Physiological Study of Lipoprotein Lipase Gene Pvu II Polymorphism in Cases of Obesity in Egypt
Genetic predisposition has been implicated in obesity. Lipoprotein lipase (LPL) gene, the main lipase of chylomicrons and Low Density Lipoproteins (LDL), has a fundamental role in the transport and metabolism of plasma cholesterol. The present study was undertaken to test for the association of the LPL gene Pvu II polymorphism with obesity with or without hypertension and diabetes and dyslipidemia among affected Egyptian cases. This study has included 120 subjects affected with obesity; 57 of them were affected with metabolic syndrome (with diabetes, dyslipidemia and hypertension) while the other 63 cases were not complicated and were termed simple obesity. These cases were compared to 83 healthy non-obese controls. Body Mass Index (BMI), Waist Hip Ration (WHR) and serum lipid levels were measured. The LPL gene polymorphic alleles were determined by PCR-RFLP that includes polymerase chain reaction for gene amplification followed by digestion with Pvu II enzyme and analysis according to the size of digested amplified DNA. Obesity cases had a significantly higher frequency of the homozygous mutated LPL Pvu II (+/+) genotype and also of the (+) allele particularly among metabolic syndrome cases compared to controls. Cases with the (+/+) homozygous genotype showed significantly higher frequency of diabetes, lower frequency of positive family history and lower values for waist hip ratio than those with the (+/-) and (-/-) genotypes. These cases have showed also higher levels of total cholesterol and LDL-C, yet not reaching statistical significance. This study showed a significant association between the LPL Pvu II gene polymorphism and obesity among Egyptian cases particularly when complicated with the metabolic syndrome.
Received: December 13, 2011;
Accepted: January 24, 2012;
Published: March 08, 2012
Obesity has become an extensive public health concern because its prevalence
has increased to epidemic proportions. Many gene variants are involved in the
development of obesity and body weight regulation (Rampersaud
et al., 2008). The biological factors significantly associated with
overweight and obesity were increasing age, being female and parental obesity.
Also, non-biological factors including, physical inactivity, non-healthy diet,
lower family monthly income and being non-smoker (Suleiman
et al., 2009). The metabolic syndrome is a common multi-component
condition including abdominal obesity, dyslipidemia, hypertension and hyperglycemia.
It is associated with an increased risk of cardiovascular disease and type 2
diabetes (Teran-Garcia and Bouchard, 2007).
Lipoprotein lipase (LPL) enter in the metabolism of the core triglycerides
of circulating Very Low Density Lipoproteins (VLDL) by hydrolyzing them and
chylomicrons, thereby delivering lipoprotein derived fatty acids to adipose
tissues for storage or oxidation in muscle (Garfinkel and
Schotz, 1987). Obesity in humans is a result of abnormal adipose tissue
LPL activity (Lithell et al., 1978). LPL gene
is located in chromosome 8p22 (Sparkes et al., 1987)
and consist of 10 exons which is about 30 kbp (Monsalve
et al.,1990). Taking into account that polymorphisms in a number
of candidate genes have been reported to be associated with obesity; this study
focused on LPL Pvu II gene polymorphism among Egyptian cases with simple and
complicated obesity. This was done using the molecular biology techniques, utilizing
the simple and rapid PCR (polymerase chain reaction) coupled with Restriction
Fragment Length Polymorphism analysis (RFLP). Correlation of detected mutational
types of LPL gene with the clinical severity of obesity and lipid profile was
MATERIALS AND METHODS
This study has included 120 subjects affected with obesity with BMI (Body Mass
Index) level being at least 30. They were selected from the Outpatient's Clinic,
Department of Obesity and Diabetes, Specialized Internal Medicine Hospital,
Mansoura University, Egypt between the times of January 2010 to January 2011.
Their age Mean±SD was 31.5±11.2 years ranging from 13-61 years.
They were in the form of 21 (17.5%) males and 99 (82.5%) females. Of them, 21
(17.5%) were positive for parental consanguinity, 73 (60.3%) had positive family
history. Of these case, 57 (47.5%) had complications of diabetes, hypertension
or dyslipidemia conforming with the definition of metabolic syndrome
(Alberti and Zimmet, 1998; Grundy
et al., 2005; Alberti et al., 2005)
while the rest of cases (63; 52.5%) were not complicated and were termed simple
obesity. These cases were compared to 83 (9 males and 74 females) normal
healthy controls from the same locality of an age Mean±SD of 29.62±9.73
years. They were taken from blood donors after confirming that they are free
from obesity, hypertension or other cardiovascular disorders in addition to
a negative family history of similar conditions.
