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A novel mutation in GJB1 (c.212T>G) in a Chinese family with X-linked Charcot–Marie–Tooth disease
Journal of Clinical Neuroscience 22 (2015) 513–518
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
A novel mutation in GJB1 (c.212T>G) in a Chinese family with X-linked Charcot–Marie–Tooth disease Fei Xiao, Jia-ze Tan, Xu Zhang, Xue-Feng Wang ⇑ Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, 1 You Yi Road, Chongqing 400016, China
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 4 August 2014
Keywords: Connexin32 Gap junction protein beta 1 GJB1 Hereditary sensory motor neuropathy X-linked Charcot–Marie–Tooth disease
a b s t r a c t Gap junction protein beta 1 (GJB1) gene mutations lead to X-linked Charcot–Marie–Tooth (CMTX) disease. We investigated a Chinese family with CMTX and identified a novel GJB1 point mutation. Clinical and electrophysiological features of the pedigree were examined, and sequence alterations of the coding region of GJB1 that encode connexin32 were determined by direct sequencing. Sequence alignment of the mutation site was performed using Clustal W. Mutation effects were analysed using PolyPhen-2, SIFT and Mutation Taster software. The three-dimensional structures of the mutant and wild-type proteins were predicted by modeling with SWISS MODEL online software. The affected family members displayed typical Charcot–Marie–Tooth phenotypes, but phenotypic heterogeneity was observed. Nerve conduction velocities of all affected patients were slow. Sequencing of GJB1 revealed a heterozygous T>G missense mutation at nucleotide 212 in the proband, the proband’s mother and the proband’s daughter. The affected male sibling of the proband displayed a hemizygous missense mutation with T>G transition at the identical position on the GJB1 gene. This mutation resulted in an amino acid change from isoleucine to serine that was predicted to lead to tertiary structural alterations that would disrupt the function of the GJB1 protein. A novel point mutation in GJB1 was detected, expanding the spectrum of GJB1 mutations known to be associated with CMTX. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Charcot–Marie–Tooth disease (CMT) is the most common hereditary motor and sensory neuropathy, affecting 1 in 2500 people worldwide [1]. Based on the motor nerve conduction velocity (MNCV) of the median nerve, CMT can be further classified into the demyelinating (CMT1) and axonal (CMT2) forms. The median MNCV is <38 m/s in CMT1 and >38 m/s in CMT2 [2]. The typical clinical features of CMT include progressive muscular weakness and atrophy of limbs, especially distally. Patients with CMT may have a family history of the disease, but CMT is clinically and genetically heterogeneous, even within a family [3]. A defect in the peripheral myelin protein 22 (PMP22) is the most common cause of CMT [4]. Thus, the PMP22 gene is generally the first gene to be examined in patients with CMT [5]. X-linked Charcot–Marie–Tooth disease (CMTX) is the second most frequent type of CMT, accounting for 10–20% of patients [6]. CMTX usually results from mutations in the gene encoding gap junction protein beta 1 (GJB1), and these mutations cause
⇑ Corresponding author. Tel./fax: +86 23 8901 2878. E-mail address: [email protected] (X.-F. Wang). http://dx.doi.org/10.1016/j.jocn.2014.08.028 0967-5868/Ó 2014 Elsevier Ltd. All rights reserved.
amino acid changes which alter the structure of the encoded protein, connexin 32. Here we report a Chinese family with X-linked dominant CMT and identify a novel GJB1 point mutation within the family.
2. Methods 2.1. Study participants A pedigree was drawn up for a Chinese family with CMTX who were referred to the Department of Neurology at the First Affiliated Hospital of Chongqing Medical University, Chongqing, China, in April 2012 (Fig. 1A). A detailed medical history was obtained for all family members. Healthy control subjects (n = 100; 50% male) were recruited from the outpatient clinic of the First Affiliated Hospital of Chongqing Medical University between May 2013 and July 2013. Members of the CMTX pedigree and control subjects were all of Chinese ancestry and resided in China. All family members underwent clinical and electrophysiological testing, including standardised electromyography and nerve conduction velocity (NCV) studies. CMT neuropathy scores [7] were determined by an experienced peripheral nerve clinician in order to assess the
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severity of the disease in the affected family members. The daughter of the proband (IV-2) refused electrophysiological inspection. MRI of the brain was conducted for the affected pedigree members. Subjects included in this study provided written informed consent, and the study protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.
