Genetic heterogeneity of familial hyperkalaemic hypertension.

BACKGROUND: Familial hyperkalaemic hypertension (FHH) is a Mendelian form of low-renin hypertension characterized by hyperkalaemia and hyperchloraemic acidosis despite a normal glomerular filtration rate. To date, three different loci have been identified, on chromosomes 1, 17 and 12. OBJECTIVE: To test for genetic linkage between the three FHH loci and three new affected kindreds. DESIGN AND METHODS: Clinical, biological and genetic analyses were made of three kindreds, including 11 affected individuals among 25 members. Genotyping was performed using four series of microsatellite markers spanning the chromosomes 1, 17 and 12 loci, and the thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene. RESULTS: Segregation of the trait in each kindred was compatible with an autosomal transmission, the affected individuals displaying reasonably consistent biochemical abnormalities and the expected variability in arterial hypertension. Multipoint linkage analysis excluded linkage with the four candidate loci in kindreds 1 and 2, but not with the chromosome 1 locus in kindred 3. CONCLUSION: These results demonstrate further genetic heterogeneity and that a fourth gene is responsible for FHH in at least two unrelated kindreds. They suggest a variety of molecular defects leading to FHH.

A new locus on chromosome 12p13.3 for pseudohypoaldosteronism type II, an autosomal dominant form of hypertension.

Pseudohypoaldosteronism type II (PHA2) is a rare autosomal dominant form of volume-dependent low-renin hypertension characterized by hyperkalemia and hyperchloremic acidosis but also by a normal glomerular filtration rate. These features, together with the correction of blood pressure and metabolic abnormalities by small doses of thiazide diuretics, suggest a primary renal tubular defect. Two loci have previously been mapped at low resolution to chromosome 1q31-42 (PHA2A) and 17p11-q21 (PHA2B). We have now analyzed a new, large French pedigree, in which 12 affected members over three generations confirmed the autosomal dominant inheritance. Affected subjects had hypertension together with long-term hyperkalemia (range 5.2-6.2 mmol/liter), hyperchloremia (range: 100-109 mmol/liter), normal plasma creatinine (range: 63-129 mmol/liter) and low renin levels. Genetic linkage was excluded for both PHA2A and PHA2B loci (all LOD scores Z<-3.2 at recombination fraction [theta] 0), as well as for the thiazide-sensitive sodium-chloride cotransporter gene. A genome-wide scan using 383 microsatellite markers showed a strong linkage with the chromosome 12p13 region (maximum LOD score Z=6.18, straight theta=0, at D12S99). Haplotype analysis using 10 additional polymorphic markers led to a minimum 13-cM interval flanked by D12S1652 and D12S336, thus defining a new PHA2C locus. Analysis of two obvious candidate genes (SCNN1A and GNb3) located within the interval showed no deleterious mutation. In conclusion, we hereby demonstrate further genetic heterogeneity of this Mendelian form of hypertension and identify a new PHA2C locus, the most compelling and precise linkage interval described to date.

Mapping of a first locus for autosomal dominant myxomatous mitral-valve prolapse to chromosome 16p11.2-p12.1.

Myxomatous mitral-valve prolapse (MMVP), also called Barlow disease, is a common cardiac abnormality and affects up to 5% of the population. It is characterized by an excess of tissue that leads to billowing of the mitral leaflets, sometimes complicated by prolapse. Typical histological findings include myxomatous degeneration and degradation of collagen and elastin. Previous reports have proposed an autosomal dominant inheritance of the trait, with age- and sex-dependent expression. By systematic echocardiographic screening of the first-degree relatives of 17 patients who underwent mitral-valve repair, we have identified four pedigrees showing such an inheritance. Genomewide linkage analysis of the most informative pedigree (24 individuals, three generations) showed a significant linkage for markers mapping to chromosome 16p, with a two-point maximum LOD score for D16S3068 (Zmax=3.30 at straight theta=0). Linkage to D16S3068 was confirmed in a second family (Zmax=2.02 at straight theta=0) but was excluded for the two remaining families, thus demonstrating the genetic heterogeneity of the disease. Multipoint linkage analysis performed, with nine additional markers, on the two families with linkage gave maximum multipoint LOD scores of 5.45 and 5.68 for D16S3133, according to a conservative and a stringent model, respectively. Haplotype analysis defined a 5-cM minimal MMVP-1 locus between D16S3068 (16p11.2) and D16S420 (16p12. 1) and a 34-cM maximal interval between D16S404 and D16S3068 when recombination events were taken into account only in affected individuals. The identification of this locus represents a first step toward a new molecular classification of mitral-valve prolapse.

Polymorphisms of the gamma subunit of the epithelial Na+ channel in essential hypertension.

