Primary coenzyme Q10 deficiency-7: expanded phenotypic spectrum and a founder mutation in southern Chinese

Primary coenzyme Q10 deficiency-7 (COQ10D7) is a rare mitochondrial disease caused by biallelic mutations in COQ4. Here we report the largest cohort of COQ10D7 to date, with 11 southern Chinese patients confirmed with biallelic COQ4 mutations. Five of them have the classical neonatal-onset encephalo-cardiomyopathy, while the others have infantile onset with more heterogeneous clinical presentations. We also identify a founder mutation COQ4 (NM_016035.5): c.370G>A, p.(Gly124Ser) for COQ10D7, suggesting a higher chance of occurrence in the southern Chinese. This study helps improve understanding of the clinical spectrum of this disorder.


INTRODUCTION
Coenzyme Q10 (CoQ), also known as ubiquinone, is crucial for the function of mitochondrial respiratory chain complexes. Currently, the pathway of CoQ biosynthesis is known to involve at least 18 proteins. Primary CoQ deficiencies in humans involve genetic mutations in COQ2, PDSS1, PDSS2, COQ8A, COQ9, COQ6, COQ4, or COQ7. 1 The primary CoQ10 deficiency-7 (COQ10D7, MIM: 616276) is caused by autosomal-recessive mutations in COQ4. COQ4 is hypothesized to take part in stabilizing the CoQ complex. 2 To date, biallelic COQ4 mutations have been described in 16 patients from 11 unrelated families. They were reported in two case series (with five to six cases each) and three case reports. The patients described had common features of cardiomyopathy, encephalopathy, lactic acidosis often with a neonatal onset, and death in the neonatal or infantile period. [2][3][4] Two siblings of childhood onset presenting with spinocerebellar ataxia and stroke-like episodes 5 and two Chinese siblings with neonatal onset of dystonia, seizures, lactic acidosis, and cerebellar atrophy were described in recent case reports. 6 In this report, we have 11 patients (4 males and 7 females) from 9 unrelated families who were managed by the Medical Genetics division of the Department of Paediatrics and Adolescent Medicine at the University of Hong Kong and the Department of Medical Genetics and Pediatrics at the National Taiwan University Hospital in the period of 2014-2018. They presented with two overlapping phenotypes: the classical neonatal-onset encephalocardiomyopathy and infantile-onset encephalopathy with or without cardiomyopathy. They were diagnosed as COQ10D7 due to homozygous or compound heterozygous COQ4 mutations. All genetic diagnoses were made by whole-exome sequencing (WES) except Patient 4, Patient 5, and Patient 11 due to their known family history and recognizable features of COQ10D7. More importantly, we have identified a Chinese-specific COQ4 founder mutation in 10 subjects, 5 of whom are homozygous for that mutation.

CASE REPORTS
The study was approved by the institutional review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW12-211) and the National Taiwan University Hospital (201703073RINB). Written informed consent was obtained from subjects or their parents. A summary of the clinical, biochemical, and radiological characteristics of the 11 patients with COQ4 mutations identified is presented in Table 1. Patients 1-5 had the classical neonatal-onset phenotype described by Brea-Calvo et al. and Chung et al., 2,3 whereas Patients 6-11 had later onset and more heterogeneous features. The frequency of distinct phenotypes compared with previous studies is summarized in Table 2. The corresponding MRI (magnetic resonance imaging) images are presented in Fig. 1. The pedigrees of the nine families are presented in Fig. 2.    MRI of the brain at 3 weeks of age showed symmetrical T1 and T2 hyperintensity with restricted diffusion at bilateral lentiform nuclei. Foci of restricted diffusion were also detected at bilateral frontal white matter (Fig. 1a-c). Magnetic resonance spectroscopy (MRS) showed raised lactate peaks at bilateral basal ganglia and cerebral white matter. Subsequent follow-up MRI showed established infarcts with cystic changes at bilateral lentiform nuclei (Fig. 1d)

Pathogenicity of the COQ4 variants
We analyzed the COQ4 variants identified in our cohort by previously reported literatures, ClinVar, population frequency in gnomAD, 7 conversation score by Combined Annotation-Dependent Depletion, 8 in silico prediction by Rare Exome Variant Ensemble Learner, 9 and protein stability change prediction by STRUM 10 (Table 3). All variants demonstrated a deleterious effect. Together with the reduced level of CoQ10 of the patients, the pathogenicity of these variants is strongly supported by the biochemical findings of the patients.
Founder mutation analysis Among these 11 patients, we identified the same missense mutation c.370G>A, p.(Gly124Ser) in 10 of them. This missense mutation is a rare variant with a population frequency of 0.001118 and it is exclusively found in South East Asians in the gnomAD database. 7 Further analysis of the DNA of the five homozygous patients using Infinium OmniZhongHua-8 BeadChip SNP array showed a common haplotype of 0.464-3.290 cM implying that the mutation was inherited from a common ancestor 27 generations ago (Fig. 3). In addition, by principal component analysis), our SNP array data are clustered with Chinese and Japanese of HapMap Phase II (n = 270) and a local southern Chinese database (n = 612). Furthermore, the pathogenicity of this mutation has been established via the reduced CoQ level in the skin fibroblasts in Patients 7 and 9 who have homozygous c.370G>A mutation (Table  4). Altogether our study confirms that the missense mutation c.370G>A, p.(Gly124Ser) represents a pathogenic founder mutation in the southern Chinese population.

DISCUSSION
To our knowledge, this is the largest case series of primary COQ10D7 reported. In the literature, primary COQ10D7 cases have been described predominantly with a neonatal onset, with only two cases of childhood onset. [2][3][4][5]8 In this study, we have expanded the phenotypic spectrum of primary COQ10D7 from neonatal to infantile onset.
We have five patients exhibiting the well-described neonatal presentation of COQ10D7 as in the literature, characterized by respiratory distress, encephalopathy, seizures, hypotonia, and cardiomyopathy. Previously, it was believed that phenotypes from affected males with COQ4 mutation would be more severe and highly likely lethal. 3,6 However, in our study the male-to-female death ratio was 2:3.
We have six patients with infantile-onset phenotypes. Unlike those of neonatal onset, MRI brain for those infantile-onset patients did not show characteristic basal ganglia lesions. Dystonia was observed in two out of the six patients with infantile-onset presentation in our cohort, and it was also observed in the two neonatal-onset cases reported by Lu et al. 8 but not reported in non-Chinese patient.
A summary of the predominant phenotypes in the spectrum of neonatal, infantile, and childhood onset of COQ10D7 is shown in Fig. 4. The variegated symptoms and disease onsets explain the frequent delay of diagnoses of COQ10D7. This also highlights the importance of the complementarity of biochemical screening for children with unexplained neurological disturbances and the prompt application of WES in order to reach a genetic diagnosis that has an impact on patient management.
Functional analysis to demonstrate the deficiency of CoQ should be carefully examined because mitochondrial enzymology can be tissue specific. In this study, among the five patients (Patients 1, 6,7,8,9) with ETC chain analysis in the skin fibroblast, all of them shown a reduced level of succinate:cytochrome c oxidoreductase (complex II+III). Measurement of CoQ level was also found significantly decreased. For Patients 7 and 8, complex II +III analysis and CoQ level measurement were also performed in the muscle. Interestingly, the CoQ level from the muscle is normal but that from skin fibroblasts was reduced. From the Genotype-Tissue Expression (GTex) data, the COQ4 median expression in the muscle is 7.58 transcripts per million (TPM) while in the skin it is 44.14 TPM, demonstrating a 6-fold lower expression in the muscle.