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Assessment of Targeted Next-Generation Sequencing as a Tool for the Diagnosis of Charcot-Marie-Tooth Disease and Hereditary Motor Neuropathy

2016; Elsevier BV; Volume: 18; Issue: 2 Linguagem: Inglês

10.1016/j.jmoldx.2015.10.005

ISSN

1943-7811

Autores

Vincenzo Lupo, Francisco García‐García, Paula Sancho, Cristina Tello, Mar García-Romero, Liliana Villarreal, Antonia Albertí, Rafael Sivera, Joaquı́n Dopazo, Samuel Ignacio Pascual Pascual, C. Márquez Infante, Carlos Casasnovas, Teresa Sevilla, Carmen Espinós,

Tópico(s)

Hippo pathway signaling and YAP/TAZ

Resumo

Charcot-Marie-Tooth disease is characterized by broad genetic heterogeneity with >50 known disease-associated genes. Mutations in some of these genes can cause a pure motor form of hereditary motor neuropathy, the genetics of which are poorly characterized. We designed a panel comprising 56 genes associated with Charcot-Marie-Tooth disease/hereditary motor neuropathy. We validated this diagnostic tool by first testing 11 patients with pathological mutations. A cohort of 33 affected subjects was selected for this study. The DNAJB2 c.352+1G>A mutation was detected in two cases; novel changes and/or variants with low frequency ( A mutation was also detected in three additional families. On haplotype analysis, all of the patients from these five families shared the same haplotype; therefore, the DNAJB2 c.352+1G>A mutation may be a founder event. Our gene panel allowed us to perform a very rapid and cost-effective screening of genes involved in Charcot-Marie-Tooth disease/hereditary motor neuropathy. Our diagnostic strategy was robust in terms of both coverage and read depth for all of the genes and patient samples. These findings demonstrate the difficulty in achieving a definitive molecular diagnosis because of the complexity of interpreting new variants and the genetic heterogeneity that is associated with these neuropathies. Charcot-Marie-Tooth disease is characterized by broad genetic heterogeneity with >50 known disease-associated genes. Mutations in some of these genes can cause a pure motor form of hereditary motor neuropathy, the genetics of which are poorly characterized. We designed a panel comprising 56 genes associated with Charcot-Marie-Tooth disease/hereditary motor neuropathy. We validated this diagnostic tool by first testing 11 patients with pathological mutations. A cohort of 33 affected subjects was selected for this study. The DNAJB2 c.352+1G>A mutation was detected in two cases; novel changes and/or variants with low frequency ( A mutation was also detected in three additional families. On haplotype analysis, all of the patients from these five families shared the same haplotype; therefore, the DNAJB2 c.352+1G>A mutation may be a founder event. Our gene panel allowed us to perform a very rapid and cost-effective screening of genes involved in Charcot-Marie-Tooth disease/hereditary motor neuropathy. Our diagnostic strategy was robust in terms of both coverage and read depth for all of the genes and patient samples. These findings demonstrate the difficulty in achieving a definitive molecular diagnosis because of the complexity of interpreting new variants and the genetic heterogeneity that is associated with these neuropathies. Charcot-Marie-Tooth (CMT) disease is the most frequently inherited neurological disorder and has a prevalence of 1 in 2500 population.1Skre H. Genetic and clinical aspects of Charcot–Marie–Tooth's disease.Clinical Genetic. 