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.

Main Text

1 Skre H. Genetic and clinical aspects of Charcot-Marie-Tooth’s disease. 2 Harel T.

Lupski J.R. Charcot-Marie-Tooth disease and pathways to molecular based therapies. , 3 Reilly M.M.

Shy M.E. Diagnosis and new treatments in genetic neuropathies. , 4 Saporta M.A.

Shy M.E. Inherited peripheral neuropathies. , 5 Shy M.E. Inherited peripheral neuropathies. , 6 Timmerman V.

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Züchner S. Genetics of Charcot-Marie-Tooth (CMT) Disease within the Frame of the Human Genome Project Success. 2 Harel T.

Lupski J.R. Charcot-Marie-Tooth disease and pathways to molecular based therapies. , 3 Reilly M.M.

Shy M.E. Diagnosis and new treatments in genetic neuropathies. , 4 Saporta M.A.

Shy M.E. Inherited peripheral neuropathies. , 5 Shy M.E. Inherited peripheral neuropathies. , 6 Timmerman V.

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Züchner S. Genetics of Charcot-Marie-Tooth (CMT) Disease within the Frame of the Human Genome Project Success. , 7 Bombelli F.

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Patzkó A. Axonal Charcot-Marie-Tooth disease. 10 Timmerman V.

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Reid E. Overlapping molecular pathological themes link Charcot-Marie-Tooth neuropathies and hereditary spastic paraplegias. , 11 Roberts R.C. The Charcot-Marie-Tooth diseases: how can we identify and develop novel therapeutic targets?. Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disorder of the peripheral nervous system with an estimated prevalence of 1 in 2,500 individuals.Clinical manifestations of CMT include slowly progressive distal weakness, wasting, and sensory loss, which spreads proximally as the disease progresses. Clinically, CMT can be divided into two major phenotypic types: a demyelinating form (CMT type 1 [CMT1]) and an axonal form (CMT type 2 [CMT2]).Mutations in 15 unique genes have so far been identified as causing CMT2. Despite this significant progress, about 70% of people with CMT2 do not have a genetic diagnosis.The identification of the remaining CMT2 genes is expected to yield important insights into the disease pathways and pathophysiology associated with axonal degeneration. In addition, it is becoming evident that the phenotypic and genotypic intersection of CMT2 with related motor neuron disorders of axonal degeneration and other neuromuscular diseases is more extensive than previously thought, increasing the importance of gene identification and characterization in this area.

