The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract

Abstract

Genome-wide linkage analysis, followed by targeted deep sequencing, in a Danish multigeneration family with juvenile cataract revealed a region of chromosome 17 co-segregating with the disease trait. Affected individuals were heterozygous for two potentially protein-disrupting alleles in this region, in ACACA and UNC45B. As alterations of the UNC45B protein have been shown to affect eye development in model organisms, effort was focused on the heterozygous UNC45B missense mutation. UNC45B encodes a myosin-specific chaperone that, together with the general heat shock protein HSP90, is involved in myosin assembly. The mutation changes p.Arg805 to Trp in the UCS domain, an amino acid that is highly conserved from yeast to human. UNC45B is strongly expressed in the heart and skeletal muscle tissue, but here we show expression in human embryo eye and zebrafish lens. The zebrafish mutant steif, carrying an unc45b nonsense mutation, has smaller eyes than wild-type embryos and shows accumulation of nuclei in the lens. Injection of RNA encoding the human wild-type UNC45B protein into the steif homozygous embryo reduced the nuclei accumulation and injection of human mutant UNC45B cDNA in wild-type embryos resulted in development of a phenotype similar to the steif mutant. The p.Arg805Trp alteration in the mammalian UNC45B gene suggests that developmental cataract may be caused by a defect in non-muscle myosin assembly during maturation of the lens fiber cells. Keywords: congenital cataract; UNC45B; non-muscle myosin; epithelial lens cell; zebrafish steif mutant INTRODUCTION Congenital/infantile cataract (CC) is a developmental anomaly characterized by opacities in the crystal lens of the eye and is a common cause of restricted vision and blindness in children. Environmental and intrinsic factors are involved, including metabolic and genetic causes for CC. Mendelian forms of CC comprise a broad spectrum of syndromic and nonsyndromic phenotypes characterized by a set of associated ocular and/or systemic abnormalities. More than 35 loci, including at least 25 known genes, have been associated with nonsyndromic cataract, the majority showing autosomal dominant inheritance with high penetrance (ADCC). 1,2 Mutations in crystallins, particularly CRYAA, CRYBB2, and CRYGD and the connexin genes GJA3 and GJA8 comprise the largest group of loci causing ADCC, but mutations are also found in the membrane proteins MIP, LIM2, TMEM114 and CHMP4B, in cytoskeleton proteins (BSFP1 and BSFP2) and in transcription factors (HSF4 and MAF). 1,2 In a recent study, 3 we reported that these loci represented most of the causative mutations in 19 out of 28 unrelated Danish CC families. Among the unsolved families was one in which genome-wide linkage analysis revealed two candidate loci at 2q32.3-q33.3 and 17q11.2-q21.2, respectively. 3 The family comprised three generations with nine

The UNC-45 Myosin Chaperone

“…Intriguingly, Arg805Trp, the sole identified human mutation in UNC-45b maps to ARM repeat 19 (helix 2) of the UCS domain (Hansen et al, 2014), which is the equivalent of C. elegans Arg819 discussed above. Hence this would be expected to disrupt the very same ionic interaction that is affected by the C. elegans Glu781Lys mutation, further emphasizing its critical nature.…”

“…The introduction of a long charged side group could interfere with substrate binding. Further, the zebrafish steif mutation introduces a stop codon after residue 788 in helix 3 of ARM repeat 18 (Etard et al, 2007), which is two residues C-terminal to the dicky ticker mutation (Hansen et al, 2014). These observations indicate that ARM repeats 18 and 19 in the UCS domain are critical for UNC-45 function.…”

“…Hence this would be expected to disrupt the very same ionic interaction that is affected by the C. elegans Glu781Lys mutation, further emphasizing its critical nature. Surprisingly, the human mutation results in dominant juvenile cataracts but does not appear to affect skeletal or cardiac muscle function (Hansen et al, 2014). This is more fully discussed in section 4.3.…”

“…UNC-45 homologues have been found in all Bilateria examined thus far including worms, flies, frogs, fish, mice and humans (Epstein and Thomson, 1974; Etheridge et al, 2002; Geach and Zimmerman, 2010; Lee et al, 2011b; Price et al, 2002) as well as various other species (Hansen et al, 2014). Invertebrates such as worms and flies have one UNC-45 protein (Barral et al, 1998; Lee et al, 2011b; Venolia et al, 1999).…”

The zebrafish eye—a paradigm for investigating human ocular genetics

“…Heterozygous mutations in the myosin chaperone UNC45B have recently been associated with congenital cataract. 62 Zebrafish steif mutants carrying an unc45b nonsense mutation present with a small lens and lens fibre cell nuclear retention, where degradation of all cytoplasmic organelles within the lens fibre cells is necessary for the establishment and maintenance of normal lens transparency. Injection of RNA encoding the human UNC45B protein into homozygous mutant zebrafish embryos rescued the ectopic nuclei phenotype, highlighting the potential of novel therapeutic targets to treat ocular pathologies.…”