1974; 6: 98-118Crossref PubMed Scopus (712) Google Scholar CMT displays broad genetic heterogeneity with a common clinical phenotype. Because both motor and sensory nerves are affected, CMT is also categorized as a hereditary motor and sensory neuropathy. When only motor nerves are affected, it is called a hereditary motor neuropathy (HMN), which corresponds to the pure motor forms. CMT can be subclassified into three types. The first is demyelinating CMT (CMT1), in which median motor nerve conduction velocities (MMNCVs) are slowed ( 38 meters per second) and which largely results in axonal loss. The third is intermediate CMT, for which the MMNCVs range from 25 to 45 meters per second and the nerve pathology shows signs of demyelinating and/or axonal features.2Dyck P. Lambert E.H. Lower motor and primary sensory neuron diseases with peroneal muscular atrophy. I. Neurologic, genetic, and electrophysiologic findings in hereditary polyneuropathies.Arch Neurol. 1968; 18: 603-618Crossref PubMed Scopus (527) Google Scholar, 3Pareyson D. Marchesi C. Diagnosis, natural history, and management of Charcot-Marie-Tooth disease.Lancet Neurol. 2009; 8: 654-667Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 4Szigeti K. Lupski J.R. Charcot-Marie-Tooth disease.Eur J Hum Genet. 2009; 17: 703-710Crossref PubMed Scopus (136) Google Scholar The list of genes involved in CMT is ever-growing and currently comprises >50 genes (Neuromuscular Disease Center, http://neuromuscular.wustl.edu/time/hmsn.html, last accessed October 5, 2015). There is a clear overlap between HMN and CMT, and the same mutation in a gene can cause both phenotypes. Nearly 22 known genes are associated with HMN, and mutations in at least eight of them are related to CMT.5Rossor A.M. Kalmar B. Greensmith L. Reilly M.M. The distal hereditary motor neuropathies.J Neurol Neurosurg Psychiatry. 2012; 83: 6-14Crossref PubMed Scopus (165) Google Scholar All of the Mendelian patterns of inheritance are observed in CMT/HMN diseases. Sporadic cases may occur as the consequence of a de novo mutation and, therefore, do not exhibit a family history of neuropathy.5Rossor A.M. Kalmar B. Greensmith L. Reilly M.M. The distal hereditary motor neuropathies.J Neurol Neurosurg Psychiatry. 2012; 83: 6-14Crossref PubMed Scopus (165) Google Scholar, 6Saporta A.S. Sottile S.L. Miller L.J. Feely S.M. Siskind C.E. Shy M.E. Charcot-Marie-Tooth disease subtypes and genetic testing strategies.Ann Neurol. 2011; 69: 22-33Crossref PubMed Scopus (388) Google Scholar, 7Murphy S.M. Laura M. Fawcett K. Pandraud A. Liu Y.T. Davidson G.L. Rossor A.M. Polke J.M. Castleman V. Manji H. Lunn M.P. Bull K. Ramdharry G. Davis M. Blake J.C. Houlden H. Reilly M.M. Charcot-Marie-Tooth disease: frequency of genetic subtypes and guidelines for genetic testing.J Neurol Neurosurg Psychiatry. 2012; 83: 706-710Crossref PubMed Scopus (260) Google Scholar Molecular diagnosis is a relevant and integral part of clinical diagnosis. The successful diagnosis of hereditary neuropathies and other Mendelian diseases has greatly improved over the past 5 years. These advances are mainly due to next-generation sequencing, which has resulted in the discovery of hundreds of genes involved in human diseases. Approximately 80% of CMT1 patients can now receive an accurate molecular diagnosis. There is a high percentage of CMT2 (between 25% and 43%) in unresolved clinical cases.6Saporta A.S. Sottile S.L. Miller L.J. Feely S.M. Siskind C.E. Shy M.E. Charcot-Marie-Tooth disease subtypes and genetic testing strategies.Ann Neurol. 2011; 69: 22-33Crossref PubMed Scopus (388) Google Scholar, 7Murphy S.M. Laura M. Fawcett K. Pandraud A. Liu Y.T. Davidson G.L. Rossor A.M. Polke J.M. Castleman V. Manji H. Lunn M.P. Bull K. Ramdharry G. Davis M. Blake J.C. Houlden H. Reilly M.M. Charcot-Marie-Tooth disease: frequency of genetic subtypes and guidelines for genetic testing.J Neurol Neurosurg Psychiatry. 