Published Ahead of Print on January 16, 2015 as 10.1212/WNL.0000000000001220
Recessive truncating IGHMBP2 mutations presenting as axonal sensorimotor neuropathy Gudrun Schottmann, MD Heinz Jungbluth, MD, PhD Ulrike Schara, MD Ellen Knierim, MD Susanne Morales Gonzalez Esther Gill Franziska Seifert Fiona Norwood, MD Charu Deshpande, MD Katja von Au, MD Markus Schuelke, MD* Jan Senderek, MD*
Correspondence to Prof. Schuelke: [email protected]
ABSTRACT
Objective: To identify the cause of sensorimotor neuropathy in a cohort of patients with genetically unsolved neuropathies (57 families with a total of 74 members) in whom hitherto known disease genes had been excluded.
Methods: We used autozygosity mapping or haplotype analysis to delineate potential disease loci in informative families. For mutation detection, we used either whole-exome sequencing or Sanger sequencing of positional candidates. Subsequently, a larger cohort was specifically screened for IGHMBP2 mutations. The pathogenicity of a splice-site mutation was verified in cultured patient skin fibroblasts on the messenger RNA level and by Western blot. Results: We report on 5 patients with neuropathy from 3 families who carried truncating mutations in IGHMBP2. Contrary to the “classic” phenotype, they did not manifest with respiratory distress, but with progressive sensorimotor neuropathy. Only one patient required nocturnal mask ventilation, while 4 others maintained normal respiratory function by the age of 14, 18, 22, and 37 years. Three patients were still able to walk independently. All patients had a predominantly axonal sensorimotor neuropathy with subsequent muscle atrophy, but without obvious sensory symptoms. Two patients had signs of autonomic neuropathy.
Conclusions: Mutations in IGHMBP2 should be considered in the molecular genetic workup of patients with hereditary sensorimotor neuropathies, even in the absence of respiratory symptoms. Neurology® 2015;84:1–9 GLOSSARY bp 5 base pair; HMN 5 hereditary motor neuropathy; HMSN 5 hereditary motor-sensory neuropathy; IGHMBP2 5 immunoglobulin m binding protein 2; SMARD1 5 spinal muscular atrophy with respiratory distress type 1; SNP 5 single-nucleotide polymorphism.
Sensorimotor neuropathies, usually referred to as Charcot-Marie-Tooth disease, are a heterogeneous group of disorders caused by more than 80 different gene defects associated with all different modes of mendelian inheritance. Defective proteins implicated in Charcot-Marie-Tooth disorders maintain a large range of cellular functions including myelin formation and stability, axonal transport, “housekeeping” functions (e.g., transfer RNA synthesis, heat shock proteins, membrane transport, transcription factors), as well as energy metabolism (reviewed in references 1 and 2). In contrast, mutations in IGHMBP2 have been mainly associated with autosomal recessive spinal muscular atrophy with respiratory distress type 1 (SMARD1, OMIM #604320).3 IGHMBP2 encodes the immunoglobulin m binding protein 2, which belongs to the family of adenosine triphosphate–dependent helicases and has a role in translation and RNA trafficking.4,5 SMARD1 is characterized by sudden onset of respiratory distress due to diaphragmatic palsy and distal motor weakness during infancy and is caused by degeneration of anterior Supplemental data at Neurology.org *These two authors jointly directed this work. From the Department of Neuropediatrics and the NeuroCure Clinical Research Center (G.S., E.K., S.M.G., E.G., F.S., M.S.), and Department of Neuropediatrics/SPZ (K.v.A.), Charité–Universitätsmedizin Berlin, Germany; Department of Paediatric Neurology–Neuromuscular Service (H.J.), Evelina Children’s Hospital, St Thomas’ Hospital, the Randall Division of Cell and Molecular Biophysics, Muscle Signalling Section and the Department of Clinical Neuroscience, IoP, King’s College, London, UK; Department of Neuropediatrics, Developmental Neurology and Social Pediatrics (U.S.), University of Essen, Germany; Department of Neurology (F.N.), King’s College Hospital, London; Department of Clinical Genetics (C.D.), Guy’s Hospital, London, UK; and Friedrich Baur Institute (J.S.), Department of Neurology, Ludwig Maximilians University Munich, Germany. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. © 2015 American Academy of Neurology
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horn cells.6 Respiratory failure requires mechanical ventilation in all patients reported to date.3,7 Apart from the typical infantile presentation associated with a rather poor prognosis, rarely patients have been reported with milder disease courses, all of whom, however, ultimately required mechanical ventilation.8–10 Herein, we report on 5 adult patients from 3 unrelated families with a progressive sensorimotor neuropathy of varying severity due to recessive mutations in the IGHMBP2 gene. Until now, 4 patients have maintained normal respiratory function within the period of observation. METHODS Mutation detection in family A. For this and for 18 other families (n 5 24 patients), we performed a candidate gene approach, first applying autozygosity mapping followed by whole-exome sequencing. For autozygosity mapping, we investigated both parents (A:I-1, A:I-2) and 2 affected (A:II-1, A:II-2) and 2 unaffected (A:II-3, A:II-4) siblings using the Affymetrix GeneChip Human Mapping 250K NSP singlenucleotide polymorphism (SNP) array (Affymetrix, Santa Clara, CA).11 With the help of the HomozygosityMapper2012 software (http://www.homozygositymapper.org), we singled out those genomic loci that were homozygous only in the patients but not in unaffected individuals.12 The exomic sequences of patient A:II-1 were captured with the SeqCap EZ Human Exome Library v3.0 (NimbleGen, Basel, Switzerland), sequenced on an Illumina HiSeq 2000 machine (Illumina, Inc., San Diego, CA) yielding 32.5 Mio 90–base pair (bp) paired-end reads and aligned to the human GRCh37.p11 (hg19/Ensembl 72) genomic sequence.13 After fine-adjustment, the raw alignments were called for deviations from the GRCh37.p11 reference in all coding exons 620-bp flanking regions.14,15 The VCF (variant call format) file was sent to the MutationTaster Query Engine software (http://www.mutationtaster.org/StartQueryEngine.html) for assessment of all variants for potential pathogenicity.16,17 The filtering options of MutationTaster were set to “autozygous region,” “homozygosity,” “coverage .103,” “removal of all variants occurring .43 in the 1000 Genomes Project in homozygous state.” Ensuing variants were tested for segregation in the family by Sanger sequencing.
Families B and C. For these 2 families as well as for 36 other families (n 5 50 patients), genetic studies comprised a combination of targeted linkage and autozygosity analysis followed by Sanger sequencing. DNA samples from index patients and informative family members were analyzed with short tandem repeat markers on 15 loci for hereditary motor neuropathy (HMN) and hereditary motor-sensory neuropathy (HMSN), including the SMARD1 locus on Chr11q13.3. Details of primer sequences, PCR conditions, detection of alleles, and haplotype reconstruction are available on request. HMN and HMSN genes located within the regions of autozygosity or possible linkage were analyzed by Sanger sequencing. Reverse transcription–PCR on the complementary DNA level. Patients’ (A:II-1, A:II-2) fibroblasts and 2 control fibroblast lines were grown to confluency and trypsinized, and total RNA was isolated using the RNAeasy Kit (Qiagen, Venlo, 2
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the Netherlands) reagent as described.9 RNA was reversely transcribed with SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA) and random hexamers. Oligonucleotide complementary DNA primers flanking exon 3 (forward: 59-ATGGCCTCGGCAGCTGTGGAGA-39; reverse: 59-GAATGTCAGCGGGTGTATTC-39) were used for PCR, and the resulting products were separated by agarose electrophoresis, extracted, and subjected to bidirectional automatic Sanger sequencing.11
Western blot of cultured fibroblasts. Protein was extracted from cultured fibroblasts of patients (A:II-1, A:II-2) and healthy controls after homogenization in lysis buffer with a proteinase inhibitor cocktail (Complete; Roche Diagnostics, Basel, Switzerland).18 One hundred micrograms of protein was separated through denaturating sodium dodecyl sulfate– polyacrylamide gel electrophoresis and blotted on a nitrocellulose membrane. The blots were probed with the 481/2 polyclonal rabbit anti-IGHMBP2 antibody (dilution 1:200)4 followed by the secondary goat anti-rabbit immunoglobulin G peroxidase conjugated antibody (dilution: 1:2,000). Bands were visualized by chemiluminescence. The density of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) bands was used for loading control. Standard protocol approvals, registrations, and patient consents. The study was approved by the local ethical review board of the Charité (EA1/228/08). All patients provided written informed consent according to the Declaration of Helsinki for all aspects of the study.