Table 1 Primers used to amplify exons 1–3 of the gap junction beta 1 (GJB1) gene Region amplified
Primer sequences
Exon 1
F 50 -CCATAGGGAGCTCAGAGGATCAAT-30 R 50 -TGAAGGCGGAGGTCAGTAGAAG-30 F 50 -GTGTGTAGGGTGGGCGGAAGTC -30 R 50 -CTTCTTCGCTTTCATCCAACAT -30 (1) F 50 -AGTGCTATGGCGCCCGACTTT-30 R 50 -ATGAAGACGGCCTCAAACAAC-30 (2) F 50 -AAGAGGCACAAGGTCCACATC-30 R 50 -CTCACTCAGCAGCTTGTTGAT-30 (3) F 50 -CATCTGCATCATCCTCAATGTG-30 R 50 -CTCTAGCTCCCCACCCCTCCAG-30
Exon 2 Exon 3
2.2. Mutation analysis Genomic DNA was extracted from peripheral leukocytes of fresh blood samples taken from the proband (III-3), the elder brother (III-2), the parents (II-5 and II-6) and daughter (IV-2) of the proband, and from healthy control subjects, using a standard proteinase K digestion and phenol–chloroform extraction method. PMP22 duplication and mutation analysis were performed as described elsewhere [8]. All three exons of GJB1 were amplified by polymerase chain reaction (PCR) using the GeneAmp PCR System 9700 thermal cycler (Perkin Elmer, Shelton, CT, USA). The primer pairs (Table 1) were designed for exons 1–3 of GJB1 using Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA), according to the previously published sequence (GenBank RefSeq (Nucleotide) NG_008357; http://www.ensembl.org, [9]). Each 25 ll PCR reaction contained 50 ng of genomic DNA, 10 pmol of each forward and reverse primer, 5 mmol dNTP and 2.5 U of Taq polymerase (Takara Biotechnology, Dalian, China) in reaction buffer (Takara Biotechnology). The PCR cycles consisted of denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30–40 s, 57–63°C
F = forward, R = reverse.
for 30 s, and 72°C for 30–40 s (depending on which segment in GJB1 was being amplified) and a final elongation step at 72°C for 10 minutes. PCR products were purified by 1.5% agarose gel electrophoresis and directly sequenced with an ABI PRISM 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The data were analyzed with Chromas 2.22 chromatogram file editor software (Technelysium Pty Ltd, Tewantin, QLD, Australia). The base mutations in GJB1 were described and numbered according to criteria provided by the Ensembl genome browser [9]. 2.3. In silico analyses Multiple GJB1 protein sequence alignment across species was performed using Clustal W [10], and the alignment results were
Fig. 1. Pedigree diagram and clinical pictures of the family. (A) Pedigree of the Chinese family with Charcot–Marie–Tooth disease (CMT). Squares = males, circles = females, filled squares and circles = affected patients, grey circle = mutation carrier, arrow = proband, oblique line = deceased. (B) Image showing atrophy in the distal lower limbs of the proband (III-3). (C) Image showing the more severe atrophy in the distal lower limbs of III-2. (D) Image showing pes cavus of II-5.
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visualized with Jalview software (http://www.jalview.org/) [11]. Exome Variant Server (evs.gs.washington.edu/) [12] was used to check if the variant is found in the normal population. The potential functional impacts of mutations within GJB1 were predicted using Polymorphism Phenotyping 2 (PolyPhen-2) software (http:// geneics.bwh.harvard.edu/pph2/) [13], Sorting Intolerant from Tolerant (SIFT) software (http://sift.jcvi.org/) [14] and Mutation Taster (http://www.mutationtaster.org/) [15]. PolyPhen-2 classifies predicted effects of amino acid substitutions on the function of human proteins as ‘‘benign’’, ‘‘possibly damaging’’, ‘‘probably damaging’’, or ‘‘unknown’’. Functional impact of the mutation was predicted as ‘‘tolerated’’ or ‘‘damaging’’ for SIFT and as ‘‘polymorphism’’ or ‘‘disease causing’’ for Mutation Taster. To further investigate the effect of the novel missense mutation within GJB1, we modeled the three-dimensional structures of GJB1 with and without the mutation using SWISS-MODEL online software (http://beta.swissmodel.expasy.org/) [16]. The results were displayed with Molsoft ICM software (http://www.molsoft.com/ icm_browser_pro.html) [17].
