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Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease
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Case report
Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease R. Kulshrestha a,*, S. Burton-Jones b, T. Antoniadi c, M. Rogers d, Z. Jaunmuktane e, S. Brandner e, N. Kiely a, R. Manuel f, T. Willis a a
Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, UK b University Hospital Bristol NHS Trust, Bristol, UK c West Midlands Molecular Genetics Lab, Birmingham, UK d Cardiff and Vale UHB – Medical Genetics, UK e UCL Institute of Neurology, London, UK f Royal Stoke University Hospital, Newcastle Road, Stoke-on-Trent, UK Received 23 December 2016; received in revised form 23 April 2017; accepted 1 May 2017
Abstract X-linked Charcot-Marie-Tooth disease (CMT) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene. This gene has nerve specific P2 promoter that work synergistically with SOX10 and EGR2 genes to initiate transcription. Mutation in this region is known to cause Schwann cell dysfunction. A single large family of X linked peripheral neuropathy was identified in our practice. Next generation sequencing for targeted panel assay identified an upstream exon-splicing deletion identified extending from nucleotide c.-5413 to approximately – c.-49. This matches the sequence of 32 nucleotides at positions c.*218–*249 in the 3’UTR downstream of the GJB1 gene. The deleted fragment included the entire P2 promoter region. The deletion segregated with the disease. To our knowledge a deletion of the P2 promoter alone as a cause of CMT has not been reported previously. © 2017 Elsevier B.V. All rights reserved. Keywords: Charcot-Marie-Tooth disease; X-linked; GJB1; Connexin 32; Deletion; P2 promoter; Schwann cell dysfunction
1. Introduction X linked Charcot-Marie-Tooth (CMT) disease is second most common cause of CMT caused by mutation in gap junction beta 1 (GJB1) gene which code for connexin 32 (CX32) [1]. Clinically, patients present with distal muscle weakness, wasting and sensory loss [2]. The connexin 32 messenger RNA and protein are expressed in Schwann cells in peripheral nervous system [3], confined to Schmidt-Lantermann incisures and paranodal segment of nodes of Ranvier [1]. GJB1 gene has nerve tissue specific P2 promoter [4,5] that is crucial in initiating transcription [6]. Mutations in the P2 promoter have been reported to cause X linked CMT suggesting its impairment leading to Schwann cell dysfunction [7,8]. The loss of connexin 32 expression is secondary to failed activation of its promoter by SOX10 and EGR2 which work synergistically for GJB1 gene
* Corresponding author. Robert Jones and Agnes Hunt Orthopaedic Hospital, Gobowen, Oswestry SY10 7AG, UK. Fax: +01691404629. E-mail address: [email protected] (R. Kulshrestha).
transcription [9]. In this case series, we report a novel mutation of P2 promoter region of GJB1 gene segregating with the disease. 2. Patients and method In our practice, we identified a large family of X linked inherited peripheral neuropathy with unconfirmed molecular genetics. Genomic DNA was extracted from peripheral blood according to standard methods. Analysis of 56 genes associated with inherited peripheral neuropathy (IPN) was achieved using a custom-designed next generation sequencing (NGS) targeted panel assay. Raw data analysis and filtering was performed using a bespoke open-source pipeline with UCSC hg19 human genome as the reference. Basic copy number enumeration was performed using the CONTRA tool as a component of the analysis pipeline [10]. Clinical details including onset of symptoms, signs, neurophysiology and histology features of affected family members were obtained from the case records with the consent of participants for this publication.
