Clinical and Molecular Characteristics of Mitochondrial DNA Depletion Syndrome Associated with Neonatal Cholestasis and Liver Failure Abdulrahman Al-Hussaini, MD1, Eissa Faqeih, MD2, Ayman W. El-Hattab, MD2, Majid Alfadhel3, Ali Asery, MD1, Badr Alsaleem, MD1, Eman Bakhsh, MD4, Ashraf Ali, MD5, Ali Alasmari, MD2, Khurram Lone, MD1, Ahmed Nahari, MD1, Wafaa Eyaid, MD3, Mohammed Al Balwi, MD5,6, Kate Craig, PhD7, Anna Butterworth, BSc7, Langping He, PhD7, and Robert W. Taylor, PhD, FRCPath7 Objective To determine the frequency of mitochondrial DNA depletion syndrome (MDS) in infants with cholestasis and liver failure and to further clarify the clinical, biochemical, radiologic, histopathologic, and molecular features associated with MDS due to deoxyguanosine kinase (DGUOK) and MPV17 gene mutations. Study design We studied 20 infants with suspected hepatocerebral MDS referred to our tertiary care center between 2007 and 2013. Genomic DNA was isolated from blood leukocytes, liver, and/or skeletal muscle samples by standard methods. Mitochondrial DNA copy number relative to nuclear DNA levels was determined in muscle and/ or liver DNA using real-time quantitative polymerase chain reaction and compared with age-matched controls. Nuclear candidate genes, including polymerase g, MPV17, and DGUOK were sequenced using standard analyses. Results We identified pathogenic MPV17 and DGUOK mutations in 11 infants (6 females) representing 2.5% of the 450 cases of infantile cholestasis and 22% of the 50 cases of infantile liver failure referred to our center during the study period. All of the 11 patients manifested cholestasis that was followed by a rapidly progressive liver failure and death before 2 years of life. Mitochondrial DNA depletion was demonstrated in liver or muscle for 8 out of the 11 cases where tissue was available. Seven patients had mutations in the MPV17 gene (3 novel mutations), 4 patients had DGUOK mutations (of which 2 were novel mutations). Conclusion Mutations in the MPV17 and DGUOK genes are present in a significant percentage of infants with liver failure and are associated with poor prognosis. (J Pediatr 2014;164:553-9).
M
itochondria are the main source of the high-energy phosphate molecule adenosine triphosphate (ATP), which is essential for all active intracellular processes. The liver is highly dependent on ATP for biosynthetic and detoxifying properties. Therefore, disorders of mitochondrial function commonly cause liver dysfunction. Mitochondria contain a separate genome, mitochondrial DNA (mtDNA), which is distinct from that of the nucleus.1 The respiratory chain peptide components are encoded by both nuclear and mtDNA genes. Nuclear genes encode more than 70 respiratory chain subunits and an array of enzymes and cofactors required to replicate and maintain mtDNA,2,3 including DNA polymerase g, thymidine kinase 2, and deoxyguanosine kinase (DGUOK). Mitochondrial DNA depletion syndrome (MDS) is clinically a heterogeneous group of disorders characterized by a severe reduction of mtDNA content and insufficient synthesis of respiratory chain complexes I, III, IV, and V in different tissues.4,5 Liver, heart, skeletal muscle, and brain are among the most energy-dependent tissues of the body and, therefore, they are vulnerable to mtDNA depletion. The clinical phenotypes of MDS are, therefore, highly variable, leading to 3 main clinical presentations: myopathic, encephalomyopathic, and hepatocerebral. The hepatocerebral form is associated with mutations in the DGUOK gene,6 the polymerase g 1 gene,7 the MPV17 gene,8 or more rarely, the Twinkle helicase gene.9 Mutations in DGUOK and MPV17 have been identified in an increasing number of patients.10-15 Given the common underlying pathology of mtDNA depletion and the clinical manifestations overlapping both DGUOK and MPV17 groups, the majority of patients have developed liver disease within a few months after birth, with rapid deterioration, and some patients have shown relatively slow progresFrom the Divisions of Pediatric Gastroenterology and sion of liver disease or neurologic regression. However, the number of reported Medical Genetics, The Children’s Hospital, King Fahad Medical City, College of Medicine, King Saud bin patients with MPV17 mutations is still small, and the clinical courses according Abdulaziz University for Health Sciences; Division of Genetics, Department of Pediatrics, King Saud bin to the mutations or the genotype–phenotype correlation remain unclear. The Abdulaziz University for Health Sciences, King Abdulaziz aims of our study were to determine the frequency of MDS as a cause of infantile Medical City; Department of Radiology, King Saud bin Abdulaziz University for Health Sciences, King Fahad cholestasis and liver failure and to further clarify the clinical, biochemical, Medical City; Department of Pathology, King Abdullah 1
2
3
4
5
ATP DGUOK MDS MRI mtDNA
Adenosine triphosphate Deoxyguanosine kinase Mitochondrial DNA depletion syndrome Magnetic resonance imaging Mitochondrial DNA
6
International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Riyadh, Saudi Arabia; and 7Newcastle Mitochondrial Highly Specialized Services Diagnostic Laboratory, Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, United Kingdom The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2014 Mosby Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2013.10.082
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radiologic, histopathologic, and molecular features associated with MDS due to DGUOK and MPV17 gene mutations. We report 11 cases from 9 families with MPV17 and DGUOK mutations. Included in our series are 3 newly-identified mutations in the MPV17 gene and 2 novel mutations in DGUOK gene. The correlation between the type of mutation in MPV17 and DGUOK and the phenotypic presentation of MDS is also discussed.
