Brain Advance Access published December 10, 2015 doi:10.1093/brain/awv362
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LETTER TO THE EDITOR Autosomal recessive cerebellar ataxia caused by a homozygous mutation in PMPCA Karine Choquet,1,2 Olga Zurita-Rendo´n,1 Roberta La Piana,1 Sharon Yang,1 Marie-Jose´e Dicaire,1 Care4Rare Consortium, Kym M. Boycott,3 Jacek Majewski,2,4 Eric A. Shoubridge,1,2 Bernard Brais1,2 and Martine Te´treault2,4 Montreal Neurological Institute, McGill University, Montreal, Que´bec, Canada Department of Human Genetics, McGill University, Montreal, Que´bec, Canada Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, Canada McGill University and Genome Quebec Innovation Centre, Montreal, Que´bec, Canada
Correspondence to: Martine Te´treault, McGill University and Genome Quebec Innovation Centre, 740 Dr. Penfield Avenue, Montreal, Quebec, H3A 0G1, Canada E-mail: [email protected]
Sir, Recently, Jobling et al. (2015) reported the identification of mutations in PMPCA in 17 patients from four families affected with autosomal recessive non-progressive cerebellar ataxia. A homozygous missense mutation in PMPCA (c.1129G4A, p.Ala377Thr) was uncovered in three families of Christian Lebanese Maronite origin, while compound heterozygous mutations (c.287C4T, p.Ser96Leu; c.1543G4A, p.Gly515Arg) were identified in a French patient (Jobling et al., 2015). PMPCA encodes the alpha subunit of the mitochondrial processing peptidase (MPP), which cleaves the targeting peptide of nuclear-encoded mitochondrial precursor proteins upon their import into mitochondria (Teixeira and Glaser, 2013). Jobling et al. (2015) showed compelling evidence for the causality of PMPCA variants. First, they demonstrated co-segregation of the variants with the disease among the four families, including a large consanguineous family of 32 individuals. Moreover, they observed decreased levels of MPPa in lymphoblasts from two affected members compared to heterozygote carriers and healthy controls. Interestingly, they also showed impaired processing of frataxin (FXN), a mitochondrial protein that is a known substrate of MPP (Cavadini et al., 2000; Schmucker et al., 2008). The paper by Jobling et al. (2015) is the first to associate defects in PMPCA with a human disease, which suggests a new mechanism for the pathogenesis of nonprogressive cerebellar ataxias.
We wish to complement this study with our own identification of two brothers affected by a juvenile-onset recessive cerebellar ataxia caused by a homozygous mutation in PMPCA. These two patients were born of French Canadian parents who are distantly related (Fig. 1A). Considerable clinical variability was present between the two affected cases. Patient II.2 started developing impaired gait, dysarthria, dysmetria and mild distal atrophy during adolescence. He was diagnosed with a slowly progressive spinocerebellar ataxia. He did not have intellectual deficiency but had learning difficulties in school. Patient II.3 had an earlier onset at 5 years of age and a more severe form of the disease than his older brother. He presented for consultation at the age of 15 with ataxia, hemidystonia, dysarthria and neurosensorial hearing loss. The MRI exams of both siblings (Fig. 1D–G) documented the presence of global cerebellar atrophy, involving both the vermis and the cerebellar hemispheres. In addition, MRI of one of the two brothers (Patient II.2) demonstrated a bilateral and symmetric T2 hyperintense signal at the level of the deep cerebellar white matter. No structural or signal abnormalities were noted in the supratentorial structures. To uncover the genetic cause of disease in this family, we performed whole-exome sequencing on Patient II.3 (SureSelect All Exon v.5 and Illumina HiSeq 2000 with 100-bp paired-end sequencing reads). We filtered for nonsynonymous, splicing and coding indel variants that were
Received September 23, 2015. Revised October 23, 2015. Accepted October 26, 2015. ß The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
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Letter to the Editor
Genomic sequence chromatograms of one affected, one heterozygote carrier and one unrelated control. The arrow indicates the substitution of the G for an A at position 266 of the coding sequence of PMPCA. (C) Amino acid conservation for the PMPCA p.Val256Met variant. MRI findings in subjects with PMPCA mutations. Sagittal T1- (D) and T2- (E) weighted images of Patients II.3 and II.2, respectively showing the presence of cerebellar atrophy and cerebellar white matter abnormalities (E). Axial T2-weighted images showing no supratentorial abnormalities (F) and hyperintense signal at the level of the peri-dentate cerebellar white matter (G) in Patient II.2.
