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Topics {✒️}

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         headline:A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR
         description:The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion.
         datePublished:
         dateModified:
         pageStart:49
         pageEnd:55
         sameAs:https://doi.org/10.1007/s004390050909
         keywords:
            Androgen
            Androgen Receptor
            Bisulfite
            Sodium Bisulfite
            Differential Methylation
            Human Genetics
            Molecular Medicine
            Gene Function
            Metabolic Diseases
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               name:T. Kubota
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                     address:
                        name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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                        name:Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan, , JP
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                        name:Department of Genomics, Mitsubishi Chemistry, Bio-clinical Laboratories, Tokyo, Japan, , JP
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      headline:A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR
      description:The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion.
      datePublished:
      dateModified:
      pageStart:49
      pageEnd:55
      sameAs:https://doi.org/10.1007/s004390050909
      keywords:
         Androgen
         Androgen Receptor
         Bisulfite
         Sodium Bisulfite
         Differential Methylation
         Human Genetics
         Molecular Medicine
         Gene Function
         Metabolic Diseases
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         name:Springer-Verlag
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      author:
            name:T. Kubota
            affiliation:
                  name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619
                  address:
                     name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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            name:Shigeaki Nonoyama
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                  address:
                     name:Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan, , JP
                     type:PostalAddress
                  type:Organization
            type:Person
            name:Hidefumi Tonoki
            affiliation:
                  name:Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan
                  address:
                     name:Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan, , JP
                     type:PostalAddress
                  type:Organization
            type:Person
            name:Mitsuo Masuno
            affiliation:
                  name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan
                  address:
                     name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan, , JP
                     type:PostalAddress
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            type:Person
            name:Kiyoshi Imaizumi
            affiliation:
                  name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan
                  address:
                     name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan, , JP
                     type:PostalAddress
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            name:Makiko Kojima
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                     name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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                     name:Department of Genomics, Mitsubishi Chemistry, Bio-clinical Laboratories, Tokyo, Japan, , JP
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      name:Hidefumi Tonoki
      affiliation:
            name:Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan
            address:
               name:Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan, , JP
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            address:
               name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan, , JP
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      name:Kiyoshi Imaizumi
      affiliation:
            name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan
            address:
               name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan, , JP
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            address:
               name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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            address:
               name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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            address:
               name:Department of Genomics, Mitsubishi Chemistry, Bio-clinical Laboratories, Tokyo, Japan, , JP
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      name:Yoshimitsu Fukushima
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            address:
               name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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      name:Department of Pediatrics, Tokyo Medical and Dental University, Tokyo, Japan, , JP
      name:Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan, , JP
      name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan, , JP
      name:Division of Medical Genetics, Kanagawa Children’s Medical Center, Yokohama, Japan, , JP
      name:Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto-shi, Nagano 390-8621, Japan e-mail: [email protected], Tel.: +81-263-37-2618, Fax: +81-263-37-2619, , JP
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