HGNC Approved Gene Symbol: KLHL3
Cytogenetic location: 5q31.2 Genomic coordinates (GRCh38) : 5:137,617,500-137,736,089 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 5q31.2 | Pseudohypoaldosteronism, type IID | 614495 | Autosomal dominant; Autosomal recessive | 3 |
KLHL3 belongs to a family of proteins that function as substrate adaptors for cullin-3 (CUL3; 603136)-dependent E3 ubiquitin ligase complexes. Substrates for KLHL3-CUL3-mediated ubiquitination and degradation include WNK1 (605232) and WNK4 (601844) (Susa et al., 2014).
Using a fragment derived from the commonly deleted region in malignant myeloid disorders within 5q31 from a PAC contig, EST database searching, 5-prime RACE, and primers designed for RT-PCR with human bone marrow mRNA as a template, Lai et al. (2000) cloned a homolog of Drosophila kelch, designated KLHL3, that has a single open reading frame and 3 isoforms with variable first exons. KLHL3a encodes the deduced full-length protein of 587 amino acids, whereas KLHL3b encodes a 555-amino acid protein and KLHL3c encodes a 505-amino acid protein. Instead of generating proteins with unique N-terminal sequences, alternative usage of the promoters affects only the translation initiation site used. Lai et al. (2000) also identified alternative polyadenylation sites and alternative splicing. The KLHL3 protein shares 77% sequence similarity with Drosophila kelch and 89% similarity with human KLHL2 (605774). Like kelch and KLHL2, KLHL3 contains a poxvirus and zinc finger (POZ) domain in the N terminus and 6 tandem repeats (kelch repeats) in the C terminus. Northern blot analysis detected expression of a 6.5-kb mRNA in fetal liver, thymus, and lymph node; the transcript was barely detectable in spleen and appeared to be absent in peripheral blood and bone marrow. Among 50 human tissues examined by dot-blot analysis, cerebellum and pituitary gland gave the strongest signal; all other tissues were positive but at low intensities.
Using quantitative RT-PCR, Louis-Dit-Picard et al. (2012) demonstrated that KLHL3 is widely expressed, with the highest level of expression being detected in the cerebellum. Quantitative RT-PCR on segments isolated from mouse cortical nephron showed that Klhl3 is highly expressed in the distal collecting tubule (DCT) and, to a lesser extent, in the connecting tubule. Immunohistochemistry studies on serial mouse kidney sections confirmed that the major site of Klhl3 expression was in the DCT and showed that Klhl3 protein was mainly present on the apical side of the DCT cells.
Lai et al. (2000) determined that the KLHL3 gene has 17 exons spanning about 120 kb of genomic DNA.
Gross (2014) mapped the KLHL3 gene to chromosome 5q31.2 based on an alignment of the KLHL3 sequence (GenBank AF208068) with the genomic sequence (GRCh38).
In both HEK 293T cells and mouse distal collecting tubule (mDCT) cells, Louis-Dit-Picard et al. (2012) demonstrated that inhibition of KLHL3 by RNA interference produced an increase in NCC (SLC12A3; 600968) membrane expression similar to that induced by exposure to a hypotonic low-chloride medium, indicating that KLHL3 inhibits NCC membrane expression. Coimmunoprecipitation studies in HEK 293T cells showed interaction between KLHL3 and NCC, suggesting that KLHL3 might be partially responsible for direct regulation of NCC surface expression. Immunofluorescence experiments in HEK 293T cells showed that hypotonicity decreased the expression of endogenous KLHL3 at the plasma membrane by 30%. These data suggested that decreased KLHL3 expression in the membrane compartment is required to increase NCC surface localization.
WNK4 is an integrative regulator of renal electrolyte transport whose main target is the thiazide-sensitive Na-Cl cotransporter NCC. By injecting constructs encoding human KLHL3, WNK4, and CUL3 and mouse Ncc into Xenopus oocytes, Wu and Peng (2013) found that KLHL3 inhibited the positive effect of WNK4 on Ncc by decreasing WNK4 protein abundance via WNK4 ubiquitination and degradation. KLHL3 had no effect on Ncc or on the coexpressed downstream kinase OSR1 (OXSR1; 604046). Wu and Peng (2013) concluded that KLHL3 is an important adaptor for ubiquitination and proteasome-mediated downregulation of WNK4 and regulation of renal Na+ reuptake.
Among 52 PHAII kindreds including 126 affected subjects, Boyden et al. (2012) identified both dominant and recessive mutations in the KLHL3 gene resulting in PHA2D (614495). Whereas recessive KLHL3 mutations were distributed throughout the encoded protein, dominant KLHL3 mutations showed marked clustering. Nine of 16 dominant mutations altered 1 of the last 4 amino acids of the 6 'd-a' loops that connect the outermost (d) beta-strand of 1 kelch propeller blade to the innermost (a) beta-strand of the next blade. Two others were in 'b-c' loops. These dominant PHAH mutations lay near the hub of the propeller at or near sites implicated in substrate binding in paralogs. Three other dominant mutations clustered within the BTB domain, at or near sites implicated in cullin binding in paralogs. Boyden et al. (2012) inferred that dominant mutations in KLHL3 probably impair binding either to specific substrates or to CUL3.
