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Constitutively Active Mutant of Rod A-Transducin in Autosomal Dominant

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Constitutively Active Mutant of Rod A-Transducin in Autosomal Dominant HUMAN MUTATION Mutation in Brief #970 (2007) Online MUTATION IN BRIEF p.Gln200Glu, a Putative Constitutively Active Mutant of Rod α-Transducin (GNAT1) in Autosomal Dominant Congenital Stationary Night Blindness Viktoria Szabo1,2†, Hans-Jürgen Kreienkamp1†, Thomas Rosenberg3, and Andreas Gal1* 1Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany; 3Gordon Norrie Centre for Genetic Eye Diseases, The National Eye Clinic for the Visually Impaired, Hellerup, Denmark; 2Permanent address: Department of Ophthalmology, Semmelweis University, Budapest, Hungary *Correspondence to: A. Gal, Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; Tel.: 49-40-42803-2120; Fax: 49-40-42803-5138; E-mail: [email protected] Grant sponsor: Recognition Award of the Alcon Research Institute (Fort Worth, TX, to A.G.) and the German Academic Exchange Service (to V.S.). †Viktoria Szabo and Hans-Jürgen Kreienkamp contributed equally to this work. Communicated by Mark H. Paalman Congenital stationary night blindness (CSNB) is a non-progressive Mendelian condition resulting from a functional defect in rod photoreceptors. A small number of unique missense mutations in the genes encoding various members of the rod phototransduction cascade, e.g. rhodopsin (RHO), cGMP phosphodiesterase β-subunit (PDE6B), and transducin α-subunit (GNAT1) have been reported to cause autosomal dominant (ad) CSNB. While the RHO and PDE6B mutations result in constitutively active proteins, the only known adCSNB-associa- ted GNAT1 change (p.Gly38Asp) produces an α-transducin that is unable to activate its downstream effector molecule in vitro. In a multigeneration Danish family with adCSNB, we identified a novel heterozygous C to G transversion (c.598C>G) in exon 6 of GNAT1 that should result in a p.Gln200Glu substitution in the evolutionarily highly conserved Switch 2 region of α-transducin, a domain that has an important role in binding and hydrolyzing GTP. Computer modeling based on the known crystal structure of transducin suggests that the p.Gln200Glu mutant exhibits impaired GTPase activity, and thereby leads to constitutive activation of phototransduction. This assumption is in line with our results of trypsin protection assays as well as previously published biochemical data on mutants of this glutamine in the GTPase active site of α-transducin following in vitro expression, and observations that inappropriately activating mutants of various members of the rod phototransduction cascade represent one of the major molecular causes of adCSNB. © 2007 Wiley-Liss, Inc. KEY WORDS: night blindness; CSNB; transducin; GNAT1; constitutive activation; phototransduction INTRODUCTION Night blindness, reduced or absent dark adaptation, is a typical and early sign of various forms of retinal dystrophies. While night blindness as disease symptom is usually progressive and parallels the disintegration of rod photoreceptors, congenital stationary night blindness (CSNB; e.g. MIM# 163500) is a rare, non-progressive Mendelian condition due to a functional disorder of rod photoreceptors. CSNB is heterogeneous as to the mode of Received 7 July 2006; accepted revised manuscript 12 April 2007. © 2007 WILEY-LISS, INC. DOI: 10.1002/humu.9499 2 Szabo et al. inheritance (autosomal dominant [ad], autosomal recessive, and X-linked), pattern of electroretinogram (Riggs or Schubert-Bornschein types), refractive error, and fundus appearance in the probands (for a recent review see Dryja 2000). Several large families with adCSNB have been documented (Carr 1974, al-Jandal et al. 1999), including the French Nougaret genealogy first described 1838 (Cunier 1838) and reinvestigated in 1907 (Nettleship 1907), and an extended Danish pedigree first published 1909 by Rambusch (Rambusch 1909) and rediscovered 1991 (Rosenberg et al. 1991). Affected family members in these two pedigrees present with identical electrophysiolo- gical and psychophysical findings. At the molecular level, two basic mechanisms of opposite nature have been suggested to explain the dominant phenotype; constitutive activation of various members of the rod phototransduction cascade, or inability to activate the cascade. Abnormally prolonged activation of photosignaling may mimic a weak background light, desensitizing the visual system. Dryja et al. (1993) suggested that a heterozygous p.Ala292Glu change of rod opsin resulted in an inappropriate continuous activity of the mutant protein, even without chromophore, in a patient with clinical features compatible with CSNB. Two further missense mutations, p.Thr94Ile of the gene (RHO) encoding rhodopsin (al-Jandal et al. 1999), and