A fatal case of mitochondrial DNA depletion syndrome with novel compound heterozygous variants in the deoxyguanosine kinase gene

The deoxyguanosine kinase (DGUOK) gene controls mitochondrial DNA (mtDNA) maintenance, and variation in the gene can alter or abolish the anabolism of mitochondrial deoxyribonucleotides. A Chinese female infant, whose symptoms included weight stagnation, jaundice, hypoglycemia, coagulation disorders, abnormal liver function, and multiple abnormal signals in the brain, died at about 10 months old. Genetic testing revealed a compound heterozygote of alleles c.128T>C (p.I43T) and c.313C>T (p.R105*) of the DGUOK gene. c.128T>C (p.I43T) is a novel variant located in exon 1 (NM_080916) in the first beta sheet of DGUOK. Her mother was an allele c.313C>T (p.R105*) heterozygote, which is located in DGUOK exon 2 (NM_080916) between the third and fourth alpha helixes. c.313C>T (p.R105*) is predicted to result in a 173 amino acid residue truncation at the C terminus of DGUOK. There are as many as 112 infantile mtDNA depletion syndrome (MDS) cases in the literature related to DGUOK gene variants. These variants include missense mutations, nucleotide deletion, nucleotide insertion, and nucleotide duplication. Integrated data showed that mutations affected both conserved and non-conserved DGUOK amino acids and are associated with patient deaths.


INTRODUCTION
Mitochondrial DNA (mtDNA) is a 16.6 kb circular double-stranded DNA encoding the protein subunits of mitochondrial respiratory chain complexes I, III, IV, and V and which functions in energy production [1,2]. All of the mtDNA replication, transcription factors, and the primary components of the mitochondrial translation machinery are encoded by nuclear genes [1,2]. Mutations in nuclear genes that function in either mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis or mtDNA replication can lead to a severe reduction in cellular mtDNA content in affected organs, which is termed mtDNA depletion syndrome (MDS) [1,2]. Thymidine kinase 2, adenosine diphosphate-forming succinyl CoA ligase beta subunit, guanosine diphosphateforming succinyl CoA ligase alpha subunit, ribonucleotide reductase M2 B subunit, thymidine phosphorylase, and deoxyguanosine kinase (DGUOK) encode proteins that maintain the mitochondrial dNTP pool, and mutations in any of these genes can result in mtDNA depletion [2]. DNA polymerase gamma and Twinkle are essential for www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 48), pp: 84309-84319 Research Paper mtDNA replication, and mutations in these genes can result in inadequate mtDNA synthesis [2].
Clinically, MDS is classified into myopathic, encephalomyopathic, neurogastrointestinal, and hepatocerebral forms that are associated with mutations in the above genes [2]. Of the four forms of MDS, hepatocerebral MDS is associated with DGUOK gene mutations and occurs during the neonatal period . The common clinical features of hepatocerebral MDS include hepatic dysfunction, psychomotor delay, hypotonia, rotary nystagmus developing into opsoclonus, lactic acidosis, and hypoglycemia . Here, we report a fatal case of MDS with a novel compound heterozygous mutation in the DGUOK gene as well as a systemic literature review.

Clinical findings
Physical examination at 9 months and 4 days of age revealed slight jaundice, with a head circumference, chest circumference, height, weight, temperature, heart rate, and respiration rate of 40.0 cm, 41.0 cm, 69.0 cm, 7.0 kg, 37.2°C, 134 beats per minute, and 32 times per minute, respectively (Table 1). No obvious psychomotor delay, hypotonia, or rotary nystagmus was observed. Visual inspection and palpation found no obvious abnormalities except for splenomegaly.
Abdominal ultrasonography showed multiple focal liver lesions, hepatic fibrosis, and massive ascites. Brain magnetic resonance imaging (MRI) showed higher DWI and T2WI signals in the bilateral frontal, temporal, parietal, and occipital lobes, the corpus callosum, internal and external capsules, and the brain stem ( Figure 1A). In addition, a high T2WI signal also was observed in the bilateral globus pallidus ( Figure 1A). Proton magnetic resonance spectroscopic imaging showed normal N-acetylaspartate, choline and creatine peaks in the basal ganglia and thalamus. The results suggested that the patient had hepatocerebral MDS and was thus given symptomatic and supportive treatment for eight days in our hospital. The girl died one and a half months after release from the hospital.

Genetic findings
Genetic testing revealed that this girl was a compound heterozygote for DGUOK gene alleles c.128T>C (p.I43T) and c.313C>T (p.R105 * ) ( Table 2). Her father was a heterozygous carrier of allele c.128T>C (p.I43T). c.128T>C (p.I43T), a novel variant located in exon 1 (National Center for Biotechnology Information ID: NM_080916) that encoded an amino acid in the first beta sheet of the DGUOK protein (Table 2 and Figure 1B). c.128T>C (p.I43T) has been included in the Human Gene Mutation Database, the NHLBI GO Exome Sequencing Project, or the 1000 Genomes Project to date, and has not been previously reported ( Table 2). The patient's mother was a heterozygous carrier of allele c.313C>T (p.R105 * ). c.313C>T (p.R105 * ) is located in exon 2 (NM_080916) and encodes amino acids located in the third and fourth alpha helixes of the DGUOK protein (Table 2 and Figure  1B). c.313C>T (p.R105 * ) is predicted to result in a 173 amino acid residue truncation at the C terminus of the DGUOK protein [18]. No prior cases of MDS were found in the family histories of either parent.
One previous study has reported DGUOK mutations in adults [12]. The subjects ranged in age from 23 to 80 years old. Clinical presentations were variable, and included mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis, and adult-onset lower motor neuron syndrome with mild cognitive impairment [12]. The genotypes of six individuals in that study were c.130G>A/c.462T>A, c.186C>A/c.509A>G, c.444-11C>G/c.509A>G, c.605_606delGA/c.462T>A, and c.605_606delGA/c.137A>G ( Table 2). As the authors indicated, the mutations identified in adults were associated with an infantile hepatocerebral form of MDS [12]. It is also difficult to find adult mtDNA multiple deletion-specific variants.
Taken together, these studies suggest that DGUOK variants can occur anywhere in the DGUOK gene, and