http://dx.doi.org/10.1016/j.nmd.2017.05.001 0960-8966/© 2017 Elsevier B.V. All rights reserved. Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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3. Results 3.1. Genetics In the female index patient no pathogenic sequence variants were detected on testing for panel genes including SOX10 and EGR2. However on review, target coverage analysis of the gene panel data indicated a possible heterozygous partial deletion in the GJB1 gene region. This deletion was evident on MLPA dosage analysis13 using MRC Holland probe mix P405-A1, which showed abnormal dosage of a single probe, indicating a heterozygous deletion within or including the exon 1B (noncoding) region of the GJB1 gene at Xq13.1, and corresponding to the P2 promoter. Direct PCR (using primers flanking the deletion break points) and Sanger sequence analysis was subsequently used to confirm and characterise this deletion further. The identified deletion in the proband removed 5365 nucleotides and extends from position c.-5413 (5769 bases upstream of the ATG start codon) to nucleotide c.-49 (405 bases upstream of the ATG) inclusive. Additionally, a 32 base pair sequence is inserted between these break points; a BLAST search [http://blast.ncbi.nlm.nih.gov/Blast.cgi] indicated that this matches the sequence of 32 nucleotides at positions c.*218_*249 in the 3’ untranslated region, downstream of the GJB1 gene. Sanger sequence analysis has revealed that it is likely this sequence is duplicated and inserted between the break points. The deleted fragment in this patient (c.-5413_-49) includes the entire P2 promoter (Fig. 1). This deletion is confirmed to be present in all affected family members. 3.2. Clinical features Affected individuals showed clinical evidence of a length dependent peripheral neuropathy. Clinical features of affected individuals were variable. They had clawing of toes, feet deformity (cavovarus and planovalgus), wasting of small muscles of hand (Fig. 2), absent reflexes and a glove and stocking distribution of sensory involvement. Clinical features are summarized in Table 1. Males had earlier onset of symptoms than
females. Neuropathy was inherited in X linked dominant fashion with no male-to-male transmission (Fig. 3). Five affected family members had neurophysiology assessment. They had sensorimotor demyelinating (four patients) and axonal (one patient) neuropathy. Nerve conduction velocities were considerably slowed (<38 m/sec), suggesting serious demyelinating component in four patients (Table 2). They did not have central nervous system features hence magnetic resonance imaging of brain and spine was not performed. 3.3. Histology The sural nerve biopsy was assessed in one patient by light microscopy and ultrastructuraly by electron microscopy (Fig. 4). Immunohistochemical (IHC) shows prominent loss of large myelinated fibres (IHC for myelin basic protein, SMI94 antibody), whilst unmyelinated and small myelinated fibres are better preserved (immunostaining for neurofilaments, SMI31 antibody). Ultrastructural examination highlights the regeneration clusters but shows no signs of active macrophage-associated demyelination or chronic demyelinating/re-myelinating process. The nerve biopsy excluded inflammation, showed chronic axonal neuropathy with no evidence of inflammatory vasculitic process. 4. Discussion GJB1 gene, a common cause of X linked CMT maps to chromosome Xq13.1 and is 10 kb in length. This gene encodes 238-amino acid gap junction, beta 1/connexin 32 protein that is involved to form hemichannel connexins as diffusion pathway between Schwann cell nucleus and axon [11]. The gene has nerve specific P2 promoter [4,5], coding region (exon 2) and three non-coding exons (1,1A and 1B). Pathogenic mutations are described in both coding and non-coding region of the gene (http://www.molgen.ua.ac.be/CMTMutations/). Similarly pathogenic variants in P2 promoter region have been reported [7,8]. In our family, the deleted fragment (c.-5413_-49) includes the entire P2 promoter region of the gene and segregated with the disease. All affected family members have typical features
Fig. 1. Map of GJB1 gene region showing location of P2 promoter and deletion identified in this study. Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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A
B
3
D
E
C
Fig. 2. Clinical features. A,B: 36-year-old female, distal muscle wasting, clawing of toes and cavus deformity of foot. C: wasting of small muscles of hand in same patient. D,E: 20-year-old male, varus and cavus deformity of the foot with clawing of toes.