3. Results 3.1. Clinical findings All affected individuals in the pedigree showed varying degrees (mild to severe) of muscle atrophy, diminished reflexes and foot drop. The CMT neuropathy scores of the proband, the proband’s mother, and the proband’s elder brother were 11, 7, and 21, respectively. The proband began to notice weakness and atrophy in the distal lower extremities at 26 years of age and first visited the neurology department because of weakness and atrophy of the distal muscles of both upper and lower limbs at age 34. Neurological examination revealed weakness and atrophy of the distal muscles of the lower limbs bilaterally (Fig. 1B) without obvious involvement of the proximal muscles, and pes cavus was also observed. The power values were 4/5 for the finger abductor muscles, 4/5 for the gastrocnemius muscle, and 3/5 for the anterior tibialis muscle, based on the Medical Research Council scale. The patient’s vibration sense was severely impaired, and deep tendon reflexes were absent in all extremities. The proband’s elder brother (III-2, aged 36) had experienced similar symptoms from the age of 15. At the time of this study, however, his muscle weakness and atrophy in the distal lower extremities were more severe than the proband. He experienced fatigue when walking and had obvious atrophy of the distal muscles of the limbs (Fig. 1C) and pes cavus. His power values were 3/5 for the finger abductor muscles, 3/5 for the gastrocnemius muscle, and 3/5 for the anterior tibialis muscle. Deep tendon reflexes were absent in all extremities. The proband’s mother (II5) had relatively mild CMT symptoms compared to the proband. The mother first noticed mild weakness and atrophy in the distal forearm and pes cavus (Fig. 1D) in her thirties, but weakness and
atrophy were not observed in the distal lower extremities and she had no difficulty walking. For all affected pedigree members, no enhanced reflexes were observed. However, the father (II-6), nephew (IV-1) and daughter (IV-2) of the proband showed no clinical symptoms of CMT. Brain MRI of the affected individuals were normal. However, NCV were reduced or not detectible in all affected pedigree members. The NCV were lower in the affected male (III-2) than in the two affected females (II-5 and III-3), with reduced amplitude and prolonged latency. NCV in the median motor nerve of all affected patients were <38 m/s, which is in accordance with CMT1. Table 2 summarises the electrophysiological data of the family members. 3.2. Mutational analysis of PMP22 and GJB1 We did not find any copy number abnormalities or mutations in the coding region of PMP22 of the proband. Bidirectional sequencing of the coding region containing exons 1–3 of GJB1 revealed a heterozygous T>G missense mutation at nucleotide position 212 in exon 3 in the proband (c.212T>G; Fig. 2A). The same heterozygous mutation was also detected in the mother and daughter of the proband (Fig. 2B, C). In addition, the affected male sibling of the proband was hemizygous for the missense mutation at this position (Fig. 2D). The c.212T>G mutation would result in an amino acid change from isoleucine (Ile) to serine (Ser) at codon 71 (p.Ile71Ser). The c.212T>G mutation was absent in the father of the proband (Fig. 2E) and in the 100 healthy control subjects (50% male; Fig. 2F). Fifteen single nucleotide polymorphisms of GJB1 have been reported in the Exome Variant Server database, however to our knowledge this mutation has not been reported as a polymorphism, suggesting that it is not common and does not exist in the normal population. No mutations were detected in exons 1 or 2 of GJB1 in any members of the target family or controls. 3.3. Predictions of functional impacts of the c.212T>G mutation on the GJB1 protein Alignment of the amino acid sequences surrounding the Ile at position 71 in several distantly related species showed that this Ile is located in a highly conserved region of GJB1 in humans and other species (Fig. 3), indicating its functional importance. The p.Ile71Ser substitution was predicted to be ‘‘probably damaging’’ and disrupt the function of GJB1 by the PolyPhen-2 software (score 0.993; sensitivity 0.70, specificity 0.97). Mutation Taster and SIFT predicted that the p.Ile71Ser mutation is functionally ‘‘disease causing’’ and ‘‘damaging’’ (SIFT score of 0), respectively. The three-dimensional structure of the mutant GJB1 was also predicted to be altered. Substitution of the nonpolar Ile with the polar Ser, likely disrupts the a-helical structure of the protein. Comparison of the predicted tertiary structures of the wild-type and mutant proteins revealed that p.Ile71Ser at the start of an
Table 2 Electrophysiological data of the affected patients Patient
II-5 III-2 III-3
Age, years
Sex
59 36 34
F M F
Median
Median
Ulnar
Peroneal
Sural
Motor CMAP (mV)
MNCV (m/s)
SNAP (lV)
SNCV (m/s)
Motor CMAP (mV)
MNCV (m/s)
Motor CMAP (mV)
MNCV (m/s)
SNAP (lV)
SNCV (m/s)
3.7 0.6 1.1
37.9 26.3 30.4
11 NR 8.2
37 NR 25.7
4.1 1.9 2.6
39.5 28.8 35.2
1.2 NR NR
36.3 NR NR
4.6 NR 3.1
34.9 NR 19.5
CMAP = compound muscle action potential, F = female, M = male, MNCV = motor nerve conduction velocity, NR = not recordable, SNAP = sensory nerve action potential, SNCV = sensory nerve conduction velocity.
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Fig. 2. DNA sequencing chromatographs of exon 3 of the gap junction beta 1 (GJB1) gene. (A) Nucleotide sequence of this region in the proband (III-3), (B) the daughter of the proband (IV-2), (C) the mother of the proband (II-5), (D) the affected brother of the proband (III-2), (E) the father of the proband (II-6), and (F) a control subject. The heterozygous missense mutation (arrow), c.212T>G, was identified in the proband, the daughter and mother of the proband, and the hemizygous mutation was identified in the brother of the proband. This mutation was absent in the father of the proband and the control subjects.
a-helix led to strengthening of the hydrogen bond between Ser 71 and tyrosine (Y) 157, shortening the distance between the two residues (Fig. 4). Because Y157 is at the end of another a-helix, this conformational change could potentially affect the overall a-helical folding of the protein.
4. Discussion The understanding of the genetic basis of CMT has increased greatly over the past 20 years. The most frequent cause of CMT is a duplication abnormality in the PMP22 gene. However, we did
F. Xiao et al. / Journal of Clinical Neuroscience 22 (2015) 513–518
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Fig. 3. Conservation of the GJB1 amino acid sequence among different species. Sequence alignment showing that isoleucine 71 (arrow) and its neighboring sequences are highly conserved in a variety of species.