Figure 2 Functional analysis of mitochondrial processing peptidase in patient lymphoblasts. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of whole cell protein extracts in lymphoblast from controls (Control-1 and Control-2), carriers (Patients I-1, I-2 and II-1) and patients (Patients II-2 and II-3). Immunoblots show the protein steady state levels of the MPP alpha (MPPa) and beta (MPPb) subunits, frataxin isoform-1 (FXN42210) and frataxin isoform-2 (FXN81-210), malate dehydrogenase (MDH2) and actin (loading control).
present at a minor allele frequency 55% in 1000 Genomes, EVS and ExAC or present in less than 30 inhouse exomes. Using this filtering strategy, we identified nine rare homozygous variants. According to its gene function, the homozygous missense variant c.766G4A (p.Val256Met) in PMPCA (NM_015160) stood out as a good candidate. This variant is predicted to be pathogenic and is present at a conserved position (Fig. 2C). In addition, it is located in a large homozygous region of 6.5 Mb and Sanger sequencing showed that it co-segregates with disease status in the family (Fig. 2B). To further link this variant to the ataxia in this family, we assessed the functional impact of the p.Val256Met mutation on MPP function. We performed immunoblots on protein extracts from immortalized lymphoblasts of the two patients, their parents, a sibling and two unrelated healthy controls. Steady-state levels of MPPa were comparable amongst patients, carriers and controls. Using a commercial antibody (14147-1-AP, Proteintech) to detect the steady state levels of both human frataxin isoforms
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
Figure 1 Genetic and MRI analysis. (A) Pedigree of the family. The red arrow indicates the individual sent for whole-exome sequencing. (B)
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proceeds normally until the generation of FXN42-210, which cannot be efficiently recognized for the second cleavage step, resulting in its accumulation. As suggested for the two missense variants found in Patient F4-II.1, the p.Val256Met may affect protein folding or stability, given the longer side chain of methionine compared to valine. The different functional impact could potentially explain the milder phenotype observed in Patient F4-II.1 and ours. In this and in other studies where the MPP activity is impaired (Greene et al., 2012; Jobling et al., 2015), the analysis of the processing profile of several mitochondrial proteins, other than frataxin, remained normal. Based on the evidence presented here and in Jobling et al. (2015), the kinetics of processing are likely to be substrate-specific. Thus in the context of a mutant form of the protein, some substrates may be more easily processed than others. The challenge that remains is to identify the substrates that are poorly processed in affected cell types that are relevant to the clinical phenotype. In fact, as suggested by Horvath and Chinnery (2015), the biochemical defect caused by the PMPCA mutation p.Val256Met may be mild enough that it only affects selected cell types or tissues. For instance, decreased levels of MPPa and processing of frataxin and/or other mitochondrial proteins may be more severely altered in the cerebellum. This mild impact may also be consequential only during earlier brain development, when the demand for mitochondrial processing is higher (Horvath and Chinnery, 2015). Both alternatives would explain the absence of a strong biochemical phenotype in adult patient lymphoblasts. In conclusion, we report an additional family, from an independent study, with a mutation in PMPCA, confirming the implication of this gene in cerebellar ataxias. Our report highlights key clinical and radiological features associated with PMPCA-related ataxias and also the presence of clinical heterogeneity by extending the phenotype to slowly progressive cerebellar ataxias. We believe these findings will help clinicians to make an accurate diagnosis and that PMPCA should be considered for both nonprogressive and slowly progressive cerebellar ataxias.