2012; 83: 706-710Crossref PubMed Scopus (260) Google Scholar, 8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google Scholar, 9Fridman V. Bundy B. Reilly M.M. Pareyson D. Bacon C. Burns J. Day J. Feely S. Finkel R.S. Grider T. Kirk C.A. Herrmann D.N. Laurá M. Li J. Lloyd T. Sumner C.J. Muntoni F. Piscosquito G. Ramchandren S. Shy R. Siskind C.E. Yum S.W. Moroni I. Pagliano E. Züchner S. Scherer S.S. Shy M.E. on behalf of the Inherited Neuropathies ConsortiumCMT subtypes and disease burden in patients enrolled in the Inherited Neuropathies Consortium natural history study: a cross-sectional analysis.J Neurol Neurosurg Psychiatry. 2015; 86: 873-878Crossref PubMed Scopus (198) Google Scholar Additionally, 80% of HMN patients remain molecularly undiagnosed.5Rossor A.M. Kalmar B. Greensmith L. Reilly M.M. The distal hereditary motor neuropathies.J Neurol Neurosurg Psychiatry. 2012; 83: 6-14Crossref PubMed Scopus (165) Google Scholar Determining which gene needs to be tested in each patient is difficult, and usually only the most common genes are analyzed. The turnaround time and the cost of the tests are also important factors. We have designed a panel based on targeted next-generation sequencing for the molecular diagnosis of CMT and HMN. The panel contains 56 genes involved in CMT/HMN and provides a cost-efficient alternative to conventional Sanger-based methods. Forty-four unrelated patients with a diagnosis of CMT or HMN were selected. These patients were evaluated by neurologists at the Spanish Consortium on CMT [TREAT-CMT, http://www.treat-cmt.es/db (login required), last accessed July 5, 2015].8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google Scholar Based on their clinical history and electrophysiological and histopathological criteria, patients were subclassified into one of four groups: HMN, CMT1, CMT2, or intermediate CMT. Whenever possible, relatives of the patients were studied for segregation analysis. All of the patients and relatives included in this study gave informed consent, and the research protocols were approved by the Institutional Review Boards or the ethics committees of the Hospital Universitario La Paz (Madrid, Spain), the Hospital Universitario Virgen del Rocío (Seville, Spain), the Hospital de Bellvitge (Barcelona, Spain), and the Hospital Universitari i Politècnic La Fe (Valencia, Spain). The 44 patients were divided into two groups. The first included 11 patients with known disease-causing mutations and was used as a control group to verify the reliability of our custom panel diagnostic strategy. It also included 33 patients without a genetic diagnosis. The control group included 11 carriers of 14 different types of mutations (indels, duplications, missense, frameshifts, and regulatory variants) located in several genes involved in CMT1 or CMT2 (Table 1).8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google Scholar, 10Genari A.B. Borghetti V.H. Gouvea S.P. Bueno K.C. dos Santos P.L. dos Santos A.C. Barreira A.A. Lourenço C.M. Marques Jr., W. Characterizing the phenotypic manifestations of MFN2 R104W mutation in Charcot-Marie-Tooth type 2.Neuromuscul Disord. 2011; 21: 428-432Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 11Sevilla T. Jaijo T. Nauffal D. Collado D. Chumillas M.J. Vílchez J.J. Muelas N. Bataller L. Domenech R. Espinós C. Palau F. Vocal cord paresis and diaphragmatic dysfunction are severe and frequent symptoms of GDAP1-associated neuropathy.Brain. 2008; 131: 3051-3061Crossref PubMed Scopus (82) Google Scholar, 12Kabzinska D. Kochanski A. Drac H. Ryniewicz B. Rowinska-Marcinska K. Hausmanowa-Petrusewicz I. Autosomal recessive axonal form of Charcot-Marie-Tooth Disease caused by compound heterozygous 3'-splice site and Ser130Cys mutation in the GDAP1 gene.Neuropediatrics. 2005; 36: 206-209Crossref PubMed Scopus (11) Google Scholar, 13Claramunt R. Sevilla T. Lupo V. Cuesta A. Millán J.M. Vílchez J.J. Palau F. Espinós C. The p.R1109X mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4.Clin Genet. 