Table 2, Figure 1 Photographs of CMT2 Individuals with IGHMBP2 Mutations Show full caption (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 Show full caption (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). Table 1 List of IGHMBP2 Mutations Found in Individuals with Axonal Neuropathy Family Ethnicity Sex Diagnosis Age at Onset Current Age Protein Change Nucleotide Change A English Female CMT2 7 years 43 years p.Cys46∗ + p.Arg971Glufs∗4 c.138T>A + c.2911_2912delAG A English Female CMT2 6 years 40 years p.Cys46∗ + p.Arg971Glufs∗4 c.138T>A + c.2911_2912delAG B English Male CMT2 5 years 23 years p.Cys46∗ + p.Arg971Glufs∗4 c.138T>A + c.2911_2912delAG C Serbian Male CMT2 2 years 14 years p.Cys46∗ + p.Phe202Val c.138T>A + c.604T>G C Serbian Female CMT2 2 years 15 years p.Cys46∗ + p.Phe202Val c.138T>A + c.604T>G D Pakistani Female CMT2 + Down Syndrome 7 years 20 years p.Pro531Thr + p.Val580Ile c.1591C>A + c.1738G>A E Vietnamese Female CMT2 3 years 39 years p.Arg605∗ + p.His924YTyr c.1813C>T + c.2770C>T F English Male CMT2 4 years 15 years p.Ser80Gly + p.Cys496∗ c.238A>G + c.1488C>A G USA Female CMT2 6 years 10 years p.Trp386Arg + p.Arg971Glufs∗4 c.1156T>C + c.2911_2912delAG H Polish Female CMT2 4 years 28 years p.990_994del (Hom) c.2968_2980del (Hom) I Italian Female CMT2 1 years 12 years p.Val373Gly + p.Ala528Thr c.1118T>G + c.1582G>A I Italian Male CMT2 1 years 6 years p.Val373Gly + p.Ala528Thr c.1118T>G + c. 1582G>A J Korean Male CMT2 5 years 41 years p.Asn245Ser (Het) c.734A>G (Het) K English Male CMT2 7 years 20 years p.Arg605∗ (Het) + deletion c.1813C>T (Het) + deletion K English Female CMT2 10 years 18 years p.Arg605∗ (Het) + deletion c.1813C>T (Het) + deletion Hom, homozygous; Het, Heterozygous. Table 2 Electrophysiology Data for the Individuals with CMT2 Individual 1 2 3 4 5 6 7 8 9 10 12 13 11 14 15 Family no. A A B C C D E F G H I I J K K Sex/age (y) F/43 F/40 M/23 M/14 F/15 F/19 F/39 M/15 F/10 F/28 F/12 M/6 M/41 M/20 F/18 Ethnicity English English English Serbian Serbian Pakistani Vietnam English USA Polish Italian Italian Korean English English Age at first symptoms 7 years 6 years <5 years <2 years <2 years <10 years <3 years 4 years 6 years 4 years 1 years 1 years 5 years 7 years 10 years First symptoms Toe walking Toe walking Difficulty walking Delayed milestones Delayed walking hypotonia, foot drop Delayed milestones Foot drop Foot drop Hand weakness Limb weakness equino-varus Gait difficulty Foot drop Feet deformity Weakness a a Weakness: N, normal; + > 4, distal muscles, ++ < 4, distal muscles, +++, proximal weakness (knee flexion and extension, elbow flexion and extension or above). UL +++ +++ ++ +++ +++ +++ +++ ++ N ++ +++ + + ++ + LL +++ +++ ++ +++ +++ +++ +++ ++ ++ +++ +++ ++ ++ ++ + Pinprick b b Pinprick and vibration sensation: N, normal; +, reduced below wrist/ankle; ++, reduced below knee/elbow; +++, reduced at or above elbow/knee. UL N N + N N n/a N n/a N + + n/a + N N LL + N + N N n/a N n/a N n/a + n/a + N N Vibration c c Reflexes: N, normal/present; ++, brisk; +++, brisk with extensor plantars; +/−, present with reinforcement; abs, absent; abs (AJ), absent ankle jerks only. UL N N N N N n/a N n/a N n/a n/a n/a ++ N N LL + N + N N n/a N n/a N n/a n/a n/a ++ N N Reflexes UL Abs Abs Abs Abs Abs Abs Abs n/a + +/− Abs abs abs N N LL Abs Abs Abs Abs Abs Abs Abs n/a Abs (AJ) Abs Abs abs abs AJ +/− Bulbar Rhomboid tongue Wasted tongue No No No Wasted tongue No n/a No No No No No No No Respiratory support No No No No No No No No No No No No No No No Overall maximal function Independent ambulation Independent ambulation Independent ambulation n/a n/a Independent ambulation n/a n/a n/a n/a Independent ambulation Walking with stick Independent ambulation Independent ambulation Independent ambulation Walking aids AFO AFO (past) n/a WC WC WC WC since 16 AFO AFO WC WC since age 5 years Bilateral support AFO AFO+Crutches No AFO, ankle-foot orthosis; n/a, not available; LL, lower limbs; UL, upper limbs; WC, wheelchair. Table 3 Electrophysiology Data from the Individuals from Our CMT2 Cohort Individual 1 2 3 4 5 6 7 11 12 13 14 15 Family no. A A B C D D E I I J K K Age at examination (y) 17 25 16 20 13 7 32 7 1.5 40 12 10 Radial n. Sensory Amp 2 μV Abs Abs 13 μV NT NT n/a Abs n/a NT 16 μV n/a Sensory CV 50 m/s Abs Abs 69 m/s NT NT n/a Abs n/a NT 63 m/s n/a Median n. Motor DML NT Abs Abs 3.5 ms 5.1 ms 3.1 ms Abs Abs n/a 6 ms 2.8 ms 3.2 ms Motor Amp NT Abs Abs 5.7 mV 0.02 mV 2.8 mV Abs Abs n/a 0.7 mV 18.8 mV 21.8 mV Motor CV NT Abs Abs 46 m/s 30 m/s 42 m/s Abs Abs n/a 33.6 m/s 58 m/s 58 m/s Sensory Amp Abs Abs Abs 6 μV Abs 20 μV 2.2 uV Abs Abs Abs 32 μV 26 μV Sensory CV Abs Abs Abs 45 m/s Abs 49 m/s 59.8 m/s Abs Abs Abs 60 m/s 52 m/s Ulnar n. Motor DML 3.8 ms 3.3 ms 4.3 ms 3.5 ms NT NT Abs n/a n/a 3.1 2.8 ms 3.2 ms Motor Amp 0.8 mV 3.7 mV 5.7 mV 2.9 mV NT NT Abs n/a n/a 14.3 8.9 mV 12.8 mV Motor CV 51 m/s 51 m/s 45 m/s 46 m/s NT NT Abs n/a 55 m/s 41.1 58 m/s 62 m/s Sensory Amp NT Abs Abs Abs Abs 12 μV 2.0 uV n/a n/a Abs 16 μV 14 μV Sensory CV NT Abs Abs Abs Abs 48 m/s 50.3 m/s n/a n/a Abs 67 m/s 53 m/s Peroneal n. Motor DML NT Abs Abs NT NT Abs n/a Abs Abs Abs Abs 4.9 ms Motor Amp NT Abs Abs NT NT Abs n/a Abs Abs Abs Abs 4.6 mV Motor CV NT Abs Abs NT NT Abs n/a Abs Abs Abs Abs 51 m/s Tibial n. Motor DML 9.3 ms Abs Abs NT Abs Abs n/a n/a Abs Abs 6.3 ms 4.3 ms Motor Amp 0.08 mV Abs Abs NT Abs Abs n/a n/a Abs Abs 2 mV 8.2 mV Motor CV 34 m/s Abs Abs NT Abs Abs n/a n/a Abs Abs 46 m/s 50 m/s Sural n. Sensory Amp Abs Abs Abs Abs Abs Abs n/a Abs Abs Abs 38 μV 35 μV Sensory CV Abs Abs Abs Abs Abs Abs n/a Abs Abs Abs 59 m/s 49 m/s Abs, absent; NT, not tested. We initially studied a family where two siblings were affected with CMT2. The onset was in late childhood, with slowly progressive disease and parents that were clinically and electrically unaffected (family A). The proband is currently 43 (family A, individual 1) and her sister is 40 years of age (family A, individual 2), both work, are able to drive, and use a stick to walk with silicon ankle foot orthosis. Examination of the index case at 43 years of age revealed bilateral foot drop, distal weakness, and wasting in the upper and lower limbs, with mild proximal lower limb weakness ( Figure 1 ). Reflexes were absent and there was sensory loss in the feet and hands. Cranial nerves were normal apart from a trombone-shaped tongue ( Figure 1 ). There were no respiratory problems. Chest X-ray and sleep study was normal; nerve conduction studies and sural nerve biopsy indicated an axonal neuropathy ( Figure 2 ). Her sister had milder clinical features, and examination findings at the age of 40 years revealed bilateral foot drop, distal weakness, and wasting in the upper and lower limbs and areflexia. There were no respiratory problems and an axonal neuropathy was seen on nerve conduction studies ( Table 1 Table 3 ; see also Table S1 available online).