Methods At King Fahad Medical City, since 2007, we have adopted a strategy for careful evaluation for mitochondrial hepatopathy in infants presenting with cholestasis and neurologic manifestations (hypotonia, seizure, nystagmus), elevated plasma lactate (>2.2 mmol/L), hypoglycemia (plasma glucose A (p.Arg206Lys), which affects a highly conserved residue within the protein and is predicted to be pathogenic based on the in silico prediction tools. Mitochondrial Respiratory Chain Analyses and mtDNA Depletion in Tissue Samples Analysis of mtDNA copy number was performed in 7 infants (Table II). Severe mtDNA depletion (5%-10%) was demonstrated in 5 infants (patients 3, 5, 6, 7, and 10) where liver was available. In contrast, mtDNA copy number was variable across the cases (patients 2, 6, and 8) where muscle tissue was available (10%-75%). Muscle was analyzed for mitochondrial respiratory chain function in only 3 patients: patient 2 had combined deficiency of respiratory chain complexes I, III, and IV, patient 6 had normal activity of all complexes (muscle tissue), whereas patient 8 had mild deficiency of complex I (muscle tissue). In the 9 infants who were negative for mitochondrial disease workup, mtDNA copy number was normal in 4 infants where liver tissue was available. Analysis of mitochondrial respiratory chain function in muscle tissue in 2 of the 9 infants was normal. Histopathologic Findings The histopathologic examination of the available 6 liver biopsies constantly showed steatosis, cholestasis, and fibrosis at variable degrees of severity (Figure 2; available at www. jpeds.com). Microsteatosis was characterized by small intracytoplasmic vacuoles of irregular sizes with the nucleus still in the center of the hepatocytes (Figure 2, A). Morphology in patient 4 was different from the other 5 cases because of extreme ballooning of hepatocytes and extensive irregular foamy microsteatosis (Figure 2, B). Canalicular and hepatocellular cholestasis was mild to moderate in most of the cases. No bile plugs were seen in the bile ducts or cholangioles. Bile ductular (cholangiolar) proliferation was minimal to mild (Figure 2, C). Lobular and portal inflammation was minimal to absent, with occasional hepatocyte syncytial cells (Figure 2, B). Portal tracts, assessed on Masson trichrome stain, showed mild to severe portal fibrosis and mild perisinusoidal fibrosis (Figure 2, D). Hemosiderin granules were present in Kupffer cells in the portal areas and some at the parenchyma, and these were generally of mild degree (Grade I). In the 9 infants who were negative for mitochondrial disease workup, the histopathologic examination of the available 4 liver biopsies showed giant cell hepatitis in 3 and cirrhosis in 1 infant, but no evidence of steatosis. Neuroradiologic Findings All brain MRI films were obtained during infancy and were read by a single pediatric neuroradiologist blinded to clinical 556
Vol. 164, No. 3 data and diagnosis. Seven of the 11 patients showed abnormal findings on brain MRI (64%; Table II). An abnormal T2 hyperintensity extending from the reticulospinal tracts at the cervicomedullary junction up to the reticular formation of the medulla and pons was seen in 4 patients (patients 1, 2, 6, and 8; Figure 3). Five patients (patients 2, 3, 4, 6, and 8) showed confluent T2 hyperintensities involving the corona radiate of both cerebral hemispheres extending to involve the subcortical U-fibers and the external capsules with diffusion restriction suggesting demyelination (Figure 3). Another 3 patients (patient 4, 6, and 10) showed abnormal T2 hyperintensity in the globus pallidi bilaterally (Figure 4). All of our patients demonstrated absence of lactate peak on the hydrogen 1 magnetic resonance spectroscopy. Brain MRI was done in 6 of the 9 infants with negative mitochondrial disease workup; white matter hypomyelination and mild cortical atrophy was seen in 2 infant and the remaining 4 infants had normal brain MRI.
Discussion Our report characterizes 11 infants with hepatocerebral mtDNA depletion: 7 infants had mutations in the MPV17 gene (3 novel mutations) and 4 had mutations in the DGUOK gene (including 2 cases with novel mutations). These mutations were associated with a severe clinical phenotype characterized by infantile onset of liver failure and death before 24 months of age. A significant percentage of infantile liver failure in our series was caused by MDS (22%). Hepatocerebral MDS can be confused with other severe hepatopathies of infancy, such as tyrosinemia, neonatal hemochromatosis, bile acid synthesis disorders, hemophagocytic lymphohistiocytosis, and infectious and endocrine causes. The liver failure caused by these pathologies may be reversible upon prompt diagnosis and early initiation of appropriate therapy. Therefore, these treatable conditions should be ruled out while investigating for mitochondrial disorders. Because the clinical, laboratory, and histopathologic findings can be variable and nonspecific for MDS, there must be a high index of suspicion for these diseases to initiate the genetic testing that is necessary for diagnosis. Therefore, characteristic MRI findings that could alert a physician to these disorders would have diagnostic utility. The MRI literature describing MDS is sparse and nonspecific with variable findings that ranged from normal to diffuse white matter abnormalities, which may resemble a leukodystrophy or hypomyelination.8,13,15 There are few reports that described specific regional abnormalities on brain MRI in patients with MPV17 or DGUOK mutations.20-22 Abnormalities within the reticular formation of the lower brain stem and within the reticulospinal tracts at the cervicocranial junction on MRI have been reported in patients with MPV17,20,21 similar to neuroimaging findings in 3 of our 7 patients with MPV17. We have observed this finding in DGUOK mutation as well (patient 8); this was not reported previously. Al-Hussaini et al
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Figure 3. Confluent T2 hyperintensities involving the corona radiata of the cerebral hemispheres extending to involve the A, subcortical U-fibers and B, external capsules (arrow). A, The globus pallidus is involved bilaterally (arrows).
