De Novo Mutations in <i>POLG</i> Presenting with Acute Liver Failure or Encephalopathy (2009)

Historically, mitochondrial respiratory chain disorders have been recognized as a multisystemic disease with predominantly neuromuscular dysfunction (1). To date, most patients with mutations in POLG have presented with predominantly neurological features of headaches or focal seizures (2,3). In 1 study, end-state hepatic disease developed in only 2 of 26 cases (2). In another study, the only patients who experienced hepatic disease had been exposed to sodium valproate (3,4). Viral infection may be temporally associated with the development of acute liver failure in pediatric patients (5). However, the majority of children who contract viral infections do not experience liver failure. This suggests that there must be host factors that differentiate those children who do experience liver failure. CASE 1 An infant girl was born at term with a normal birth weight to nonconsanguineous white parents. At 2 months of age she had feeding difficulties and hypotonia. Consequently, she underwent an extensive medical evaluation that included determination of plasma electrolytes, lactate/pyruvate, plasma amino acids, urine organic acids, chromosome karyotyping, fluorescence in situ hybridization for DiGeorge deletion, Angelman/Prader-Willi methylation studies, and magnetic resonance imaging (MRI) of the brain. The results of these tests were normal. Although the result of her initial newborn hearing screening was normal, subsequent testing showed her auditory brainstem response to be abnormal. At 4.5 months of age the patient contracted an influenza A infection. Concurrently she experienced hepatomegaly associated with severe hypoglycemia and elevations of plasma tyrosine (203 μmol/L, normal range [NR] 35–126), glutamine (994 μmol/L, NR 353–883), and alanine (1048 μmol/L, NR 152–459). Her serum lactate was increased to 9.8 mmol/L (NR <2.2). The markedly increased lactate-to-pyruvate ratio (43.9, NR <15) suggested a defect in the electron transport chain or tricarboxylic acid cycle (6). Abnormalities in liver synthetic function were demonstrated by an elevated prothrombin time of 23 seconds (NR 12.4–15.1) and a partial thromboplastin time of 58 seconds (NR 25–39). Her conjugated bilirubin increased to 1.89 mg/dL (NR 0.0–0.3), suggesting a cholestatic process. Conversely, there were only modest elevations in liver hepatocellular enzymes (aspartate aminotransferase [AST] 260 U/L, NR 16–46; alanine transaminase [ALT] 313 U/L, NR 29–46) without significant elevations in creatinine kinase. Ammonia was normal until the final stages of her illness, when it rose to 76 μmol/L (normal <50). Her neurological status did not show further decline. She did not experience seizures or nystagmus. However, a lumbar puncture 2 days after her second admission for worsening liver failure showed elevated cerebrospinal fluid (CSF) protein (550 mg/dL, NR 15–40) with a nearly acellular tap (13 white blood cells, 0 red blood cells). MRI of the brain showed nonspecific extraaxial fluid collection over the frontal cortex and also thickening and enhancement of the nerve roots of the cauda equina. These changes, sometimes seen in demyelinating polyneuropathies, suggested a possible neurodegenerative disorder. The results of other studies, including lysosomal enzymes, plasma very-long-chain fatty acids, and screening for common mutations of mitochondrial deoxyribonucleic acid (mtDNA) were normal. She showed signs of ongoing respiratory difficulty, pancreatitis, and renal tubulopathy. Despite aggressive medical therapy, the patient died at the age of 5.5 months. Liver biopsy revealed marked panlobular accumulation of neutral lipid within hepatocytes, with a predominantly microvesicular pattern (Fig. 1A). Moderate subacute portal inflammation, mild canalicular and hepatocellular cholestasis, and mild pseudoacinar formation were also noted. Glycogen staining was normal. Electron microscopy confirmed the microvesicular steatosis but did not demonstrate any structural abnormalities of mitochondria. The muscle biopsy revealed prominent accumulation of neutral lipid within myocytes, especially in the subsarcolemmal region, corresponding to a “lipid myopathy” (Fig. 1B). The myofibers were abnormally small, measuring from 5 to 10 μm in cross-sectional diameter. There were no ragged-red fibers or cyclooxygenase-negative fibers. Glycogen staining was normal. Electron microscopy confirmed the excessive accumulation of lipid and demonstrated small subsarcolemmal collections of mitochondria in occasional myofibers. Most mitochondria in the specimen contained matrix granules. Some cristae were slightly curved or dilated, but there were no unique structural abnormalities