A total of 57 families with 74 affected members with genetically unsolved neuropathies were screened for IGHMBP2 mutations. In 5 patients, we detected truncating mutations in IGHMBP2. In them, axonal sensorimotor neuropathy could be verified by electrophysiology. Cognitive development was normal. With the exception of patient A:II-1, who had to be ventilated noninvasively with a mask, all other patients maintained independent breathing. The main clinical features are summarized in the table.
RESULTS Clinical description of the patients.
Family A. The parents were first-degree cousins from
Lebanon. The boy (A:II-1) was born at term after a normal pregnancy. Generalized hypotonia and motor developmental delay became evident during infancy. Slowly progressive distal atrophy and weakness of arms and legs (figure 1, D and E) led to wheelchair dependence by the age of 6 years. Respiratory insufficiency required nocturnal noninvasive mask ventilation from the age of 15 years (figure 1B, figure e-1 on the Neurology® Web site at Neurology.org). A recent sonographic investigation of the diaphragm verified immobility and the x-ray showed bilateral eventration (figure 1A). Repeated echocardiographic investigations were normal. The motor development of his younger sister (A:II-2) was moderately delayed. Distal muscle atrophy and weakness (figure 1, C and F) without sensory
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Excluded (sonography) 9 y; 1.47 L (1.64 L) 5 89.5%
15 y; 2.56 L (3.10 L) 5 85.3% 18 y; 3.41 L (3.37 L) 5 101.2%
Male
34
0.5
Generalized muscular hypotonia
Absent
6
Present
Verified (sonography)
15 y, before start of ventilation; 0.83 L (3.31 L) 5 25.1%
17 y; 0.96 L (3.93 L) 5 27.0%
18 y; 0.78 L (3.93 L) 5 20.3%
Sex
Age at last assessment, y
Clinical onset, y
Presenting symptoms
Deep tendon reflexes
Loss of free ambulation, y
Symptoms of respiratory insufficiency
Diaphragmatic paralysis
Age at investigation; FVC patient value (normal value)
No desaturation or hypercapnia (under noninvasive mask ventilation), normal sleep pattern (34 y)
Present
Bladder and gastrointestinal dysfunction, achalasia
Absent
Polysomnography (age at investigation)
Scoliosis
Signs of autonomic neuropathy
Sensory symptoms
30 y; 0.69 L (3.93 L) 5 17.5%
Unknown ND
Absent
A:II-1
Patient
Absent
Unknown
Bladder and gastrointestinal dysfunction
Absent
Absent
ND
Absent
Still able to walk
Absent
Bilateral pes cavus
2
18
Male
B:II-1
p.Gly871Aspfs*6
Present
No desaturation or hypercapnia, normal sleep pattern (19 y)
21 y; 3.05 L (3.37 L) 5 90.4%
10
Absent
Delayed motor development, distal muscular weakness
2
22
Female
A:II-2
p.Lys150Asnfs*0
IGHMBP2 mutation protein
Homozygous c.278411G.T
Homozygous c.44911G.Tjc.449_450ins100bp
Yes
Yes
Family B
IGHMBP2 mutation cDNA
Family A
Overview of clinical, neurophysiologic, and molecular data of 5 patients from 3 families
Parental consanguinity
Table
Absent
Unknown
Present
ND
ND
Unknown
Absent
Still able to walk
Absent
Bilateral pes cavus, thenar atrophy
3.5
14
Female
B:II-2
Absent
Absent
Absent
ND
ND
Continued
Unknown
Absent
Still able to walk
Absent
Toe walking
6
37
Female
C:II-1
p.Cys46*jp. Arg971fs*4
c.138T.Ajc.29112912delAG
No
Family C
cDNA 5 complementary DNA; CMAP 5 compound muscle action potential; FVC 5 forced vital capacity; IGHMBP2 5 immunoglobulin m binding protein 2; N 5 normal; NCV 5 nerve conduction velocity; ND 5 not done; SNAP 5 sensory nerve action potential.
ND
ND ND Neurogenic changes
ND
ND
ND
Neurogenic changes
Incomplete myelination, acute disaggregation of myelin fibers
Muscle biopsy
Sural nerve biopsy
ND
23 y; median nerve: no SNAPs; sural nerve: no SNAPs 18 y; median nerve: no SNAPs 14 y; median nerve: no SNAPs; sural nerve: no SNAPs ND Sensory NCV
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Age at investigation; nerve
18 y; median nerve: no CMAPs; tibial nerve: no CMAPs
7.5 y; median nerve: no SNAPs
7.5 y; tibial nerve: 30.9 m/s (0.1 mV) [N: .43.4 m/s, 5.8 6 1.9 mV] 12 y; tibial nerve: 29.5 m/s (0.2 mV) [N: .43.4 m/s, 5.8 6 1.9 mV] 22 y; median nerve: no CMAPs; tibial nerve: no CMAPs; peroneal nerve: no CMAPs
23 y; ulnar nerve: 40 m/s (4.5 mV) [N: .50.1 m/s, 5.7 6 2.0 mV] 3.5 y; median nerve: 35.8 m/s (ND) [N: .48 m/s]; ulnar nerve: 46.5 m/s (ND) [N: .48.3 m/s]; tibial nerve: 27.8 m/s (0.2 mV) [N: .42.0 m/s, 9.6 6 3.5]; peroneal nerve: 40.3 m/s (0.1 mV) [N: .44.9 m/s, 4.3 6 1.6 mV] 7 y; median nerve: 35.1 m/s (0.1 mV) [N: .49.6 m/s, 7.0 6 3.0 mV); ulnar nerve: 28 m/ s (1.2 mV) [N: .50.1 m/s, 5.7 6 2.0 mV]; tibial nerve: no CMAPs; peroneal nerve: no CMAP Motor NCV
Age at investigation; nerve: velocity (CMAP amplitude) [normal values]
1.7 y; tibial nerve: 23 m/s (ND) [N: .38.2 m/s]; peroneal nerve: no CMAPs
14 y; median nerve: 35.1 m/s (0.1 mV) [N: .50.9 m/s, 7.0 6 3.0 mV]; tibial nerve: no CMAPs; peroneal nerve: no CMAPs
Neurogenic
Family C
ND Neurogenic ND
Family B Family A
Neurogenic EMG
Continued Table 4
symptoms manifested in the third year of her life. In contrast to her elder brother, her clinical course was milder. She maintained full independent ambulation until the age of 10 years and presently at the age of 22 years she is still able to walk some steps with assistance. Her respiratory function as assessed by whole-body plethysmography (figures 1B and e-1) and polysomnography was normal. A recent sonographic investigation of the diaphragm confirmed normal mobility. Echocardiography revealed grade 1 mitral regurgitation but excluded cardiomyopathy. Both siblings had progressive scoliosis and autonomic symptoms including gastrointestinal and bladder dysfunction. Patient A:II-1 had to be operated for achalasia at the age of 18 years while urinary retention required regular catheterization. Family B. The parents are first-degree cousins from
Turkey. The index patient (B:II-1) started walking at the age of 10 months, but remained unsteady with a clumsy gait. Bilateral cavovarus foot deformity became apparent at 2 years of age and required corrective surgery 3 years later. He developed slowly progressive atrophy and weakness of distal hand and leg muscles, with preserved strength in the limb girdle. When last seen at the age of 18 years, he was able to walk independently with ankle-foot orthoses and to climb stairs with support. His respiratory function was always normal. His younger sister (B:II-2) initially developed normally, before she presented with high-arched feet and atrophy of the small hand muscles at the age of 3.5 years. Later, bilateral talipes cavovarus deformity developed and her gait became unsteady with frequent stumbling. When she was last seen at the age of 14 years, she was able to walk with anklefoot orthoses and breathe independently. Family C. The index patient (C:II-1) is 1 of 2 affected sisters, the only children of a healthy, nonconsanguineous couple from the United Kingdom. After an initially normal psychomotor development, toe walking was noted from the age of 6 years. She was never able to run but managed to participate in sports. Later, slowly progressive muscle wasting and weakness of both the distal arms and legs became apparent. She never developed any respiratory problems. Involvement of the lower cranial nerves was suspected at her last assessment at the age of 37 years. MRI of brain, brainstem, and cervical spine was normal. The patient still has an unlimited walking distance on a flat surface. Her sister (C:II-2) was reported to be similarly affected without any respiratory involvement but could not be contacted for follow-up.