Measurements of lipids: After obtaining informed consent, blood samples were obtained from all cases and controls in the morning after fasting for 12 h. Immediately following clotting, serum was separated by centrifugation for 15 min at 3000 rpm.. The levels of TC (Total cholestrol), TG (Triglyceride), HDL-C (High Density Lipoprotein Cholesterol) and LDL-C (Low Density Lipoprotein Cholesterol) in samples were determined by enzymatic methods with commercially available kits, Tcho-1, TG-LH (RANDOX Laboratories Ltd., Ardmore, Diamond Road, Crumlin Co. Antrim, United Kingdom, BT29 4QY), Cholestest N HDL. and Cholestest LDL (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), respectively.
DNA extraction, purification and amplification: Another venous blood samples (3 mL) were collected on EDTA (ethylenediamine tetra acetate) containing tubes, DNA was extracted promptly using DNA extraction and purification Kit (Gentra Systems, USA) according to manufacturer's instructions and then stored at -20°C till use.
LPL gene amplification was carried out by PCR using the selected sequences
for 5 and 3 primers: SB-75: 5-ATG GCACCC ATG TGT AAG GTG-3
and SB-76: 5GTG AAC TTC TGA TAA CAA TCT C-3 (Georges
et al., 1996). Quality analysis of the PCR products (430 bp-long)
was performed by electrophoresis with a 50 bp marker (Pharmacia Biotech, Uppsala,
Sweden) on 1.5% agarose gel.
Restriction fragment polymorphism analysis (RFLP) of amplified DNA: Samples of PCR products (8 uL) were then incubated with Pvu II restriction endonuclease (Boehringer) overnight at 37°C. The 430 bp-long product was digested to 320 and 110 bp-long products if there was a Pvu II restriction site (+) and remain as it is if it is absent (-). The digested DNA was electrophoresed on a 2% agarose gel stained with ethidium bromide (90 V/1 h), visualized under UV light and photographed. The length of each amplified DNA fragment was determined by comparing migration of a sample with that of standard DNA marker (Fig. 1, 2).
||Amplification product (430 bp) of LPL gene using Primer 1,
Primer 2 M and molecular marker bp base pair. lane 1: -/- (no digestion)
which include 2. lane 2: -/+ (digestion of one allele) which include 1,5,
7.lane 3: +/+ (digestion of both allele) which include 3,4,6. M molecular
marker bp base pair
||Digestion products of amplified segment of LPL gene (430 bp)
using PVU II restriction enzyme showing lane 1: -/- (no digestion) which
include 2. lane 2: -/+ (digestion of one allele) which include 1,5, 7. lane
3: +/+ (digestion of both allele) which include 3,4,6. M molecular marker
bp base pair
Statistical analysis: Statistical analysis of data was done using the
software statistical package SPSS program version 17 (Chicago, USA). Student
t-test was used to compare the numerical values related to lipid profile, body
mass index and waist hip ratio, whereas Chi-square, Fisher exact and odds ratio
with 95% confidence interval were used to compare frequencies of different genotypes
and alleles among cases and controls.
Table 1 shows comparison between BMI, waist hip ratio and lipid profile among studied cases of obesity compared to controls. The BMI of total cases was significantly higher than that of the controls that was more obvious among metabolic syndrome cases. Similarly, the waist hip ratio of total cases was significantly higher that of the controls that was more obvious among simple obesity cases that were also significantly higher than metabolic syndrome cases. Lipid profile TC, TG, HDL-C and LDL-C of total cases was significantly higher than that of the controls. On the other hand, metabolic syndrome cases showed significantly higher TG with HDL-C levels than simple obesity cases.
