Novel homozygous mutations in Pakistani families with recessive Charcot-Marie-Tooth disease

Novel homozygous mutations in Pakistani families with recessive Charcot-Marie-Tooth disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Novel homozygous mutations in Pakistani families with recessive Charcot-Marie-Tooth disease Sumaira Kanwal, Yu Jin Choi, Si On Lim, Hee Ji Choi, Jin Hee Park, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-279595/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Charcot-Marie-Tooth disease (CMT) is a group of genetically and clinically heterogeneous peripheral nervous disorders. Few studies have identified genetic causes in the Pakistani CMT patients. Methods This study was performed to identify pathogenic mutations in five consanguineous Pakistani CMT families negative for PMP22 duplication. Genomic screening was performed by application of whole exome sequencing Results We identified five pathogenic or likely pathogenic homozygous mutations in four genes: c.2599C > T (p.Gln867*) and c.3650G > A (p.Gly1217Asp) in SH3TC2 , c.19C > T (p.Arg7*) in HK1 , c.247delG (p.Gly83Alafs*44) in REEP1 , and c.334G > A (p.Val112Met) in MFN2 . All the mutations were not reported in the CMT patients. Mutations in the SH3TC2 , HK1 , REEP1 , and MFN2 have been reported to be implicated to CMT4C, CMT4G, dHMN5B (DSMA5B), and CMT2A, respectively. The genotype-phenotype correlations were confirmed in all the examined families. We also confirmed that both alleles from the homozygous variants were originated from a single founder using homozygosity mapping. Conclusions This study found five novel mutations as the underlying causes of CMT. Pathogenic mutations in SH3TC2, HK1 , and REEP1 have been reported rarely in other populations, suggesting ethnic-specific distribution. This study will be useful for the exact molecular diagnosis and treatment in the Pakistani CMT patients. Epigenetics & Genomics Charcot-Marie-Tooth disease (CMT) Consanguinity Homozygosity Pakistan Whole exome sequencing Figures Figure 1 Figure 2 Figure 3 Background Charcot-Marie-Tooth disease (CMT) and related neuropathies are a group of genetically and clinically quite heterogeneous rare peripheral neuropathies with the prevalence of approximately 1 in 2,500 people. CMT, also called hereditary motor and sensory neuropathy (HMSN), is impaired in both sensory and motor nerves, whereas, both distal hereditary motor neuropathy (dHMN) and hereditary sensory and autonomic neuropathy (HSAN) only affect motor and sensory nerves, respectively. Their common clinical phenotypes include progressive distal muscle weakness and atrophy, loss of sensation, and anti-reflection symptom of the upper and lower limbs [ 1 ]. CMT is commonly divided into demyelinating type of CMT1 with the reduced motor nerve conduction velocity (NCV) of less than 38 m/s, axonal type of CMT2 with normal or slightly reduced NCV of 38 m/s or more, and intermediate CMT type [ 2 , 3 ]. CMT is often viewed as a monogenic Mendelian disease; however, mutations in more than 130 genes are associated with the development of peripheral neuropathies in an autosomal or X-linked dominant or recessive manner [ 4 ]. Several studies have attempted to determine the underlying causes of CMT. In particular, recent application of next generation sequencing (such as whole exome or targeted sequencing) has enhanced unveiling of the genetic pathogenicity. However, limited studies have been performed to determine the genetic causes of CMT and related peripheral neuropathies in Pakistan [ 5 – 8 ]. Pakistani patients whose genetic causes were identified showed unusually high rate of recessive homozygous mutations. Pedurupillay et al. reported three patients with CMT2S or spinal muscular atrophy with respiratory distress type 1 (SMARD1) with IGHMBP2 mutations [ 6 ]. Two of them presented with homozygous mutations. Wright et al. reported a homozygous Fig. 4 variant in four independent patients with combined phenotypes of CMT4J and Yunis-Varón syndrome [ 8 ]. Houlden et al. reported several patients with HSPB1 mutations which contained a homozygous mutation in addition to heterozygous mutations [ 5 ]. Zambon et al. reported a patient with CMT4B1 with homozygous MTMR2 mutation [ 7 ]. The high rates of homozygous mutations in Pakistani patients can be attributed to the relatively frequent consanguineous marriages. This study aimed to determine the genetic causes in Pakistani patients with CMT or related neuropathies using whole exome sequencing (WES) and subsequent filtering process of called variants. We identified five pathogenic or likely pathogenic homozygous mutations in the CMT-related genes. Additionally, we determined that all the observed homozygous mutations originated from a single founder through homozygosity mapping. Methods Subjects This study examined five CMT patients and 15 of their unaffected familial members that originated from consanguineous Pakistani pedigrees, negative for the duplication or deletion of the 17p12 chromosomal region including PMP2 2 gene (Fig. 1). All participants were recruited from Care Hospital Sahiwal, Pakistan, and provided written informed consent. For the minors involved in the study, consent was provided by their parents. This study was approved by the Institutional Review Boards for Kongju National University (KNU_IRB_2018-06) and Sungkyunkwan University, Samsung Medical Center (2014-08-057-002). Clinical and electrophysiological assessments Motor and sensory impairments, deep tendon reflexes, and muscle atrophy were measured as the clinical information. Onset age was determined through patient interviews about when symptoms such as distal muscle weakness, foot deformity, or sensory change first appeared. Disease severity was determined using the functional disability scale (FDS). Motor and sensory conduction velocities of median, ulnar, peroneal, tibial, and sural nerves were determined by surface stimulation and recording electrodes. Motor nerve conduction velocities (MNCVs) of the median and ulnar nerves were determined by stimulating at the elbow and wrist while recording compound muscle action potentials (CMAPs) over the abductor pollicis brevis and adductor digiti quinti, respectively. In the same way, the NCVs of peroneal and tibial nerves were determined by stimulating at the knee and ankle, while recording CMAPs over the extensor digitorum brevis and adductor hallucis, respectively. Sensory nerve conduction velocities (SNCVs) and sensory nerve action potential (SNAP) amplitudes were obtained over a finger-wrist segment from the median and ulnar nerves by orthodromic scoring, and were also recorded for sural nerves. Electromyography was tested for investigation of neuromuscular disease. MRIs of the brain and spinal cord were obtained using a 1.5-T system (Siemens Healthineers, Erlangen, Germany). DNA purification and paternity testing Genomic DNA was purified from whole blood by using the HiGene Genomic DNA Prep Kit (Biofact, Daejeon, Korea). Paternity was confirmed for all the examined families by PCR amplification of STR markers using the PowerPlex Fusion System (Promega, Wisconsin-Madison, USA) and resolution of the PCR products by the SeqStudio genetic analyzers (Life Technologies-Thermo Fisher Scientific, Foster City, CA, USA). Exome sequencing and filtering WES was performed for patients in the five examined families. Exome was captured using the SureSelect Human All Exon 50M kit (Agilent Technologies, Santa Clara, CA, USA), and sequencing was performed using the HiSeq 2000 Genome Analyzer (Illumina, San Diego, CA, USA). The UCSC assembly hg19 (GRCh37) was used as the reference sequence (http://genome.ucsc.edu). Small nucleotide variants (SNVs) were called using the programs of GATK (https://software.broad institute.org/gatk/) and SAMtools (http://samto ols.sourceforge.net/). Rare alleles with minor allele frequencies (MAFs) of < 0.1 were obtained from the 1000 Genomes Project (1000G, http://www.1000genomes.org/), and the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org/). Candidate variants for the genetic causes were also checked in the dbSNP (http://www.ncbi.nlm.nih.gov/snp) and the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). Pathogenicity of the variants was evaluated into five grades (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) based on the guidelines of the American College of Medical Genetics and Genomics (ACMG) [9]. Pathogenic candidate variants were confirmed by Sanger sequencing using the SeqStudio genetic analyzers (Life Technologies-Thermo Fisher Scientific). In silico prediction and conservation analysis In silico analyses to predict the mutation effect were performed using the programs of MUpro (http://www.ics.uci.edu/~baldig/mutation), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and PROVEAN (http://provean.jcvi.org/). Conservation analysis of the mutation sites were performed by the MEGA-X software, ver. 5.05 (http://www.megasoftware.net/). Genomic evolutionary rate profiling score (GERP) were determined by the GERP++ program (http://mendel.stanford.edu/SidowLab/downloads/gerp/). Homozygosity mapping For the putative pathogenic homozygous variants, homozygosity mapping was performed to determine whether two same alleles originated from a single founder. Homozygosity mapping was achieved through haplotyping of SNPs distributed around the corresponding mutations from the WES data of the affected persons by using the method outlined by Park et al. [10]. Results 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 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. Discussion From the genetic screening of the consanguineous Pakistani CMT families, we identified five homozygous mutations in SH3TC2, HK1, REEP1 , and MFN2 as the underlying causes. All the identified homozygous mutations were not reported in the CMT patients. SH3TC2 which encodes a protein of SH3 domain and tetratricopeptide repeats containing protein 2, expressed in Schwann cells of peripheral nerves, suggesting a possible role in myelination [ 24 ]. Mutations in SH3TC2 cause recessive CMT4C usually concurrent with scoliosis, with the onset ranging from infancy to early teens [ 11 ], however, cases with late onset (≤ 30 years) were also reported [ 25 ]. Mutations in HK1 cause recessive CMT4G (HMSNR), mostly found in the Spanish Gypsy patients [ 15 ]. Hexokinase 1 encoded by HK1 catalyzes the phosphorylation of glucose. HK1 localizes at the outer membrane of mitochondria (OMM) through a porin-binding domain, and it was suggested that the involvement of