Fig. 4. SWISS-MODEL generated three-dimensional models of the portion of gap junction beta 1 (GJB1) encoded by exon 3 [16]. Hydrogen bond distances between the amidogen group of serine (Ser) 71 and the hydroxyl group in the benzene ring of tyrosine 157 are different, at (A) 3.5 angstrom in the wild-type and (B) 2.9 angstrom in the mutant p.Ile71Ser protein.
not detect duplications or mutations in PMP22 in the proband of our pedigree. Phenotypic heterogeneity was observed within this family, with males tending to be more severely affected than females according to CMT neuropathy score assessment. Pedigree analysis showed that the inheritance mode was X-linked dominant (CMTX), and therefore there was no father-to-son transmission. Our findings are consistent with the fact that CMTX-affected women usually have a milder phenotype than men at the same age [18]. The identical c.212T>G mutation in GJB1 was detected in the daughter of the proband, but no clinical symptoms were observed in this individual. As CMT onset usually occurs after adolescence, and the daughter was just 10 years old at the time of this study, it is likely that clinical symptoms had not yet manifested in this individual. However, because she refused electrophysiological examination, any sub-clinical indicators of CMT could not be examined.
GJB1 belongs to a highly conserved family of proteins, which form gap junctions in vertebrates and are widely expressed in myelinating Schwann cells [19]. Different GJB1 mutations have been reported [20]. Mutations at different locations in the protein cause different functional changes [21] and gap junctions formed with mutant GJB1 lead to altered morphological and biophysical characteristics [22]. In addition, different mutations at the same site in GJB1 may have different impacts on channel function. For example, the arginine 15 to glutamine and the histidine 94 to glutamine mutants form normal, functional channels, whereas the arginine 15 to tryptophan and histidine 94 to Y mutants do not [23]. The c.210_211insC insertion in the GJB1 coding region causes a frame shift and a stop at codon position 109 (I71fsX109) [24]. However, the c.212T>G mutation in GJB1 that we identified was present in all affected members of a Chinese CMTX pedigree but was absent in the control subjects, suggesting that the c.212T>G alteration is not a polymorphism. Ile 71 of GJB1 is highly conserved among many animal species implying important structural and/or functional roles. This implication is supported by the fact that the tertiary structure of the mutant GJB1 was predicted to be altered. Ile 71 is located in the first extracellular loop of GJB1, and our data suggested that the substitution at this site may affect the overall protein conformation. Therefore, we speculate that the mutant GJB1 fails to form functional gap junctions, or forms gap junctions with altered biophysical properties. However, we did not perform additional laboratory studies to assess of the function of the mutant connexin 32 protein. Thus, we should regard the mutation as putatively pathogenic. Our results expand the spectrum of mutations in GJB1 known to be associated with CMTX, and may contribute to the diagnosis of CMT and clinical genetic counseling. Although the tertiary structure of GJB1 appears to be modified by p.Ile71Ser, the mechanism by which this substitution facilitates disease development remains to be elucidated. Therefore, future research on the c.212T>G mutation in GJB1 should focus on understanding its role in the pathogenesis of CMTX. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Acknowledgements The authors thank all of the subjects for their participation in the study.
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References [1] Szigeti K, Lupski JR. Charcot–Marie–Tooth disease. Eur J Hum Genet 2009;17:703–10. [2] Pareyson D. Charcot–Marie–Tooth disease and related neuropathies: molecular basis for distinction and diagnosis. Muscle Nerve 1999;22:1498–509. [3] Reilly MM, Shy ME. Diagnosis and new treatments in genetic neuropathies. J Neurol Neurosurg Psychiatry 2009;80:1304–14. [4] Li J, Parker B, Martyn C, et al. The PMP22 gene and its related diseases. Mol Neurobiol 2013;47:673–98. [5] Siskind CE, Panchal S, Smith CO, et al. A review of genetic counseling for Charcot Marie Tooth disease (CMT). J Genet Couns 2013;22:422–36. [6] Dubourg O, Tardieu S, Birouk N, et al. Clinical, electrophysiological and molecular genetic characteristic of 93 patients with X-linked CMT disease. Brain 2001;124:1958–67. [7] Shy ME, Blake J, Krajewski K, et al. Reliability and validity of the CMT neuropathy score as a measure of disability. Neurology 2005;64:1209–14. [8] Miltenberger-Miltenyi G, Schwarzbraun T, Löscher WN, et al. Identification and in silico analysis of 14 novel GJB1, MPZ and PMP22 gene mutations. Eur J Hum Genet 2009;17:1154–9. [9] Fernández-Suárez XM, Schuster MK. Using the ensembl genome server to browse genomic sequence data. Curr Protoc Bioinformatics 2010(Suppl. 30):1–48. [10] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80. [11] Troshin PV, Procter JB, Barton GJ. Java bioinformatics analysis web services for multiple sequence alignment–JABAWS:MSA. Bioinformatics 2011;27:2001–2. [12] Carter H, Douville C, Stenson PD, et al. Identifying Mendelian disease genes with the variant effect scoring tool. BMC Genomics 2013;14:S3.
