PMC:5069600 / 24277-26357 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/5069600","sourcedb":"PMC","sourceid":"5069600","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5069600","text":"RNA Expression Analysis and Haplotype Analysis of MME \nRNA analysis obtained from 6 patients (P1, P3, P5, P7, P8, and P10) and a P1 family member revealed abnormal transcripts. In P1 and P3 with the c.654+1G\u003eA mutation at the splicing donor site of intron 7 of the MME gene, agarose gel electrophoresis of the RT‐PCR products obtained with primer pair 1 showed a band smaller than the 350‐bp band observed in the healthy normal control (NC; Fig 4D). Sequences of the RT‐PCR products in P1 and P3 revealed aberrantly spliced mRNA lacking exon 7, resulting in a 231‐bp fragment (Fig 4G). The lack of exon 7 creates a frameshift and, consequently, premature termination within exon 8; therefore, the c.654+1G\u003eA mutation was designed as p.Gly179AspfsX2 at the protein level. In P5 with the c.661C\u003eT nonsense mutation, RT‐PCR products were not detected (Fig 4D), suggesting the occurrence of nonsense‐mediated decay of MME mRNA. In P8 and P10 with the c.655−2A\u003eG mutation at the splicing acceptor site of intron 7, the RT‐PCR products showed a smaller band than the 350‐bp band of the NC (Fig 4E). The sequences of the RT‐PCR products in P10 revealed aberrantly spliced mRNA lacking exon 8, resulting in a 284‐bp fragment (Fig 4H). The lack of exon 8 lead to an in‐frame deletion of 22 amino acids from the catalytic (extracellular) domain of NEP (p.Ile219_Glu240del). In P7 and P8 with the c.439+2T\u003eA mutation at the splicing donor site of intron 5, the RT‐PCR products obtained with primer pair 2 showed a band smaller than the 344‐bp band of the NC (Fig 4F). Sequences of the RT‐PCR products revealed aberrantly spliced mRNA lacking exon 5, resulting in a 263‐bp fragment (Fig 4I). The lack of exon 5 lead to an in‐frame deletion of 27 amino acids from the catalytic domain of NEP (p.Asp120_Glu146del; Asp120Ala).\nHaplotype analysis in 3 patients (P1–P3) with the same homozygous c.654+1G\u003eA mutation revealed that P1 and P3, but not P2, shared the same haplotype for the two closest markers (D3S3509 and D3S1275) and four SNPs (rs12493885, rs12497267, rs9438, and rs358733; Fig 4J).","divisions":[{"label":"Title","span":{"begin":0,"end":54}}],"tracks":[]}