2007; 71: 343-349Crossref PubMed Scopus (37) Google Scholar, 14Sevilla T. Martínez-Rubio D. Márquez C. Paradas C. Colomer J. Jaijo T. Millán J.M. Palau F. Espinós C. 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: 565-570Crossref PubMed Scopus (25) Google Scholar, 15Houlden H. Laura M. Wavrant-De Vrieze F. Blake J. Wood N. Reilly M.M. Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2.Neurology. 2008; 71: 1660-1668Crossref PubMed Scopus (159) Google Scholar These mutations were identified by Sanger sequencing of the codified regions of the respective genes.Table 1Control Group: Clinical Form and Genetic CharacteristicsID no.Clinical formInheritanceCarrier statusGeneNucleotide changeAmino acid changeReferenceSGT-036CMT2ADHeterozygosisMFN2c.310C>Tp.R104W10Genari A.B. Borghetti V.H. Gouvea S.P. Bueno K.C. dos Santos P.L. dos Santos A.C. Barreira A.A. Lourenço C.M. Marques Jr., W. Characterizing the phenotypic manifestations of MFN2 R104W mutation in Charcot-Marie-Tooth type 2.Neuromuscul Disord. 2011; 21: 428-432Abstract Full Text Full Text PDF PubMed Scopus (17) Google ScholarDNA_121CMT1ADHeterozygosisMPZc.21_26dupTGCCCCp.P9_A10dup8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google ScholarDNA_837CMT2X-linkedHemizygosisGJB1c.44_45delinsTTp.R15L8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google ScholarDNA_872CMT2X-linkedHemizygosisGJB1c.-540G>CNo aa change8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google ScholarDNA_554CMT1ARCompound heterozygosisPRXc.642insCp.R215QfsX88Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google Scholarc.589G>Tp.E197X8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google ScholarDNA_571CMT1ARHomozygosisFGD4c.1886delGAAAp.K630NfsX58Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google ScholarDNA_223CMT1ADHeterozygosisGARSc.1171C>Tp.R391C8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google ScholarDNA_708CMT2ARCompound heterozygosisGDAP1c.172_173delCTinsTTAp.P59AfsX411Sevilla T. Jaijo T. Nauffal D. Collado D. Chumillas M.J. Vílchez J.J. Muelas N. Bataller L. Domenech R. Espinós C. Palau F. Vocal cord paresis and diaphragmatic dysfunction are severe and frequent symptoms of GDAP1-associated neuropathy.Brain. 2008; 131: 3051-3061Crossref PubMed Scopus (82) Google Scholarc.311-1G>ANo aa change12Kabzinska D. Kochanski A. Drac H. Ryniewicz B. Rowinska-Marcinska K. Hausmanowa-Petrusewicz I. Autosomal recessive axonal form of Charcot-Marie-Tooth Disease caused by compound heterozygous 3'-splice site and Ser130Cys mutation in the GDAP1 gene.Neuropediatrics. 2005; 36: 206-209Crossref PubMed Scopus (11) Google ScholarSGT-047CMT1ARHomozygosisHK1g.9712G>CNo aa change13Claramunt R. Sevilla T. Lupo V. Cuesta A. Millán J.M. Vílchez J.J. Palau F. Espinós C. The p.R1109X mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4.Clin Genet. 2007; 71: 343-349Crossref PubMed Scopus (37) Google Scholar, 14Sevilla T. Martínez-Rubio D. Márquez C. Paradas C. Colomer J. Jaijo T. Millán J.M. Palau F. Espinós C. 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: 565-570Crossref PubMed Scopus (25) Google ScholarHeterozygosisSH3TC2c.3325C>Tp.R1109X13Claramunt R. Sevilla T. Lupo V. Cuesta A. Millán J.M. Vílchez J.J. Palau F. Espinós C. The p.R1109X mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4.Clin Genet. 2007; 71: 343-349Crossref PubMed Scopus (37) Google Scholar, 14Sevilla T. Martínez-Rubio D. Márquez C. Paradas C. Colomer J. Jaijo T. Millán J.M. Palau F. Espinós C. 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: 565-570Crossref PubMed Scopus (25) Google ScholarSGT-044CMT1ARCompound heterozygosisSH3TC2c.3325C>Tp.R1109X13Claramunt R. Sevilla T. Lupo V. Cuesta A. Millán J.M. Vílchez J.J. Palau F. Espinós C. The p.R1109X mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4.Clin Genet. 