Wong Clifford, Mahapatra Nitish R, Chitbangonsyn Surin, Mahboubi Payam, Mahata Manjula, Mahata Sushil K, O'Connor Daniel T
Department of Medicine and Center for Molecular Genetics, University of California, and Veterans Affairs San Diego Healthcare System, San Diego, California 92161, USA.
Physiol Genomics. 2003 Jun 24;14(1):83-93. doi: 10.1152/physiolgenomics.00162.2002.
Hypertension is a complex trait with multiple genetic determinants. A previous genome-wide linkage study of systolic blood pressure in a mouse genetic backcross implicated a region of chromosome 13 (LOD = 3.3 at 16.0 cM) as a determinant of blood pressure differences between a hereditary low blood pressure strain of Mus musculus (BPL/1) and Mus spretus (SPRET); at this locus, the unexpected effect of the BPL/1 allele was to increase blood pressure. A plausible candidate locus encoding angiotensin II receptor isoform 1a (Agtr1a) is also located at 16.0 cM on chromosome 13. We therefore investigated structural and functional differences at Agtr1a between BPL/1 and SPRET, as well as the BPH/2 strain. Resequencing Agtr1a in the three strains established the exon/intron and proximal promoter structure of the mouse gene. Coding exon 3 spanned 1,960 bp (with 26 SNPs), including the 1,077-bp/359-amino acid ORF (with 5 cSNPs, all of which were synonymous). Promoter sequences revealed a consensus TATA box, conserved G/C-rich regions, and a striking, lengthy simple sequence repeat region, composed of di-, tri-, tetra-, and penta-nucleotide repeats, whose overall length varied markedly among the strains. Twenty-five other SNPs and three single nucleotide deletions differentiated the strains' promoters, six of which were in likely functional promoter motifs. Agtr1a mRNA abundance in the adrenal gland in vivo was greater (P < 0.05) in BPL/1 than SPRET, consistent with the predicted effect of the BPL/1 allele to confer higher blood pressure. When Agtr1a promoters were subcloned into luciferase reporter plasmids and transfected into PC12 chromaffin cells, basal promoter expression was higher (P < 0.001) in BPL/1 than in SPRET, consistent with the endogenous mRNA results. In summary, Agtr1a on chromosome 13 is highly polymorphic between mouse strains, although the amino acid sequence specified by the ORF is invariant, even across mouse species. We conclude that polymorphisms in the Agtr1a promoter account for differences in gene expression in vivo between BPL/1 and SPRET, in a way consistent with the effects of alleles at this locus on chromosome 13 to affect blood pressure in the mouse genome-wide linkage study.
高血压是一种具有多种遗传决定因素的复杂性状。先前在小鼠遗传回交中对收缩压进行的全基因组连锁研究表明，13号染色体上的一个区域（在16.0厘摩处的对数优势比为3.3）是小家鼠（BPL/1）的遗传性低血压品系与西班牙小鼠（SPRET）之间血压差异的一个决定因素；在这个基因座上，BPL/1等位基因产生了意想不到的效果，即升高血压。一个编码血管紧张素II受体亚型1a（Agtr1a）的可能候选基因座也位于13号染色体上的16.0厘摩处。因此，我们研究了BPL/1、SPRET以及BPH/2品系之间Agtr1a在结构和功能上的差异。对这三个品系的Agtr1a进行重测序，确定了小鼠基因的外显子/内含子和近端启动子结构。编码外显子3跨度为1960碱基对（有26个单核苷酸多态性），包括1077碱基对/359个氨基酸的开放阅读框（有5个编码单核苷酸多态性，均为同义突变）。启动子序列显示有一个共有TATA框、保守的富含G/C区域以及一个由二核苷酸、三核苷酸、四核苷酸和五核苷酸重复组成的显著且冗长的简单序列重复区域，其总长度在各品系间有明显差异。另外25个单核苷酸多态性和3个单核苷酸缺失区分了各品系的启动子，其中6个位于可能具有功能的启动子基序中。在体内，BPL/1肾上腺中Agtr1a mRNA丰度高于SPRET（P < 0.05），这与BPL/1等位基因赋予更高血压的预测效果一致。当将Agtr1a启动子亚克隆到荧光素酶报告质粒中并转染到PC12嗜铬细胞中时，BPL/1中的基础启动子表达高于SPRET（P < 0.001），这与内源性mRNA结果一致。总之，13号染色体上的Agtr1a在小鼠品系间具有高度多态性，尽管开放阅读框指定的氨基酸序列是不变的，甚至在不同小鼠物种间也是如此。我们得出结论，Agtr1a启动子中的多态性导致了BPL/1和SPRET在体内基因表达的差异，其方式与该基因座在小鼠全基因组连锁研究中对13号染色体上等位基因影响血压的作用一致。