The reticular formation and reticulospinal tracts are responsible for tone and posture; therefore, lesions involving such locations may correspond with the neurologic presentation of MPV17 and DGUOK mutations, which often involves hypotonia. Another region in the brain that was reported to be involved in 2 patients with DGUOK included globus pallidi.22 We have observed bilateral globus pallidi involvement in DGUOK mutation (patient 11) and in MPV17 mutations (patient 2 and 6). Although these neuroimaging findings overlap among MPV17 and DGUOK mutations, lesions in the reticular formation and globus pallidi are characteristic
of both mutations and, in the right clinical setting, might serve as a neuroimaging clue to the diagnosis of MPV17 or DGUOK mutations. Infants with MDS could have normal brain MRI despite clinically evident neurologic impairment. Others have reported normal initial brain MRI during infancy that have shown abnormal findings on a repeated MRI after infancy.20,23 These data emphasize the importance of ongoing neurodevelopmental assessment and careful search for a neuroimaging abnormality on a follow-up MRI after infancy, especially in an infant with isolated liver dysfunction and under consideration of liver transplantation.
Figure 4. A, Axial T2 and B, axial inversion recovery images demonstrate abnormal T2 signal in the posterior aspect of the medulla extending up to the pones clearly seen in the inversion recovery images (arrows). Clinical and Molecular Characteristics of Mitochondrial DNA Depletion Syndrome Associated with Neonatal Cholestasis and Liver Failure
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The diagnosis of MDS could be hampered by the difficulty of performing a liver biopsy because of coagulopathy or because of the tendency of physicians to use conventional diagnostic approaches that focus on less invasive techniques, particularly in infants, such as cultured skin fibroblasts or skeletal muscle. In our cohort, mtDNA content and respiratory chain analysis was variable across the 3 cases where muscle tissue was available. In contrast, severe mtDNA depletion (5%-10%) was demonstrated in 5 cases where liver tissue was available. Our results are consistent with previously described patients with MPV17 and DGUOK mutations who had mtDNA depletion that was most pronounced in liver and a less severe depletion or normal mtDNA content in skeletal muscle.14,15,24,25 Collectively, data from our study and others demonstrate the importance of studying the most affected tissues whenever possible because the biochemical defect may not be apparent in unaffected tissues and the mitochondrial etiology of the disease might be overlooked by studies confined to skeletal muscle. The severe mtDNA depletion in liver in our patients correlated with the severe clinical phenotype. The tendency that patients with the most severe mtDNA depletion in liver or muscle present and die earlier than patients with less severe depletion has been reported previously in certain MPV17 and DGUOK mutations.12,15 Other prognostic factors included presence or absence of neurologic involvement. Our data and others support the observation that early onset of neurologic symptoms had been associated with severe disease, early mortality, and poorer outcome after liver transplantation.12,25 Certain MPV17 and DGUOK mutations had been associated with an isolated hepatic phenotype that was characterized by a more benign course, and better outcome post-liver transplantation. Understanding the molecular etiology of this disease, its phenotypic spectrum, and identification of genotype–phenotype correlations might help in predicting the natural history of disease progression and the potential therapeutic decisions surrounding liver transplantation.4 Large scale, multicenter, and longitudinal prospective studies are needed to address these objectives. There is no clear evidence to suggest benefit of any medical therapies in mitochondrial disorders.4 At present, there is no consensus on the role of liver transplantation in hepatocerebral form of MDS. A review of the literature showed a mixed outcome, with a survival rate of less than 50%.4 The presence of significant neuromuscular involvement, as evident in our cohort of 11 patients, is an absolute contraindication to liver transplantation. The variable time of onset and severity of neurologic deterioration merit frank discussion with parents in the course of evaluating a child for transplantation candidacy. In conclusion, mutations in MPV17 and DGUOK genes appear to be a significant cause of infantile liver failure. During evaluation of an infant for suspected hepatocerebral MDS form, whenever possible, a study of liver tissue is important because the liver consistently shows depletion of mtDNA compared with skeletal muscle. Lesions in reticular formation or globus pallidi on brain MRI in an infant with liver 558
Vol. 164, No. 3 failure could be a clue to the diagnosis of MDS. However, normal brain MRI in an infant with MDS does not exclude neurologic involvement. n Submitted for publication Jul 11, 2013; last revision received Sep 23, 2013; accepted Oct 29, 2013. Reprint requests: Abdulrahman Al-Hussaini, MD, Division of Pediatric Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital, King Fahad Medical City, College of Medicine, King Saud bin Abdulaziz University for Health Sciences, PO box 59046, Riyadh 11525, Kingdom of Saudi Arabia. E-mail: [email protected]
References 1. Johns DR. Mitochondrial DNA and disease. N Engl J Med 1995;333: 638-44. 2. Chinnery PF, DiMauro S. Mitochondrial hepatopathies. J Hepatol 2005; 43:207-9. 3. DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003;348:2656-68. 4. Lee WS, Sokol RJ. Mitochondrial hepatopathies: Advances in genetics, therapeutic approaches, and outcomes. J Pediatr. In press. 5. Spinazzola A, Invernizzi F, Carrara F, Lamantea E, Donati A, DiRocco M, et al. Clinical and molecular features of mitochondrial DNA depletion syndromes. J Inherit Metab Dis 2009;32:143-58. 6. Mandel H, Szargel R, Labay V, Elpeleg O, Saada A, Shalata A, et al. The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 2001;29: 337-41. 7. Ferrari G, Lamantea E, Donati A, Filosto M, Briem E, Carrara F, et al. Infantile hepatocerebral syndromes associated with mutations in the mitochondrial DNA polymerase-gamma A. Brain 2005;128:723-31. 8. Spinazzola A, Viscomi C, Fernandez-Vizarra E, Carrara F, D’Adamo P, Calvo S, et al. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat Genet 2006;38:570-5. 9. Hakonen AH, Isohanni P, Paetau A, Herva R, Suomalainen A, L€ onnqvist T. Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion. Brain 2007;130:3032-40. 10. Wang L, Eriksson S. Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome. FEBS Lett 2003;554:319-22. 11. Wang L, Limongelli A, Vila MR, Carrara F, Zeviani M, Eriksson S. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes. Mol Genet Metab 2005;84:75-82. 12. Dimmock DP, Zhang Q, Dionisi-Vici C, Shieh J, Tang L-Y, Truong C, et al. Clinical and molecular features of mitochondrial DNA depletion due to mutations in deoxyguanosine kinase. Hum Mutat 2008;29:330-1. 13. Freisinger P, Futterer N, Lankes E, Gempel K, Berger TM, Spalinger J, et al. Hepatocerebral mitochondrial DNA depletion syndrome caused by deoxyguanosine kinase (DGUOK) mutations. Arch Neurol 2006;63: 1129-34. 14. Wong LJ, Brunetti-Pierri N, Zhang Q, Yazigi N, Bove KE, Dahms BB, et al. Mutations in the MPV17 gene are responsible for rapidly progressive liver failure in infancy. Hepatology 2007;46:1218-27. 15. El-Hattab AW, Li FY, Schmitt E, Zhang S, Craigen WJ, Wong LJ. MPV17-associated hepatocerebral mitochondrial DNA depletion syndrome: new patients and novel mutations. Mol Genet Metab 2010;99: 300-8. 16. Poulton J, Sewry C, Potter CG, Bougeron T, Chretien D, Wijburg FA, et al. Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome? J Inherit Metab Dis 1995;18:4-20. 17. Morten KJ, Ashley N, Wijburg F, Hadzic N, Parr J, Jayawant S, et al. Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion. Mitochondrion 2007;7:386-95.
