Figure 4  Localization and conservation of MME mutations, haplotype analysis, and RNA analysis. (A and B) Schematic representation of the MME gene and neprilysin. Red arrows indicate the location of mutations in the extracellular domain. N = N‐glycosylation sites; TM = transmembrane domain. (C) Conservation analysis. Glutamine 221, cysteine 411, tryptophan 606, cysteine 621, and canonical GT‐AG nucleotides (c.439+2t, c.654+1g, c.655‐2a) of the splice donor and acceptor junctions in the MME gene were highly conserved among species. (D) Agarose gel electrophoresis of cDNA fragments obtained from RT‐PCR of P1, his family member (IV‐4, V‐2, IV‐9, and V‐5), P3 with the c.654+1G>A mutation, P5 with c.661C>T mutation, and a normal control (NC). The P1, IV‐4 (affected), IV‐9 (affected), and P3 lanes showed a 231‐bp band, which is smaller than the 350‐bp band in the NC lane. The V‐2 and V‐5 (unaffected heterozygous carrier) lanes showed a 231‐bp band and a 350‐bp band. The P5 lane showed no band. (E) Agarose gel electrophoresis of cDNA fragments obtained from RT‐PCR of P8, P10 with the c.655‐2A>G mutation, and the NC. The P10 lane showed a 284‐bp band, which is smaller than the 350‐bp band in the NC lane. The P8 lane showed a 284‐bp band and a 350‐bp band. (F) Agarose gel electrophoresis of cDNA fragments obtained from RT‐PCR of P7, P8 with the c.439+2T>A mutation, and the NC. The P7 lane showed a 263‐bp band, which is smaller than the 344‐bp band in the NC lane. The P8 lane showed a 263‐bp band and a 344‐bp band. (G– I) Sequence chromatogram of the RT‐PCR product from P1, P10, and P7 showing exon 7 skipping and premature termination within exon 8 (G), exon 8 skipping (H), and exon 5 skipping (I) as schematically shown in the lower panel, respectively. (J) Haplotype analysis in P1 to P3. Shared haplotypes are shown in the gray box.