Large-scale search for genes predisposing to essential hypertension.
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Genetic Predisposition to Disease↗
Biomedical subjects
Jean-Marc Lalouel
Publications and source records attributed to Jean-Marc Lalouel.
Molecular cloning and functional analysis of a factor that binds to the proximal promoter of human angiotensinogen.
A significant association has been reported between a common variant in the angiotensinogen gene (AGT), allele T235, and essential hypertension. In subsequent work, it was found that another variant, the presence of an adenine instead of a guanine 6bp upstream from the initiation site of transcription, was in absolute linkage disequilibrium with T235. The nucleotide substitution at the -6 position affected the formation of DNA-protein complexes in gel mobility shift assays and the basal transcription of AGT in transactivation experiments. We have further examined the potential impact of this polymorphism on AGT promoter function. In ultraviolet cross-linking analysis. 150- and 75-kDa proteins bound to the AGT proximal promoter. The possible involvement of factors that bind to GC-rich domains, including Sp1, Sp3, and AP2, was not supported by gel mobility shift assays. Screening an expression library with a double-stranded DNA segment centered on -6 led to the isolation of cDNA clones encoding the YB1 protein. The specificity of the interaction of YB1 with the proximal promoter of AGT was verified by Southwestern blotting and gel mobility shift assays. In cotransfection experiments, YB1 reduced basal AGT promoter activity in a dose-dependent manner. Although these observations suggest a possible role for YB1 in modulating AGT expression, this function is likely to occur in the context of complex interactions involving other nuclear factors. The work illustrates the challenge of developing a molecular understanding of the relationship between common genetic variants and conditions that are only partly caused by them.
Angiotensinogen↗
Contribution of Sp1 to initiation of transcription of angiotensinogen.
Several genetic polymorphisms have been identified in the proximal promoter of angiotensinogen ( AGT). Gene titration experiments in transgenic animals have demonstrated that small increases in the basal expression of AGT can lead to elevated blood pressure. The direct proof that promoter variants of AGT can lead to elevated blood pressure will ultimately require the development of specific animal models. Before such work can be contemplated, however, a formal understanding of the mechanisms controlling transcriptional activation of AGT needs to be developed. Analysis of DNA-protein interactions in vitro and transactivation experiments in cultured cells reveal the critical role of an Sp1 binding site immediately upstream of the TATA box of AGT in both mouse and human. Both sites are required for transcription initiation in the mouse. By contrast, a minimal human AGT promoter can initiate transcription in the absence of either this Sp1 site or the TATA box, albeit at a lower level. Further analysis and consideration of these interspecific differences will be essential for the development of meaningful animal models to probe the mechanism by which AGT may predispose to human essential hypertension.
Angiotensinogen↗
Common variant of human NEDD4L activates a cryptic splice site to form a frameshifted transcript.
The ubiquitin ligase NEDD4L is a candidate gene for essential hypertension on both functional and genetic grounds. By targeting the epithelial sodium channel (ENaC) for degradation, NEDD4L is a significant determinant of sodium reabsorption in the distal nephron. Genetic linkage has been reported to a region of chromosome 18q harboring the gene, with phenotypes that include a rare orthostatic hypotension disorder, essential hypertension, and postural change in systolic blood pressure. A systematic search for genetic polymorphisms by resequencing exons and intron boundaries in 48 Caucasians yielded 38 variants. Among these, variant 13 is common, with either G (70%) or A (30%) as the last nucleotide of a putative exon 1. This mutation could affect the generation of a previously unrecognized splice isoform. In subsequent experiments, (1) we confirmed the presence of this putative isoform in both kidney and adrenals; (2) we established that variant 13-A leads to the systematic use of an alternative splice site, generating a transcript encoding a nonfunctional protein; and (3) we demonstrated differences in tissue-specific expression of the novel isoform relative to its previously reported counterpart. Variant 13-A precludes the formation of a transcript encoding a full-length Ca2+-dependent lipid-binding (C2) domain with very high evolutionary conservation among NEDD4L orthologs. A similar C2 domain in the paralogous NEDD4 gene plays a significant role in the transfer of its product to the apical membrane of epithelial cells. Differential function of NEDD4L isoforms could prove significant in blood pressure regulation through an effect on ENaC-dependent sodium reabsorption.
Alternative Splicing↗
Power and replication in case-control studies.