Acknowledgements We thank the patients and their relatives who accepted to partake in this study. We would also like to thank Dr Grazia Isaya for kindly providing us with the frataxin antibody PAC2518.
Funding This work was supported by the Fondation Groupe Monaco and the Care4Rare Canada Consortium, funded in part by Genome Canada, the Canadian Institutes of Health Research (CIHR), the Ontario Genomics Institute, Ontario Research Fund, Genome Quebec and the
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
(FXN81-210, FXN42-210), we showed that all family members accumulate FXN42-210 compared to controls. Moreover, both patients presented a slight increase in FXN42-210 compared to heterozygous carriers (Fig. 2). These results were further validated using a second antibody (Dr Isaya PAC2518, Supplementary Fig. 1A). In addition, we did not observe a specific pattern of accumulation in the steady state levels of FXN1-210 or FXN81-210 (Fig. 2 and Supplementary Fig. 1A). We also evaluated the processing profile of the malate dehydrogenase (MDH2), an established substrate of the MPP (Ogishima et al., 1995), which remained unaffected in all samples. To further investigate the effect of MPPa depletion, we used a small interfering RNA construct to knock-down MPPa for 2, 3 and 4 days in a control fibroblast cell line. Western blot analysis of these cell lines showed that the complete loss of MPPa at 4 days resulted in a significant accumulation of FXN42210 and depletion of FXN81-210 (Supplementary Fig. 1B). As mentioned above, recessive mutations in PMPCA have recently been associated with non-progressive cerebellar ataxia. Considering the clinical and MRI similarities between our patients and the ones reported in Jobling et al. (2015), this homozygous variant in PMPCA appears to be the cause of cerebellar ataxia in this family. Although interand intra-familial variability is observed, there is a clear clinical overlap between the previously reported patients and ours. Gait ataxia, dysarthria and dysmetria are observed in all PMPCA patients. However, intellectual disability was not observed in our patients, although it is a frequent phenotype in the previously reported patients. In contrast to the majority of patients described by Jobling et al., our patients seem to have a milder course of disease, resembling Patient F4-II.1 in their study. It is important to point out that the cerebellar ataxia phenotype is slowly progressing in our patients in comparison to the report from Jobling et al., extending the phenotype associated with PMPCA mutations to slowly progressive cerebellar ataxia. The variability in phenotype could potentially be explained by a different functional impact of the mutations. The previously reported p.Ala377Thr mutation is in close proximity to the glycine-rich loop, which is the most conserved part of MPPa and is crucial for initial interaction with and binding to the substrate (Nagao et al., 2000; Dvorakova-Hola et al., 2010; Kucera et al., 2013). Jobling et al. (2015) showed that the mutation resulted in a decreased amount of MPPa in patients and that it altered the processing of frataxin. The other two mutations (p.Ser96Leu and p.Gly515Arg) are located in two other conserved regions of the peptidase. The variant p.Val256Met identified in the two affected siblings reported here also localizes to a conserved region away from the glycine-rich loop. Immunoblot analysis in our patients did not reveal decreased levels of MPPa; however, we observed a consistent increase in FXN42-210 with no obvious changes of FXN81-210. Together, these results imply that the initial cleavage event in the processing of frataxin
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Children’s Hospital of Eastern Ontario Foundation. KC and RLP received a Doctoral Award from the Fonds de recherche du Que´bec - Sante´ (FRQS). MT received a post-doctoral award from the Re´seau de Me´decine Ge´ne´tique Applique´e, FRQS and CIHR.
Supplementary material Supplementary material is available at Brain online.
