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Division of Endocrinology, Diabetes and Metabolism, Department of Medicine (M.O.G) and Medical Genetics Institute (M.O.G., K.D.T., X.G., H.J.A., H.Y., J.I.R.), Cedars-Sinai Medical Center, Los Angeles, California 90048; the Lipid Research Laboratory, Veterans Affairs Greater Los Angeles Healthcare Center (H.W.) and the Department of Medicine (H.W.), University of California, Los Angeles, California 90073; and Divisions of Endocrinology, Diabetes and Hypertension (M.J.Q., W.A.H.) and Cardiology (L.W.C.), Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90025
Address all correspondence and reprint requests to: Mark O. Goodarzi, M.D., Ph.D., Cedars-Sinai Medical Center Division of Endocrinology, Diabetes and Metabolism, 8700 Beverly Boulevard., Becker B-128, Los Angeles, California 90048. E-mail: mark.goodarzi{at}cshs.org.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Objective: Based on rodent studies, we hypothesized that these haplotypes are in linkage disequilibrium with functional variants in the 3' untranslated region of LPL, which is encoded by exon 10, and that these variants influence phenotype by altering LPL expression.
Design: We sequenced exon 10 in subjects with divergent insulin sensitivity and divergent haplotypes. We also sequenced the other common LPL haplotypes. Variants identified by sequencing were genotyped in a large, family-based population along with the six SNPs spanning intron 7 to intron 9. We tested the potential functional significance of variation in exon 10 by evaluating association of haplotypes with post-heparin plasma LPL activity.
Setting: The study took place within the general community, with the Mexican-American Coronary Artery Disease Project cohort.
Participants: Participants included 847 subjects from 163 families.
Main Outcome Measures: We determined LPL haplogenotype and post-heparin plasma LPL activity.
Results: Exon 10 sequencing identified 15 variants. Thirteen of these variants were genotyped in large-scale along with the six SNPs spanning intron 7 to intron 9. LPL haplotypes and their relative frequencies in Mexican-Americans were determined. The fourth most common haplotype based on 19 SNPs (haplotype 19-4) was associated with increased LPL activity as well as multiple phenotypes related to the metabolic syndrome.
Conclusions: These results support the possibility that variation in the 3' untranslated region of LPL affects LPL expression and activity, consequently influencing risk of atherosclerosis and insulin resistance, and provides important tools for further dissection of LPL regulation.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our work has focused on the 3' end of the LPL gene, distal to a recombination hot spot located in intron 6 (13). We characterized the haplotype structure of the gene composed of variants spanning intron 6 to intron 9; six single nucleotide polymorphisms (SNPs) were sufficient to uniquely identify the common haplotypes (11). Among Mexican-American individuals ascertained by a family history of coronary artery disease (CAD) (from the Mexican-American Coronary Artery Disease Project), these LPL 3'-end haplotypes were associated with CAD (11). We subsequently demonstrated that these haplotypes were associated with insulin sensitivity/resistance, directly quantified using the euglycemic-hyperinsulinemic clamp in this cohort (5). A consistent picture arose wherein the most common haplotype (haplotype 1) was associated with insulin sensitivity and protection against CAD, and the fourth most common haplotype (haplotype 4) was associated with insulin resistance and a tendency to increased risk of CAD. These studies suggested that LPL is a possible genetic link underlying the often-observed co-occurrence of insulin resistance and coronary disease.
