Alternative titles; symbols
HGNC Approved Gene Symbol: KLHL40
Cytogenetic location: 3p22.1 Genomic coordinates (GRCh38) : 3:42,685,537-42,692,544 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 3p22.1 | Nemaline myopathy 8, autosomal recessive | 615348 | Autosomal recessive | 3 |
Ravenscroft et al. (2013) reported that the 621-amino acid KLHL40 protein has an N-terminal BTB/BACK domain and 5 C-terminal Kelch repeats predicted to form a beta-propeller structure. RT-PCR analysis of human tissues detected highest expression in adult skeletal muscle, with much lower expression in heart, and very weak expression in pancreas. No expression was detected in brain, placenta, lung, liver, and kidney. In human fetal tissues, KLHL40 was detected in striated muscle only. Western blot analysis of human skeletal muscle showed that KLHL40 was more abundant in fetal muscle than in postnatal muscle. Western blot analysis of mouse tissues detected Klhl40 in skeletal muscle only. Expression of Klhl40 increased with differentiation in cultured mouse C2C12 myoblasts. Immunohistochemical analysis of human fetal skeletal muscle localized KLHL40 to the A-band.
By Northern blot analysis of mouse tissues, Garg et al. (2014) detected high Klhl40 expression in skeletal muscle, lower expression in heart, and little to no expression in other tissues examined. Electroporation of flexor digitorum brevis of adult mice showed localization of fluorescence-tagged Klhl40 at both the I band and A band.
Ravenscroft et al. (2013) determined that the KLHL40 gene contains 6 coding exons.
Hartz (2013) mapped the KLHL40 gene to chromosome 3p22.1 based on an alignment of the KLHL40 sequence (GenBank AK056577) with the genomic sequence (GRCh37).
Garg et al. (2014) found that, rather than promote proteasome-mediated protein degradation like other BTB-BACK-Kelch family proteins, mouse Klhl40 stabilized skeletal muscle Neb (161650) and Lmod3 (616112). Yeast 2-hybrid analysis of a human skeletal muscle cDNA library, followed by coimmunoprecipitation analysis, showed that Klhl40 interacted directly with NEB and LMOD3. In transfected COS7 cells, coexpression of Klhl40 stabilized NEB and LMOD3 proteins comparably to a proteasome inhibitor. Klhl40 reduced polyubiquitination of lys48 of LMOD3, which specifically marks a protein for proteasome-mediated degradation.
By whole-exome sequencing, Ravenscroft et al. (2013) identified homozygous or compound heterozygous mutations (see, e.g., 615340.0001-615340.0003) in the KLHL40 gene in affected members from 6 families with autosomal recessive severe nemaline myopathy-8 (NEM8; 615348). The mutations segregated with the disorder and were not found in several large control databases. Subsequent screening of KLHL40 by Sanger sequencing in additional probands with severe NEM resulted in the identification of a total of 19 variants in 28 (19.6%) of 143 families affected by severe NEM. There were 4 frameshifts, 12 missense mutations, 2 nonsense mutations, and 1 splice site mutation, and the mutations were scattered throughout all exons. One mutation (E528K; 615340.0001) was identified to be a founder mutation among Japanese individuals. Structural analysis of the missense mutations indicated that they would most likely destabilize bonds or hydrophobic cores, resulting in protein instability. In addition, 129 probands with a milder phenotype were screened, but no KLHL40 mutations were identified in this cohort, confirming that KLHL40 mutations are most likely exclusive to severe cases. The phenotype was severe and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties apparent at birth. The average age at death was 5 months. Skeletal muscle biopsy showed absence of KLHL40 even in patients with 2 missense mutations, consistent with a loss of function mechanism, and up to 20% of patients had virtually no normal myofibrils ('miliary NEM'). Ravenscroft et al. (2013) suggested that the KLHL40 gene should be screened in individuals with autosomal recessive NEM who present with prenatal symptoms and/or contractures, as well as in all Japanese individuals with severe NEM.
By analyzing muscle samples from patients with nemalin myopathy and mutations in KLHL40, Garg et al. (2014) found that 2 patients with severe muscle pathology and complete deficiency of KLHL40 expression had marked reduction in LMOD3 and NEB. A third patient with less severe muscle pathology and residual KLHL40 had relatively normal LMOD3 and NEB content.
