Detection of dinucleotide repeat polymorphism in exon 18 of LDL receptor gene for determining predisposition to obesity

A method detects whether an individual is predisposed to obesity. The method includes the steps of (i) obtaining a sample containing human genomic DNA from the individual; (ii) detecting whether the genomic DNA in the sample has a 7 AT tandem repeat in exon 18 of the low density lipoprotein receptor gene on one or both chromosomes; and (iii) correlating the absence of the 7 AT tandem repeat on the chromosome or chromosomes with a predisposition to obesity in the individual.

BACKGROUND OF THE INVENTION 
1. Technical Field 
This invention relates to a method for detecting individuals who are 
predisposed to obesity. This invention further relates to genetic 
techniques for detecting a low density lipoprotein receptor gene (LDLR) 
microsatellite polymorphism. 
2. Background 
Obesity is a common nutritional disorder that affects approximately 30% of 
adults in the Western world. Obesity is a multifactorial condition in 
which both environmental and genetic factors are important determinants in 
susceptibility to body fat accumulation (Despres et al., 1992, Molecular 
and Cellular Biochemistry, 113, 151-169). Adoption studies have implicated 
genetic control, rather than childhood environment, as the main influence 
on the development of adult obesity (Sorensen, T. I. & Stunkard, A. J., 
1993, Acta Psychiatrica Scandinavica, 370, 67-72). Obesity, essential 
hypertension, impaired glucose tolerance, non-insulin-dependent diabetes 
mellitus and dyslipidaemia tend to cluster in families. Collectively, 
these abnormalities constitute the multiple metabolic syndrome or Syndrome 
X, which is associated with cardiovascular disease (Kesaniemi et. al., 
1992, Annals of Medicine (Helsinki), 24, 461-464). 
Lipida and cholesterol ingested in an individuals' diet are essential for 
body maintenance. These molecules are transported through the body in 
lipoproteins. There are four types of lipoproteins each responsible for 
transporting varying amounts of lipid and cholesterol. It has been shown 
that lipoprotein concentration in the blood is proportional to abdominal 
fat disposition in obese individuals (Nishina et al., 1992, Proceeding of 
the National Academy of Science U.S.A., 89, 708-712). The low density 
lipoprotein (LDL) receptor is responsible for regulating LDL levels and 
hence cholesterol and plasma lipids in the blood. The gene encoding the 
LDL receptor (LDLR) is located at chromosome 19 position p13.2. 
LDLR is approximately 45 kb in length and contains 18 exons (Sudhof et al., 
1985, Science, 228, 815-822). The ApaLI LDLR polymorphism detects a 
nucleotide substitution in intron 15 of this gene. 
Studies of the ApaLI restriction fragment length polymorphism (RFLP) of 
LDLR showed an association with obesity in essential hypertensives. 
However, no association was shown between the ApaLI RFLP and hypertension 
(Zee et al., 1992, Biochemical Biophysical Research Communications, 189, 
965-971). In a further study with a population of 70 normotensives, the 
ApaLI polymorphism did not show a significant association with obesity 
(Morris et al., 1994, Clinical Science, 86, 583-592). Similarly, a HincII 
LDLR polymorphism which detects a substitution in exon 12 showed a 
significant association with obesity in essential hypertensives but not in 
normotensives (Zee et al., 1995, Clinical Genetics, 47, 118-121). Both of 
these polymorphisms are located towards the 5' end of the gene. 
There appears to be no reports associating obesity in normotensives with 
the LDLR gene. Furthermore, there is no known test of identifying such 
individuals who may be predisposed to obesity. If these individuals were 
aware that they were predisposed to obesity, they could be treated or 
follow dietary programs to prevent the onset of an obesity problem or 
treat an existing obesity problem. 
SUMMARY OF THE INVENTION 
The present invention results from the surprising discovery that a LDLR 
microsatellite marker located towards the 3' end of the gene showed a 
significant association with obesity in a mixed population of lean and 
obese individuals. The LDLR microsatellite marker is located in exon 18 
and is a dinucleotide AT tandem repeat region. Individuals who had an AT 
tandem repeat number of 7 in exon 18 of the LDLE gene on both chromosomes 
had a statistical predisposition to being lean. Hence, individuals who did 
not have an AT tandem repeat of 7 in exon 18 of the LDLR gene on one 
chromosome had a statistical predisposition to obesity. 