the non-OMM-binding HK1 protein in the CMT4G pathogenesis [ 15 ]. Several HK1 mutations are also associated with autosomal dominant retinitis pigmentosa-79 (RP79, MIM 617460), which exhibits variable phenotype with ages of onset ranging from childhood to 70 years [ 26 ]. Here, the affected 11-year-old boy with the HK1 mutation did not show retinitis pigmentosa symptom until his examined age. REEP1 encodes a receptor accessory protein 1 that suggested to have a role in facilitating endoplasmic reticulum (ER)-mitochondrial interactions [ 27 ]. It is known that the REEP1 mutations exhibited considerable phenotypic heterogeneity [ 28 ]. The dominant REEP1 mutations have been reported to cause dHMN5B (DSMA5B) and SPG21 with the onset ages falling in either the first or second decades [ 17 , 18 ]. Recently, a recessive REEP1 mutation was reported in a Lebanese 5-year-old boy with a SMARD-similar phenotype [ 19 ]. The affected boy presented foot deformity and contractures of the distal phalanges at birth. This case was similar to our PaC6 case having a p.Gly83Alafs*44 mutation in view of premature termination, onset age, and some clinical symptoms. In the nerve conduction studies, all motor nerves were not evoked at all, but all sensory nerves showed normal SNAPs and SNCVs. From the clinical and NCV findings, this patient's symptoms are apparently similar with SMARD. MFN2 encodes mitofusin 2 which plays an important role maintaining equilibrium between mitochondrial fusion and fission [ 29 ]. Most MFN2 mutations have been reported to cause dominant CMT2A2A [ 20 ]. However, some homozygous or compound heterozygous mutations cause recessive CMT2A2B (MIM 617087) with more severe and earlier onset phenotypes. Nicholson et al. suggested that CMT2A2B may semidominant and carriers with a single mutant allele may show weak phenotype with incomplete penetrance [ 22 ]. Our case with the homozygous MFN2 mutations showed relatively early onset (5 years old) and severe phenotypes, which are matched with the characteristics of CMT2A2B. Additionally, the vocal cord paralysis seen in the affected boy has been occasionally reported in CMT2A patients with MFN2 mutations [ 30 , 31 ]. The patient showed no symptom of optic atrophy which is a characteristic of CMT6A until his examined age (7 years old). His parents were apparently seemed to be unaffected; however, exact clinical and electrophysiological tests were not done. Although a small number of CMT cases were investigated in this study, the incidence of the recessive patients with homozygous mutations was certainly frequent compared to other populations. Homozygosity mapping showed that both alleles of the homozygous mutations identified in each family originated from a single founder. Mutations in MFN2 are well known as the cause of dominant CMT2, however, recessive homozygous mutations have been rarely reported. This suggests an increased risk of consanguinity prone to develop rare recessive genetic diseases. Conclusions In conclusion, we identified five pathogenic or likely pathogenic mutations in the consanguineous Pakistani families with early onset CMT. All the mutations were novel, and the genotype-phenotype correlations were confirmed. We believe that our findings will contribute to expanding understanding of the genetic basis of peripheral neuropathy, improving molecular diagnostics and treatment options. Abbreviations ACMG: American College of Medical Genetics and Genomics; CMAP: Compound muscle action potential; CMT: Charcot-Marie-Tooth disease; dHMN: Distal hereditary motor neuropathy; dSMA5B: Distal spinal muscular atrophy type 5B; ER: Endoplasmic reticulum; FDS: Functional disability scale; GERP: Genomic evolutionary rate profiling score; HB: Homozygous block; HMSN: Hereditary motor and sensory neuropathy; HSAN: Hereditary sensory and autonomic neuropathy; MAF: Minor allele frequency; MNCV: Motor nerve conduction velocity: NCV: Nerve conduction velocity; OMM: Outer membrane of mitochondria; SMARD: Spinal muscular atrophy with respiratory distress; SNAP: Sensory nerve action potential; SNCV: Sensory nerve conduction velocity; TPR: tetratricopeptide repeat; WES: Whole exome sequencing Declarations A cknowledgements We would like to thank the patients and their families for their consent of participation and sample donation. A uthor contributions Conceptualization: Chung KW, Kanwal S; Sampling: Kanwal S, Nuzhat R, Khan A, Perveen S; Genetic investigation: Choi YJ, Lim SO, Choi HJ, Park JH, Son WS; Clinical data analysis: Choi BO; Funding acquisition: Chung KW, Choi BO; Writing original draft preparation: Son WS, Choi BO, Chung KW; Review and editing: all authors. Funding This work was supported by grants from the National Research Foundation (2019R1A2C1087547, 2020M3H4A1A03084600, and 2021R1A4A2001389) and the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI14C3484 and HI20C0039), Republic of Korea. Availability of data and materials All pathogenic variants and data from this study are available upon reasonable request. Raw data on the exome sequencing are available upon request. Ethics approval and consent to participate This study was approved by the Institutional Review Boards for Kongju National University (KNU_IRB_2018-06) and Sungkyunkwan University, Samsung Medical Center (2014-08-057-002). All participants were recruited from Care Hospital Sahiwal, Pakistan, and provided written informed consent. For the minors involved in the study, the consent was provided by their parents. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests References Saporta MA, Shy ME. Inherited peripheral neuropathies. Neurol Clin. 2013;31:597-619. Harding AE, Thomas PK. Genetic aspects of hereditary motor and sensory neuropathy (types I and II). J Med Genet. 1980;17:329-36. Fridman V, Bundy B, Reilly MM, Pareyson D, Bacon C, Burns J, et al. CMT subtypes and disease burden in patients enrolled in the Inherited Neuropathies Consortium natural history study: a cross-sectional analysis. 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Clinical and genetic diversities of Charcot-Marie-Tooth disease with MFN2 mutations in a large case study. J Peripher Nerv Syst. 2017;22:191-9. Tables Table 1. Clinical characterization of the five Pakistani CMT patients Item ＼ Patient (sex) PaC2:IV-1 (male) PaC3:IV-1 (male) PaC4:IV-1 (male) PaC6:IV-2 (female) PaC14:IV-2 (male) Gene: mutation SH3TC2 : p.Q867* SH3TC2 : p.G1217D HK1 : p.R7* REEP1 : p.G83Afs*44 MFN2 : p.V112M Type CMT4C CMT4C CMT4G dHMN5B/SMARD1 CMT2A2B Ages at exam/onset (yr) 6/3 10/3 11/1 2.5/< 1 7/5 Muscle atrophy Yes Yes Yes Yes Yes FDS 3 3 3 3 3 Sensory loss Yes Yes Yes No Yes DTR, ankle Absent Absent Absent Decreased Absent Foot deformities Yes Yes Yes Yes Yes Brain/Spine MRI ND Normal brain Normal spine Normal brain ND Other symptoms Scoliosis Scoliosis, short stature - Mild respiratory distress Vocal cord hoarseness Motor nerve conduction studies Median CMAP (mV) 1.3 Absent ND Absent ND Median MNCV (m/s) 14.1 Absent ND Absent ND Ulnar CMAP (mV) 0.8 4.1 ND Absent ND Ulnar MNCV (m/s) 12.8 25.0 ND Absent ND Peroneal CMAP (mV) 0.8 Absent ND Absent ND Peroneal MNCV (m/s) 14.9 Absent ND Absent ND Sensory nerve conduction studies Median SNAP (μV) Absent 10.6 ND 22.0 ND Median SNCV (m/s) Absent 26.0 ND 51.3 ND Ulnar SNAP (μV) Absent ND ND 22.2 ND Ulnar SNCV (m/s) Absent ND ND 39.5 ND Sural SNAP (μV) Absent 8.8 ND 16.3 ND Sural SNCV (m/s) Absent 32.0 ND 39.8 ND CMAP: compound muscle action potential, DTR: deep tendon reflexes, FDS: functional disability scale, MNCV: motor nerve conduction velocity, ND: not done, SNAP: sensory nerve action potential, SNCV: sensory nerve conduction velocity. Table 2. Homozygous mutations and clinical phenotypes in the Pakistani CMT patients Family ID Gene Mutation a Type Onset age (yr) Other symptom Allele frequency GERP In silico prediction b Note 1000G ExAC PP2 MU PRO PaC2 SH3TC2 c.2599C>T;p.Q867* CMT4C 3 Scoliosis UR UR 2.17 - - - P PaC3 SH3TC2 c.3650G>A;p.G1217D CMT4C 3 Scoliosis, short stature UR 1.6E-5 6.17 1.00 * 0.10 -5.79 * P PaC4 HK1 c.19C>T;p.R7* CMT4G 1 UR 1.7E-5 1.13 - - - P PaC6 REEP1 c.247delG;p.G83Afs*44 SMARD/dHMN5B A;p.V112M CMT2A2B 5 Vocal cord hoarseness UR 1.6E-5 4.70 1.00 * 0.32 -2.76 * P 1000G: 1000 Genomes Project, CMT: Charcot-Marie-Tooth disease, ExAC: Exome Aggregation Consortium, GERP: genomic evolutionary rate profiling score, P: pathogenic, LP: likely pathogenic, UR: unreported. a Reference DNA and protein sequences: SH3TC2 : NM_024577.4 and NP_078853.2, HK1 : NM_033498.3 and NP_277033.1, REEP1 : NM_022912.3 and NP_075063.1, MFN2 : NM_014874.3 and NP_055689.1 . b Scores of PolyPhen-2 (PP2) ~1, MUpro (MU) <0, and PROVEAN (PRO) <-2.5 indicate pathogenic prediction ( * denotes a pathogenic prediction). Table 3. Rare variants observed in the CMT-related genes from patients of five Pakistani CMT families Family Gene Variant ClinVar dbSNP151 Allele frequency GERP In silico analysis Note Nucleotide Amino acid 1000G ExAC PP2 PRO PaC2 KIF1B c.3209C>T p.A1070V UR rs768176241 UR 1.7E-5 4.82 0.02 -0.01 Nonsegregation, LB DST [c.7252G>A + c.7765A>G] [p.V2418I + p.I2589V] UR rs62621210 0.0400 0.0356 4.80 0.00 0.76 Cis , nonsegregation, LB B,LB rs150191284 0.0102 0.0239 4.45 0.02 0.07 MYH14 c.3748G>T p.V1250L LB rs202242879 0.0006 0.0009 3.78 0.11 -0.39 Nonsegregation, LB SCN11A c.1732T>A p.F578I UR rs772393665 UR 0.0001 5.58 0.98* -4.61* Nonsegregation, LB PaC3 KIF1B [c.2107T>C] + [c.2455A>C] [p.W703R] + [p.S819R] B,LB rs551543997 0.0054 0.0033 5.32 0.99* -10.0* Trans , nonsegregation, LB LB rs140015591 0.0002 0.0005 -2.96 0.02 -2.19 NTRK1 [c.2339G>A + c.2360C>T] [p.R780Q + p.A787V] B,LB rs35669708 0.0038 0.0064 4.10 0.87* -1.25 Cis , nonsegregation, LB UR rs761207548 UR 7.E-05 4.07 0.36 -3.00* NAGLU c.2209C>A p.R737S B rs86312 0.0116 0.0192 4.01 0.47* -0.19 Nonsegregation, LB SCN10A c.3887G>T p.S1296I LB rs779527264 UR 0.0002 4.88 1.00* -5.79* Nonsegregation, LB PaC4 DST [c.1933A>G] + [c.1933A>G] [p.I645V] + [p.I645V] UR rs754692637 UR 8.E-06 2.51 0.00 -0.20 Homozygous, cosegregation, VUS ARHGEF10 c.2566G>A p.V856I UR rs773521162 UR 0.0003 4.25 0.87* -0.62 Nonsegregation, LB PaC6 TFG c.175A>G p.K59E UR rs1232918261 UR UR 5.90 1.00* -3.02* Nonsegregation, LB PaC14 SETX c.2385_2387delTTT p.I795_K796delinsM UR rs755971927 UR 6.E-05 - - - Nonsegregation, LB 1000G: 1000 Genomes Project, ExAC: Exome Aggregation Consortium, B: benign, LB: likely benign, VUS: variant of uncertain significance, trans : trans arrangement of variants in homologous chromosomes (bi-alleles), cis : cis arrangement of variants in a chromosome, UR: unreported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 06 Apr, 2021 Review # 4 received at journal 30 Mar, 2021 Review # 2 received at journal 30 Mar, 2021 Review # 3 received at journal 29 Mar, 2021 Reviewer # 4 agreed at journal 20 Mar, 2021 Reviewer # 3 agreed at journal 18 Mar, 2021 Reviewer # 2 agreed at journal 18 Mar, 2021 Review # 1 received at journal 18 Mar, 2021 Reviewer # 1 agreed at journal 06 Mar, 2021 Editor assigned by journal 22 Feb, 2021 Reviewers invited by journal 22 Feb, 2021 Submission checks completed at journal 22 Feb, 2021 Editor invited by journal 22 Feb, 2021 First submitted to journal 14 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Genotypes of pathogenic or likely pathogenic mutations are indicated at the bottom of all the examined family members. Black and white symbols represent affected and unaffected individuals, respectively. The affected individuals subjected to whole exome sequencing are indicated by an asterisk (□: male, and ○: female). (A) PaC2 family with c.2599C\u003eT (p.Gln867*) in SH3TC2, (B) PaC3 family with c.3650G\u003eA (p.Gly1217Asp) mutation in SH3TC2, (C) PaC4 family with c.19C\u003eT (p.Arg7*) mutation in HK1, (D) PaC6 family with c.247delG (p.Gly83Alafs*44) mutation in REEP1, and (E) PaC14 family with c.334G\u003eA (p.Val112Met) mutation in MFN2.