A cohort of 28 children with mitochondrial encephalopathy and liver failure was qualified for retrospective POLG
mutations screening. Availability of the patient’s frozen tissue sample was the only inclusion criteria. The samples were used for assessment of mtDNA/nDNA ratio and/or DNA isolation. One of the patients was included in the reported Polish-Czech cohort of COX deficiency [25
The search for POLG gene mutations revealed 2 patients bearing the p.W748S mutation. Clinical, biochemical and morphological documentation of the patients was re-analyzed in detail.
The study protocol was approved by the Bioethics Commission at the Children’s Memorial Health Institute.
This female patient, born in 2002, was first admitted to our hospital at the age of 12 months. She was the second child born to a non-consanguineous couple. Her older brother was healthy. Pregnancy, delivery and neonatal period were uneventful. The girl was born with Apgar score 10, and birth weight 3800 g. At the age of 2 months she had a short episode of diarrhea with mild hypertansaminasemia (50–80 U/l). Her psychomotor development was normal up to the age of 7 months.
At the age of 7 months, she developed focal status epilepticus involving her left limbs, followed by postictal hemiparesis. Partial and generalized seizures recurred, progressing commonly to status epilepticus, and were often followed by postictal palsy. Laboratory investigations revealed elevated blood lactate concentration (4.1, 4.09 mmol/l., control <2.0 mmol), increased excretion of lactate in urine, and high alanine concentration in plasma (548.2 umol/l). Several antiepileptic drugs were implemented, including sodium valproate, but satisfactory seizure control was not achieved.
Upon initial admission to our institute, the child was drowsy and anxious. Significant psychomotor delay was noted – the child could not sit and raise her head. Muscle tone was decreased and tendon reflexes were very weak or absent. Her EEG recording showed diffuse, irregular slowing of the background and focal delta activity over the left fronto-temporal region. The delta activity correlated with clinical clonic seizures recorded continuously at the same time. In subsequent EEG examination, progressing deterioration was noted. Brain CT scans showed diffuse atrophy.
Liver failure developed during observation, with hypertransaminasemia (GGTP 164 u/l, AspAT 351 u/l, AlAT 303 u/l), severe clotting abnormalities, jaundice (bilirubin concentration increased from 1 mg% to 11.3 mg%), and ascites. Metabolic workup revealed relatively low ceruloplasmin level (13 mg%), and a slight increase in alfa-fetoprotein concentration (115 IU). Triglyceride accumulation was found by liver biopsy (by thin layer chromatography). Tyrosinemia type I was excluded by the absence of succinylacetone excretion. Organic acids profile showed nonspecific dicarboxylic aciduria (C6-C10), hydroxyisovaleric aciduria and ketonuria. Biotinidase activity was normal.
Mitochondrial disorder with Alpers-Huttenlocher syndrome phenotype was established, and additional CMV infection was suspected depending on positive IgM test. Liver transplantation was decided against because of the clearly poor prognosis. The girl died within 3 months from the onset.
Autopsy of the patient was refused by the parents. Liver and muscle biopsies were performed after death, with the shortest acceptable delay of 2 hours.
The girl was born in 2004 with weight 4470 g and Apgar score 8/9/10, and developed normally. At the age of 18 months she presented with recurrent complex partial seizures. Transaminases were not increased at that point. Electroencephalography showed generalized epileptic discharges. MRI examination performed at the age of 20 months revealed delayed myelination in the occipital and parietal regions. Epilepsy was diagnosed and valproic acid treatment was started. No psychomotor delay was observed at that time.
Five months later, she was admitted to the hospital again due to vomiting and progressive liver failure symptoms. Her seizure control was not satisfactory. EEG recording performed at that point showed slow activity and almost continuous spike waves over the central, parietal, and occipital regions of both hemispheres. Metabolic testing revealed high tyrosine and methionine levels (125 and 848 umol/l, respectively), normal iron concentration (108 ug%), and slightly abnormal transferrin glycosylation pattern (15.2%, 12.8% and 12.2%, control value <7.6%), assessed as secondary to liver failure.
Liver transplantation from the mother was considered. After administration of prednisone and gancyclovir, a clinical remission appeared. The girl was discharged home for some weeks before planned MRS brain imaging. The patient was lost from our further observation and died at the local hospital.
Real-Time PCR quantification
Total DNA from patient’s liver and muscle samples was extracted using QIAamp DNA Mini Kit and protocol (Qiagen Inc.). DNA concentration was determined by using a microtiter plate reader/spectrophotometer (Perkin Elmer), and DNA was diluted in ddH2O for mtDNA and nDNA amplification. Quantification of the mtDNA copy number was performed using real-time PCR amplification on Light Cycler (Roche Diagnostics) and Light Cycler FastStart DNA Master SYBR green I (Roche Diagnostics) following the instructions of the manufacturer.
