Clinical manifestations
This study examined five consanguineous families with CMT. CMT types and clinical phenotypes are provided in Table 1.
In the PaC2 family, a 6-year-old boy was born with a full term pregnancy to healthy parents. He showed delayed development and frequent fall during walking since early onset of 3 years old. He did not complain of sensory symptoms, however, his vibration sense were reduced. Deep tendon reflex at the knee was absent. Scoliosis, and foot deformities were observed. No family history of such complaints was recorded. Motor nerves conduction studies showed prolonged distal latencies and low distal CMAP amplitudes with no reproducible f-wave latencies and wave forms. He showed absent SNAP amplitudes in upper and lower extremities. These findings are compatible with demyelinating CMT neuropathy.
In the PaC3 family, a 10-year-old boy born full term from healthy normal parents had demyelinating CMT. At the age of 3, he was noticed frequent fall and difficulty in standing from sitting position. In addition to CMT phenotype, he showed the scoliosis and short stature. He had walking difficulty but still possible unaided. Motor nerves conduction studies of median and peroneal nerves showed prolonged distal latencies and low distal CMAP amplitudes of median and peroneal nerves were absent, and those of ulnar and tibial nerves were decreased. Motor and sensory NCVs were decreased on upper and lower extremities. Brain MRI showed no area of abnormal signal intensity.
In the PaC4 family, an 11-year-old boy was the product of a normal full-term pregnancy from healthy parents. At the age of 15 months, he was unable to walk without support. His parents first noticed gait disturbance at the age of 2.5 years. No family history of such complaints was recorded. Neurologic examination revealed decreased vibration and pain sense, which were consistent with the results of sensory nerve conduction study. Deep tendon reflex at the knee was absent, and foot deformities were observed. Lumbo-sacral spine MRI showed no noticeable abnormal signal.
In the PaC6 family, a 2.5-year-old girl born full term from unaffected parents had congenital motor neuropathy. She showed delayed development. The affected girl showed foot deformity and contractures of the distal phalanges before 6 months old, and her parents noticed the neuromuscular defect before 1 years. She had frequent fall during walking, and mild respiratory distress. Deep tendon reflex at the knee was decreased, and foot deformities were observed. No family history of such complaints was recorded. CMAPs of median, ulnar, peroneal and tibial nerves were not evoked, at all. But, normal SNAPs and SNCVs were observed in the sensory median, ulnar and sural nerves.
In the PaC14 family, a 7-year-old boy showed axonal CMT with onset of 5 years old. He had vocal cord hoarseness as the additional symptom. He showed delayed development. At 5 years, he showed gait disturbances, and frequent fall during walking. He did not complain of sensory symptoms, however, his vibration and position sense were reduced. Deep tendon reflex at the knee was absent, and foot deformities were observed. No family history of such complaints was recorded.
Identification of novel homozygous pathogenic mutations
This study identified five pathogenic or likely pathogenic homozygous mutations from SH3TC2, HK1, REEP1, and MFN2 genes in the examined families by the WES and subsequent annotation and filtering processes (Table 2). All the candidate pathogenic mutations were confirmed by Sanger sequencing (Fig. 2a).
Mutations in the SH3TC2 (MIM 608206) are implicated to the recessive CMT4C (MIM 601596) [11] and the relatively mild dominant mononeuropathy of the median nerve (MNMN, MIM 613353) [12]. We identified two novel or rare homozygous variants of SH3TC2 in two families. As the first mutation, a novel homozygous c.2599C>T mutation which results in a stop-gain mutation (p.Gln867*) was identified in a 6-year-old boy (family ID: PaC2). The SH3TC2 mutation was heterozygous in both unaffected parents and a brother (Fig. 1a). This mutation has not been reported as pathogenic, nor has it been registered in the public databases of dbSNP, 1000G, and ExAC. The p.Gln867* mutation is expected to produce a truncated protein of which many tetratricopeptide repeat (TPR) domains are deleted. Although the p.Gln867* was not reported yet, several stop-gain mutations, such as p.Gln892*, p.Arg904*, and p.Tyr943*, have been reported to the underlying causes of the patients with CMT4C [11, 13, 14]. As the second SH3TC2 mutation, a c.3650G>A resulting p.Gly1217Asp was identified in a 10 years old boy (family ID: PaC3). This mutation was heterozygous in the unaffected parents and two siblings (Fig. 1b). The homozygous p.Gly1217Asp was still not reported as pathogenic, although the same heterozygous variant was recently registered as “uncertain significance” in the ClinVar database. It was registered in the dbSNP (rs758669363) and ExAC with a very low allele frequency (1.6E-5). The p.Gly1217Asp mutation was located at a highly conserved TPR domain which has a putative function for protein-protein interactions (Fig. 2b, 2c), and was suggested to be pathogenic by the PolyPhen-2 and PROVEAN in silico prediction programs.