Table 1 Clinical features of affected and genetically confirmed family members. Gender Decade Age in Presenting features of onset years
Foot deformity
M
First
65
F
Second
35
F
Fourth
46
Bilateral cavovarus feet with fixed flexion N N contractures of toes (hammer toes) Right hind foot equinus, surgically N N corrected Bilateral planovalgus feet N N
F
Second
56
Planovalgus feet
M
First
51
M
First
19
M
First
19
F
Second
26
Bilateral cavovarus deformity with hammer toes Hind foot varus and cavus deformity of foot Cavovarus deformity, surgical correction of left side Bilateral planovalgus feet
F F
Second Third
15 37
F
Second
38
F F M
Third Third First
33 24 19
M
First
12
M
First
5
Pain in legs and progressive foot deformity Pain in calves, clawing of toes, deterioration of hand function Altered gait pattern, unsteady at walking, struggling with hand function, cramps Loss of balance, frequent falls and leg cramps. Walking difficulty, altered shape of feet Abnormal shape of feet and frequent falls Abnormal shape of feet, pain, cramps and frequent falls Frequent falls, trips, pain in feet, struggle with hand function, Pain in feet, cramps and clawing of toes Unsteady gait, tripping and pain Pain in leg, frequent trips and falls, struggle with hand function Pain in feet, cramps Pain in leg, tripping, poor hand function Ankles give way on running, pes cavus, struggle with hand function Frequent falls, pain in calf muscles, difficulty at running Slow at running, frequent falls and pain in legs
S
HD Weakness UL+LL
CNS or other features
Distal UL+LL
N
Distal UL+LL
N
Distal UL+LL
N
N N
Distal LL
N
N N N N
Distal UL +LL Positive sensory symptoms Distal LL N
N N
Distal UL+LL
N N
Distal UL +LL Tremors
Clawing of toes Hind foot varus with loading on lateral border. Normal shape
N N N N
Distal LL Distal LL
N N
Distal LL
Cavovarus feet and clawing of toes Hind foot varus deformity Cavovarus feet with clawing
N N N N N N
Positive sensory symptoms Distal LL N Distal UL +LL N Distal UL +LL N
Bilateral cavovarus feet
N N
Distal UL+LL
Tremors
Planovalgus feet
N N
Distal LL
N
N
N N
Y: yes; N: no; LL: lower limb; UL: upper limb; S: scoliosis; HD: hip dysplasia; CNS: central nervous system. Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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Fig. 3. Family tree: generation I-VI, no male to male transmission. Table 2 Neurophysiology results. Nerve
Normal values
F
M
F
F
M
Median SNCV (m/s) Ulnar SNCV (m/s) Radial SNAP (µV) Radial SNCV (m/s) Sural SNAP (µV) Sural SNCV (m/s) Median CMAP (mV) Median MNCV (m/s) Ulnar CMAP (mV) Ulnar MNCV (m/s) Peroneal CMAP (mV) Peroneal MNCV (m/s) Tibial CMAP (mV) Tibial MNCV (m/s)
>56 >55 >14 >50 >6 >43 >4 >48 >6 >51 >4 >43 >4 >41
35 38 ND ND ND ND 5 35 7 32 ND ND ND ND
32.1 32.8 ND ND 7.6 35.3 ND 33.3 ND ND 0.5 43.3 0.7 42.5
33.5 29.9 ND ND ND ND ND ND ND 33.3 ND ND ND ND
34 ND ND ND ND ND ND 29 ND 31 ND ND ND ND
37.5 34 16.6 29.8 7.4 36.6 7.7 44.9 4.6 43.6 3.9 42.9 11.0 45.7
SNCV: sensory nerve conduction velocity; MNCV: motor nerve conduction velocity; SNAP: sensory nerve action potential; CMAP: compound muscle action potential; F: female; M: male; ND: not done.
