Mutations that impair interaction properties of TRIM32 associated with limb-girdle muscular dystrophy 2H

Abstract

TRIM32 belongs to a large family of proteins characterized by a tripartite motif, possibly involved in the ubiquitination process, acting as an E3 ligase. In addition, TRIM32 has six NHL repeats with putative interaction properties. A homozygous mutation at the third NHL repeat (D487N) has been found in patients with limb girdle muscular dystrophy 2H (LGMD2H). This mutation was only identified in the inbred Manitoba Hutterite or their descendants. Interestingly, a mutation in the B-box domain of TRIM32 cosegregates with Bardet-Biedl syndrome type 11 (BBS11). The signs of BBS11 include obesity, pigmentary and retinal malformations, diabetes, polydactyly, and no muscular dystrophy, suggesting an alternative disease mechanism. We aim to ascertain whether D487N is the only pathological LGMD2H allele, limited to Hutterites. We studied the TRIM32 gene in 310 LGMD patients with no mutations at the other known loci. We identified four patients with novel mutated alleles. Two mutations were homozygous and missing in controls. These mutations also clustered at the NHL domain, suggesting that a specific (interaction) property might be abolished in LGMD2H patients. No mutations were found at the B-box region where the BBS11 mutation is found. We tested TRIM32 and its mutants by yeast-two-hybrid assay, developing an interaction test to validate mutations. All LGMD2H mutants, but not the BBS11, lost their ability to self-interact. The interaction of TRIM32 mutants with E2N, a protein involved in the ubiquitination process, was similarly impaired. In conclusion, the mutations here reported may cause muscular dystrophy by affecting the interaction properties of TRIM32.

Detection of TRIM32 deletions in LGMD patients analyzed by a combined strategy of CGH array and massively parallel sequencing

“…All the reported single nucleotide variations are located in the C-terminal part of the protein, in one of the six NHL domains. 1,9,15,18,19 We report here the eighth point mutation in TRIM32. Interestingly, this mutation, p.Leu535Serfs*21, is also located in a NHL domain.…”

“…7,8 TRIM32 contains a RING-finger, a B-box motif, a coiledcoil region and a C terminal domain constituted of six NHL repeats. This last domain is predicted to form a six-bladed beta-propeller structure 9 and is known to be involved in protein-protein interactions. 1,10 In particular, TRIM32 interacts with myosin and is able to ubiquitinate actin, suggesting its participation in myofibrillar turnover.…”

Satellite cell senescence underlies myopathy in a mouse model of limb-girdle muscular dystrophy 2H

“…LGMD2H biopsies show myopathic features of central nucleation, fiber splitting, Z-line streaming, and a dilated sarcotubular system with vacuoles (4,5,8,(13)(14)(15)(16). In the Trim32 -/-mouse model, the absence of TRIM32 resulted in a similar myopathic phenotype to that described in LGMD2H/STM biopsies, demonstrating similar muscle morphology and muscle weakness on grip strength and wire hang testing.…”

“…In agreement with our findings in the mouse, neuronal involvement was also confirmed in some patients with LGMD2H/STM. These LGMD2H-associated neurogenic features included mixed neuropathic and myopathic elements revealed by electromyography, muscle weakness, paresis, paresthesia, hypoactive or absent tendon reflexes, a slight dominance of type I slow muscle fibers, and decreased motor and sensory nerve conduction velocities (5,7,8,14). Therefore, the Trim32 -/-mouse replicates the LGMD2H phenotype and provides an excellent model system for elucidating functions of TRIM32 in vivo.…”

“…Although the TRIM32 protein is ubiquitously expressed, different mutations in the Trim32 gene result in several different inherited diseases, with disparate phenotypes that affect both muscle and nonmuscle tissues. For example, all mutations that result in muscle phenotypes (limb-girdle muscular dystrophy 2H [LGMD2H] and sarcotubular myopathy [STM]) occur in the C-terminal NHL domain of TRIM32 (4)(5)(6)(7)(8).…”