PatientsThree female siblings (Fig. 1A), born to non-consanguineous parents of Iraqi-Jewish descent, presented to our center with varying degrees of cerebellar ataxia, encephalopathy, generalized tonic clonic seizures and developmental delay. The proband (III.4), the fourth of seven children, presented in early childhood with short stature and moderate delay in motor and cognitive milestones, followed by non-progressive cerebellar ataxia, dysarthria, mild-moderate cognitive disability, and behavioral problems including impulsivity, short attention span and oppositional characteristics. At approximately 12 years of age, she had myoclonic jerks which were more noticeable during voluntary motor movements. At age 17, she experienced several generalized tonic-clonic seizures. Epilepsy and myoclonus were well controlled by valproic acid combined with low dose clonazepam. On physical examination, the patient showed no dysmorphic features. Her head circumference was 54.5cm (35th percentile), and her height was 150cm (−2 Standard Deviations). She had mild horizontal nystagmus, slow saccades with saccadic pursuit and apraxic gaze, normal fundoscopy, dysarthric cerebellar speech, dysmetria in finger-to-nose test, a mild postural and intention tremor, ataxic gait and inability to perform tandem walking. She also showed mild lower limb spasticity with brisk tendon joint reflexes and negative Babinski sign.Open in a separate windowFig. 1Patients included in the study(A) Family pedigree. Patients who are affected are shown in filled circles. Male individuals are represented by squares, while female individuals are represented by circles. (B) SNP data analysis of all members of the family shows a single homozygous region, from 118.5Mb-121Mb on chromosome 12 (hg19) (arrow).(C) Whole genome sequencing on III.4 was inconclusive, but analysis of Bam file shows an increase of the number of reads in the candidate locus on chromosome 12 between positions ~120,940,150 and ~120,949,950, with an average number of reads of ~60X compared to the rest of the region (~30X). A cartoon below the Bam file shows the duplication in tandem, breakpoints confirmed by Sanger sequencing), between an AluYc (chr12:120939934–120940228) and an AluY (chr12:120949733–120950042). The encircled numbers below the gene show the positions of the different Taqman probes used for testing the copy number of the duplicated region, which is shown in (A) as the number below each box or circle.Brain MRI at 8 and 16 years of age showed mild non-progressive cerebellar atrophy; EEG revealed normal background activity and infrequent generalized polyspike-wave (PSW) discharges; nerve conduction study and cardiac echocardiogram were both normal. Complete blood count, electrolyte levels, CPK, liver and renal function tests, carnitine and acyl-carnitine, copper, ceruloplasmin, thyroid function tests, lactate, pyruvate, ammonia, blood amino acid profile, VLCFA, phytanic acid, homocysteine, isoelectric focusing of transferrin, alpha –FP, quantitative immunoglobulin levels, vitamin E levels, as well as urine for protein and organic acids, were within normal limits.The proband’s older sister (III.3) presented in early childhood (following identification of cerebellar ataxia in her younger sister) with mild static gait ataxia, mild dysarthria, and borderline intelligence (requiring special school only at high school). In her 20’s, the patient had myoclonic jerks followed by a single generalized tonic-clonic seizure at the age 22; there was no seizure recurrence on low dose valproic acid, which was later replaced by lamotrigine. The patient gave birth to a healthy baby with no signs of deterioration during her pregnancy. Upon physical examination, she showed mild gait ataxia with inability to perform tandem walking, mild dysmetria and oculomotor apraxia and horizontal nystagmus. A brain MRI showed mild cerebellar atrophy and EEG showed infrequent generalized PSW discharges.The youngest of the affected siblings, III.6, exhibited mild motor delay followed by mild learning difficulties in school, characterized by slow response rate, relatively poor verbal skills