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d’Ydewalle C.

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Jablonka S.

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. Two compound heterozygous mutations were identified in the affected individuals in immunoglobulin helicase μ-binding protein 2 (IGHMBP2 [MIM 600502 ]; RefSeq: NM_002180.2), a nonsense 5′ mutation (c.138T>A: p.Cys46) and a 3′ frameshift mutation in the last exon of the gene (c.2911_2912delAG: p.Arg971Glufs4). The mother and father were heterozygous for the c.138T>A and c.2911_2912delAG mutations, respectively. These mutations were absent from the 1000 Genomes database (healthy controls) and our in-house exome database of 480 clinically and neuropathologically normal controls. Mutations in IGHMBP2 have previously been associated with a different phenotype, spinal muscular atrophy with respiratory distress type 1 (SMARD1 [MIM: 604320]), a devastating neuromuscular disorder with muscle weakness and atrophy severely affecting the diaphragm.SMARD1 mutations are typically missense in the helicase domain or mutations where both alleles are loss-of-function, usually in the 5′ region of the gene.The onset of this condition is usually in the first few weeks of life with early respiratory failure and death in infancy, typically before 12 months of age.The longest surviving children reported were 13 and 15 years of age, they had profound upper and lower limb muscle and trunk weakness and respiratory compromise.Three children have been reported with delayed onset of respiratory distress of between 4 and 10 years old and designated juvenile SMARD1.

