Abstract
Objective: Idiopathic peripheral neuropathy is common and likely due to genetic factors that are not detectable using standard linkage analysis. We initiated a candidate gene approach to study the genetic influence of the small heat shock protein (sHSP) gene family on an axonal motor and motor/sensory neuropathy patient population.
Methods: The promoter region and all exonic and intronic sequences of the 10 sHSP genes (HSPB1-HSPB10) were screened in a cohort of presumed nonacquired, axonal motor and motor/sensory neuropathy patients seen at the Ohio State University Neuromuscular Clinic.
Results: A missense mutation in the gene encoding small heat shock protein B3 (HSPB3, also called HSP27, protein 3) was discovered in 2 siblings with an asymmetric axonal motor neuropathy. Electrophysiologic studies revealed an axonal, predominantly motor, length-dependent neuropathy. The mutation, HSPB3(R7S), is located in the N-terminal domain and involves the loss of a conserved arginine.
Conclusions: The discovery of an HSPB3 mutation associated with an axonal motor neuropathy using a candidate gene approach supports the notion that the small heat shock protein gene family coordinately plays an important role in motor neuron viability.
Glossary
- CMT=
- Charcot-Marie-Tooth disease;
- EDTA=
- ethylenediaminetetraacetic acid;
- HMN=
- hereditary motor neuropathy;
- mRNA=
- messenger RNA;
- sHSP=
- small heat shock protein.
Peripheral neuropathy is extremely common, with a prevalence of 2% to 8%.1,2 Despite intensive investigation, the etiology of neuropathy cannot be determined in up to 40% of patients.3–6 Although hereditary neuropathies have been reported to account for 14% of hospital-referred idiopathic neuropathies,6 this is likely an underestimation, because large families in whom linkage analysis can be performed are rare and the current list of genes associated with hereditary neuropathy is incomplete. Moreover, it is not practical or economically feasible to perform genetic analysis on every patient with an idiopathic neuropathy.
To study the genetic influence on a complex phenotype such as peripheral neuropathy, a candidate gene identification approach can be applied when a group of genes is selected based on an a priori hypothesis about their etiologic role in the disease.7,8 As part of our efforts to study the etiology of axonal forms of predominantly motor neuropathies, with or without sensory involvement (i.e., Charcot-Marie-Tooth disease [CMT] type 2, hereditary motor neuropathy [HMN], or chronic idiopathic axonal polyneuropathy), we selected the small heat shock protein (sHSP) gene family as a candidate group of genes to sequence in a well-defined population of patients with idiopathic neuropathy.
The small heat shock proteins (sHSP) are multifunctional, widely distributed, and collaborative proteins. Eleven sHSP genes (HSPB1-11) have been described in humans, and they freely interact with one another to form large hetero-oligomeric complexes in cells.9,10 Mutations in HSPB1 and HSPB8 result in HMN and in forms of CMT that have minimal sensory involvement.11–18 We report here a novel mutation in HSPB3 that is associated with a hereditary axonal motor-predominant neuropathy linking a third member of the sHSP family to motor neuron vulnerability.
METHODS
Subjects.
Patients seen at the Ohio State University Medical Center Department of Neurology, Neuromuscular Medicine and Muscular Dystrophy Association Clinics with clinical findings consistent with a peripheral predominantly motor neuropathy, with or without sensory symptoms, and axonal features on electrophysiologic testing were recruited sequentially. A pilot cohort of 28 patients was enrolled over a 6-month period. Patients were excluded if they had abnormal serum glucose, lead, serum protein electrophoresis, or glucose tolerance testing. Those with clear demyelinating physiology (e.g., CMT type 1) were excluded. Patients were recruited irrespective of whether there was a positive family history for neuropathy.
Standard protocol approvals, registrations, and patient consents.
The study was approved by the institutional review board at the Ohio State University Medical Center, and written informed consent was obtained from all participants.
Study protocol and sHSP sequencing.
A single peripheral blood sample was obtained, and DNA was extracted from 2 to 5 mL of peripheral blood using a standard salting out method.19 The DNA was resuspended in TE buffer (10 mM Tris, 0.1 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0). Spectrophotometry was used for concentration estimation, and all samples were diluted to 1,000 ng/μL.
