Introduction
Liver failure is a rare but life-threatening critical illness requiring intensive care that occurs when large parts of the liver become severely damaged resulting in severe liver dysfunction. Symptoms include jaundice, encephalopathy, bleeding problems, fatigue and lactic acidosis. Treatment of liver failure is symptomatic, however, transplantation can be lifesaving in severe cases (Chinnery and DiMauro
2005; Fellman and Kotarsky
2011).
Although the cause of liver failure is often unknown, inherited disorders of mitochondrial oxidative phosphorylation, fatty acid oxidation, the urea cycle or glycogen storage may be responsible, especially in childhood (Casey et al
2012). Other causes of liver failure include biliary atresia, cirrhosis, tumours, intoxications and infections such as cytomegalovirus, adenovirus and hepatitis A, B and C (Iwama et al
2011).
As the liver is a vital organ with a wide range of functions it is highly dependent on ATP and a functioning oxidative phosphorylation system (Chinnery and DiMauro
2005). In general, when liver disease occurs with extra-hepatic involvement there is more reason to suspect a mitochondrial condition (Rahman
2013), however, isolated hepatic failure may also be related to mitochondrial dysfunction (Tables
1 and
2). Mitochondrial hepatopathies are most frequently caused by defects of mitochondrial DNA (mtDNA) maintenance such as mtDNA deletion (Pearson syndrome) and depletion (Rahman
2013). Genetic forms of mtDNA depletion are associated with a predominant hepatopathy, however, other organs (including muscle and brain) may also be involved (Fellman and Kotarsky
2011), such as in epileptic encephalopathy, liver failure and visual impairment in Alpers-Huttenlocher syndrome due to autosomal recessive
POLG mutations (Naviaux and Nguyen
2004). Other defects of
POLG are associated with valproate induced liver failure, further in support that mtDNA replication is essential for optimal hepatocyte function. Deoxyguanosine kinase (
DGUOK) deficiency causes mtDNA depletion with a predominant liver phenotype though neurological features (nystagmus, muscular hypotonia, psychomotor retardation) often accompany this condition (Dimmock et al
2008). In patients carrying
MPV17 mutations hepatopathy is present with poor feeding, hypoglycaemia, hypotonia and faltering growth and central nervous system involvement usually appears later in the disease course (Uusimaa et al
2014).
C10orf2 and
SUCLG1 deficiency may also result in an early-onset multisystem mitochondrial hepatoencephalomyopathy with hepatic mtDNA depletion (Fellman and Kotarsky
2011; Van Hove et al
2010). Dysfunction of mitochondrial translation may also account for severe infantile liver failure (Kemp et al
2011) caused by defects in mitochondrial translation elongation factors (
GFM1,
TSFM) (Balasubramaniam et al
2012; Vedrenne et al
2012). A unique reversible infantile hepatopathy has been shown in association with mutations in the mitochondrial tRNA modifying factor
TRMU (Zeharia et al
2009; Schara et al
2011). In addition, liver dysfunction has been associated with defects in mitochondrial proteins involved in single respiratory chain complexes, such as
SCO1 (complex IV assembly factor) and
BCS1L (complex III assembly factor) (Rahman
2013). The high number of mitochondrial disease genes affecting the liver highlights the importance of mitochondria in liver cell function.