References
Greene AW, Grenier K, Aguileta MA, Muise S, Farazifard R, Haque ME, et al. Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment. EMBO Rep 2012; 13: 378–85. Horvath R, Chinnery PF. Nuclear-mitochondrial proteins: too much to process? Brain 2015; 138: 1451–3. Jobling RK, Assoum M, Gakh O, Blaser S, Raiman JA, Mignot C, et al. PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia. Brain 2015; 138: 1505–17. Kucera T, Otyepka M, Matusˇkova´ A, Samad A, Kutejova´ E, Janata J. A computational study of the glycine-rich loop of mitochondrial processing peptidase. PLoS One 2013; 8; e74518. Nagao Y, Kitada S, Kojima K, Toh H, Kuhara S, Ogishima T, et al. Glycine-rich region of mitochondrial processing peptidase alphasubunit is essential for binding and cleavage of the precursor proteins. J Biol Chem 2000; 275: 34552–6. Ogishima T, Niidome T, Shimokata K, Kitada S, Ito A. Analysis of elements in the substrate required for processing by mitochondrial processing peptidase. J Biol Chem 1995; 270: 30322–6. Schmucker S, Argentini M, Carelle-Calmels N, Martelli A, Puccio H. The in vivo mitochondrial two-step maturation of human frataxin. Hum Mol Genet 2008; 17: 3521–31. Teixeira PF, Glaser E. Processing peptidases in mitochondria and chloroplasts. Biochim Biophys Acta 2013; 1833: 360–70.
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
Cavadini P, Adamec J, Taroni F, Gakh O, Isaya G. Two-step processing of human frataxin by mitochondrial processing peptidase. Precursor and intermediate forms are cleaved at different rates. J Biol Chem 2000; 275: 41469–75. Dvorakova-Hola K, Matuskova´ A, Kubala M, Otyepka M, Kucera T, Vecer J, et al. Glycine-rich loop of mitochondrial processing peptidase alpha-subunit is responsible for substrate recognition by a mechanism analogous to mitochondrial receptor Tom20. J Mol Biol 2010; 396: 1197–210.
Letter to the Editor
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LETTER TO THE EDITOR Autosomal recessive cerebellar ataxia caused by a homozygous mutation in PMPCA Karine Choquet,1,2 Olga Zurita-Rendo´n,1 Roberta La Piana,1 Sharon Yang,1 Marie-Jose´e Dicaire,1 Care4Rare Consortium, Kym M. Boycott,3 Jacek Majewski,2,4 Eric A. Shoubridge,1,2 Bernard Brais1,2 and Martine Te´treault2,4 Montreal Neurological Institute, McGill University, Montreal, Que´bec, Canada Department of Human Genetics, McGill University, Montreal, Que´bec, Canada Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, Canada McGill University and Genome Quebec Innovation Centre, Montreal, Que´bec, Canada
Correspondence to: Martine Te´treault, McGill University and Genome Quebec Innovation Centre, 740 Dr. Penfield Avenue, Montreal, Quebec, H3A 0G1, Canada E-mail: [email protected]
Sir, Recently, Jobling et al. (2015) reported the identification of mutations in PMPCA in 17 patients from four families affected with autosomal recessive non-progressive cerebellar ataxia. A homozygous missense mutation in PMPCA (c.1129G4A, p.Ala377Thr) was uncovered in three families of Christian Lebanese Maronite origin, while compound heterozygous mutations (c.287C4T, p.Ser96Leu; c.1543G4A, p.Gly515Arg) were identified in a French patient (Jobling et al., 2015). PMPCA encodes the alpha subunit of the mitochondrial processing peptidase (MPP), which cleaves the targeting peptide of nuclear-encoded mitochondrial precursor proteins upon their import into mitochondria (Teixeira and Glaser, 2013). Jobling et al. (2015) showed compelling evidence for the causality of PMPCA variants. First, they demonstrated co-segregation of the variants with the disease among the four families, including a large consanguineous family of 32 individuals. Moreover, they observed decreased levels of MPPa in lymphoblasts from two affected members compared to heterozygote carriers and healthy controls. Interestingly, they also showed impaired processing of frataxin (FXN), a mitochondrial protein that is a known substrate of MPP (Cavadini et al., 2000; Schmucker et al., 2008). The paper by Jobling et al. (2015) is the first to associate defects in PMPCA with a human disease, which suggests a new mechanism for the pathogenesis of nonprogressive cerebellar ataxias.