Forty-three amplicons, exons and noncoding sequences, of 10 sHSP genes (HSPB1-10) were amplified (table e-1 on the Neurology® Web site at www.neurology.org). The PCR reaction consisted of 67 mM Tris pH 8.0, 10 mM β-mercaptoethanol, 17 mM ammonium sulfate, 67 μM EDTA, 500 μM deoxyribonucleotide triphosphates, 1 U Taq polymerase (ABI, Carlsbad, CA), and 2.5 μM each F and R m13 tailed primer in a total volume of 25 μL. The magnesium chloride and dimethyl sulfoxide concentrations in addition to the annealing temperatures and cycle numbers were reaction specific. The PCR reactions were cleaned of primer dimer with exonuclease/bovine alkaline phosphatase. Five microliters of the cleaned product was used in the Big Dye Terminator V1.1 Cycle Sequencing (ABI) reaction. The sequencing reaction was cleaned on a Performa DTR 96-well plate (Edge BioSystems, Gaithersburg, MD) and electrophoresed on a 3130 Genetic Analyzer (ABI). The results were analyzed using Mutation Surveyor software version 3.24 (SoftGenetics LLC, State College, PA).
RESULTS
Detection of a missense mutation in HSPB3 using a candidate gene approach.
DNA from 28 patients was analyzed initially. The proband had a missense mutation, c.21 G>T p.R7S, in the gene encoding small heat shock protein B3 (HSPB3, also called HSP27, protein 3, Online Mendelian Inheritance in Man 604624). The mutation was located in the N-terminal domain and involved the loss of a conserved arginine. The variation was not found in 200 normal alleles. The mutation was analyzed using Align GVGD (International Agency for Research on Cancer, Lyon, France) and gave a GD = 109.21, a class C65 variation (most likely to interfere with function) (figure, A). The proband had a sister with similar symptoms and clinical findings who also carried the HSPB3 c.21 G>T p.R7S mutation. The affected sibling declined electrophysiologic testing. Two unaffected siblings did not harbor the mutation (figure, B). Comparison of the currently recognized sHSP mutations associated with HMN and CMT indicates that the HSPB3(R7S) mutation does not occur in a conserved amino acid and is not within the α-crystallin domain (figure, C).
Figure HSPB3 mutation associated with hereditary motor neuropathy
(A) Mutation Surveyor (SoftGenetics LLC, State College, PA) summary of c.21 G>T p.R7S heterozygote. (B) HSPB3(R7S) pedigree. The proband (arrow) and her sister had a history of asymmetric weakness and limb muscle atrophy (dark circles). They had 2 unaffected sibling (clear circle and square). The mutation leading to a serine at amino acid position 7 (p7S) was found in the proband and her affected sister. The unaffected siblings were found to have the wild-type arginine at position 7 (p7R). The mother of the proband also has lower extremity weakness and atrophy but declined genetic testing and was not examined. (C) Alignment of HSPB3 with HSPB1 and HSB8 was performed using the Cobalt alignment tool (National Center for Biotechnology Information, Bethesda, MD). The location of mutations in these proteins that result in hereditary motor neuropathy are underlined and emphasized with a diamond. The mutation described here is emphasized with a circle.
Clinical and electrophysiologic characterization of patient with HSPB3 mutation.
The proband was a 58-year-old woman who presented with a history of slowly progressive distal arm and leg weakness. Her symptoms began in her early 20s, when she noticed easy fatigability in her legs, frequent ankle turning, and instability going up and down stairs. Her symptoms were progressive, but were restricted to the legs until 5 years before presentation, when she first noticed bilateral hand weakness. She had been evaluated 25 years before presentation with electrodiagnostic studies and a sural nerve biopsy and had been told she had “an axonal hereditary neuropathy.” Her medical history was otherwise benign. Her family history revealed a 51-year-old sister who was similarly affected, and a second sister and 2 brothers who were unaffected. Her father was healthy without neuromuscular symptoms, and she had 2 healthy children. Although her mother was also affected by history, she was not available for examination.
Examination showed normal mentation, speech, and cranial nerves. The patient had atrophy of the intrinsic foot and hand muscles without pes cavus or hammer toe deformities. Her strength was normal (Medical Research Council grade 5) in all upper extremity and proximal lower groups. The ankle dorsiflexors, evertors, invertors, and plantar flexors all were grade 0 to 1. She had a marked steppage gait and could not walk on her heels or toes. Sensory examination revealed mild loss of light touch over the toes to nylon hair testing, but otherwise normal sensation to pin, position, and vibration. Her deep tendon reflexes were 2+ throughout except for the ankle jerks, which were absent. Nerve conduction studies revealed normal sural sensory responses, and reduced compound motor action potentials in the peroneal and tibial nerves (table 1). The needle electrode examination demonstrated abnormal spontaneous activity in the hand and distal leg muscles with signs of chronic denervation in all lower limb and hand muscles.
