Mutational Analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth Neuropathy Patients

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1 HUMAN MUTATION Mutation in Brief #732 (2004) Online MUTATION IN BRIEF Mutational Analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean CharcotMarieTooth Neuropathy Patients ByungOk Choi 1, Mi Sun Lee 2, Sang Hee Shin 2, Jung Hee Hwang 2, KyoungGyu Choi 1, WonKi Kim 3, Il Nam Sunwoo 4, Nam Keun Kim 5, and Ki Wha Chung 2 * 1 Department of Neurology and Ewha Medical Research Center and 3 Department of Pharmacology and Ewha Institute of Neuroscience, Ewha Womans University College of Medicine, Seoul , Korea; 2 Department of Biological Science, Kongju National University, 182 Kongju, Chungnam , Korea; 4 Department of Neurology, Yonsei University College of Medicine, Seoul , Korea; 5 Department of Biochemistry and Institute of Clinical Research, College of Medicine, Pochon CHA University, Sungnam , Korea *Correspondence to: Ki Wha Chung, PhD, Department of Biological Science, Kongju National University, 182 Kongju, Chungnam , Korea; Tel.: ; Fax: ; kwchung@kongju.ac.kr Grant sponsor: Korea Science and Engineering Foundation; Grant numbers: R , 04B9003 Communicated by Jurgen Horst We examined A duplication of 17p11.2p12, mutations of PMP22, MPZ (P0), GJB1 (Cx32), EGR2 and NEFL genes in 57 Korean families with patients diagnosed as having CharcotMarieTooth (CMT) disease. The A duplication was present in 53.6% of 28 CMT type 1 patients. In the 42 CMT families without A duplication, 10 pathogenic mutations were found in 9 families. The 10 mutations were not detected in 105 healthy controls. Seven mutations (c.318delt (p.ala106fs) in PMP22, c.352g>a (p.asp118asn), c.4491g>t (3 splice site), c.706a>g (p.lys236glu) in MPZ, c.408t>c (p.val136ala), c.502t>c (p.cys168arg) in GJB1, and c.1001t>c (p.leu334pro) in NEFL) were determined to be novel. The mutation frequencies of PMP22 and MPZ were similar to those found in several European populations, however, it appeared that mutations in GJB1 are less frequent in East Asian CMT patients than in European patients. We described the identified mutations and phenotypegenotype correlations based on nerve conduction studies WileyLiss, Inc. KEY WORDS: CharcotMarieTooth disease; A; PMP22; MPZ; GJB1; EGR2; NEFL; Korean INTRODUCTION CharcotMarieTooth (CMT) disease is the most common form of inherited motor and sensory neuropathy. CMT is usually classified as a type 1, the demyelinating form () and a type 2, the axonal form (). patients have particularly reduced nerve conduction velocities (NCV). Histopathological examinations of peripheral nerve biopsies have frequently revealed extensive segmental demyelination and remyelination with 'onion bulb' formation. The primary defect of patients is neuronal (Harding and Thomas, 1980). Received 20 January 2004; accepted revised manuscript 12 May WILEYLISS, INC. DOI: /humu.9261

2 2 Choi et al. CMT is a genetically heterogeneous disorder of the peripheral nervous system, thus many genes have been identified as CMTcausative genes (Berger et al., 2002). Tandem duplication of the A region within chromosome 17p11.2p12 including peripheral myelin protein 22 (PMP22) gene is the most frequent cause of CMT type 1. This duplication is caused by an unequal crossover event between two homologous repetitive elements, which flank the 1.4 Mb region (Lupski et al., 1991). Patients carrying one extra copy of PMP22 develop A, whereas entire or even partial deletion of the 17p11.2p12 region causes hereditary neuropathy with liability to pressure palsies (HNPP). Mutations in PMP22 (MIM# ), myelin protein zero (MPZ/P0) in 1q23 (MIM# ), early growth response 2 (EGR2/Krox20) in 19q21.1q22.1 (MIM# ), gap junction protein beta1 (GJB1/Cx32) in Xq13.1 (MIM# ) and neurofilament light chain (NEFL) gene in 8p21 (MIM# ) frequently provide the genetic causes of hereditary peripheral neuropathies (Bird et al., 1982; Timmerman et al., 1996; Warner et al., 1998; Mersiyanova et al., 2000a). Mutations in PMP22 gene encoding fourtransmembrane myelin protein lead to variable clinical phenotypes including A, DejerineSottas syndrome (DSS) and HNPP (Mostacciuolo et al., 2001; Numakura et al., 2002). MPZ gene, which encodes a major component of myelin, is frequently