Ethnic Variation and In Vivo Effects of the −93t→g Promoter Variant in the Lipoprotein Lipase Gene
Abstract Recently, a (t→g) transition at nucleotide −93 in the lipoprotein lipase (LPL) gene promoter has been observed in Caucasians. Here, we have compared the frequency of the −93g carriers in three distinct populations (Caucasians, South African Blacks, and Chinese). The carrier frequency in the Caucasian population was 1.7% (4/232), which was in contrast to the South African Black population, which had a frequency for this allele of 76.4% (123/161) of the individuals tested. This transition was not observed in the Chinese population under study. Near complete linkage disequilibrium between the −93g and the previously described D9N mutation was observed in the Caucasian population but not in South African Blacks. To further assess the ancestral origins of these DNA changes, DNA haplotyping using a CA repeat 5′ to these substitutions was performed. The −93t allele was associated with only a few specific dinucleotide repeat sizes. In contrast, the −93g allele occurred on chromosomes with many different repeat lengths. The broad distribution of repeats on −93g carrying chromosomes, their high frequency in the South African Black population, and the conservation of the −93g allele among different species may suggest that the −93g allele is the ancestral allele on which a transition to t and the D9N mutations arose. The very high frequency of the −93g allele distinct from the N9 allele in a cohort of Black South Africans allowed us to specifically assess the phenotypic effects of the −93g allele on lipids. Individuals homozygous for the g allele at −93 showed mildly decreased triglycerides compared with individuals homozygous for the t allele (1.14±0.66 mmol/L versus 0.82±0.3; P=.04). Thus, the −93g allele in this cohort is associated with low plasma triglyceride levels.
- Received January 28, 1997.
- Accepted June 10, 1997.
Lipoprotein lipase (LPL) is crucial for lipoprotein metabolism when its main function is the hydrolysis of the triglyceride core of very low-density lipoproteins and chylomicrons.1 This results in the release of free fatty acids, which either undergo oxidation for use as energy or re-esterification for storage in adipocytes. LPL is synthesized in parenchymal cells, primarily in muscle and adipose tissue, after which it is transported to its site of action, the intimal surface of the vascular endothelium, to which it is anchored by heparan sulfate proteoglycan side chains.1
Complete enzyme deficiency caused by homozygosity or compound heterozygosity for mutations in the LPL gene results in familial chylomicronemia. This is a rare (approximately 1/106) autosomal recessive disorder characterized by massive elevation of plasma triglycerides, recurrent bouts of abdominal pain and/or pancreatitis, and the deposition of lipids in various body sites.1 Over 60 mutations in the coding region of the LPL gene causing complete enzyme deficiency have thus far been described.1 2 3 The carrier frequency for these mutations has been estimated to be 1 in 500 individuals.1
More recently, however, mutations in the LPL gene, which occur with significantly higher frequencies and result in a partially defective LPL protein, have been described in Caucasian populations.4 5 6 7 8 9 10 The N291S mutation has been identified in the Caucasian population with a carrier frequency of 2 to 5%.4 9 10 The D9N mutation in exon 2 has been identified in Caucasian populations with carrier frequencies of approximately 1 to 3%.5 6 This mutation results in a mild defect in LPL lipolytic function as assessed both in vitro5 11 and in vivo.5 8 These mutations have generally been associated with elevated triglyceride and/or reduced high-density lipoprotein (HDL) cholesterol levels.4 5 8 9
In addition to these mutations in the coding region of the LPL gene, three promoter mutations at positions −93, −53, and −39 have recently been identified in patients with familial combined hyperlipidemia.12 13 This disorder is characterized by elevated plasma levels of total cholesterol, triglycerides, or both in multiple individuals of the affected family.14 Although the substitutions at −53 and −39 are rare, the transition at −93 was reported to occur in 3 of 183 (1.6%) control individuals and at an increased carrier frequency of 5.2% (6/115) in a cohort of patients with coronary artery disease,13 which suggested that this particular mutation may contribute to dyslipidemia in atherosclerosis.13 Both decreased and increased LPL transcriptional activity have been suggested12 13 15 as the functional effects in vitro of the −93g allele.