Comparison between cases with obesity and healthy controls regarding their
genotype and allelic distribution of LPL Gene Pvu II polymorphism is shown in
Table 2. From this Table 2 it is noted that
obesity cases had a significantly higher frequency of the homozygous mutated
(+/+)genotype of LPL Gene Pvu II polymorphism particularly among cases with
metabolic syndrome compared to controls (p = 0.001, OR = 6.6 and p<0.0001,
OR = 10.4 respectively).
||Comparison between cases with obesity and healthy controls
regarding their genotype and allelic distribution of LPL Gene Pvu II polymorphism
|*p significant = 0.05, **p significant <0.001 OR (95% CI)
= Odds ratio (95% confidence interval)
|| Body mass index (BMI), waist hip ratio, age of onset and
lipid profile among cases of obesity related to their LPL genotypes
|*p significant = 0.05
Cases of obesity had also a significantly higher frequency of (+) allele of
LPL Pvu II gene polymorphism particularly also the metabolic syndrome cases
compared to controls (p = 0.005, p<0.0001, respectively).
Also from this Table 2 it is noted that simple obesity cases showed also a higher frequency of the homozygous mutated (+/+) genotype of LPL Pvu II gene polymorphism yet this was statistically near significant (p = 0.05, OR = 3.9).
On other hand, by comparing metabolic syndrome cases to simple obesity cases it noted that metabolic syndrome cases had a significantly higher frequency of the homozygous mutated (+/+) genotype of LPL Gene Pvu II polymorphism (p= 0.04, OR = 2.7).
Table 3 shows a comparison between BMI, waist hip ratio, age of onset, Family History, Gender, Consanguinity, Hypertension, Diabetes and lipid profile among cases of obesity related to their LPL genotypes. From this Table 3, it is noted that Cases with the (+/+) homozygous genotype showed significantly higher frequency of diabetes, lower frequency of positive family history and lower values for waist hip ratio than those with the (+/-) and (-/-) genotypes. These cases have showed also higher levels of total cholesterol and LDL-C, yet not reaching statistical significance.
Excess adipose tissue lead to Obesity (Spiegelman and Flier,
2001). Obesity cause morbidity because of hypertension, type 2 diabetes
mellitus, dyslipidemia, endocrinal abnormalities besides it can cause mortality
due to some cancers like esophagus, colon, rectum and breast (Spiegelman
and Flier, 2001). Obesity can be combined with metabolic syndrome which
includes a group of conditions as increased blood pressure, elevated insulin
levels, excess body fat around the waist or abnormal cholesterol levels that
stimulate the risk of heart disease, stroke and diabetes. Lipoprotein lipase
(LPL) is a key enzyme in lipoprotein metabolism through hydrolysis of triglyceride-rich
particles in muscles, adipose tissues and macrophages, thereby generating free
fatty acids and glycerol for energy utilization and storage (Goldberg,
In this work, we explored the association of genetic polymorphisms of lipoprotein lipase gene Pvu II site with the development of either simple obesity or metabolic syndrome among Egyptian cases, correlating it to other clinical variables including gender, age of onset, Body Mass Index (BMI), waist hip ratio, diabetes, hypertension in addition to lipid profile and glucose. Our study results showed that the frequency of homozygous mutated (+/+) genotype and mutant (+) allele of LPL Pvu II gene polymorphism were significantly higher among cases of obesity associated with metabolic syndrome compared to controls. Thus (+/+) genotype and (+) allele may be considered as genetic risk factors for complicated obesity. This might support the autosomal recessive mode of the effect of this gene so that the homozygous mutant (+/+) is associated with the manifestation of complicated obesity.
These results are in agreement with results of (Sertic et
al., 2009) who stated that LPL genetic polymer variants could represent
predictive genetic risk markers for obesity-related metabolic disorders in young
healthy subjects. Also these results are in agreement with results of Liu
et al. (2005) who stated that, LPL Pvu II polymorphisms are determinants
of plasma LPL concentration among Chinese population. These results are in agreement
with results of with Zhu et al. (2003) who stated
that the variants of LPL-Pvu II locus were important determinants of variation
in serum cholesterol response to dietary change in hyperlipidemia population
among Chinese population and in agreement with results of Wang
et al. (1996) who stated that, Our patients with the Pvu II (+/+)
genotype were significantly more likely to have diabetes. As far as we are aware,
this has not been reported previously among Chinese population and with results
of with Das et al. (2009) who stated that, LPL
may have strong genetic association with hypertensive individuals among India
population, and Wang et al. (2011) who studies
LPL Pvu II polymorphism LPL gene and measured the serum lipid levels in a case-control
study among preschool Chinese children. The variant genotypes of LPL Pvu II
CC (+/+) were associated with a significantly increased risk of childhood obesity.