of peripheral neuropathy with varying severity suggesting absence of P2 in causing pathogenic nerve dysfunction. Male members reported early onset of symptoms. None of the affected individuals had hip dysplasia which is an association described with CMT [11]. They did not have central nervous
involvement as reported with some X linked CMT families [12,13]. Electrophysiology was consistent with peripheral nerve dysfunction and nerve biopsy in one individual excluded inflammatory cause of neuropathy. P2 promoter region is important for gene function as reduced or absent (as in our family) activity will cause a reduction/loss of messenger RNA transcript, hence reduced GJB1 protein [9]. Functional analysis of promoter region by Houlden et al. (2004) identified its role to cause Schwann cell dysfunction by interacting with SOX10 and EGR2 genes [9]. Interaction between them is complex. SOX10 and EGR2 bind to the P2 promoter region of GJB1 gene at S1-S2 and E2-E3 sites respectively for transcription [14,15]. This interaction between GJB1 P2 promoter, SOX10 and EGR2 can be interrupted by either mutations in SOX10 [16] and EGR2 [17] genes or mutation in P2 promoter region of GJB1 [9]. Our patients did not have mutations in SOX10 and EGR2 genes supporting restriction of transcription by latter mechanism. This observation strengthens the hypothesis by Houlden et al. that P2 promoter is central in function of Schwann cell in peripheral nerve. 5. Conclusion We have described a novel mutation deleting entire P2 promoter of GJB1 gene in a single large family with CMT.
Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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Fig. 4. Sural nerve biopsy features. Immunohistochemical (IHC) examination (A and B) shows prominent loss of large myelinated fibres (A: IHC for myelin basic protein, SMI94 antibody), whilst unmyelinated and small myelinated fibres are better preserved (B: immunostaining for neurofilaments, SMI31 antibody). Semi-thin resin section stained with methylene blue azure – basic fuchsin (C) confirms markedly reduced numbers of large normally myelinated fibres, with relatively frequent small and medium sized thinly myelinated fibres of which many are associated with regeneration clusters (arrow) but there is no active axonal degeneration. Ultrastructural examination (D) highlights the regeneration clusters (arrow) but shows no signs of active macrophage-associated demyelination or chronic demyelinating/re-myelinating process. Scale bar: 30 µm in A and B, 15 µm in C, 2 µm in D.
Inheritance and phenotype of affected individuals had classical features of X linked peripheral neuropathy. This study affirms the role of P2 promoter being vital for Schwann cell function.
References [1] Bergoffen J, Scherer SS, Wang S, Scott MO, Bone LJ, Paul DL, et al. Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science 1993;262:2039–42. [2] Pareyson D, Marchesi C. Diagnosis, natural history, and management of Charcot-Marie-Tooth disease. Lancet Neurol 2009;8:654–67. [3] Scherer SS, Deschenes SM, Xu YT, Grinspan JB, Fischbeck KH, Paul DL. Connexin32 is a myelin-related protein in the PNS and CNS. J Neurosci 1995;15:8281–94. [4] Söhl G, Gillen C, Bosse F, Gleichmann M, Müller HW, Willecke K. A second alternative transcript of the gap junction gene connexin 32 is expressed in murine Schwann cells and modulated in injured sciatic nerve. Eur J Cell Biol 1996;69:267–75. [5] Neuhaus IM, Dahl G, Werner R. Use of alternate promoters for tissue-specific expression of the gene coding for connexin 32. Gene 1995;158:257–62. [6] Neuhaus IM, Bone L, Wang S, Ionasescu V, Werner R. The human connexin 32 gene is transcribed from two tissue specific promoters. Biosci Rep 1996;16:239–48. [7] Murphy SM, Polke J, Manji H, Blake J, Reiniger L, Sweeney M, et al. A novel mutation in the nerve-specific 5’UTR of the GJB1 gene causes X-linked Charcot-Marie-Tooth disease. J Peripher Nerv Syst 2011;16: 65–70.