Thus far, the frequency of the −93g allele in the LPL gene has not been examined in populations other than Caucasians. In the study contained herein, we sought to investigate the ethnic variation in frequency of this mutation. We studied the frequency of this DNA change in three ethnically diverse populations (Caucasian, South African Black, and Chinese) and show marked differences in the frequency of this DNA change in different populations. In particular, the −93g allele was not detected in persons of Chinese descent but, in contrast, this allele was wild-type (predominant) in South African Blacks. Haplotype analysis revealed that the −93g allele was almost always seen in association with the D9N mutation in Caucasians but not in Blacks. As the D9N mutation has been shown to be associated with the disturbances in lipids and increased progression of atherosclerosis, the question, therefore, remained as to whether the D9N mutation alone caused the phenotypic effects previously reported or whether the promoter mutation in almost complete nonrandom association might be contributing to this finding.
The high frequency of −93gg homozygotes and −93tt homozygotes in the absence of the D9N mutation in the South African Black population allowed us to directly address the phenotypic effects of this DNA transition. Herein, we show that the −93g allele had only a mild effect on lipid levels resulting in lowered triglyceride levels in these carriers.
To assess the frequency of the −93g allele in a Caucasian population, 232 unrelated male subjects <60 years of age, ascertained from a large Dutch population-based risk factor study were examined.16 All subjects were normolipidemic and had no history of coronary artery disease. No subject had any disease known to affect lipid metabolism including diabetes mellitus, hypertension, or thyroid, renal, or liver disease, and none were taking medications known to effect lipoprotein metabolism (diuretics, β-blockers, calcium channel blockers, and steroids including hormone replacement therapy and oral contraceptives).
To assess the chromosomal relationship between the −93g allele and the D9N mutation, a larger cohort of Caucasian carriers of these mutations was obtained for analysis of flanking short sequence repeats. Seventy-six subjects from the Framingham Offspring Study cohort consisting of individuals with mixed European descent (Reference 1717 and S.E. Gagné, unpublished data) were ascertained. This cohort comprised 39 D9N and −93g carriers and 37 subjects who did not carry the −93g allele, −53, −39, D9N, or the N291S mutations. The D9N and −93g carriers did not carry the −53, −39, or N291S mutations.
South African Blacks
One hundred sixty-one unrelated Black South African subjects were ascertained for these frequency studies, including volunteers from the hospital staff and patients from the Medical Outpatient Department at the Red Cross Childrens Hospital, Cape Town.18 All 161 individuals were analyzed for the presence of the −93g allele and the −53, −39, D9N, and N291S mutations.
The influence of the −93g allele on lipid levels independent of the D9N mutation was assessed in 92 male subjects for whom fasting lipid profiles were available. All were from the Venda tribe, in rural areas of South Africa, and were between 18 and 70 years of age. None of these subjects were taking medications known to affect lipid metabolism or were consuming alcohol in excess (i.e., >3 drinks/day), and none were carriers of the −53, −39, or N291S mutations.
One hundred thirty unrelated individuals of Chinese (Cantonese) ancestry recruited from six Chinese family physician practices in Vancouver were screened for the −93g allele and the D9N mutation. Subjects were selected as consecutive unrelated patients being assessed for a routine physical examination and were part of a more detailed study identifying coronary risk factors in the Cantonese-speaking Chinese population. Only individuals 20 to 70 years of age were included in the study. Thirty-eight of these individuals were ascertained for CA repeat frequencies. In addition, frequencies of the −93g allele and the −53, −39, D9N, and N291S mutations were analyzed.