The case-control study design, a common staple of epidemiology, is increasingly used to test for genetic association. The simplicity of the design accounts for both its appeal and its limitations. Too often, however, apparent controversy arises for lack of appreciation of basic tenets underlying statistical testing. Power and replication are two concepts most commonly ignored in evaluating such studies. We review the basic principles of statistical testing, recall simple means to calculate power, and provide numerical examples pertaining to the association between angiotensinogen and essential hypertension.
Alleles↗
Effects of dietary sodium and genetic background on angiotensinogen and Renin in mouse.
Elements of a renin-angiotensin system expressed along the entire nephron, including angiotensinogen secreted by proximal tubule and renin expressed in connecting tubule, may participate in the regulation of sodium reabsorption at multiple sites of the nephron. The response of this tubular renin-angiotensin system to stepwise changes in dietary sodium was investigated in 2 mouse strains, the sodium-sensitive inbred C57BL/6 and the sodium-resistant CD1 outbred. Plasma angiotensinogen was not affected by sodium regimen, whereas plasma renin increased 2-fold under low sodium. In both strains, the variation in urinary parameters did not parallel the changes observed in plasma. Angiotensinogen and renin excretion were significantly higher under high sodium than under low sodium. Water deprivation, by contrast, induced significant activation in the tubular expression of angiotensinogen and renin. C57BL/6 exhibited significantly higher urinary excretion of angiotensinogen than did CD1 animals under both conditions of sodium intake. The extent to which these urinary parameters reflect systemic or tubular responses to challenges of sodium homeostasis may depend on the relative contribution of sodium restriction and volume depletion.
Aldosterone↗
Nucleotide diversity and haplotype structure of the human angiotensinogen gene in two populations.
Variation in the angiotensinogen gene, AGT, has been associated with variation in plasma angiotensinogen levels. In addition, the T235M polymorphism in the AGT product is associated with an increased risk of essential hypertension in multiple populations, making AGT a good example of a quantitative-trait locus underlying susceptibility to a common disease. To better understand genetic variation in AGT, we sequenced a 14.4-kb genomic region spanning the entire AGT and identified 44 single-nucleotide polymorphisms (SNPs). Forty-two SNPs were observed both in 88 white and in 77 Japanese unselected subjects. Six major haplotypes accounted for most of the variation in this region, indicating less allelic complexity than in many other genomic regions. Although the two populations were found to share all of the major AGT haplotypes, there were substantial differences in haplotype frequencies. Pairwise linkage disequilibrium (LD), measured by the D', r(2), and d(2) statistics, demonstrated a general pattern of decline with increasing distance, but, as expected in a small genomic region, individual LD values were highly variable. LD between T235M and each of the other 39 SNPs was assessed in order to model the usefulness of LD to detect a disease-associated mutation. Among the Japanese subjects, 13 (33%) of the SNPs had r(2) values >0.1, whereas this statistic was substantially higher for the white subjects (occurring in 35/39 [90%]). LD between a hypertension-associated promoter mutation, A-6G, and 39 SNPs was also measured. Similar results were obtained, with 33% of the SNPs showing r(2)>0.1 in the Japanese subjects and 92% of the SNPs showing r(2)>0.1 in the white subjects. This difference, which occurs despite an overall similarity in LD patterns in the two populations, reflects a much higher frequency of the M235-associated haplotype in the white sample. These results have important implications for the usefulness of LD approaches in the mapping of genes underlying susceptibility to complex diseases.
Angiotensinogen↗
Angiotensinogen in essential hypertension: from genetics to nephrology.
There is general consensus that genetic variation accounts in part for individual susceptibilities to essential hypertension. In marked contrast to classic mendelian disorders, in which genetic alterations produce a gain or loss of function, genetic determinants of essential hypertension, high blood pressure of unknown cause, are expected to be small, achieving significance through the cumulative effects of environmental exposure over the course of a lifetime. Whether and how genetic factors that contribute to common diseases can be identified remain unclear. Research on a link between angiotensinogen and essential hypertension illustrates a path that began in genetics and is now leading toward nephrology. Various challenges encountered along the way may prove to be characteristic features of genetic investigations of the pathogenesis of common diseases. The implication of a gene by statistical analysis is only the beginning of a protracted process of functional analysis at increasing levels of biologic integration. The ultimate goal is to develop an understanding of the manner in which genetic variation at a locus can affect a physiologic parameter and to extract from this inference new knowledge of significance for the prevention or treatment of disease.
Alleles↗