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Melissa Lees - One of the best experts on this subject based on the ideXlab platform.

  • Miller genee wiedemann Syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of dhodh
    Human Molecular Genetics, 2012
    Co-Authors: Joe Rainger, Hemant Bengani, Leigh Campbell, Eve Anderson, Kishan Sokhi, Wayne Lam, Angelika Riess, Morad Ansari, Sarah F. Smithson, Melissa Lees
    Abstract:

    Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genee-Weidemann or postaxial acrofacial dysostosis) Syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller Syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

  • Miller (Genée–Wiedemann) Syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH
    Human molecular genetics, 2012
    Co-Authors: Joe Rainger, Hemant Bengani, Leigh Campbell, Eve Anderson, Kishan Sokhi, Wayne Lam, Angelika Riess, Morad Ansari, Sarah F. Smithson, Melissa Lees
    Abstract:

    Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genee-Weidemann or postaxial acrofacial dysostosis) Syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller Syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

Kazuaki Nonaka - One of the best experts on this subject based on the ideXlab platform.

  • dihydro orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction
    Bioscience Reports, 2013
    Co-Authors: Jing Xian Fang, Mikako Yagi, Rie Amamoto, Shinya Takazaki, Kazuaki Nonaka, Shinya Matsumoto, Takeshi Uchiumi, Haruyoshi Yamaza, Dongchon Kang
    Abstract:

    Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller Syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller Syndrome in part through somehow altered mitochondrial function.

  • protein instability and functional defects caused by mutations of dihydro orotate dehydrogenase in Miller Syndrome patients
    Bioscience Reports, 2012
    Co-Authors: Jing Xian Fang, Mikako Yagi, Rie Amamoto, Shinya Takazaki, Shinya Matsumoto, Takeshi Uchiumi, Haruyoshi Yamaza, Toshiro Saito, Tomotake Kanki, Kazuaki Nonaka
    Abstract:

    Miller Syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller Syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller Syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller Syndrome phenotype.

Takeshi Uchiumi - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial nucleic acid binding proteins associated with diseases.
    Frontiers in Bioscience, 2017
    Co-Authors: Takeshi Uchiumi, Dongchon Kang
    Abstract:

    : Mammalian mitochondrial DNA (mtDNA) exists in structures called nucleoids, which correspond to the configuration of nuclear DNA. Mitochondrial transcription factor A (TFAM), first cloned as an mtDNA transcription factor, is critical for packaging and maintaining mtDNA. To investigate functional aspects of TFAM, we identified many RNA-binding proteins as candidate TFAM interactors, including ERAL1 and p32. In this review, we first describe the functions of TFAM, replication proteins such as polymerase gamma and Twinkle, and mitochondrial RNA binding proteins. We describe the role of mitochondrial nucleic acid binding proteins within the mitochondrial matrix and two oxidative phosphorylation-related proteins within the mitochondrial intermembrane space. We then discuss how mitochondrial dysfunction is related to several diseases, including mitochondrial respiratory disease, Miller Syndrome and cancer. We also describe p32 knockout mice, which are embryonic lethal and exhibit respiratory chain defects. Miller Syndrome is a recessive disorder characterized by postaxial acrofacial dysostosis and caused by a mutation in DHODH. Finally, we explain that p32 and mitochondrial creatine kinase may be novel markers for the progression of prostate cancer.

  • dihydro orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction
    Bioscience Reports, 2013
    Co-Authors: Jing Xian Fang, Mikako Yagi, Rie Amamoto, Shinya Takazaki, Kazuaki Nonaka, Shinya Matsumoto, Takeshi Uchiumi, Haruyoshi Yamaza, Dongchon Kang
    Abstract:

    Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller Syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller Syndrome in part through somehow altered mitochondrial function.

  • protein instability and functional defects caused by mutations of dihydro orotate dehydrogenase in Miller Syndrome patients
    Bioscience Reports, 2012
    Co-Authors: Jing Xian Fang, Mikako Yagi, Rie Amamoto, Shinya Takazaki, Shinya Matsumoto, Takeshi Uchiumi, Haruyoshi Yamaza, Toshiro Saito, Tomotake Kanki, Kazuaki Nonaka
    Abstract:

    Miller Syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller Syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller Syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller Syndrome phenotype.

Jing Xian Fang - One of the best experts on this subject based on the ideXlab platform.

  • dihydro orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction
    Bioscience Reports, 2013
    Co-Authors: Jing Xian Fang, Mikako Yagi, Rie Amamoto, Shinya Takazaki, Kazuaki Nonaka, Shinya Matsumoto, Takeshi Uchiumi, Haruyoshi Yamaza, Dongchon Kang
    Abstract:

    Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller Syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller Syndrome in part through somehow altered mitochondrial function.

  • protein instability and functional defects caused by mutations of dihydro orotate dehydrogenase in Miller Syndrome patients
    Bioscience Reports, 2012
    Co-Authors: Jing Xian Fang, Mikako Yagi, Rie Amamoto, Shinya Takazaki, Shinya Matsumoto, Takeshi Uchiumi, Haruyoshi Yamaza, Toshiro Saito, Tomotake Kanki, Kazuaki Nonaka
    Abstract:

    Miller Syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller Syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller Syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller Syndrome phenotype.

Joe Rainger - One of the best experts on this subject based on the ideXlab platform.

  • Miller genee wiedemann Syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of dhodh
    Human Molecular Genetics, 2012
    Co-Authors: Joe Rainger, Hemant Bengani, Leigh Campbell, Eve Anderson, Kishan Sokhi, Wayne Lam, Angelika Riess, Morad Ansari, Sarah F. Smithson, Melissa Lees
    Abstract:

    Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genee-Weidemann or postaxial acrofacial dysostosis) Syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller Syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

  • Miller (Genée–Wiedemann) Syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH
    Human molecular genetics, 2012
    Co-Authors: Joe Rainger, Hemant Bengani, Leigh Campbell, Eve Anderson, Kishan Sokhi, Wayne Lam, Angelika Riess, Morad Ansari, Sarah F. Smithson, Melissa Lees
    Abstract:

    Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genee-Weidemann or postaxial acrofacial dysostosis) Syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller Syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.