OBJECTIVE: The gamma subunit of the epithelial Na channel (gammaENaC) has been implicated in Liddle's syndrome. The objective of this study was to examine its status in essential hypertension. DESIGN AND METHODS: The search for molecular variants was performed using the SSCP technique after determination of the intron-exon boundaries of the transcribed sequence. We found an additional 205 bp intron splitting the published exon 10 in two. The last exon of gammaENaC was tested with samples from a series of 245 normotensive patients and 453 hypertensive subjects (383 Caucasians, 70 Afro-Caribbeans), all probands of hypertensive families in the HYPERGENE data set. The search was extended to the other 11 transcribed exons in a subset of 65 patients with low-renin profile. RESULTS: Four neutral polymorphisms were detected, three in the third exon of the gene (T387C, T474C, C549T) and one in the last exon (C1990G). These four variants were found with similar frequencies in hypertensive and normotensive Caucasian subjects as well as in patients with low-renin profile. Hypertensive Caucasians and hypertensive subjects of African ancestry also had similar frequencies. Lastly, we found two rare mutations, one the insertion of a proline residue at position 594 of the mature protein (594insP), the other an Arg-to-His substitution at position 631 (R631H). Compared to wild-type (1.00 +/- 0.42, n = 26), expression of the 594insP (1.10 +/- 0.43, n = 26) and R631H (0.97 +/- 0.43, n = 26) variants in Xenopus oocytes produced no significant increase in Na+ current. CONCLUSIONS: Screening of the entire coding sequence of gammaENaC does not suggest that this subunit is frequently involved in essential hypertension.

Genetic analysis of the beta subunit of the epithelial Na+ channel in essential hypertension.

Mutations of the last exon of the beta subunit of the amiloride-sensitive epithelial Na+ channel (betaENaC) can lead to Liddle's syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of betaENaC could be implicated in essential hypertension. After determination of the betaENaC coding gene organization (12 exons spanning 23.5 kb), a systematic screening of the last exon of the gene was performed in 525 subjects (475 whites, 50 Afro-Caribbeans), all probands of hypertensive families. This search was extended to the remaining 11 exons in a subset of 101 probands with low-renin hypertension. Seven amino acid changes were detected: G589S, T594M, R597H, R624C, E632G (last exon), G442V, and V434M (exon 8). These genetic variants were more frequent in subjects of African origin (44%) than in whites (1%). The functional properties of the variants were analyzed in Xenopus oocytes by two independent techniques, ie, electrophysiology and 22Na+ uptake. Small but not significant differences were observed between the variants and wild type. The clinical evaluation of the family bearing the G589S variant, which provided the highest relative ENaC activity, did not show a cosegregation between the mutation and hypertension. The present study illustrates the difficulty in establishing a relation of causality between a susceptibility gene and hypertension. Furthermore, it does not favor a substantial role of the betaENaC gene in essential hypertension.

Renin gene expression in the aging kidney: effect of sodium restriction.

The activity of the renin-angiotensin system as well as the ability of the kidney to retain sodium following salt restriction are reduced with age. The relationship between these age-related changes in renal function and the renin gene expression was presently investigated. The concentrations of renin and its mRNA were measured in kidney of 10- and 30-month-old control female WAG/Rij rats and of animals which were salt restricted for 4 days. In the senescent rats, the kidney renin concentration, like the plasma concentration of angiotensin II, was half that in adult rats. The intrarenal content of renin mRNA did not differ between 10- and 30-month-old animals, suggesting that the transcriptional rate of the renin gene is unchanged with age. During the early phase of adaptation to sodium depletion, the systemic angiotensin II concentration was not modified in either age groups. Four-days salt restriction did not significantly change the renal storage of renin. In contrast, this short term salt restriction induced a 2.3-fold increase in the renin mRNA in adult kidney, and a 1.9-fold increase in the senescent kidney. These data suggest that the age-related decrease in renal concentration of renin is linked to a modification in the rate of translation of renin mRNA, or to an alteration in the protein maturation. The difference in adaptation to the early phase of salt restriction with age should not be linked to changes in renin gene transcription, but more likely to a change in the tissue response to the local renin-angiotensin system.

A mutant renin gene in familial elevation of prorenin.

A case of familial elevation of plasma prorenin levels was discovered during an epidemiological survey of a Dutch population. Trypsin-activated prorenin was elevated in the 58-year-old father, his son, and one of his sisters. All family members were normotensive and had normal plasma renin activities. Exon sequencing of the renin gene of the proband and of his son after polymerase chain reaction amplification identified a point mutation in the last exon of the gene (exon 10). A cytosine to thymine transition creates a premature stop codon at position 387 resulting in a truncated form of renin with 20 amino acids deleted from the carboxyl terminus. All family members presenting high levels of plasma prorenin were heterozygous for the mutation. Expression vectors carrying normal or mutated renin cDNA were transiently transfected into AtT-20 cells to test in vitro the functional consequences of this mutation. Measurements of renin activity and pulse-chase experiments indicated that the truncated renin is inactive and not secreted from transfected cells. We hypothesize that the abnormal gene product of the mutated allele alters renin sorting and propose that plasma prorenin elevation may result from a compensatory mechanism.