In affected individuals from 16 of 45 families with hyperkalemic hypertension, Louis-Dit-Picard et al. (2012) identified missense mutations in the KLHL3 gene, present in heterozygosity in 12 of the families (see, e.g., 605775.0003, 605775.0004, 605775.0008, 605775.0010, and 605775.0011) and in homozygosity in 4 (see, e.g., 605775.0012). On average, the recessive cases were diagnosed at an earlier age (2.5 months to 17 years) than those with heterozygous mutations (15 to 56 years) and had a more severe phenotype. Screening of the KLHL3 gene in 1,232 individuals with essential hypertension (see 145000) revealed only 1 deleterious mutation in a hypertensive patient with mild hyperkalemia; Louis-Dit-Picard et al. (2012) concluded that missense mutations causing hyperkalemic hypertension are rare in the general hypertensive population of European descent. In addition, analysis of a total of 794 SNPs spread over 1 Mb of KLHL3 in 69,000 individuals showed no significant association with systolic or diastolic blood pressure or with both traits combined when results were adjusted for age, gender, and body mass index, suggesting that common variation in KLHL3 has no significant impact on blood pressure regulation in healthy populations.
Wu and Peng (2013) found that wildtype KLHL3 reduced the protein content of epitope-tagged human WNK4 following coexpression in Xenopus oocytes, whereas KLHL3 with any 1 of 5 representative mutations showed higher WNK4 protein content. Wildtype KLHL3 exhibited robust inhibition of Na+ uptake by oocytes coexpressing mouse Ncc. In contrast, all 5 KLHL3 mutants more weakly inhibited Ncc-dependent Na+ uptake in coexpressed oocytes. Wu and Peng (2013) concluded that PHA2D-causing mutations in KLHL3 interfere with the role of KLHL3 as an adaptor for ubiquitination and proteasome-mediated downregulation of WNK4.
Exclusion Studies
Lai et al. (2000) detected no inactivating mutations of KLHL3 in malignant myeloid disorders with loss of 5q.
Using transgenic mice, Susa et al. (2014) found that heterozygous expression of human KLHL3 with the arg528-to-his (R528H; 605775.0004) mutation caused a high salt-dependent elevation in systolic blood pressure compared with control mice or KLHL3(R528H/+) mice on a low-salt diet. Transgenic KLHL3(R528H/+) mice also showed hyperkalemia and metabolic acidosis under both low- and high-salt conditions. Homozygous KLHL3(R528H/R528H) mice showed similar salt-sensitive hypertension, hyperkalemia, and metabolic acidosis. KLHL3(R528H/+) mice had elevated renal expression of the kinases Wnk1 (605232) and Wnk4 (601844) in distal convoluted tubules, resulting in increased phosphorylation of the Wnk targets Osr1 and Spak (STK39; 607648) and of the sodium-chloride channel Ncc. Susa et al. (2014) proposed that the R528H mutation interferes with binding of KLHL3 to WNK1 and WNK4, impairing ubiquitination and degradation of the kinases and resulting in activation of a phosphorylation cascade that elevates activity of sodium channels, such as ENaC (see 600228), and sodium-chloride channels, such as NCC, at the distal convoluted tubule.
In all affected members of a family with pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified homozygosity for a G-to-A transition in the KLHL3 gene that resulted in a trp-to-ter substitution at codon 470 (W470X). Affected individuals were sibs.
In 1 family with pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified compound heterozygosity for missense mutations in the KLHL3 gene, one a T-to-G transversion resulting in a phe-to-cys substitution at codon 322 (F322C).
In 1 family with pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified compound heterozygosity for missense mutations in the KLHL3 gene; one was a C-to-T transition resulting in a ser-to-leu substitution at codon 410 (S410L). See also 605775.0007.
In a French male proband diagnosed at 29 years of age with hyperkalemic hypertension (PHAII), Louis-Dit-Picard et al. (2012) identified heterozygosity for the S410L substitution, which occurs at a conserved residue in the KLHL3 gene. The mutation was not found in 800 normotensive controls.
In 2 families segregating autosomal dominant pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified a G-to-A transition in the KLHL3 gene resulting in an arg-to-his substitution at codon 528 (R528H).
In 3 French families with hyperkalemic hypertension (PHAII), Louis-Dit-Picard et al. (2012) identified heterozygosity for a 1583G-A transition in the KLHL3 gene, resulting in the R528H substitution, located at a conserved residue in the V-VI interblade d-a loop that was predicted to interact with N529 (see 605775.0011). The mutation segregated with disease in each family and was not found in 800 normotensive controls.
Using transgenic mice expressing human KLHL3 with the R528H mutation, Susa et al. (2014) presented evidence that R528H causes PHA2D by interfering with KLHL3-dependent binding and degradation of the kinases WNK1 (605232) and WNK4 (601844), leading to excessive phosphorylation and activation of sodium channels, such as ENaC (see 600228), and sodium-chloride channels, such as NCC (SLC12A3; 600968), in distal convoluted tubules.
