TY - JOUR

T1 - Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia

AU - Grau, Tanja

AU - Artemyev, Nikolai O

AU - Rosenberg, Thomas

AU - Dollfus, Hélène

AU - Haugen, Olav H

AU - Cumhur Sener, E

AU - Jurklies, Bernhard

AU - Andreasson, Sten

AU - Kernstock, Christoph

AU - Larsen, Michael

AU - Zrenner, Eberhart

AU - Wissinger, Bernd

AU - Kohl, Susanne

PY - 2011/2/15

Y1 - 2011/2/15

N2 - 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 P¿. 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 P¿ inhibition. The p.E790K mutant, with an IC50 value of 2.7 nm is 20.7-fold more sensitive for P¿ inhibition, whereas the p.Y323N mutant with an IC50 of 158 nm is 3-fold less sensitive when compared with the wildtype control.

AB - 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 P¿. 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 P¿ inhibition. The p.E790K mutant, with an IC50 value of 2.7 nm is 20.7-fold more sensitive for P¿ inhibition, whereas the p.Y323N mutant with an IC50 of 158 nm is 3-fold less sensitive when compared with the wildtype control.

U2 - 10.1093/hmg/ddq517

DO - 10.1093/hmg/ddq517

M3 - Journal article

C2 - 21127010

VL - 20

SP - 719

EP - 730

JO - Human Molecular Genetics

JF - Human Molecular Genetics

SN - 0964-6906

IS - 4

ER -