Abstract
purpose. Mutations in the MERTK gene are responsible for retinal degeneration in the Royal College of Surgeons (RCS) rat and are a cause of human autosomal recessive retinitis pigmentosa (RP). This study reports the identification and functional analysis of novel MERTK mutations to provide information regarding whether they are causative of severe rod–cone degeneration in a young patient.
methods. MERTK missense variants identified by single-strand conformational polymorphism (SSCP) and sequence analysis were introduced into expression constructs and used to transfect HEK293T cells. Recombinant protein expression was assayed with anti-MERTK and anti-phosphotyrosine antibodies. Protein turnover was assayed in pulse–chase studies of 35S-methionine incorporation. Transcript levels were determined by quantitative RT-PCR.
results. Three MERTK sequence variants were identified in a patient with rod–cone dystrophy: R722X in exon 16 and R865W in exon 19 on the paternal allele and R844C in exon 19 on the maternal allele. The R844C sequence change affects an evolutionarily conserved amino acid residue and was not detected in unaffected individuals. In transfected HEK293Tcells, wild-type (wt) and W865 MERTK were expressed at equivalent levels and present in the plasma membrane, stimulated tyrosine phosphorylation, and induced significant rounding of the cell bodies. In contrast, C844 MERTK was expressed at low levels and did not stimulate tyrosine phosphorylation. In addition, the relative stability of C844 MERTK was significantly less than wt in assays of protein turnover. At age 13, the patient had 20/60 and 20/200 acuities, tunnel vision of 5° centrally, and a far temporal peripheral crescent bilaterally, and ERGs were nondetectable. The fundi showed bull’s-eye macular atrophy and widespread RPE thinning.
conclusions. The present study reports the identification of R844C, the first putative pathogenic MERTK missense mutation that results in severe retinal degeneration with childhood onset when in compound heterozygous form with a R722X allele. The loss of function of C844 MERTK is probably due to decreased protein stability.
The retinal degeneration phenotype of the Royal College of Surgeons (RCS) rat results from a 1.5- to 2-kb deletion in the
Mertk gene
1 that impairs the ability of the retinal pigment epithelium (RPE) to phagocytose membranes shed from photoreceptor outer segments (OS) during their renewal.
2 3 4 Shed OS membranes are bound, but not ingested by the RPE, resulting in a debris field that disrupts the vital flow of nutrients, vitamin A, and oxygen between the RPE and retina.
5 6 As one of the best-characterized animal models of retinal dystrophy, the RCS rat has been the subject of intense study and testing of various therapeutic strategies.
7 A corresponding mouse model also exists—created by targeted disruption of c-
Mer (the mouse orthologue of
Mertk) by using homologous recombination—that has a retinal degeneration phenotype similar to that of the RCS rat.
8 9
Mertk (Tyro12)
10 11 is a member of the receptor tyrosine kinase family of cell-surface receptors that includes Axl (Tyro7) and Rse (Sky, Tyro3) and consists of an intracellular kinase-containing domain, a transmembrane region, and a cell-adhesion molecule–related extracellular domain.
12 13 14 15 Mertk is expressed in a number of tissues outside the eye, including macrophages, epithelia, and reproductive tissue.
10 It is required for clearance of apoptotic cells by mononuclear phagocytes in mice,
16 with its absence resulting in progressive lupuslike autoimmunity.
17 It also plays a role in blocking lipopolysaccharide-induced endotoxic shock through inhibition of TNF-α production.
8 Growth-arrest–specific protein 6 (Gas6) is an activating ligand for Mertk and the other receptors in this family,
18 19 and stimulates OS phagocytosis by RPE cells in culture.
20 21 Gas6 may facilitate phagocytosis by binding to phosphatidylserine residues exposed on the outer leaflet of spent OS membranes, thus promoting association with the RPE.
1 22 23 Ligand activation of Mertk is predicted to result in the formation of receptor homodimers, activation of receptor kinase activity, and phosphorylation of tyrosine residues in the receptor intracellular domain.