[13] Adzhubei I, Jordan DM, Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet 2013(Suppl. 76):1–41. [14] Sim NL, Kumar P, Hu J, et al. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res 2012;40:W452–7. [15] Schwarz JM, Rödelsperger C, Schuelke M, et al. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010;7:575–6. [16] Schwede T, Kopp J, Guex N, et al. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 2003;31:3381–5. [17] Raush E, Totrov M, Marsden BD, et al. A new method for publishing threedimensional content. PLoS One 2009;4:e7394. [18] Birouk N, LeGuern E, Maisonobe T, et al. X-linked Charcot–Marie–Tooth disease with connexin 32 mutations: clinical and electrophysiologic study. Neurology 1998;50:1074–82. [19] Menichella DM, Goodenough DA, Sirkowski E, et al. Connexins are critical for normal myelination in the CNS. J Neurosci 2003;13:5963–73. [20] Scherer SS, Kleopa KA. X-linked Charcot–Marie–Tooth disease. J Peripher Nerv Syst 2012;17:9–13. [21] Kleopa KA, Scherer SS. Molecular genetics of X-linked Charcot–Marie–Tooth disease. Neuromolecular Med 2006;8:107–22. [22] Abrams CK, Oh S, Ri Y, et al. Mutations in connexin 32: the molecular and biophysical bases for the X-linked form of Charcot–Marie–Tooth disease. Brain Res Rev 2000;32:203–14. [23] Abrams CK, Freidin MM, Verselis VK, et al. Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot–Marie– Tooth disease. Brain Res 2001;900:9–25. [24] Haites NE, Nelis E, Van Broeckhoven C. 3rd workshop of the European CMT consortium: 54th ENMC international workshop on genotype/phenotype correlations in Charcot–Marie–Tooth type 1 and hereditary neuropathy with liability to pressure palsies, 28–30 November 1997, Naarden, The Netherlands. Neuromuscul Disord 1998;8:591–603.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
A novel mutation in GJB1 (c.212T>G) in a Chinese family with X-linked Charcot–Marie–Tooth disease Fei Xiao, Jia-ze Tan, Xu Zhang, Xue-Feng Wang ⇑ Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, 1 You Yi Road, Chongqing 400016, China
a r t i c l e
i n f o
Article history: Received 15 May 2014 Accepted 4 August 2014
Keywords: Connexin32 Gap junction protein beta 1 GJB1 Hereditary sensory motor neuropathy X-linked Charcot–Marie–Tooth disease
a b s t r a c t Gap junction protein beta 1 (GJB1) gene mutations lead to X-linked Charcot–Marie–Tooth (CMTX) disease. We investigated a Chinese family with CMTX and identified a novel GJB1 point mutation. Clinical and electrophysiological features of the pedigree were examined, and sequence alterations of the coding region of GJB1 that encode connexin32 were determined by direct sequencing. Sequence alignment of the mutation site was performed using Clustal W. Mutation effects were analysed using PolyPhen-2, SIFT and Mutation Taster software. The three-dimensional structures of the mutant and wild-type proteins were predicted by modeling with SWISS MODEL online software. The affected family members displayed typical Charcot–Marie–Tooth phenotypes, but phenotypic heterogeneity was observed. Nerve conduction velocities of all affected patients were slow. Sequencing of GJB1 revealed a heterozygous T>G missense mutation at nucleotide 212 in the proband, the proband’s mother and the proband’s daughter. The affected male sibling of the proband displayed a hemizygous missense mutation with T>G transition at the identical position on the GJB1 gene. This mutation resulted in an amino acid change from isoleucine to serine that was predicted to lead to tertiary structural alterations that would disrupt the function of the GJB1 protein. A novel point mutation in GJB1 was detected, expanding the spectrum of GJB1 mutations known to be associated with CMTX. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Charcot–Marie–Tooth disease (CMT) is the most common hereditary motor and sensory neuropathy, affecting 1 in 2500 people worldwide [1]. Based on the motor nerve conduction velocity (MNCV) of the median nerve, CMT can be further classified into the demyelinating (CMT1) and axonal (CMT2) forms. The median MNCV is <38 m/s in CMT1 and >38 m/s in CMT2 [2]. The typical clinical features of CMT include progressive muscular weakness and atrophy of limbs, especially distally. Patients with CMT may have a family history of the disease, but CMT is clinically and genetically heterogeneous, even within a family [3]. A defect in the peripheral myelin protein 22 (PMP22) is the most common cause of CMT [4]. Thus, the PMP22 gene is generally the first gene to be examined in patients with CMT [5]. X-linked Charcot–Marie–Tooth disease (CMTX) is the second most frequent type of CMT, accounting for 10–20% of patients [6]. CMTX usually results from mutations in the gene encoding gap junction protein beta 1 (GJB1), and these mutations cause
⇑ Corresponding author. Tel./fax: +86 23 8901 2878. E-mail address: [email protected] (X.-F. Wang). http://dx.doi.org/10.1016/j.jocn.2014.08.028 0967-5868/Ó 2014 Elsevier Ltd. All rights reserved.
amino acid changes which alter the structure of the encoded protein, connexin 32. Here we report a Chinese family with X-linked dominant CMT and identify a novel GJB1 point mutation within the family.