[8] Tsai PC, Chen CH, Liu AB, Chen YC, Soong BW, Lin KP, et al. Mutation analysis of the 5’non-coding region of GJB1 in a Taiwanese cohort with Charcot-Marie-Tooth neuropathy. J Neurol Sci 2013;332:51–5. [9] Houlden H, Girard M, Cockerell C, Ingram D, Wood NW, Goossens M, et al. Connexin 32 promoter P2 mutations: a mechanism of peripheral nerve dysfunction. Ann Neurol 2004;56:730–4. [10] Li J, Lupat R, Amarasinghe K, Thompson ER, Doyle MA, Ryland GL, et al. CONTRA: copy number analysis for targeted resequencing. Bioinformatics 2012;28(10):1307–13. [11] Evans WH. Cell communication across gap junctions: a historical perspective and current developments. Biochem Soc Trans 2015;43(3): 450–9. [12] Panas M, Kalfakis N, Karadimas C, Vassilopoulos D. Episodes of generalized weakness in two sibs with the C164T mutation of the connexion 32 gene. Neurology 2001;57:1906–8. [13] Taylor RA, Simon EM, Marks HG, Scherer SS. The CNS phenotype of X-linked Charcot-Marie-Tooth disease: more than a peripheral problem. Neurology 2003;61:1475–8. [14] Boundurand N, Girard M, Pingault V, Lemort N, Dubourg O, Goossens M. Human connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10. Hum Mol Genet 2011;10:2783–95. [15] Musso M, Balestra P, Bellone L, Cassandrini D, Di Maria E, Doria LL, et al. The D355V mutation decreases EGR2 binding to an element within the Cx32 promoter. Neurobiol Dis 2001;8:700–6. [16] Inoue K, Tanabe Y, Lupski JR. Myelin deficiencies in both the central and the peripheral nervous system associated with a SOX10 mutation. Ann Neurol 1999;46:313–18. [17] Warner LE, Mancias P, Butler IJ, McDonald CM, Keppen L, Koob KG, et al. Mutations in the early growth response 2 (EGR2) gene are associated with hereditary myelinopathies. Nat Genet 1998;18:382–4.
Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
ScienceDirect Neuromuscular Disorders ■■ (2017) ■■–■■ www.elsevier.com/locate/nmd
Case report
Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease R. Kulshrestha a,*, S. Burton-Jones b, T. Antoniadi c, M. Rogers d, Z. Jaunmuktane e, S. Brandner e, N. Kiely a, R. Manuel f, T. Willis a a
Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, UK b University Hospital Bristol NHS Trust, Bristol, UK c West Midlands Molecular Genetics Lab, Birmingham, UK d Cardiff and Vale UHB – Medical Genetics, UK e UCL Institute of Neurology, London, UK f Royal Stoke University Hospital, Newcastle Road, Stoke-on-Trent, UK Received 23 December 2016; received in revised form 23 April 2017; accepted 1 May 2017
Abstract X-linked Charcot-Marie-Tooth disease (CMT) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene. This gene has nerve specific P2 promoter that work synergistically with SOX10 and EGR2 genes to initiate transcription. Mutation in this region is known to cause Schwann cell dysfunction. A single large family of X linked peripheral neuropathy was identified in our practice. Next generation sequencing for targeted panel assay identified an upstream exon-splicing deletion identified extending from nucleotide c.-5413 to approximately – c.-49. This matches the sequence of 32 nucleotides at positions c.*218–*249 in the 3’UTR downstream of the GJB1 gene. The deleted fragment included the entire P2 promoter region. The deletion segregated with the disease. To our knowledge a deletion of the P2 promoter alone as a cause of CMT has not been reported previously. © 2017 Elsevier B.V. All rights reserved. Keywords: Charcot-Marie-Tooth disease; X-linked; GJB1; Connexin 32; Deletion; P2 promoter; Schwann cell dysfunction
1. Introduction X linked Charcot-Marie-Tooth (CMT) disease is second most common cause of CMT caused by mutation in gap junction beta 1 (GJB1) gene which code for connexin 32 (CX32) [1]. Clinically, patients present with distal muscle weakness, wasting and sensory loss [2]. The connexin 32 messenger RNA and protein are expressed in Schwann cells in peripheral nervous system [3], confined to Schmidt-Lantermann incisures and paranodal segment of nodes of Ranvier [1]. GJB1 gene has nerve tissue specific P2 promoter [4,5] that is crucial in initiating transcription [6]. Mutations in the P2 promoter have been reported to cause X linked CMT suggesting its impairment leading to Schwann cell dysfunction [7,8]. The loss of connexin 32 expression is secondary to failed activation of its promoter by SOX10 and EGR2 which work synergistically for GJB1 gene
* Corresponding author. Robert Jones and Agnes Hunt Orthopaedic Hospital, Gobowen, Oswestry SY10 7AG, UK. Fax: +01691404629. E-mail address: [email protected] (R. Kulshrestha).