Thus, it is an object of the present invention to provide a method for 
detecting whether an individual may be predisposed to obesity. Further, 
individuals who had a tandem repeat of 8 or 10 in exon 18 of the LDLR gene 
On one chromosome and a tandem repeat of 8 or 10 in exon 18 of the LDLR 
gene on the other chromosome had a statistical predisposition to obesity. 
In one aspect, the invention resides in a method for detecting whether an 
individual is disposed to obesity including the steps of: 
(i) obtaining a sample containing human genomic DNA from said individual; 
and 
(ii) detecting whether said genomic DNA in said sample has a 7 AT tandem 
repeat in exon 18 of the low density lipoprotein receptor gene on one or 
both chromosomes, wherein the absence of the 7 AT tandem repeat indicates 
said individual is predisposed to obesity, lean being defined as having a 
body mass index less than 26 kg/m.sup.2 and obese being defined as having 
a body mass index equal to or greater than 26 kg/m.sup.2. 
The presence of the 7 AT tandem repeat in exon 18 of the LDLR gene 
indicates said individual is predisposed to being lean. 
The sample may be any suitable tissue or body fluid. These samples are 
preferably blood containing leukocyte(s). 
The detecting of the AT tandem repeats preferably involves PCR 
amplification using suitable primers. The choice of primers will determine 
the size of the FCR product and hence the manner of distinguishing and 
identifying products. Preferably the primers disclosed in Zuliani and 
Hobbs et al., 1990, Nucleic Acids Research, 18, 4300 are used. These 
primers are also shown in FIG. 1. After PCR amplification the amplified 
products may be determined by sequence analysis or by size separation. 
Size separation is preferably achieved by electrophoresis of the PCR 
products in a suitable agarose or polyacylamide gel. 
The number of tandem repeats in exon 18 of the LDLR gene may vary. Where an 
individual has a genotype with 7 tandem repeats in the LDLR gene on both 
chromosomes, or 7 tandem repeats in the LDLR gene on one chromosome and 8 
or 10 tandem repeats in the LDLR gene on the other chromosome, then the 
individual has a predisposition to being lean. Where an individual has a 
genotype with 8 tandem repeats in the LDLR gene on both chromosomes, 10 
tandem repeats in the LDLR gene on both chromosomes, or 8 tandem repeats 
in the LDLR gene on one chromosome and 10 tandem repeats in the LDLR gene 
on the other chromosome, then the individual has a predisposition to 
obesity. 
In summary, the following results of the method can be achieved: 
(i) 7/7 (AT tandem repeat number in exon of the LDLR gene on one 
chromosome/AT tandem repeat number in exon 18 of the LDLR gene on the 
other chromosome) indicates the individual is predisposed to being lean; 
(ii) 7/8 (AT tandem repeat number in exon 18 of the LDLR gene on one 
chromosome/AT tandem repeat number in exon 18 of the LDLR gene on the 
other chromosome) indicates the individual is predisposed to being lean; 
(iii) 7/10 (AT tandem repeat number in exon 18 of the LDLE gene on one 
chromosome/AT tandem repeat number in exon 18 of the LDLR gene on the 
other chromosome) indicates the individual is predisposed to being lean; 
(iv) 8/8 (AT tandem repeat number in exon 18 of the LDLR gene on One 
chromosome/AT tandem repeat number in exon 18 of the LDLR gene on the 
other chromosome) indicates the individual is predisposed to obesity; 
(v) 8/10 (AT tandem repeat number in exon 18 of the LDLR gene on one 
chromosome/AT tandem repeat number in exon 18 of the LDLR gene on the 
other chromosome) indicates the individual is predisposed to obesity; and 
(vi) 10/10 (AT tandem repeat number in exon 18 of the LDLE gene on one 
chromosome/AT tandem repeat number in exon 18 of the LDLR gene on the 
other chromosome) indicates the individual is predisposed to obesity. 
Support for the determinations of whether an individual is predisposed to 
being lean or obese is provided in the statistical analysis of the results 
shown in Examples 2 and 3. 
The results of this method also appears to be independent of whether an 
individual is hypertensive or normotensive. Therefore, the method can be 
used for hypertensive and normotensive individuals. 