and poor writing. She was first examined at the age of 14 years, when mild cerebellar ataxia with abnormal tandem walking were identified, as well as brisk deep tendon reflexes, negative Babinski, no tremor or dysmetria but mild cerebellar dysarthria and horizontal nystagmus with hypometric saccades.Next generation sequencing identifies duplication in COQ5Because of the Iraqi-Jewish origin of both parents, a homozygote recessive mode of inheritance was suspected. A SNP array study, performed on the parents and all the siblings revealed several areas of homozygous regions (Supp. Table S1), conforming consanguinity in the family; however, there was one homozygous region, from 118.5Mb-121Mb on chromosome 12 (Chr37) (Fig. 1B), which was observed only in the affected children. Whole exome sequencing (WES), performed on patient III.4, did not identify a potential pathogenic variant (data not shown). Whole genome sequencing (WGS) was then pursued on the same individual, but variant analysis again failed to reveal potential pathogenic variant. Analysis of the Bam files showed a significant increase in the number of reads in the candidate locus on chromosome 12 between positions ~120,940,150 and ~120,949,950. The average number of reads in this region (~60X) compared to the rest of the region (~30X) suggested the presence of 4 copies, i.e., a homozygote duplication) (Fig. 1C).Sanger sequencing confirmed the breakpoints of this duplication and showed that it was in tandem (Fig. 1C). This duplication, exactly 9,590bp [Chr12(GRCh37):120940098–120949687], is between an AluYc (chr12:120939934–120940228) and an AluY (chr12:120949733–120950042); these two Alus have 84% identity, potentially making this duplication the result of an unbalanced crossing-over between these two Alus. The duplication spanned the last 4 exons of the COQ5 gene. COQ5 encodes for 2-hexaprenyl-6-methoxy-1,4-benzoquinone methyltransferase, a C-methyltransferase required in the biosynthesis of CoQ10. This duplication does not alter the original COQ5 ORF and is located about 1kb after the DNA sequence that encodes the COQ5 3′UTR (Fig. 1C).Copy number (CN) analysis, performed on all the family members for whom DNA was available, revealed that the duplication segregated with the disease based on a homozygote recessive model. Indeed, all the affected individuals were homozygous for the duplication (CN = 4), the parents were both carriers (CN = 3), and all the unaffected members were either carriers or homozygous not duplicated (CN = 2) (Fig. 1A). The duplication was analyzed in a cohort of 92 healthy individual from the same ethnic background, and 3 carriers were identified. These results reveal that, in this population, the duplication has a minor allele frequency of approximately 1.5%.To investigate the potential effect of this duplication on COQ5, we first measured the mRNA expression level. In patient fibroblasts, qRT-PCR using two different probe sets revealed COQ5 mRNA at a level of only 2–4% of that in control fibroblasts (Fig. 2A). We then demonstrated reduced COQ5 protein expression in patient fibroblasts by western blotting (Fig. 2B). Sequencing of the cDNA demonstrated that part of the 3′UTR has been spliced off and replaced by the duplicated part of the gene (exon 4 to 7), generating a longer alternative 3′UTR (Fig. 2C).Open in a separate windowFig. 2Effects of COQ5 mutation in RNA and protein from fibroblasts(A) Quantitative real-time PCR results for the expression of three COQ5 mRNA isoforms in fibroblasts from patient compared with control. Values are percentage expression of COQ5 in patient fibroblasts compared with control fibroblasts, normalized to POLR2A (error bars represent S.D., n=3). (B) Quantification of the expression of COQ5 protein in patient fibroblasts and control fibroblasts. Values are relative expression of COQ5 normalized to the loading control ACTB (error bars represent S.D., n=3 from two independent experiments). Next to the histogram is the immunoblot of fibroblast whole cell lysates of patient and control, showing the decreased expression of COQ5 in the patient. (C) A diagram of the known COQ5 isoform ({"type":"entrez-nucleotide","attrs":{"text":"NM_032314.3","term_id":"116063535","term_text":"NM_032314.3"}}NM_032314.3) is shown, and regions amplified by qPCR analysis in (A) are marked with arrows (red represents Probe 1, and violet represents Probe 2). Below the normal COQ5 isoform is a diagram depicting a new isoform generated. Sequencing of the cDNA from fibroblasts of patient III.4 shows an abnormal 3′UTR because of an abnormal splicing event: part of the original 3′UTR has been spliced off and replaced by the duplicated part of the gene (Exon 4 to 7), depicted in the figure as “New mRNA”. At the bottom of the figure is a chromatogram that shows the nucleotide sequence between the 3′UTR and the duplicated part of the gene, beginning from exon 4.COQ10 levels are reduced in patient’s leukocytes and muscle tissueTo determine whether the COQ5 deficiency could affect CoQ10 synthesis, we examined CoQ10 levels of affected and unaffected family members. CoQ10 levels were mildly reduced in all three affected patients (III.3, III.4, and III.6), as compared to their unaffected mother (II.2) (Fig. 3A). We then collected a muscle specimen from III.4, that showed increased type 2C muscle fibers (Fig. 3B), which comprised ~34% of all the myofibers; in comparison, type 2C fibers usually comprise 0–6.5% of the total number of fibers in unaffected young women (Staron, et al., 2000). There were no signs of myopathy, ragged red fibers, mitochondrial dysfunction or structural abnormalities on histochemical analysis (Fig. 3B), but Type 2C muscle fibers were 34% the total (normal in young women is 0–6.5% [4]). CoQ10 levels obtained from the skeletal muscle biopsy was reduced, confirming primary CoQ10 deficiency (Fig. 3A; full blot shown as Supp. Figure S1).Open in a separate windowFig. 3COQ5 mutation reduces CoQ10 levels and lead to abnormal mitochondrial respiration(A) Table showing CoQ10 levels in leukocytes from patients III.3, III.4, and III.6. Baseline levels and levels at 3 months after initiation of oral CoQ10 treatment are shown. Range of normal values was the average from 14 individuals with no known mitochondrial defect or mutations in the COQ genes. Level of Coenzyme Q10 levels in skeletal muscle in II.4 is also shown. (B) Representative muscle histopathology slides showing hematoxylin and eosin (H&E), Gomori trichrome (GT), nicotinamide adenine dinucleotide (NADH), cytochrome oxidase (COX) and succinate dehydrogenase (SDH) stains. The morphology of muscle fibers appears normal. Myosin adenosine tri-phosphatase (ATPase) activity staining at pH 4.3 identifies type 2C (immature) muscle fibers with an intermediate staining intensity between dark, type 1 (slow, oxidative) and light, type 2 (fast, glycolytic) fibers. Many type 2C muscle fibers are seen (asterisks). Scale bar represents 50 μm.(C) Cellular oxygen consumption rates (OCR) for fibroblast cell lines from wild-type (WT) and COQ5 patient (III.4) were measured by XF24 extracellular flux analysis. Oligomycin (1.25μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.2μM), and antimycin A (AA,1.8μM) together with Rotenone (Rtn,1μM) were injected after 20,50 and 80 minutes, respectively (n 4 plates; * p. value<0.05).(D) Clinical scoring (ICARS) before and during CoQ10 treatment.Measurement of oxygen consumption and activity of the respiratory complex chain in human fibroblastsTo understand the metabolic effects in the mitochondria due to the COQ5 mutation, we used the Seahorse analyzer to measure the cellular oxygen consumption rate (OCR) in cultured fibroblasts. The mutation in COQ5 significantly reduced uncoupled respiration compared to normal (Fig. 3C). Similarly, high resolution respirometry in fibroblasts showed a reduced uncoupling ratio and increased net routine flux control ratio, consistent with decreased reserve capacity (Supp. Table S2). In addition, the phosphorylation respiratory control ratio was increased, indicating that a higher proportion of oxidative phosphorylation is activated to drive ATP synthesis. We also measured the activity of the individual complexes of the respiratory complex chain including the combined activity