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March 2014 18. Blakely E, He L, Gardner JL, Hudson G, Walter J, Hughes I, et al. Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy. Neuromuscul Disord 2008;18:557-60. 19. Uusimaa J, Evans J, Smith C, Butterworth A, Craig K, Ashley N, et al. Clinical, biochemical, cellular, and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene. Eur J Hum Genet. In press. 20. Brunetti-Pierri N, Selby K, O’Sullivan M, Hendson G, Truong C, Waters PJ, et al. Rapidly progressive neurological deterioration in a child with Alpers syndrome exhibiting a previously unremarkable brain MRI. Neuro Pediatr 2008;39:179-83. 21. Vu TH, Sciacco M, Tanji K, Nichter C, Bonilla E, Chatkupt S, et al. Clinical manifestations of mitochondrial DNA depletion. Neurology 1998; 50:1783-90.
ORIGINAL ARTICLES 22. Merkle AN, Nascene DR, McKinney AM. MR imaging findings in the reticular formation in siblings with MPV17-related mitochondrial depletion syndrome. Am J Neuroradiol 2012;33:E34-5. 23. DeBruyn JC, Chan AK, Bhargava R, Idikio H, Huynh HQ. Liver failure in mitochondrial DNA depletion syndrome: the importance of serial neuroimaging in liver transplantation evaluation. J Pediatr Gastroenterol Nutr 2007;45:252-6. 24. Brahimi N, Jambou M, Sarzi E, Serre V, Boddaert N, Romano S, et al. The first founder DGUOK mutation associated with hepatocerebral mitochondrial DNA depletion syndrome. Mol Genet Metab 2009;97:221-6. 25. Labarthe F, Dobbelaere D, Devisme L, De Muret A, Jarde C, Taanman J, et al. Clinical, biochemical, and morphological features of hepatocerebral syndrome with mitochondrial DNA depletion due to deoxyguanosine kinase deficiency. J Hepatol 2005;43:333-41.
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Table II. Molecular, histopathologic, and neuroradiologic characteristics of the 11 infants with hepatocerebral mtDNA depletion Liver histopathology Gene Patient defect
Gene mutation
1
MPV17 c.62T>G (p.Leu21Arg), homozygous
2†
MPV17 c.278A>C (p. Gln93Pro), homozygous
3†
MPV17 c.278A>C (p.Gln93Pro), homozygous MPV17 c.278A>C (p.Gln93Pro), homozygous MPV17 c.279+1G>T (splicing site), homozygous MPV17 c.279+1G>T (splice site), homozygous
4 5 6
7 8
9 10 11
MPV17 c.279+1G>T (splicing site), homozygous DGUOK c.223T>A (p. Trp75Arg), homozygous
% of mtDNA copy number
Brain MRI findings (age*)
High T2 signal in the posterior medulla and pons (5 mo) 10% (muscle) Delayed myelination, High T2 signal in the posterior medulla and Pons, bilateral involvement of globus pallidi (6 mo) 10% (liver) Delayed myelination (6 mo)
Steatosis
Hepatocyte Bile duct ballooning Cholestasis proliferation Fibrosis
ND
ND 5% (liver)
Delayed myelination (3 mo) Normal (4.5 mo)
10% (liver) Delayed myelination, High T2 75% (muscle) signal in the posterior medulla and Pons, , bilateral involvement of globus pallidi (12 mo) 10% (liver) Normal (4 mo)
++ (micro) + (macro) +++ (micro) + (macro) ++ (micro) + (macro) + (micro) 0 (macro)
+ (micro) + (macro)
30% (muscle) Delayed myelination, High T2 signal in the posterior medulla and Pons (10 mo) DGUOK c.766_767insGATT(p.Phe256*), ND Normal (2.5 mo) homozygous DGUOK c.766_767insGATT(p.Phe256*), 5% (liver) Normal (4 mo) ++ (micro) homozygous + (macro) DGUOK c. 617G>A (p.Arg206Lys) ND Bilateral involvement of Globus pallidi (12 mo)
+ +++ ++ +
+ (Hep) + (Can) ++ (Hep) + (Can) + (Hep) ++ (Can) + (Hep) +++ (Can)
+
+
+
++
+
+++
++
++
++
+ (Hep) ++ (Can)
0
+
+
+ (Hep) 0 (Can)
++
++
Can, canalicular; Hep, hepatocellular; macro, macrovesicular steatosis; micro, microvesicular steatosis. *Age at time of performance of brain MRI; 0 = absent; + = mild; ++ = moderate; +++ = severe. †Patients 2 and 3 are siblings.
