A mutation of COX6A1 causes a recessive axonal or mixed form of Charcot-Marie-Tooth disease

Abstract

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6delCACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.

Figure 1

Genomic Analyses in the CMT Families (A) Pedigrees of two consanguineous CMT families, from which a total of ten members in broken line boxes were sampled. (B) Multipoint LOD scores on chromosome 12 using these ten members from two families with the HumanLinkage V SNP chip. We extracted genomic DNA from peripheral blood using the QIAamp DNA spin columns (QIAGEN) and quantified them using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). Genotyping was performed with the GoldenGate Genotyping Universal-32 kit on a BeadArray Reader System (Illumina) according to the manufacturer’s assay guide. Genotype calls were made using the Genotyping module of the GenomeStudio v2009.1 software (Illumina). A multipoint linkage analysis was performed with Allegro v.2 and a human genetic map based on NCBI dbSNP Build 123 (see also Figure S1). (C) Gene map in the significant linkage region on 12q24. These physical coordinates are taken from UCSC Genome Browser build hg19, RefSeq, and dbSNP 138 (UCSC Genome Browser database: 2014 update). (D) A disease-specific 5 bp deletion (c.247−10_247−6delCACTC) in the pyrimidine tract near to the splicing acceptor near to the third exon of COX6A1. (E) Validation and distribution of the 5 bp deletion among the CMT family members by Sanger-based PCR direct sequencing (see also Table S10).

Figure 2

COX6A1 Expression and COX Activity Levels (A and B) COX6A1 expression in fresh whole-blood from a control and the two affected individuals from family 1 (A) and in EBV-transformed B cell lines from four controls and the two affected individuals (B). Lymphoblastoid cells from two affected members in the family 1 were immortalized by infection with the Epstein-Barr virus (VR-1492; American Type Culture Collection). Immortalized cells from the four healthy Japanese individuals (HEV0031, HEV0032, HEV0038, and HEV0041) were provided by RIKEN BioResource Center. Total RNA was extracted using QIAamp RNA Blood Mini Kit or AllPrep DNA/RNA Kit (QIAGEN) according to the manufacturer’s instructions with on-column DNase I treatment. After determining RNA concentrations using Quant-iT RiboGreen RNA Assay Kit (Life Technologies), 400 ng of total RNA per 40 μl reaction was used to synthesize cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) with random primers. Absolute quantification for COX6A1 was performed using a custom TaqMan assay (Table S12). PCR products were ligated into pGEM-T easy vector (Promega) and isolated plasmid DNA was then linearized by EcoRI digestion. Before use, plasmid concentration was determined by Quant-iT PicoGreen dsDNA Assay Kit and serial dilutions were performed to generate standard curve. Real-time PCR was conducted using TaqMan Universal Master Mix II (Life Technologies) with a 7500 Fast real-time PCR system (Life Technologies). Each reaction was run in triplicate and contained 2 μl of cDNA template in a final reaction volume of 20 μl and data were analyzed with 7500 Software v.2.0.2. (C and D) COX activity (C) and ATP amount (D) in mitochondrial fractions from the same four controls and two affected individuals’ cell lines. For the determination of COX activity, we used a Cytochrome c Oxidase Assay kit (Sigma-Aldrich). Mitochondrial fractions were obtained from cells by homogenization in homogenization buffer (20 mM HEPES-KOH [pH 7.4], 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 1× protease inhibitor). The determination of COX activity was based on a colorimetric assay that quantifies the oxidation of ferrocytochrome c to ferricytochrome c via cytochrome c oxidase, a reaction that results in a decrease in absorbance at 550 nm. After measurement of absorbance, COX activity was calculated according to manufacturer’s instructions. The amount of ATP was measured using a Lumino assay detecting cellular-ATP kit (CA100; Wako Pure Chemical Industries). The collected cells (1,000 cells per well) were used according to manufacturer’s instructions. All experiments were triplicated per sample and tested using t test for the difference between controls mean and each affected individual mean. ^∗∗∗p < 0.001. The error bars represent the standard deviation.