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Figure 1
Clinical images of patients from family A
(A) The chest x-ray of patient A:II-1 at the age of 24 years shows bilateral diaphragmatic eventration. (B) Whole-body plethysmography traces of the 2 siblings at the age of 18 years. The integral below the red triangle represents 100% of the body height–related forced vital capacity (FVC). Distally marked muscle atrophy in the hands and legs of patient A:II-1 at the age of 34 years (D, E) and his sister A:II-2 at the age of 22 years (C, F). FEV1 5 forced expiratory volume in the first second of expiration.
Identification of IGHMBP2 mutations. Autozygosity
mapping in family A revealed several regions on chromosomes 6, 7, 10, 11, 12, and 16 (538.2 Mbp) comprising 594 genes. Whole-exome sequencing revealed 6 homozygous variants in genes within the autozygous region that were predicted to be potentially disease-causing by the MutationTaster2 software (FADD, IGHMBP2, SNX19, CNTNAP4, COBL, RAB6A). The intronic variants of the last 4 genes could be found in homozygous and heterozygous state in the 1000 Genome Project, had already been assigned an rs number, and could thus be ruled out. The variant (p.V177L) in FADD could be ruled out as well, because a completely different phenotype had been described for mutations in this gene comprising recurrent infection with encephalopathy, hepatic dysfunction, and cardiovascular malformations
(FADD deficiency OMIM #613759). The effect of the splice donor site mutation in IGHMBP2 at position c.44911G.T was verified on the complementary DNA level (figure 2C). The mutation led to the activation of a cryptic downstream splice site at position c.4491101 (GTjGTGGGT) thus retaining 100 bp of intron 3, which led to a premature termination codon at the first triplet into intron 3. Neither full-length nor truncated IGHMBP2 protein could be found in fibroblasts of both patients (figure 2D). The c.44911G.T mutation segregated with the phenotype in family A. Both parents and the healthy younger sister were heterozygous; the younger brother had 2 wild-type alleles (figure 2A). It was not found in the public SNP databases such as the 1000 Genome Project (http://www.1000Genomes.gov), the Exome Variant Server (http://evs.gs.washington.edu/EVS/), as Neurology 84
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Figure 2
Molecular genetic findings
(A) Pedigrees of the families, 2 of them consanguineous. The genotypes of the affected and unaffected family members are provided below the symbols. (B) Electrophoretic traces of the Sanger sequencing from affected individuals and controls. The codons are depicted below the traces. (C) Reverse transcription–PCR on complementary DNA from cultured fibroblasts with oligonucleotide primers that flank IGHMBP2 exon 3 shows the retention of 100 base pairs (bp) of intron 3 through activation of a downstream cryptic splice donor site in the presence of the 44911G.T mutation. (D) Absence of the 110-kDa IGHMBP2 protein band in both patients from family A in comparison to 2 controls. GAPDH band density was used as loading control. Co 5 control; GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase; IGHMBP2 5 immunoglobulin m binding protein 2; NTK 5 no template control; wt 5 wild-type.
well as dbSNP138 (http://www.ncbi.nlm.nih.gov/ projects/SNP) and in 130 ethnically matched inhouse whole exomes. The public resources allow interrogating exome data of .10,000 individuals. Family B. Linkage analysis was compatible with the SMARD1 region. Two other loci could not be excluded by linkage analysis, but were unlikely candidates because we did not observe homozygous haplotypes in these regions. Sequencing of the 6
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IGHMBP2 gene revealed the homozygous mutation c.278411G.T (figure 2B), which segregated with the phenotype (figure 2A). This mutation is predicted to lead to loss of the splice-donor signal of the 14th intron of the IGHMBP2 transcript leading to a C-terminally truncated protein (p.Gly871Aspfs*6) or nonsense-mediated messenger decay. This mutation was absent from all public SNP databases (see above). A benign population-specific genetic
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variant was excluded by testing DNAs of 96 healthy Turkish control individuals. Compatible with its localization in the invariant splice donor site, comparison of the IGHMBP2 genes in humans and other species showed complete conservation of the guanine nucleotide at position c.278411. Family C. Linkage analysis was compatible with linkage to 4 of 15 HMN and HMSN loci including the SMARD1 region. The 2 patients in family C were found to carry compound heterozygous mutations c.138T.A (p.Cys46*) and c.2911_2912delAG (p.Arg971Glufs*4) in the IGHMBP2 gene (figure 2, A and B), while no pathogenic variants were found in the 3 other genes. Both variants in the IGHMBP2 gene truncate the protein and represent bona fide pathogenic mutations. The p.Cys46* mutation has already been reported in SMARD1 patients.19 None of these changes was found in public SNP databases (see above).