Figure 1. The DGUOK gene comprises 7 exons. The 2 novel p.Trp75Arg and p.Arg206Lys missense mutations are shown. The c.766_767insGATT (p.Phe256*) insertion mutation is predicted to result in a frameshift and premature truncation of the protein. 559.e1
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Figure 2. A, Hepatocytes in zone 3 with microvesicular steatosis (arrow head) and canalicular cholestasis (arrow) (hematoxylin and eosin staining 40 magnification). B, Ballooned hepatocytes with extensive irregular foamy microvesicular steatosis in case 4. A syncytial cell is noted in this field (arrow) (hematoxylin and eosin staining 40 magnification). C, Portal tract with prominent cholangiolar proliferation (arrows) and interface hepatocytes with microvesicular steatosis (hematoxylin and eosin staining 40 magnification). D, Masson trichrome stain showing portal fibrosis with thin septal formation (arrow head), mild pericellular fibrosis (arrow), and mild macro- and microvesicular steatosis (hematoxylin and eosin staining 40 magnification). CV, central vein.
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M
itochondria are the main source of the high-energy phosphate molecule adenosine triphosphate (ATP), which is essential for all active intracellular processes. The liver is highly dependent on ATP for biosynthetic and detoxifying properties. Therefore, disorders of mitochondrial function commonly cause liver dysfunction. Mitochondria contain a separate genome, mitochondrial DNA (mtDNA), which is distinct from that of the nucleus.1 The respiratory chain peptide components are encoded by both nuclear and mtDNA genes. Nuclear genes encode more than 70 respiratory chain subunits and an array of enzymes and cofactors required to replicate and maintain mtDNA,2,3 including DNA polymerase g, thymidine kinase 2, and deoxyguanosine kinase (DGUOK). Mitochondrial DNA depletion syndrome (MDS) is clinically a heterogeneous group of disorders characterized by a severe reduction of mtDNA content and insufficient synthesis of respiratory chain complexes I, III, IV, and V in different tissues.4,5 Liver, heart, skeletal muscle, and brain are among the most energy-dependent tissues of the body and, therefore, they are vulnerable to mtDNA depletion. The clinical phenotypes of MDS are, therefore, highly variable, leading to 3 main clinical presentations: myopathic, encephalomyopathic, and hepatocerebral. The hepatocerebral form is associated with mutations in the DGUOK gene,6 the polymerase g 1 gene,7 the MPV17 gene,8 or more rarely, the Twinkle helicase gene.9 Mutations in DGUOK and MPV17 have been identified in an increasing number of patients.10-15 Given the common underlying pathology of mtDNA depletion and the clinical manifestations overlapping both DGUOK and MPV17 groups, the majority of patients have developed liver disease within a few months after birth, with rapid deterioration, and some patients have shown relatively slow progresFrom the Divisions of Pediatric Gastroenterology and sion of liver disease or neurologic regression. However, the number of reported Medical Genetics, The Children’s Hospital, King Fahad Medical City, College of Medicine, King Saud bin patients with MPV17 mutations is still small, and the clinical courses according Abdulaziz University for Health Sciences; Division of Genetics, Department of Pediatrics, King Saud bin to the mutations or the genotype–phenotype correlation remain unclear. The Abdulaziz University for Health Sciences, King Abdulaziz aims of our study were to determine the frequency of MDS as a cause of infantile Medical City; Department of Radiology, King Saud bin Abdulaziz University for Health Sciences, King Fahad cholestasis and liver failure and to further clarify the clinical, biochemical, Medical City; Department of Pathology, King Abdullah 1
2
3
4
5
ATP DGUOK MDS MRI mtDNA
Adenosine triphosphate Deoxyguanosine kinase Mitochondrial DNA depletion syndrome Magnetic resonance imaging Mitochondrial DNA
6
International Medical Research Center, King Saud bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Riyadh, Saudi Arabia; and 7Newcastle Mitochondrial Highly Specialized Services Diagnostic Laboratory, Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, United Kingdom The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2014 Mosby Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2013.10.082
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radiologic, histopathologic, and molecular features associated with MDS due to DGUOK and MPV17 gene mutations. We report 11 cases from 9 families with MPV17 and DGUOK mutations. Included in our series are 3 newly-identified mutations in the MPV17 gene and 2 novel mutations in DGUOK gene. The correlation between the type of mutation in MPV17 and DGUOK and the phenotypic presentation of MDS is also discussed.