Given the association of LPL 3'-end haplotypes with CAD and insulin resistance, we hypothesized that these haplotypes are in linkage disequilibrium with functional polymorphisms in the 3' untranslated region (UTR) of LPL, encoded by exon 10. The 3' UTR comprises about half of the LPL mRNA, and studies in rodents have demonstrated that sequences in the 3' UTR of LPL may regulate LPL translation (14, 15, 16). In this study, we 1) sequenced LPL exon 10 from insulin-sensitive and insulin-resistant individuals with relevant LPL haplotypes to describe the polymorphism and haplotype structure of the 3' UTR of LPL, and 2) compared the post-heparin plasma LPL activity of subjects with different haplotypes to examine the potential functional significance of this LPL variation. To provide additional confirmation of the physiological relevance of LPL haplotypes, secondary association analyses were conducted with multiple phenotypes related to the metabolic syndrome.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The University of California, Los Angeles/Cedars-Sinai Mexican-American Coronary Artery Disease (MACAD) Project enrolled families ascertained through a proband (parent) with CAD, determined by evidence of myocardial infarction on electrocardiogram or hospital record, evidence of atherosclerosis on coronary angiography, or history of coronary artery bypass graft or angioplasty (5, 11). Two generations were enrolled into the study: 1) the proband and proband spouses (parental generation) and 2) their adult (age 18 yr or older) offspring and the spouses of those offspring (offspring generation). All subjects were genotyped, and only the offspring generation was phenotyped. By design, the offspring were free of diabetes and clinically manifest cardiovascular disease, thus avoiding secondary changes in phenotype caused by overt disease. In the present study, 891 subjects from 163 families were genotyped; of these, 497 adult offspring and offspring spouses had undergone assessment of post-heparin plasma LPL activity at the time of the analyses reported herein. Insulin sensitivity was determined using the euglycemic-hyperinsulinemic clamp, which yields the M value; higher M values indicate higher sensitivity to insulin, and lower M values indicate insulin resistance (5, 17).
All studies were approved by Human Subjects Protection Institutional Review Boards at University of California, Los Angeles, and Cedars-Sinai Medical Center. All subjects gave informed consent before participation.
Subject selection for sequencing
We initially planned to sequence exon 10 in a minimum of 12 subjects from the MACAD population, to give us a 99% power to detect at least one polymorphism with an allele frequency of 10%. Ideally, we would have selected four insulin-resistant subjects with haplogenotype 4/4, four insulin-sensitive subjects with haplogenotype 4/4, and four insulin-sensitive subjects with haplogenotype 1/1. However, the offspring generation contained no haplotype 4/4 homozygotes. Therefore, we initially sequenced exon 10 in four insulin-sensitive subjects of haplogenotype 1/1 (mean M, 9.17 mg/kg·min), four insulin-resistant subjects of haplogenotype 1/4 (mean M, 2.23 mg/kg·min), and four insulin-sensitive subjects with haplogenotype 1/4 (mean M, 8.46 mg/kg·min); subjects were unrelated. This strategy of sequencing subjects with divergent genotypes and divergent phenotypes was chosen to maximize the chance of identifying variation with a functional impact. Six parents of four individuals were sequenced to facilitate identification of the haplotype phase of identified variants.
To completely characterize the haplotype structure of LPL exon 10, we also sequenced subjects carrying haplotypes 2, 3, and 5 (without consideration of phenotype). Haplotypes 2 and 3 were sufficiently common in the cohort to provide homozygotes for sequencing. Two haplotype 2 homozygotes and three haplotype 3 homozygotes were sequenced. To sequence the rarer haplotype 5 (founder frequency of 2.4%) (5), we selected three subjects of haplogenotype 1/5.
Sequencing methodology
We sequenced the 1949-bp sequence of exon 10 as well as 65 bp of upstream and 63 bp of downstream genomic sequence. This sequence was divided into four overlapping segments. PCR was used to amplify each fragment; the PCR primer sequences are as follows: forward 5'-CAGGCGGGAATTGTAAAACA-3' and reverse 5'-TTGACGTCTGGACCACATTC-3'; forward 5'-CTGGATCTTTCGGACTGAGG-3' and reverse 5'-CAGGAACCTCTCCACCCTTT-3'; forward 5'-TTCCAGTGCGTCTCTTTTGTT-3' and reverse 5'-ATTCCAAGCCTGATGATGTT-3'; and forward 5'-TTGTTCCTGATGTGCCAGAA-3'and reverse 5'-TGCTGAGTGAATCTGACCTAAGAA-3'. Each amplified segment was sequenced in both directions using BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems, Foster City, CA). Sequence was determined on an ABI 377 automated sequencer. Variation was identified by comparison to the reference sequence from GenBank (NT_030737). At this time, the National Center for Biotechnology Information’s dbSNP build 123 (http://www.ncbi.nlm.nih.gov/SNP/) lists 21 SNPs in LPL exon 10.
