locus have been implicated in the S2-associated hypertriglyceridemia, we determined apoC-III haplotypes comprising promoter polymorphisms at -935, -641, -630, -625, -482, -455, as well as the SstI sites and a BbvI site, both located in the 3' untranslated region. None of these polymorphisms nor any haplotype exhibited a perfect association with allele-specific expression, but variation at the T-482C site correlated in four of five subjects with the relative allele abundance.
Conclusion: These data provide preliminary evidence for allele-specific differences in apoC-III mRNA expression in vivo and suggest that such differences may contribute to associations of apoC-III gene polymorphisms with hypertriglyceridemia. ©1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidemiological studies have shown an association between an SstI polymorphic site in the 3' untranslated region (UTR) of the apoC-III gene and HTG in several populations (3)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). A less-established association may exist between a polymorphism located at position +3293, which leads to the deletion of a BbvI restriction site, and HTG (30)(31). In addition, six polymorphic sites in the promoter of the apoC-III gene have been found, which were in strong linkage disequilibrium with each other and the variant SstI site (16)(17). Two of the polymorphic nucleotides, at position -455 and -482 relative to the transcription start site, reside within a putative negative insulin-response element (IRE) (32), originally described within the promoter of the phosphoenolpyruvate carboxykinase (PEPCK) gene (33)(34). Transfection studies in HepG2-cells showed that insulin down-regulates transcriptional activity of reporter constructs containing the wild-type apoC-III sequences within the putative IRE (32), whereas the presence of variant nucleotides within the IRE abrogated the transcriptional repression by insulin. These findings suggest that the presence of variant nucleotides within the IRE leads to a loss of insulin repression of apoC-III gene transcription and causes constitutive overexpression of apoC-III, thereby contributing to HTG in some subjects. This hypothesis has been challenged by two recent studies, both showing a stronger association of HTG or TG concentrations with the variant SstI site than with the polymorphisms in the promoter region (13)(16).
To gain insight into the in vivo importance of sequence variations at the apoC-III gene locus, we determined the abundance of allele-specific apoC-III mRNA in liver biopsies of five subjects heterozygous for the variant SstI site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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laboratory methods
After an overnight fast, venous blood was collected into tubes
containing EDTA. Plasma glucose was measured by a
hexokinase/glucose-6-phosphate dehydrogenase method (Boehringer
Mannheim). Plasma insulin was measured by immunoassay (Abbott
Laboratories). Plasma cholesterol and TG were measured by enzymatic
procedures using a Hitachi 717 analyzer (Boehringer Mannheim) and the
respective enzymatic kits (Boehringer Mannheim). HDL-cholesterol was
determined in supernates after precipitation of plasma with
phosphotungstic acid/magnesium chloride (Boehringer Mannheim). apoB and
apoA-I concentrations were determined using nephelometric procedures
(Array 360; Beckman). Plasma leptin was measured with an RIA kit (Linco
Inc.).
SstI TYPING
Genomic DNA was extracted from white blood cells using the QIAamp
blood kit (Qiagen). For typing of the SstI polymorphism in
the 3'-UTR of the apoC-III gene, the conditions described by Hixson et
al. (35) were used. The alleles were defined as S1 and S2
based on absence or presence of the SstI restriction site.
allele-specific gene expression studies by restriction isotyping
Total liver RNA was isolated from 200 mg of liver tissue according
to the method of Chomczynski and Sacchi (36). The integrity
of the RNA was ascertained by the electrophoretic patterns of rRNA in
formaldehyde gels. After DNase I digestion (Boehringer Mannheim), RNA
concentrations were determined by absorbance measurements at 260 nm.
Total liver RNA (1 µg) was reverse-transcribed using 200 U of Moloney
murine leukemia virus reverse transcriptase (Life Technologies), 10
mmol/L Tris, pH 8.3, 50 mmol/L KCl, 5 mmol/L
MgCl2, 0.5 µmol/L random hexamers, 1 mmol/L
dNTP, and 20 units of RNasin (Promega) in a total volume of 20 µL.
