Truncating and missense mutations in IGHMBP2 cause Charcot-Marie Tooth disease type 2

Abstract

Using a combination of exome sequencing and linkage analysis, we investigated an English family with two affected siblings in their 40s with recessive Charcot-Marie Tooth disease type 2 (CMT2). Compound heterozygous mutations in the immunoglobulin-helicase-μ-binding protein 2 (IGHMBP2) gene were identified. Further sequencing revealed a total of 11 CMT2 families with recessively inherited IGHMBP2 gene mutations. IGHMBP2 mutations usually lead to spinal muscular atrophy with respiratory distress type 1 (SMARD1), where most infants die before 1 year of age. The individuals with CMT2 described here, have slowly progressive weakness, wasting and sensory loss, with an axonal neuropathy typical of CMT2, but no significant respiratory compromise. Segregating IGHMBP2 mutations in CMT2 were mainly loss-of-function nonsense in the 5' region of the gene in combination with a truncating frameshift, missense, or homozygous frameshift mutations in the last exon. Mutations in CMT2 were predicted to be less aggressive as compared to those in SMARD1, and fibroblast and lymphoblast studies indicate that the IGHMBP2 protein levels are significantly higher in CMT2 than SMARD1, but lower than controls, suggesting that the clinical phenotype differences are related to the IGHMBP2 protein levels.

Figure 1

Photographs of CMT2 Individuals with IGHMBP2 Mutations (A) Legs and feet of family A, with individual II.1 also showing silicon ankle foot orthosis. (B) Hands of family A, individual II.1. (C) Left hand of family B, individual II.2. (D) Right foot of family B, individual II.1. (E) Trombone-shaped tongue of family A, individual II.1. (F) Left hand of family B, individual II.1.

Figure 2

Morphological Appearances of the Sural Nerve Biopsy in the Individual with IGHMBP2 Mutation, Healthy Age-Matched Control and Individual with MFN2 Mutation (A, D, and G) Sural nerve biopsy of a healthy age-matched control. (B, E, and H) Sural nerve biopsy of a patient with IGHMBP2 mutation. (C, F, and I) Sural nerve biopsy of an individual with known MFN2 mutation. Semithin resin sections stained with toluidine blue (A, healthy age-matched control; C, individual with known MFN2 mutation) and methylene blue azure–basic fuchsin (MBA-BF) (B, individual with IGHMBP2 mutation). When compared with the control (A), the biopsy of the individual with IGHMBP2 mutation (B) shows a moderate reduction in density of the large myelinated fibers, whereas the small myelinated fibers are well preserved and regeneration clusters is not a feature. In contrast, in the individual with MFN2 mutation (C), there is near complete loss of large fibers and severe widespread loss of small myelinated fibers. Ultrastructural assessment reveals occasional actively degenerating axonal profiles (E, red arrowhead) in the individual with IGHMBP2 mutation. In the individual with MFN2 mutation rare regeneration clusters are seen (F, brown arrowhead). The thickness and configuration of the myelin sheaths of remaining large (D and E, blue arrowheads) and small myelinated fibers (G, H, and I, green arrowheads) are similar to that seen in a healthy age-matched control. Scale bar represents 35 μm in (A)–(C) and 5 μm in (D)–(I).

Figure 3

Pedigrees of Four CMT2 Families Affected by IGHMBP2 Mutations and Chromatograms of These Mutations (A–D) Pedigrees of family A (A), family B (B), family C (C), and family D (D). (E) A schematic of IGHMBP2 (993 amino acids) showing the helicase, R3H, and ZnF domains. The relative base pair positions of the identified mutations are located. Mutations in bold are nonsense or frameshift and result in an altered transcript. ^∗ = pathogenic mutations found before in SMARD1 patients. (F) Conservation of the missense mutations found in IGHMBP2. A selected subset of 9 species were chosen, representing the 100 species available at the USCS browser. Red boxes indicate the location of the amino acid changed due to the mutation.

Figure 4

Localization of the IGHMBP2 Protein in Fibroblasts Scale bar represents 44.00 μm. Green represents IGHMBP2; blue represents 4’,6-diamidino-2-phenylindole (DAPI) staining for the nucleus. No difference in clustering of the truncated protein is found between the control and both the affected individuals and the carrier. SMARD1 = p.Gly98Fs; CMT2 = p.Cys46^∗ + p.Arg971Glufs^∗4; Carrier = p.Arg971Glufs^∗4. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.05% Triton X-100 and blocked in 10% FBS. Coverslips were incubated with a 1:1000 dilution of primary antibody (Millipore) for 60 min, washed with PBS and incubated with a 1:2000 dilution of goat anti-mouse immunoglobulin G Alexa Fluor 488-A11008 secondary antibody (Invitrogen) for 60 min. Following, the coverslips were washed with PBS and mounted on microscope slides with Prolong Gold Antifade with DAPI and imaged using a Zeiss 710 confocal microscope (Carl Zeiss AG) with the 63× oil immersion objective.

Figure 5

Protein Levels of IGHMBP2 Normalized Against an Actin Control in All Individuals for Both Fibroblasts and Lymphoblastoid Cell Lines (A) Protein levels in fibroblast cell lines of families A, B, and C. (B) Protein levels in lymphoblastoid cell lines of families A and B. Family A consists of two affected individuals (II.1 + II.2) with lower levels of the protein in fibroblasts in comparison with the carrier of the p.Arg971Glufs^∗4 mutation (I.1). This is consistent in lymphoblasts, where individual II.2 has lower levels than the carrier (I.1). Family B consists of one affected individual (I.1) with lower levels of the IGHMBP2 protein in comparison with the carriers of the p.Cys46^∗ mutation (II.2, I.1) or the p.Arg971Glufs^∗4 mutation (I.2). These all have lower levels than the unaffected sibling of the patient (II.3). This is consistent in the lymphoblasts. For family C, only two patient fibroblasts cell lines were available, both showing reduced levels in comparison with controls. All SMARD1 fibroblasts and lymphoblasts have lower levels than any of the individuals. ^∗ = individuals with CMT2. (C) Protein levels of the IGHMBP2 protein normalized against actin in controls, CMT2 individuals, and SMARD1 individuals. There is a significant difference between all groups. All samples were standardized against two controls: C1 and C2. Data are presented as mean ± SEM. Statistical analysis was performed with Bonferroni’s multiple comparison test. ^∗p < 0.05; ^∗∗∗p < 0.0001. (D) Existence of a degradation band around 70–80 kDa in individuals with CMT2 and a combination of the p.Cys46^∗ and p.Arg971Glufs^∗4 mutations. Cells were lysed in 50 μl of NP40 buffer (150 mM Tris (pH 8), 1 mM EDTA, 150 mM NaCl, 0.5% NP40) containing 1× complete protease inhibitor cocktail (Roche). 80 μg of protein was run on a 4%–12% Bis-Tris gel, blocked in 5% (w/v) milk for 1 hr at room temperature. Membranes were incubated overnight with the primary antibody (Millipore) at 4°C. β-actin (Sigma) was used as a loading control.