Table 1
Summary of mitochondrial causes of liver failure with respiratory chain deficiency
Disorders of mtDNA maintenance | Hepatocerebral mitochondrial disease |
DGUOK, MPV17, POLG, SUCLG1, C10ORF2
| Combined RC defect |
Pearson syndrome | Single mtDNA deletion | Combined RC defect |
Alpers-Huttenlocher syndrome |
POLG
| Combined RC defect or normal |
Disorders of mitochondrial protein synthesis | Reversible infantile mitochondrial hepatopathy |
TRMU
| Combined RC defect |
Mitochondrial tRNA synthetase defects |
EARS2, FARS2
| |
Nuclear translation initiation-elongation factors |
GFM1, TSFM
| Combined RC defect |
Defects of OXPHOS complex assembly | Complex III assembly |
BCS1L
| Complex III |
Complex IV assembly |
SCO1
| Complex IV |
Table 2
Number of patients with deficiencies on BN PAGE, respiratory chain enzyme activities and mtDNA copy numbers in the cohort of 45 patients
Acute liver failure (3) | 3/3 (100 %) | 3/3 (100 %) | 3/3 (100 %) |
Biliary atresia (9) | 1/8 (13 %) | 5/9 (56 %) | 2/9 (22 %) |
Cirrhosis (11) | 3/10 (30 %) | 6/11 (55 %) | 2/11 (18 %) |
Tumour (6) | 3/5 (60 %) | 2/6 (33 %) | 2/6 (33 %) |
Other (16) | 0 | 4/16 (25 %) | 1/16 (6 %) |
Total | 10/40 (25 %) | 20/45 (44 %) | 10/45 (22 %) |
Patients with mitochondrial liver diseases usually present with defects of the respiratory chain enzymes in liver tissue. However, the role of mitochondrial dysfunction in the pathomechanism of severe liver disease of non-mitochondrial or unknown origin leading to severe liver failure has not been investigated in detail before. This study investigates mitochondrial function in liver in a large cohort of 45 patients undergoing liver transplantation due to severe liver disease of various aetiologies.
Discussion
End stage liver disease can be caused by a variety of conditions including viral infections, inherited metabolic diseases, drug toxicity, defects of biliary ducts, cholestasis or tumours, and in many cases the aetiology remains unclear. Even in conditions with known causes such as tumours and progressive familial intrahepatic cholestasis the reasons for progression to end stage liver disease involve various pathomechanisms. We studied liver tissue samples in a large, representative cohort of 45 patients undergoing liver transplantation for various forms of liver disease and detected mitochondrial abnormalities (mitochondrial respiratory chain deficiency and/or abnormal mtDNA copy number) in several cases. This cohort is representative of a wide range of severe liver conditions and is the first to systematically investigate mitochondrial function in severely affected liver at the time of transplantation. All patients had a severe, isolated liver manifestation and no involvements of other organs supporting mitochondrial disease such as brain and skeletal muscle were noted at the time of transplantation.
In acute liver failure all three patients had abnormal mitochondrial function supported by reduced complexes on BN PAGE, decreased activities of complexes II and IV and low mtDNA copy numbers. These results indicate that mtDNA depletion may lead to mitochondrial dysfunction and acute liver cell death in these patients. Although screening for known genetic causes of hepatic mtDNA depletion did not reveal the primary molecular defect in our patients, it is still possible that mutations in novel genes or in known genes where a different clinical presentation is expected are underlying mtDNA depletion in these cases. However we cannot exclude that the detected mitochondrial abnormalities in these patients are due to a low number of mitochondria in the diseased liver cells as a result of a sudden unknown disease mechanism.
Helbling et al
2013 studied mtDNA copy numbers in 244 patients with various forms of liver disease leading to hepatic failure requiring liver transplantation. They detected low mtDNA copy numbers in 66 % of the cases and in half of these patients the mtDNA copy number was in the range of definite mtDNA depletion (Helbling et al
2013). Screening for mutations in known genes associated with mtDNA depletion revealed heterozygous variants in
POLG and
DGUOK, however, a causative effect of these variants to cause mtDNA depletion and liver failure has not been shown, and no other mitochondrial studies were performed in support of a mitochondrial aetiology of liver dysfunction. The authors suggest that patients with acute liver failure in association with mtDNA depletion may have an underlying genetic predisposition or a mitochondrial disease, however, no experimental evidence has been shown in support of this hypothesis.
MtDNA depletion has been detected in 50 % (50/100) of children with multiple respiratory chain enzyme deficiency and most of these patients (32/50; 64 %) presented with severe neonatal onset liver involvement (Sarzi et al
2007). However, the causative mutations could not be identified in half of these cases, illustrating further genetic heterogeneity (Sarzi et al
2007). Another study performed whole exome sequencing in three children with acute liver failure and identified pathogenic mutations in
MPV17, SERAC1 and
NOTCH2, despite the lack of characteristic clinical phenotypes for these genes (Vilarinho et al
2014). Based on our data and previous reports rare genetic causes may be responsible for acute liver failure in a number of patients and next generation sequencing studies may define further novel mitochondrial phenotypes.