Reference may now be made to various preferred embodiments of the 
invention. Example 1 is a preferred embodiment of the method of the 
present invention. Examples 2 and 3 are examples of the use of the method 
and provide supporting evidence for the method. The preferred embodiments 
are described by way of example only.

EXAMPLE 1 
The preferred method of determining whether an individual is predisposed to 
obesity is described below. 
A sample of 20 to 40 mls of blood was extracted from individuals. The 
sample was centrifuged at 3800 rpm for 10 minutes to separate the blood 
cells from the serum. The serum was stored at -70.degree. C. 
DNA was extracted from isolated blood cells using an adapted salting out 
method (Miller et al., 1988, Nucleic Acids Research, 16, 1215). To 10 mls 
of blood, NKM (140 mM NaCl, 30 mM KCl, 3 mM MgCl.sub.2) was mixed to give 
a final volume of 25 mL and vigorously shaken for 10 seconds to lyse the 
cells. After centrifugation at 4800 rpm for 25 minutes, the cell pellet 
was washed with 25 mL RSB (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM 
MgCl.sub.2). The mixture was centrifuged at 4000 rpm with 15 minutes and 
the pellet was resuspended in 1 ml of RSB followed by the addition of 4 mL 
of lympholysis buffer (1% SDS, 50 mM Tris-HCl, pH 7.5, 50 mM EDTA, 0.15 mM 
NaCl) and 0.5 mg/mL of proteinase K. The mixture was incubated overnight 
at 37.degree. C. in a shaking water bath. After the incubation, 2 mL of a 
saturated NaCl solution was added and the mixture shaken vigorously for 15 
seconds. The proteins were removed by centrifuging for 15 minutes at 2500 
rpm. Following the addition of a further 2 mL of saturated NaCl solution 
to the supernatant and centrifugation at 2500 rpm for minutes, two volumes 
of absolute ethanol were added to the supernatant and inverted to mix. The 
clump of precipitated DNA was removed using a disposable inoculation loop 
and placed in 2 mL of TE buffer and heated at 37.degree. C. for 2 hours to 
dissolve the DNA. 
The LDLR dinucleotiae AT repeat polymorphism was amplified using primers, 
GZ-7 and GZ-8 (FIG. 1), in the Polymerase Chain Reaction (PCR). The PCR 
amplification involved final concentrations of 200 .mu.M deoxynucleotides 
(dATP, dCTP, dGTP and dTTP), 1.75 mM MgCl.sub.2, 5 .mu.l of 10 times 
buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton) and 1.2 units of 
Taq Polymerase together and adding an aliquot to 150 ng purified DNA in a 
thin walled PCR tube under a biohazard flow hood. The cycling conditions 
for the PCR amplification were an initial 94.degree. C. denaturing period 
of four minutes, followed by 35 cycles of 94.degree. C. for 40 seconds; 
60.degree. C. for 9 seconds and a final extension period of 72.degree. C. 
for 120 seconds). 
Detection of PCR products involved electrophoresis on a 6% polyacrylamide 
denaturing gel and analysis using the Applied Biosystems 373 DNA sequencer 
with Genescan software. Using laser technology to excite the LDLR 
microsatellite primers, the allele sizes were determined by comparison to 
fluorescent internal lane standards. Genescan eletrophoretograms and 
spreadsheets were used to identify genotypes and to determine allele 
frequencies. 
The following results were achievable: 
(i) Where only a 106 bp PCR product was detected and identified, it was 
held that the individual was predisposed to being lean. A 106 bp PCR 
product represents a 7 tandem repeat in exon 18 of the LDLR gene. 
(ii) Where a 106 bp PCR product and a 108 bp PCR product were detected and 
identified, it was held that the individual was predisposed to being lean. 
A 108 bp PCR product represents an 8 tandem repeat in exon 18 of the LDLR 
gene. 
(iii) Where a 106 bp PCR product and a 112 bp PCR product were detected and 
identified, it was held that the individual was predisposed to being lean. 
A 112 bp PCR product represents a 10 tandem repeat in exon 18 of the LDLR 
gene. 
(iv) Where only a 108 bp PCR product was detected and identified, it was 
held that the individual was predisposed to obesity. 
(v) Where a 108 bp PCR product and a 112 bp PCR product were detected and 
identified, it was held that the individual was predisposed to obesity. 
(vi) Where only a 112 bp PCR product was detected and identified, it was 
held that individual was predisposed to obesity. 