associated with B, and DSS (Marrosu et al., 1998; Huehne et al., 2003). Mutations in GJB1, which encodes a gapjunction protein in the peripheral nervous system, lead to CMTX (Silander et al., 1998; Nelis et al., 1999). EGR2 gene that encodes a zincfinger transcription factor plays an important role in the peripheral myelination. Mutations in EGR2 have been found in patients with CMT type 1, DSS and congenital hypomyelination (Warner et al., 1998; Bellone et al., 1999; Timmerman et al., 1999). NEFL gene encoding the neurofilament light chain plays an important role in axonal structure that includes an extensive fibrous network in the cytoplasm of the neuron. Recently, mutations in NEFL gene have been found in patients with E (Mersiyanova et al., 2000a) and early onset severe CMT phenotypes (Jordanova et al., 2003). We examined the A duplication of 17p11.2p12, mutations in PMP22, MPZ, GJB1, EGR2 and NEFL in 57 Korean families with patients diagnosed as having CMT or with related peripheral neuropathy phenotypes. In the present study we detected 7 novel and 3 reported mutations associated with CMT disease. MATERIALS AND METHODS Patient Samples The study included a total of 238 patients from 57 unrelated CMT families of Korean origin. In 1993 we started to collect blood samples from patients who were diagnosed with CMT at both Yonsei University hospital and Ewha Womans University hospital (Seoul, Korea). Based on electrophysiological criteria, 28 families were diagnosed with, 18 families with, 4 families with DSS and 7 families with other CMT type based on clinical records. For the diagnosis of, we used the clinical guidelines of the European CMT consortium (De Jonghe et al. 1998). A group of 105 healthy controls were recruited from the Neurological Department after careful clinical and electrophysiological examination. All participants gave written informed consent according to the protocol approved by the Ethics Committee of Yonsei University Hospital and Ewha Womans University Hospital. DNA Extraction and Duplication Screen DNA extraction from blood samples was performed using a genomic DNA isolation kit (CoreBio, Korea). Seven microsatellite markers (D17S921, D17S1358, D1S9B, D17S9A, D17S918, D17S122 and D17S4A) within the 1.4 Mb duplication/deletion region of chromosome 17p11.2p12 were amplified by the general PCR method using FAM or HEXlabeled primers. PCR products were resolved using ABI 3100 automatic sequencer (Applied Biosystems, USA). Genotyping analysis was supported using the GENOTYPER (Ver. 3.7) program. The duplication was deduced by the presence of three distinct peaks or two peaks with a clear dosage difference. Mutation Screen The coding regions and some promoters of PMP22, MPZ, GJB1, EGR2 and NEFL were amplified by the general PCR method. The primer sequences as well as PCR conditions are available upon request. Exons 2, 3, 4 and 5 of PMP22 were separately amplified. For the amplification of MPZ, the promoter region (up to 650 bp from the start codon) to exon 1 and exon 4 to 5 were amplified together. Exons 2, 3 and 6 were separately amplified.

3 Mutations in Korean CMT Patients 3 Exon 2 of GJB1 was amplified with three overlapping fragments. Exon 1 was amplified separately and was amplified with four overlapping fragments for EGR2. For NEFL, exon 1, including the proximal promoter region (up to 280 bp from the start codon), was amplified with three overlapping fragments, and exons 2, 3 and 4 were amplified separately. The PCR products were purified using a PCR product purification kit (CoreBio, Korea), and then they were directly sequenced using the ABI 3100 automatic sequencer (Applied Biosystems, USA). Sequencing variations were confirmed by sequencing both strands. RESULTS A Duplication From the genotyping of 7 microsatellite markers located within the chromosome 17p11.2p12 region, the A duplication was detected in 53.6% of 28 families, a lower level than that found by the European Collaborative Study (70.7%) (Nelis et al., 1996). However, the results were similar to those of Italian (57.6%) (Mostacciuolo et al., 2001) and Russian (53.7%) (Mersiyanova et al., 2000b). Mutations in Myelin Genes The mutation screen in PMP22, MPZ, GJB1, EGR2 and NEFL was performed in subjects from 42 families that did not have the A duplication (Table 1). We found 4 variations in PMP22 by screening exons 2, 3, 4, 5 and the surrounding intron regions (c.318delt, c a>g, c.17983t>c and c.542a>c). The c.318delt (p.ala106fs) frameshift mutation, not listed in the European CMT Consortium Mutations Update database ( March 2004), was detected in a family with phenotype (familial ID: FC35). This frameshift mutation was not detected among the 105 healthy controls. In the pedigree analysis of FC35, the Ala106fs mutation was found in two affected members, but not detected in unaffected members. It was probably transmitted from father to daughter. Another 3 variations (c a>g and c.17983t>c at intron 3; c.542a>c at 3'UTR) were located in the noncoding region and were determined to be polymorphisms, thus they could not be regarded as causative mutations. Four variations were detected in MPZ. Three of them, c.352g>a (p.asp118asn), c.4491g>t (intron 3, 3' splice site) and c.706a>g (p.lys236glu), were considered as causative mutations. The three mutations were novel and were not detected in the 105 healthy controls. The p.asp118asn missense mutation was identified in a family (FC26). In the FC26 pedigree, the proband and her affected mother carried the missense mutation, whereas the unaffected father and sister did not. The c.4491g>t 3'splice site mutation was found in a family (FC19). In the pedigree analysis, the mutation was well segregated to 4 affected persons. A similar c.4491g>c mutation was previously described in a patient (Bort et al., 1997). The p.lys236glu missense mutation was found in an isolated patient. This mutation causes the replacement of a basic by an acidic amino acid, and was therefore considered as the causative mutation. The c.600g>a (p.gly200gly) variation was not considered as a causative mutation, as it was reported as a polymorphism (Bort et al., 1997). Five variations were found in GJB1. Three mutations, c.408t>c (p.val136ala), c.491g>a (p.arg164gln) and c.502t>c (p.cys168arg), were considered as causative mutations, and were not detected among the 105 healthy controls. The c.408t>c (p.val136ala) and c.502t>c (p.cys168arg) missense mutations have not been reported in the CMT mutations database. The p.val136ala was found in a family (FC2). The p.val136ala was determined to be a de novo mutation, because it was only detected in a severe female patient, but not in her parents. Paternity and maternity were confirmed by the genotyping of 12 autosomal STR markers. The p.cys168arg mutation was observed in a family (FC22). In the pedigree analysis, the p.cys168arg mutation was found in two affected members, but not detected in unaffected members. The p.arg164gln mutation was observed in an isolated patient. Several previous studies have reported this mutation mostly in demyelinating patients (Mersiyanova et al., 2000b; Yoshihara et al., 2000; Huehne et al., 2003). The c.30c>t (p.leu10leu) mutation was detected in two CMT patients, but not detected in the 105 controls. This situation was very similar to the Japanese population as reported by Numakura et al (2002), who observed this mutation in patients but not in controls. The c.594t>a (p.ser198ser) was only found in a family and not among the

4 4 Choi et al. 105 controls. However, it does not cause amino acid replacement and was not segregated with affected members in the family, therefore, it could not be regarded as a causative mutation. Mutations in EGR2 and NEFL Two variations, c.1075c>t (p.arg359trp) and c.1086a>c (p.arg362arg), were detected in EGR2. The p.arg359trp in the zincfinger domain was considered to be causative. The p.arg359trp was detected in the FC2 family who also showed the p.val136ala mutation in GJB1. The p.arg359trp mutation was found in two affected members, proband with an additional p.val136ala in GJB1 and her father. Gene/ exon PMP22 intron 3 intron 3 exon 4 exon 5 MPZ exon 3 intron 3 exon 5 exon 6 GJB1 EGR2 NEFL exon 1 exon 1 intron 1 exon 3 TABLE 1. Mutations Found in PMP22, MPZ, Cx32, GJB1 and NEFL Domain a Nucleotide Amino acid Inheritance Phenotype References change a change TM3 3'UTR EC IC IC IC1 TM3 EC2 EC2 TM4 Zn1 Zn1 Head ROD Tail c a>g c.17983t>c c.318delt c.542a>c c.352g>a c.4491g>t c.600g>a c.706a>g c.30c>t c.408t>c c.491g>a c.502t>c c.594t>a c.1075c>t c.1086a>c c.276g>a c.1001t>c c inst c.1189g>a