These results are in agreement with results of Smart et
al. (2010) who demonstrated that these common variants were apparently
impacting the lipid levels in a healthy paediatric cohort, suggesting that even
in these young children there may be potential in predicting their lifelong
exposure to an adverse lipid profile. These results are in agreement with results
of Voruganti et al. (2010) who stated there is
strong genetic influence on plasma fatty acid distribution and that genetic
variation in LPL that may play role in plasma fatty acid distribution among
American studied subjects. Also, Kisfali et al. (2010)
stated that, the number of candidate genes in metabolic syndrome and coronary
heart disease susceptibility increases very rapidly from the growing spectrum
of the genes influencing lipid metabolism like the LPL. Conversely, these results
were in disagreement with results of Jemaa et al.
(1995) who stated that, The Pvu II polymorphisms did not exhibit any significant
association with the biochemical traits (total cholesterol, low-density lipoprotein
(LDL) cholesterol, high-density lipoprotein) among French population. Also,
these results were in disagreement with the results of Shen
et al. (2000), who stated that,: The LPL Pvu II is not significantly
associated with type 2 diabetes mellitus in Chinese population and Comparing
cases-subgroups according to their family history as regard their studied genotypes,
it is noted the cases with positive family history of obesity has a significantly
higher frequency of the heterozygous mutant (+/-) genotype, whereas cases with
negative family history showed a significantly higher frequency of the wild
type or normal (-/-) genotype. This supports the familial nature of obesity
particularly when associated with the (+) mutant allele. Interestingly, however,
the (+/+) genotype was not significantly different when comparing cases with
positive to that with negative family history. This may be due to the fact that
most of our cases were taken from the outpatient ambulatory cases when it is
expected that most of the cases with the (+/+) genotype will have a severe form
of complicated obesity that requires inpatient or intensive unit (ICU) care.
So, we recommend taking a wider scale sample of cases including inpatient and
ICU cases to get a proper picture of the familial pattern of the disease.
BMI regarding their LPL genotypes, although, there are significant differences between cases and healthy controls (p<0.001) as being very high regardless of their genotypes, cases did not show a significant difference in between each other.
This study showed a significant association between the LPL Pvu II gene polymorphism and obesity particularly when complicated with metabolic syndrome among Egyptian cases. The genotype (+/+) was mostly associated with the risk of complicated obesity. Although, lipid profile was significantly higher among obesity cases compared to controls irrespective of the LPL genotype variants, it was non-significantly different between cases subgroups related to different LPL genotypes.
1: Suleiman, A.A., O.K. Alboqai, N. Yasein, J.M. El-Qudah, M.F. Bataineh and B.A. Obeidat, 2009. Prevalence of and factors associated with overweight and obesity among Jordan University Students. J. Biol. Sci., 9: 738-745.
CrossRef | Direct Link |
2: Alberti, K.G.M.M., P. Zimmet and J. Shaw, 2005. The metabolic syndrome-a new worldwide definition. Lancet, 366: 1059-1062.
3: Alberti, K.G.M.M. and P.Z. Zimmet, 1998. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus. Provisional report of a WHO consultation. Diabetic Med., 15: 539-553.
CrossRef | PubMed | Direct Link |
4: Das, B., N. Pawar, D. Saini and M. Seshadri, 2009. Genetic association study of selected candidate genes (ApoB, LPL, Leptin) and telomere length in obese and hypertensive individuals. BMC Med. Genet., Vol. 10.
CrossRef | Direct Link |
5: Garfinkel, A.S. and M.C. Schotz, 1987. In Plasma Lipoproteins. In: Lipoprotein Lipase, Gotto A.M. (Ed.). Elsevier, New York, pp: 335-357
6: Georges, J.L., A. Regis-Bailly, D. Salah, R. Rakotovao, G. Siest, S. Visvikis and L. Tiret, 1996. Family study of lipoprotein lipase gene polymorphisms and plasma triglyceride levels. Genet. Epidemiol., 13: 179-192.
7: Goldberg, I.J., 1966. Lipoprotein lipase and lipolysis: Central roles in lipoprotein metabolism and atherogenesis. J. Lipd Res., 37: 693-707.
8: Grundy, S.M., J.I. Cleeman, S.R. Daniels, K.A. Donato and R.H. Eckel et al., 2005. Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung and Blood Institute Scientific statement. Circulation, 112: 2735-2752.