Methods At King Fahad Medical City, since 2007, we have adopted a strategy for careful evaluation for mitochondrial hepatopathy in infants presenting with cholestasis and neurologic manifestations (hypotonia, seizure, nystagmus), elevated plasma lactate (>2.2 mmol/L), hypoglycemia (plasma glucose A (p.Arg206Lys), which affects a highly conserved residue within the protein and is predicted to be pathogenic based on the in silico prediction tools. Mitochondrial Respiratory Chain Analyses and mtDNA Depletion in Tissue Samples Analysis of mtDNA copy number was performed in 7 infants (Table II). Severe mtDNA depletion (5%-10%) was demonstrated in 5 infants (patients 3, 5, 6, 7, and 10) where liver was available. In contrast, mtDNA copy number was variable across the cases (patients 2, 6, and 8) where muscle tissue was available (10%-75%). Muscle was analyzed for mitochondrial respiratory chain function in only 3 patients: patient 2 had combined deficiency of respiratory chain complexes I, III, and IV, patient 6 had normal activity of all complexes (muscle tissue), whereas patient 8 had mild deficiency of complex I (muscle tissue). In the 9 infants who were negative for mitochondrial disease workup, mtDNA copy number was normal in 4 infants where liver tissue was available. Analysis of mitochondrial respiratory chain function in muscle tissue in 2 of the 9 infants was normal. Histopathologic Findings The histopathologic examination of the available 6 liver biopsies constantly showed steatosis, cholestasis, and fibrosis at variable degrees of severity (Figure 2; available at www. jpeds.com). Microsteatosis was characterized by small intracytoplasmic vacuoles of irregular sizes with the nucleus still in the center of the hepatocytes (Figure 2, A). Morphology in patient 4 was different from the other 5 cases because of extreme ballooning of hepatocytes and extensive irregular foamy microsteatosis (Figure 2, B). Canalicular and hepatocellular cholestasis was mild to moderate in most of the cases. No bile plugs were seen in the bile ducts or cholangioles. Bile ductular (cholangiolar) proliferation was minimal to mild (Figure 2, C). Lobular and portal inflammation was minimal to absent, with occasional hepatocyte syncytial cells (Figure 2, B). Portal tracts, assessed on Masson trichrome stain, showed mild to severe portal fibrosis and mild perisinusoidal fibrosis (Figure 2, D). Hemosiderin granules were present in Kupffer cells in the portal areas and some at the parenchyma, and these were generally of mild degree (Grade I). In the 9 infants who were negative for mitochondrial disease workup, the histopathologic examination of the available 4 liver biopsies showed giant cell hepatitis in 3 and cirrhosis in 1 infant, but no evidence of steatosis. Neuroradiologic Findings All brain MRI films were obtained during infancy and were read by a single pediatric neuroradiologist blinded to clinical 556
Vol. 164, No. 3 data and diagnosis. Seven of the 11 patients showed abnormal findings on brain MRI (64%; Table II). An abnormal T2 hyperintensity extending from the reticulospinal tracts at the cervicomedullary junction up to the reticular formation of the medulla and pons was seen in 4 patients (patients 1, 2, 6, and 8; Figure 3). Five patients (patients 2, 3, 4, 6, and 8) showed confluent T2 hyperintensities involving the corona radiate of both cerebral hemispheres extending to involve the subcortical U-fibers and the external capsules with diffusion restriction suggesting demyelination (Figure 3). Another 3 patients (patient 4, 6, and 10) showed abnormal T2 hyperintensity in the globus pallidi bilaterally (Figure 4). All of our patients demonstrated absence of lactate peak on the hydrogen 1 magnetic resonance spectroscopy. Brain MRI was done in 6 of the 9 infants with negative mitochondrial disease workup; white matter hypomyelination and mild cortical atrophy was seen in 2 infant and the remaining 4 infants had normal brain MRI.
Discussion Our report characterizes 11 infants with hepatocerebral mtDNA depletion: 7 infants had mutations in the MPV17 gene (3 novel mutations) and 4 had mutations in the DGUOK gene (including 2 cases with novel mutations). These mutations were associated with a severe clinical phenotype characterized by infantile onset of liver failure and death before 24 months of age. A significant percentage of infantile liver failure in our series was caused by MDS (22%). Hepatocerebral MDS can be confused with other severe hepatopathies of infancy, such as tyrosinemia, neonatal hemochromatosis, bile acid synthesis disorders, hemophagocytic lymphohistiocytosis, and infectious and endocrine causes. The liver failure caused by these pathologies may be reversible upon prompt diagnosis and early initiation of appropriate therapy. Therefore, these treatable conditions should be ruled out while investigating for mitochondrial disorders. Because the clinical, laboratory, and histopathologic findings can be variable and nonspecific for MDS, there must be a high index of suspicion for these diseases to initiate the genetic testing that is necessary for diagnosis. Therefore, characteristic MRI findings that could alert a physician to these disorders would have diagnostic utility. The MRI literature describing MDS is sparse and nonspecific with variable findings that ranged from normal to diffuse white matter abnormalities, which may resemble a leukodystrophy or hypomyelination.8,13,15 There are few reports that described specific regional abnormalities on brain MRI in patients with MPV17 or DGUOK mutations.20-22 Abnormalities within the reticular formation of the lower brain stem and within the reticulospinal tracts at the cervicocranial junction on MRI have been reported in patients with MPV17,20,21 similar to neuroimaging findings in 3 of our 7 patients with MPV17. We have observed this finding in DGUOK mutation as well (patient 8); this was not reported previously. Al-Hussaini et al
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Figure 3. Confluent T2 hyperintensities involving the corona radiata of the cerebral hemispheres extending to involve the A, subcortical U-fibers and B, external capsules (arrow). A, The globus pallidus is involved bilaterally (arrows).
The reticular formation and reticulospinal tracts are responsible for tone and posture; therefore, lesions involving such locations may correspond with the neurologic presentation of MPV17 and DGUOK mutations, which often involves hypotonia. Another region in the brain that was reported to be involved in 2 patients with DGUOK included globus pallidi.22 We have observed bilateral globus pallidi involvement in DGUOK mutation (patient 11) and in MPV17 mutations (patient 2 and 6). Although these neuroimaging findings overlap among MPV17 and DGUOK mutations, lesions in the reticular formation and globus pallidi are characteristic
of both mutations and, in the right clinical setting, might serve as a neuroimaging clue to the diagnosis of MPV17 or DGUOK mutations. Infants with MDS could have normal brain MRI despite clinically evident neurologic impairment. Others have reported normal initial brain MRI during infancy that have shown abnormal findings on a repeated MRI after infancy.20,23 These data emphasize the importance of ongoing neurodevelopmental assessment and careful search for a neuroimaging abnormality on a follow-up MRI after infancy, especially in an infant with isolated liver dysfunction and under consideration of liver transplantation.