Standard DNA curves for quantization of the products were used. Both mitochondrial (16S rDNA) and nuclear (β-globin gene) target sequences were PCR amplified. The primers used to amplify the mtDNA were as follows: forward, 5′-CGA AAG GAC AAG AGA AAT AAG G, and reverse, 5≥-CTG TAA AGT TTT AAG TTT TAT GCG. Total DNA quantity was corrected by simultaneous measurement of the amount of β-globin gene, using oligonucleotides: 5≥-CAA CTT CAT CCA CGT TCA CC-3≥ and 5≥-GAA GAG CCA AGG ACA GGT AC-3′ as primers. The PCR products were purified with Clean-UP kit (A&A Biotechnology) and were subjected to precise estimation of DNA concentration. Serial dilutions were made from products, and PCR reactions were performed to construct the standard curve for mitochondrial and nuclear DNAs. Standard curves were generated using 5 10-fold serial-dilutions (10–100 000 copies) of the 152 bp PCR product of mtDNA, and 268 bp PCR product of nDNA. The PCR conditions were as follows: 95°C for 10 minutes and 45 cycles at 95°C for 6 sec, 53°C for 6 sec, 72°C for 4 sec, and a final extension step at 72°C for 7 minutes, for 16S rDNA, and 95°C for 10 minutes followed by 45 cycles at 95°C for 4 sec, 56°C for 4 sec, 72°C for 12 sec, and a final extension step at 72°C for 7 minutes, for the β-globin gene. The standard curves were saved as external standard curves and were later used to quantify the mtDNA and nuclear DNA after each run. Samples were run in duplicate. PCR products of mtDNA and nDNA were quantified by using the corresponding external standard.
During creation of standard curves, amplifications of external standards were performed with the same primers and conditions as those used for further patient’s mtDNA and nDNA amplifications. Conditions of the mtDNA and nDNA amplifications were adjusted in order to assess the same efficiency of both reactions. The threshold cycle or CT value within the linear exponential phase was used to construct the standard curve and to measure the original copy number of DNA template.
PCR reactions were set up according to the manufacturer’s recommendations, with final amounts and/or concentrations: ready Master Mix (containing FastStart Taq DNA Polymerase, reaction buffer, MgCl2, SYBR Green I dye and dNTP mix) 1 μl, 0.5 μM of each primer, 1 μl of the extracted DNA (10 ng/μl) or 1 μl of Standard and water to the final volume of 10 μl. The reactions were performed under the following conditions: for mtDNA amplification: initial denaturing at 95°C for 10 minutes and 40 cycles at 95°C for 6 sec, 53°C for 6 sec, 72°C for 4 sec; for nDNA: 95°C for 10 minutes and 50 cycles at 95°C for 6 sec, 60°C for 5 sec. The SYBR Green fluorescence was read at the end of each extension step for mtDNA amplification and annealing step for nDNA amplification. A melting curve was systematically analyzed in order to check for the absence of contamination and quality of amplification. Real-time PCR was performed in triplicate for each amplicon.
DNA extracted from skeletal muscle and from liver samples was used as a template to amplify the selected regions of the POLG
gene, and two most frequently occurring mutations were assessed as described [26
]. We used, as reference for POLG nucleotide positions, the cDNA sequence corresponding to GenBank ID NM_002693.1. Sequence analysis was performed on PCR products previously purified by ExoSAP-IT treatment (USB Corp.), using the BigDye terminator Ready Reaction Kit v.3 on a 3730 Genetic Analyzer Automatic Sequencer (Applied Biosystems). Sequencing data were analyzed using the ChromasLite2.01 software.
Morphological and histochemical study
Skeletal muscle samples obtained during open surgical biopsy of the vastus lateralis was snap frozen in isopentane cooled with liquid nitrogen. Myopathology panel of stains and reactions of frozen sections comprised: hematoxylin and eosin; modified Gomori trichrome; oil red O; succinate dehydrogenase; NADH dehydrogenase; cytochrome c oxidase; acid phosphatase; and myosin ATP-ase at pH 4,3/4,6/9,4.
Liver core needle biopsy fixed in 4% buffered formalin was processed routinely for paraffin sections stained with: hematoxylin and eosin, periodic acid Shiff (PAS), PAS after diastase digestion, AZAN for collagen fibers, and silver impregnation for reticulin fibers.
Small tissue blocks for transmission electron microscopy were fixed in 2.5% cold glutaraldehyde for 1 hour, washed in cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated in graded alcohols and embedded in Epon 812. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined in a JEOL 1200EX electron microscope.
The parents of Patient 1 made their agreement to perform post mortem study limited to liver and muscle biopsies, only. The specimens were taken 2 hours after death.