Evidence for a role of LPGAT1 in influencing BMI and percent body fat in Native Americans

Abstract

Objective: A genome-wide association study (GWAS) was recently completed in 1120 Pima Indians to identify loci that influence BMI. Among the top 100 signals were three variants that mapped within the lysophosphatidylglycerol acyltransferase 1 (LPGAT1) gene. LPGAT1 belongs to a large family of acyltransferases, which are involved in a variety of biological processes including pathways that regulate energy homeostasis and body weight. Therefore LPGAT1 was analyzed as a candidate gene for obesity in Pima Indians.

Design and methods: Variants (n = 26) located within and adjacent to LPGAT1 including a novel 27bp deletion in the 5'-untranslated region identified by sequencing were genotyped in a population-based sample of 3,391 full-heritage Pima Indians living in the Gila River Indian Community. Replication of selected variants was assessed in a second sample of 3,327 mixed-heritage Native Americans from the same community.

Results: Variants with nominal associations with BMI in each of the two independent samples (tagged by rs112662024 and rs12058008) had associations of P = 1-4 × 10(-5) in the combined sample (n = 6718). A haplotype that includes the novel 27bp deletion, which does not occur in Caucasians, showed the strongest association with BMI in the full-heritage Pima Indians. In vitro functional studies provided suggestive evidence that this 27bp deletion may affect transcriptional or posttranscriptional regulation. Analysis of LPGAT1 cDNA from human preadipocytes identified an additional exon whose sequence could potentially serve as a mitochondrial targeting peptide.

Conclusions: LPGAT1 is a novel gene that influences BMI in Native Americans.

FIGURE 1

LPGAT1 expression levels based on the copy number of haplotype 2 in (a) muscle and (b) adipose. Means are indicated by the horizontal lines. P values were adjusted for age, sex, and Pima heritage. LPGAT1, lysophosphatidylglycerol acyltransferase 1.

FIGURE 2

Identification of an alternative exon 2 in LPGAT1. (a) Gel image showing the results for the PCR reactions. PCR reactions were done using subcutaneous preadipocyte cDNA isolated from 10 Pima Indian subjects. (b) Schematic showing the alignment of the sequenced cDNA PCR products (aligned sequence) with the first three exons of the LPGAT1.cApr07 partial transcript (AceView Gene Models track, UCSC Genome Browser). The alternative exon identified by sequencing, which is also present in the LPGAT1.cApr07 transcript is highlighted by the gray shaded rectangle. For the LPGAT1.cApr07, isoform A, and isoform B transcripts the thicker bars indicate regions that are translated and the thinner bars indicate regions that are not translated. Horizontal arrows in exons 1 and 2 of isoform B show the locations of the primers used to screen the preadipocyte cDNA. (c) Amino acid sequences for the translated exons shown in (b). Single underline indicates the putative mitochondrial targeting peptide (exon 2 of LPGAT1.cApr07). The signal peptide was predicted using TargetP 1.1(25) and iPSORT(26) subcellular localization prediction programs. Double underline indicates the endoplasmic reticulum signal peptide for both isoforms A(15) and B. Gt, genotype; L, DNA ladder; LPGAT1, lysophosphatidylglycerol acyltransferase 1; wt, wild-type (no deletion).

FIGURE 3

Functional studies to examine whether the 27 bp deletion affects transcriptional regulation. (a) Diagram showing the LPGAT1–4^wt and LPGAT1–4^{−27 bp} regions (dashed lines) that were cloned into the promoterless pGL4.17 vector. Relative luciferase activities for the LPGAT1–4^wt and LPGAT1–4^{−27 bp} isoform A and isoform B promoter constructs in (b) HepG2, (c) CaCo-2, (d) 3T3-L1, and (e) G8 myoblast cell lines. Transfections were done with equal amounts of plasmids. The constructs were co-transfected with pGL4.74[hRluc/TK] and luciferase levels are expressed as the relative activities and are shown as the means ± s.d. for six replicates. LPGAT1, lysophosphatidylglycerol acyltransferase 1; pGL3-BV, pGL3-basic vector no insert control; wt, wild-type (no deletion).

FIGURE 4

Functional studies to examine whether the 27 bp deletion affects posttranscriptional regulation. (a) Diagram showing the two LPGAT1 5′-UTRs (isoform B and LPGAT1.cApr 07) that were cloned into the pGL3-promoter vector. Open boxes indicate the regions that were cloned. Arrows indicate the location of the 27 bp deletion. Relative luciferase activities for the LPGAT1.5UTR-B^wt, LPGAT1.5UTR-B^{−27 bp}, LPGAT1.cApr07^wt, and LPGAT1.cApr07^{−27 bp} constructs in (b) HepG2, (c) Caco-2, (d) 3T3-L1, and (e) G8 myoblast cell lines. Transfections were performed with equal amounts of plasmids. The constructs were co-transfected with pGL4.74[hRluc/TK] and luciferase assays were done in replicates of six and the results are shown as means ± s.d. LPGAT1, lysophosphatidylglycerol acyltransferase 1; pGL3-PV, pGL3-promoter vector no insert control; UTR, untranslated region; wt, wild-type (no deletion).

FIGURE 5

Predicted RNA secondary structures for both normal (no deletion) and mutant (27 bp deletion) transcripts. The 27 bp deletion eliminated a double-stem loop structure and a smaller single doublelooped region formed (arrows). Only the region that was altered is shown. The alteration was the same for both sets of transcripts (isoform B and LPGAT1.cApr 07). LPGAT1, lysophosphatidylglycerol acyltransferase 1; UTR, untranslated region.