Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy

Abstract

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

“…Less severely affected patients often have difficulties walking and swallowing and require nocturnal respiratory support. Nine genes have been linked to NM to date; NEB, ACTA1, TPM3, TPM2, CFL2, and TNNT1 all encode components of the sarcomeric thin filament in skeletal muscle, while KBTBD13, KLHL40, and KLHL41, which belong to the BTB-BACK-kelch (BBK) protein family, are thought to be involved in ubiquitination and protein degradation (2,3). However many patients with NM do not have identifiable mutations in these genes, suggesting there is further genetic heterogeneity.…”

KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

“…To better understand how loss of KLHL40 from muscle causes lethality, we analyzed the subcellular localization of KLHL40 in mature myofibers. Two previous reports on the localization of KLHL40 reported conflicting data, with one study showing A band localization of KLHL40 while another showed localization to the I band using a commercial antibody on fixed tissue sections (12,13). To determine KLHL40 localization by an alternative method, we electroporated the flexor digitorum brevis of adult mice with KLHL40 C-terminally fused with EGFP (KLHL40-EGFP).…”

“…In NM, it is believed that mutations disrupting thin filament proteins result in reduced force generation and subsequent myopathy (10). However, mutations in 3 proteins with no established association with the sarcomere thin filament, kelch repeat and BTB (POZ) domain containing 13 (KBTBD13), kelch-like family member 40 (KLHL40), and KLHL41, have been shown recently to cause NM in patients by an unknown mechanism (11)(12)(13). Notably, KLHL40 mutations occur frequently in severe forms of autosomal recessive NM (12).…”

Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice

“…These findings demonstrate that the cytoplasmic Klhl31 protein predominantly localizes to the Z-disc in skeletal muscle. This localization differs from that of Klhl40, another muscle-specific kelch-like protein, which was reported to localize to the A-and I-bands (6)(7)(8). Dysregulated sarcomeric, mitochondrial, and SR proteins in Klhl31-KO mice.…”