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-279595/v1/ebc032355d2191d8e3496238.png"},{"id":6624294,"identity":"fb1c6c7c-95aa-4e31-8e71-bf0cfe51be91","added_by":"auto","created_at":"2021-03-04 21:41:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94771,"visible":true,"origin":"","legend":"Identification of novel homozygous variants thought to be the underlying causes of CMT. a. Sequencing chromatograms of c.2599C\u003eT and c.3650G\u003eA in SH3TC2, c.19C\u003eT in HK1, c.247delG in REEP1, and c.334G\u003eA in MFN2. b. Conservation of two missense mutation sites. The amino acids at the mutation sites are highly conserved among vertebrate species. c. Domain structure and location of the mutations of SH3TC2, and MFN2. The p.Gly1217Asp in SH3TC2, and the p.Val112Met in MFN2 are located in the tetratricopeptide repeats (TPR) and GTPase domains, respectively. ","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-279595/v1/bdd047f77e2d857b6cc19847.png"},{"id":6624639,"identity":"d32cf6cc-c6cd-4f14-9121-43e9aa48b60d","added_by":"auto","created_at":"2021-03-04 21:44:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51689,"visible":true,"origin":"","legend":"Homozygosity mapping of the chromosomal regions around the pathogenic mutations for the affected individuals from the consanguineous Pakistani CMT families. Heterozygous blocks (HBs) were observed from all the examined chromosomes. a. 16 Mbp HB from FGF1 to THG1L in the PaC2 family with SH3TC2 mutation. b. 12 Mbp HB from PKD2L2 to SLC6A7 in the PaC3 family with SH3TC2 mutation. c. 38 Mbp HB from PPYR1 to NRG3 in the PaC4 family with HK1 mutation. d. 53 Mbp HB from CTNNA2 to MZT2A in the PaC6 family with REEP1 mutation. e. 14 Mbp HB from NADK to CLCNKB in the PaC14 family with MFN2 mutation.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-279595/v1/6578034f11aa3d11b621261f.png"},{"id":13675415,"identity":"daa605e9-454c-49e6-841b-096087e2ed2e","added_by":"auto","created_at":"2021-09-17 11:26:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":463778,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-279595/v1/66ead6c6-61ac-436b-aa16-cd44921dec77.pdf"}],"financialInterests":"","formattedTitle":"Novel homozygous mutations in Pakistani families with recessive Charcot-Marie-Tooth disease","fulltext":[{"header":"Background","content":" \u003cp\u003eCharcot-Marie-Tooth disease (CMT) and related neuropathies are a group of genetically and clinically quite heterogeneous rare peripheral neuropathies with the prevalence of approximately 1 in 2,500 people. CMT, also called hereditary motor and sensory neuropathy (HMSN), is impaired in both sensory and motor nerves, whereas, both distal hereditary motor neuropathy (dHMN) and hereditary sensory and autonomic neuropathy (HSAN) only affect motor and sensory nerves, respectively. Their common clinical phenotypes include progressive distal muscle weakness and atrophy, loss of sensation, and anti-reflection symptom of the upper and lower limbs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. CMT is commonly divided into demyelinating type of CMT1 with the reduced motor nerve conduction velocity (NCV) of less than 38 m/s, axonal type of CMT2 with normal or slightly reduced NCV of 38 m/s or more, and intermediate CMT type [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. CMT is often viewed as a monogenic Mendelian disease; however, mutations in more than 130 genes are associated with the development of peripheral neuropathies in an autosomal or X-linked dominant or recessive manner [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies have attempted to determine the underlying causes of CMT. In particular, recent application of next generation sequencing (such as whole exome or targeted sequencing) has enhanced unveiling of the genetic pathogenicity. However, limited studies have been performed to determine the genetic causes of CMT and related peripheral neuropathies in Pakistan [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Pakistani patients whose genetic causes were identified showed unusually high rate of recessive homozygous mutations. Pedurupillay et al. reported three patients with CMT2S or spinal muscular atrophy with respiratory distress type 1 (SMARD1) with \u003cem\u003eIGHMBP2\u003c/em\u003e mutations [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Two of them presented with homozygous mutations. Wright et al. reported a homozygous \u003cem\u003eFig.\u0026nbsp;4\u003c/em\u003e variant in four independent patients with combined phenotypes of CMT4J and Yunis-Var\u0026oacute;n syndrome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Houlden et al. reported several patients with \u003cem\u003eHSPB1\u003c/em\u003e mutations which contained a homozygous mutation in addition to heterozygous mutations [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Zambon et al. reported a patient with CMT4B1 with homozygous \u003cem\u003eMTMR2\u003c/em\u003e mutation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The high rates of homozygous mutations in Pakistani patients can be attributed to the relatively frequent consanguineous marriages.\u003c/p\u003e \u003cp\u003eThis study aimed to determine the genetic causes in Pakistani patients with CMT or related neuropathies using whole exome sequencing (WES) and subsequent filtering process of called variants. We identified five pathogenic or likely pathogenic homozygous mutations in the CMT-related genes. Additionally, we determined that all the observed homozygous mutations originated from a single founder through homozygosity mapping.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSubjects \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study examined five CMT patients and 15 of their unaffected familial members that originated from consanguineous Pakistani pedigrees, negative for the duplication or deletion of the 17p12 chromosomal region including \u003cem\u003ePMP2\u003c/em\u003e2 gene (Fig. 1). All participants were recruited from Care Hospital Sahiwal, Pakistan, and provided written informed consent. For the minors involved in the study, consent was provided by their parents. This study was approved by the Institutional Review Boards for Kongju National University (KNU_IRB_2018-06) and Sungkyunkwan University, Samsung Medical Center (2014-08-057-002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical and electrophysiological assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotor and sensory impairments, deep tendon reflexes, and muscle atrophy were measured as the clinical information. Onset age was determined through patient interviews about when symptoms such as distal muscle weakness, foot deformity, or sensory change first appeared. Disease severity was determined using the functional disability scale (FDS). Motor and sensory conduction velocities of median, ulnar, peroneal, tibial, and sural nerves were determined by surface stimulation and recording electrodes. Motor nerve conduction velocities (MNCVs) of the median and ulnar nerves were determined by stimulating at the elbow and wrist while recording compound muscle action potentials (CMAPs) over the abductor pollicis brevis and adductor digiti quinti, respectively. In the same way, the NCVs of peroneal and tibial nerves were determined by stimulating at the knee and ankle, while recording CMAPs over the extensor digitorum brevis and adductor hallucis, respectively. Sensory nerve conduction velocities (SNCVs) and sensory nerve action potential (SNAP) amplitudes were obtained over a finger-wrist segment from the median and ulnar nerves by orthodromic scoring, and were also recorded for sural nerves. Electromyography was tested for investigation of neuromuscular disease. MRIs of the brain and spinal cord were obtained using a 1.5-T system (Siemens Healthineers, Erlangen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA purification and paternity testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was purified from whole blood by using the HiGene Genomic DNA Prep Kit (Biofact, Daejeon, Korea). Paternity was confirmed for all the examined families by PCR amplification of STR markers using the PowerPlex Fusion System (Promega, Wisconsin-Madison, USA) and resolution of the PCR products by the SeqStudio genetic analyzers (Life Technologies-Thermo Fisher Scientific, Foster City, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExome sequencing and filtering\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWES was performed for patients in the five examined families. Exome was captured using the SureSelect Human All Exon 50M kit (Agilent Technologies, Santa Clara, CA, USA), and sequencing was performed using the HiSeq 2000 Genome Analyzer (Illumina, San Diego, CA, USA). The UCSC assembly hg19 (GRCh37) was used as the reference sequence (http://genome.ucsc.edu). Small nucleotide variants (SNVs) were called using the programs of GATK (https://software.broad institute.org/gatk/) and SAMtools (http://samto ols.sourceforge.net/). Rare alleles with minor allele frequencies (MAFs) of \u0026lt; 0.1 were obtained from the 1000 Genomes Project (1000G, http://www.1000genomes.org/), and the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org/). Candidate variants for the genetic causes were also checked in the dbSNP (http://www.ncbi.nlm.nih.gov/snp) and the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). Pathogenicity of the variants was evaluated into five grades (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) based on the guidelines of the American College of Medical Genetics and Genomics (ACMG) [9]. Pathogenic candidate variants were confirmed by Sanger sequencing using the SeqStudio genetic analyzers (Life Technologies-Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e prediction and conservation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e analyses to predict the mutation effect were performed using the programs of MUpro (http://www.ics.uci.edu/~baldig/mutation), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and PROVEAN (http://provean.jcvi.org/). Conservation analysis of the mutation sites were performed by the MEGA-X software, ver. 5.05 (http://www.megasoftware.net/). Genomic evolutionary rate profiling score (GERP) were determined by the GERP++ program (http://mendel.stanford.edu/SidowLab/downloads/gerp/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHomozygosity mapping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the putative pathogenic homozygous variants, homozygosity mapping was performed to determine whether two same alleles originated from a single founder. Homozygosity mapping was achieved through haplotyping of SNPs distributed around the corresponding mutations from the WES data of the affected persons by using the method outlined by Park et al. [10].