In 3 affected sibs from a family with autosomal recessive pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified a C-to-T transition resulting in an arg-to-ter substitution at codon 240 (R240X) in compound heterozygosity with a missense mutation (see 605775.0006).
In a family with autosomal recessive pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified compound heterozygosity for a G-to-T transversion in the KLHL3 gene resulting in an arg-to-ile substitution at codon 336 (R336I) and a nonsense mutation (see 605775.0005).
In a single proband with pseudohypoaldosteronism type II (PHA2D; 614495), Boyden et al. (2012) identified compound heterozygosity for the S410L mutation in KLHL3 (605775.0003) and an A-to-G transition resulting in a tyr-to-cys substitution at codon 557 (Y557C).
In a family segregating autosomal dominant pseudohypoaldosteronism type IID (PHA2D; 614495), Boyden et al. (2012) identified a C-to-T transition in the KLHL3 gene resulting in an arg-to-cys substitution at codon 528 (R528C).
In a 21-year-old Canadian male proband with hyperkalemic hypertension (PHAII), Louis-Dit-Picard et al. (2012) identified heterozygosity for a 1582C-T transition in the KLHL3 gene, resulting in the R528C substitution, located at a conserved residue in the V-VI interblade d-a loop that was predicted to interact with N529 (see 605775.0011). The mutation was not found in 800 normotensive controls.
In 2 independent pedigrees segregating autosomal dominant pseudohypoaldosteronism, type IID (PHA2D; 614495), Boyden et al. (2012) identified heterozygosity for a G-to-A transition in the KLHL3 gene resulting in an ser-to-asn substitution at codon 433 (S433N).
In affected members of a large 4-generation French family segregating autosomal dominant hyperkalemic hypertension (PHA2D; 614495), Louis-Dit-Picard et al. (2012) identified heterozygosity for a 1193C-T transition in the KLHL3 gene, resulting in an ala398-to-val (A398V) substitution at a conserved residue. The mutation was not found in unaffected family members or in 800 normotensive controls. The phenotype was mild in the 14 affected family members, only 3 of whom had hypertension, with a modest but significant increase in serum potassium present in the remaining affected individuals compared to unaffected family members.
In a 47-year-old French man with hyperkalemic hypertension (PHA2D; 614495), Louis-Dit-Picard et al. (2012) identified heterozygosity for a 1587C-A transversion in the KLHL3 gene, resulting in an asn529-to-lys (N529K) substitution at a conserved residue in the V-VI interblade d-a loop that was predicted to interact with R528 (see 605775.0004 and 605775.0008) and Y577. The mutation was not found in 800 normotensive controls.
In 2 male probands from unrelated consanguineous families with hyperkalemic hypertension (PHA2D; 614495), Louis-Dit-Picard et al. (2012) identified homozygosity for a 1277C-T transition in the KLHL3 gene, resulting in a pro426-to-leu (P426L) substitution at a conserved residue in the III-IV interblade d-a loop. The mutation was not found in 800 normotensive controls.
Boyden, L. M., Choi, M., Choate, K. A., Nelson-Williams, C. J., Farhi, A., Toka, H. R., Tikhonova, I. R., Bjornson, R., Mane, S. M., Colussi, G., Lebel, M., Gordon, R. D., and 34 others. Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities. Nature 482: 98-102, 2012. [PubMed: 22266938] [Full Text: https://doi.org/10.1038/nature10814]
Gross, M. B. Personal Communication. Baltimore, Md. 11/24/2014.
Lai, F., Orelli, B. J., Till, B. G., Godley, L. A., Fernald, A. A., Pamintuan, L., Le Beau, M. M. Molecular characterization of KLHL3, a human homologue of the Drosophila kelch gene. Genomics 66: 65-75, 2000. [PubMed: 10843806] [Full Text: https://doi.org/10.1006/geno.2000.6181]
Louis-Dit-Picard, H., Barc, J., Trujillano, D., Miserey-Lenkei, S., Bouatia-Naji, N., Pylypenko, O., Beaurain, G., Bonnefond, A., Sand, O., Simian, C., Vidal-Petiot, E., Soukaseum, C., and 27 others. KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron. Nature Genet. 44: 456-460, 2012. Note: Erratum: Nature Genet. 44: 609 only, 2012. [PubMed: 22406640] [Full Text: https://doi.org/10.1038/ng.2218]
Susa, K., Sohara, E., Rai, T., Zeniya, M., Mori, Y., Mori, T., Chiga, M., Nomura, N., Nishida, H., Takahashi, D., Isobe, K., Inoue, Y., Takeishi, K., Takeda, N., Sasaki, S., Uchida, S. Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice. Hum. Molec. Genet. 23: 5052-5060, 2014. [PubMed: 24821705] [Full Text: https://doi.org/10.1093/hmg/ddu217]
Wu, G., Peng, J.-B. Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation. FEBS Lett. 587: 1717-1722, 2013. [PubMed: 23665031] [Full Text: https://doi.org/10.1016/j.febslet.2013.04.032]