Detection of mutations in KLHL3 and CUL3 in families with FHHt (familial hyperkalaemic hypertension or Gordon's syndrome)

Abstract

The study of families with rare inherited forms of hypo- and hyper-tension has been one of the most successful strategies to probe the molecular pathophysiology of blood pressure control and has revealed dysregulation of distal nephron Na+ reabsorption to be a common mechanism. FHHt (familial hyperkalaemic hypertension; also known as Gordon's syndrome) is a salt-dependent form of hypertension caused by mutations in the regulators of the thiazide-sensitive Na+-Cl- co-transporter NCC [also known as SLC12A3 (solute carrier family 12 member 3)] and is effectively treated by thiazide diuretics and/or dietary salt restriction. Variation in at least four genes can cause FHHt, including WNK1 [With No lysine (=K) 1] and WNK4, KLHL3 (kelch-like family member 3), and CUL3 (cullin 3). In the present study we have identified novel disease-causing variants in CUL3 and KLHL3 segregating in 63% of the pedigrees with previously unexplained FHHt, confirming the importance of these recently described FHHt genes. We have demonstrated conclusively, in two unrelated affected individuals, that rare intronic variants in CUL3 cause the skipping of exon 9 as has been proposed previously. KLHL3 variants all occur in kelch-repeat domains and so probably disrupt WNK complex binding. We have found no evidence of any plausible disease-causing variants within SLC4A8 (an alternative thiazide-sensitive sodium transporter) in this population. The results of the present study support the existing evidence that the CUL3 and KLHL3 gene products are physiologically important regulators of thiazide-sensitive distal nephron NaCl reabsorption, and hence potentially interesting novel anti-hypertensive drug targets. As a third of our non-WNK FHHt families do not have plausible CUL3 or KLHL3 variants, there are probably additional, as yet undiscovered, regulators of the thiazide-sensitive pathways.

Figure 1. Demonstration that the CUL3 variants result in splice variation leading to a loss of exon 9 in affected individuals from pedigree 1 (Ped1) and pedigree 2 (Ped2)

The affected individuals sequenced are highlighted by * in Supplementary Figure S3 (at http://www.clinsci.org/cs/126/cs1260721add.htm). (A) Reverse transcription–PCR of CUL3 from peripheral blood mononuclear cells demonstrated an additional (smaller) cDNA band only in the affected individuals. The size of the smaller band was consistent with a deficiency of exon 9 (difference in band size=171 bp). PCR primers RT1 (random primers) and RT2 (a CUL3-specific primer) are detailed in the Materials and methods section. The molecular size is given on the left-hand side in bp. (B) Sanger sequencing of CUL3 cDNA from the smaller 167 bp band confirmed that exon 9 is skipped in individuals from both pedigrees. Sequence excerpts from the larger 338 bp band are shown for the wild-type (WT) individual for comparison, demonstrating the wild-type exon boundaries. Sequencing chromatograms are shown together with the DNA sequence and amino acid codons above.