Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia

Tanja Grau; Nikolai O Artemyev; Thomas Rosenberg; Hélène Dollfus; Olav H Haugen; E Cumhur Sener; Bernhard Jurklies; Sten Andreasson; Christoph Kernstock; Michael Larsen; Eberhart Zrenner; Bernd Wissinger; Susanne Kohl

doi:10.1093/hmg/ddq517

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Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia

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Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia

Tanja Grau, Nikolai O Artemyev, Thomas Rosenberg, Hélène Dollfus, Olav H Haugen, E Cumhur Sener, Bernhard Jurklies, Sten Andreasson, Christoph Kernstock, Michael Larsen, …

Human molecular genetics, Vol.20(4), pp.719-730

02/15/2011

DOI: 10.1093/hmg/ddq517

PMCID: PMC3269206

PMID: 21127010

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Abstract

Mutations in the gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase ( PDE6C ) have been recently reported in patients with autosomal recessive inherited achromatopsia (ACHM) and early-onset cone photoreceptor dysfunction. Here we present the results of a comprehensive study on PDE6C mutations including the mutation spectrum, its prevalence in a large cohort of ACHM/cone dysfunction patients, the clinical phenotype and the functional characterization of mutant PDE6C proteins. Twelve affected patients from seven independent families segregating PDE6C mutations were identified in our total patient cohort of 492 independent families. Eleven different PDE6C mutations were found including two nonsense mutations, three mutations affecting transcript splicing as shown by minigene assays, one 1 bp-insertion and five missense mutations. We also performed a detailed functional characterization of six missense mutations applying the baculovirus system to express recombinant mutant and wildtype chimeric PDE6C/PDE5 proteins in Sf9 insect cells. Purified proteins were analyzed using Western blotting, phosphodiesterase (PDE) activity measurements as well as inhibition assays by zaprinast and Pγ. Four of the six PDE6C missense mutations led to baseline PDE activities and most likely represent functional null alleles. For two mutations, p.E790K and p.Y323N, we observed reduction in PDE activity of approximately 60% and 80%, respectively. We also observed differences for Pγ inhibition. The p.E790K mutant, with an IC 50 value of 2.7 n m is 20.7-fold more sensitive for Pγ inhibition, whereas the p.Y323N mutant with an IC 50 of 158 n m is 3-fold less sensitive when compared with the wildtype control.

Details

Title: Subtitle: Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia
Creators: Tanja Grau - University Clinics Tuebingen
Nikolai O Artemyev - University of Iowa College of Medicine
Thomas Rosenberg - Kennedy Center
Hélène Dollfus - Hôpitaux Universitaires de Strasbourg
Olav H Haugen - Haukeland University Hospital
E Cumhur Sener - Hacettepe University
Bernhard Jurklies - University Eye Hospital Essen
Sten Andreasson - University Hospital of Lund
Christoph Kernstock - University Tuebingen
Michael Larsen - University of Copenhagen
Eberhart Zrenner - University Tuebingen
Bernd Wissinger - University Clinics Tuebingen
Susanne Kohl - University Clinics Tuebingen
Resource Type: Journal article
Publication Details: Human molecular genetics, Vol.20(4), pp.719-730
Publisher: Oxford University Press
DOI: 10.1093/hmg/ddq517
PMID: 21127010
PMCID: PMC3269206
ISSN: 0964-6906
eISSN: 1460-2083
Language: English
Date published: 02/15/2011
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9984025402502771

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