Genotyping and haplotype determination
We designed PCR primers and TaqMan minor groove binder (MGB) (Applied Biosystems) probes to genotype the following exon variants identified from sequencing exon 10: rs11570891, rs4922115, rs3289, rs11570892, rs1803924, rs1059507, rs3735964, rs3200218, rs13702, rs1059611, rs10645926, rs15285, and rs3866471 (Table 1
). We were unable to design suitable probes for the SNP rs3208305 because it is immediately adjacent to a polyadenine (polyA) tract. We also could not design probes for the uncharacterized insertion/deletion. Thus, of the 15 variants identified by sequencing exon 10, we genotyped 13.
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Haploview 3 was used to determine haplotypes as well as delineate haplotype blocks (21). Haploview constructs haplotypes by using an accelerated expectation maximization algorithm similar to the partition/ligation method (22), which creates highly accurate population frequency estimates of the phased haplotypes based on the maximum likelihood derived from the unphased input genotypes. Of the 847 subjects genotyped at all 19 LPL variants, 810 were assigned a haplogenotype (i.e. two haplotypes). Haploview was used to calculate linkage disequilibrium (LD, the D' statistic) between each pairwise combination of all 19 SNPs used in haplotype block determination. Haploview was then used to assign haplotype blocks using a variant of the four-gamete rule (23).
Phenotyping
For post-heparin lipase activity determination, subjects were asked to come to the General Clinical Research Center after fasting for 12 h. Subjects who had evidence of anemia on complete blood count, evidence of hematuria on urinalysis, or a positive pregnancy test were excluded. Eligible subjects received an iv bolus of heparin (60 U/kg), followed by collection of post-heparin blood 10 min later. Administration of heparin to subjects releases both LPL and hepatic lipase (HL) from capillary endothelial cells, allowing their collection in peripheral blood. Blood samples are then assayed for lipase activity by measuring the lipolysis of a radiolabeled triolein substrate, yielding activity resulting from the action of both LPL and HL (24). Because high-salt conditions inhibit LPL activity but not HL activity, LPL activity is then derived from the difference of total lipase activity and that activity determined in the presence of 1 M NaCl. When this study was performed, 497 subjects from the offspring generation had undergone post-heparin lipase activity determination. Of these, 397 subjects from 112 families had been haplotyped at the 19 LPL markers.
Phenotypes relevant to the metabolic syndrome, including body mass index (BMI), waist and hip measurements, fasting lipid profile, apolipoproteins, fasting insulin, fasting and postprandial glucose measurements, and hyperinsulinemic-euglycemic clamp were performed as previously described (5, 25).
Association analysis
Association of lipase activity and metabolic phenotypes with LPL 3'-end variants was evaluated using a robust variance estimation approach, employing the generalized estimating equation (GEE1) ((26) to test hypothesized associations between phenotypes and haplotypes while accounting for familial correlations present in the family data. The PROC GENMOD procedure in SAS (version 8.0; SAS Institute, Cary, NC) was used for the analysis using the GEE1 model. Family was taken as the cluster factor; i.e. members from the same family were assumed to be correlated, and those from different families were assumed to be independent. Age, sex, and BMI were included as covariates in all analyses, except when indices of adiposity were being analyzed for association, in which case only age and sex were taken as covariates. Quantitative trait values were log or square root transformed as appropriate to reduce skewness.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Sequencing of 12 individuals (and six parents) with haplotypes 1 and 4 and extremes of insulin sensitivity identified 10 variants, composed of eight SNPs (rs11570891, rs3289, rs3208305, rs1803924, rs3735924, rs13702, rs1059611, and rs15285), one two-nucleotide insertion/deletion (rs10645926), and another insertion/deletion. The latter insertion/deletion could not be characterized because it was not present in the homozygous state in any of the subjects sequenced. One SNP (rs11570891) was 11 bp proximal to exon 10.