DNA was extracted from leukocytes by standard procedures.19 The D9N and N291S mutations were detected as previously described.5 7 Polymerase chain reaction (PCR) amplifications of genomic DNA for promoter mutation analysis and allelic variation in the 5′ region and intron 6 were performed in 25-μL reactions in the presence of 0.3 mmol/L specific primer, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl pH 8.4 at 70°C, 0.1% Triton X-100, 0.2 mmol/L of each dNTP, and 1.25 units of Taq DNA polymerase (GIBCO BRL). The promoter variants at −93 and −53 were analyzed by amplification with primer prLPL-8 (5′-GTGTTTGGTGCTTAGACAGG, located at positions −258 to −239) and primer prLPL-1 (5′-GCTAGAAGTGGGCAGCTTTC, located at positions +37 to +56). The analysis of the −39 mutation was performed using a mismatch primer, −39prLPL (5′-AATAGGTGATGAGGTTTATTTGTA, located at positions −63 to −40) and primer prLPL-1. The reactions were incubated for 5 minutes at 96°C, followed by 40 cycles at 96°C for 45 seconds, 57°C for 30 seconds, and 72°C for 45 seconds. The PCR products were digested with 10 units of HaeIII for detection of the −93 substitution, with 10 units of BclI for −53 detection, with 10 units of RsaI for detection of the −39 mutation. The digested fragments were separated on 3.5% agarose gel.
Allelic variation in the 5′ region was determined by PCR amplifications with oligonucleotides described previously.20 The reactions specific for the CA repeat were incubated for 5 minutes at 96°C, followed by 20 cycles at 96°C for 60 seconds, 60°C for 30 seconds, and 72°C for 60 seconds. Polymerase chain reaction products were run on a 6% denaturing polyacrylamide gel. The sizes of alleles were determined by comparison with an M13 sequencing reaction as a standard.
Triglycerides and total cholesterol were measured using commercially available kits (Boehringer Mannheim). HDL cholesterol was measured following precipitation of apolipoprotein B-containing lipoproteins with polyethylene glycol using a commercially available kit (Boehringer Mannheim). LDL cholesterol was calculated according to the Friedewald formula.21 22
Significance of frequency distributions of mutations/alleles between and within populations were determined using chi-squared analysis and Fisher’s exact two-tail probability when appropriate. Investigated mutations are in Hardy-Weinberg equilibrium. Allele sizes of the simple sequence repeat were grouped until a minimum of one allele in each group was obtained. Linkage disequilibrium is presented as a D value,23 and the corresponding significance was assessed by Fisher’s exact two-tail probability test using the maximum likelihood estimated haplotype frequencies. In the South African Black population, group differences in biochemical parameters were determined using an analysis of variance between the following pairs: −93tt/DD versus −93gg/DD; −93gg/DD versus −93gg/DN; and −93gt/DD versus −93gt/DN. All pairs were matched for age, systolic and diastolic blood pressure, mean alcohol consumption, and smoking behavior. The first two of these pairs were also matched for body mass index (BMI), but small differences in BMI were seen between the −93gt/DD and the −93gt/DN groups (P=.03). Statistics for triglyceride levels were performed on log-transformed data. Statistical analysis of the data was carried out using All Stats (UBC) and Systat (SPSS Inc), version 5.0 for Windows.
Frequency of the −93g Allele in Different Populations
The frequency of the sequence substitution in the promoter region at −93, described as a t→g substitution12 was investigated in three ethnically distinct populations: Caucasian (n=232), South African Black (n=161), and Chinese (n=130). Significant differences in the frequency of the −93g allele were observed between the populations (Table 1⇓). The g allele at −93 was identified in 5 of 232 Caucasians (carrier frequency, 2.2%). In the South African Black population, however, this allele was identified in 76.4% of the individuals (123/161). In contrast, this mutation was not identified in the Chinese population.
Furthermore, when the genotypic information obtained for the Caucasian population was compared with that of previous studies regarding the D9N and N291S mutations on this cohort,4 8 the −93g allele was observed to be in almost complete linkage disequilibrium with the D9N mutation (D=0.0085;23 P=8×10−9). The D9N mutation was identified in four individuals from this Dutch cohort allele, representing a carrier frequency of 1.7% (4/232), all of whom were heterozygous for the −93g allele. One individual homozygous for the −93g allele without the D9N mutation was also identified in the Dutch population. The D9N mutation was not found in the absence of the −93g allele in the Caucasian population. In contrast, of the 123 Black subjects carrying the −93g allele, only 20 were carrying the D9N mutation. Similar to the −93g allele, the D9N mutation was not identified in the 130 Chinese subjects (Table 1⇑).
