Arachidonate 5-Lipoxygenase Variants in Atherosclerosis, Obesity, and Bone Traits
To the Editor:
I write regarding publications by Mehrabian et al1,2 appearing in Circulation Research and a subsequent publication by Kuhn et al3 appearing in Arteriosclerosis, Thrombosis, and Vascular Biology. The key implication from the combination of these reports was that amino acid substitutions in arachidonate 5-lipoxygenase (ALOX5) could account for differences in susceptibility to atherosclerosis between mouse strains CAST/EiJ and C57BL/6J. The two putative amino acid substitutions reported by Mehrabian et al to be present in strain CAST/EiJ1 were at positions 645 and 646, which are conserved across human, mouse, rat, dog, cow, and chicken. Indeed, when Kuhn et al3 mutated either or both of these residues in the human cDNA, ALOX5 activity was markedly impaired suggesting a key role for one or both of these residues in protein function. Recently, I determined that the coding sequences reported in the first article and implemented in the third article are incorrect.
We detected QTLs for cholesterol traits on Chr 6 in crosses between strains CAST/EiJ and 129S1/SvImJ4 and between strains CAST/EiJ and DBA/2J5,6 that colocalized with Alox5. The coding region of Alox5 was sequenced among these inbred mouse strains. On comparison of all sequences, we made four observations. (1) Our deduced amino acid sequence derived from strain CAST/EiJ (genomic DNA and cDNA) was different to that reported by Mehrabian et al.1 (2) The amino acid sequence for strain CAST/EiJ was identical to strain C57BL/6J. (3) Residue 645 was identical among all the mouse strains. (4) Strains 129S1/SvImJ and DBA/2J exhibited ALOX5 amino acid identity, but differed from strains CAST/EiJ and C57BL/6J at residue 646 (129S1/SvImJ=DBA/2J, isoleucine; CAST/EiJ=C57BL/6J, valine).
The amino acid sequence identity of ALOX5 between strains CAST/EiJ and C57BL/6J precludes amino acid substitutions in ALOX5 from an atheroprotective role in the reported B6.CAST-(D6Mit102-D6Mit198) congenic mice.2 Because the authors also showed altered Alox5 mRNA and ALOX5 protein expression in the congenic mice,1 it is more likely that the effect arises from altered gene transcription or protein processing than altered protein function. In their more recent publication, Mehrabian et al7 provided convincing evidence that Alox5 affected multiple metabolic traits that differed between strains C57BL/6J and DBA/2J. In this instance it is plausible that the single amino acid substitution at residue 646 was responsible for the differences in the measured traits of interest. Given that DBA/2J harbors the 646I residue that renders ALOX5 nonfunctional, it is not surprising that Alox5 knockout mice resembled (C57BL/6J × DBA/2J)F2 mice that were homozygous for the DBA/2J Alox5 allele.7 Interestingly, because 129S1/SvImJ also expresses the nonfunctional ALOX5 protein, one may predict that any B6.129 knockout-congenic strain that possesses the 129 Alox5 allele in the region flanking the targeted gene also will be essentially an Alox5 knockout and behave similarly to the Alox5 knockout mouse itself.
Overall, the implication of these findings is that this is a good example of a QTL arising both from altered protein function (C57BL/6J versus DBA/2J) and from altered transcriptional or translational efficiency (C57BL/6J versus CAST/EiJ). This highlights the appearance in different ancestral populations (eg, Mus mus musculus, M. m. domesticus, M. m. castaneus) of alternative variants that arise within a single gene that act via different modes of activity, but exhibit similar phenotypic results. Furthermore, these observations demonstrate the ability of QTL mapping approaches to detect both types of genetic variation when they occur within genes whose products are key regulators of biological processes.
Mehrabian M, Allayee H, Wong J, Shi W, Wang XP, Shaposhnik Z, Funk CD, Lusis AJ, Shih W. Identification of 5-lipoxygenase as a major gene contributing to atherosclerosis susceptibility in mice. Circ Res. 2002; 91: 120–126.
Mehrabian M, Wong J, Wang X, Jiang Z, Shi W, Fogelman AM, Lusis AJ. Genetic locus in mice that blocks development of atherosclerosis despite extreme hyperlipidemia. Circ Res. 2001; 89: 125–130.
Kuhn H, Anton M, Gerth C, Habenicht A. Amino acid differences in the deduced 5-lipoxygenase sequence of cast atherosclerosis-resistance mice confer impaired activity when introduced into the human ortholog. Arterioscler Thromb Vasc Biol. 2003; 23: 1072–1076.
Lyons MA, Wittenburg H, Li R, Walsh K, Leonard MR, Churchill GA, Carey MC, Paigen B. New quantitative trait loci that contribute to cholesterol gallstone formation detected in an intercross of CAST/Ei and 129S1/SvImJ inbred mice. Physiol Genomics. 2003; 14: 225–239.
Lyons MA, Wittenburg H, Li R, Walsh K, Churchill GA, Carey MC, Paigen B. Quantitative trait loci that determine lipoprotein cholesterol levels in DBA/2J and CAST/Ei inbred mice. J Lipid Res. 2003; 44: 953–967.
Lyons MA, Wittenburg H, Li R, Walsh K, Leonard MR, Korstanje R, Churchill GA, Carey MC, Paigen B. Lith6: A new QTL for cholesterol gallstones from an intercross of CAST/Ei and DBA/2J inbred mouse strains. J Lipid Res. 2003; 44: 1763–1771.
Mehrabian M, Allayee H, Stockton J, Lum PY, Drake TA, Castellani LW, Suh M, Armour C, Edwards S, Lamb J, Lusis AJ, Schadt EE. Integrating genotypic and expression data in a segregating mouse population to identify 5-lipoxygenase as a susceptibility gene for obesity and bone traits. Nat Genet. 2005; 37: 1224–1233.