Sequencing common LPL haplotypes other than 1 and 4
Sequencing of haplotypes 2, 3, and 5 yielded five additional SNPs that were not identified on haplotypes 1 or 4. Of the LPL exon 10 SNPs listed in dbSNP, seven were not found on chromosomes defined by the most common LPL 3'-end (intron 7 to intron 9) haplotypes 1 through 5 in this population.
Haplotype structure
Table 2 shows the frequency and position information of the 19 LPL variants based on genotyping in the 847 subjects genotyped for all 19 variants. The haplotypes constructed based on these 19 variants [six original LPL SNPs and the 13 exon 10 variants (12 SNPs and the TT insertion)] are listed in Table 3, along with their respective frequencies. These haplotypes are labeled 19-1, 19-2, 19-3, etc. to denote that they are based on a total of 19 polymorphisms, and to avoid confusion with the original six-SNP-based haplotypes 1, 2, 3, 4, etc. The haplotypes (19-1 to 19-7) occurring at a frequency of greater than approximately 1% were identical to those predicted from the exon 10 sequencing. These seven haplotypes together comprise 94% of the haplotypes found in this population. The original six-SNP-based haplotypes are also listed in Table 3, in rows corresponding to the new 19-SNP-based haplotypes. The latter shows that haplotype 1 was subdivided into three new haplotypes; however, one haplotype (19-1) remained common, whereas the new sub-haplotypes (19-5 and 19-7) occurred with low frequency. The other common haplotypes (3, 4, and 5) were extended 3' into exon 10 without generating new haplotypes. Linkage disequilibrium (D') ranged from 0.04–1, and haplotype block determination showed that the 19 SNPs lie in two haplotype blocks (Fig. 1). The exon 10 variants are entirely included in the large 3'-end block.
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Of the common haplotypes, haplotype 19-4 showed association with LPL activity (P = 0.025). Figure 2 shows the mean LPL activity level for carriers of each of the most common haplotypes. Haplotype 19-4 is associated with elevated LPL activity. The mean LPL activity level in the whole haplotyped population was 24.4 mU/ml (1 U = 1 µmol of FFA released per minute). Carriers of haplotype 19-4 had a mean post-heparin LPL activity level of 31.8 mU/ml, whereas subjects without haplotype 19-4 had a mean LPL activity of 23.4 mU/ml.
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) suggests that the variant(s) that increase lipase activity lie on the distal haplotype of 19-4. Association of LPL haplotypes with metabolic traits
Given the association of haplotype 19-4 with LPL activity, we next evaluated the physiological relevance of this finding by assessing the association of haplotype 19-4 with multiple lipid, apolipoprotein, adiposity, insulin resistance, and blood pressure traits. Haplotype 19-4 was associated with increased BMI, high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I, apolipoprotein A-II, and systolic blood pressure and with decreased insulin sensitivity (M value) (Table 4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The LPL gene is approximately 30 kb in genomic length and is composed of 10 exons (nine introns). The 10th exon is large (1949 bp) and codes for an approximately 2-kb (1948-bp) 3' UTR, which makes up over half of the mature LPL mRNA. The LPL enzyme hydrolyzes triglycerides in circulating very low-density lipoprotein (VLDL) cholesterol and chylomicrons, providing FFAs and monoacylglycerol for use by the surrounding target tissues (27). LPL is located in capillary endothelium and is most abundant in adipose tissue and cardiac and skeletal muscle but is also found in other tissue types of relevance, such as vascular wall monocytes.
Our efforts were focused on the 3' end of the LPL gene because polymorphisms in the 3' end, such as HindIII, have been associated with insulin resistance and with atherosclerosis (3, 4, 10). We previously took advantage of the well-characterized catalog (20) of SNPs in the 3' end of the LPL gene (intron 6 to intron 9), to construct haplotypes to use in association analysis against insulin resistance and atherosclerosis (5, 11). Once associated haplotypes were found, we searched for functional variants by sequencing exon 10 in subjects with these haplotypes. When our study began, exon 10 had not been as rigorously sequenced as the region from exon 4 to exon 9 (20). Our hypothesis is that functional variation in the 3' UTR (encoded by exon 10) may influence these phenotypes by altering translation of LPL. The high degree of sequence homology (75%) between human and mouse 3' UTR suggests that the LPL 3' UTR has functional significance (28).