Figure 3 Pedigrees of Four CMT2 Families Affected by IGHMBP2 Mutations and Chromatograms of These Mutations Show full caption (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. IGHMBP2 was Sanger sequenced in a cohort of 85 likely recessive CMT2 families, and CMT exome sequence data was analyzed from the Hussman Institute for Human Genomics. A total of 11 CMT2 families with IGHMBP2 mutations were identified ( Table 1 ). All families with CMT2 and IGHMBP2 mutations showed an autosomal recessive pattern of inheritance but in two families only one heterozygous pathogenic mutation was identified ( Table 1 Figure 3 ). The phenotype consisted of childhood onset, progressive CMT2 with mild proximal weakness and scoliosis in some cases. Sensory involvement was mild glove and stocking and electrophysiology indicated a sensory and motor axonal neuropathy in all cases ( Tables 2 and 3 ). Two further cases had unusually shaped tongues ( Figure 1 ); none of the cases had significant respiratory compromise, recurrent chest infections or previous ventilator assistance or sleep apnea. One case had trisomy 21 and Down syndrome ( Tables 2 and 3 ).

∗ nonsense variant in combination with either an AG deletion, causing a p.Arg971Glufs∗4 frameshift in the last exon (family A and B), or a novel p.Phe202Val variant (family C). Haplotype analysis indicated that a common founder was unlikely in the individuals with p.Cys46∗ variants ( 40 Lim S.C.

Bowler M.W.

Lai T.F.

Song H. The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1. 40 Lim S.C.

Bowler M.W.

Lai T.F.

Song H. The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1. Three families (five individuals) carried the p.Cys46nonsense variant in combination with either an AG deletion, causing a p.Arg971Glufs4 frameshift in the last exon (family A and B), or a novel p.Phe202Val variant (family C). Haplotype analysis indicated that a common founder was unlikely in the individuals with p.Cys46variants ( Figure S1 Table S2 ). In the IGHMBP2 helicase domain(PDB code 4B3G), Cys46 is located in the β-barrel of domain 1B and the side chain does not interact with any neighboring residues. Phe202 is part of an α-helix in domain 1A and is pathogenic, but not central to the protein structure, suggesting a milder phenotype. Family 4 has compound heterozygous missense mutations and presents with a known severe pathogenic variant p. Val580Ile and a novel variant p.Pro531Thr. Pro531 lies in a loop region and is exposed to the solvent region on the protein surface. The side chain of the residue does not interact with neighboring residues and will likely have a milder phenotype ( Figure S2 ). Val580 lies near a β strand in the core of domain 2A and interacts with Ser539 on a neighboring strand to stabilize the RecA-like fold. Mutating Val580 to isoleucine, which has a longer side chain, likely disrupts the formation of the β sheet and hence destabilizes domain 2A ( Figure S2 ). Similarly, in family G (p.Trp386Arg), mutating a hydrophobic residue to a positively charged residue can result in protein instability due to the loss of some favorable van der Waals contacts with neighboring hydrophobic residues. The other missense mutations at Asn245, Val373, and Ala528 (families I and J) are also predicted to cause protein instability, resulting in loss of functional protein Figure S2 ). In the two families with a single IGHMBP2 mutation and recessive CMT2 phenotype, we additionally analyzed the 5′ promoter region and the exome BAM files for sequencing coverage and carried out IGHMBP2 cDNA sequencing in the two affected individuals from family K. The cDNA analysis identified that the stop mutation was hemizygous, suggesting a deletion on the other allele ( Figure S3 Table 1 ).

41 Pitt M.

Houlden H.

Jacobs J.

Mok Q.

Harding B.

Reilly M.

Surtees R. Severe infantile neuropathy with diaphragmatic weakness and its relationship to SMARD1. The neurophysiological pattern in individuals with CMT2 and IGHMBP2 mutations was consistent with a mild motor and sensory axonal polyneuropathy (velocities 40–50 m/s) ( Table 3 ). This is in contrast to SMARD1 with markedly reduced motor conduction velocities (around 20 m/s), particularly in the legs, and a very marked reduction or loss of the compound muscle action potential.Nerve biopsy in CMT2 family A, individual 1 showed a moderate reduction in density of the large myelinated fibers, whereas the small myelinated fibers are well preserved. This is in contrast with the individual with a MFN2 mutation where there is near complete loss of large fibers and severe widespread loss of small myelinated fibers. Ultrastructural assessment revealed occasional actively degenerating axonal profiles in CMT2 with an IGHMBP2 mutation, but these were rare in MFN2 patients ( Figure 2 ).