Figure 4. A, Axial T2 and B, axial inversion recovery images demonstrate abnormal T2 signal in the posterior aspect of the medulla extending up to the pones clearly seen in the inversion recovery images (arrows). Clinical and Molecular Characteristics of Mitochondrial DNA Depletion Syndrome Associated with Neonatal Cholestasis and Liver Failure
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The diagnosis of MDS could be hampered by the difficulty of performing a liver biopsy because of coagulopathy or because of the tendency of physicians to use conventional diagnostic approaches that focus on less invasive techniques, particularly in infants, such as cultured skin fibroblasts or skeletal muscle. In our cohort, mtDNA content and respiratory chain analysis was variable across the 3 cases where muscle tissue was available. In contrast, severe mtDNA depletion (5%-10%) was demonstrated in 5 cases where liver tissue was available. Our results are consistent with previously described patients with MPV17 and DGUOK mutations who had mtDNA depletion that was most pronounced in liver and a less severe depletion or normal mtDNA content in skeletal muscle.14,15,24,25 Collectively, data from our study and others demonstrate the importance of studying the most affected tissues whenever possible because the biochemical defect may not be apparent in unaffected tissues and the mitochondrial etiology of the disease might be overlooked by studies confined to skeletal muscle. The severe mtDNA depletion in liver in our patients correlated with the severe clinical phenotype. The tendency that patients with the most severe mtDNA depletion in liver or muscle present and die earlier than patients with less severe depletion has been reported previously in certain MPV17 and DGUOK mutations.12,15 Other prognostic factors included presence or absence of neurologic involvement. Our data and others support the observation that early onset of neurologic symptoms had been associated with severe disease, early mortality, and poorer outcome after liver transplantation.12,25 Certain MPV17 and DGUOK mutations had been associated with an isolated hepatic phenotype that was characterized by a more benign course, and better outcome post-liver transplantation. Understanding the molecular etiology of this disease, its phenotypic spectrum, and identification of genotype–phenotype correlations might help in predicting the natural history of disease progression and the potential therapeutic decisions surrounding liver transplantation.4 Large scale, multicenter, and longitudinal prospective studies are needed to address these objectives. There is no clear evidence to suggest benefit of any medical therapies in mitochondrial disorders.4 At present, there is no consensus on the role of liver transplantation in hepatocerebral form of MDS. A review of the literature showed a mixed outcome, with a survival rate of less than 50%.4 The presence of significant neuromuscular involvement, as evident in our cohort of 11 patients, is an absolute contraindication to liver transplantation. The variable time of onset and severity of neurologic deterioration merit frank discussion with parents in the course of evaluating a child for transplantation candidacy. In conclusion, mutations in MPV17 and DGUOK genes appear to be a significant cause of infantile liver failure. During evaluation of an infant for suspected hepatocerebral MDS form, whenever possible, a study of liver tissue is important because the liver consistently shows depletion of mtDNA compared with skeletal muscle. Lesions in reticular formation or globus pallidi on brain MRI in an infant with liver 558
Vol. 164, No. 3 failure could be a clue to the diagnosis of MDS. However, normal brain MRI in an infant with MDS does not exclude neurologic involvement. n Submitted for publication Jul 11, 2013; last revision received Sep 23, 2013; accepted Oct 29, 2013. Reprint requests: Abdulrahman Al-Hussaini, MD, Division of Pediatric Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital, King Fahad Medical City, College of Medicine, King Saud bin Abdulaziz University for Health Sciences, PO box 59046, Riyadh 11525, Kingdom of Saudi Arabia. E-mail: [email protected]
References 1. Johns DR. Mitochondrial DNA and disease. N Engl J Med 1995;333: 638-44. 2. Chinnery PF, DiMauro S. Mitochondrial hepatopathies. J Hepatol 2005; 43:207-9. 3. DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003;348:2656-68. 4. Lee WS, Sokol RJ. Mitochondrial hepatopathies: Advances in genetics, therapeutic approaches, and outcomes. J Pediatr. In press. 5. Spinazzola A, Invernizzi F, Carrara F, Lamantea E, Donati A, DiRocco M, et al. Clinical and molecular features of mitochondrial DNA depletion syndromes. J Inherit Metab Dis 2009;32:143-58. 6. Mandel H, Szargel R, Labay V, Elpeleg O, Saada A, Shalata A, et al. The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 2001;29: 337-41. 7. Ferrari G, Lamantea E, Donati A, Filosto M, Briem E, Carrara F, et al. Infantile hepatocerebral syndromes associated with mutations in the mitochondrial DNA polymerase-gamma A. Brain 2005;128:723-31. 8. Spinazzola A, Viscomi C, Fernandez-Vizarra E, Carrara F, D’Adamo P, Calvo S, et al. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat Genet 2006;38:570-5. 9. Hakonen AH, Isohanni P, Paetau A, Herva R, Suomalainen A, L€ onnqvist T. Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion. Brain 2007;130:3032-40. 10. Wang L, Eriksson S. Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome. FEBS Lett 2003;554:319-22. 11. Wang L, Limongelli A, Vila MR, Carrara F, Zeviani M, Eriksson S. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes. Mol Genet Metab 2005;84:75-82. 12. Dimmock DP, Zhang Q, Dionisi-Vici C, Shieh J, Tang L-Y, Truong C, et al. Clinical and molecular features of mitochondrial DNA depletion due to mutations in deoxyguanosine kinase. Hum Mutat 2008;29:330-1. 13. Freisinger P, Futterer N, Lankes E, Gempel K, Berger TM, Spalinger J, et al. Hepatocerebral mitochondrial DNA depletion syndrome caused by deoxyguanosine kinase (DGUOK) mutations. Arch Neurol 2006;63: 1129-34. 14. Wong LJ, Brunetti-Pierri N, Zhang Q, Yazigi N, Bove KE, Dahms BB, et al. Mutations in the MPV17 gene are responsible for rapidly progressive liver failure in infancy. Hepatology 2007;46:1218-27. 15. El-Hattab AW, Li FY, Schmitt E, Zhang S, Craigen WJ, Wong LJ. MPV17-associated hepatocerebral mitochondrial DNA depletion syndrome: new patients and novel mutations. Mol Genet Metab 2010;99: 300-8. 16. Poulton J, Sewry C, Potter CG, Bougeron T, Chretien D, Wijburg FA, et al. Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome? J Inherit Metab Dis 1995;18:4-20. 17. Morten KJ, Ashley N, Wijburg F, Hadzic N, Parr J, Jayawant S, et al. Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion. Mitochondrion 2007;7:386-95.
