The former is in the C terminal part of helix a 1 or in a loop between a 1 and

Methods Muscle biopsy Muscle samples were obtained from the quadriceps of each patient. Standard histochemical stains including hematoxylin and eosin, Gomori trichrome, NADH tetrazolium reductase, succinate dehydrogenase, periodic acid Schiff, acid phosphatase, phosphatase 阻害剤 adenosine triphosphatase at pH 4. 3 and 9. 4, cytochrome c oxidase, and alkaline phosphatase were conducted. Immunohistochemical stains for and B dystroglycan, B, and sarco glycan; dystrophin; and merosin were performed using their respective monoclonal antibodies, Gene analysis Under an institutionally approved protocol, DNA was extracted from blood samples that were obtained from the probands and all available family members. For exam ination of the SGCG gene, eight sets of primers were designed to amplify eight exons, with the amplified PCR products then directly sequenced.<br><br> Reverse tran scription PCR was used to analyze the SGCG mRNA expressed in lymphocytes or skeletal muscle, as previously described, Full length SGCG cDNA was amplified as two separate, partially overlapping frag ments by using two sets of primers, The ampli fied products were then directly sequenced. Quantitative PCR Genomic dosage of the exons of the SGCG gene was assessed Lenalidomide 価格 by a semiquantitative multiplex PCR, as previ ously described, Eight fragments encompassing exons 1 to 8 of the SGCG gene and one fragment encom passing exon 2 of the dystroglycan gene were co ampli fied using two PCR reactions that employed six sets of primers, PCR products were separated by capil lary electrophoresis, The amount of PCR product derived from the SGCG exons was quantified by measuring their peak areas followed by calculating the ratio of these areas to that found for the dystroglycan exon 2.<br><br> Results The two male patients with clinical diagnosis of DMD were incidentally found to have marked elevations of serum CK levels in early childhood, despite a negative family history for muscular supplier LY2603618 dystrophy. To confirm the clinical diagnosis of DMD, dystrophin gene mutations were extensively searched for using not only genomic DNA but also mRNA. However, no mutations could be identified, even when we included a deep intron mutation, To clarify pathological changes in these two patients, muscle biopsies were performed. In patient 1, H E staining revealed marked replacement of mus cle by adipose tissue along with increases in endomysial connective tissue.<br><br> We also found a few muscle fibers that were remarkably different in size, In spite of the clinical diagnosis of DMD, the immunostain ing pattern for both dystrophin and merosin staining was completely normal. Unexpectedly, there was no staining for sarcoglycan, while there was only a mild reduction for sarcoglycan and almost normal results for B and sarcoglycan, In order to confirm the sarco glycan deficiency, we looked for mutations in the SGCG gene. When PCR was used to amplify the eight exons that encompassed the regions of the SGCG gene, all regions other than exon 6 could be obtained. This suggests a homozygous deletion of exon 6, To confirm this, we used RT PCR to analyze the SGCG mRNA from the patients muscle. Amplification of the fragment encompassing exons 5 to 8 resulted in a small sized prod uct.