Figure 3

Characterization of Cox6a1 Knockout Mice (A) Immunoblot of COX6A1 and voltage-dependent anion channel (VDAC) as control in a wild-type and Cox6a1 knockout null mice. Mice aged 7–8 weeks, three (one male and two female) knockout and three (two male and one female) wild-type were anesthetized and perfused with 10 mM PBS. Mitochondria fractions were obtained from liver tissue and applied to immunoblot. 20 μg of protein was loaded into a SDS-PAGE and transferred to the polyvinylidene difluoride membrane. The primary antibody for COX6A1 (mouse monoclonal, ab110265; abcam) is diluted 1:1,000 and secondary antibody for anti-COX6A1 (anti-mouse IgG, HRP-conjugated, 315-035-003; Jackson ImmunoResearch) is diluted 1:10,000. All blotting is carried out in 5% skim-milk/TBS solutions at room temperature for 1 hr. (B) Motor coordination and balance was assessed as the latency to fall in the rota-rod. Mice aged 7–8 weeks, four (two male and two female) knockout and four (two male and two female) wild-type were used. Each mouse underwent the same 4 day procedure on a rota-rod (MK-660A; Muromachi Kikai). The first 3 days were used to train the mice (four sessions of 60 s each, walking at 20 rpm). The test sessions were run on the last day. The mice performed two series of three trials (10, 15, and 20 rpm) at each speed, with a 10 min rest period between trials. The latency to fall was recorded with a cut-off at 120 s. The difference between the wild-type and knockout null mice means were tested using t test. ^∗∗∗p < 0.001. The error bars represent the standard deviation. (C and D) Histological examinations by toluidine blue staining sections of mice sciatic nerves at lower magnification (C) and hematoxylin-eosin staining sections of mice lower limb muscles (D). Arrow indicates a smaller number of fibers are involved in small group atrophy and arrowhead indicates small angular fibers despite the limited numbers. Mice aged 7–8 weeks, four (two male and two female) knockout and four (two male and two female) wild-type were anesthetized and perfused with 10 mM PBS, followed by a fixative of 4% paraformaldehyde (for leg muscle) or 2.5% glutaraldehyde (for sciatic nerve) in 0.1 M phosphate buffer. Sciatic nerve specimens were fixed in 2.5% glutaraldehyde in phosphate buffer for 2 hr at room temperature. After postfixation with 1% OsO₄, the tissues were embedded in epoxy resin. Tissue blocks were sectioned at 1 mm thickness and stained with toluidine blue for light microscopic examination. For the histological analysis of leg muscle, mice tissues were postfixed in 4% paraformaldehyde for 48 hr at 4°C. The muscle tissues were dissected out and then incubated overnight in 10% sucrose in phosphate buffer. After snap freezing with CO₂ gas, tissue blocks were sectioned at 20 μm thickness in a cryostat and stained with hematoxylin and eosin.

Figure 4

COX Activity and Electrophysiological Analysis in Mice (A and B) COX activity in mitochondrial fractions from livers (A) and ATP amount in liver homogenates (B) from three wild-type and knockout null mice, respectively. Experiments for COX activity and ATP amount were triplicated per sample and all experiments were tested using t test for the difference between the wild-type and knockout mice means. (C) Nerve conduction velocity of sciatic nerve from mice. Studies were demonstrated at 7–10 weeks of age, six (three male and three female) wild-type and seven (five male and two female) null mice with Sapphire (Medelec) under anesthesia of pentobarbital sodium (5 mg/kg i.p.). At the right dorsal femoral, sciatic nerve was exposed by opening up overlying skin and electrically stimulated using needle electrodes at the sciatic notch and at the knee joint level under 37°C ± 0.5°C. The compound muscle action potential (CMAP) evoked by the two parts of stimuli were recorded at gastrocnemius. Motor nerve conduction velocity (NCV) was calculated with dividing the distance between the sciatic notch and knee joint level by the delta latency between the two CMAP curves. Asterisks indicate the level of statistical significance (^∗∗p < 0.01; ^∗∗∗p < 0.001). The error bars represent the standard deviation.