PubMed | Direct Link |
9: Jemaa, R., S. Tuzet, C. Portos, D. Betoulle, M. Apfelbaum and F. Fumeron, 1995. Lipoprotein lipase gene polymorphisms: Associations with hypertriglyceridemia and body mass index in obese people. Int. J. Obes. Relat. Metab. Disord., 19: 270-274.
PubMed | Direct Link |
10: Kisfali, P., N. Polgar, E. Safrany, K. Sumegi and B.I. Melegh et al., 2010. Triglyceride level affecting shared susceptibility genes in metabolic syndrome and coronary artery disease. Curr. Med. Chem., 17: 3533-3541.
11: Lithell, H., J. Boberg, K. Hellsing, G. Lundqvist and B. Vessby, 1978. Lipoprotein-lipase activity in human skeletal muscle and adipose tissue in the fasting and the fed states. Aterosclerosis, 30: 89-94.
12: Liu, J., D. Zhao, J. Liu, S. Liu, L.P. Qin and Z.S Wu., 2005. The effect of lipoprotein lipase (LPL) polymorphism on plasma LPL concentration and triglyceride. Zhonghua Yi Xue Za Zhi, 85: 1339-1343.
13: Monsalve, M.V., H. Henderson, G. Roederer, P. Julien and S. Deeb et al., 1990. Amissense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries. J. Clin. Invest., 86: 728-734.
CrossRef | Direct Link |
14: Rampersaud, E., B.D. Mitchell, T.I. Pollin, M. Fu and H. Shenet al., 2008. Physical activity and the association of common FTO gene variants with body mass index and obesity. Arch. Int. Med., 168: 1791-1797.
Direct Link |
15: Sertic, J., L. Juricic, H. Ljubic, T. Bozina and J. Lovric et al., 2009. Variants of ESR1, APOE, LPL and IL-6 loci in young healthy subjects: Association with lipid status and obesity. BMC Res. Notes, Vol. 2.,
CrossRef | Direct Link |
16: Shen, H., S. Yu, Y. Xu, R. Yu, W. Jiang and W. Chen, 2000. DNA polymorphism of Pvu II site in the lipoprotein lipase gene in patients with type 2 diabetes mellitus. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 17: 24-27.
17: Smart, M.C., G. Dedoussis, E. Louizou, M. Yannakoulia and F. Drenos et al., 2010. APOE, CETP and LPL genes show strong association with lipid levels in Greek children. Nutr. Metab. Cardiovasc. Dis., 20: 26-33.
CrossRef | Direct Link |
18: Sparkes, R.S., S. Zollman, I. Klisak, T.G. Kirchgessnerb and M.C. Komaromy et al., 1987. Human genes involved in lipolysis of plasma lipoproteins: mapping of loci for lipoprotein lipase to 8p22 and hepatic lipase to 15q21. Genomics, 1: 138-144.
19: Spiegelman, B.M. and J.S. Flier, 2001. Obesity and the regulation of energy balance. Cell, 104: 531-543.
CrossRef | PubMed | Direct Link |
20: Teran-Garcia, M. and C. Bouchard, 2007. Genetics of the metabolic syndrome. Applied Physiol. Nutr. Metab., 321: 89-114.
21: Voruganti, V.S., S.A. Cole, S.O. Ebbesson, H.H. Goring and K. Haack et al., 2010. Genetic variation in APOJ, LPL and TNFRSF10B affects plasma fatty acid distribution in Alaskan Eskimos. Am. J. Clin. Nutr., 91: 1574-1583.
Direct Link |
22: Wang, L.N., Q. Yu, Y. Xiong, L.F. Liu and Z. Zhang et al., 2011. Lipoprotein lipase gene polymorphisms and risks of childhood obesity in Chinese preschool children. Eur. J. Pediatr, 170: 1309-1316.
23: Wang, X.L., R.M. McCredie and D.E.L. Wilken, 1996. Common DNA polymorphism at the lipoprotein lipase gene association with severity of CAD and diabetes. Circulation, 93: 1339-1345.
Direct Link |
24: Zhu, W., Z. Zhang, J. Wang and Z. Qi, 2003. Relations of lipoprotein lipase gene polymorphism at Pvu II locus and dietary intervention predisposition in hyperlipidemia population. Wei Sheng Yan Jiu, 32: 147-149.