and no paracrystalline inclusions. The mitochondria ranged from 105 to 510 (mean 262) nm in cross-sectional diameter. Abnormally enlarged mitochondria were not present. The lipid droplets ranged from 260 to 1464 nm in diameter. The activities of liver mitochondrial enzymes, including respiratory chain complexes, are summarized in Table 1.FIG. 1: (A) Liver shows marked accumulation of lipid (microsteatosis; trichrome stain, original magnification ×40). (B) Longitudinal section of skeletal muscle shows marked accumulation of subsarcolemmal neutral lipid; mitochondria are unremarkable (electron microscopy, bar equals 1 μm).TABLE 1: Mitochondrial respiratory chain activityThree heterozygous POLG mutations; c.752C>T (p.T251I), c.1760C>T (p.P587L), and c.3572A>G (p.K1191R), were identified. The p.T251I and p.P587L missense mutations have been frequently reported in cis(7–9). The novel missense variant, c.3572A>G (p.K1191R), predicts a relatively conservative change of lysine to arginine; however, a p.K1191N mutation at the same amino acid residue has been found in a patient with Alper syndrome (2). Thus, the p.K1191R alteration is likely to be pathogenic. Testing of the parents' DNA showed that the mother was heterozygous for the p.T251I and p.P587L missense mutations, confirming her carrier status. Test results from the father were negative for all of the mutations. Paternity was confirmed by comparative analysis using 15 unlinked polymorphic markers. Measurement of the mtDNA content in liver using qPCR revealed a severely reduced mtDNA (3% of age-matched mean), consistent with 2 mutated alleles in the POLG gene. CASE 2 A white infant boy was born to healthy unrelated parents. At 18 months of age, he had speech and motor delay. He was able to crawl but not walk. MRI of the spinal cord showed an asymptomatic syrinx between T11 and L1. While at home with his family, he had a new-onset seizure that included eye deviation, jaw clenching, and hypotonia of the trunk and extremities. He experienced repetitive generalized tonic-clonic seizures that evolved into refractory status epilepticus. No precipitating event or ingestion was identified, and he was not ill or febrile before the onset of seizures. Standard treatment with phenobarbital, fosphenytoin, midazolam, lorazepam, and diazepam was unsuccessful. Therefore, the child underwent pentobarbital coma for seizure control. He was not treated with valproic acid. After 30 days of this treatment, his seizures resolved and the pentobarbital was discontinued. He was left with a severe encephalopathy characterized by choreo-athetoid movements, corticovisual impairment, diffuse hypotonia, and severe swallowing dysfunction. The initial laboratory tests revealed elevated plasma lactate (3.2 mmol/L, NR 0.5–2.0), AST (85 U/L, NR 16–46), and ALT (69 U/L, NR 29–46). Ammonia was within normal range. A lumbar puncture was performed. This did not suggest an infectious cause because the CSF was colorless, with a few white cells (18 white blood cells, 4 red blood cells). However, CSF lactate (3.4 mmol/L, NR 0.6–2.2) and protein (46 mg/dL, NR 15–40) were both elevated. Cultures of the blood, urine, and CSF were negative for bacterial or viral processes. Titers for multiple causes of viral encephalitis and herpes simplex virus polymerase chain reaction were negative. Liver transaminases remained mildly elevated throughout his hospitalization. AST peaked at 195 U/L and ALT at 200 U/L; however, liver synthetic dysfunction was not significantly disturbed, with a normal coagulation profile (prothrombin time 13.1, international normalized ratio 1.0, partial thromboplastin time 28), bilirubin (maximum total bilirubin 1.1 mg/dL, NR 0–1.5), and maximum direct bilirubin (0.6 mg/dL, NR 0.1–0.5). Magnetic resonance imaging of the brain showed abnormal restricted diffusion involving the subcortical white matter in the left posterior parietal and occipital lobes, consistent with acute infarction or ischemia. Abnormally increased T2-weighted image and fluid attenuated inversion recovery (FLAIR) signal were seen in the bilateral thalami. He had normal biotinidase, very-long-chain fatty acids, carbohydrate-deficient transferrin, urine organic acids, plasma amino acids, plasma acylcarnitine profile, palmitoyl-protein thioesterase 1, and tripeptidyl-peptidase 1. Subsequently, he has remained neurologically static, with 1 admission for seizures. He has not had any subsequent elevation in his liver enzymes. Sequence analysis revealed 2 mutations in POLG: p.A467T mutation and a novel splice site mutation, c.2157+5_6GC>AG, in the child. The mother carried the p.A467T mutation. Test results from the father, however, were negative for both mutations, despite 15 unlinked markers being consistent