p.His258Asn of the rod cGMP phosphodiesterase β-subunit gene (PDE6B; Gal et al. 1994) were suggested to result in constitutive activation of the mutant protein and continuous phototransduction in two families with adCSNB. In contrast, a heterozygous missense mutation (p.Gly38Asp) in GNAT1 (MIM# 139330) (Dryja et al. 1996), identified in the Nougaret family, was shown to result in inability of the mutant α-transducin to activate its downstream effector, rod cGMP-specific phosphodiesterase (PDE). In the light of these findings, the question arises whether adCSNB-associated transducin mutations differ principally in their mode of action from those of other genes. Here we report a novel heterozygous missense mutation of the α- transducin gene that is likely to result in constitutive activation of phototransduction. MATERIALS AND METHODS The Danish pedigree studied here consists of 9 family members presenting with typical symptoms of CSNB in three generations suggesting an autosomal dominant trait (Fig. 1). In addition to standard ophthalmologic exami- nation, dynamic visual field measurement with a Goldmann apparatus (object size I/4e and IV/4e), colour vision testing (Ishihara, AOHRR, Farnsworth D-15, and Nagel anomaloscope), dark adaptometry a.m. Goldmann- Weekers with a diffuse light stimulus (integral technique), and full-field ERG following the ISCERG recommen- dations were performed. Informed consent was obtained from all human subjects included in this study. For linkage analysis, fluorescence-tagged microsatellite markers were analysed on an ABI PRISM 310 automated DNA sequencer (Applied Biosystems, Forster City, USA). For GNAT1 mutation numbering GenBank reference cDNA sequence NM_144499.1 was used with +1 corresponding to the A of the ATG translation initiation codon, which is codon 1. Protein expression. A His6-tagged expression clone for the transducin/Gai-chimera (chimera 8; Skiba et al., 1996) was kindly provided by Dr. H. Hamm (Vanderbilt University, USA). The p.Q200E mutation was introduced by site-directed mutagenesis, using appropriate PCR primers. For expression in bacteria, plasmids were transformed into BL21 cells; cells were cultured in terrific broth media including phosphate salts. After induction with IPTG overnight at 16°C, cells were lysed and recombinant proteins were purified essentially as described by Skiba et al. (1996). After elution from the Ni-NTA agarose with 100 mM imidazole, purified protein was dialyzed against 50 mM Tris-HCl; pH 8.0; 50 mM NaCl; 5 mM MgCl2 containing 50 μM GDP and stored at -80°C until further use. Trypsin protection assay. Purified protein was diluted to a final concentration of 0.1 mg/ml in 20 mM - Tris/HCl (pH 8.0) and 50 μM GDP. The AlF4 complex was generated by adding 1 mM NaF and 50 μM AlCl3. After incubation at room temperature for 60 min, trypsin was added to a final concentration of 0.01 mg/ml. Samples were incubated for 10 min at room temperature, followed by boiling in SDS sample buffer and SDS- polyacrylamide electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue. Molecular modeling. Molecular modeling was performed using the SWISS-Model server (Guex and Peitsch, 1997) at http://swissmodel.expasy.org. Modeling was based on the transducin/Gαi-chimera in complex with PDE- - - γ, RGS9 and GDP.AlF4 (Slep et al., 2001; template file: 1fqjA.pdb), and also on the isolated transducin/GDP.AlF4 -complex (template file 1tadA.pdb; Sondek et al., 1994). Structures were visualized using the PDBviewer software (Guex and Peitsch, 1997). p.Gln200Glu in Rod α-Transducin 3 RESULTS AND DISCUSSION Of the nine night blind family members, five underwent detailed clinical investigations. All but one (II-2, see later) had experienced non-progressive night blindness from early infancy. The latter four family members (II-4, III-2, III-4, and III-7, aged, respectively, 53, 37, 31, and 25 years at the time of first clinical assessment in 1991) had normal visual acuity and colour vision, visual fields were unconstricted, and the fundi unremarkable. Refrac- tive values were –8.50 and –7.00 (spherical equivalent) in subject III-2, and –2.00 and –3.50 in subject III-4. Dark adaptometry confirmed that the three of them were completely night blind with an appr. 100-fold reduction in rod sensitivity. In the youngest subject, however, an initial cone phase of 2.0 log units was succeeded by a small rod deflection and additional 0.6 log increase in sensitivity. Scotopic electroretinograms (ERG) showed an absent rod b-wave in response to dim flashes of light and a cone-like response only to bright flashes. In light-adapted state, cone responses of normal-to-moderately decreased amplitudes and normal implicit time were found. This ERG pattern (Riggs-type ERG) suggests a presynaptic defect in rod phototransduction. Re-examination
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