Experimental evidence that the 3' UTR plays a role in regulating translation of LPL comes from studies in rodents. Kern and colleagues implicated a role for the 3' UTR in LPL translation regulation in the setting of diabetes. Induction of diabetes by streptozocin treatment in rats led to a 75% decrease in LPL activity and synthesis with no change in the mRNA level and no change in the amount of mRNA associated with polysomes (no inhibition of translation initiation) (14). Cytoplasmic extracts from diabetic rat adipose tissue inhibited in vitro translation if 1818–2000 of the LPL 3' UTR was present (14). Gel shift studies suggested a protein in diabetic adipose extract binds there and inhibits LPL translation (14). Insulin-treated rats had increased LPL toward normal.
Indirect evidence that the 3' UTR may affect LPL translation in humans comes from studies in transgenic mice expressing human LPL specifically in adipose tissue. Two transgenic constructs were used, one containing the entire coding sequence of human LPL and the 3' UTR, and another containing the coding sequence of human LPL with only the distal approximately 750 nucleotides of the 3' UTR (15). The proximal 3' UTR was deleted in light of the above experimental evidence on the importance of this region in LPL translation regulation. Both sets of transgenic mice overexpressed human LPL 2-fold, but the 3' UTR deletion mice did so with a much lower level of human LPL mRNA, suggesting that deletion of the 3' UTR resulted in a translationally unrepressed LPL (15). Whether the 3' UTR truly plays a role in humans remains to be determined. We investigated this question with a population genetic, haplotype-based approach.
Haplotypes reflect global gene structure, encompassing chromosomal blocks that have remained unbroken by recombination during the population history of the gene. Thus, haplotypes capture the majority of common variation in a gene; consequently, the use of haplotypes is more likely to identify disease variation associations than is the use of a random single polymorphism. Identification of a haplotype associated with increased or decreased disease risk should facilitate identification of the actual functional variant that affects disease risk, because this variant should lie on chromosomes identified by that haplotype (29, 30).
We found that haplotype 19-4 was associated with post-heparin LPL activity. The minor alleles of six variants (rs328, rs11570891, rs1803924, rs3735964, rs1059611, and rs10645926), the latter five of which were identified by sequencing exon 10, are found uniquely on haplotype 19-4. These 3' UTR variants are candidates for variants that alter the expression of LPL and thus influence LPL activity. Some investigators (31) but not others (32, 33) have successfully identified association of LPL polymorphisms with post-heparin LPL activity. Our data do not allow us to distinguish whether one of these SNPs has a functional impact on LPL expression or whether several or all the SNPs acting together affect LPL expression. Ser447Stop (rs328 or 9040), the rare allele of which is found only on haplotype 19-4 (Table 3), has in many but not all studies been associated with increased LPL activity both in population genetic studies (31) and in in vitro experimentation that has suggested either increased specific activity (34) or increased secretion of catalytically normal LPL (35). At this time, researchers do not agree on the exact molecular consequences of the 447Stop variant, which truncates only the last two amino acids from the enzyme. We suggest that effects of this SNP on LPL activity may be a manifestation of the linkage disequilibrium with functional variants in the 3' UTR. The finding of normal enzyme activity with increased production (35) is consistent with increased translation mediated by 3' UTR variation that is linked to 447Stop (in a model where 447Stop is nonfunctional).
Additional evidence of the physiological relevance of haplotype 19-4 came from secondary analyses demonstrating association with multiple metabolic traits. As would be predicted on the basis of increased LPL activity, haplotype 19-4 was associated with increased HDL-C and a trend to decreased triglycerides. Apolipoproteins A-I and A-II, the major lipoproteins of HDL particles, were both increased by haplotype 19-4, with a decreased A-I/A-II ratio (Table 4). The relative increase in apolipoprotein A-II may explain the association of haplotype 19-4 with insulin resistance and atherosclerosis, despite the higher HDL-C levels. Transgenic mice overexpressing apolipoprotein A-II have skeletal muscle insulin resistance and accelerated atherosclerosis; HDL particles from these mice exhibit proinflammatory and prooxidant properties (36, 37).