Allele Distribution of a Highly Polymorphic Marker Upstream of the LPL Coding Region
To further investigate the chromosomal origins of both the −93g allele and D9N mutation in the LPL gene, the CA repeat polymorphism located approximately 5-kilobases upstream of the transcription start site20 was analyzed. To assess whether the N9 and the −93g allele were in complete linkage disequilibrium and to obtain larger numbers of Caucasian D9N carriers for dinucleotide repeat analysis, 39 individuals identified as heterozygous D9N carriers in the Framingham Offspring study (S.E. Gagné, unpublished data) were also screened for the presence of the −93g allele. All 39 people were also found to be heterozygous carriers of the −93g allele. These 39 individuals were typed for the CA repeat, as were Black subjects with genotype −93 gg/DD (n=17), −93gt/DN (n=2) and −93gg/DN (n=3). In addition, 5′ CA repeat lengths in 37 Caucasians, 17 South African Blacks, and 38 Chinese subjects with the genotype −93tt/DD were assessed (Table 2⇓).
Repeat lengths varied from 14 to 28 repeats.20 Because of the small numbers for each repeat size, allele sizes were grouped until the minimum number of alleles in all categories was at least one. Significant differences in allele distributions between all three populations were observed (Table 2⇑; P=.0003 to .05). Thus, further analysis of the alleles was performed in each population separately.
In Black subjects with the −93gg/DD genotype the −93g allele is seen on chromosomes with many different CA repeat alleles (Table 2⇑; Fig 1B⇓), whereas the −93t allele in subjects with the −93tt/DD genotype was primarily associated with CA alleles containing 16 or 17 repeats (Table 2⇑; Fig 1A⇓). Furthermore, repeat lengths 16 and 17 are the predominant CA alleles in both Caucasian (Table 2⇑; Fig 1E⇓) and Chinese (Fig 1C⇓) populations in whom the −93t allele is predominant. Thus, as the −93t allele is primarily associated with the CA allele of 16 repeats in all three populations (Fig 1A⇓, C, and E), whereas no such associations are seen for the −93g allele. This is compatible with a common origin of the carriers of the −93t allele.
The D9N mutation is associated with the 23 repeat CA allele in the Black population (Fig 1D⇑). Similarly, in the Caucasian population, a significant shift (P<10−6; Table 2⇑) toward CA repeats of particularly 24 to 25 repeats in size are seen in subjects of the −93gt/DN genotype compared with individuals carrying the −93tt/DD genotype (Fig 1E⇑ and F). This suggests that the D9N mutation is associated with longer CA repeat lengths such as those associated with chromosomes carrying the −93g allele (Fig 1D⇑ and F) as opposed to the highly prevalent −93t allele seen in Caucasians. Taken together these data would suggest that the −93t allele and the D9N mutation each occurred independently on distinct chromosomal backgrounds.
Phenotypic Effects of the −93g Allele
In Caucasians, as the D9N mutation is in linkage disequilibrium with the −93g allele, the effects of the D9N mutation and −93g allele cannot be distinguished in this population. Furthermore, it is unclear whether either one or both of these changes may contribute to the phenotypic effects previously described in Caucasian D9N carriers.5 8 The presence of the −93g allele at high frequency and independent of the N9 allele in the South African Black population represents an ideal opportunity to study the phenotypic effects of this DNA substitution.
Lipid profiles were assessed in 92 Black rural South African males originating from Venda (Table 3⇓). Individuals with the −93gg/DD genotype (n=20) showed significantly lower triglyceride levels compared with 21 subjects carrying the −93tt/DD genotype (0.82±0.3 mmol/L versus 1.14±0.66; P=0.04). No significant differences were found for total HDL or LDL cholesterol levels (Table 3⇓). Carriers of the −93gg/DN (n=8) had a trend to higher triglyceride levels compared with persons without the D9N mutation (−93gg/DD), but this did not reach significance because of variability in the results and small sample size of the N9 carriers (n=8; Table 3⇓).