24 The role of Mertk in OS phagocytosis may also involve interactions with other receptors involved in OS recognition and binding, including αvβ5 integrin, type B scavenger receptor CD36, and mannose receptors.
25 26 27 28
MERTK was established as a human retinal dystrophy gene by the finding of disease-associated mutations in three patients with autosomal recessive retinitis pigmentosa (RP).
29 The mutations identified were presumed loss-of-function alleles predicted to result in truncated protein lacking the intracellular region. A number of apparently nonpathogenic
MERTK sequence variants were also identified, suggesting that
MERTK tolerates a significant number of amino acid substitutions. The present study reports the identification of novel disease-associated
MERTK mutations in a young retinal dystrophy patient. Our findings establish that the amino acid substitution found can disrupt MERTK signaling by decreasing protein stability and provide a description of the associated phenotype that may be helpful in identifying additional patients with
MERTK mutations.
A human cDNA encoding MERTK (including 70-bp 5′- and 273-bp 3′-untranslated sequence) was obtained by screening a kidney cDNA library and cloned into pcDNA3.1 (Invitrogen, San Diego, CA). The c.2530C→T (R844C) and c.2593C→T (R865W) sequence variants were introduced by site-directed mutagenesis (QuikChange Kit; Stratagene, La Jolla, CA). Constructs were verified by DNA sequencing, and two independent isolates of each construct were obtained and used in replicate experiments.
HEK293T cells grown in DMEM containing 1 mM sodium pyruvate and 10% FBS were transiently transfected with
MERTK constructs using a lipophilic transfection agent (1μg DNA/4 μL reagent; Lipofectamine Plus; Invitrogen) according to the manufacturer’s instructions. A lacZ encoding expression construct (pCMV-βgal) was included in each
MERTK DNA sample (10% of total DNA) to control for transfection efficiency assayed by β-galactosidase staining. MERTK expression was evaluated with a rabbit polyclonal antiserum raised against a fusion protein containing the 103 carboxyl-terminal amino acids of rat Mertk.
31 The specificity of this antiserum for the rat protein has been demonstrated
31 and confirmed for the human protein by comparison of cells transfected with either empty pcDNA3.1 or pcDNA3.1 containing
MERTK cDNA, by using immunohistochemical and Western analysis of MERTK expression.
For immunohistochemical analysis, HEK293T cells grown and transfected on plastic chamber slides were incubated with a 1:100 dilution of Mertk antiserum and then with Cy3-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR). Cells were viewed and photographed with a microscope (Eclipse E800; Nikon, Melville, NY) equipped with a digital camera (DMX1200; Nikon) and the manufacturer’s data acquisition software.
For Western analysis, transfected HEK293T cells were dissociated in 2% SDS-containing sample buffer, electrophoresed on precast Bis-Tris 10% acrylamide gels (NuPage; Novex, San Diego, CA) in the 3-(N-morpholino)propanesulfonic acid (MOPS) electrophoresis buffer system, and transferred to nitrocellulose membranes (Protran BA85; Schleicher & Schuell, Keene, NH). Equivalent protein loading was verified by Coomassie blue staining. Blots were incubated with a 1:1000 dilution of Mertk antiserum, then with alkaline phosphatase-conjugated anti-rabbit IgG (Promega, Madison, WI). Protein molecular masses were estimated by comparison of mobility relative to prestained standards (SeeBlue Plus2; Invitrogen) calibrated for the MOPS electrophoresis buffer system used.
For analysis of the glycosylation status of recombinant MERTK, lysates from transfected HEK293T cells in 1× denaturing buffer were treated with either Endo H or PNGase F endoglycosidases, according to the manufacturer’s instructions (New England Biolabs), and Western analysis was performed using Mertk antiserum as described earlier.
For detection of phosphotyrosine-modified proteins in MERTK-transfected HEK293T cells, Western analysis was performed on protein samples electrophoresed as described earlier, and probed using a chemiluminescence kit (Anti-pTyr Immunoblotting and ECL Detection kit; Upstate Biotechnology, Lake Placid, NY).