Post-heparin plasma LPL activity measures whole-body LPL activity and therefore does not provide information on tissue-specific effects of haplotype 19-4. LPL is known to have complex, tissue-specific regulation; for example, in the fed state, adipose LPL activity is increased and muscle LPL activity is decreased (38). We hypothesize that haplotype 19-4 promotes insulin resistance by increasing LPL activity and FFA uptake in skeletal muscle, promotes atherosclerosis and hypertension by increasing LPL activity in vessel wall macrophages, and increases adipose tissue LPL activity promoting fat storage and obesity; however, specific study of LPL activity in such tissues isolated from carriers of haplotype 19-4 will be needed to confirm these hypotheses.
Although not allowing us to determine which exon 10 variant or combination thereof on haplotype 19-4 affects LPL expression, our data do exclude the first four (introns 7 to 8) variants of the 5'-end haplotype block because subjects who differed only at this haplotype segment (those with 19-3 and 19-6) had similar post-heparin lipase activities (Fig. 2). The 3'-end haplotype segment of 19-4 appeared uniquely associated with elevated lipase activity. Ser447Stop and the five exon 10 variants unique to haplotype 19-4 lie on this distal segment.
In an additional effort to assess the potential functional relevance of the five exon 10 variants unique to haplotype 19-4, we determined whether these polymorphisms lie in regions that display conservation with exon 10 across species, because conserved sequences are likely to be functionally important. We used the VISTA Browser, accessed from the Berkeley Programs for Genomic Applications (PGA) website (http://pipeline.lbl.gov/cgi-bin/gateway2), to identify conserved regions between the human LPL 3' UTR and that of the mouse, rat, and chicken. SNPs rs3735964 and rs1059611 and the dinucleotide insertion rs10645926 were conserved across species.
The sequence context of the SNPs found by sequencing was examined for known functional motifs from other genes whose expression is regulated by the 3' UTR, using the database of functional UTR motifs available at UTRdb (http://bighost.area.ba.cnr.it/BIG/UTRHome/) (39). None of the SNPs we identified by sequencing exon 10 was found in a known functional motif. We also did not find the occurrence of these exon 10 variants in the polyadenylation signals (the hexanucleotide AAUAAA) or polyadenylation sites in exon 10.
A final indication that variation in the LPL 3' UTR may have a functional impact comes from RNA secondary structure prediction. We compared the secondary structure of the reference sequence (major allele at all SNP sites) with that of haplotype 19-4 using the Vienna RNA Package (http://www.tbi.univie.ac.at/
ivo/RNA/) (40) and found a clear difference in secondary structure between the two (Fig. 3). This raises the possibility that an entire collection of variants may influence LPL translation by altering access of RNA binding proteins to the 3' UTR.
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In summary, this work extends our previous studies demonstrating association of LPL 3'-end (intron 7 to intron 9) haplotypes with insulin resistance and CAD, wherein haplotype 1 was protective and haplotype 4 predisposing to these conditions. To maximize our discovery of biologically significant variation, we selected subjects with divergent insulin sensitivity and haplotypes 1 and 4 for sequencing in exon 10. We characterized the haplotype structure of exon 10 and provide preliminary evidence of the functionality of these haplotypes by demonstrating association of haplotype 19-4 with post-heparin plasma LPL activity and multiple metabolic phenotypes, thus providing a potential molecular basis for the population clinical and phenotypic associations. Knowledge of functional 3' UTR variants may allow development of LPL-specific, tissue-specific therapy that may provide new avenues to treat and prevent insulin resistance, the metabolic syndrome, and ultimately atherosclerosis.
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Footnotes |
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First Published Online May 31, 2005
Abbreviations: BMI, Body mass index; CAD, coronary artery disease; FFA, free fatty acids; HDL-C, high-density lipoprotein cholesterol; HL, hepatic lipase; LPL, lipoprotein lipase; MACAD, Mexican-American Coronary Artery Disease; MGB, minor groove binder; SNP, single nucleotide polymorphism; UTR, untranslated region.
Received February 22, 2005.
Accepted May 24, 2005.
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