2. Methods 2.1. Study participants A pedigree was drawn up for a Chinese family with CMTX who were referred to the Department of Neurology at the First Affiliated Hospital of Chongqing Medical University, Chongqing, China, in April 2012 (Fig. 1A). A detailed medical history was obtained for all family members. Healthy control subjects (n = 100; 50% male) were recruited from the outpatient clinic of the First Affiliated Hospital of Chongqing Medical University between May 2013 and July 2013. Members of the CMTX pedigree and control subjects were all of Chinese ancestry and resided in China. All family members underwent clinical and electrophysiological testing, including standardised electromyography and nerve conduction velocity (NCV) studies. CMT neuropathy scores [7] were determined by an experienced peripheral nerve clinician in order to assess the
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severity of the disease in the affected family members. The daughter of the proband (IV-2) refused electrophysiological inspection. MRI of the brain was conducted for the affected pedigree members. Subjects included in this study provided written informed consent, and the study protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.
Table 1 Primers used to amplify exons 1–3 of the gap junction beta 1 (GJB1) gene Region amplified
Primer sequences
Exon 1
F 50 -CCATAGGGAGCTCAGAGGATCAAT-30 R 50 -TGAAGGCGGAGGTCAGTAGAAG-30 F 50 -GTGTGTAGGGTGGGCGGAAGTC -30 R 50 -CTTCTTCGCTTTCATCCAACAT -30 (1) F 50 -AGTGCTATGGCGCCCGACTTT-30 R 50 -ATGAAGACGGCCTCAAACAAC-30 (2) F 50 -AAGAGGCACAAGGTCCACATC-30 R 50 -CTCACTCAGCAGCTTGTTGAT-30 (3) F 50 -CATCTGCATCATCCTCAATGTG-30 R 50 -CTCTAGCTCCCCACCCCTCCAG-30
Exon 2 Exon 3
2.2. Mutation analysis Genomic DNA was extracted from peripheral leukocytes of fresh blood samples taken from the proband (III-3), the elder brother (III-2), the parents (II-5 and II-6) and daughter (IV-2) of the proband, and from healthy control subjects, using a standard proteinase K digestion and phenol–chloroform extraction method. PMP22 duplication and mutation analysis were performed as described elsewhere [8]. All three exons of GJB1 were amplified by polymerase chain reaction (PCR) using the GeneAmp PCR System 9700 thermal cycler (Perkin Elmer, Shelton, CT, USA). The primer pairs (Table 1) were designed for exons 1–3 of GJB1 using Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA), according to the previously published sequence (GenBank RefSeq (Nucleotide) NG_008357; http://www.ensembl.org, [9]). Each 25 ll PCR reaction contained 50 ng of genomic DNA, 10 pmol of each forward and reverse primer, 5 mmol dNTP and 2.5 U of Taq polymerase (Takara Biotechnology, Dalian, China) in reaction buffer (Takara Biotechnology). The PCR cycles consisted of denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30–40 s, 57–63°C
F = forward, R = reverse.
for 30 s, and 72°C for 30–40 s (depending on which segment in GJB1 was being amplified) and a final elongation step at 72°C for 10 minutes. PCR products were purified by 1.5% agarose gel electrophoresis and directly sequenced with an ABI PRISM 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The data were analyzed with Chromas 2.22 chromatogram file editor software (Technelysium Pty Ltd, Tewantin, QLD, Australia). The base mutations in GJB1 were described and numbered according to criteria provided by the Ensembl genome browser [9]. 2.3. In silico analyses Multiple GJB1 protein sequence alignment across species was performed using Clustal W [10], and the alignment results were
Fig. 1. Pedigree diagram and clinical pictures of the family. (A) Pedigree of the Chinese family with Charcot–Marie–Tooth disease (CMT). Squares = males, circles = females, filled squares and circles = affected patients, grey circle = mutation carrier, arrow = proband, oblique line = deceased. (B) Image showing atrophy in the distal lower limbs of the proband (III-3). (C) Image showing the more severe atrophy in the distal lower limbs of III-2. (D) Image showing pes cavus of II-5.
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visualized with Jalview software (http://www.jalview.org/) [11]. Exome Variant Server (evs.gs.washington.edu/) [12] was used to check if the variant is found in the normal population. The potential functional impacts of mutations within GJB1 were predicted using Polymorphism Phenotyping 2 (PolyPhen-2) software (http:// geneics.bwh.harvard.edu/pph2/) [13], Sorting Intolerant from Tolerant (SIFT) software (http://sift.jcvi.org/) [14] and Mutation Taster (http://www.mutationtaster.org/) [15]. PolyPhen-2 classifies predicted effects of amino acid substitutions on the function of human proteins as ‘‘benign’’, ‘‘possibly damaging’’, ‘‘probably damaging’’, or ‘‘unknown’’. Functional impact of the mutation was predicted as ‘‘tolerated’’ or ‘‘damaging’’ for SIFT and as ‘‘polymorphism’’ or ‘‘disease causing’’ for Mutation Taster. To further investigate the effect of the novel missense mutation within GJB1, we modeled the three-dimensional structures of GJB1 with and without the mutation using SWISS-MODEL online software (http://beta.swissmodel.expasy.org/) [16]. The results were displayed with Molsoft ICM software (http://www.molsoft.com/ icm_browser_pro.html) [17].