The detection of mitochondrial abnormalities in a relatively large number of patients with a wide range of diagnoses raises the possibility that oxidative phosphorylation may be a secondary result of a more complex cellular phenotype and can contribute to the hepatocellular dysfunction in various conditions. In biliary atresia we detected variable mitochondrial alterations (low complex IV activity, decreased complex I and V on BN PAGE, low mtDNA copy number) in 20–50 % of patients. However the lack of consequent findings in this disease group did not allow a better understanding of their role in the pathomechanism of the disease. It is possible that they are linked with certain stages of hepatocellular dysfunction, as supported by the detection of low mtDNA copy numbers in leukocytes of patients with early stage biliary atresia, suggesting a role of inflammatory reaction and secondary mitochondrial DNA damage in this condition (Tiao et al
2007).
Variable findings were detected in our study in patients with cirrhosis. About half of cirrhosis patients did not show any abnormalities, however, others had defects in one or more complexes or showed copy number abnormalities. The decreased activity of complex II was quite common in cirrhosis (55 %). It may be related to the disease progression, however, currently we have no explanation why complex II is most affected in this disease group. Chronic ethanol consumption has been shown to affect mitochondrial function by altering the mitochondrial permeability transition pore in the liver, suggesting a potential mechanism (King et al
2014).
In support of the secondary aetiology of mitochondrial alterations in cirrhosis, mitochondrial DNA re-arrangements and low copy numbers have been previously detected in patients with alcohol-induced end stage liver disease and their role has been suggested in the pathophysiology of the disease (Tang et al
2012).
We detected very diverse findings in liver tumours. While five out of six patients with tumours had deficient respiratory chain enzymes, one patient showed increased complexes on BN PAGE. Contrasting results were detected in the different tumours in the activity assays and two patients had lower and one patient very high mtDNA copy numbers on quantitative PCR analysis compared to the healthy tissue from the same liver, suggesting that cancer cells have altered mitochondrial metabolism. The role of mitochondria has been intensively studied in cancer and the pharmacological inhibition of mitochondrial metabolism is emerging as a potential therapeutic strategy in some cancers (Ahn and Metallo
2015). In support of our findings, somatic mtDNA mutations and decreased mtDNA copy number have been detected in hepatocellular carcinoma, suggesting that a mitochondrial dysfunction-activated signalling cascade may play an important role in the disease progression (Hsu et al
2013). A possible link between mtDNA depletion and tumorigenesis has been suggested by the detection of hepatocellular carcinoma in a patient with
DGUOK deficiency (Freisinger et al
2006).
Our data highlight that mitochondrial dysfunction may be secondary in a wide range of liver diseases of non-mitochondrial aetiology, although exclusion of primary mitochondrial causes has only been performed in a few selected cases. Detection of respiratory chain dysfunction in liver disease requiring transplantation is not sufficient to make the diagnosis of a mitochondrial disease, and should not restrict the inclusion of patients for liver transplantation. Furthermore, recent data suggest that liver transplantation may provide clinical benefit for patients with primary mitochondrial disease, especially when the clinical presentation is likely to be restricted to liver (Dimmock et al
2008; Hynynen et al
2014).
In conclusion, this study provides good evidence that mitochondrial dysfunction is present in patients undergoing transplantation due to various types of primary liver disease. We suggest that mitochondrial disease should be investigated in patients with acute liver failure of unknown cause, although finding the molecular cause can be difficult due to genetic heterogeneity. Although mitochondrial respiratory chain deficiencies and mtDNA copy number abnormalities are helpful to identify patients with a potentially primary mitochondrial liver disease, a critical interpretation of these data is needed. Further investigation of the role of mitochondrial dysfunction in end stage liver disease in other, non-mitochondrial hepatic disorders may reveal novel pathways, which may be targeted to improve mitochondrial function and to prevent or ameliorate disease progression.
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