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical manifestations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study examined five consanguineous families with CMT. CMT types and clinical phenotypes are provided in Table 1.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e\n\u003cp\u003eIn 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of novel homozygous pathogenic mutations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study identified five pathogenic or likely pathogenic homozygous mutations from \u003cem\u003eSH3TC2, HK1, REEP1\u003c/em\u003e, and \u003cem\u003eMFN2 \u003c/em\u003egenes 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).\u003c/p\u003e\n\u003cp\u003eMutations in the \u003cem\u003eSH3TC2\u003c/em\u003e (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 \u003cem\u003eSH3TC2 \u003c/em\u003ein two families. As the first mutation, a novel homozygous c.2599C\u0026gt;T mutation which results in a stop-gain mutation (p.Gln867*) was identified in a 6-year-old boy (family ID: PaC2). The \u003cem\u003eSH3TC2\u003c/em\u003e 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 \u003cem\u003eSH3TC2 \u003c/em\u003emutation, a c.3650G\u0026gt;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 \u0026ldquo;uncertain significance\u0026rdquo; 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 \u003cem\u003ein silico\u003c/em\u003e prediction programs.\u003c/p\u003e\n\u003cp\u003eFew cases with homozygous mutations in \u003cem\u003eHK1\u003c/em\u003e 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\u0026gt;T (p.Arg7*) in \u003cem\u003eHK1\u003c/em\u003e (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 \u003cem\u003eHK1\u003c/em\u003e 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).\u003c/p\u003e\n\u003cp\u003eA small number of mutations in \u003cem\u003eREEP1\u003c/em\u003e (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 \u003cem\u003eREEP1 \u003c/em\u003emutation 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.\u003c/p\u003e\n\u003cp\u003eMost mutations in \u003cem\u003eMFN2\u003c/em\u003e (MIM 608507) are relevant with autosomal dominant CMT2A2A (MIM 609260) [20] and CMT6A (MIM 601152) [21], whereas, recessive \u003cem\u003eMFN2\u003c/em\u003e 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\u0026gt;A (p.Val112Met) in \u003cem\u003eMFN2\u003c/em\u003e. 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 \u003cem\u003ein silico\u003c/em\u003e analysis using the PolyPhen-2 and PROVEAN programs, and was located at the highly conserved GTPase domain among vertebrate species (Fig. 2b, 2c).\u003c/p\u003e\n\u003cp\u003eFrom the filtering of the WES data for the affected individuals of five families, several rare functionally significant variants (MAF \u0026lt; 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\u0026gt;G (p.Ile645Val) variant in \u003cem\u003eDST\u003c/em\u003e was observed in the PaC4 patient. The \u003cem\u003eDST\u003c/em\u003e mutation was cosegregated with the affected individual. However, \u003cem\u003ein silico\u003c/em\u003e analyses with PolyPhen-2 and PROVEAN programs predicted it to be nonpathogenic. \u003cem\u003eDST\u003c/em\u003e mutations have been reported to be implicated in HSAN6 (MIM 614653) [23], thus, we classified this homozygous variant as \u0026lsquo;variant of uncertain significance (VUS)\u0026rdquo;. All other rare variants were considered as nonpathogenic because they were either nonsegregated with affected individuals or did not fit the inheritance modes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHomozygosity mapping \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHomozygous 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 \u003cem\u003eFGF1\u003c/em\u003e to \u003cem\u003eTHG1L\u003c/em\u003e in the PaC2 family with \u003cem\u003eSH3TC2\u003c/em\u003e mutation, 12 Mbp HB from \u003cem\u003ePKD2L2\u003c/em\u003e to \u003cem\u003eSLC6A7 \u003c/em\u003ein the PaC3 family with \u003cem\u003eSH3TC2\u003c/em\u003e mutation, 38 Mbp HB from \u003cem\u003ePPYR1\u003c/em\u003e to \u003cem\u003eNRG3 \u003c/em\u003ein the PaC4 family with \u003cem\u003eHK1\u003c/em\u003e mutation, 53 Mbp HB from \u003cem\u003eCTNNA2\u003c/em\u003e to \u003cem\u003eMZT2A \u003c/em\u003ein the PaC6 family with \u003cem\u003eREEP1\u003c/em\u003e mutation, and 14 Mbp HB from \u003cem\u003eNADK\u003c/em\u003e to \u003cem\u003eCLCNKB \u003c/em\u003ein the PaC14 family with \u003cem\u003eMFN2\u003c/em\u003e mutation. This homozygosity mapping suggests that both homozygous alleles in each family originated from a single founder.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eFrom the genetic screening of the consanguineous Pakistani CMT families, we identified five homozygous mutations in \u003cem\u003eSH3TC2, HK1, REEP1\u003c/em\u003e, and \u003cem\u003eMFN2\u003c/em\u003e as the underlying causes. All the identified homozygous mutations were not reported in the CMT patients.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSH3TC2\u003c/em\u003e which encodes a protein of SH3 domain and tetratricopeptide repeats containing protein 2, expressed in Schwann cells of peripheral nerves, suggesting a possible role in myelination [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Mutations in \u003cem\u003eSH3TC2\u003c/em\u003e cause recessive CMT4C usually concurrent with scoliosis, with the onset ranging from infancy to early teens [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], however, cases with late onset (\u0026le;\u0026thinsp;30 years) were also reported [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMutations in \u003cem\u003eHK1\u003c/em\u003e cause recessive CMT4G (HMSNR), mostly found in the Spanish Gypsy patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hexokinase 1 encoded by \u003cem\u003eHK1\u003c/em\u003e catalyzes the phosphorylation of glucose. HK1 localizes at the outer membrane of mitochondria (OMM) through a porin-binding domain, and it was suggested that the involvement of the non-OMM-binding HK1 protein in the CMT4G pathogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Several \u003cem\u003eHK1\u003c/em\u003e mutations are also associated with autosomal dominant retinitis pigmentosa-79 (RP79, MIM 617460), which exhibits variable phenotype with ages of onset ranging from childhood to 70 years [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Here, the affected 11-year-old boy with the \u003cem\u003eHK1\u003c/em\u003e mutation did not show retinitis pigmentosa symptom until his examined age.\u003c/p\u003e \u003cp\u003e \u003cem\u003eREEP1\u003c/em\u003e encodes a receptor accessory protein 1 that suggested to have a role in facilitating endoplasmic reticulum (ER)-mitochondrial interactions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is known that the \u003cem\u003eREEP1\u003c/em\u003e mutations exhibited considerable phenotypic heterogeneity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The dominant \u003cem\u003eREEP1\u003c/em\u003e mutations have been reported to cause dHMN5B (DSMA5B) and SPG21 with the onset ages falling in either the first or second decades [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recently, a recessive \u003cem\u003eREEP1\u003c/em\u003e mutation was reported in a Lebanese 5-year-old boy with a SMARD-similar phenotype [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The affected boy presented foot deformity and contractures of the distal phalanges at birth. This case was similar to our PaC6 case having a p.Gly83Alafs*44 mutation in view of premature termination, onset age, and some clinical symptoms. In the nerve conduction studies, all motor nerves were not evoked at all, but all sensory nerves showed normal SNAPs and SNCVs. From the clinical and NCV findings, this patient's symptoms are apparently similar with SMARD.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMFN2\u003c/em\u003e encodes mitofusin 2 which plays an important role maintaining equilibrium between mitochondrial fusion and fission [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Most \u003cem\u003eMFN2\u003c/em\u003e mutations have been reported to cause dominant CMT2A2A [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, some homozygous or compound heterozygous mutations cause recessive CMT2A2B (MIM 617087) with more severe and earlier onset phenotypes. Nicholson et al. suggested that CMT2A2B may semidominant and carriers with a single mutant allele may show weak phenotype with incomplete penetrance [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our case with the homozygous \u003cem\u003eMFN2\u003c/em\u003e mutations showed relatively early onset (5 years old) and severe phenotypes, which are matched with the characteristics of CMT2A2B. Additionally, the vocal cord paralysis seen in the affected boy has been occasionally reported in CMT2A patients with \u003cem\u003eMFN2\u003c/em\u003e mutations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The patient showed no symptom of optic atrophy which is a characteristic of CMT6A until his examined age (7 years old). His parents were apparently seemed to be unaffected; however, exact clinical and electrophysiological tests were not done.