RNA Expression Analysis and Haplotype Analysis of MME 
RNA 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>A 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>A mutation was designed as p.Gly179AspfsX2 at the protein level. In P5 with the c.661C>T 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>G 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>A 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).
Haplotype analysis in 3 patients (P1–P3) with the same homozygous c.654+1G>A 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).