with the stated paternity. The patient has an asymptomatic older brother who carries the p.A467T mutation. DISCUSSION In the past few years there has been a substantial increase in the understanding of the molecular basis of infantile liver failure caused by mtDNA depletion. At least 3 nuclear genes are known to be responsible of the hepatic form of mtDNA depletion. These are POLG, deoxyguanosine kinase (DGUOK), and the recently discovered MPV 17(10). In general, CNS symptoms are the predominant clinical features of cases with mutations in POLG(11); infantile liver failure, or Navajo neurohepatopathy in MPV 17 deficiency (10,12), whereas DGUOK mutations may present with either liver and neurological symptoms or isolated liver failure without CNS symptoms (13,14). Alper syndrome is a severe autosomal recessive disorder caused by mutations in the POLG gene. The minimum diagnostic criteria for Alper syndrome are refractory seizures, psychomotor regression, and characteristic hepatic disease with or without acute liver failure (15). The majority of patients present with central nervous system disease, most notably headaches or seizures. In 1 series, only 9 of 26 patients experienced liver failure, and in the majority of those it was precipitated by sodium valproate exposure (3). A similar relation to sodium valproate was seen in another series (2). In the context of isolated liver failure without neurological signs, the clinical diagnosis of Alper syndrome cannot be established. Influenza A viral infection has been associated with hepatic decompensation in adults with preexisting liver disease (16). Similarly, acute hepatic disease was observed concurrent with influenza A infection in 4 apparently healthy children, 1 of whom was subsequently suspected to have a mitochondrial cytopathy (4). Inasmuch as the 13,000 other children from Colorado with influenza infection during the time of that study did not experience liver failure, the postulates of Kock were not fulfilled. It is most likely that this difference reflects a host susceptibility. We believe that case 1 in our report represents another example in which an underlying genetic susceptibility leads to acute liver failure in the face of a hepatotropic viral infection. Additionally, we recently described twin boys, with mutations in DGUOK, 1 of whom experienced acute liver failure apparently precipitated by a herpesvirus infection. The condition of this patient acutely deteriorated, and he died at 9 months of age (17). His twin brother, aside from modest elevation in his AST and ALT, remains well at 3.5 years with the same mutations. These cases may begin to answer the question why only some children experience acute liver failure in the face of viral infections whereas most do not. Furthermore, they suggest that evaluation for the mtDNA depletion syndromes should be considered in patients with infantile acute liver failure even in the presence of an apparent viral trigger. Case 2 demonstrates that the full triad of Alper syndrome may not always be seen at presentation. Therefore, POLG testing should be considered in patients with intractable seizures even without liver failure. Both individuals seem to have a mutation in both copies of POLG consistent with autosomal recessive disease. The p.K1191R and c.2157+5_6GC>AG mutations have not been reported previously. The p.K1191R affects a highly conserved amino acid residue, and the c.2157+5_6GC>AG splice site mutation produces a truncated polypeptide. Furthermore, they are not present in 250 individuals whose POLG gene has been sequenced in our laboratory. These results, in the context of the clinical presentation, strongly support the pathogenic role of these mutations. Comparative analysis on both fathers and their affected children at 15 unlinked polymorphic markers is consistent with the stated paternity. Thus, the data support the presence of a de novo mutation in both cases. It is likely that both novel mutations occur in the paternally derived chromosome. This observation is consistent with those in many other diseases, such as achondroplasia, in which paternal chromosomes are the source of de novo point mutations, as reviewed by Crow (18). This is the first report of de novo mutations in the POLG gene. To date, more than 100 mutations in POLG have been found in patients with a broad clinical spectra of mitochondrial diseases (19). Contrary to a recent report (20), the discovery of 2 novel de novo mutations in a total of 28 patients with Alper syndrome identified in our laboratory suggests that the POLG gene may be susceptible to de novo or somatic mutations. Acknowledgment The authors thank Melissa S. Holzapfel, BS, for the electron microscopy of muscle and preparation of electron micrographs.