\u003c/p\u003e \u003cp\u003eAlthough a small number of CMT cases were investigated in this study, the incidence of the recessive patients with homozygous mutations was certainly frequent compared to other populations. Homozygosity mapping showed that both alleles of the homozygous mutations identified in each family originated from a single founder. Mutations in \u003cem\u003eMFN2\u003c/em\u003e are well known as the cause of dominant CMT2, however, recessive homozygous mutations have been rarely reported. This suggests an increased risk of consanguinity prone to develop rare recessive genetic diseases.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, we identified five pathogenic or likely pathogenic mutations in the consanguineous Pakistani families with early onset CMT. All the mutations were novel, and the genotype-phenotype correlations were confirmed. We believe that our findings will contribute to expanding understanding of the genetic basis of peripheral neuropathy, improving molecular diagnostics and treatment options.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eACMG: American College of Medical Genetics and Genomics; CMAP: Compound muscle action potential; CMT: Charcot-Marie-Tooth disease; dHMN: Distal hereditary motor neuropathy; dSMA5B: Distal spinal muscular atrophy type 5B; ER: Endoplasmic reticulum; FDS: Functional disability scale; GERP: Genomic evolutionary rate profiling score; HB: Homozygous block; HMSN: Hereditary motor and sensory neuropathy; HSAN: Hereditary sensory and autonomic neuropathy; MAF: Minor allele frequency; MNCV: Motor nerve conduction velocity: NCV: Nerve conduction velocity; OMM: Outer membrane of mitochondria; SMARD: Spinal muscular atrophy with respiratory distress; SNAP: Sensory nerve action potential; SNCV: Sensory nerve conduction velocity; TPR: tetratricopeptide repeat; WES: Whole exome sequencing\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003ecknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the patients and their families for their consent of participation and sample donation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Chung KW, Kanwal S; Sampling: Kanwal S, Nuzhat R, Khan A, Perveen S; Genetic investigation: Choi YJ, Lim SO, Choi HJ, Park JH, Son WS; Clinical data analysis: Choi BO; Funding acquisition: Chung KW, Choi BO; Writing original draft preparation: Son WS, Choi BO, Chung KW; Review and editing: all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Research Foundation (2019R1A2C1087547, 2020M3H4A1A03084600, and 2021R1A4A2001389) and the Korean Health Technology R\u0026amp;D Project, Ministry of Health and Welfare (HI14C3484 and HI20C0039), Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll pathogenic variants and data from this study are available upon reasonable request. Raw data on the exome sequencing are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Boards for Kongju National University (KNU_IRB_2018-06) and Sungkyunkwan University, Samsung Medical Center (2014-08-057-002). All participants were recruited from Care Hospital Sahiwal, Pakistan, and provided written informed consent. For the minors involved in the study, the consent was provided by their parents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSaporta MA, Shy ME. 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Clinical and radiological characterization of novel FIG4-related combined system disease with neuropathy.\u0026nbsp;Clin Genet. 2020;98:147-54.\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405-24.\u003c/li\u003e\n\u003cli\u003ePark HR, Kanwal S, Lim SO, Nam DE, Choi YJ, Chung KW. Homozygous mutations in Pakistani consanguineous families with prelingual nonsyndromic hearing loss. Mol Biol Rep. 2020;47:9979-85.\u003c/li\u003e\n\u003cli\u003eSenderek J, Bergmann C, Stendel C, Kirfel J, Verpoorten N, De Jonghe P, et al. Mutations in a gene encoding a novel SH3/TPR domain protein cause autosomal recessive Charcot-Marie-Tooth type 4C neuropathy. Am J Hum Genet. 2003;73:1106-19.\u003c/li\u003e\n\u003cli\u003eLupski JR, Reid JG, Gonzaga-Jauregui C, Rio Deiros D, Chen DCY, Nazareth L, et al. Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. New Engl J Med. 2010;362:1181-91.\u003c/li\u003e\n\u003cli\u003eAzzedine H, Ravis\u0026eacute; N, Verny C, Gabr\u0026euml;els-Festen A, Lammens M, Grid D, et al. Spine deformities in Charcot-Marie-Tooth 4C caused by SH3TC2 gene mutations. Neurology. 2006;67:602-6.\u003c/li\u003e\n\u003cli\u003eYger M, Stojkovic T, Tardieu S, Maisonobe T, Brice A, Echaniz-Laguna A, et al. Characteristics of clinical and electrophysiological pattern of Charcot-Marie-Tooth 4C. J Peripher Nerv Syst. 2012;17:112-22.\u003c/li\u003e\n\u003cli\u003eHantke J, Chandler D, King R, Wanders RJA, Angelicheva D, Tournev I, et al. A mutation in an alternative untranslated exon of hexokinase 1 associated with hereditary motor and sensory neuropathy-Russe (HMSNR). Eur J Hum Genet. 2009;17:1606-14.\u003c/li\u003e\n\u003cli\u003eSevilla T, Martinez-Rubio D, Marquez C, Paradas C, Colomer J, Jaijo T, et al. Genetics of the Charcot-Marie-Tooth disease in the Spanish Gypsy population: the hereditary motor and sensory neuropathy-Russe in depth. Clin Genet. 2013;83:565-70.\u003c/li\u003e\n\u003cli\u003eBeetz C, Pieber TR, Hertel N, Schabhuttl M, Fischer C, Trajanoski S, et al. Exome sequencing identifies a REEP1 mutation involved in distal hereditary motor neuropathy type V. Am J Hum Genet. 2012;91:139-45.\u003c/li\u003e\n\u003cli\u003eZ\u0026uuml;chner S, Wang G, Tran-Viet K-N, Nance MA, Gaskell PC, Vance JM, et al. Mutations in the novel mitochondrial protein REEP1 cause hereditary spastic paraplegia type 31. Am J Hum Genet. 2006;79:365-9.\u003c/li\u003e\n\u003cli\u003eSchottmann G, Seelow D, Seifert F, Morales-Gonzalez S, Gill E, von Au K, et al. Recessive REEP1 mutation is associated with congenital axonal neuropathy and diaphragmatic palsy. Neurol Genet. 2015;1:e32.\u003c/li\u003e\n\u003cli\u003eZ\u0026uuml;chner S, Mersiyanova IV, Muglia M, Bissar-Tadmouri N, Rochelle J, Dadali EL, et al. Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat Genet. 2004;36:449-51.\u003c/li\u003e\n\u003cli\u003eZ\u0026uuml;chner S, De Jonghe P, Jordanova A, Claeys KG, Guergueltcheva V, Cherninkova S, et al. Axonal neuropathy with optic atrophy is caused by mutations in mitofusin 2. Ann Neurol. 2006;59:276-81.\u003c/li\u003e\n\u003cli\u003eNicholson GA, Magdelaine C, Zhu D, Grew S, Ryan MM, Sturtz F, et al. Severe early-onset axonal neuropathy with homozygous and compound heterozygous MFN2 mutations. Neurology. 2008;70:1678-81.\u003c/li\u003e\n\u003cli\u003eEdvardson S, Cinnamon Y, Jalas C, Shaag A, Maayan C, Axelrod FB, Elpeleg O. Hereditary sensory autonomic neuropathy caused by a mutation in dystonin. Ann Neurol. 2012;71:569-72.\u003c/li\u003e\n\u003cli\u003eArnaud E, Zenker J, de Preux Charles A-S, Stendel C, Roos A, Medard J-J, et al. SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the peripheral nervous system. Proc. Natl Acad Sci USA. 2009;106:17528-33.\u003c/li\u003e\n\u003cli\u003eColomer J, Gooding R, Angelicheva D, King RHM, Guillen-Navarro E, Parman Y, et al. Clinical spectrum of CMT4C disease in patients homozygous for the p.Arg1109X mutation in SH3TC2. Neuromuscul Disord. 2006;16:449-53.\u003c/li\u003e\n\u003cli\u003eSullivan LS, Koboldt DC, Bowne SJ, Lang S, Blanton SH, Cadena E, et al. A dominant mutation in hexokinase 1 (HK1) causes retinitis pigmentosa. Invest Ophthal Vis Sci. 2014;55:7147-58.\u003c/li\u003e\n\u003cli\u003eLim Y, Cho I-T, Schoel LJ, Cho G, Golden JA. Hereditary spastic paraplegia-linked REEP1 modulates endoplasmic reticulum/mitochondria contacts. Ann Neurol. 2015;78:679-96.\u003c/li\u003e\n\u003cli\u003eMaroofian R, Behnam M, Kaiyrzhanov R, Salpietro V, Salehi M, Houlden H. Further supporting evidence for REEP1 phenotypic and allelic heterogeneity. Neurol Genet. 2019;5:e379.\u003c/li\u003e\n\u003cli\u003eSantel A, Fuller MT. Control of mitochondrial morphology by a human mitofusin. J Cell Sci. 2001;114:867-74.\u003c/li\u003e\n\u003cli\u003eBombelli F, Stojkovic T, Dubourg O, Echaniz-Laguna A, Tardieu S, Larcher K, et al. Charcot-Marie-Tooth disease type 2A: from typical to rare phenotypic and genotypic features. JAMA Neurol. 2014;71:1036-42.\u003c/li\u003e\n\u003cli\u003eAndo M, Hashiguchi A, Okamoto Y, Yoshimura A, Hiramatsu Y, Yuan J, et al. Clinical and genetic diversities of Charcot-Marie-Tooth disease with MFN2 mutations in a large case study. J Peripher Nerv Syst. 2017;22:191-9.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Clinical characterization of the five Pakistani CMT patients\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eItem ＼ Patient (sex)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003ePaC2:IV-1 (male)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003ePaC3:IV-1 (male)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003ePaC4:IV-1 (male)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003ePaC6:IV-2 (female)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003ePaC14:IV-2 (male)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eGene: mutation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e\u003cem\u003eSH3TC2\u003c/em\u003e: p.Q867*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e\u003cem\u003eSH3TC2\u003c/em\u003e: p.G1217D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e\u003cem\u003eHK1\u003c/em\u003e: p.R7*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e\u003cem\u003eREEP1\u003c/em\u003e: p.G83Afs*44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e\u003cem\u003eMFN2\u003c/em\u003e: p.V112M\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eType\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eCMT4C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eCMT4C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eCMT4G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003edHMN5B/SMARD1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eCMT2A2B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eAges at exam/onset (yr)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e6/3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e10/3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e11/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e2.5/\u0026lt; 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e7/5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eMuscle atrophy\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eFDS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eSensory loss\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eDTR, ankle\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eDecreased\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eFoot deformities\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eBrain/Spine MRI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eNormal brain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eNormal spine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eNormal brain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eOther symptoms\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eScoliosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eScoliosis, short stature\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eMild respiratory distress\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eVocal cord hoarseness\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" width=\"928\"\u003e\n\u003cp\u003eMotor nerve conduction studies\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eMedian CMAP (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e1.