Herein, we report 5 patients from 3 families with mutations in the IGHMBP2 gene who presented with predominantly axonal sensorimotor neuropathy. To our knowledge, no patients have been described to date with genetically proven IGHMBP2 mutations, who present without signs of respiratory problems beyond the first decade of life. Of our 5 patients, only one developed respiratory insufficiency by the age of 14 years, which is much later than previously reported for any patient with SMARD1. These findings expand the phenotypic spectrum of IGHMBP2-associated neuromuscular disorders. A protracted onset of respiratory insufficiency between 3.2 and 9.0 years of age has rarely been described9,20,21 and in all patients ultimately diaphragmatic palsy was recorded. The onset of diaphragmatic dysfunction in patient A:II-1 could not be precisely determined, because it started gradually and was thus solely attributed to the weakness of the intercostal muscles and his severe scoliosis. The gradual onset of respiratory insufficiency in our patient is in contrast to the often sudden and dramatic respiratory presentation in “classic” SMARD1, which often requires emergency intubation, tracheostomy, and permanent mechanical ventilation. Of note, his 22year-old sister (A:II-2) continues to have an entirely normal respiratory function and no signs of diaphragmatic dysfunction on ultrasound. This illustrates a considerable intrafamilial phenotypic heterogeneity as seen in a report of 2 siblings with an identical IGHMBP2 mutation but a different phenotype: one child died at the age of 6 months of respiratory failure, while the other showed only limb weakness and mild nocturnal hypoventilation by the age of 12 years. It is DISCUSSION
thus obvious that additional factors may come into play and modify the phenotype. The patients belonging to families A and C are aged 22, 34, and 37 years, thus are the oldest patients alive with pathogenic IGHMBP2 mutations. The oldest SMARD1 patient reported to date was 20 years old. However, in contrast to our patients, he had been typically affected from early infancy and had survived long term on fulltime ventilation, despite a complicated disease course.22 There is an ongoing debate, whether SMARD1 should be considered a subtype of spinal muscular atrophy or rather a severe peripheral neuropathy with diaphragmatic palsy.23 Electrophysiologic findings in various patients have demonstrated a severe peripheral neuropathy rather than evidence of an isolated anterior horn cell disease.23 In addition, electrophysiologic and histopathologic studies indicate the involvement of the sensory system, even in the absence of clinical symptoms.21,24 The latter finding is in keeping with the clinical phenotype seen in our patients, who did not report any sensory symptoms despite electrophysiologic evidence of a severely impaired sensory nerve function. The discrepancy between clinical findings and neurophysiologic results cannot be readily explained. Clear genotype-phenotype relations for the known IGHMBP2 mutations have not been established. For some mutations, a potential link has been demonstrated between a milder clinical course and higher levels of residual IGHMBP2 protein function.4,7,20 Of the 4 mutations presented here, only one heterozygous mutation found in family C (c.138T.A, p.Cys46*) has been described before.19 Both this mutation and the splice-site mutation identified in family A (c.44911G.T, p.Lys150Asnfs*0) cause a premature termination codon or frameshift, leading to the absence of functional IGHMBP2 protein, which could be verified by Western blot in family A. The remaining mutations (family C: c.2911_2912delAG, p.Arg971Glufs*4; and family B: c.278411G.T) affect the last exons of the gene and may thus result in truncated but stable proteins (figure 2B). However, it remains speculative whether such a mechanism might be related to the patients’ clinical presentation. A modifying effect of the genetic background on the phenotypical expression of Ighmbp2 mutations has been demonstrated through a backcross of the nmd mutation25 of the mouse model of human SMARD1, from the original CAST/Ei strain into the BL6 strain. Genetic quantitative trait locus analysis of the B6.CAST backcross showed that CASTderived modifiers on mouse chromosomes 9, 10, and 16 were able to mitigate the nmd-related cardiomyopathy and increase the lifespan.26 Neurology 84
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Our findings expand the phenotypical spectrum of IGHMBP2-associated neuromuscular disorders and emphasize that IGHMBP2 mutations should be considered in the molecular genetic workup of patients presenting with isolated peripheral neuropathy even without additional respiratory involvement.
5.
6.
AUTHOR CONTRIBUTIONS Gudrun Schottmann: recruited, investigated, and phenotyped the patients, performed neurophysiologic investigations, validated the results of the whole-exome sequencing, wrote the first draft of the manuscript. Heinz Jungbluth: recruited, investigated, and phenotyped the patients, wrote the first draft of the manuscript. Ulrike Schara: recruited, investigated, and phenotyped the patients. Ellen Knierim: validated the results of the whole-exome sequencing. Susanne Morales Gonzalez: performed the Western blotting and reverse transcription–PCR. Esther Gill: performed and analyzed the Sanger sequencing. Franziska Seifert: performed and analyzed the Sanger sequencing. Fiona Norwood: recruited, investigated, and phenotyped the patients, performed neurophysiologic investigations. Charu Deshpande: recruited, investigated, and phenotyped the patients. Katja von Au: recruited, investigated, and phenotyped the patients, provided reagents and materials. Markus Schuelke: recruited, investigated, and phenotyped the patients, performed the autozygosity mapping and haplotype analysis, validated the results of the wholeexome sequencing, supervised the work and obtained funding support, wrote the first draft of the manuscript. Jan Senderek: performed the autozygosity mapping and haplotype analysis, supervised the work and obtained funding support, wrote the first draft of the manuscript. All authors read the final version of the manuscript for intellectual content and gave their permission for publication.
ACKNOWLEDGMENT The authors thank the patients and their parents for participation in the study, Dr. med. Walter Stäblein (Charité Berlin) for doing the ultrasound investigations, and Prof. Utz Fischer (University Würzburg) for providing the 481/2 polyclonal rabbit anti-IGHMBP2 antibody. The excellent technical assistance of Angelika Zwirner (Charité Berlin) is much appreciated.
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STUDY FUNDING The project was funded by the Deutsche Forschungsgemeinschaft (SFB 665 TP C4) to M.S. and the NeuroCure Center of Excellence (Exc 257) to M.S.
DISCLOSURE
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The authors report no disclosures relevant to the manuscript. Go to Neurology.org for full disclosures.
17. Received June 12, 2014. Accepted in final form October 6, 2014. REFERENCES 1. Timmerman V, Strickland AV, Züchner S. Genetics of Charcot-Marie-Tooth (CMT) disease within the frame of the human genome project success. Genes 2014;5: 13–32. 2. Bird TD. Charcot-Marie-Tooth hereditary neuropathy overview. In: Pagon RA, Adam MP, Ardinger HH, et al, editors. GeneReviews [Internet]. Seattle: University of Washington; 1993. Available at: http://www.ncbi.nlm. nih.gov/books/NBK1358/. Accessed December 24, 2014. 3. Grohmann K, Schuelke M, Diers A, et al. Mutations in the gene encoding immunoglobulin mu-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1. Nat Genet 2001;29:75–77. 4. Guenther UP, Handoko L, Laggerbauer B, et al. IGHMBP2 is a ribosome-associated helicase inactive in 8
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the neuromuscular disorder distal SMA type 1 (DSMA1). Hum Mol Genet 2009;18:1288–1300. Lim SC, Bowler MW, Lai TF, Song H. The Ighmbp2 helicase structure reveals the molecular basis for diseasecausing mutations in DMSA1. Nucleic Acids Res 2012; 40:11009–11022. Grohmann K, Rossoll W, Kobsar I, et al. Characterization of Ighmbp2 in motor neurons and implications for the pathomechanism in a mouse model of human spinal muscular atrophy with respiratory distress type 1 (SMARD1). Hum Mol Genet 2004;13:2031–2042. Eckart M, Guenther UP, Idkowiak J, et al. The natural course of infantile spinal muscular atrophy with respiratory distress type 1 (SMARD1). Pediatrics 2012;129:e148–e156. Joseph S, Robb SA, Mohammed S, et al. Interfamilial phenotypic heterogeneity in SMARD1. Neuromuscul Disord 2009;19:193–195. Guenther UP, Schuelke M, Bertini E, et al. Genomic rearrangements at the IGHMBP2 gene locus in two patients with SMARD1. Hum Genet 2004;115:319–326. Messina MF, Messina S, Gaeta M, et al. Infantile spinal muscular atrophy with respiratory distress type I (SMARD 1): an atypical phenotype and review of the literature. Eur J Paediatr Neurol 2012;16:90–94. von Renesse A, Petkova MV, Lützkendorf S, et al. POMK mutation in a family with congenital muscular dystrophy with merosin deficiency, hypomyelination, mild hearing deficit and intellectual disability. J Med Genet 2014;51:275–282. Seelow D, Schuelke M. HomozygosityMapper2012: bridging the gap between homozygosity mapping and deep sequencing. Nucleic Acids Res 2012;40:W516–W520. Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. 2013. Report 1303.3997. Available at: http://arxiv.org/abs/1303.3997. Accessed December 24, 2014. DePristo MA, Banks E, Poplin R, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011;43:491–498. McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010;20:1297–1303. Schwarz JM, Rödelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010;7:575–576. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods 2014;11:361–362. Rajab A, Straub V, McCann LJ, et al. Fatal cardiac arrhythmia and long-QT syndrome in a new form of congenital generalized lipodystrophy with muscle rippling (CGL4) due to PTRF-CAVIN mutations. PLoS Genet 2010;6:e1000874. Grohmann K, Varon R, Stolz P, et al. Infantile spinal muscular atrophy with respiratory distress type 1 (SMARD1). Ann Neurol 2003;54:719–724. Guenther UP, Handoko L, Varon R, et al. Clinical variability in distal spinal muscular atrophy type 1 (DSMA1): determination of steady-state IGHMBP2 protein levels in five patients with infantile and juvenile disease. J Mol Med 2009;87:31–41. Blaschek A, Gläser D, Kuhn M, et al. Early infantile sensory-motor neuropathy with late onset respiratory distress. Neuromuscul Disord 2014;24:269–271.