2007; 71: 343-349Crossref PubMed Scopus (37) Google Scholar, 14Sevilla T. Martínez-Rubio D. Márquez C. Paradas C. Colomer J. Jaijo T. Millán J.M. Palau F. Espinós C. 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: 565-570Crossref PubMed Scopus (25) Google Scholarc.2211_2213delCCCp.C737_P738delinsX13Claramunt R. Sevilla T. Lupo V. Cuesta A. Millán J.M. Vílchez J.J. Palau F. Espinós C. The p.R1109X mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4.Clin Genet. 2007; 71: 343-349Crossref PubMed Scopus (37) Google Scholar, 14Sevilla T. Martínez-Rubio D. Márquez C. Paradas C. Colomer J. Jaijo T. Millán J.M. Palau F. Espinós C. 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: 565-570Crossref PubMed Scopus (25) Google ScholarDNA_621CMT1ADHeterozygosisHSPB1c.418C>Gp.R140G15Houlden H. Laura M. Wavrant-De Vrieze F. Blake J. Wood N. Reilly M.M. Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2.Neurology. 2008; 71: 1660-1668Crossref PubMed Scopus (159) Google ScholarDNAs indicated with the code SGT or DNA were studied for segregation analysis.AD, autosomal dominant; AR, autosomal recessive; CMT1, Charcot-Marie-Tooth disease type 1; Charcot-Marie-Tooth disease type 2. Open table in a new tab DNAs indicated with the code SGT or DNA were studied for segregation analysis. AD, autosomal dominant; AR, autosomal recessive; CMT1, Charcot-Marie-Tooth disease type 1; Charcot-Marie-Tooth disease type 2. The group of affected individuals without a molecular diagnosis included 33 CMT or HMN patients, distributed as follows: two CMT1, 20 CMT2, nine HMN, and two intermediate CMT. In these patients, the CMT1A duplication was verified by multiplex ligation–dependent probe amplification (Salsa Kit P033B CMT1/HNPP region; MRC-Holland, Amsterdam, the Netherlands) before testing and was subsequently discarded. For the majority of patients, at least mutations in the genes that are frequently involved in CMT disease (PMP22, MPZ, GJB1, GDAP1, and MFN2) were also ruled out by Sanger sequencing of exons and their intronic flanking sequences. Table 2 shows the 56 genes included in our panel. All of these genes are involved in CMT and/or HMN. The clinical and genetic features of 54 genes have been described by the Neuromuscular Disease Center (http://neuromuscular.wustl.edu/time/hmsn.html, last accessed January 20, 2015). The features of two genes, MICAL1 and TUBA8, were communicated at the Fifth International CMT Meeting,16Lassuthová P, Safka Brozkova D, Krutova M, Zavad'akova P, Rivolta C, Ivanek R, Mazanec R, Haberlová J, Speziani F, González MA, Züchner S, Seemen P: Identifying a new gene for CMT2 in a Polish family using whole exome sequencing. Presented at the 5th European and North American Charcot-Marie-Toot Consortium Meeting, 2013 June 25–27, Antwerp, Belgium.Google Scholar, 17Kennerson ML, Pérez-Siles G, Kochanski A, Kidambi A, Drew AP, Kosinska J, Kabzinska D, Ploski R, Menezes M, Hausmanowa-Petrusewicz I, Züchner S, Nicholson GA: Stop-mutation in the MICAL1 gene identified by exome sequencing in combination with a useful previous linkage analysis in a Czech HMSN II family. Presented at the 5th European and North American Charcot-Marie-Toot Consortium Meeting, 2013 June 25–27, Antwerp, Belgium.Google Scholar and these genes were also included because they were reported to be involved in CMT disease.Table 2Target Genes Included in the PanelGeneRef sequenceMIM No.RegionGeneRef sequenceMIM No.RegionAARSNM_001605.260106520LITAFNM_004862.36037954ATP7ANM_000052.630001122LMNANM_170707.215033014BICD2NM_015250.36152908LRSAM1NM_138361.561093324BSCL2NM_001122955.360615811MARSNM_004990.315656021DCTN1NM_004082.460114332MED25†Only founder mutations were