For immunoprecipitation experiments, cells grown and transfected in six-well plates were lysed in 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 8.0), and 1% NP-40, containing protease inhibitors (Complete; Roche Diagnostics, Indianapolis, IN). Lysates were cleared by low-speed centrifugation. Supernates were incubated overnight with Mertk antiserum (10 μL) and then for 2 hours with protein-A agarose beads. Bound proteins were eluted with 2% SDS sample buffer, electrophoresed as described earlier, and transferred to nitrocellulose. Blots were probed for phosphotyrosine-modified proteins, using the chemiluminescence kit (Anti-pTyr Immunoblotting and ECL Detection kit; Upstate Biotechnology) or for MERTK expression as described earlier.
For pulse–chase analysis of protein turnover, at 16 hours after transfection, cells were incubated for 1 hour in serum-free and methionine/cysteine (met/cys)–deficient medium, then for 5 hours in deficient medium supplemented with 35S-met/cys promix (Amersham, Arlington Heights, IL) and then returned to standard serum-containing medium and incubated for various times. Cells were harvested, and recombinant protein was immunoprecipitated, eluted with SDS sample buffer, electrophoresed on 10% Tris-glycine acrylamide gels, and analyzed by fluorography.
For quantitation of MERTK transcripts, total RNA was isolated from transfected cells (RNeasy kit; Qiagen, Valencia, CA), and first-strand cDNAs were synthesized according to the manufacturer’s protocol (Amersham). PCR products (226 bp) were amplified with the following primers: forward (exon 18), 5′-CAGAACCATGAGATGTATGACTATC-3′, and reverse (exon 19), 5′-TCTCCAGCAACTGTGTATTGAC-3′. Control reactions were performed with primers for human hypoxanthine phosphoribosyltransferase (HPRT): forward (exon 4), 5′-GTGGAGATGATCTCTCAACTTTAACTG-3′, and reverse (exon 8), 5′-CATTATAGTCAAGGGCATATCCTACAAC-3′ (236-bp product). Reaction progress was monitored in real-time with a thermocycler (Rotorgene Thermocycler RG3000; Corbett Research, Mortlake, NSW, Australia) and the manufacturer’s software.
The patient was a young girl whose parents first noticed her vision problems when she was 3 years old and began to trip over objects in front of her. When she was 8 years of age, advanced rod–cone dystrophy was diagnosed, and she had surgery to correct strabismus. At age 9 her visual acuities were 20/60 +2 OD and 20/60 +2 OS, with the myopia corrected by spectacles. She had no color discrimination. Nystagmus was present. Her lenses and media were clear and the pupils round and reactive to light. Her fundus showed considerable disc pallor from gliosis and retinal vessels constricted to approximately half normal size. Both eyes showed macular atrophy, fine bone spicule pigment in all quadrants, dense parafoveal pigmentation, and heavy RPE granularity throughout the fundus. Visual fields were considerably limited in both eyes, with a “tunnel” of 20° central detection and a peripheral crescent of detection in the far periphery of both eyes. Dark-adapted threshold sensitivity was elevated by 4 log units in both eyes. The ERG showed total loss of response to all rod and cone stimuli (>99%, to the limit of noise alone).
By age 13, the patient’s best corrected visual acuity had decreased to 20/200, and her visual fields to the largest test target (V4e) had constricted to less than 5° central detection with a narrow peripheral crescent remaining for both eyes. Her condition qualified as a severe, early-onset rod–cone degeneration, with widespread loss of visual function at a young age. She used a white cane for mobility, attended regular school with services from the visually impaired program, and although intelligent, struggled with her studies as she continued to rely on vision for reading rather than learning Braille. At age 15, both of her fundi showed a pattern of bull’s-eye macular atrophy with widespread RPE thinning in the periphery
6 . Her medical history included kidney reflux diagnosed at 13 months of age and managed with antibiotics. Both parents, one sibling 2 years older, and two half-siblings 4 years younger, had normal vision.