EXAMPLE 2 
METHODS: 
Subjects 
Twenty millilitre blood samples were collected from normotensives for the 
present cross-sectional association study. Individuals were classified as 
normotensive if their blood pressure was less than 140/90 mmHg, they were 
not on hypertensive medication and they had no family history of 
hypertension, diabetes or heart disease. The population was divided into 
lean and obese categories on the basis of body mass index (BMI); obese 
individuals had a BMI of .gtoreq.26 kg/m.sup.2 and individuals were 
classified as lean if they had a BMI of &lt;26 kg/m.sup.2. 
Polymerase chain reaction analysis 
DNA was extracted from white blood cells as previously described (Zee, R. 
Y. L. et al., 1992, supra) and alleles for an LDLR dinucleotide repeat 
polymorphism were determined using fluorescently labelled primers (FIG. 1) 
and polymerase chain reaction (PCR) amplification. Polymerase chain 
reactions were performed as described previously (Zuliani, G. & Hobbs, H. 
H., 1990, supra), but modified to use a 94.degree. C. initial denaturing 
step for 3 minutes followed by 35 cycles of denaturation for 40s at 
94.degree. C., annealing and extension at 60.degree. C. for 1 minute and a 
final extension for 2 minutes at 72.degree. C. Polymerase chain reaction 
products were fractionated on 6% polyacrylamide denaturing gels and 
alleles were determined by comparison to size standards using an Applied 
Biosystems DNA sequencer with Genescan software (Perkin-Elmer), as 
described by Ziegle et al., 1992, Genomics, 14, 1026-1031. 
Analysis of data 
Electrophoretograms and spreadsheets were used to determine the genotypes 
for each subject tested. The total for each genotype was tabulated and 
allele frequencies calculated. Differences in frequencies were tested by 
Chi-squared analysis with two degrees of freedom. 
RESULTS 
Genotypes for the LDLR dinucleotide tandem repeat were determined for 83 
normotensives, 33 of whom were obese and 50 lean. The polymorphic repeat 
marker has been localised to exon 18 of LDLR. Polymerase chain reaction 
amplification using the GZ-7 and GZ-8 LDLR oligonucleotide primers detects 
three DNA fragments, 106, 108, and 112 bp, containing 7, 8, and 10 (TA) 
repeats, respectively (Zuliani, G. & Hobbs, H. H., 1990, supra). Genotypes 
were determined after fractionation of amplified, fluorescently labelled 
PCR products on polyacrylamide denaturing gels and comparison of fragment 
sizes to labelled standards using Genescan software. The total number of 
alleles was determined from genotype results and statistical analysis 
performed (Table 1). Statistical analysis of these results indicated that 
there was a significant difference between the lean and obese groups of 
normotensives (X.sup.2 =9.8; p=0.008). 
EXAMPLE 3 
METHODS 
Subjects 
Categorising obese subjects required a body mass index of 26 kg/m.sup.2 or 
greater whilst lean subjects required a body mass index less than 26 
kg/m.sup.2. Individuals were excluded from the study if they had a family 
history of diabetes or thyroid disease. These conditions were determined 
by a detailed questionnaire on each of the 92 obese and 158 lean 
consenting subjects. If individuals satisfied the criteria, 40 mL of blood 
was extracted from the antecubital fossa of each non-fasting subject and 
placed in a lithium heparin tube. 
Lipid Analysis 
Blood being used in the study was transported on ice to the laboratory. The 
blood sample was centrifuged at 3800 rpm for 10 minutes to separate the 
blood cells from the serum which was stored at -70.degree. C. The serum 
was analysed for total cholesterol, high density lipoprotein cholesterol 
(HDL-cholesterol) and triglyceride. 
Cholesterol was analysed on a BM Hitachi 747-200 analyser using the 
CHOD-PAP enzymatic colorimetric method (Siedel et al., 1983, Clin. Chem, 
29, 1075). HDL-cholesterol was precipitated with polyethylene glycol. 
Triglyceride was assayed on the same analyser using the GPO-PAP enzymatic 
colorimetric method (Wahlefeld, A. W., 1974, Methods of enzymatic 
analysis, 2nd edition, p 1831. Verlag Chemie Weinheim and Academic Press, 
Inc. New York). 