analyzed.NM_030973.36101971DHTKD1NM_018706.661498417MFN2NM_014874.360850717DNAJB2NM_006736.56041399MICAL1NM_001286613.160712924DNM2NM_001005360.260237822MPZNM_000530.61594407DYNC1H1NM_001376.460011278MTMR2NM_016156.560355715EGR2NM_000399.31290102NDRG1NM_001135242.160526215FBLN5NM_006329.360458011NEFLNM_0061581622804FGD4NM_139241.261110415PDK3NM_001142386.230090612FIG4NM_014845.560939023PLEKHG5NM_198681.361110125GANNM_022041.360537911PMP22NM_000304.26010974GARSNM_002047.260028717PRPS1NM_002764.33118507GDAP1NM_018972.26065986PRXNM_181882.26057254GJB1∗Promoter sequence included.NM_000166.53040402RAB7ANM_004637.56022985GNB4NM_021629.36108639SBF1NM_002972.260356041HARSNM_002109.514281013SBF2NM_030962.360769740HINT1NM_0053406013143SETXNM_015046.560846524HK1†Only founder mutations were analyzed.NM_000188.21426001SH3TC2NM_024577.360820617HSPB1NM_001540.36021953SLC12A6NM_133647.160487826HSPB3NM_014365.26046241TDP1NM_018319.360719815HSPB8NM_014365.26080143TFGNM_006070.56024987IGHMBP2NM_002180.260050215TRIM2NM_015271.461414112KARSNM_001130089.160142115TRPV4NM_021625.460542715KIF1BNM_015074.360599547TUBA8NM_018943.26057425KIF5ANM_004984.260282128YARSNM_003680.360362313MIM, Mendelian Inheritance in Man.∗ Promoter sequence included.† Only founder mutations were analyzed. Open table in a new tab MIM, Mendelian Inheritance in Man. The panel of genes was generated using Agilent's SureDesign tool (Agilent Technologies Inc., Santa Clara, CA). For the capture design, we included all exons plus 25 bp of intronic flanking regions of the genes, taking into account different isoforms, except for two genes, HK1 and MED25. For both of these, we exclusively covered the analysis of the amplicon that contains the founder mutation described for them (HK1 g.9712G>C and MED25 p.A335V). Finally, we also added the promoter region of the GJB1 gene, because four causative mutations have been reported for it.8Sivera R. Sevilla T. Vílchez J.J. Martínez-Rubio D. Chumillas M.J. Vázquez J.F. Muelas N. Bataller L. Millán J.M. Palau F. Espinós C. Charcot-Marie-Tooth disease: genetic and clinical spectrum in a Spanish clinical series.Neurology. 2013; 81: 1617-1625Crossref PubMed Scopus (109) Google Scholar, 18Ionasescu V.V. Searby C. Ionasescu R. Neuhaus I.M. Werner R. Mutations of the noncoding region of the connexin32 gene in X-linked dominant Charcot-Marie-Tooth neuropathy.Neurology. 1996; 47: 541-544Crossref PubMed Scopus (90) Google Scholar, 19Houlden H. Girard M. Cockerell C. Ingram D. Wood N.W. Goossens M. Walker R.W. Reilly M.M. Connexin 32 promoter P2 mutations: a mechanism of peripheral nerve dysfunction.Ann Neurol. 2004; 56: 730-734Crossref PubMed Scopus (51) Google Scholar Taken together, we generated a panel of 56 genes comprising 57 targets that are divided into 862 regions with 8383 of total amplicons and a size of 186.34 Kbp. The theoretical target coverage was 99.98%. DNA from patients and relatives was previously extracted from blood samples using a Gentra Puregene blood kit (Qiagen, Venlo, the Netherlands). All DNA samples were repurified and re-eluted in nuclease-free water using the QIAamp DNA micro kit (Qiagen). The quantity and quality of the genomic DNA were determined using both the NanoDrop and the Qubit dsDNA BR in a Qubit 2.0 fluorometer (all from Thermo Fisher Scientific Inc., Rochester, NY). Agarose gel electrophoresis was used for validating the integrity of DNA. Sequence capture was performed using the HaloPlex Target Enrichment System (protocol version D.5; Agilent Technologies Inc.) for Illumina Sequencing (Illumina Inc., San Diego, CA). Approximately 300 ng of each genomic DNA sample was digested. The genomic DNA fragments were then hybridized to the HaloPlex probe capture library and Illumina sequencing motifs including index sequences. Subsequently, target DNA-HaloPlex probe hybrids were biotinylated and captured on