Aliquots (1 µL) of the cDNA were subjected to PCR using the primers
described by Hixson et al. (35). PCR reactions contained
0.17 µmol/L each upstream and downstream primer, 0.2 mmol/L each
dNTP, 1 µCi of 
32P-dCTP (3000
Ci/mmol; Amersham), 10 mmol/L Tris, pH 8.3, 50 mmol/L KCl, 2.5 mmol/L
MgCl2, and 1 U of Taq polymerase (Perkin-Elmer)
in a volume of 30 µL. Samples were processed through initial
denaturation for 3 min at 96 °C; 28 cycles of amplification, each
consisting of 30 s at 60 °C (annealing), 1 min at 72 °C
(extension), and 30 s at 96 °C (denaturation); and a final
extension at 72 °C for 5 min. For restriction digestion, 20 U of
SstI (New England Biolabs) was added directly to the PCR
tubes along with 5 µL of buffer supplied by the manufacturer and 15
µL of H2O and incubated for 8 h at
37 °C. Aliquots of digestion mixtures were applied to denaturing 8%
polyacrylamide gels using 100 mmol/L Tris, 30 mmol/L taurine, 0.5
mmol/L EDTA, pH 9.2, as a buffer system to provide glycerol tolerance.
Polynucleotide calibrators (100-bp ladder; MBI-Fermentas) were
end-labeled with 
32P-dATP (3000 Ci/mmol;
Amersham). After removal of urea, gels were dried and exposed to x-ray
film (X-Omat AR; Eastman Kodak Co.). Autoradiograms were analyzed by
quantitative scanning densitometry using a Model GS-700 Imaging
densitometer and the Molecular Analyst software (Bio-Rad).
For assay standardization, genomic DNA from an SstI heterozygote patient was amplified by PCR using Pfu polymerase (Stratagene) and cloned into pGEM3Zf(+) (Promega). Sequence-verified clones containing the variant SstI site or devoid of it were used to ascertain the adequacy of digestion by SstI. In addition, various mixtures of clones containing the S1 or S2 allele as insert as well as allele-specific PCR products served as templates for amplification and SstI digestion to relate signal intensities in patient samples to relative molar abundance.
For intron-spanning allele-specific expression studies, cDNA aliquots were subjected to PCR using 5'-GTTACATGAAGCACGCCACCA-3' (+1169 to +1192, GenBank accession no. X03120) as the sense primer and 5'-GGTAGGAGAGCACTGAGAATACTG-3' (+3318 to +3341) as the antisense primer. Assay conditions were exactly the same as described above.
allele-specific gene expression studies by primer extension
Transcription of apoC-III mRNA from the wild-type allele and from
the variant allele that harbored a C/G substitution at position +3262
(GenBank accession no. X03120) was also performed by primer extension
as described (37). Aliquots of cDNA were subjected to PCR
using the primer pair described for intron-spanning PCR, except that
the forward exon 3 primer (+1169 to +S1192) was 5'-biotinylated. The
primer for mutant allele-specific extension was 5'-CAGGACCCAAGGAGC-3'
(+3264 to +3278), and extension reactions were performed in the
presence of 0.1 mmol/L each dNTP (dGTP, dATP, and dTTP) and 0.25 mmol/L
ddCTP as described (37). Reactions were subjected to
electrophoresis on 8% denaturing polyacrylamide gels using 100 mmol/L
Tris, 30 mmol/L taurine, 0.5 mmol/L EDTA, pH 9.2, as buffer. Dried gels
were exposed to x-ray film, and autoradiograms were analyzed by
quantitative scanning. For assay standardization, the cDNA of two
subjects carrying either two wild-type or two mutant alleles was cloned
using the ZERO-Background(TM) (Invitrogen) cloning system. Various
mixtures of plasmids containing the wild-type and mutant allele as
inserts served as templates for PCR amplification and subsequent primer
extension to generate a calibration curve that compared molar
ratios of templates with their signal intensity ratios.