The variant at nucleotide −93 in the LPL promoter was originally described in a Caucasian population as a t to g substitution, with a carrier frequency of 1.6%.12 In the study contained herein, we sought to determine its frequency in populations of different ethnic origins.
The frequency of this mutation varied widely between different ethnic groups. The carrier frequency of the −93g allele was 1.7% in the Dutch Caucasian population studied, similar to prior reports. However, in contrast, in the South African Black population, the carrier frequency was 76.4%, and the −93g allele was not found in 130 Chinese individuals screened. Of further interest, was the finding of near complete linkage disequilibrium between the −93g allele and the D9N mutation in Caucasians (D=0.0085;23 P=8×10−9). In contrast, within the Black population, most carriers of the g allele at −93 did not carry the D9N mutation.
To further study the origin of the −93g allele and its association with the D9N mutation, analysis of a highly polymorphic CA repeat 5′ to the LPL gene was performed. Interestingly, the −93g allele was seen across many different CA alleles in the South African Blacks (Fig 1B⇑). In contrast, the −93t allele was seen predominantly in association with CA alleles of 16 repeats in size in all three populations (Fig 1A⇑, C, and E). This raises the question as to whether the −93g or the −93t allele is the original ancestral allele. The data presented here suggest that the −93t allele, reported as the wild-type allele in Caucasians,12 may be derived from the −93g allele, which is most frequent in Blacks, and that the −93g allele might in fact be the ancestral allele. In further support of this hypothesis is the fact that the −93g and not the −93t allele is conserved among other species including the mouse,24 25 chicken,26 and cat (K.A. Excoffon, unpublished observation), again suggesting that the −93g allele is the more ancient allele. Thus, the data suggest that a mutation arose early at position −93, resulting in a g→t substitution on a single or few chromosomes carrying the CA allele with the size of 16 repeats. The European and Asian populations27 derived later from Africa and because of a possible founder effect would, therefore, be expected to carry predominantly the CA allele of 16 repeats in size and the −93t allele, which is evident.
The fact that the D9N mutation is seen associated with CA alleles with 23 to 25 repeats in size suggests it arose on a specific −93g allele carrying (Table 2⇑, Fig 1D⇑ and F) larger CA repeat sizes. Furthermore, the absence of the D9N mutation in the Chinese population suggests that this mutation occurred after branching off from the Asian population approximately 50 000 years ago.27 This could then explain the linkage disequilibrium between the −93g allele and the D9N mutation in the Caucasian population and the apparent absence of both from the Chinese population. However, because of the relatively small number of Chinese individuals screened for the −93g allele and the D9N mutation, these DNA changes may still be present but at reduced frequencies.
To further explore the frequency and possible genetic relationships between both the promoter mutations and common coding mutations, all subjects were also screened for the mutations at −39 and −53 in the promoter12 and the N291S mutation (Reference 44 ; data not shown). In this study, the nucleotide substitution at −39 was not detected in either Caucasian (control n=232), Black South African (n=161), or Chinese (n=130) individuals. The mutation at −53 was found in 2 of 232 controls of Dutch origin but not in either the Black (n=161) or Chinese populations (n=130). Thus, in contrast to the −93 substitution, the −39 and −53 promoter mutations appear to be rare in all populations. The N291S mutation was present in only the Caucasian population, with a carrier frequency of 4.3% (10/232). None of these mutations appeared to be in linkage disequilibrium.