Ala106fs Asp118Asn 3'splice site Gly200Gly Lys236Glu Leu10Leu Val136Ala Arg164Gln Cys168Arg Ser198Ser Arg359Trp Arg362Arg Gln92Gln Leu334Pro Glu397Lys Isolated De novo Isolated Xlinked dominant Isolated 2 cases 1 case Bort et al., 1997 Numakura et al., 2002 Mersiyanova et al., 2000b Yoshihara et al., 2000 Huehne et al., 2003 Taroni et al., 1999 Timmerman et al., 1999 Boerkoel et al., 2001 Timmerman et al., case Yoshihara et al., 2002 Zuchner et al., 2004 IC, intracellular; EC, extracellular; TM, transmembrane; Zn, Zincfinger domain. a Nucleotide numbering: the A of ATG translation initiation site as +1. Reference sequence accession numbers: PMP22= NM_000304; MPZ= NM_000530; GJB1= NM_000166; EGR2= NM_000399; NEFL= NM_ b Unreported in the CMT Consortium database ( The p.arg359trp mutation has been frequently reported in DSS patients with sporadic inheritance, however, our patients showed a CMT type 1 phenotype with autosomal dominant inheritance (Taroni et al., 1999; Timmerman et al., 1999; Boerkoel et al., 2001). It was not detected among the 105 healthy controls. The c.1086a>c (p.arg362arg) was determined to polymorphic, as described by Timmerman et al. (1999). Four variations, c.1001t>c (p.leu334pro), c.1189g>a (p.glu397lys), c.276g>a (p.gln92gln) and c inst were observed in NEFL. Of them, c.1001t>c (p.leu334pro) and c.1189g>a (p.glu397lys) were considered as the causative

5 Mutations in Korean CMT Patients 5 mutations. These two mutations were not detected among the 105 healthy controls. The p.glu397lys mutation was found in a family (FC7). In the pedigree analysis, the mutation was detected from two affected members, proband and his daughter. The p.glu397lys was recently reported by Zuchner et al (2004). The p.leu334pro mutation was found in an isolated patient, thus no pedigree analysis was performed. The p.leu334pro has not been reported in the CMT mutations database. The c.276g>a (p.gln92gln) was determined to be a polymorphism, as reported by Yoshihara et al. (2002). The c inst was observed in two patients, however it was not found in 105 controls. It could not be regarded as a causative mutation because it was located on the noncoding region (intron 1). DISCUSSION Most molecular genetic studies of CMT have been performed in European populations and little data is available on Asian CMT patients. In the present study, we investigated genetic causes pertaining to CMT or related neuropathies in subjects with 57 Korean families. We first examined the A duplication by genotyping seven microsatellites within the 17p11.2p12 region, and found 15 A duplication families among 28 cases. The duplication frequency (53.6%) in Korean patients is slightly lower than, or similar to those of European cases: 70.7% in an Europian collaborative study (Nelis et al., 1996), 57.6% in Italy (Mostacciuolo et al., 2001) and 53.7% in Russia (Mersiyanova et al., 2000b). However the frequency is higher than the level of 31.2% in Japanese patients (Ikegami et al., 1997). CMT type DSS Other CMT Total TABLE 2. Overview of Mutations in Korean CMT Families Number A Nonduplicated cases duplication PMP22 MPZ GJB1 EGR2 NEFL Uninformative TABLE 3. Mutation Frequencies in CMT Patients According to Genes Ethnic group PMP22 MPZ GJB1 EGR2 NEFL Uninformative Total a References Asians Korea Japan Japan Europeans Russia Germany Spain Finland Belgium b Italy 1(2.4) 4(2.5) 2(1.7) 1(2.4) 1(2.1) 1(1.6) 2(2.8) 3(7.1) 8(5.6) 5(7.1) 6(5.2) 3(7.1) 5(10.6) 3(4.8) 4 (5.6) 3(7.1) 9(5.6) 4(5.7) 15(13.0) 5(11.9) 10(21.3) 12(19.0) 12(16.7) 1(2.4) 0(0.0) 2(4.8) 7(2.2) 33(78.6) 140(87.0) 61(87.1) 92(80.0) 33(78.6) 31(66.0) 47(74.6) 316(97.8) 54(75.0) a Patients with the A duplication were excluded. b Patients neither A duplication nor causative mutation in PMP22, MPZ and GJB1. This study Numakuma et al., 2002 Yoshihara et al., 2000 Mersiyanova et al., 2000b Huehne et al., 2003 Bort et al., 1997 Silander et al., 1998 Jordanova et al., 2003 Mostacciuolo et al., 2001 Fortytwo families without A duplication were further screened for mutations in PMP22, MPZ, GJB1, EGR2 and NEFL. We found 10 CMTcausative mutations in nine families: one PMP22 mutation (2.4%), three MPZ mutations (7.1%), three GJB1 mutations (7.1%), one EGR2 mutation (2.4%) and two NEFL mutations (4.8%), respectively (Table 2, 3).