sequencing of cDNA ALLELES
Near full-length apoC-III cDNA was prepared using 3' rapid
amplification of cDNA ends combined with reverse transcription
(RT)-PCR (38). Human liver RNA (1 µg) was
reverse-transcribed using SuperScript(TM) II Reverse Transcriptase (Life
Technologies) and 5'-GAGGACTCGAGCTCAAGCT (20)-3'
as adapter-primer. After RNase H (Boehringer Mannheim) digestion, the
first-strand cDNA was subjected to PCR using 5'-TCATCCCTAGAGGCAGCTG-3'
(+267 to +285) as apoC-III gene-specific sense and
5'-GAGGACTCGAGCTCAAGC-3' as anchor-primer, respectively. PCR products
were diluted and reamplified using 5'-CTAGAGGCAGCTGCTCCAG-3' (+273 to
+291) as nested gene-specific primer and the anchor-primer described
above. Products of 3' rapid amplification of cDNA ends were gel
purified and cloned using the ZERO-Background cloning system.
Three clones, each containing either the S1 or the S2 allele, were
sequenced using the PRISM(TM) Ready Reaction dRhodamine-Terminator
kit and an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Applied
Biosystems).
haplotype determination by cloning and sequencing
To determine the haplotypes spanning the promoter region and the
3'-UTR, 4.35-kb apoC-III gene fragments (-1255 to +3090, relative to
the transcriptional start site) of study subjects were cloned into
pGEM3Zf(+). The fragments were amplified by PCR using
5'-CGCCGGTACCAGGAGGGAGAGGGAGGTGTGAGTC-3' (+157 to +180, GenBank
accession no. M60674) as the sense primer and
5'-GGCGGGTACCTGAGGTGGGGTAGGAGAGCACTGA-3' (+3326 to +3349, GenBank
accession no. X03120) as the antisense primer, respectively, with each
primer containing an engineered KpnI restriction site. PCR
was carried out in a total volume of 100 µL, using the Expand High
Fidelity kit (Boehringer Mannheim), 200 ng of DNA, 0.30 µmol/L each
primer, and 0.2 mmol/L each dNTP. Samples were processed through
initial denaturation for 3 min at 94 °C and 35 cycles of
amplification, each consisting of 30 s denaturation at 94 °C,
30 s annealing at 60 °C, and 3 min extension at 68 °C. The
extension time was increased by 15 s each cycle starting with
cycle 15, and the final extension time was 10 min. PCR products were
cloned into the KpnI site of pGEM3Zf(+). Allele-specific
clones were identified by colony PCR screening followed by
SstI digestion. Allele-specific plasmids were sequenced by
primer walking.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. All subjects were heterozygous for the variant SstI
site in the 3'-UTR of the apoC-III mRNA. Three of the patients
exhibited fasting HTG, and one subject had overt type 2 diabetes
mellitus. Histological examination of the liver biopsies revealed
steatosis in all five patients.
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allele-specific apoC-III mRNA QUANTIFICATION
To study allele-specific hepatic expression of apoC-III alleles in
individual patients, we used restriction isotyping of RT-PCR products
harboring the SstI polymorphic site. To evaluate the
reliability of our assay for quantification of mRNA transcribed from
different alleles, we cloned, from a heterozygous patient, a 233-bp
fragment of S1 and S2 alleles located in exon 4. Plasmids harboring S1
or S2 alleles were mixed at different weight ratios, amplified by PCR
in the presence of 32P-dCTP, digested with
SstI, and subjected to denaturing polyacrylamide gel
electrophoresis. An autoradiograph of this experiment as well as
its densitometric evaluation is shown in Fig. 1
A. After correction of signal intensities of fragments
representing the S1 or S2 allele for their G/C content, a linear
relationship was obtained between the input molar ratio of S1 and S2
plasmids and the signal intensity ratio of S1- and S2-specific bands
(r = 0.99). This experiment also showed complete
digestion of S2 PCR products, whereas digestion of S1 PCR products
generated no fragments. The intraassay variability of signal intensity
ratios averaged 3%. In addition, we verified these results with RT-PCR
products to eliminate the possibility that PCR products exhibited
intrinsic resistance to SstI digestion. Hence, the
restriction isotyping method selected for allele-specific expression
studies was accurate and precise over a wide range of wild-type and
variant DNA mixtures.
