Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities - PubMed (original) (raw)

. 2012 Jan 22;482(7383):98-102.

doi: 10.1038/nature10814.

Murim Choi, Keith A Choate, Carol J Nelson-Williams, Anita Farhi, Hakan R Toka, Irina R Tikhonova, Robert Bjornson, Shrikant M Mane, Giacomo Colussi, Marcel Lebel, Richard D Gordon, Ben A Semmekrot, Alain Poujol, Matti J Välimäki, Maria E De Ferrari, Sami A Sanjad, Michael Gutkin, Fiona E Karet, Joseph R Tucci, Jim R Stockigt, Kim M Keppler-Noreuil, Craig C Porter, Sudhir K Anand, Margo L Whiteford, Ira D Davis, Stephanie B Dewar, Alberto Bettinelli, Jeffrey J Fadrowski, Craig W Belsha, Tracy E Hunley, Raoul D Nelson, Howard Trachtman, Trevor R P Cole, Maury Pinsk, Detlef Bockenhauer, Mohan Shenoy, Priya Vaidyanathan, John W Foreman, Majid Rasoulpour, Farook Thameem, Hania Z Al-Shahrouri, Jai Radhakrishnan, Ali G Gharavi, Beatrice Goilav, Richard P Lifton

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Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities

Lynn M Boyden et al. Nature. 2012.

Abstract

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Recessive and dominant KLHL3 mutations in PHAII kindreds

a–b, Representative kindreds demonstrating recessive (a) and dominant (b) KLHL3 mutations (all 24 kindreds are shown in Supplementary Figs. 3–4). Affected, unaffected, and phenotype-undetermined subjects are denoted by black, white, and gray symbols, respectively. KLHL3 alleles are denoted by ‘+’ (wild-type), ‘d’ (recessive mutation), and ‘D’ (dominant mutation). Sequence traces show wild-type (wt) and mutant (*) alleles and encoded amino acids. c, KLHL3 protein sequence. Colored bars indicate BTB domain (lavender), BACK domain (peach), and Kelch propeller blades (B1-B6, gray) with β-strands ‘a’–‘d’ in yellow, red, green, and blue respectively. Recessive (aqua) and dominant (pink) mutations are shown; recurrences indicated by numbers. d, Kelch propeller schematic, from KLHL2 crystal structure. β-strands colored as in c; dominant mutations indicated. e, CRL schematic, comprising a BTB-Kelch protein (KLHL3), CUL3, and a ubiquitin transfer-mediating RING protein, with substrate bound via the Kelch propeller. Complex shown as a dimer.

Figure 2

Figure 2. Dominant CUL3 mutations in PHAII kindreds cause skipping of exon 9

a, Representative kindreds demonstrating CUL3 mutations, depicted as in Fig. 1 (all 17 kindreds are shown in Supplementary Fig. 6). b, CUL3 mutation locations. Consensus splicing sequences, and corresponding wildtype CUL3 sequences within intron 8, exon 9, and intron 9 are shown; invariant bases (green) and consensus homology (yellow) are indicated. Positions numbered relative to splice sites and first base of the exonic splice (ES) enhancer. Mutations shown in red; recurrences indicated by numbers. c, RT-PCR of spliced RNA. Wild-type CUL3 constructs produce a single product including exons 8, 9, and 10 (844 bp); all nine mutants tested produce a predominant product that skips exon 9 (673 bp). d, Representative RT-PCR sequences. Wild-type construct produces cDNA with properly spliced junctions between exons 8–9 (top) and 9–10 (middle), while mutant construct [splice donor g(+1)c] produces cDNA joining exon 8 to exon 10 (bottom).

Figure 3

Figure 3. KLHL3 expression in the kidney

Mouse kidney sections stained with antibodies to KLHL3 (red), TRPM6 (a marker of the DCT, green) and AQP2 (a marker of the CD, blue). Scale bars 25 μm. a–c, Staining for KLHL3 (a), TRPM6 (b), and the merged image (c) demonstrates KLHL3 expression in the DCT with apical localization (arrowheads). d–f, Staining for KLHL3 (d), AQP2 (e), and the merged image (f) demonstrates KLHL3 expression in CD.

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