A novel H101Q mutation causes PKCγ loss in spinocerebellar ataxia type 14

Alonso, Isabel; Costa, Cristina; Gomes, André Raeli; Ferro, Anabela; Seixas, Ana I.; Silva, Sérgio; Cruz, Vítor Tedim; Coutinho, Paula; Sequeiros, Jorge; Silveira, Isabel

doi:10.1007/s10038-005-0287-z

Cited by 32 publications

(21 citation statements)

References 21 publications

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“…( A ) The Histidine at position 36 is located in the first cysteine-rich domain. The equivalent Histidine H101, located in the second cysteine rich-domain is associated with several known spinocerebellar ataxia 14 (SCA14) mutations (Alonso et al , 2005). ( B ) Comparison of the 3D structure of the cysteine-rich domains in PRKCG shows the located of H36R and other known mutations including H101Y, H101Q and H101R.…”

Section: Resultsmentioning

confidence: 99%

“…Affected mother reported to have a rapidly progressive disorder. Dominant FH.Cerebellar atrophy on MRIMultiple out-patient investigations. PRKCG , (SCA14) (Chen et al , 2003; Yabe et al , 2003; Alonso et al , 2005; Seki et al , 2005): heterozygote: H36RNovel Bioinformatics: PolyPhen2 Probably damaging, MutPred Probably damagingNo other family members available for segregation analysis. Extensive bioinformatics and functional assays required to confirm pathogenicity. In vitro analysis ; see Fig.…”

Section: Resultsmentioning

confidence: 99%

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Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model

Németh

Kwasniewska

Lise

et al. 2013

Brain

152

139

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Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model

Németh

Kwasniewska

Lise

et al. 2013

Brain

152

139

View full text Add to dashboard Cite

show abstract

“…H 2 O 2 , through oxidation of the C1B domain . Recently, missense mutations in the PKC␥ C1B domain have been identified as causing a type of autosomal dominant neurodegenerative disorder, spinocerebellar ataxia type 14 (SCA-14) (Chen et al, 2003;Chen et al, 2005;Alonso et al, 2005;Seki et al, 2005). However, it is still unclear how these C1B mutations lead to loss of Purkinje neuron and cerebellar dysfunction during Fig.·6.…”

Section: Discussionmentioning

confidence: 99%

PKCγ knockout mouse lenses are more susceptible to oxidative stress damage

Lin

Barnett

Lobell

et al. 2006

Journal of Experimental Biology

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SUMMARY Cataracts, or lens opacities, are the leading cause of blindness worldwide. Cataracts increase with age and environmental insults, e.g. oxidative stress. Lens homeostasis depends on functional gap junctions. Knockout or missense mutations of lens gap junction proteins, Cx46 or Cx50, result in cataractogenesis in mice. We have previously demonstrated that protein kinase Cγ (PKCγ) regulates gap junctions in the lens epithelium and cortex. In the current study, we further determined whether PKCγ control of gap junctions protects the lens from cataractogenesis induced by oxidative stress in vitro, using PKCγ knockout and control mice as our models. The results demonstrate that PKCγ knockout lenses are normal at 2 days post-natal when compared to control. However, cell damage, but not obvious cataract, was observed in the lenses of 6-week-old PKCγ knockout mice,suggesting that the deletion of PKCγ causes lenses to be more susceptible to damage. Furthermore, in vitro incubation or lens oxidative stress treatment by H2O2 significantly induced lens opacification (cataract) in the PKCγ knockout mice when compared to controls. Biochemical and structural results also demonstrated that H2O2 activation of endogenous PKCγ resulted in phosphorylation of Cx50 and subsequent inhibition of gap junctions in the lenses of control mice, but not in the knockout. Deletion of PKCγaltered the arrangement of gap junctions on the cortical fiber cell surface,and completely abolished the inhibitory effect of H2O2on lens gap junctions. Data suggest that activation of PKCγ is an important mechanism regulating the closure of the communicating pathway mediated by gap junction channels in lens fiber cells. The absence of this regulatory mechanism in the PKCγ knockout mice may cause those lenses to have increased susceptibility to oxidative damage.

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“…Whereas many types of SCA are caused by coding polyglutamine repeat expansion mutations in the encoding proteins, missense mutations in PKCγ were identified to cause SCA14 in families with different genetic backgrounds (Alonso et al, 2005;Chen et al, 2003;Dalski et al, 2006;Fahey et al, 2005;Klebe et al, 2005;Stevanin et al, 2004;van de Warrenburg et al, 2003;Vlak et al, 2006;Yabe et al, 2003). Up to now, 23 different mutations have been identified throughout the coding region of PKCγ, although about 75% of all SCA14 mutations are located in the highly conserved C1B subdomain of the regulatory domain of PKCγ.…”

Section: Introductionmentioning

confidence: 99%

PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

Verbeek

Goedhart

Bruinsma

et al. 2008

Journal of Cell Science

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Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKCγ) gene. Since most mutations causing SCA14 are located in the PKCγ C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKCγ in living cells. SCA14 mutant PKCγ proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKCγ. The mutations led to a decrease in intramolecular FRET of PKCγ, suggesting that they `open' PKCγ protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKCγ showed reduced kinase activity as measured by phosphorylation of PKC reporter MyrPalm-CKAR, as well as downstream components of the MAPK signaling pathway. Together, these results show that SCA14 mutations located in the C1B subdomain `open' PKCγ protein conformation leading to increased C1 domain accessibility, but inefficient activation of downstream signaling pathways.

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A novel H101Q mutation causes PKCγ loss in spinocerebellar ataxia type 14

Cited by 32 publications

References 21 publications

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model

PKCγ knockout mouse lenses are more susceptible to oxidative stress damage

PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

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