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Several precautions were taken to quantify the relative expression of
the S1 and S2 alleles in patient RNA. To exclude effects of DNA that
would have reduced possible allele-specific differences in mRNA
expression, all RNA samples were subjected to DNase I digestion. This
treatment effectively eliminated amplification of target sequences by
PCR without prior reverse transcription (not shown). In addition, a
mixture of DNA of several heterozygotes was analyzed in triplicate
within each assay to assure precision and accuracy of signal
intensities. Moreover, wild-type and variant apoC-III plasmid DNA as
well as RT-PCR products were included in each assay to assure the
adequacy of SstI digestion. An autoradiogram of
allele-specific gene expression studies by restriction isotyping in our
five patients is given in Fig. 1B
. The adequacy of restriction
digestion is demonstrated in lanes 1 and 2, representing variant and
wild-type alleles, respectively. In lanes 3–7, the results of our five
study subjects are shown, whereas lane 8 depicts a DNA pool of
SstI heterozygote subjects. To further support these
results, we performed allele-specific gene expression studies based on
restriction isotyping assays using intron-spanning primers. Results of
these experiments are given in Table 2
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To corroborate differences in allele-specific expression by a method
independent of SstI restriction digestion, we used
quantitative allele-specific primer extension (Fig. 2
). Mixtures consisting of different molar ratios of plasmids
harboring the wild-type or variant cDNA were used to establish that the
primer extension method could reliably quantitate different amounts of
variant and wild-type templates. Analysis of RNA samples isolated from
liver tissues of our five study subjects revealed an excellent
agreement among the three methods (Table 2
). When these analytical
control measures were used, the average expression of the S2 allele,
which was calculated from the three different analytical methods,
exceeded that of the S1 allele by 29%, 26%, and 14% in patients 2,
3, and 4, respectively. This corresponds to variant apoC-III mRNA
concentrations, expressed as percentage of total apoC-III mRNA, of
56.7%, 55.7%, and 53.3%. Consideration of the threefold SD interval
calculated from the three different assays indicated that the abundance
of the variant in patients 2, 3, and 4 was significantly different from
the 50% level expected for an equal expression of apoC-III alleles
(Table 2
). In the two remaining patients, the difference in relative
expression between wild-type and variant alleles did not exceed the
analytical variance. Consistent with the low assay variability, these
results were highly reproducible in several independent determinations.
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allele-specific expression patterns and sequence variations at the
apoC-III GENE LOCUS
To study a possible relation of polymorphisms in the apoC-III gene
with relative expression of the S1 and S2 alleles, we determined six
promoter polymorphisms and the SstI and BbvI
polymorphisms, both located in the 3'-UTR of the apoC-III gene. These
polymorphisms, their location within the apoA-I/C-III/A-IV
gene cluster, and their notation as used in our study are given in Fig. 3
. For exact haplotype determination, we cloned 4.35-kb fragments
spanning the apoC-III gene locus of our five study subjects and
sequenced the promoter and 3'-UTR harboring totally eight variant sites
previously implicated in apoC-III gene expression (Table 2
and Fig. 3
).