Table Electrophysiologic measurements
Examination of the affected sister showed normal mentation, speech, and cranial nerves. She had distal upper and lower limb weakness with atrophy. She had a steppage gait, and sensory examination was normal to light touch, pin, position, and vibration. Her deep tendon reflexes were 2+ in the upper extremity but absent at the knees and ankles. The unaffected siblings had no symptoms of weakness and had normal gaits but were not examined.
DISCUSSION
The most described function of sHSPs is that of protein chaperone. Chaperones ensure the correct 3-dimensional conformation of polypeptides under normal conditions and protect cells from errant protein folding under conditions of cell stress.20,21 sHSPs form dimers that are the basic building blocks for large oligomeric complexes, and interact with each other in complex ways to form heterodimers and mixed heterooligomers, a property that is not well understood.22,23 The sHSP family does not require adenosine triphosphate for protein chaperone activity, in contrast to the major heat shock protein families, yet they bind to substrate proteins and maintain them in a refolding competent state.24,25
Little is known about the function of HSPB3 in cells. It is the only member of the sHSP family to be encoded by a single exon and contains 150 amino acids.26 In the mouse, hspb3 messenger RNA (mRNA) (mouse homolog to human HSPB3) is expressed in the gray matter of the spinal cord, including large cells in the ventral horn that are morphologically motor neurons (Allen Spinal Cord Atlas).27 The expression of human HSPB3 mRNA is less well characterized; however, mRNA may be enriched in pituitary gland and nerve.28 HSPB3 constructs have been expressed in HeLa cells, and the protein was distributed throughout the cytoplasm and nucleus and not associated with nuclear bodies unless cells were exposed to heat shock.28 HSPB3 has also been shown to interact with HSPB2 and to be highly expressed in muscle.29 The HSPB2/HSPB3 complex associates with HSPB8 in vitro.30
Many of the HSPB1 and HSPB8 mutations that cause HMN form intracellular aggregates when expressed in neurons, including mouse motor neurons in primary culture and are toxic to neurons when expressed at high levels.22,30–33 Aggregate formation is not a marker for protein chaperone activity; however, this observation makes clear that a functional consequence of HMN mutations is to alter how the protein interacts with other proteins, perhaps by making proteins insoluble. Seven of the 11 currently reported mutations in HSPB1 target positively charged arginines or lysines that are critical for the structural and functional integrity of the α-crystallin domain and seem to alter sHSP chaperone activity as measured by the ability of the protein, in vitro, to refold a denatured protein substrate.12,34,35 These findings suggest that dominant mutants of HSPB1 and HSPB8 interact via a common pathway. The HSPB3 mutation discovered in our patient is also an arginine substitution, suggesting that it may be similarly involved in this pathway. Future work will be needed to characterize the functional consequences of the HSPB3 mutation on protein binding and to determine the sHSP function or functions that are relevant to motor neuron survival. Given the involvement of multiple members of this collaborative set of sHSPs in axonal motor or motor/sensory neuropathy, individual mutations in multiple sHSPs may have a combinatorial effect on sHSP function as a whole, leading to an increased susceptibility to motor neuron dysfunction.
ACKNOWLEDGMENT
The authors thank the affected individuals and their relatives for participating in this research project.
DISCLOSURE
Dr. Kolb, Dr. Snyder, Mr. Poi, Ms. Renard, Ms. Bartlett, Dr. Gu, Mr. Sutton, and Dr. Arnold report no disclosures. Dr. Freimer receives research support from Alexion Pharmaceuticals, Inc. Dr. Lawson reports no disclosures. Dr. Kissel receives research support from Alexion Pharmaceuticals, Inc., Acceleron Pharma, and Abbott; serves as a consultant for Genzyme Corporation and Alexion Pharmaceuticals, Inc.; serves on the editorial board of Muscle & Nerve; and receives publishing royalties for Diagnosis and Management of Peripheral Nerve Disorders Contemporary Neurology Series (Oxford Press, 2004). Dr. Prior reports no disclosures.
Footnotes
-
Supplemental data at www.neurology.org
Study funding: Sponsored by start-up funds from The Ohio State University Medical Center (S.J.K.).
Disclosure: Author disclosures are provided at the end of the article.
Received August 1, 2009. Accepted in final form November 5, 2009.
REFERENCES
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