Expanding the Clinical and Genetic Spectrum of RAB28-Related Cone-Rod Dystrophy: Pathogenicity of Novel Variants in Italian Families

Abstract

The small Ras-related GTPase Rab-28 is highly expressed in photoreceptor cells, where it possibly participates in membrane trafficking. To date, six alterations in the RAB28 gene have been associated with autosomal recessive cone-rod dystrophies. Confirmed variants include splicing variants, missense and nonsense mutations. Here, we present a thorough phenotypical and genotypical characterization of five individuals belonging to four Italian families, constituting the largest cohort of RAB28 patients reported in literature to date. All probands displayed similar clinical phenotype consisting of photophobia, decreased visual acuity, central outer retinal thinning, and impaired color vision. By sequencing the four probands, we identified: a novel homozygous splicing variant; two novel nonsense variants in homozygosis; a novel missense variant in compound heterozygous state with a previously reported nonsense variant. Exhaustive molecular dynamics simulations of the missense variant p.(Thr26Asn) in both its active and inactive states revealed an allosteric structural mechanism that impairs the binding of Mg2+, thus decreasing the affinity for GTP. The impaired GTP-GDP exchange ultimately locks Rab-28 in a GDP-bound inactive state. The loss-of-function mutation p.(Thr26Asn) was present in a compound heterozygosis with the nonsense variant p.(Arg137*), which does not cause mRNA-mediated decay, but is rather likely degraded due to its incomplete folding. The frameshift p.(Thr26Valfs4*) and nonsense p.(Leu13*) and p.(Trp107*) variants, if translated, would lack several key structural components necessary for the correct functioning of the encoded protein.

Dawn and dusk peaks of outer segment phagocytosis, and visual cycle function require Rab28

“…These data agree with previous immunofluorescence data suggesting that zebrafish Rab28 is a non‐canonical Rab in its behavior and function 27 . Notably, the recently reported Rab28 T26N variant reported in a CRD family showed normal peak levels of OSP in the null background 19 . Although a slight reduction in RPE phagosomes was observed in the “nucleotide empty” eGFP‐Rab28 T26N retinas compared with wild‐type siblings, this was not statistically significant (Figure 4B,F).…”

“…Notably, the Rab28 T26N variant was recently reported as pathogenic in CRD patients. 19 A slight reduction in RPE phagosomes was observed in the eGFP-Rab28 T26N retinas, however, this was not statistically significant (Figure 4B,F). We also observed an increase in the amount of RPE phagosomes with eGFP-Rab28 Q72L in comparison with the wild-type variant, albeit again this difference did not show statistical significance (Figure 4B,G).…”

“…RAB28 null and hypomorphic alleles cause autosomal recessive cone-rod dystrophy (arCRD) with eight pathogenic alleles attributed to date. [18][19][20][21][22][23] In Caenorhabditis elegans, we have shown that Rab28 is an intraflagellar transport (IFT) and BBSome-associated ciliary protein, 24 which regulates extracellular vesicle biogenesis in a subset of ciliated neurons. 25 In addition, we employed zebrafish knockout and transgenic reporter models to probe Rab28's localization and, we previously described the localization, GTP/GDP nucleotide regulation, and a protein interactome of Rab28 in cone photoreceptors.…”

“…In 2012, the small ciliary GTPase Rab28 was identified as the first Rab GTPase associated with human cone‐rod dystrophy (CRD). RAB28 null and hypomorphic alleles cause autosomal recessive cone‐rod dystrophy (arCRD) with eight pathogenic alleles attributed to date 18‐23 . In Caenorhabditis elegans , we have shown that Rab28 is an intraflagellar transport (IFT) and BBSome‐associated ciliary protein, 24 which regulates extracellular vesicle biogenesis in a subset of ciliated neurons 25 .…”

Prediction of local convergent shifts in evolutionary rates with phyloConverge characterizes the phenotypic associations and modularity of regulatory elements

“…Indeed, the first cytoplasmic domain of GluA1 contains a residue S567 that serves as a substrate for CAMKII in the regulation of AMPA receptor trafficking (Lu et al 2010). STRING also identifies interaction between CAMKIID and RAB28 , a RAS-related small GTPase that is highly expressed in photoreceptors and has been associated with cone-rod dystrophies (Iarossi et al 2020), as well as genes involved in myelination ( QK1 (Wu et al 2002)) and synaptic development ( CNKSR2 (Zhang et al 2020), IL1RAP1 and PTPRD (Yamagata et al 2015)).…”