Intragenic exonic deletions, which cannot be detected by direct DNA sequencing, are a common cause of Mendelian disease. Array-based comparative genomic hybridisation (aCGH) is now widely used for the clinical diagnosis of large chromosomal deletions, but not small deletions or analysis of the mitochondrial genome. An oligonucleotide-based microarray that provides high-density coverage of the entire mitochondrial genome and nuclear genes related to mitochondrial disorders has been developed. In this report, the case of an infant referred with tyrosinaemia on newborn screening who developed liver failure is presented. DNA sequencing revealed a heterozygous missense mutation (c.679G>A, p.E227K) in the deoxyguanosine gene (DGUOK). Oligonucleotide aCGH allowed simultaneous detection of an intragenic heterozygous deletion of exon 4 of DGUOK and mitochondrial DNA depletion in blood and liver. Screening of the parents’ DNA samples indicated that the patient was compound heterozygous for these mutations. An older sibling who had died from liver failure was then retrospectively diagnosed with the same mutations. This report shows the clinical utility of this oligoarray in the detection of changes in DNA copy number in both the mitochondrial and nuclear genomes, thus greatly improving the molecular diagnosis of mitochondrial disorders caused by nuclear genes involved in mitochondrial DNA biosynthesis.
Statistics from Altmetric.com
The mitochondrial DNA (mtDNA) depletion syndromes (MIM 251880) are a genetically and clinically heterogeneous group of autosomal recessive diseases characterised by a reduction in tissue-specific mtDNA copy number. The main clinical presentations of mtDNA depletion include the myopathic, encephalomyopathic and hepatocerebral forms.1 Most patients die in infancy, but some reported cases have survived into the teenage years. In the hepatocerebral form, progressive liver failure, hypoglycaemia, lactic acidaemia and neurological abnormalities are often present in early life.1–3 However, several recent papers have described patients with significantly longer survival.4 5
More than 38 different mutations in the DGUOK gene have been detected by genomic sequencing.4 However, conventional PCR-based sequencing analysis does not detect heterozygous intragenic deletions. To overcome this problem, we designed an oligonucleotide array that includes nuclear genes responsible for mtDNA depletion syndrome and the 16.6 kb mitochondrial genome for array-based comparative genomic hybridisation (oligonucleotide aCGH) analysis.
DGUOK was sequenced as previously described.4 6 mtDNA and nuclear DNA copy numbers were determined by real-time quantitative PCR. The mtDNA content of samples was then compared with the mtDNA content of five age-matched control samples as previously described.4 6 7
A clinical oligoarray was made in collaboration with Agilent’s microarray research group (Agilent Technologies, Santa Clara, California, USA). This array contained coverage for ∼130 nuclear genes that are related to mitochondrial biogenesis or function, including TYMP, POLG, DGUOK, TK2, SUCLA2, MPV17, SUCLG1, RRM2B and C10orf2, at an average probe spacing of about 250 bp/oligonucleotide. In addition, there were 6400 overlapping oligonucleotide probes covering the 16.6 kb mitochondrial genome in both forward and reverse directions.
The proband, the second child of non-consanguineous Chinese parents, was born at 38 weeks’ gestation by caesarean section because of breech presentation. No perinatal insult was noted, but he had mild growth retardation, with a birth weight of 2528 g. After birth, mild hypoglycaemia (41–48 mg/dl) with recovery after feeding was noted. At 3 days of age, he was admitted to the hospital with decreased activity, a weak cry and hypothermia. He was not hypotonic and had an otherwise normal examination. On admission, he was coagulopathic (prothrombin time 28.6 s (normal range 8.0–12.2 s); international normalised ratio 2.86; activated partial thromboplastin time >200 s (normal range 26.9–36.3 s)) with lactic acidosis (10.43 mM, normal range <2 mM), mild hepatomegaly and hyperammonaemia (133 μM, normal range <40 μM). He was provided with supportive care. His ammonia normalised, but lactic acid remained slightly raised (2–3 mM). Newborn screening using tandem mass spectroscopy revealed significantly raised tyrosine (801 μM; cutoff 181 μM) and alanine (883 μM; cutoff 511 μM) concentrations and a mild generalised increase in other amino acids. Subsequent plasma amino acid analysis was consistent with significant increases in tyrosine (1180 μM, reference range 20–96 μM), alanine (1800 μM, reference range 148–420 μM) and glutamine (1670 μM, reference range 238–842 μM). By 2 months, the amino acid profile had normalised, except for a residual modest increase in tyrosine (131 μM) with normal alanine concentration (161 μM). However, significant increases in tyrosine (919 μM) and alanine (1100 μM) recurred at 4 months of age, in association with significant worsening of liver function.
Given this clinical situation and as the proband’s older sibling had died from liver failure without a definitive diagnosis, a liver biopsy was performed at 10 days of age. Histological examination revealed panlobular microvesicular steatosis (∼70% of the specimen area). Haemosiderin deposits were seen in hepatocytes, Kupffer cells and macrophages in the portal areas. Focal bile plugging, oncocytic change and mild extramedullary haematopoiesis were also present (fig 1A). Although minimal, occasional spotty necrosis was discernible. No giant cell hepatocytes, portal inflammation, piecemeal necrosis, or portal fibrosis was observed. Periodic acid–Schiff stain revealed glycogen depletion and steatosis. Electron microscopy showed elongated and angulated mitochondria with focal loss of cristae (fig 1B). Dense matrix granules were absent. No mitochondrial crowding or concentric laminated cytoplasmic bodies was observed.