DISCUSSION
MDS is an autosomal recessive disorder. In this report, we describe a fatal MDS case with novel compound heterozygous variants of the DGUOK gene, c.128T>C(p.I43T)/c.313C>T(p.R105 * ). To date, two additional reports have described the c.313C>T(p.R105 * ) variant [5,18]. The patients in which these mutations were found were both boys, and one was from Germany and one was suspected to be French. Both boys died before 1 year of age. c.313C>T is predicted to result in a truncated DGUOK protein lacking the 173 amino acid residue at the C terminus. c.128T>C (p.I43T) is a novel variant. Because the girl died before reaching 1 year of age, this allele is likely also lethal. Thus, this finding should be considered during pre-pregnancy checkups.
Hepatocerebral MDS always leads to hepatic failure and a variable neurological phenotype [1][2][3]. Our patient manifested jaundice, hepatosplenomegaly, abnormal hepatic function, and multiple abnormal signals in the brain. It is worth mentioning that the alpha-fetoprotein levels in all reports was extremely high [7,11,16]. In our patient, levels were as high as 29,090 ng/mL, which may occur due to the liver regeneration process or the early onset of hepatocellular carcinoma.
Since the first report of MDS by Moraes et al. in 1991 [1], the number of cases has increased, and now stand at more than 100. There is confusion in the position and nomenclature of MDS-related DGUOK variants, and many studies have either not provided a reference sequence or have adopted different sequence nomenclature. In Table 2, we list the original expression of variants. A pathogenic map of the DGUOK gene needs to be created. Therefore, it is important to follow Human Genome Variation Society (http://varnomen.hgvs. org) recommendations to standardize the location and nomenclature of MDS-related DGUOK variants going forward.
The main limitation to this study is that the patient was critically ill, which prevented us from performing biopsies for complete respiratory chain analysis and mtDNA quantification. Therefore, we were unable to provide any molecular evidence regarding the influence of the c.128T>C(p.I43T) variant on DGUOK expression or activity.
In conclusion, our report shows that diversified DGUOK variants can occur anywhere in the DGUOK gene, and that mutations affect both conserved and nonconserved DGUOK amino acids are associated with fatalities.

MATERIALS AND METHODS
This study was approved by the Research Ethics Committee of Children's Hospital of Fudan University and was conducted under the Declaration of Helsinki ethical principles for medical research involving human subjects. Informed consent was obtained from the child's parents.

Patient
A Chinese girl, aged 9 months and 4 days, visited our hospital on February 17, 2016, due to jaundice. The patient was a gravidity 3 and parity 1 (G3P1) infant of unrelated parents. The mother was 31 years old and had three pregnancies, with pregnancy terminations occurring at 1 and 3 months due to fetal growth arrest. The father was 32 years old. The patient was delivered by cesarean section at 37 weeks and 2 days due to a breech position, and had a birth weight of 2,550 g. Eight days after birth, she was hospitalized due to recurrent hypoglycemia (1.0-4.1 mmol/L). Physical and laboratory examinations found jaundice, coagulation disorders, and abnormal liver function. After her release from the hospital, the patient visited multiple hospitals for jaundice, weight stagnation, hepatomegaly, and abnormal liver function. Clinical laboratory and imaging examinations were performed in the corresponding clinical departments in our hospital.

DGUOK gene detection
During multiple hospitalizations, the patient was screened by whole exome sequencing (WES) and multigene panels on several occasions to find risk gene variants. WES was performed by an authorized independent gene testing company (Jinyu, Changsha, China). The testing process is briefly described as follows: WES was initiated by preparing a library using 3 μg purified DNA; enrichment was achieved using the SOLiDoptimized Sure Select All Human Exon Kit (Agilent, Technologies, Santa Clara, CA); sequencing was performed using 5500XL sequencers (Life Technologies, Carlsbad, CA). Quality control parameters were strictly observed throughout the WES workflow, and samples failing quality testing were either restarted completely or restarted from an earlier point. Sequence reads were aligned to hg19 using Lifescope v2.1 software (Life Technologies, Shanghai, China) followed by variant analysis of the aligned sequence. Variants were annotated using a custom analysis pipeline. A minimum of 30×median coverage per sample was required for sufficient data quality.

Literature search
To understand the relationships between MDS and DGUOK gene variants, we systematically searched all published studies in PubMed. The latest published data were updated in April 2017. The following terms were used to search titles and abstracts: (mitochondrial DNA depletion syndrome or MDS or OMIM 251880) and (deoxyguanosine kinase or DGUOK or dGK). In total, 31 multi-case and single-case reports were reviewed in this study.