streptavidin beads. The captured target library was amplified according to the manufacturer's instructions and subsequently purified using AMPure XP beads (Beckman Coulter Inc., Pasadena, CA). Before sample pooling and sequencing, quality-control stops were included to evaluate and control for possible contamination and errors: the success of genomic DNA restriction digestion using an enrichment control DNA, and the validation and quantification of the enriched target DNA in each library sample, the amplicons of which should have ranged from 175 to 625 bp in length, with the majority of products sized 225 to 525 bp. Both of them were performed using a Bioanalyzer High Sensitivity DNA Kit and the 2100 Bioanalyzer with 2100 Expert software version B.02.08SI648 (Agilent Technologies Inc.). An enrichment control DNA sample was used during the procedure. Finally, four different runs were processed using a 300-cycle MiSeq Reagent Kit version 2 (Illumina Inc.) on an Illumina sequencing platform. The read length was 150 bp. For each run, 11 samples were pooled for multiplexed sequencing. Sequence data have been deposited into the Sequence Read Archive repository (http://www.ncbi.nlm.nih.gov/sra; accession number SRP061110). To evaluate the sequencing coverage, we used BAM files to generate coverage indicators from the Genome Analysis Toolkit (GATK) version 3.0 (https://www.broadinstitute.org/gatk).20McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar Clustering and principal component methods were performed to determine the coverage data for all of the samples. Boxplots, scatterplots, and statistics were used for describing coverage by gene and by regions. Bar graphs described the mean coverage for each region and each gene. The statistical software R version 3.2.0 was used for performing this analysis (http://www.r-project.org). Quality metrics for sequence processing, mapping, and calling variants were calculated using the FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), SAMStat (http://samstat.sourceforge.net, all last accessed September 16, 2015), and Variant tools.21Medina I. De Maria A. Bleda M. Salavert F. Alonso R. Gonzalez C.Y. Dopazo J. VARIANT: Command Line, Web service and Web interface for fast and accurate functional characterization of variants found by Next-Generation Sequencing.Nucleic Acids Res. 2012; 40: W54-W58Crossref PubMed Scopus (34) Google Scholar Data were analyzed using a platform provided by DNAnexus (Mountain View, CA). Annotated variants that had a quality value of ≥250 and a percentage of heterozygosity of ≥30% of the reads were selected. To filter out common single-nucleotide polymorphisms (SNPs) and indels with allele frequency cutoffs of 0.01, we used the following databases: dbSNP (http://www.ncbi.nlm.nih.gov/SNP), ESP6500 (http://evs.gs.washington.edu/EVS), GEM.app (https://genomics.med.miami.edu), ExAC (http://exac.broadinstitute.org), and CSVS (http://csvs.babelomics.org, all last accessed September 16, 2015). Variant annotations of interest were performed according to the gene reference sequence reported in Table 2. All changes detected with a minor allele frequency of <1% were validated by Sanger sequencing on a 3730xl DNA analyzer (Applied Biosystems Inc., Foster City, CA). Whenever possible, segregation analysis was performed. In silico analysis was performed to predict the phenotypical consequences of the novel and low-frequency variants, using the SIFT (http://sift.bii.a-star.edu.sg) and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2) algorithms. Moreover, possible splicing process alterations were evaluated using NNSPLICE version 0.9 (http://www.fruitfly.org/seq_tools/splice.html), Human Splicing Finder (http://www.umd.be/HSF), and RESCUE-ES

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