We wish to complement this study with our own identification of two brothers affected by a juvenile-onset recessive cerebellar ataxia caused by a homozygous mutation in PMPCA. These two patients were born of French Canadian parents who are distantly related (Fig. 1A). Considerable clinical variability was present between the two affected cases. Patient II.2 started developing impaired gait, dysarthria, dysmetria and mild distal atrophy during adolescence. He was diagnosed with a slowly progressive spinocerebellar ataxia. He did not have intellectual deficiency but had learning difficulties in school. Patient II.3 had an earlier onset at 5 years of age and a more severe form of the disease than his older brother. He presented for consultation at the age of 15 with ataxia, hemidystonia, dysarthria and neurosensorial hearing loss. The MRI exams of both siblings (Fig. 1D–G) documented the presence of global cerebellar atrophy, involving both the vermis and the cerebellar hemispheres. In addition, MRI of one of the two brothers (Patient II.2) demonstrated a bilateral and symmetric T2 hyperintense signal at the level of the deep cerebellar white matter. No structural or signal abnormalities were noted in the supratentorial structures. To uncover the genetic cause of disease in this family, we performed whole-exome sequencing on Patient II.3 (SureSelect All Exon v.5 and Illumina HiSeq 2000 with 100-bp paired-end sequencing reads). We filtered for nonsynonymous, splicing and coding indel variants that were
Received September 23, 2015. Revised October 23, 2015. Accepted October 26, 2015. ß The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
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| BRAIN 2015: Page 2 of 4
Letter to the Editor
Genomic sequence chromatograms of one affected, one heterozygote carrier and one unrelated control. The arrow indicates the substitution of the G for an A at position 266 of the coding sequence of PMPCA. (C) Amino acid conservation for the PMPCA p.Val256Met variant. MRI findings in subjects with PMPCA mutations. Sagittal T1- (D) and T2- (E) weighted images of Patients II.3 and II.2, respectively showing the presence of cerebellar atrophy and cerebellar white matter abnormalities (E). Axial T2-weighted images showing no supratentorial abnormalities (F) and hyperintense signal at the level of the peri-dentate cerebellar white matter (G) in Patient II.2.
Figure 2 Functional analysis of mitochondrial processing peptidase in patient lymphoblasts. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of whole cell protein extracts in lymphoblast from controls (Control-1 and Control-2), carriers (Patients I-1, I-2 and II-1) and patients (Patients II-2 and II-3). Immunoblots show the protein steady state levels of the MPP alpha (MPPa) and beta (MPPb) subunits, frataxin isoform-1 (FXN42210) and frataxin isoform-2 (FXN81-210), malate dehydrogenase (MDH2) and actin (loading control).