Fibroblast and lymphoblastoid cell lines from families 1 and 2 were used to investigate whether the c.138T>A mutation resulted in nonsense mediated decay. The presence of both wild-type (WT) and mutant mRNA persisted in carriers and affected individuals, indicating that NMD has not been activated ( Figure S3 ). Because the c.2911_2912del mutation is located in the last exon of the gene, we would not expect nonsense-mediated decay to occur. Fibroblasts from individuals with SMARD1 with heterozygous or homozygous frameshift mutations also failed to show NMD ( Figure S3 ), suggesting that IGHMBP2 is protected from NMD and likely produces truncated protein products.

Figure 4 Localization of the IGHMBP2 Protein in Fibroblasts Show full caption 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. Considering the presumed existence of a truncated protein in the CMT2 cell lines, and for the missense mutations, immunofluorescence experiments were performed to detect changes in the localization or potential clustering of the truncated protein. Misfolded or mislocalized proteins can interact inappropriately with other cellular factors to cause toxicity. However, results show no difference between fibroblast lines of individuals with SMARD1 or CMT2 in comparison with controls and carriers ( Figure 4 Figure S4 ).

∗ and p.Arg971Glufs∗4 combination of variants, a band was detected between 70–80 kDa. This band was not observed in any other affected individuals, carriers or controls (∗ variant at 52 kDa, whereas the p.Arg971Glufs∗4 frameshift results in a protein of 109 kDa. In previous experiments, physicochemical properties of the WT protein in comparison with the p.Thr493Ile variant, known to cause SMARD1, have been investigated. Results showed a degradation band at 75 kDa that was primarily present in the p.Thr493Ile transfected cells and comprises the N-terminal amino acid residues 1–674. 8 Wee C.D.

Kong L.

Sumner C.J. The genetics of spinal muscular atrophies. ∗ variant or the p.Arg971Glufs∗4 frameshift in these individuals could alter the physicochemical properties of the protein and results in a degradation product at 75 kDa. Because neither of the carriers with either the p.Cys46∗ and p.Arg971Glufs∗4 variant show a band at this molecular weight, it could be hypothesized that the lower levels of functioning protein in the compound heterozygous individuals activate a feedback mechanism that preserved any residual truncated protein. Figure 5 Protein Levels of IGHMBP2 Normalized Against an Actin Control in All Individuals for Both Fibroblasts and Lymphoblastoid Cell Lines Show full caption (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. Protein quantification was estimated in both fibroblast and lymphoblastoid cell lines from IGHMBP2-associated CMT2, SMARD1, carriers, and controls to investigate whether abundance of residual protein correlates with the severity of the disease ( Table S3 ). When comparing the fibroblast lines of six CMT2 and two SMARD1 individuals against controls, a clear difference in protein levels can be observed ( Figure 5 ). Looking at the levels of the protein in all fibroblast and lymphoblastoid cell lines, single heterozygous carriers of IGHMBP2-associated CMT2 mutations were found to have intermediate IGHMBP2 protein levels in between affected and control individuals ( Figure 5 ). Interestingly, in the three individuals with the p.Cys46and p.Arg971Glufs4 combination of variants, a band was detected between 70–80 kDa. This band was not observed in any other affected individuals, carriers or controls ( Figure 5 ). Using online tools, we estimated the molecular weight of the truncated protein resulting from the p.Cys46variant at 52 kDa, whereas the p.Arg971Glufs4 frameshift results in a protein of 109 kDa. In previous experiments, physicochemical properties of the WT protein in comparison with the p.Thr493Ile variant, known to cause SMARD1, have been investigated. Results showed a degradation band at 75 kDa that was primarily present in the p.Thr493Ile transfected cells and comprises the N-terminal amino acid residues 1–674.Similar to this variant, the p.Cys46variant or the p.Arg971Glufs4 frameshift in these individuals could alter the physicochemical properties of the protein and results in a degradation product at 75 kDa. Because neither of the carriers with either the p.Cys46and p.Arg971Glufs4 variant show a band at this molecular weight, it could be hypothesized that the lower levels of functioning protein in the compound heterozygous individuals activate a feedback mechanism that preserved any residual truncated protein.

42 Trabzuni D.

Ryten M.

Walker R.

Smith C.

Imran S.

Ramasamy A.

Weale M.E.

Hardy J. Quality control parameters on a large dataset of regionally dissected human control brains for whole genome expression studies. 42 Trabzuni D.