At 4 months of age, he was growing well with mild cholestasis (total bilirubin 2.94 mg/dl, and direct bilirubin 1.81 mg/dl) and raised liver enzymes (alanine aminotransferase 58 U/l and aspartate aminotransferase 119 U/l; normal range <40 U/l). However, at this point, he developed nystagmus without hypotonia and has failed to attain further developmental milestones.
The older sibling had a similar early course, with hypoglycaemia and hypothermia shortly after birth. He was admitted to the hospital at 3 days of age because of a seizure. Expanded newborn screening using tandem mass spectroscopy had revealed increase tyrosine (620 μM) and alanine (2100 μM) concentrations. This was confirmed in plasma with a tyrosine concentration of 981 μM, alanine concentration of 3340 μM and glycine concentration of 1188 μM. Tyrosinaemia was suspected initially, but no succinylacetone was detected. Lactic acidosis (14 mM), hyperammonaemia (205 μM), raised liver enzymes (alanine aminotransferase 46 U/l, aspartate aminotransferase 180 U/l) and severe coagulopathy (prothrombin time >60 s, partial thromboplastin time >120 s) were also apparent in this child, without evidence of infection. Nystagmus was noted at 1 month of age. The liver failure progressed in spite of supportive therapy with ursodeoxycholic acid and vitamin K1. He died at 4 months of age. His parents declined a post-mortem examination.
Sequence analysis of the proband revealed a heterozygous missense mutation, c.679G>A (p.E227K) in DGUOK. This mutation has previously been described in other patients with hepatocerebral mtDNA depletion.8 No mutations were found in POLG1 or MPV17 genes to explain the hepatocerebral phenotype6 9 The father was subsequently found to be a carrier of this mutation.
Oligonucleotide aCGH analysis
DNA from the patient’s blood was analysed on the oligoarray to test for intragenic heterozygous deletions. A heterozygous intragenic deletion of ∼1.8 kb covered by 11 probes encompassing exon 4 of the DGUOK gene was detected in the affected child and the mother (fig 2A). This was confirmed by sequencing across the breakpoint (fig 2B).
The probes covering the entire mitochondrial genome revealed about a 50% reduction in mtDNA copy number in the blood sample and severe mtDNA depletion in liver after normalisation to matched tissue control (fig 2C). Quantitative PCR analysis confirmed this depletion in the liver (6.5% of control mean) and blood (22% of control mean) of the proband, who had normal leucocyte and platelet counts at the time of analysis (table 1). This is consistent with previously reported cases.4
After the molecular diagnosis had been made in the proband, DNA was retrieved from a newborn screening dried blood spot on the deceased sibling. Molecular analysis revealed that he was also compound heterozygous for the c.679G>A (p.E227K) mutation and the exon 4 deletion in the DGUOK gene.
mtDNA depletion resulting in liver failure has been associated with bi-allelic mutations in four nuclear genes (POLG, DGUOK, MPV17, C10orf2 (TWINKLE)). Identification of the specific deficiency carries important prognostic information. Unfortunately, conventional sequencing technologies will not identify heterozygous intragenic deletions which are a common cause of many diseases, including Duchenne muscular dystrophy, cystic fibrosis and neurofibromatosis (NF1).
Traditionally, intragenic deletions have been detected by a variety of tedious methods such as Southern analysis, customised fluorescent in situ hybridisation and multiplex ligation-dependent probe amplification.10–13 In our experience, the initial set up and performance of these assays is time consuming and requires costly validation. Conversely, the availability of well-characterised oligonucleotides covering the whole human genome from established databases reduces the time to create and validate probes. In addition, this highly multiplexed technology allows us to detect deletions in flanking introns and partial exon deletions14 in multiple genes, resulting in the same clinical phenotype. More significantly, in this case, the availability of probes targeted across the whole mitochondrial genome enabled us to definitively diagnose this patient’s condition with mtDNA depletion. Such whole-genome testing negates the possibility of a deletion in the position of a real-time quantitative PCR probe or a point mutation in a restriction site giving a false-positive result, as has previously been described.15
Consistent with recently published experience, we noted an increase in plasma tyrosine on expanded newborn screening in the patient and his deceased brother, which resolved.4 This may be the first indication of liver dysfunction in patients with mtDNA depletion syndrome. The resolution of this abnormality carries significant implications for those following patients with raised tyrosine concentrations, which may be incorrectly ascribed to transient tyrosinaemia of the newborn.
In conclusion, we have shown that an oligonucleotide array can simultaneously detect a heterozygous intragenic deletion in a nuclear gene, DGUOK, and mtDNA depletion in blood or liver samples.
We thank the members of the family for their cooperation in the study, and Drs Hey-Chin Hsu, Yen-Hsuan Ni and Yi-Ning Su at the National Taiwan University Hospital for help with this study and the management of these cases. The study was supported in part by an NIH career development award (K12RR17665 to DD).
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.