present at a minor allele frequency 55% in 1000 Genomes, EVS and ExAC or present in less than 30 inhouse exomes. Using this filtering strategy, we identified nine rare homozygous variants. According to its gene function, the homozygous missense variant c.766G4A (p.Val256Met) in PMPCA (NM_015160) stood out as a good candidate. This variant is predicted to be pathogenic and is present at a conserved position (Fig. 2C). In addition, it is located in a large homozygous region of 6.5 Mb and Sanger sequencing showed that it co-segregates with disease status in the family (Fig. 2B). To further link this variant to the ataxia in this family, we assessed the functional impact of the p.Val256Met mutation on MPP function. We performed immunoblots on protein extracts from immortalized lymphoblasts of the two patients, their parents, a sibling and two unrelated healthy controls. Steady-state levels of MPPa were comparable amongst patients, carriers and controls. Using a commercial antibody (14147-1-AP, Proteintech) to detect the steady state levels of both human frataxin isoforms
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
Figure 1 Genetic and MRI analysis. (A) Pedigree of the family. The red arrow indicates the individual sent for whole-exome sequencing. (B)
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proceeds normally until the generation of FXN42-210, which cannot be efficiently recognized for the second cleavage step, resulting in its accumulation. As suggested for the two missense variants found in Patient F4-II.1, the p.Val256Met may affect protein folding or stability, given the longer side chain of methionine compared to valine. The different functional impact could potentially explain the milder phenotype observed in Patient F4-II.1 and ours. In this and in other studies where the MPP activity is impaired (Greene et al., 2012; Jobling et al., 2015), the analysis of the processing profile of several mitochondrial proteins, other than frataxin, remained normal. Based on the evidence presented here and in Jobling et al. (2015), the kinetics of processing are likely to be substrate-specific. Thus in the context of a mutant form of the protein, some substrates may be more easily processed than others. The challenge that remains is to identify the substrates that are poorly processed in affected cell types that are relevant to the clinical phenotype. In fact, as suggested by Horvath and Chinnery (2015), the biochemical defect caused by the PMPCA mutation p.Val256Met may be mild enough that it only affects selected cell types or tissues. For instance, decreased levels of MPPa and processing of frataxin and/or other mitochondrial proteins may be more severely altered in the cerebellum. This mild impact may also be consequential only during earlier brain development, when the demand for mitochondrial processing is higher (Horvath and Chinnery, 2015). Both alternatives would explain the absence of a strong biochemical phenotype in adult patient lymphoblasts. In conclusion, we report an additional family, from an independent study, with a mutation in PMPCA, confirming the implication of this gene in cerebellar ataxias. Our report highlights key clinical and radiological features associated with PMPCA-related ataxias and also the presence of clinical heterogeneity by extending the phenotype to slowly progressive cerebellar ataxias. We believe these findings will help clinicians to make an accurate diagnosis and that PMPCA should be considered for both nonprogressive and slowly progressive cerebellar ataxias.
Acknowledgements We thank the patients and their relatives who accepted to partake in this study. We would also like to thank Dr Grazia Isaya for kindly providing us with the frataxin antibody PAC2518.
Funding This work was supported by the Fondation Groupe Monaco and the Care4Rare Canada Consortium, funded in part by Genome Canada, the Canadian Institutes of Health Research (CIHR), the Ontario Genomics Institute, Ontario Research Fund, Genome Quebec and the
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