3. Results 3.1. Clinical findings All affected individuals in the pedigree showed varying degrees (mild to severe) of muscle atrophy, diminished reflexes and foot drop. The CMT neuropathy scores of the proband, the proband’s mother, and the proband’s elder brother were 11, 7, and 21, respectively. The proband began to notice weakness and atrophy in the distal lower extremities at 26 years of age and first visited the neurology department because of weakness and atrophy of the distal muscles of both upper and lower limbs at age 34. Neurological examination revealed weakness and atrophy of the distal muscles of the lower limbs bilaterally (Fig. 1B) without obvious involvement of the proximal muscles, and pes cavus was also observed. The power values were 4/5 for the finger abductor muscles, 4/5 for the gastrocnemius muscle, and 3/5 for the anterior tibialis muscle, based on the Medical Research Council scale. The patient’s vibration sense was severely impaired, and deep tendon reflexes were absent in all extremities. The proband’s elder brother (III-2, aged 36) had experienced similar symptoms from the age of 15. At the time of this study, however, his muscle weakness and atrophy in the distal lower extremities were more severe than the proband. He experienced fatigue when walking and had obvious atrophy of the distal muscles of the limbs (Fig. 1C) and pes cavus. His power values were 3/5 for the finger abductor muscles, 3/5 for the gastrocnemius muscle, and 3/5 for the anterior tibialis muscle. Deep tendon reflexes were absent in all extremities. The proband’s mother (II5) had relatively mild CMT symptoms compared to the proband. The mother first noticed mild weakness and atrophy in the distal forearm and pes cavus (Fig. 1D) in her thirties, but weakness and
atrophy were not observed in the distal lower extremities and she had no difficulty walking. For all affected pedigree members, no enhanced reflexes were observed. However, the father (II-6), nephew (IV-1) and daughter (IV-2) of the proband showed no clinical symptoms of CMT. Brain MRI of the affected individuals were normal. However, NCV were reduced or not detectible in all affected pedigree members. The NCV were lower in the affected male (III-2) than in the two affected females (II-5 and III-3), with reduced amplitude and prolonged latency. NCV in the median motor nerve of all affected patients were <38 m/s, which is in accordance with CMT1. Table 2 summarises the electrophysiological data of the family members. 3.2. Mutational analysis of PMP22 and GJB1 We did not find any copy number abnormalities or mutations in the coding region of PMP22 of the proband. Bidirectional sequencing of the coding region containing exons 1–3 of GJB1 revealed a heterozygous T>G missense mutation at nucleotide position 212 in exon 3 in the proband (c.212T>G; Fig. 2A). The same heterozygous mutation was also detected in the mother and daughter of the proband (Fig. 2B, C). In addition, the affected male sibling of the proband was hemizygous for the missense mutation at this position (Fig. 2D). The c.212T>G mutation would result in an amino acid change from isoleucine (Ile) to serine (Ser) at codon 71 (p.Ile71Ser). The c.212T>G mutation was absent in the father of the proband (Fig. 2E) and in the 100 healthy control subjects (50% male; Fig. 2F). Fifteen single nucleotide polymorphisms of GJB1 have been reported in the Exome Variant Server database, however to our knowledge this mutation has not been reported as a polymorphism, suggesting that it is not common and does not exist in the normal population. No mutations were detected in exons 1 or 2 of GJB1 in any members of the target family or controls. 3.3. Predictions of functional impacts of the c.212T>G mutation on the GJB1 protein Alignment of the amino acid sequences surrounding the Ile at position 71 in several distantly related species showed that this Ile is located in a highly conserved region of GJB1 in humans and other species (Fig. 3), indicating its functional importance. The p.Ile71Ser substitution was predicted to be ‘‘probably damaging’’ and disrupt the function of GJB1 by the PolyPhen-2 software (score 0.993; sensitivity 0.70, specificity 0.97). Mutation Taster and SIFT predicted that the p.Ile71Ser mutation is functionally ‘‘disease causing’’ and ‘‘damaging’’ (SIFT score of 0), respectively. The three-dimensional structure of the mutant GJB1 was also predicted to be altered. Substitution of the nonpolar Ile with the polar Ser, likely disrupts the a-helical structure of the protein. Comparison of the predicted tertiary structures of the wild-type and mutant proteins revealed that p.Ile71Ser at the start of an
Table 2 Electrophysiological data of the affected patients Patient
II-5 III-2 III-3
Age, years
Sex
59 36 34
F M F
Median
Median
Ulnar
Peroneal
Sural
Motor CMAP (mV)
MNCV (m/s)
SNAP (lV)
SNCV (m/s)
Motor CMAP (mV)
MNCV (m/s)
Motor CMAP (mV)
MNCV (m/s)
SNAP (lV)
SNCV (m/s)
3.7 0.6 1.1
37.9 26.3 30.4
11 NR 8.2
37 NR 25.7
4.1 1.9 2.6
39.5 28.8 35.2
1.2 NR NR
36.3 NR NR
4.6 NR 3.1
34.9 NR 19.5
CMAP = compound muscle action potential, F = female, M = male, MNCV = motor nerve conduction velocity, NR = not recordable, SNAP = sensory nerve action potential, SNCV = sensory nerve conduction velocity.