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eMedian MNCV (m/s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e14.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eUlnar CMAP (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e0.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e4.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eUlnar MNCV (m/s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e12.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e25.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003ePeroneal CMAP (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e0.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003ePeroneal MNCV (m/s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003e14.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" width=\"928\"\u003e\n\u003cp\u003eSensory nerve conduction studies\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eMedian SNAP (\u0026mu;V)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e10.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e22.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eMedian SNCV (m/s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e26.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e51.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eUlnar SNAP (\u0026mu;V)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e22.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eUlnar SNCV (m/s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e39.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eSural SNAP (\u0026mu;V)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e8.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e16.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"172\"\u003e\n\u003cp\u003eSural SNCV (m/s)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"144\"\u003e\n\u003cp\u003eAbsent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"162\"\u003e\n\u003cp\u003e32.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"143\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"153\"\u003e\n\u003cp\u003e39.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"154\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCMAP: compound muscle action potential, DTR: deep tendon reflexes, FDS: functional disability scale, MNCV: motor nerve conduction velocity, ND: not done, SNAP: sensory nerve action potential, SNCV: sensory nerve conduction velocity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Homozygous mutations and clinical phenotypes in the Pakistani CMT patients\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 38.1505px;\"\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eFamily ID\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"67\"\u003e\n\u003cp\u003eGene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"146\"\u003e\n\u003cp\u003eMutation\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"125\"\u003e\n\u003cp\u003eType\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"67\"\u003e\n\u003cp\u003eOnset age\u003c/p\u003e\n\u003cp\u003e(yr)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"135\"\u003e\n\u003cp\u003eOther symptom\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38.1505px;\" colspan=\"2\" width=\"106\"\u003e\n\u003cp\u003eAllele frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"56\"\u003e\n\u003cp\u003eGERP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38.1505px;\" colspan=\"3\" width=\"125\"\u003e\n\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e prediction\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.1505px;\" rowspan=\"2\" width=\"46\"\u003e\n\u003cp\u003eNote\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003e1000G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"58\"\u003e\n\u003cp\u003eExAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003ePP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003eMU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003ePRO\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"57\"\u003e\n\u003cp\u003ePaC2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"67\"\u003e\n\u003cp\u003e\u003cem\u003eSH3TC2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"146\"\u003e\n\u003cp\u003ec.2599C\u0026gt;T;p.Q867*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"125\"\u003e\n\u003cp\u003eCMT4C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"67\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"135\"\u003e\n\u003cp\u003eScoliosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"58\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"56\"\u003e\n\u003cp\u003e2.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"46\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 38px;\"\u003e\n\u003ctd style=\"height: 38px;\" width=\"57\"\u003e\n\u003cp\u003ePaC3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"67\"\u003e\n\u003cp\u003e\u003cem\u003eSH3TC2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"146\"\u003e\n\u003cp\u003ec.3650G\u0026gt;A;p.G1217D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"125\"\u003e\n\u003cp\u003eCMT4C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"67\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"135\"\u003e\n\u003cp\u003eScoliosis, short stature\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"48\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"58\"\u003e\n\u003cp\u003e1.6E-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"56\"\u003e\n\u003cp\u003e6.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"38\"\u003e\n\u003cp\u003e1.00\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"38\"\u003e\n\u003cp\u003e0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"48\"\u003e\n\u003cp\u003e-5.79\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"46\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"57\"\u003e\n\u003cp\u003ePaC4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"67\"\u003e\n\u003cp\u003e\u003cem\u003eHK1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"146\"\u003e\n\u003cp\u003ec.19C\u0026gt;T;p.R7*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"125\"\u003e\n\u003cp\u003eCMT4G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"67\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"135\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"58\"\u003e\n\u003cp\u003e1.7E-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"56\"\u003e\n\u003cp\u003e1.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"46\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"57\"\u003e\n\u003cp\u003ePaC6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"67\"\u003e\n\u003cp\u003e\u003cem\u003eREEP1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"146\"\u003e\n\u003cp\u003ec.247delG;p.G83Afs*44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"125\"\u003e\n\u003cp\u003eSMARD/dHMN5B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"67\"\u003e\n\u003cp\u003e\u0026lt; 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"135\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"58\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"56\"\u003e\n\u003cp\u003e5.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"38\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"48\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"46\"\u003e\n\u003cp\u003eLP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 38px;\"\u003e\n\u003ctd style=\"height: 38px;\" width=\"57\"\u003e\n\u003cp\u003ePaC14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"67\"\u003e\n\u003cp\u003e\u003cem\u003eMFN2\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"146\"\u003e\n\u003cp\u003ec.334G\u0026gt;A;p.V112M\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"125\"\u003e\n\u003cp\u003eCMT2A2B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"67\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"135\"\u003e\n\u003cp\u003eVocal cord hoarseness\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"48\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"58\"\u003e\n\u003cp\u003e1.6E-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"56\"\u003e\n\u003cp\u003e4.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"38\"\u003e\n\u003cp\u003e1.00\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"38\"\u003e\n\u003cp\u003e0.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"48\"\u003e\n\u003cp\u003e-2.76\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" width=\"46\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e1000G: 1000 Genomes Project, CMT: Charcot-Marie-Tooth disease, ExAC: Exome Aggregation Consortium, GERP: genomic evolutionary rate profiling score, P: pathogenic, LP: likely pathogenic, UR: unreported.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eReference DNA and protein sequences:\u003cem\u003e SH3TC2\u003c/em\u003e: NM_024577.4 and NP_078853.2, \u003cem\u003eHK1\u003c/em\u003e: NM_033498.3 and NP_277033.1, \u003cem\u003eREEP1\u003c/em\u003e: NM_022912.3 and NP_075063.1, \u003cem\u003eMFN2\u003c/em\u003e: NM_014874.3 and NP_055689.1\u003cem\u003e. \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eScores of PolyPhen-2 (PP2) ~1, MUpro (MU) \u0026lt;0, and PROVEAN (PRO) \u0026lt;-2.5 indicate pathogenic prediction (\u003csup\u003e*\u003c/sup\u003e denotes a pathogenic prediction).