Functional study of the germinal angiotensin I-converting enzyme promoter.

Polymerase chain amplification experiments indicate that the germinal specific promoter of the angiotensin I-converting enzyme (ACE) is completely extinguished in somatic tissues. Despite this very strict specificity of expression, the germinal ACE promoter is active in transient transfection experiments in two somatic cell lines and one cell line of germinal origin. The analysis of the promoter shows the existence two regulatory elements within the first 350 bp: a proximal positive element and a distal negative element.

Sib pair linkage analysis of renin gene haplotypes in human essential hypertension.

Although essential arterial hypertension is believed to have a strong genetic predisposition, the gene(s) responsible are unknown. The mechanisms underlying the regulation of blood pressure and experimental studies place the renin gene among the main candidate genes that need to be tested in humans. We tested the hypothesis of a linkage between the renin gene and essential hypertension using the affected sib pair method. Siblings (133 subjects, 52.1 +/- 10.9 years) from 57 families were selected for sustained hypertension (160.7 +/- 22.9/99.5 +/- 12.8 mmHg with 80% of patients under antihypertensive treatment), of early onset (40.7 +/- 12.0 years), in the absence of obesity, diabetes mellitus, and secondary hypertension. Eight renin haplotypes were generated from three diallelic renin restriction fragment length polymorphisms (RFLPs) (TaqI, HinfI, HindIII) located throughout the renin gene. The allelic concordance between the sib pairs was analyzed by identity by state relationships for 98 sib pairs (41 for 41 couples, 39 for 13 trios, 18 for 3 quartets). Allelic frequencies in the 57 hypertensive probands were similar to those observed among 102 hypertensive subjects studied previously. Six of eight possible haplotypes were observed, the informativity of the marker corresponded to 70% of heterozygosity. Allelic concordance for all sib pairs according to sibship size was not significantly different from that expected under the hypothesis of no linkage (t = 0.52, P = 0.15) reflecting only a small excess of renin alleles shared by the hypertensive sibs (1.44 +/- 0.6 vs 1.36 +/- 0.6). Likewise the linkage hypothesis was unsupported by weighted estimates to correct for possible bias due to large sibship size. Thus, the sib pair analysis suggests that the renin gene does not have a frequent role in the pathogenesis of essential hypertension; further more powerful linkage studies or other approaches will be needed to detect contributions at the renin locus to the heritability of essential hypertension.

Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene.

Overlapping genomic clones containing the entire sequence of the human angiotensin I-converting enzyme (ACE) gene were isolated from a lamda phage human DNA library. This gene spans 21 kilobases (kb) and comprises 26 exons, ranging in size from 88 to 481 base pairs. Intron-exon boundaries were sequenced and the relative positions of the exons were mapped. The two different mRNAs transcribed from the ACE gene were assigned to their respective exons. The large endothelial type ACE mRNA (4.3 kb long) is transcribed from exon 1 to exon 26, excluding exon 13. The 3-kb long testicular ACE mRNA is transcribed from exon 13 to exon 26. Exon 13 encodes for the 67 amino acids of the NH2-terminal region of the testicular ACE, whereas downstream exons encode a sequence common to both isozymes. The gene duplication suggested by the internal homology of the endothelial ACE mRNA is now confirmed by the presence of two homologous clusters of eight exons (exons 4-11 and exons 17-24) having similar sizes and codon phases at exon-intron boundaries. The presence of two alternate promoters was investigated by ribonuclease protection assays. The different 5' ends of the two ACE transcripts revealed a promoter for the endothelial ACE mRNA in the 5'-flanking region of the first exon and a promoter for the testicular ACE mRNA situated in intron 12.

Similar frequencies of renin gene restriction fragment length polymorphisms in hypertensive and normotensive subjects.