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Fig. 2. DNA sequencing chromatographs of exon 3 of the gap junction beta 1 (GJB1) gene. (A) Nucleotide sequence of this region in the proband (III-3), (B) the daughter of the proband (IV-2), (C) the mother of the proband (II-5), (D) the affected brother of the proband (III-2), (E) the father of the proband (II-6), and (F) a control subject. The heterozygous missense mutation (arrow), c.212T>G, was identified in the proband, the daughter and mother of the proband, and the hemizygous mutation was identified in the brother of the proband. This mutation was absent in the father of the proband and the control subjects.
a-helix led to strengthening of the hydrogen bond between Ser 71 and tyrosine (Y) 157, shortening the distance between the two residues (Fig. 4). Because Y157 is at the end of another a-helix, this conformational change could potentially affect the overall a-helical folding of the protein.
4. Discussion The understanding of the genetic basis of CMT has increased greatly over the past 20 years. The most frequent cause of CMT is a duplication abnormality in the PMP22 gene. However, we did
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Fig. 3. Conservation of the GJB1 amino acid sequence among different species. Sequence alignment showing that isoleucine 71 (arrow) and its neighboring sequences are highly conserved in a variety of species.
Fig. 4. SWISS-MODEL generated three-dimensional models of the portion of gap junction beta 1 (GJB1) encoded by exon 3 [16]. Hydrogen bond distances between the amidogen group of serine (Ser) 71 and the hydroxyl group in the benzene ring of tyrosine 157 are different, at (A) 3.5 angstrom in the wild-type and (B) 2.9 angstrom in the mutant p.Ile71Ser protein.
not detect duplications or mutations in PMP22 in the proband of our pedigree. Phenotypic heterogeneity was observed within this family, with males tending to be more severely affected than females according to CMT neuropathy score assessment. Pedigree analysis showed that the inheritance mode was X-linked dominant (CMTX), and therefore there was no father-to-son transmission. Our findings are consistent with the fact that CMTX-affected women usually have a milder phenotype than men at the same age [18]. The identical c.212T>G mutation in GJB1 was detected in the daughter of the proband, but no clinical symptoms were observed in this individual. As CMT onset usually occurs after adolescence, and the daughter was just 10 years old at the time of this study, it is likely that clinical symptoms had not yet manifested in this individual. However, because she refused electrophysiological examination, any sub-clinical indicators of CMT could not be examined.
GJB1 belongs to a highly conserved family of proteins, which form gap junctions in vertebrates and are widely expressed in myelinating Schwann cells [19]. Different GJB1 mutations have been reported [20]. Mutations at different locations in the protein cause different functional changes [21] and gap junctions formed with mutant GJB1 lead to altered morphological and biophysical characteristics [22]. In addition, different mutations at the same site in GJB1 may have different impacts on channel function. For example, the arginine 15 to glutamine and the histidine 94 to glutamine mutants form normal, functional channels, whereas the arginine 15 to tryptophan and histidine 94 to Y mutants do not [23]. The c.210_211insC insertion in the GJB1 coding region causes a frame shift and a stop at codon position 109 (I71fsX109) [24]. However, the c.212T>G mutation in GJB1 that we identified was present in all affected members of a Chinese CMTX pedigree but was absent in the control subjects, suggesting that the c.212T>G alteration is not a polymorphism. Ile 71 of GJB1 is highly conserved among many animal species implying important structural and/or functional roles. This implication is supported by the fact that the tertiary structure of the mutant GJB1 was predicted to be altered. Ile 71 is located in the first extracellular loop of GJB1, and our data suggested that the substitution at this site may affect the overall protein conformation. Therefore, we speculate that the mutant GJB1 fails to form functional gap junctions, or forms gap junctions with altered biophysical properties. However, we did not perform additional laboratory studies to assess of the function of the mutant connexin 32 protein. Thus, we should regard the mutation as putatively pathogenic. Our results expand the spectrum of mutations in GJB1 known to be associated with CMTX, and may contribute to the diagnosis of CMT and clinical genetic counseling. Although the tertiary structure of GJB1 appears to be modified by p.Ile71Ser, the mechanism by which this substitution facilitates disease development remains to be elucidated. Therefore, future research on the c.212T>G mutation in GJB1 should focus on understanding its role in the pathogenesis of CMTX. Conflicts of Interest/Disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication. Acknowledgements The authors thank all of the subjects for their participation in the study.
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