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Rare variants observed in the CMT-related genes from patients of five Pakistani CMT families\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"50\"\u003e\n\u003cp\u003eFamily\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"76\"\u003e\n\u003cp\u003eGene\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"246\"\u003e\n\u003cp\u003eVariant\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"57\"\u003e\n\u003cp\u003eClinVar\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"85\"\u003e\n\u003cp\u003edbSNP151\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"113\"\u003e\n\u003cp\u003eAllele frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"47\"\u003e\n\u003cp\u003eGERP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"104\"\u003e\n\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003eNote\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003eNucleotide\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003eAmino acid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e1000G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eExAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003ePP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003ePRO\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"5\" width=\"50\"\u003e\n\u003cp\u003ePaC2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eKIF1B\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.3209C\u0026gt;T\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.A1070V\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers768176241\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e1.7E-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eDST\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e[c.7252G\u0026gt;A + c.7765A\u0026gt;G]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e[p.V2418I + p.I2589V]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers62621210\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0400\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0356\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e, nonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eB,LB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers150191284\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0102\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0239\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eMYH14\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.3748G\u0026gt;T\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.V1250L\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eLB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers202242879\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0006\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0009\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e3.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-0.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eSCN11A\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.1732T\u0026gt;A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.F578I\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers772393665\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e5.58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.98*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-4.61*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"6\" width=\"50\"\u003e\n\u003cp\u003ePaC3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eKIF1B\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e[c.2107T\u0026gt;C] + [c.2455A\u0026gt;C]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e[p.W703R] + [p.S819R]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eB,LB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers551543997\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0054\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0033\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e5.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.99*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-10.0*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eTrans\u003c/em\u003e, nonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eLB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers140015591\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e-2.96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-2.19\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eNTRK1\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e[c.2339G\u0026gt;A + c.2360C\u0026gt;T]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"123\"\u003e\n\u003cp\u003e[p.R780Q + p.A787V]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eB,LB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers35669708\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0038\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0064\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.87*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-1.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e, nonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers761207548\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e7.E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-3.00*\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eNAGLU\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.2209C\u0026gt;A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.R737S\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers86312\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0116\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0192\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.47*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eSCN10A\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.3887G\u0026gt;T\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.S1296I\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eLB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers779527264\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.88\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e1.00*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-5.79*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"50\"\u003e\n\u003cp\u003ePaC4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eDST\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e[c.1933A\u0026gt;G] + [c.1933A\u0026gt;G]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e[p.I645V] + [p.I645V]\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers754692637\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e8.E-06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e2.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-0.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eHomozygous, cosegregation, VUS\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eARHGEF10\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.2566G\u0026gt;A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.V856I\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers773521162\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e0.0003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e4.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e0.87*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-0.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"50\"\u003e\n\u003cp\u003ePaC6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eTFG\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.175A\u0026gt;G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.K59E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers1232918261\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e5.90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e1.00*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-3.02*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"50\"\u003e\n\u003cp\u003ePaC14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e\u003cem\u003eSETX\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ec.2385_2387delTTT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003ep.I795_K796delinsM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003ers755971927\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003eUR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e6.E-05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"47\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"57\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"132\"\u003e\n\u003cp\u003eNonsegregation, LB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e1000G: 1000 Genomes Project, ExAC: Exome Aggregation Consortium, B: benign, LB: likely benign, VUS: variant of uncertain significance,\u003cem\u003e trans\u003c/em\u003e: \u003cem\u003etrans\u003c/em\u003e arrangement of variants in homologous chromosomes (bi-alleles), \u003cem\u003ecis\u003c/em\u003e: \u003cem\u003ecis\u003c/em\u003e arrangement of variants in a chromosome, UR: unreported.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Charcot-Marie-Tooth disease (CMT), Consanguinity, Homozygosity, Pakistan, Whole exome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-279595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-279595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCharcot-Marie-Tooth disease (CMT) is a group of genetically and clinically heterogeneous peripheral nervous disorders. Few studies have identified genetic causes in the Pakistani CMT patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study was performed to identify pathogenic mutations in five consanguineous Pakistani CMT families negative for \u003cem\u003ePMP22\u003c/em\u003e duplication. Genomic screening was performed by application of whole exome sequencing\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe identified five pathogenic or likely pathogenic homozygous mutations in four genes: c.2599C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Gln867*) and c.3650G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Gly1217Asp) in \u003cem\u003eSH3TC2\u003c/em\u003e, c.19C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Arg7*) in \u003cem\u003eHK1\u003c/em\u003e, c.247delG (p.Gly83Alafs*44) in \u003cem\u003eREEP1\u003c/em\u003e, and c.334G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Val112Met) in \u003cem\u003eMFN2\u003c/em\u003e. All the mutations were not reported in the CMT patients. Mutations in the \u003cem\u003eSH3TC2\u003c/em\u003e, \u003cem\u003eHK1\u003c/em\u003e, \u003cem\u003eREEP1\u003c/em\u003e, and \u003cem\u003eMFN2\u003c/em\u003e have been reported to be implicated to CMT4C, CMT4G, dHMN5B (DSMA5B), and CMT2A, respectively. The genotype-phenotype correlations were confirmed in all the examined families. We also confirmed that both alleles from the homozygous variants were originated from a single founder using homozygosity mapping.