transcription [9]. In this case series, we report a novel mutation of P2 promoter region of GJB1 gene segregating with the disease. 2. Patients and method In our practice, we identified a large family of X linked inherited peripheral neuropathy with unconfirmed molecular genetics. Genomic DNA was extracted from peripheral blood according to standard methods. Analysis of 56 genes associated with inherited peripheral neuropathy (IPN) was achieved using a custom-designed next generation sequencing (NGS) targeted panel assay. Raw data analysis and filtering was performed using a bespoke open-source pipeline with UCSC hg19 human genome as the reference. Basic copy number enumeration was performed using the CONTRA tool as a component of the analysis pipeline [10]. Clinical details including onset of symptoms, signs, neurophysiology and histology features of affected family members were obtained from the case records with the consent of participants for this publication.
http://dx.doi.org/10.1016/j.nmd.2017.05.001 0960-8966/© 2017 Elsevier B.V. All rights reserved. Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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3. Results 3.1. Genetics In the female index patient no pathogenic sequence variants were detected on testing for panel genes including SOX10 and EGR2. However on review, target coverage analysis of the gene panel data indicated a possible heterozygous partial deletion in the GJB1 gene region. This deletion was evident on MLPA dosage analysis13 using MRC Holland probe mix P405-A1, which showed abnormal dosage of a single probe, indicating a heterozygous deletion within or including the exon 1B (noncoding) region of the GJB1 gene at Xq13.1, and corresponding to the P2 promoter. Direct PCR (using primers flanking the deletion break points) and Sanger sequence analysis was subsequently used to confirm and characterise this deletion further. The identified deletion in the proband removed 5365 nucleotides and extends from position c.-5413 (5769 bases upstream of the ATG start codon) to nucleotide c.-49 (405 bases upstream of the ATG) inclusive. Additionally, a 32 base pair sequence is inserted between these break points; a BLAST search [http://blast.ncbi.nlm.nih.gov/Blast.cgi] indicated that this matches the sequence of 32 nucleotides at positions c.*218_*249 in the 3’ untranslated region, downstream of the GJB1 gene. Sanger sequence analysis has revealed that it is likely this sequence is duplicated and inserted between the break points. The deleted fragment in this patient (c.-5413_-49) includes the entire P2 promoter (Fig. 1). This deletion is confirmed to be present in all affected family members. 3.2. Clinical features Affected individuals showed clinical evidence of a length dependent peripheral neuropathy. Clinical features of affected individuals were variable. They had clawing of toes, feet deformity (cavovarus and planovalgus), wasting of small muscles of hand (Fig. 2), absent reflexes and a glove and stocking distribution of sensory involvement. Clinical features are summarized in Table 1. Males had earlier onset of symptoms than
females. Neuropathy was inherited in X linked dominant fashion with no male-to-male transmission (Fig. 3). Five affected family members had neurophysiology assessment. They had sensorimotor demyelinating (four patients) and axonal (one patient) neuropathy. Nerve conduction velocities were considerably slowed (<38 m/sec), suggesting serious demyelinating component in four patients (Table 2). They did not have central nervous system features hence magnetic resonance imaging of brain and spine was not performed. 