Identification of MME Mutations Figure 1 shows a flow chart for the selection of patients, genetic tests, and the filtering process for homozygous variants using the ESVD system. We extracted three kinds of homozygous mutations in the MME gene in 5 patients in the first case series of patients with CMT with no pathogenic mutations in known CMT or IPN genes. In P1 to P3, we identified the same homozygous mutation in the splice donor site [c.654+1G>A] in intron 7 of MME. A novel homozygous missense mutation [c.1861T>C, p.Cys621Arg] and a nonsense mutation [c.661C>T, p.Gln221X] were found in P4 and P5, respectively. On the other hand, we could not extract presumed compound heterozygous variants or other homozygous variants in MME from the WES data of 163 AR/sporadic cases in first case series, although we identified a novel heterozygous nonsense variant of unknown significant (c.264C>A, p.Cys88X) and a rare SNV (c.1489T>C, p.Typ497His). In the second and third case series of patients with CMT with no pathogenic mutations in known CMT or IPN genes, five additional recessive mutations in the MME gene were identified (Fig 2): [c.1231_1233delTGT, p.Cys411del], [c.439+2T>A], [c.439+2T>A and c.655−2A>G (compound heterozygous mutation)], [c.1817G>A, p.Trp606X], and [c.655−2A>G] mutations in P6 to P10, respectively (Table 1). Segregation analysis from the families of P1, P4, P6, P7, P8, and P10 revealed that the MME mutations were segregated with the disease in each family (Fig 3). Glutamine 221, cysteine 411, tryptophan 606, cysteine 621, and canonical GT‐AG nucleotides of the splice donor and acceptor junctions in the MME gene were highly conserved among species (Fig 4A–C). Notably, the p.Cys621Arg mutation was classified as “pathogenic” or “deleterious” using in silico analysis (Polyphen2 score = 1.00, SIFT score = 0.00, and PROVEAN score = −11.2 [cutoff = −2.5]), and the p.Cys411del mutation was also predicted as deleterious in the PROVEAN software (PROVEAN score = −17.4 [cutoff = −2.5]). MAF of all the validated mutations in public databases, including Exome Sequencing Project (ESP) and Exome Aggregation Consortium (ExAC), and in‐house database was ≤0.002 (Supplementary Table 2). Table 1 Genetic, Clinical, and Laboratory Findings a ‘Motor’ indicates distal lower limbs weakness/atrophy or gait disturbance, and ‘Sensory’ indicates decreased superficial sensation or dysesthesia in lower limbs. b Scores indicating manual muscle testing (MMT) grade in the distal lower limbs. c Brain atrophy is evaluated by CT or MRI. d Nerve conduction study of Median nerve. Ax = axonal; CH = compound heterozygous; CT computed tomography; dCMAP = distal compound muscle action potential (mV); De = demyelinating; DL = distal latency (ms); DTRs = deep tendon reflexes; Htz = heterozygous; Hmz = homozygous; MCV = motor conduction velocity (m/s); MMSE = Mini–Mental State Examination; MRI = magnetic resonance imaging; NA = not available (not scored or not examined); NCS = nerve conduction study. Figure 3 Pedigrees and segregation analysis of MME mutations in 10 families. P1, P2, P4, P5, P7, and P10 have consanguineous parents. P3, P6, and P9 have unaffected parents with affected siblings, and P8 was sporadic. Autosomal‐recessive inheritance is presumed in all pedigrees. A DNA sample was available from individuals marked with an asterisk. Affected members have a homozygous or compound heterozygous mutation, whereas unaffected members were heterozygous or wild‐type carriers. Squares represent males and circles represent females. Filled symbols represent those affected with a similar phenotype. Oblique lines represent deceased family members. Black arrows indicate the proband (P1–P10). +/+ = homozygous for mutation; +/− = heterozygous; −/− = homozygous for wild type. 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. C