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study found five novel mutations as the underlying causes of CMT. Pathogenic mutations in \u003cem\u003eSH3TC2, HK1\u003c/em\u003e, and \u003cem\u003eREEP1\u003c/em\u003e have been reported rarely in other populations, suggesting ethnic-specific distribution. This study will be useful for the exact molecular diagnosis and treatment in the Pakistani CMT patients.\u003c/p\u003e","manuscriptTitle":"Novel homozygous mutations in Pakistani families with recessive Charcot-Marie-Tooth disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-04 21:41:47","doi":"10.21203/rs.3.rs-279595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-04-07T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-31T00:00:00+00:00","index":4,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nThis article describes the discovery of novel homozygous mutations in a Pakistani population that result in recessive Charcot-Marie-Tooth neuropathy. The candidate mutations described have strong evidence for their pathogenicity and contribute to our body of knowledge of inherited neuropathies. Overall, my suggested revisions are minor, and I would recommend this manuscript for publication.\n\nMy suggested revisions are noted below:\n\n1. Page 2, Line 10-\n\"All the mutations were not reported in the CMT patients.\" \nThis may be better phrased as being 'previously unreported in CMT patients'. 'The CMT patients' sounds like the patients you are referring to in your manuscript.\n\n2. Page 3, Line 11:\n\"CMT is often viewed as a monogenic Mendelian disease; however, mutations in more than 130 genes are associated with the development of peripheral neuropathies in an autosomal or X-linked dominant or recessive manner [4].\"\nMonogenic refers to a single gene mutation causing the disease in that individual. CMT is monogenic, although many genes have been implicated. The 'however' in this sentence is unnecessary.\n\n3. Page 5, Line 21:\n\"Rare alleles with minor allele frequencies (MAFs) of \u003c 0.1 were obtained from the 1000 Genomes Project (1000G, 23 http://www.1000genomes.org/)\"\nTypically a MAF of \u003c0.01 is used in WES filtering, which indicates variants with a frequency of 1%. Is there any justification or reference for using a higher MAF cut-off?\n\n4. Page 5 and 6:\nIn silico programs and databases typically have manuscripts associated with them that they request are cited when you use their software. The methods section should be edited to include these citations.\n\n5. Page 10, Homozygosity mapping\nThe flanking SNPs and their more specific chromosomal base pair positions in the genome should be noted.\n\n6. Page 11, Line 24:\n\"The patient showed no symptom of optic atrophy which is a characteristic of CMT6A until his examined age (7 years old)\"\nShould this be CMT2A?\n\n7. Page 11, Line 26:\n\"Although a small number of CMT cases were investigated in this study, the incidence of the recessive patients with homozygous mutations was certainly frequent compared to other populations.\"\nThis section needs a citation and some statistics to back-up this claim that you are seeing a higher frequency. Compared to which populations? What frequencies?\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons after the final decision on the manuscript has been made. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **No**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorInvitedReview","content":"","date":"2021-03-31T00:00:00+00:00","index":2,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nKanwel et al report novel mutations for CMT related genes in families of Pakistan ethnicity.\nOverall, this is an important scholarly piece of work that adds information regarding novel mutations causing recessive CMT in consanguineous families from the Pakistan population.\nThe families that are reported, do they come from a larger pool of CMT patients in Pakistan. What is the likely prevalence of CMT in Pakistan?\nThe reviewer has uploaded suggestions and comments overlayed on the original manuscript submitted.\n\nThe reviewer requests that all comments in the manuscript be addressed with a particular focus on the following points:\n1) Provide an explanation and rationale for the conclusions made regarding the homozygosity mapping. It is unclear what the authors are trying to achieve and how the conclusions of a common founder can be drawn from the data presented.\n2) The discussion needs improvement based on the questions raised by the reviewer.\n3) The manuscript would be improved by being reviewed for English and grammar.\n\n\n\n\n\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons after the final decision on the manuscript has been made. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **No**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **No**\n* Are the methods sufficiently described to allow the study to be repeated?: **No**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **No**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorInvitedReview","content":"","date":"2021-03-30T00:00:00+00:00","index":3,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nI suggest some minor modifications in the text:\nPage 3:\nLine 3: eliminate \"rare\"; Line 4: Begin the sentence with: In CMT; Line 4: Write the sentence as follows: \"...(HMSN), both sensory and..\"; Line 5: Write as follows: \"motor nerves are impaired, whereas...\"\nLines 7-8: specify the meaning of \"anti-reflection symptom...\"; Line 9: Write as follows: \"type (CMT1), with reduced..\"; Line 9: Write as follows: \"...38 m/s and axonal type; Line 10: Begin the line with: (CMT2)\nPage 5\nLine 3: replace \"scoring\" by method; Line 4: Write as follows: \"Electromyography was tested with a concentric needle electrode.\"\nPage 6\nLine 24: Write as follows: \"...sense was reduced.\" Line 2: replace \"foot\" by feet, and make the same modification whenever \"foot\" appears in theb text. Line 27: Replace \"amplitudes\" by responses.\nPage 7\nLine 1: Write as follows: \"These findings were compatible\"; Line 3: Write: , it was noticed...\"; Loine 4: Write \"...he showed scoliosis...\"; In this page in different line, the written word foot must be replaced by feet. Line 18: Write \"years.\" Line 20: Write as follows: \"...not evoked, but normal...\"* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons after the final decision on the manuscript has been made. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interess´below**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2021-03-21T00:00:00+00:00","index":4,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-03-19T01:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-03-19T00:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-19T00:00:00+00:00","index":1,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nThe manuscript by Kanwal et al. reports on new homozygous mutations underlying Pakistani cases of CMT. The manuscript is well structured and the methods are appropriate to address the research question.\n\nMajor point:\n(i) the authors conclude the study with the word 'recessive' in the title, however as they mention themselves in the discussion of the manuscript are mutations in MFN2 considered autosomal dominant with very few exceptions. As previously suggested by Nicholson et al. 2008, the homozygous cases may rather represent weakly impacting mutations in the heterozygous state that cause severe phenotypes in the homozygous state. Given that the authors were not able to clinically examine the parents of this child, the conclusion that these are recessive cases rather than mild dominant cases with a cumulative effect in the homozygous state is not sufficiently supported. Unless the researchers are able to still examine the parents and rule out any form of mild symptoms, I think the word 'recessive' would be better removed from the title.\n\nMinor points:\n(i) In the results section, the authors mention the presence of siblings for the respective cases. However, the manuscript would benefit from specification of their age relative to the affected subject. In other words, instead of writing '...and a brother' it would be informative to specify '...younger/older brother'.\n(ii) Related to this, also the age of examination of the parents would be useful to have it mentioned in the results section. In particular for cases with the possibility of weak phenotypes in the heterozygous state, this would help readers to assess the possibility of minor phenotypes to still arise later in life.\n(iii) The predicted impact of the mutations was assessed by PolyPhen-2 and PROVEAN. It would be interesting to also add the outcome of SIFT-predictions.\n(iv) Typo on page 6 (line 25): remove comma after scoliosis\n(v) Page 6 (line 23): Please use a more active tense \u003e 'since he was 3 years old' instead of 'since early onset of 3 years old'\n(vi) Page 7 (line 3): Please use a more active tense \u003e 'he was noticed to fall frequently' instead of 'he was noticed frequent fall'\n(vii) Page 7 (line 4): Please remove 'the' and correct to 'he showed scoliosis'\n(viii) Page 7 (line 23): Please correct 'as the additional symptom' to 'as an additional symptom'\n(iv) Discussion: the manuscript would benefit from a brief discussion of the REEP1 KO mouse models (published by the Hübner and Blackstone labs), as they are relevant in the context of loss-of-fucntion mutations as described here.* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons after the final decision on the manuscript has been made. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2021-03-07T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-23T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-23T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-22T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-22T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2021-02-15T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1dcb5fd-65ba-498d-bfbe-e315e841aa65","owner":[],"postedDate":"March 4th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2711336,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2021-03-04T21:41:47+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-04 21:41:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-279595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-279595","identity":"rs-279595","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}