Few cases with homozygous mutations in HK1 have been reported to recessive CMT4G (MIM 605285), also called HMSN Russe type [15, 16]. An 11 years old boy with demyelinating CMT (family ID: PaC4) showed a stop-gain mutation of c.19C>T (p.Arg7*) in HK1 (MIM 142600), which putatively resulted in a very short premature peptide. The mutation was homozygous in the affected boy and heterozygous in the unaffected father and brother (Fig. 1c). This HK1 mutation was not reported as pathogenic, although registered in the dbSNP (rs779250530) and the ExAC database with a very low allele frequency (1.7E-5).
A small number of mutations in REEP1 (MIM 609139) have been reported to cause several neuromuscular disorders, such as the dominant dHMN5B (MIM 614751), also called distal spinal muscular atrophy type 5B (dSMA5B) [17], and the dominant spastic paraplegia-31 (SPG31, MIM 610250) [18]. A homozygous splicing site mutation was also recently reported in a patient having similar symptoms of the spinal muscular atrophy with respiratory distress (SMARD), of which phenotype is similar to the SMA but with additional symptom of diaphragmatic palsy [19]. This study identified a homozygous frameshift REEP1 mutation of c.247delG in a 2.5-year-old girl with dHMN (family ID: PaC6). This deletion was expected to produce a truncated premature peptide (p.Gly83Alafs*44). The mutation was heterozygous in the unaffected parents and sister (Fig. 1d). It has not been registered in any databases, nor has it been reported as a pathogenic mutation.
Most mutations in MFN2 (MIM 608507) are relevant with autosomal dominant CMT2A2A (MIM 609260) [20] and CMT6A (MIM 601152) [21], whereas, recessive MFN2 mutations have been rarely reported with more severe and earlier onset CMT2A2B (MIM 617087) [22]. The affected 7-year-old boy in the PaC14 family revealed a homozygous c.334G>A (p.Val112Met) in MFN2. The mutation was heterozygous in the unaffected parents and a sister (Fig. 1e). The mutation was reported in the ExAC with a very low frequency (1.6E-5), and was registered as likely pathogenic in the ClinVar database. It was predicted to be pathogenic by the in silico analysis using the PolyPhen-2 and PROVEAN programs, and was located at the highly conserved GTPase domain among vertebrate species (Fig. 2b, 2c).
From the filtering of the WES data for the affected individuals of five families, several rare functionally significant variants (MAF < 0.1) were observed in the CMT-related genes, in addition to the above mentioned five pathogenic or likely pathogenic mutations (Table 3). A homozygous c.1933A>G (p.Ile645Val) variant in DST was observed in the PaC4 patient. The DST mutation was cosegregated with the affected individual. However, in silico analyses with PolyPhen-2 and PROVEAN programs predicted it to be nonpathogenic. DST mutations have been reported to be implicated in HSAN6 (MIM 614653) [23], thus, we classified this homozygous variant as ‘variant of uncertain significance (VUS)”. All other rare variants were considered as nonpathogenic because they were either nonsegregated with affected individuals or did not fit the inheritance modes.
Homozygosity mapping
Homozygous blocks (HBs) were found at the chromosomal regions including pathogenic or likely pathogenic mutations in all the five affected individuals by the SNP haplotype analysis using WES data (Fig. 3). The lengths of HBs were from approximately 12 Mbp to 53 Mbp: 16 Mbp HB from FGF1 to THG1L in the PaC2 family with SH3TC2 mutation, 12 Mbp HB from PKD2L2 to SLC6A7 in the PaC3 family with SH3TC2 mutation, 38 Mbp HB from PPYR1 to NRG3 in the PaC4 family with HK1 mutation, 53 Mbp HB from CTNNA2 to MZT2A in the PaC6 family with REEP1 mutation, and 14 Mbp HB from NADK to CLCNKB in the PaC14 family with MFN2 mutation. This homozygosity mapping suggests that both homozygous alleles in each family originated from a single founder.