(FXN81-210, FXN42-210), we showed that all family members accumulate FXN42-210 compared to controls. Moreover, both patients presented a slight increase in FXN42-210 compared to heterozygous carriers (Fig. 2). These results were further validated using a second antibody (Dr Isaya PAC2518, Supplementary Fig. 1A). In addition, we did not observe a specific pattern of accumulation in the steady state levels of FXN1-210 or FXN81-210 (Fig. 2 and Supplementary Fig. 1A). We also evaluated the processing profile of the malate dehydrogenase (MDH2), an established substrate of the MPP (Ogishima et al., 1995), which remained unaffected in all samples. To further investigate the effect of MPPa depletion, we used a small interfering RNA construct to knock-down MPPa for 2, 3 and 4 days in a control fibroblast cell line. Western blot analysis of these cell lines showed that the complete loss of MPPa at 4 days resulted in a significant accumulation of FXN42210 and depletion of FXN81-210 (Supplementary Fig. 1B). As mentioned above, recessive mutations in PMPCA have recently been associated with non-progressive cerebellar ataxia. Considering the clinical and MRI similarities between our patients and the ones reported in Jobling et al. (2015), this homozygous variant in PMPCA appears to be the cause of cerebellar ataxia in this family. Although interand intra-familial variability is observed, there is a clear clinical overlap between the previously reported patients and ours. Gait ataxia, dysarthria and dysmetria are observed in all PMPCA patients. However, intellectual disability was not observed in our patients, although it is a frequent phenotype in the previously reported patients. In contrast to the majority of patients described by Jobling et al., our patients seem to have a milder course of disease, resembling Patient F4-II.1 in their study. It is important to point out that the cerebellar ataxia phenotype is slowly progressing in our patients in comparison to the report from Jobling et al., extending the phenotype associated with PMPCA mutations to slowly progressive cerebellar ataxia. The variability in phenotype could potentially be explained by a different functional impact of the mutations. The previously reported p.Ala377Thr mutation is in close proximity to the glycine-rich loop, which is the most conserved part of MPPa and is crucial for initial interaction with and binding to the substrate (Nagao et al., 2000; Dvorakova-Hola et al., 2010; Kucera et al., 2013). Jobling et al. (2015) showed that the mutation resulted in a decreased amount of MPPa in patients and that it altered the processing of frataxin. The other two mutations (p.Ser96Leu and p.Gly515Arg) are located in two other conserved regions of the peptidase. The variant p.Val256Met identified in the two affected siblings reported here also localizes to a conserved region away from the glycine-rich loop. Immunoblot analysis in our patients did not reveal decreased levels of MPPa; however, we observed a consistent increase in FXN42-210 with no obvious changes of FXN81-210. Together, these results imply that the initial cleavage event in the processing of frataxin
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| BRAIN 2015: Page 4 of 4
Children’s Hospital of Eastern Ontario Foundation. KC and RLP received a Doctoral Award from the Fonds de recherche du Que´bec - Sante´ (FRQS). MT received a post-doctoral award from the Re´seau de Me´decine Ge´ne´tique Applique´e, FRQS and CIHR.
Supplementary material Supplementary material is available at Brain online.
References
Greene AW, Grenier K, Aguileta MA, Muise S, Farazifard R, Haque ME, et al. Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment. EMBO Rep 2012; 13: 378–85. Horvath R, Chinnery PF. Nuclear-mitochondrial proteins: too much to process? Brain 2015; 138: 1451–3. Jobling RK, Assoum M, Gakh O, Blaser S, Raiman JA, Mignot C, et al. PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia. Brain 2015; 138: 1505–17. Kucera T, Otyepka M, Matusˇkova´ A, Samad A, Kutejova´ E, Janata J. A computational study of the glycine-rich loop of mitochondrial processing peptidase. PLoS One 2013; 8; e74518. Nagao Y, Kitada S, Kojima K, Toh H, Kuhara S, Ogishima T, et al. Glycine-rich region of mitochondrial processing peptidase alphasubunit is essential for binding and cleavage of the precursor proteins. J Biol Chem 2000; 275: 34552–6. Ogishima T, Niidome T, Shimokata K, Kitada S, Ito A. Analysis of elements in the substrate required for processing by mitochondrial processing peptidase. J Biol Chem 1995; 270: 30322–6. Schmucker S, Argentini M, Carelle-Calmels N, Martelli A, Puccio H. The in vivo mitochondrial two-step maturation of human frataxin. Hum Mol Genet 2008; 17: 3521–31. Teixeira PF, Glaser E. Processing peptidases in mitochondria and chloroplasts. Biochim Biophys Acta 2013; 1833: 360–70.
Downloaded from http://brain.oxfordjournals.org/ by guest on December 11, 2015
Cavadini P, Adamec J, Taroni F, Gakh O, Isaya G. Two-step processing of human frataxin by mitochondrial processing peptidase. Precursor and intermediate forms are cleaved at different rates. J Biol Chem 2000; 275: 41469–75. Dvorakova-Hola K, Matuskova´ A, Kubala M, Otyepka M, Kucera T, Vecer J, et al. Glycine-rich loop of mitochondrial processing peptidase alpha-subunit is responsible for substrate recognition by a mechanism analogous to mitochondrial receptor Tom20. J Mol Biol 2010; 396: 1197–210.
Letter to the Editor