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Pierson TM, Tart G, Adams D, et al. Infantile-onset spinal muscular atrophy with respiratory distress-1 diagnosed in a 20-year-old man. Neuromuscul Disord 2011;21:353–355. Pitt M, Houlden H, Jacobs J, et al. Severe infantile neuropathy with diaphragmatic weakness and its relationship to SMARD1. Brain 2003;126:2682–2692. Diers A, Kaczinski M, Grohmann K, Hübner C, Stoltenburg-Didinger G. The ultrastructure of peripheral nerve, motor end-plate and skeletal muscle in patients suffering
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Recessive truncating IGHMBP2 mutations presenting as axonal sensorimotor neuropathy Gudrun Schottmann, Heinz Jungbluth, Ulrike Schara, et al. Neurology published online January 7, 2015 DOI 10.1212/WNL.0000000000001220 This information is current as of January 7, 2015 Updated Information & Services
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Neurology ® is the official journal of the American Academy of Neurology. Published continuously since 1951, it is now a weekly with 48 issues per year. Copyright © 2015 American Academy of Neurology. All rights reserved. Print ISSN: 0028-3878. Online ISSN: 1526-632X.
Recessive truncating IGHMBP2 mutations presenting as axonal sensorimotor neuropathy Gudrun Schottmann, MD Heinz Jungbluth, MD, PhD Ulrike Schara, MD Ellen Knierim, MD Susanne Morales Gonzalez Esther Gill Franziska Seifert Fiona Norwood, MD Charu Deshpande, MD Katja von Au, MD Markus Schuelke, MD* Jan Senderek, MD*
Correspondence to Prof. Schuelke: [email protected]
ABSTRACT
Objective: To identify the cause of sensorimotor neuropathy in a cohort of patients with genetically unsolved neuropathies (57 families with a total of 74 members) in whom hitherto known disease genes had been excluded.
Methods: We used autozygosity mapping or haplotype analysis to delineate potential disease loci in informative families. For mutation detection, we used either whole-exome sequencing or Sanger sequencing of positional candidates. Subsequently, a larger cohort was specifically screened for IGHMBP2 mutations. The pathogenicity of a splice-site mutation was verified in cultured patient skin fibroblasts on the messenger RNA level and by Western blot. Results: We report on 5 patients with neuropathy from 3 families who carried truncating mutations in IGHMBP2. Contrary to the “classic” phenotype, they did not manifest with respiratory distress, but with progressive sensorimotor neuropathy. Only one patient required nocturnal mask ventilation, while 4 others maintained normal respiratory function by the age of 14, 18, 22, and 37 years. Three patients were still able to walk independently. All patients had a predominantly axonal sensorimotor neuropathy with subsequent muscle atrophy, but without obvious sensory symptoms. Two patients had signs of autonomic neuropathy.
Conclusions: Mutations in IGHMBP2 should be considered in the molecular genetic workup of patients with hereditary sensorimotor neuropathies, even in the absence of respiratory symptoms. Neurology® 2015;84:1–9 GLOSSARY bp 5 base pair; HMN 5 hereditary motor neuropathy; HMSN 5 hereditary motor-sensory neuropathy; IGHMBP2 5 immunoglobulin m binding protein 2; SMARD1 5 spinal muscular atrophy with respiratory distress type 1; SNP 5 single-nucleotide polymorphism.
Sensorimotor neuropathies, usually referred to as Charcot-Marie-Tooth disease, are a heterogeneous group of disorders caused by more than 80 different gene defects associated with all different modes of mendelian inheritance. Defective proteins implicated in Charcot-Marie-Tooth disorders maintain a large range of cellular functions including myelin formation and stability, axonal transport, “housekeeping” functions (e.g., transfer RNA synthesis, heat shock proteins, membrane transport, transcription factors), as well as energy metabolism (reviewed in references 1 and 2). In contrast, mutations in IGHMBP2 have been mainly associated with autosomal recessive spinal muscular atrophy with respiratory distress type 1 (SMARD1, OMIM #604320).3 IGHMBP2 encodes the immunoglobulin m binding protein 2, which belongs to the family of adenosine triphosphate–dependent helicases and has a role in translation and RNA trafficking.4,5 SMARD1 is characterized by sudden onset of respiratory distress due to diaphragmatic palsy and distal motor weakness during infancy and is caused by degeneration of anterior Supplemental data at Neurology.org *These two authors jointly directed this work. From the Department of Neuropediatrics and the NeuroCure Clinical Research Center (G.S., E.K., S.M.G., E.G., F.S., M.S.), and Department of Neuropediatrics/SPZ (K.v.A.), Charité–Universitätsmedizin Berlin, Germany; Department of Paediatric Neurology–Neuromuscular Service (H.J.), Evelina Children’s Hospital, St Thomas’ Hospital, the Randall Division of Cell and Molecular Biophysics, Muscle Signalling Section and the Department of Clinical Neuroscience, IoP, King’s College, London, UK; Department of Neuropediatrics, Developmental Neurology and Social Pediatrics (U.S.), University of Essen, Germany; Department of Neurology (F.N.), King’s College Hospital, London; Department of Clinical Genetics (C.D.), Guy’s Hospital, London, UK; and Friedrich Baur Institute (J.S.), Department of Neurology, Ludwig Maximilians University Munich, Germany. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. © 2015 American Academy of Neurology
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1
horn cells.6 Respiratory failure requires mechanical ventilation in all patients reported to date.3,7 Apart from the typical infantile presentation associated with a rather poor prognosis, rarely patients have been reported with milder disease courses, all of whom, however, ultimately required mechanical ventilation.8–10 Herein, we report on 5 adult patients from 3 unrelated families with a progressive sensorimotor neuropathy of varying severity due to recessive mutations in the IGHMBP2 gene. Until now, 4 patients have maintained normal respiratory function within the period of observation. METHODS Mutation detection in family A. For this and for 18 other families (n 5 24 patients), we performed a candidate gene approach, first applying autozygosity mapping followed by whole-exome sequencing. For autozygosity mapping, we investigated both parents (A:I-1, A:I-2) and 2 affected (A:II-1, A:II-2) and 2 unaffected (A:II-3, A:II-4) siblings using the Affymetrix GeneChip Human Mapping 250K NSP singlenucleotide polymorphism (SNP) array (Affymetrix, Santa Clara, CA).11 With the help of the HomozygosityMapper2012 software (http://www.homozygositymapper.org), we singled out those genomic loci that were homozygous only in the patients but not in unaffected individuals.12 The exomic sequences of patient A:II-1 were captured with the SeqCap EZ Human Exome Library v3.0 (NimbleGen, Basel, Switzerland), sequenced on an Illumina HiSeq 2000 machine (Illumina, Inc., San Diego, CA) yielding 32.5 Mio 90–base pair (bp) paired-end reads and aligned to the human GRCh37.p11 (hg19/Ensembl 72) genomic sequence.13 After fine-adjustment, the raw alignments were called for deviations from the GRCh37.p11 reference in all coding exons 620-bp flanking regions.14,15 The VCF (variant call format) file was sent to the MutationTaster Query Engine software (http://www.mutationtaster.org/StartQueryEngine.html) for assessment of all variants for potential pathogenicity.16,17 The filtering options of MutationTaster were set to “autozygous region,” “homozygosity,” “coverage .103,” “removal of all variants occurring .43 in the 1000 Genomes Project in homozygous state.” Ensuing variants were tested for segregation in the family by Sanger sequencing.