The zebrafish genome contains 2 orthologs of KLHL40, Klhl40a and Klhl40b. RT-PCR detected Klhl40a prominently expressed in larval zebrafish and in adult heart and skeletal muscle, with much weaker expression in adult eye, skin, and brain. Klhl40b was prominently expressed in larval zebrafish and in adult skeletal muscle, with weaker expression in adult eye. Ravenscroft et al. (2013) found that morpholino-mediated knockdown of both Klhl40 genes in zebrafish resulted in a curved trunk and small head, with disrupted muscle patterning, gaps between myofibers, and widened Z-disks. Knockdown of either or both Klhl40 genes in zebrafish caused sporadic muscle tremors and absence of coordinated swimming.
Garg et al. (2014) found that Klhl40 -/- mice were indistinguishable from wildtype at birth, but they failed to grow, and none survived past 3 weeks of age. At 1 day of age, hindlimb skeletal muscle appeared normal, but it showed at least 50% less strength than wildtype. At 1 week of age, Klhl40 -/- skeletal muscle fibers appeared abnormal, with Z-line streaming, widened Z discs, and reduced content of Neb and Lmod3.
In affected members of 2 consanguineous families of Turkish and Kurdish origin, respectively, with nemaline myopathy-8 (NEM8; 615348), Ravenscroft et al. (2013) identified a homozygous c.1582G-A transition in exon 4 of the KLHL40 gene, resulting in a glu528-to-lys (E528K) substitution at a conserved residue in Kelch repeat domain-4. The mutation, which was identified by whole-exome sequencing and segregated with the disorder, was not found in several large control databases. Subsequent screening of this gene in a large cohort of patients with a similar phenotype identified the homozygous E528K mutation in another Kurdish family and in 9 Japanese families. The E528K mutation was found in 1 of 510 Japanese controls, yielding a frequency of 0.0098 in this population. The mutation was estimated to have arisen in the Japanese population about 4,900 years ago, consistent with a founder effect. However, haplotype analysis showed a different haplotype between a Japanese and Turkish family with the mutation, indicating that the mutation arose independently in these populations. Another Japanese family in whom exome sequencing was performed was found to be compound heterozygous for E528K and a c.1405G-T transversion in exon 3 of the KLHL40 gene, resulting in a gly469-to-cys (G469C; 615340.0002) substitution in Kelch repeat domain-3. Affected members of yet another Japanese family were homozygous for G469C. Structural modeling suggested that both mutations would destabilize hydrogen bonds and thus impair protein stability. Another Japanese and a Korean family were compound heterozygous for E528K and another pathogenic truncating mutation.
For discussion of the gly469-to-cys (G469C) mutation in the KLHL40 gene that was found in compound heterozygous state in patients with nemaline myopathy-8 (NEM8; 615348) by Ravenscroft et al. (2013), see 615340.0001.
In affected members of a Turkish family with nemaline myopathy-8 (NEM8; 615348), Ravenscroft et al. (2013) identified a homozygous c.602G-T transversion in exon 1 of the KLHL40 gene, resulting in a trp201-to-leu (W201L) substitution at a conserved residue in the BACK domain. The mutation, which was found by whole-exome sequencing, segregated with the disorder and was not found in several large control databases. Structural modeling suggested that the mutation would destabilize hydrogen bonds and thus impair protein stability.
In affected members of a consanguineous Turkish family with nemaline myopathy-8 (NEM8; 615348), Ravenscroft et al. (2013) identified a homozygous c.1612G-C transversion in exon 5 of the KLHL40 gene, resulting in an ala538-to-pro (A538P) substitution at a conserved residue in Kelch repeat domain-4. Structural modeling suggested that the mutation would destabilize hydrogen bonds and thus impair protein stability.
In affected members of a Norwegian family with nemaline myopathy-8 (NEM8; 615348), Ravenscroft et al. (2013) identified a homozygous c.602G-A transition in exon 1 of the KLHL40 gene, resulting in a trp201-to-ter (W201X) substitution in the BACK domain.
Garg, A., O'Rourke, J., Long, C., Doering, J., Ravenscroft, G., Bezprozvannaya, S., Nelson, B. R., Beetz, N., Li, L., Chen, S., Laing, N. G., Grange, R. W., Bassel-Duby, R., Olson, E. N. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy. J. Clin. Invest. 124: 3529-3539, 2014. [PubMed: 24960163] [Full Text: https://doi.org/10.1172/JCI74994]
Hartz, P. A. Personal Communication. Baltimore, Md. 7/25/2013.
Ravenscroft, G., Miyatake, S., Lehtokari, V.-L., Todd, E. J., Vornanen, P., Yau, K. S., Hayashi, Y. K., Miyake, N., Tsurusaki, Y., Doi, H., Saitsu, H., Osaka, H., and 43 others. Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy. Am. J. Hum. Genet. 93: 6-18, 2013. [PubMed: 23746549] [Full Text: https://doi.org/10.1016/j.ajhg.2013.05.004]