Ryten M.

Walker R.

Smith C.

Imran S.

Ramasamy A.

Weale M.E.

Hardy J. Quality control parameters on a large dataset of regionally dissected human control brains for whole genome expression studies. 43 Warde-Farley D.

Donaldson S.L.

Comes O.

Zuberi K.

Badrawi R.

Chao P.

Franz M.

Grouios C.

Kazi F.

Lopes C.T.

et al. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. The mRNA expression of IGHMBP2 in six brain regions was assessed in humans during development.After birth, the expression of IGHMBP2 shows an increase in the cerebellar cortex, whereas in other brain regions there is a small decrease. IGHMBP2 expression levels seem to be constant throughout adult life ( Figure S5 ). In adults, using data from ten regions of normal human postmortem brain tissue,the highest IGHMBP2 expression levels were also in the cerebellum. Expression in other body tissues was ubiquitous, with moderate expression in fibroblasts and lymphoblastoid cell lines ( Figure S5 ). These data indicate the importance of the IGHMBP2 protein in the peripheral nerve but suggest that in other tissues with high expression, such as the cerebellum, the protein has a less important function as individuals with IGHMBP2 mutations do not have cerebellar signs. CMT2 is characterized by a highly heterogeneous genotype, with mutations in several unique genes being responsible for disease. The genetic background plays an important role in the classification of the disease and is crucial to find common pathways to explain the characteristic features seen in most affected individuals. No direct interactions between IGHMBP2 and any of the CMT2 proteins have been found so far. However, with the GeneMANIA prediction server,the presence of a network of interacting proteins known in CMT2 with IGHMBP2 can be observed ( Figure S6 ). Given that many people with CMT2 are genetically undefined, and with the increasing amount of genetic data available, network analysis will be important in identifying causative and modifying gene pathways.

Together, our studies indicate that autosomal recessive IGHMBP2 mutations are a cause of CMT2. The clinical presentation is of a relatively pure form of CMT2, some more severe than others as seen in Tables 2 and 3 , and typically what is seen in a number of the other genetic causes of CMT2, such as those individual with defects in MFN2, MPZ, MED25, and Lamin A/C genes. In contrast, SMARD1 usually presents in the first few days or weeks of life and children usually die before they are 1 year old. In addition, neurophysiology is much milder in CMT2 as compared to SMARD1 ( Table 3 ), and the CMT2 sural nerve biopsy shows similar mild features ( Figure 2 ).

Tyr genes and the activator of basal transcription 1 (ABT1) gene; 29 de Planell-Saguer M.

Schroeder D.G.

Rodicio M.C.

Cox G.A.

Mourelatos Z. Biochemical and genetic evidence for a role of IGHMBP2 in the translational machinery. Previous work by Guenther and Grohmann and colleagues also quantified the residual IGHMBP2 protein levels in a mouse model of SMARD1 and in lymphoblastoid cell lines from children with SMARD1. They found significant differences in the IGHMBP2 protein levels of individuals with typical congenital SMARD1, juvenile SMARD1 (respiratory distress at 3.5 months), and controls. Despite the reduction in protein levels, IGHMBP2 mRNA levels were not decreased in individuals with SMARD1 and IGHMBP2 mutations, an identical result which we also found in individuals with CMT2 (data not shown). This suggests that defective or truncated proteins undergo posttranslational degradation. Although we have found a number of IGHMBP2 mutations associated with CMT2, and mutations are usually different to SMARD1 in type and combination and result in higher residual protein levels in CMT2 as compared with SMARD1 and controls, we are cautious whether this always correlates with the onset of disease and phenotype. Protein levels are reduced in missense and nonsense or frameshift mutations, but the numbers are too few to correlate exact figures and there might be differences between mutation types. In addition, these experiments were carried out on material such as fibroblast and lymphoblastoid cell lines, which are not primarily affected in CMT2 or SMARD1. However, IGHMBP2 mRNA is widely expressed throughout the body and it is likely that these tissues might reflect the consequences of mutations. A further genetic factor that might modify the phenotype was identified in the IGHMBP2 mouse model (nmd) and was contained within the BAC-27k3 transgene. Expression of this transgene completely rescued the reduction in the total number of myelinated axons in the nmd femoral motor nerves. The syntenic genomic area in humans contains four tRNAgenes and the activator of basal transcription 1 (ABT1) gene;no variations were found in these genes in the index cases with CMT2 studied here.