Families B and C. For these 2 families as well as for 36 other families (n 5 50 patients), genetic studies comprised a combination of targeted linkage and autozygosity analysis followed by Sanger sequencing. DNA samples from index patients and informative family members were analyzed with short tandem repeat markers on 15 loci for hereditary motor neuropathy (HMN) and hereditary motor-sensory neuropathy (HMSN), including the SMARD1 locus on Chr11q13.3. Details of primer sequences, PCR conditions, detection of alleles, and haplotype reconstruction are available on request. HMN and HMSN genes located within the regions of autozygosity or possible linkage were analyzed by Sanger sequencing. Reverse transcription–PCR on the complementary DNA level. Patients’ (A:II-1, A:II-2) fibroblasts and 2 control fibroblast lines were grown to confluency and trypsinized, and total RNA was isolated using the RNAeasy Kit (Qiagen, Venlo, 2
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the Netherlands) reagent as described.9 RNA was reversely transcribed with SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA) and random hexamers. Oligonucleotide complementary DNA primers flanking exon 3 (forward: 59-ATGGCCTCGGCAGCTGTGGAGA-39; reverse: 59-GAATGTCAGCGGGTGTATTC-39) were used for PCR, and the resulting products were separated by agarose electrophoresis, extracted, and subjected to bidirectional automatic Sanger sequencing.11
Western blot of cultured fibroblasts. Protein was extracted from cultured fibroblasts of patients (A:II-1, A:II-2) and healthy controls after homogenization in lysis buffer with a proteinase inhibitor cocktail (Complete; Roche Diagnostics, Basel, Switzerland).18 One hundred micrograms of protein was separated through denaturating sodium dodecyl sulfate– polyacrylamide gel electrophoresis and blotted on a nitrocellulose membrane. The blots were probed with the 481/2 polyclonal rabbit anti-IGHMBP2 antibody (dilution 1:200)4 followed by the secondary goat anti-rabbit immunoglobulin G peroxidase conjugated antibody (dilution: 1:2,000). Bands were visualized by chemiluminescence. The density of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) bands was used for loading control. Standard protocol approvals, registrations, and patient consents. The study was approved by the local ethical review board of the Charité (EA1/228/08). All patients provided written informed consent according to the Declaration of Helsinki for all aspects of the study.
A total of 57 families with 74 affected members with genetically unsolved neuropathies were screened for IGHMBP2 mutations. In 5 patients, we detected truncating mutations in IGHMBP2. In them, axonal sensorimotor neuropathy could be verified by electrophysiology. Cognitive development was normal. With the exception of patient A:II-1, who had to be ventilated noninvasively with a mask, all other patients maintained independent breathing. The main clinical features are summarized in the table.
RESULTS Clinical description of the patients.
Family A. The parents were first-degree cousins from
Lebanon. The boy (A:II-1) was born at term after a normal pregnancy. Generalized hypotonia and motor developmental delay became evident during infancy. Slowly progressive distal atrophy and weakness of arms and legs (figure 1, D and E) led to wheelchair dependence by the age of 6 years. Respiratory insufficiency required nocturnal noninvasive mask ventilation from the age of 15 years (figure 1B, figure e-1 on the Neurology® Web site at Neurology.org). A recent sonographic investigation of the diaphragm verified immobility and the x-ray showed bilateral eventration (figure 1A). Repeated echocardiographic investigations were normal. The motor development of his younger sister (A:II-2) was moderately delayed. Distal muscle atrophy and weakness (figure 1, C and F) without sensory
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Excluded (sonography) 9 y; 1.47 L (1.64 L) 5 89.5%
15 y; 2.56 L (3.10 L) 5 85.3% 18 y; 3.41 L (3.37 L) 5 101.2%
Male
34
0.5
Generalized muscular hypotonia
Absent
6
Present
Verified (sonography)
15 y, before start of ventilation; 0.83 L (3.31 L) 5 25.1%
17 y; 0.96 L (3.93 L) 5 27.0%
18 y; 0.78 L (3.93 L) 5 20.3%
Sex
Age at last assessment, y
Clinical onset, y
Presenting symptoms
Deep tendon reflexes
Loss of free ambulation, y
Symptoms of respiratory insufficiency
Diaphragmatic paralysis
Age at investigation; FVC patient value (normal value)
No desaturation or hypercapnia (under noninvasive mask ventilation), normal sleep pattern (34 y)
Present
Bladder and gastrointestinal dysfunction, achalasia
Absent
Polysomnography (age at investigation)
Scoliosis
Signs of autonomic neuropathy
Sensory symptoms
30 y; 0.69 L (3.93 L) 5 17.5%
Unknown ND
Absent
A:II-1
Patient
Absent
Unknown
Bladder and gastrointestinal dysfunction
Absent
Absent
ND
Absent
Still able to walk
Absent
Bilateral pes cavus
2
18
Male
B:II-1
p.Gly871Aspfs*6
Present
No desaturation or hypercapnia, normal sleep pattern (19 y)
21 y; 3.05 L (3.37 L) 5 90.4%
10
Absent
Delayed motor development, distal muscular weakness
2
22
Female
A:II-2
p.Lys150Asnfs*0
IGHMBP2 mutation protein
Homozygous c.278411G.T
Homozygous c.44911G.Tjc.449_450ins100bp
Yes
Yes
Family B
IGHMBP2 mutation cDNA
Family A
Overview of clinical, neurophysiologic, and molecular data of 5 patients from 3 families
Parental consanguinity
Table
Absent
Unknown
Present
ND
ND
Unknown
Absent
Still able to walk
Absent
Bilateral pes cavus, thenar atrophy
3.5
14
Female
B:II-2
Absent
Absent
Absent
ND
ND
Continued
Unknown
Absent
Still able to walk
Absent
Toe walking
6
37
Female
C:II-1
p.Cys46*jp. Arg971fs*4
c.138T.Ajc.29112912delAG
No
Family C
cDNA 5 complementary DNA; CMAP 5 compound muscle action potential; FVC 5 forced vital capacity; IGHMBP2 5 immunoglobulin m binding protein 2; N 5 normal; NCV 5 nerve conduction velocity; ND 5 not done; SNAP 5 sensory nerve action potential.