of II and III, on mitochondrial fractions isolated from dermal fibroblasts, following our published methods (Kirby, et al., 2007; Taylor, et al., 1993). Our analysis showed that there is a measurable defect in Complex II+III activity in the cells, using an assay that relies on endogenous CoQ10 to shuttle electrons from succinate to cytochrome c (Supp. Table S3).Clinical outcome of CoQ10 supplementation in patients with defects in COQ5The pre-treatment ICARS scoring was consistent with the relative clinical severity of the disorder for each patient; the more affected patient (III.4) scored 27/100 and the least affected patient (III.6) scored 13/100. After 3 months of CoQ10 treatment, the scores of all three patients decreased significantly (patient 1: 27 to 14.5, patient 2: 13 to 7.5, and patient 3: 23 to 8.5). In addition, the patients appeared to have a quicker response rate during conversation and better alertness under the CoQ10 supplementation. A follow-up visit was obtained six months after initiation of treatment, and two weeks after halting CoQ10 treatment. Clinical assessment showed slight deterioration in the scoring results compared to the 3-months’ assessment rates (patient 1: −22, patient 2: −8, patient 3: −11) (Fig. 3D).

The proband (III.4), the fourth of seven children, presented in early childhood with short stature and moderate delay in motor and cognitive milestones, followed by non-progressive cerebellar ataxia, dysarthria, mild-moderate cognitive disability, and behavioral problems including impulsivity, short attention span and oppositional characteristics. At approximately 12 years of age, she had myoclonic jerks which were more noticeable during voluntary motor movements. At age 17, she experienced several generalized tonic-clonic seizures. Epilepsy and myoclonus were well controlled by valproic acid combined with low dose clonazepam. On physical examination, the patient showed no dysmorphic features. Her head circumference was 54.5cm (35th percentile), and her height was 150cm (−2 Standard Deviations). She had mild horizontal nystagmus, slow saccades with saccadic pursuit and apraxic gaze, normal fundoscopy, dysarthric cerebellar speech, dysmetria in finger-to-nose test, a mild postural and intention tremor, ataxic gait and inability to perform tandem walking. She also showed mild lower limb spasticity with brisk tendon joint reflexes and negative Babinski sign.Open in a separate windowFig. 1Patients included in the study(A) Family pedigree. Patients who are affected are shown in filled circles. Male individuals are represented by squares, while female individuals are represented by circles. (B) SNP data analysis of all members of the family shows a single homozygous region, from 118.5Mb-121Mb on chromosome 12 (hg19) (arrow).(C) Whole genome sequencing on III.4 was inconclusive, but analysis of Bam file shows an increase of the number of reads in the candidate locus on chromosome 12 between positions ~120,940,150 and ~120,949,950, with an average number of reads of ~60X compared to the rest of the region (~30X). A cartoon below the Bam file shows the duplication in tandem, breakpoints confirmed by Sanger sequencing), between an AluYc (chr12:120939934–120940228) and an AluY (chr12:120949733–120950042). The encircled numbers below the gene show the positions of the different Taqman probes used for testing the copy number of the duplicated region, which is shown in (A) as the number below each box or circle.Brain MRI at 8 and 16 years of age showed mild non-progressive cerebellar atrophy; EEG revealed normal background activity and infrequent generalized polyspike-wave (PSW) discharges; nerve conduction study and cardiac echocardiogram were both normal. Complete blood count, electrolyte levels, CPK, liver and renal function tests, carnitine and acyl-carnitine, copper, ceruloplasmin, thyroid function tests, lactate, pyruvate, ammonia, blood amino acid profile, VLCFA, phytanic acid, homocysteine, isoelectric focusing of transferrin, alpha –FP, quantitative immunoglobulin levels, vitamin E levels, as well as urine for protein and organic acids, were within normal limits.