A missense mutation in ASRGL1 is involved in causing autosomal recessive retinal degeneration

Abstract

Inherited retinal dystrophies are a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. We analyzed a large five-generation pedigree with early-onset recessive retinal degeneration to identify the causative mutation. Linkage analysis and homozygosity mapping combined with exome sequencing were carried out to map the disease locus and identify the p.G178R mutation in the asparaginase like-1 gene (ASRGL1), segregating with the retinal dystrophy phenotype in the study pedigree. ASRGL1 encodes an enzyme that catalyzes the hydrolysis of L-asparagine and isoaspartyl-peptides. Studies on the ASRGL1 expressed in Escherichia coli and transiently transfected mammalian cells indicated that the p.G178R mutation impairs the autocatalytic processing of this enzyme resulting in the loss of functional ASRGL1 and leaving the inactive precursor protein as a destabilized and aggregation-prone protein. A zebrafish model overexpressing the mutant hASRGL1 developed retinal abnormalities and loss of cone photoreceptors. Our studies suggest that the p.G178R mutation in ASRGL1 leads to photoreceptor degeneration resulting in progressive vision loss.

Figure 1.

(A) Haplotype analysis and segregation of the ASRGL1 p.G178R mutation in a five-generation pedigree with recessive IRD: haplotype was constructed with the genotypes of 7 microsatellite markers and identified D11S4459 and D11S1883 markers as the boundaries of the disease interval on chromosome 11 (q12.1–q13.1). The p.G178R ASRGL1 mutation segregated with IRD in this pedigree. (B) Sequence of the region encompassing the ASRGL1 p.G178R mutation in an affected, carrier and unaffected individual from the pedigree with IRD.

Figure 2.

Fundus images of two affected and one unaffected member: Fundus images of affected members show retinal vessel attenuation, pigmentation (IV:1 30 years old and IV:8 50 years old) and a bull’s eye pattern of RPE atrophy in IV:1. Whereas, the fundus images of the unaffected member IV:3 showed no abnormality.

Figure 3.

Full field ERG response in two affected and one unaffected member of the family with IRD. Scotopic responses at 0 db and 30 Hz flicker responses of affected members IV:1 (30 years old) and IV:8 (50 years old) were undetectable suggestive of compromised rod and cone photoreceptor response while the unaffected individual (IV:3) exhibited rod and cone responses within normal ranges.

Figure 4.

Expression profile of ASRGL1 in ocular and body tissue. (A) Levels of expression of ASRGL1 transcript in 2-months-old old mouse ocular tissue as measured by quantitative real-time PCR. PE: Posterior eye cup (RPE + Choroid); ICB: Iris-Ciliary body; ON: Optic nerve. (B) Expression of ASRGL1 transcript in the retinal tissue of mice during development and aging as measured by quantitative real-time PCR. (C) Levels of expression of ASRGL1 transcript in 2-months-old mouse different body tissue as measured by quantitative real-time PCR. (D) Immunostaining of 2 months old mouse retinal sections with only secondary antibodies did not reveal positive staining in the left image. Right side image representing the localization of ASRGL1 protein to the photoreceptor inner segment region of a 2-months-old mouse retina: ASRGL1 signal was represented by green fluorescence and red fluorescence color represented S-cones. RPE: Retinal pigment epithelium; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GC: ganglion cell layer.

Figure 5.

In vitro processing of wt-ASRGL1and G178R-ASRGL1. (A) SDS-PAGE of wt-ASRGL1 and G178R-ASRGL1 following in vitro incubation at 37 °C over time. HMW species in lanes (5 and 7) loaded with the G178R-ASRGL1 are indicated with arrows. (B) Densitometry analysis of intramolecular processing of wt-ASRGL1 and G178R-ASRGL1 following in vitro incubation at 37 °C over time.

Figure 6.

Expression and localization of wt and G178R-ASRGL1 in Cos-7 cells. (A) Cos7 cells transfected with wt-ASRGL1 tagged with cMyc (green), showed the distribution of ASRGL1-cMyc fusion protein throughout the cytoplasm. (B) The cytoskeletal marker Vimentin (Red) distributed throughout the cytosol. (C) Merged image of A and B. (D) The cells transfected with G178R-ASRGL1 showed perinuclear localization of the G178R-ASRGL1-cMyc fusion protein (green). (E) Re-organization of Vimentin signal (Red) in cells transfected with G178R-ASRGL1. (F) Co-localization of G178R-ASRGL1 and Vimentin. (G) Asparaginase activity in the whole cell lysates of cells transfected with wt-ASRGL1 and G178R-ASRGL1. The asparaginase activity detected in cells expressing the G178R-ASRGL1 was significantly lower (P < 0.0001) compared with the enzymatic activity observed in cells expressing the wt-ASRGL1.

Figure 7.

Retinal maker protein expression in zebrafish injected with wt or G178R-ASRGL1 mRNA. (A) Dye-injected fish showing normal distribution of cones. (B) Fish injected with 20 pg wt-ASRGL1 mRNA showed normal pattern of cone distribution. (C) Fish injected with only 4 pg G178R-ASRGL1 mRNA showed loss of green cones. (D) Fish injected with 5 pg of G178R-ASRGL1 mRNA showed eye development abnormality. First column: Immunostaining with rod specific antiobodies (green); Second column: Immunostaining with Blue/Red/UV opsin specific antibodies (Red). The third column in each panel is the merged image of sections stained for rod and cone photoreceptors and the nuclei stained with DAPI.