ND
ND ND Neurogenic changes
ND
ND
ND
Neurogenic changes
Incomplete myelination, acute disaggregation of myelin fibers
Muscle biopsy
Sural nerve biopsy
ND
23 y; median nerve: no SNAPs; sural nerve: no SNAPs 18 y; median nerve: no SNAPs 14 y; median nerve: no SNAPs; sural nerve: no SNAPs ND Sensory NCV
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Age at investigation; nerve
18 y; median nerve: no CMAPs; tibial nerve: no CMAPs
7.5 y; median nerve: no SNAPs
7.5 y; tibial nerve: 30.9 m/s (0.1 mV) [N: .43.4 m/s, 5.8 6 1.9 mV] 12 y; tibial nerve: 29.5 m/s (0.2 mV) [N: .43.4 m/s, 5.8 6 1.9 mV] 22 y; median nerve: no CMAPs; tibial nerve: no CMAPs; peroneal nerve: no CMAPs
23 y; ulnar nerve: 40 m/s (4.5 mV) [N: .50.1 m/s, 5.7 6 2.0 mV] 3.5 y; median nerve: 35.8 m/s (ND) [N: .48 m/s]; ulnar nerve: 46.5 m/s (ND) [N: .48.3 m/s]; tibial nerve: 27.8 m/s (0.2 mV) [N: .42.0 m/s, 9.6 6 3.5]; peroneal nerve: 40.3 m/s (0.1 mV) [N: .44.9 m/s, 4.3 6 1.6 mV] 7 y; median nerve: 35.1 m/s (0.1 mV) [N: .49.6 m/s, 7.0 6 3.0 mV); ulnar nerve: 28 m/ s (1.2 mV) [N: .50.1 m/s, 5.7 6 2.0 mV]; tibial nerve: no CMAPs; peroneal nerve: no CMAP Motor NCV
Age at investigation; nerve: velocity (CMAP amplitude) [normal values]
1.7 y; tibial nerve: 23 m/s (ND) [N: .38.2 m/s]; peroneal nerve: no CMAPs
14 y; median nerve: 35.1 m/s (0.1 mV) [N: .50.9 m/s, 7.0 6 3.0 mV]; tibial nerve: no CMAPs; peroneal nerve: no CMAPs
Neurogenic
Family C
ND Neurogenic ND
Family B Family A
Neurogenic EMG
Continued Table 4
symptoms manifested in the third year of her life. In contrast to her elder brother, her clinical course was milder. She maintained full independent ambulation until the age of 10 years and presently at the age of 22 years she is still able to walk some steps with assistance. Her respiratory function as assessed by whole-body plethysmography (figures 1B and e-1) and polysomnography was normal. A recent sonographic investigation of the diaphragm confirmed normal mobility. Echocardiography revealed grade 1 mitral regurgitation but excluded cardiomyopathy. Both siblings had progressive scoliosis and autonomic symptoms including gastrointestinal and bladder dysfunction. Patient A:II-1 had to be operated for achalasia at the age of 18 years while urinary retention required regular catheterization. Family B. The parents are first-degree cousins from
Turkey. The index patient (B:II-1) started walking at the age of 10 months, but remained unsteady with a clumsy gait. Bilateral cavovarus foot deformity became apparent at 2 years of age and required corrective surgery 3 years later. He developed slowly progressive atrophy and weakness of distal hand and leg muscles, with preserved strength in the limb girdle. When last seen at the age of 18 years, he was able to walk independently with ankle-foot orthoses and to climb stairs with support. His respiratory function was always normal. His younger sister (B:II-2) initially developed normally, before she presented with high-arched feet and atrophy of the small hand muscles at the age of 3.5 years. Later, bilateral talipes cavovarus deformity developed and her gait became unsteady with frequent stumbling. When she was last seen at the age of 14 years, she was able to walk with anklefoot orthoses and breathe independently. Family C. The index patient (C:II-1) is 1 of 2 affected sisters, the only children of a healthy, nonconsanguineous couple from the United Kingdom. After an initially normal psychomotor development, toe walking was noted from the age of 6 years. She was never able to run but managed to participate in sports. Later, slowly progressive muscle wasting and weakness of both the distal arms and legs became apparent. She never developed any respiratory problems. Involvement of the lower cranial nerves was suspected at her last assessment at the age of 37 years. MRI of brain, brainstem, and cervical spine was normal. The patient still has an unlimited walking distance on a flat surface. Her sister (C:II-2) was reported to be similarly affected without any respiratory involvement but could not be contacted for follow-up.
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Figure 1
Clinical images of patients from family A
(A) The chest x-ray of patient A:II-1 at the age of 24 years shows bilateral diaphragmatic eventration. (B) Whole-body plethysmography traces of the 2 siblings at the age of 18 years. The integral below the red triangle represents 100% of the body height–related forced vital capacity (FVC). Distally marked muscle atrophy in the hands and legs of patient A:II-1 at the age of 34 years (D, E) and his sister A:II-2 at the age of 22 years (C, F). FEV1 5 forced expiratory volume in the first second of expiration.
Identification of IGHMBP2 mutations. Autozygosity
mapping in family A revealed several regions on chromosomes 6, 7, 10, 11, 12, and 16 (538.2 Mbp) comprising 594 genes. Whole-exome sequencing revealed 6 homozygous variants in genes within the autozygous region that were predicted to be potentially disease-causing by the MutationTaster2 software (FADD, IGHMBP2, SNX19, CNTNAP4, COBL, RAB6A). The intronic variants of the last 4 genes could be found in homozygous and heterozygous state in the 1000 Genome Project, had already been assigned an rs number, and could thus be ruled out. The variant (p.V177L) in FADD could be ruled out as well, because a completely different phenotype had been described for mutations in this gene comprising recurrent infection with encephalopathy, hepatic dysfunction, and cardiovascular malformations
(FADD deficiency OMIM #613759). The effect of the splice donor site mutation in IGHMBP2 at position c.44911G.T was verified on the complementary DNA level (figure 2C). The mutation led to the activation of a cryptic downstream splice site at position c.4491101 (GTjGTGGGT) thus retaining 100 bp of intron 3, which led to a premature termination codon at the first triplet into intron 3. Neither full-length nor truncated IGHMBP2 protein could be found in fibroblasts of both patients (figure 2D). The c.44911G.T mutation segregated with the phenotype in family A. Both parents and the healthy younger sister were heterozygous; the younger brother had 2 wild-type alleles (figure 2A). It was not found in the public SNP databases such as the 1000 Genome Project (http://www.1000Genomes.gov), the Exome Variant Server (http://evs.gs.washington.edu/EVS/), as Neurology 84
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5
Figure 2
Molecular genetic findings
(A) Pedigrees of the families, 2 of them consanguineous. The genotypes of the affected and unaffected family members are provided below the symbols. (B) Electrophoretic traces of the Sanger sequencing from affected individuals and controls. The codons are depicted below the traces. (C) Reverse transcription–PCR on complementary DNA from cultured fibroblasts with oligonucleotide primers that flank IGHMBP2 exon 3 shows the retention of 100 base pairs (bp) of intron 3 through activation of a downstream cryptic splice donor site in the presence of the 44911G.T mutation. (D) Absence of the 110-kDa IGHMBP2 protein band in both patients from family A in comparison to 2 controls. GAPDH band density was used as loading control. Co 5 control; GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase; IGHMBP2 5 immunoglobulin m binding protein 2; NTK 5 no template control; wt 5 wild-type.
well as dbSNP138 (http://www.ncbi.nlm.nih.gov/ projects/SNP) and in 130 ethnically matched inhouse whole exomes. The public resources allow interrogating exome data of .10,000 individuals. Family B. Linkage analysis was compatible with the SMARD1 region. Two other loci could not be excluded by linkage analysis, but were unlikely candidates because we did not observe homozygous haplotypes in these regions. Sequencing of the 6
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IGHMBP2 gene revealed the homozygous mutation c.278411G.T (figure 2B), which segregated with the phenotype (figure 2A). This mutation is predicted to lead to loss of the splice-donor signal of the 14th intron of the IGHMBP2 transcript leading to a C-terminally truncated protein (p.Gly871Aspfs*6) or nonsense-mediated messenger decay. This mutation was absent from all public SNP databases (see above). A benign population-specific genetic
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variant was excluded by testing DNAs of 96 healthy Turkish control individuals. Compatible with its localization in the invariant splice donor site, comparison of the IGHMBP2 genes in humans and other species showed complete conservation of the guanine nucleotide at position c.278411. Family C. Linkage analysis was compatible with linkage to 4 of 15 HMN and HMSN loci including the SMARD1 region. The 2 patients in family C were found to carry compound heterozygous mutations c.138T.A (p.Cys46*) and c.2911_2912delAG (p.Arg971Glufs*4) in the IGHMBP2 gene (figure 2, A and B), while no pathogenic variants were found in the 3 other genes. Both variants in the IGHMBP2 gene truncate the protein and represent bona fide pathogenic mutations. The p.Cys46* mutation has already been reported in SMARD1 patients.19 None of these changes was found in public SNP databases (see above).