It has previously been suggested that the −93g allele reflects a functional variant.13 15 However, the reports concerning the transcriptional activity in vitro for the −93g allele are conflicting.12 13 15 This allele has been shown to have a reduced (40-50% of wild-type) transcriptional activity in vitro using the human monocytic leukemic cell line, THP-1, and the mouse myoblast cell line, C2C12,12 13 whereas the promoter activity was increased with this substitution by approximately 24% using a smooth muscle cell line.15 On the other hand, the D9N mutation has been reported to have decreased secretion in vitro11 and to be associated with hypertriglyceridemia in vivo.5
The relative in vivo contribution of each of these mutations to the phenotype of hyperlipidemia cannot easily be distinguished in studies of Caucasian patients, because there is almost complete linkage disequilibrium between the −93g allele and the D9N mutation in Caucasians. However, the presence of the −93g allele alone on more than 53% of alleles in South African Blacks afforded us an ideal population in which to study the independent phenotypic effects of the −93g allele.
Within the South African Black population, we have shown that the −93g allele is associated with lower triglyceride levels compared with the −93t allele (P=.04). These results support what might be expected if the −93g allele increased transcriptional activity in vivo.15 An interesting question is thus raised in carriers of both the −93g allele and the D9N mutation as to the relative effects of both DNA changes. From the data obtained in the Black population, the −93g allele would be expected to lead to decreased triglycerides, whereas the D9N mutation has been shown to have decreased activity in vitro and in vivo and would thus be expected to result in increased triglycerides.5 11 We have previously reported the phenotypic effects of the D9N mutation in carriers8 who are also carriers of the −93g allele. In this situation, these mutations are associated with elevated triglycerides in vivo, suggesting that the D9N mutation, which results in decreased catalytic activity, is dominant in its effect over the −93g allele.
Some caveats, however, should be noted. The triglyceride and cholesterol levels in all Black individuals were generally low, compared with typical Caucasian levels. This may be attributable in part to the consumption of a rural low-fat diet by these individuals in contrast to the typical diet of Western populations and may relate to a significantly lower incidence of coronary artery disease reported in South African Black versus Caucasian populations.28 Similar low cholesterol and triglyceride concentrations have been observed in rural Chinese populations and are suggested to be in part dietary-related as increased urbanization results in significantly elevated cholesterol and triglyceride levels.29 However, when comparing lipid profiles of Black and Caucasian South Africans on the same Western diet for two years, Black South Africans were still found to have significantly lower cholesterol and triglyceride concentrations.30 The increased frequency of the −93g allele in the Black population in the absence of the D9N mutation could also contribute to the findings of lower triglyceride levels in Blacks compared with Caucasians.
Black subjects with the D9N mutation had a trend to higher mean triglyceride levels than persons without this DNA change (2.97±0.33 versus 3.81±0.39 mol/L), but this did not reach significance (P=.25). This may suggest that the phenotypic effects of the D9N mutation only become obviously apparent after environmental challenges as seen in other partial LPL catalytic defects.9 31 32 33 It also, however, should be noted that only a small cohort of D9N carriers were available for study, which may have been insufficient to reach significance.
Here, we show that the −93g allele, which occurs with high frequency, is associated with mildly lower triglycerides in a South African Black population. The −93g allele has been shown15 to be associated with increased transcriptional activity. Increased production of catalytically normal LPL would be predicted to lower triglyceride levels. However, in the company of the D9N mutation on the same allele, increased production of a catalytically defective protein might now be expected to be associated with higher triglyceride levels, as is seen in studies of Caucasians with both of these DNA changes.5 8
We thank Dr. W. Robinson for assistance with the statistical analysis, Drs. H. Pritchard and J. Frohlich for providing the Chinese subjects used in this study, and C.A. Hoogendijk for assistance with the blood sampling in Venda. This work was supported by the MRC Canada and the Heart and Stroke Foundation of British Columbia and the Yukon. Dr. E. Ehrenborg was supported by the Swedish Medical Research Council and the Henning and Johan Throne-Holst Foundation. S.M. Clee is supported by an MRC Canada Studentship. Dr. J.J.P. Kastelein is a clinical investigator of the Dutch Heart Association. Dr. M.R. Hayden is an established investigator of the British Columbia Children’s Hospital and an investigator of the Canadian Genetic Disease Network.
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