Genetic Analysis 
DNA Extraction 
DNA was extracted from isolated blood cells using an adapted salting out 
method (Miller et al., 1988, supra). To 10 ml of blood, NKM (140 mM NaCl, 
30 mM KCl, 3 mM MgCl.sub.2) was mixed to give a final volume of 25 mL and 
vigorously shaken for 10 seconds to lyse the cells. After centrifugation 
at 4800 rpm for 25 minutes, the cell pellet was washed with 25 mL RSB (10 
mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl.sub.2). The mixture was 
centrifuged at 4000 rpm with 15 minutes and the pellet was resuspended in 
1 ml of RSB followed by the addition of 4 mL of lympholysis buffer (1% 
SDS, 50 mM Tris-HCl, pH 7.5, 50 mM EDTA, 0.15 mM NaCl) and 0.5 mg/mL of 
proteinase K. The mixture was incubated overnight at 37.degree. C. in a 
shaking water bath. After the incubation, 2 mL of a saturated NaCl 
solution was added and the mixture shaken vigorously for 15 seconds. The 
proteins were removed by centrifuging for 15 minutes at 2500 rpm. 
Following the addition of a further 2 mL of saturated NaCl solution to the 
supernatant and centrifugation at 2500 rpm for 15 minutes, two volumes of 
absolute ethanol were added to the supernatant and inverted to mix. The 
clump of precipitated DNA was removed using a disposable inoculation loop 
and placed in 2 mL of TE buffer and heated at 37.degree. C. for 2 hours to 
dissolve the DNA. 
PCR amplification 
The LDLR dinucleotide AT repeat polymorphism was amplified using primers, 
GZ-7 and GZ-8 (FIG. 1), in the Polymerase Chain Reaction (PCR). The PCR 
amplification involved final concentrations of 200 .mu.M deoxynucleotides 
(dATP, dCTP, dGTP and dTTP), 1.75 mM MgCl.sub.2, 5 .mu.l of 10 times 
buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton) and 1.2 units of 
Taq Polymerase together and adding an aliquot to 150 ng purified DNA in a 
thin walled PCR tube under a biohazard flow hood. The cycling conditions 
for the PCR amplification were an initial 94.degree. C. denaturing period 
of four minutes, followed by 35 cycles of 94.degree. C. for 40 seconds; 
60.degree. C. for 9 seconds and a final extension period of 72.degree. C. 
for 120 seconds). 
Detection of PCR products involved electrophoresis on a 6% polyacrylamide 
denaturing gel and analysis using the Applied Biosystems 373 DNA sequencer 
with Genescan Software. Using laser technology to excite the LDLR 
microsatellite primers, the allele sizes were determined by comparison to 
fluorescent internal lane standards. Genescan cletrophoretograms and 
spreadsheets were used to identify genotypes and to determine allele 
frequencies. 
Statistical Analysis 
Differences in total cholesterol, triglyceride, HDL-cholesterol, 
LDL-cholesterol and VLDL-cholesterol parameters as compared to body mass 
index and genotype were determined by one-way analysis of variance 
(ANOVA). Chi-squared analysis was used to determine any differences 
between the allele frequencies in the obese and lean subjects. 
RESULTS 
Following the PCR amplifications of the labelled primers that detect the 
LDLR dinucleotide AT repeat polymorphism, the ABI DNA sequencer with 
Genescan software was used to identify fragment sizes. Genescan analysis 
on the PCR fragments resulted in detection of three alleles of 106 bp, 108 
bp and 112 bp. A combination of these alleles results in homozygote 
genotypes, as shown in FIG. 2 (106/106 bp), FIG. 3 (108 bp), FIG. 5 
(112/112 bp) and heterozygote genotypes as shown in FIG. 4 (108/112 pb). 
The results of a study on 92 obese subjects with a body mass index equal to 
or greater than 26 kg/m.sup.2 and with 158 lean subjects with a body mass 
index less than 26 kg/m.sup.2 are illustrated in Table 2. Chi-squared 
analysis on the total allele counts revealed a highly significant 
difference (X.sup.2 =7.09, P=0.0298) between the lean and obese groups. 
Furthermore, genotypes were investigated to determine the allele 
combinations that determine an obese BMI (Table 2). Genotypic analysis 
indicated that individuals with the 106 bp allele were more likely to be 
lean than individuals possessing 108 bp or 112 bp alleles. 