6 6 Choi et al. Most observed mutations were missense except for one frameshift and one 3'splice site mutation. Seven mutations (p.ala106fs in PMP22, p.asp118asn, c.4491g>t and p.lys236glu in MPZ, p.val136ala and p.cys168arg in GJB1, and p.leu334pro in NEFL) were determined to be novel. The p.val136ala in GJB1 and p.arg359trp in EGR2 were found in a severe patient (FC2). Several cases of double mutations in the same gene have been reported (Silander et al., 1998; Mersiyanova et al., 2000b), however, no case of a CMT patient with double mutations in different genes has been reported. The mutation frequencies in MPZ (7.1%) and PMP22 (2.4%) were similar to those reported in other ethnic nonduplicated CMT groups, i.e., % in MPZ and % in PMP22 (Bort et al., 1997; Silander et al., 1998; Mersiyanova et al., 2000b; Yoshihara et al., 2000; Mostacciuolo et al., 2001; Numakura et al., 2002; Huehne et al., 2003). The mutation frequency in GJB1 (7.1%) in this study was considerably lower than in several European groups, i.e., Spain 21.3% (Bort et al., 1997), Finland 19.0% (Silander et al., 1998), Russia 13.0% (Mersiyanova et al., 2000b), Italy 16.7% (Mostacciuolo et al., 2001) and Germany 11.9% (Huehne et al., 2003), but similar to that of the Japanese % (Yoshihara et al., 2000; Numakura et al., 2002). It appears that mutations in GJB1 are less frequent in East Asian CMT patients than in European patients. Population data on mutation frequencies of the NEFL and EGR2 genes are very limited. We observed two NEFL mutations with a frequency of 4.8%. Jordanova et al. (2003) reported seven NEFL mutations in 323 Caucasian CMT patients (2.2%). It seems that the NEFL mutation frequency is similar to or higher than PMP22 in nonduplicated CMT patients. We found a causative EGR2 mutation in this study. Mostacciuolo et al. (2001) found no causative EGR2 mutation in 72 Italian CMT patients, and few EGR2 mutations are contained in the CMT Consortium database, which implies that the mutation frequency of EGR2 is the lowest among the five analyzed genes. We found 9 polymorphic variations in this study. Whereas four of the variations have been reported by other studies (Bort et al., 1997; Timmerman et al., 1999; Numakura et al., 2002; Yoshihara et al., 2002), five have not been reported (c a>g, c.17983t>c and c.542a>c in PMP22, c.594t>a in Cx32, and c inst in NEFL). Of the polymorphic variations, five were found in the coding regions, but no amino acid substitution occurred. The other four variations were located on noncoding regions, e.g., intron or 3'UTR. In addition to the duplication of 17p11.2p12 and mutations in PMP22, MPZ, GJB1, EGR2 and NEFL, many other genes or chromosomal loci have been reported over recent several years as being responsible for hereditary peripheral neuropathies. We identified the causative mutations in approximately 22% of the A nonduplicated cases (Table 3). Thirtytwo cases were not found to have a causative mutation by the screening. These cases may have mutation(s) in promoter regions of the screened genes or in other CMTrelevant genes, i.e., PRX, NDRG1, GAN, GDAP1, LITAF/SIMPLE or SBF2. CMT is heterogeneous not only genetically and clinically, but also ethnically. Therefore, it is also possible that novel CMTinducible genes or loci are associated with these genetically unexplained patients. ACKNOWLEDGMENTS This study was supported by RRC for New Materials Recycling in Kongju National University (#04B9003) and grant (#R ) from the Korea Science and Engineering Foundation. REFERENCES Bellone E, Di Maria E, Soriani S, Varese A, Doria LL, Ajmar F, Mandich P A novel mutation (D305V) in the early growth response 2 gene is associated with severe CharcotMarieTooth type 1 disease. Hum Mutat 14: Berger P, Young P, Suter U Molecular cell biology of CharcotMarieTooth disease. Neurogenetics 4:115. Bird TD, Ott J, Giblett ER Evidence for linkage of CharcotMarieTooth neuropathy to the Duffy locus on chromosome 1. Am J Hum Genet 34: Boerkoel CF, Takashima H, Bacino CA, Daentl D, Lupski JR EGR2 mutation R359W causes a spectrum of Dejerine Sottas neuropathy. Neurogenetics 3:

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