A prospective study was conducted to compare the frequency of renin gene polymorphisms in normotensive and hypertensive subjects. Hypertensive (n = 102, blood pressure 168 +/- 17/103 +/- 9 mm Hg) and normotensive (n = 120, blood pressure 122 +/- 10/75 +/- 9 mm Hg) subjects were white, had similar age and sex distributions (hypertensive group, 45 +/- 10 years old and 52% female; normotensive group, 44 +/- 9 years old and 55% female) and similar body mass index (hypertensive group, 23.2 +/- 2.6; normotensive group, 22.5 +/- 2.4 kg/m2, p = 0.048). The familial susceptibility to hypertension was defined as at least one parent and one sibling who were hypertensive before age 65; subjects in the normotensive group had no familial history of hypertension. Renin gene polymorphisms located throughout the renin gene were identified by using three restriction enzymes (Taq I, HinfI, HindIII). For each polymorphic restriction site, allele frequencies were similar in the hypertensive and the normotensive groups. In the absence of parental genotypes, the haplotype frequencies combining the three restriction fragment length polymorphisms were estimated by using maximum likelihood techniques and were similar in both groups (hypertensive group, 0.429, 0.277, and 0.177; normotensive group, 0.453, 0.245, and 0.195 for the three most common haplotypes). A rare haplotype detected by Taq I/Hind III was apparently more frequent in the hypertensive than in the normotensive group (hypertensive group, tH 0.086, th 0.022; normotensive group, tH 0.038, th 0.050), but the difference was not statistically significant. In conclusion, no association between renin gene polymorphisms and essential hypertension was demonstrated in the present study.

Molecular biology as a tool for genetic research in hypertension: application to the renin gene.

Hypertension is a multifactorial and heterogeneous disease caused by the addition of environmental and genetic factors. Heritability of systolic and diastolic blood pressure has been well established. Among 6,594 hypertensive patients studied in our clinic, 42% had a history of hypertension in one of their parents and in this group, 13% also had a hypertensive sibling. Anamnestic but not clinical features of patients with familial hypertension slightly differ from patients without familial hypertension. A general strategy is proposed to test the hypothesis that genes known to regulate blood pressure are also responsible for liability to hypertension. In a preliminary report, a study of the TaqI polymorphism of the human renin gene did not reveal a significant difference between hypertensive patients with a family history of hypertension and normotensive controls. A linkage study with DNA markers in informative families is also proposed as an alternative.

Segmental homology between the promoter region of the human renin gene and the mouse ren1 and ren2 promoter regions.

We have isolated and determined the nucleotide (nt) sequence of the 5' region of the human renin gene (h-ren). Two TATA boxes and a CAAT box were found. Start point determination has shown that only the proximal TATA box was used as the transcription initiation signal, both in the kidney and in a renin-secreting tumor. Comparison of the sequence of the 500-bp region upstream from the transcription start point with the corresponding regions of the ren1 and ren2 genes of the Swiss mouse revealed no overall homology between the human and mouse renin sequences. Only very short sequences of high homology ranging in size from 10 to 18 nt were found in the sequenced regions. By hybridization experiments, we have detected a region upstream from each mouse renin gene related to the h-ren; analysis of the nt sequence of this region reveals that they belong to the Alu family of repetitive DNA.

Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture.

Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0 degree C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.

Renin biosynthesis by human tumoral juxtaglomerular cells. Evidences for a renin precursor.

Renin biosynthesis was studied in a juxtaglomerular cell tumor. The tumoral tissue had a high renin content (180 Goldblatt Units/g of tissue), was heavily stained by immunofluorescence using human renin antiserum, and exhibited numerous characteristic secretory granules by electron microscopy. In one series of experiments, renin biosynthesis was studied in tissue slices, by following the incorporation of radiolabeled amino acids into specific immunoprecipitable renin. Time course studies showed that renin was first synthesized in a high molecular weight form, 55,000 mol wt, i.e., 10,000 mol wt higher than that of active renin, and was then converted into a 44,000-mol wt form. In a second series of experiments renin tumoral cells were cultured. Small, round, birefringent cells obtained after collagenase digestion produced renin in both primary culture and subculture media. After 5 d most of the renin found in the culture medium was inactive, but could be activated by trypsin treatment. The tumoral tissue exhibited a strong renin immunofluorescence and numerous secretory granules were observed by electron microscopy. In contrast, the renin-producing cells isolated from this tumor and grown in culture showed little renin immunofluorescence and no secretory granule could be observed. The renin-producing cells in primary culture and subculture were pulsed with radiolabeled amino acids, and immunoprecipitable radiolabeled renin was found in the culture media, thus demonstrating the actual biosynthesis of the enzyme. This renin was not stored inside cultured cells but was rapidly released into the medium and had a molecular weight of 55,000. No conversion of this inactive high molecular weight renin into the active, 44,000 mol wt form of renin was observed. We postulate the existence of two pathways for the processing, packaging, and secretion of renin in the tumoral cells: in juxtaglomerular cells of tumoral tissue renin is synthesized as a preprorenin and rapidly converted into prorenin (55,000 mol wt), which is in turn packaged in secretory granules where it is processed into active renin (44,000 mol wt) and finally secreted; in the cultured tumoral cells renin is still biosynthesized as a preprorenin molecule and then converted into prorenin, but is neither stored as granules nor processed into active renin. In this case the renin is released in an inactive form.