The results of the one-way anova study comparing the relationship between 
the plasma lipid concentrations and BMI (Table 3) revealed a significant 
difference between total cholesterol, triglyceride, HDL-cholesterol, 
LDL-cholesterol and VLDL-cholesterol concentration. An association can 
therefore be drawn between obesity and high concentrations for total 
cholesterol, triglyceride and triglyceride carrying LDL-cholesterol and 
VLDL-cholesterol lipoproteins whereas obese individuals have a lower 
HDL-cholesterol concentration. 
It has been found that the results of the method of determining whether an 
individual is predisposed to obesity is independent of hypertension. Of 
the population tested in this study, 73 were hypertensive (34 lean, 39 
obese) and 175 were normotensive (128 lean, 47 obese). When the 
hypertensives were removed from the analysis and only normotensives tested 
for significant differences between lean and obese populations, the LDLR 
microsatellite marker still showed a strongly significant association with 
obesity (X.sup.2 6.07; P=0.048). Hence the association of this marker with 
obesity is independent of hypertension. 
DISCUSSION 
Results from this study indicated a significant association X.sup.2 =7.09, 
P=0.029) between the LDLR microsatellite marker located towards the 3' end 
of the gene and obesity. ANOVA analysis between the lean and obese 
individuals illustrated that the obese individuals have higher 
cholesterol, triglyceride and LDL-cholesterol levels which may arise from 
an impaired LDL receptor regulation and lower HDL-cholesterol levels than 
lean individuals. The association of LDLR and obesity appears to be 
related to the importance of the 106 bp allele. 
ANOVA analysis on actual BMI values for each genotype revealed the 106 bp 
allele increases an individuals' chance of being lean. Mean values 
obtained for BMI indicated that the 106 bp homozygote is associated with a 
lean BMI. This phenomena appears to be a result of a 106 bp allele 
dominance. 
In this study, a significant association (X.sup.2 =7.09, P=0.0298) between 
an LDLR microsatellite marker, located towards the 3' end of the gene, and 
BMI was shown from a cross-sectional analysis study in a general 
population comprised of obese and lean individuals. These results together 
with the ANOVA results confirm that there is an association between an 
LDLR microsatellite and obesity indicating that this gene may play an 
important role in obesity predisposition. 
TABLE 1 
______________________________________ 
Association analysis of a low density 
lipoprotein receptor dinucleotide repeat 
polymorphism in obese and lean normotensives 
Allele frequencies (bp) 
Total alleles (bp)* 
NT NO. 106 108 112 106 108 112 
______________________________________ 
Obese 33 0.60 0.20 0.20 40 13 13 
Lean 50 0.78 0.17 0.05 78 17 5 
______________________________________ 
*x.sup.2 = 9.8; P = 0.008 
TABLE 2 
__________________________________________________________________________ 
Chi-squared anaylsis on allele numbers in 
lean and obese subjects 
Genotype Frequency 
106/ 
106/ 
106/ 
108/ 
108/ 
112/ 
Allele Number (bp)* 
n 106 
108 112 
108 112 
112 106 
108 112 
__________________________________________________________________________ 
Obese 
92 0.33 
0.12 
0.34 
0.01 
0.08 
0.12 
104 
20 60 
Lean 
158 0.44 
0.12 
0.37 
0.01 
0.03 
0.03 
216 
26 74 
__________________________________________________________________________ 
*x.sup.2 = 7.09; P = 0.029 
TABLE 3 
______________________________________ 
Results of ANOVA analysis on plasma lipid 
parameters for the obese and lean subjects 
Statistical 
BMI Analysis 
Obese Lean ANOVA 
______________________________________ 
n 53 71 
Total cholesterol 
6.2421 +/- 1.09 
5.79 +/- 1.02 
0.0202 
Triglyceride 
2.13 +/- 1.11 1.70 +/- 1.12 
0.0351 
HDL-cholesterol 
1.38 +/- 0.42 1.66 +/- 0.57 
0.0025 
LDL-cholesterol 
3.93 +/- 1.09 3.38 +/- 0.97 
0.0056 
VLDL-cholesterol 
0.93 +/- 0.41 0.70 +/- 0.49 
0.0310 
______________________________________ 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
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(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CACTTTGTATATTGGTTGAAACTGT25 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CACTGAACAAATACAGCAACCAGGG25 
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