Herein, we report 5 patients from 3 families with mutations in the IGHMBP2 gene who presented with predominantly axonal sensorimotor neuropathy. To our knowledge, no patients have been described to date with genetically proven IGHMBP2 mutations, who present without signs of respiratory problems beyond the first decade of life. Of our 5 patients, only one developed respiratory insufficiency by the age of 14 years, which is much later than previously reported for any patient with SMARD1. These findings expand the phenotypic spectrum of IGHMBP2-associated neuromuscular disorders. A protracted onset of respiratory insufficiency between 3.2 and 9.0 years of age has rarely been described9,20,21 and in all patients ultimately diaphragmatic palsy was recorded. The onset of diaphragmatic dysfunction in patient A:II-1 could not be precisely determined, because it started gradually and was thus solely attributed to the weakness of the intercostal muscles and his severe scoliosis. The gradual onset of respiratory insufficiency in our patient is in contrast to the often sudden and dramatic respiratory presentation in “classic” SMARD1, which often requires emergency intubation, tracheostomy, and permanent mechanical ventilation. Of note, his 22year-old sister (A:II-2) continues to have an entirely normal respiratory function and no signs of diaphragmatic dysfunction on ultrasound. This illustrates a considerable intrafamilial phenotypic heterogeneity as seen in a report of 2 siblings with an identical IGHMBP2 mutation but a different phenotype: one child died at the age of 6 months of respiratory failure, while the other showed only limb weakness and mild nocturnal hypoventilation by the age of 12 years. It is DISCUSSION
thus obvious that additional factors may come into play and modify the phenotype. The patients belonging to families A and C are aged 22, 34, and 37 years, thus are the oldest patients alive with pathogenic IGHMBP2 mutations. The oldest SMARD1 patient reported to date was 20 years old. However, in contrast to our patients, he had been typically affected from early infancy and had survived long term on fulltime ventilation, despite a complicated disease course.22 There is an ongoing debate, whether SMARD1 should be considered a subtype of spinal muscular atrophy or rather a severe peripheral neuropathy with diaphragmatic palsy.23 Electrophysiologic findings in various patients have demonstrated a severe peripheral neuropathy rather than evidence of an isolated anterior horn cell disease.23 In addition, electrophysiologic and histopathologic studies indicate the involvement of the sensory system, even in the absence of clinical symptoms.21,24 The latter finding is in keeping with the clinical phenotype seen in our patients, who did not report any sensory symptoms despite electrophysiologic evidence of a severely impaired sensory nerve function. The discrepancy between clinical findings and neurophysiologic results cannot be readily explained. Clear genotype-phenotype relations for the known IGHMBP2 mutations have not been established. For some mutations, a potential link has been demonstrated between a milder clinical course and higher levels of residual IGHMBP2 protein function.4,7,20 Of the 4 mutations presented here, only one heterozygous mutation found in family C (c.138T.A, p.Cys46*) has been described before.19 Both this mutation and the splice-site mutation identified in family A (c.44911G.T, p.Lys150Asnfs*0) cause a premature termination codon or frameshift, leading to the absence of functional IGHMBP2 protein, which could be verified by Western blot in family A. The remaining mutations (family C: c.2911_2912delAG, p.Arg971Glufs*4; and family B: c.278411G.T) affect the last exons of the gene and may thus result in truncated but stable proteins (figure 2B). However, it remains speculative whether such a mechanism might be related to the patients’ clinical presentation. A modifying effect of the genetic background on the phenotypical expression of Ighmbp2 mutations has been demonstrated through a backcross of the nmd mutation25 of the mouse model of human SMARD1, from the original CAST/Ei strain into the BL6 strain. Genetic quantitative trait locus analysis of the B6.CAST backcross showed that CASTderived modifiers on mouse chromosomes 9, 10, and 16 were able to mitigate the nmd-related cardiomyopathy and increase the lifespan.26 Neurology 84
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Our findings expand the phenotypical spectrum of IGHMBP2-associated neuromuscular disorders and emphasize that IGHMBP2 mutations should be considered in the molecular genetic workup of patients presenting with isolated peripheral neuropathy even without additional respiratory involvement.
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AUTHOR CONTRIBUTIONS Gudrun Schottmann: recruited, investigated, and phenotyped the patients, performed neurophysiologic investigations, validated the results of the whole-exome sequencing, wrote the first draft of the manuscript. Heinz Jungbluth: recruited, investigated, and phenotyped the patients, wrote the first draft of the manuscript. Ulrike Schara: recruited, investigated, and phenotyped the patients. Ellen Knierim: validated the results of the whole-exome sequencing. Susanne Morales Gonzalez: performed the Western blotting and reverse transcription–PCR. Esther Gill: performed and analyzed the Sanger sequencing. Franziska Seifert: performed and analyzed the Sanger sequencing. Fiona Norwood: recruited, investigated, and phenotyped the patients, performed neurophysiologic investigations. Charu Deshpande: recruited, investigated, and phenotyped the patients. Katja von Au: recruited, investigated, and phenotyped the patients, provided reagents and materials. Markus Schuelke: recruited, investigated, and phenotyped the patients, performed the autozygosity mapping and haplotype analysis, validated the results of the wholeexome sequencing, supervised the work and obtained funding support, wrote the first draft of the manuscript. Jan Senderek: performed the autozygosity mapping and haplotype analysis, supervised the work and obtained funding support, wrote the first draft of the manuscript. All authors read the final version of the manuscript for intellectual content and gave their permission for publication.
ACKNOWLEDGMENT The authors thank the patients and their parents for participation in the study, Dr. med. Walter Stäblein (Charité Berlin) for doing the ultrasound investigations, and Prof. Utz Fischer (University Würzburg) for providing the 481/2 polyclonal rabbit anti-IGHMBP2 antibody. The excellent technical assistance of Angelika Zwirner (Charité Berlin) is much appreciated.
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STUDY FUNDING The project was funded by the Deutsche Forschungsgemeinschaft (SFB 665 TP C4) to M.S. and the NeuroCure Center of Excellence (Exc 257) to M.S.
DISCLOSURE
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The authors report no disclosures relevant to the manuscript. Go to Neurology.org for full disclosures.
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Recessive truncating IGHMBP2 mutations presenting as axonal sensorimotor neuropathy Gudrun Schottmann, Heinz Jungbluth, Ulrike Schara, et al. Neurology published online January 7, 2015 DOI 10.1212/WNL.0000000000001220 This information is current as of January 7, 2015 Updated Information & Services
including high resolution figures, can be found at: http://www.neurology.org/content/early/2015/01/16/WNL.0000000000 001220.full.html
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Supplementary material can be found at: http://www.neurology.org/content/suppl/2015/01/07/WNL.000000000 0001220.DC1.html
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