MERTK R844C Mutant Shows Reduced Phosphorylation Activity in Transfected Cells and is Associated with Severe Rod-Cone Dystrophy

Abstract

Abstract: : Purpose: Mutations in the MERTK gene encoding a receptor tyrosine kinase are responsible for retinal degeneration in the RCS rat, and are a cause of autosomal recessive RP in patients. This study reports novel MERTK mutations associated with severe rod and cone degeneration in a young patient. Methods: Missense mutations identified in the patient were introduced into MERTK expression contructs in pcDNA3.1, and these were used to transfect HEK293T cells. Recombinant MERTK was assayed using immunoprecipitation and immunoblot analysis with anti-MERTK and anti-phosphotyrosine antibodies. Transcript levels were estimated by RT-PCR. Results: By age 13 the patient had 20/60 and 20/200 acuities, with tunnel vision of 5^o centrally and a far temporal peripheral crescent bilaterally; rod and cone ERGs were non-detectable. The fundi showed "bulls eye" macular atrophy and widespread RPE thinning. Three MERTK sequence variants were identified in DNA from the patient: 2164C>T (R722X) in exon 16 and 2593C>T (R865W) in exon 19 on the paternal allele, and 2530C>T (R844C) in exon 19 on the maternal allele. The R844C variant was not detected in screens of unaffected individuals, and R844 is conserved among human, rat, mouse, and chicken. Levels of recombinant MERTK wt and R865W protein in transfected cells were approximately equal, but R844C levels were less than 5% of wt. Tyrosine phosphorylation of cellular proteins was similarly reduced in cells transfected with R844C. Transcript levels for all three constructs were approximately equal. Pulse chase studies are being performed to determine whether R844C has reduced protein stability. Conclusions: R844C present in compound heterozygous form with a presumed null mutation R722X on the other allele results in a severe retinal degeneration phenotype with early age of onset. The R844C variant represents the first putative pathogenic MERTK missense mutation identified, with loss of tyrosine kinase activity potentially due to decreased protein stability.

Keywords: retinal degenerations: hereditary • retinal pigment epithelium • proteins encoded by disease genes