3.3. Histology The sural nerve biopsy was assessed in one patient by light microscopy and ultrastructuraly by electron microscopy (Fig. 4). Immunohistochemical (IHC) shows prominent loss of large myelinated fibres (IHC for myelin basic protein, SMI94 antibody), whilst unmyelinated and small myelinated fibres are better preserved (immunostaining for neurofilaments, SMI31 antibody). Ultrastructural examination highlights the regeneration clusters but shows no signs of active macrophage-associated demyelination or chronic demyelinating/re-myelinating process. The nerve biopsy excluded inflammation, showed chronic axonal neuropathy with no evidence of inflammatory vasculitic process. 4. Discussion GJB1 gene, a common cause of X linked CMT maps to chromosome Xq13.1 and is 10 kb in length. This gene encodes 238-amino acid gap junction, beta 1/connexin 32 protein that is involved to form hemichannel connexins as diffusion pathway between Schwann cell nucleus and axon [11]. The gene has nerve specific P2 promoter [4,5], coding region (exon 2) and three non-coding exons (1,1A and 1B). Pathogenic mutations are described in both coding and non-coding region of the gene (http://www.molgen.ua.ac.be/CMTMutations/). Similarly pathogenic variants in P2 promoter region have been reported [7,8]. In our family, the deleted fragment (c.-5413_-49) includes the entire P2 promoter region of the gene and segregated with the disease. All affected family members have typical features
Fig. 1. Map of GJB1 gene region showing location of P2 promoter and deletion identified in this study. Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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A
B
3
D
E
C
Fig. 2. Clinical features. A,B: 36-year-old female, distal muscle wasting, clawing of toes and cavus deformity of foot. C: wasting of small muscles of hand in same patient. D,E: 20-year-old male, varus and cavus deformity of the foot with clawing of toes.
Table 1 Clinical features of affected and genetically confirmed family members. Gender Decade Age in Presenting features of onset years
Foot deformity
M
First
65
F
Second
35
F
Fourth
46
Bilateral cavovarus feet with fixed flexion N N contractures of toes (hammer toes) Right hind foot equinus, surgically N N corrected Bilateral planovalgus feet N N
F
Second
56
Planovalgus feet
M
First
51
M
First
19
M
First
19
F
Second
26
Bilateral cavovarus deformity with hammer toes Hind foot varus and cavus deformity of foot Cavovarus deformity, surgical correction of left side Bilateral planovalgus feet
F F
Second Third
15 37
F
Second
38
F F M
Third Third First
33 24 19
M
First
12
M
First
5
Pain in legs and progressive foot deformity Pain in calves, clawing of toes, deterioration of hand function Altered gait pattern, unsteady at walking, struggling with hand function, cramps Loss of balance, frequent falls and leg cramps. Walking difficulty, altered shape of feet Abnormal shape of feet and frequent falls Abnormal shape of feet, pain, cramps and frequent falls Frequent falls, trips, pain in feet, struggle with hand function, Pain in feet, cramps and clawing of toes Unsteady gait, tripping and pain Pain in leg, frequent trips and falls, struggle with hand function Pain in feet, cramps Pain in leg, tripping, poor hand function Ankles give way on running, pes cavus, struggle with hand function Frequent falls, pain in calf muscles, difficulty at running Slow at running, frequent falls and pain in legs
S
HD Weakness UL+LL
CNS or other features
Distal UL+LL
N
Distal UL+LL
N
Distal UL+LL
N
N N
Distal LL
N
N N N N
Distal UL +LL Positive sensory symptoms Distal LL N
N N
Distal UL+LL
N N
Distal UL +LL Tremors
Clawing of toes Hind foot varus with loading on lateral border. Normal shape
N N N N
Distal LL Distal LL
N N
Distal LL
Cavovarus feet and clawing of toes Hind foot varus deformity Cavovarus feet with clawing
N N N N N N
Positive sensory symptoms Distal LL N Distal UL +LL N Distal UL +LL N
Bilateral cavovarus feet
N N
Distal UL+LL
Tremors
Planovalgus feet
N N
Distal LL
N
N
N N
Y: yes; N: no; LL: lower limb; UL: upper limb; S: scoliosis; HD: hip dysplasia; CNS: central nervous system. Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