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When the variant site at -455 was considered specifically, only patients 2 and 4, out of the four patients examined who were heterozygous at this site, exhibited differences in S1 and S2 expression. Patient 1 showed the same alleles at -455 as patients 2 and 4, but exhibited no difference in the expression of S1 and S2 alleles, whereas patient 3, who carried two variant nucleotides at position -455, exhibited a marked difference in allele-specific expression. Regarding the -482 site, all variant SstI sites were in perfect linkage disequilibrium with the variant -482 site, and three of the four patients who were heterozygous for the -482 site displayed significant differences in allele-specific expression studies. Patient 5, who showed no difference in expression of the two alleles, was homozygous for the -482 variant site. Only patient 1 displayed similar expression of the two alleles despite being heterozygous at the -482 site. The remaining four promoter polymorphisms located farther upstream were in less linkage disequilibrium with the SstI site than the -455 and -482 sites. Heterozygosity at the -625 deletion was associated with differences in apoC-III allele-specific gene expression only in patient 4, whereas homozygosity at this locus was consistent with allele expression in patient 1. The same result was observed for the tightly linked -630 polymorphism. The -641 and -935 substitutions were both predictive in three of five patients, including patients 1 and 5, who displayed no difference in expression studies. The third patient predicted correctly by either of these two sites was patient 3 for the -935 site and patient 4 for the -641 site. Sequencing of the 3'-UTR revealed the two well-known polymorphisms, i.e., the variant SstI site and a variant T-to-G transversion at +3293, which deletes a BbvI site. However, the variant BbvI site agreed with the results of allele-specific amplification in only two patients.
Patient 3, whose TG concentration was 0.92 mmol/L (Table 1
), possessed
a putative protective apoC-III haplotype, consisting of wild-type
nucleotides at positions -482/SstI and variant nucleotides
at -625/-455 (13). In this patient, who carried the
variant nucleotide at -455 on both alleles, allele-specific expression
assays revealed a 26% higher abundance of the variant apoC-III allele.
To identify other sequence substitutions possibly contributing to
allele-specific expression differences in this patient, we cloned near
full-length cDNA and sequenced both alleles. Apart from the previously
described variant sites in the 3'-UTR, the two alleles exhibited
complete sequence homology with each other, including the variant site
at C1170T (GenBank accession no. X03120) in exon 3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because an increased expression of the SstI variant allele
was not observed in all subjects studied, we searched for stronger
correlations among other sequence substitutions at the apoC-III gene
locus. Specifically, two polymorphic sites, at -455 and -482 relative
to the transcriptional start site (17) and located within a
putative negative IRE (32), appeared as candidate loci for
providing insight into the imperfect association of the SstI
polymorphism with allele-specific mRNA expression. In addition, we also
considered a T-to-G transversion, located 31 bases downstream of the
SstI site at position +3293 (30)(31).
Using the RNA secondary structure prediction algorithm described by
Zuker et al. (39), we found that this substitution is
located within the bulb of a putative hairpin and may therefore affect
mRNA stability. To correctly assign these sequence variations to the
respective SstI allele, we cloned long-range genomic PCR
products encompassing the BbvI and the promoter
substitutions and sequenced the discriminatory regions of several
inserts in each patient. These studies revealed a high degree of
allelic heterogeneity that was not expected from previous linkage
studies (13)(17). Among the candidate sites
studied, only the -482 polymorphism was superior to the
SstI site in predicting allele-specific mRNA expression in
that four of five patients exhibited the expected pattern. The
agreement of this site with allele-specific expression would be lowered
if one takes into account that patient 2 had type 2 diabetes and
should, therefore, display similar expression of the wild-type and
variant alleles according to the IRE model. Functional insulin
deficiency in this patient would be expected to reduce or abolish
allele-specific differences in mRNA expression. However, the role of
the -482 mutation in down-regulation of apoC-III expression is
incompletely understood because specific interactions of the wild-type
and variant site with specific trans-acting factors have not been
presented. The predictive value of the -455 site was only 40% (Table 2
, patients 2 and 4) compared with 60% of the SstI site,
and this value would be further lowered upon consideration of the
diabetic patient 2. The BbvI polymorphism correlated in only
two patients with the results of the allele-specific gene expression
and was therefore less informative than the SstI site.