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Fig. 3. Family tree: generation I-VI, no male to male transmission. Table 2 Neurophysiology results. Nerve
Normal values
F
M
F
F
M
Median SNCV (m/s) Ulnar SNCV (m/s) Radial SNAP (µV) Radial SNCV (m/s) Sural SNAP (µV) Sural SNCV (m/s) Median CMAP (mV) Median MNCV (m/s) Ulnar CMAP (mV) Ulnar MNCV (m/s) Peroneal CMAP (mV) Peroneal MNCV (m/s) Tibial CMAP (mV) Tibial MNCV (m/s)
>56 >55 >14 >50 >6 >43 >4 >48 >6 >51 >4 >43 >4 >41
35 38 ND ND ND ND 5 35 7 32 ND ND ND ND
32.1 32.8 ND ND 7.6 35.3 ND 33.3 ND ND 0.5 43.3 0.7 42.5
33.5 29.9 ND ND ND ND ND ND ND 33.3 ND ND ND ND
34 ND ND ND ND ND ND 29 ND 31 ND ND ND ND
37.5 34 16.6 29.8 7.4 36.6 7.7 44.9 4.6 43.6 3.9 42.9 11.0 45.7
SNCV: sensory nerve conduction velocity; MNCV: motor nerve conduction velocity; SNAP: sensory nerve action potential; CMAP: compound muscle action potential; F: female; M: male; ND: not done.
of peripheral neuropathy with varying severity suggesting absence of P2 in causing pathogenic nerve dysfunction. Male members reported early onset of symptoms. None of the affected individuals had hip dysplasia which is an association described with CMT [11]. They did not have central nervous
involvement as reported with some X linked CMT families [12,13]. Electrophysiology was consistent with peripheral nerve dysfunction and nerve biopsy in one individual excluded inflammatory cause of neuropathy. P2 promoter region is important for gene function as reduced or absent (as in our family) activity will cause a reduction/loss of messenger RNA transcript, hence reduced GJB1 protein [9]. Functional analysis of promoter region by Houlden et al. (2004) identified its role to cause Schwann cell dysfunction by interacting with SOX10 and EGR2 genes [9]. Interaction between them is complex. SOX10 and EGR2 bind to the P2 promoter region of GJB1 gene at S1-S2 and E2-E3 sites respectively for transcription [14,15]. This interaction between GJB1 P2 promoter, SOX10 and EGR2 can be interrupted by either mutations in SOX10 [16] and EGR2 [17] genes or mutation in P2 promoter region of GJB1 [9]. Our patients did not have mutations in SOX10 and EGR2 genes supporting restriction of transcription by latter mechanism. This observation strengthens the hypothesis by Houlden et al. that P2 promoter is central in function of Schwann cell in peripheral nerve. 5. Conclusion We have described a novel mutation deleting entire P2 promoter of GJB1 gene in a single large family with CMT.
Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001
ARTICLE IN PRESS R. Kulshrestha et al. / Neuromuscular Disorders ■■ (2017) ■■–■■
5
Fig. 4. Sural nerve biopsy features. Immunohistochemical (IHC) examination (A and B) shows prominent loss of large myelinated fibres (A: IHC for myelin basic protein, SMI94 antibody), whilst unmyelinated and small myelinated fibres are better preserved (B: immunostaining for neurofilaments, SMI31 antibody). Semi-thin resin section stained with methylene blue azure – basic fuchsin (C) confirms markedly reduced numbers of large normally myelinated fibres, with relatively frequent small and medium sized thinly myelinated fibres of which many are associated with regeneration clusters (arrow) but there is no active axonal degeneration. Ultrastructural examination (D) highlights the regeneration clusters (arrow) but shows no signs of active macrophage-associated demyelination or chronic demyelinating/re-myelinating process. Scale bar: 30 µm in A and B, 15 µm in C, 2 µm in D.
Inheritance and phenotype of affected individuals had classical features of X linked peripheral neuropathy. This study affirms the role of P2 promoter being vital for Schwann cell function.
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Please cite this article in press as: R. Kulshrestha, et al., Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease, Neuromuscular Disorders (2017), doi: 10.1016/j.nmd.2017.05.001