Notwithstanding the small sample size, the low predictive value of the
-455 site argues against the importance of this site for apoC-III mRNA
expression. The fact that allele-specific differences were observed in
one patient harboring two variant nucleotides at -455 clearly
indicates that other sequence substitutions can affect apoC-III mRNA
expression, at least in some patients. Interestingly, this patient had
low plasma TG (Table 1
) and possessed the putative protective apoC-III
haplotype described recently (13)(17).
Because we excluded by cDNA sequencing mRNA substitutions other
than the variant SstI site as well as truncations that could
have led to mRNA turnover differences, other factors must have
contributed to the allele-specific differences in this patient.
Our conclusions concerning the relative importance of the various sequence substitutions on gene expression must be qualified in several regards. First, our sample size was small, and the observed associations may have arisen by chance. Second, all of our patients were heterozygous for the SstI site, which exhibits variable degrees of linkage disequilibrium with the -455 and the -482 site. The allelic frequencies of the -482 and -455 variant alleles and of the BbvI polymorphic sites each were 0.60 and exceeded only slightly the frequency of the SstI variant allele (0.50). In population samples, the frequencies of the -482, -455, and BbvI sites have been reported to be two- to threefold higher than that of the SstI polymorphism (13)(14)(15)(16)(17). The variant SstI allele was thus overrepresented in our study group because of the inclusion criteria required for mRNA quantification. Third, correlation studies of entire haplotypes with allele-specific expression differences were not possible because of the limited sample size. Thus, our method of interpretation was dependent on only one site, although more than one variant may alter expression. Fourth, all of our patients were morbidly obese, and the metabolic disturbances associated with obesity may have modulated the influence of cis-factors on mRNA expression. In addition, variable degrees of insulin resistance may have partially confounded the results in our patients. Fifth, our studies were conducted in the postabsorptive state. As exemplified by the IRE model, transcriptional regulation of the apoC-III gene may exhibit temporal variation and may be more important during the postprandial state. Measurement of mRNA concentrations in the postabsorptive state may therefore underestimate the magnitude of cis-regulatory factors on apoC-III transcription. In contrast, the influence of cis-factors on mRNA stability would be expected to continue during the postabsorptive state.
In two other recent studies, the association of HTG with SstI polymorphism was stronger than that with promoter haplotypes (13)(16). The SstI variant allele consistently has been found to be associated with increased TG (3)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) and, in some studies, with increased apoC-III concentrations (14)(15)(16)(22). It is possible that this site might be involved in mRNA stability because 3' untranslated mRNA sequences of other genes have been shown to influence mRNA turnover (40)(41). However, experimental evidence for such functionality has not been presented for the SstI site of apoC-III mRNA. As shown in the study by Shoulders et al. (16), three children carrying two variant SstI alleles had TG concentrations below the mean concentration found in children homozygous for the SstI common allele. Our data also suggest that the SstI site itself may not account for enhanced abundance of apoC-III mRNA, at least not in all subjects. Interestingly, the association of the SstI polymorphism with increased TG is observed mainly in HTG individuals (13). An interaction of the SstI site with another factor also involved in HTG may therefore be invoked to explain the imperfect association of the SstI site with differences in allelic expression.
In conclusion, our study shows that mRNA expression of apoC-III alleles can differ in human liver. Whether the imperfect association between allele-specific expression patterns and sequence substitutions at the apoC-III gene locus relates to the study population or measurements in the postabsorptive state or is the result of more complex interactions at the A-I/C-III/A-IV gene locus, as suggested by others (15)(16), remains to be determined.
This study was supported by a grant of the Medizinische Forschungsgesellschaft, Salzburg, Austria. We thank D. Breban and C. Winkler for technical assistance.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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