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March 2014 18. Blakely E, He L, Gardner JL, Hudson G, Walter J, Hughes I, et al. Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy. Neuromuscul Disord 2008;18:557-60. 19. Uusimaa J, Evans J, Smith C, Butterworth A, Craig K, Ashley N, et al. Clinical, biochemical, cellular, and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene. Eur J Hum Genet. In press. 20. Brunetti-Pierri N, Selby K, O’Sullivan M, Hendson G, Truong C, Waters PJ, et al. Rapidly progressive neurological deterioration in a child with Alpers syndrome exhibiting a previously unremarkable brain MRI. Neuro Pediatr 2008;39:179-83. 21. Vu TH, Sciacco M, Tanji K, Nichter C, Bonilla E, Chatkupt S, et al. Clinical manifestations of mitochondrial DNA depletion. Neurology 1998; 50:1783-90.
ORIGINAL ARTICLES 22. Merkle AN, Nascene DR, McKinney AM. MR imaging findings in the reticular formation in siblings with MPV17-related mitochondrial depletion syndrome. Am J Neuroradiol 2012;33:E34-5. 23. DeBruyn JC, Chan AK, Bhargava R, Idikio H, Huynh HQ. Liver failure in mitochondrial DNA depletion syndrome: the importance of serial neuroimaging in liver transplantation evaluation. J Pediatr Gastroenterol Nutr 2007;45:252-6. 24. Brahimi N, Jambou M, Sarzi E, Serre V, Boddaert N, Romano S, et al. The first founder DGUOK mutation associated with hepatocerebral mitochondrial DNA depletion syndrome. Mol Genet Metab 2009;97:221-6. 25. Labarthe F, Dobbelaere D, Devisme L, De Muret A, Jarde C, Taanman J, et al. Clinical, biochemical, and morphological features of hepatocerebral syndrome with mitochondrial DNA depletion due to deoxyguanosine kinase deficiency. J Hepatol 2005;43:333-41.
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Table II. Molecular, histopathologic, and neuroradiologic characteristics of the 11 infants with hepatocerebral mtDNA depletion Liver histopathology Gene Patient defect
Gene mutation
1
MPV17 c.62T>G (p.Leu21Arg), homozygous
2†
MPV17 c.278A>C (p. Gln93Pro), homozygous
3†
MPV17 c.278A>C (p.Gln93Pro), homozygous MPV17 c.278A>C (p.Gln93Pro), homozygous MPV17 c.279+1G>T (splicing site), homozygous MPV17 c.279+1G>T (splice site), homozygous
4 5 6
7 8
9 10 11
MPV17 c.279+1G>T (splicing site), homozygous DGUOK c.223T>A (p. Trp75Arg), homozygous
% of mtDNA copy number
Brain MRI findings (age*)
High T2 signal in the posterior medulla and pons (5 mo) 10% (muscle) Delayed myelination, High T2 signal in the posterior medulla and Pons, bilateral involvement of globus pallidi (6 mo) 10% (liver) Delayed myelination (6 mo)
Steatosis
Hepatocyte Bile duct ballooning Cholestasis proliferation Fibrosis
ND
ND 5% (liver)
Delayed myelination (3 mo) Normal (4.5 mo)
10% (liver) Delayed myelination, High T2 75% (muscle) signal in the posterior medulla and Pons, , bilateral involvement of globus pallidi (12 mo) 10% (liver) Normal (4 mo)
++ (micro) + (macro) +++ (micro) + (macro) ++ (micro) + (macro) + (micro) 0 (macro)
+ (micro) + (macro)
30% (muscle) Delayed myelination, High T2 signal in the posterior medulla and Pons (10 mo) DGUOK c.766_767insGATT(p.Phe256*), ND Normal (2.5 mo) homozygous DGUOK c.766_767insGATT(p.Phe256*), 5% (liver) Normal (4 mo) ++ (micro) homozygous + (macro) DGUOK c. 617G>A (p.Arg206Lys) ND Bilateral involvement of Globus pallidi (12 mo)
+ +++ ++ +
+ (Hep) + (Can) ++ (Hep) + (Can) + (Hep) ++ (Can) + (Hep) +++ (Can)
+
+
+
++
+
+++
++
++
++
+ (Hep) ++ (Can)
0
+
+
+ (Hep) 0 (Can)
++
++
Can, canalicular; Hep, hepatocellular; macro, macrovesicular steatosis; micro, microvesicular steatosis. *Age at time of performance of brain MRI; 0 = absent; + = mild; ++ = moderate; +++ = severe. †Patients 2 and 3 are siblings.
Figure 1. The DGUOK gene comprises 7 exons. The 2 novel p.Trp75Arg and p.Arg206Lys missense mutations are shown. The c.766_767insGATT (p.Phe256*) insertion mutation is predicted to result in a frameshift and premature truncation of the protein. 559.e1
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Figure 2. A, Hepatocytes in zone 3 with microvesicular steatosis (arrow head) and canalicular cholestasis (arrow) (hematoxylin and eosin staining 40 magnification). B, Ballooned hepatocytes with extensive irregular foamy microvesicular steatosis in case 4. A syncytial cell is noted in this field (arrow) (hematoxylin and eosin staining 40 magnification). C, Portal tract with prominent cholangiolar proliferation (arrows) and interface hepatocytes with microvesicular steatosis (hematoxylin and eosin staining 40 magnification). D, Masson trichrome stain showing portal fibrosis with thin septal formation (arrow head), mild pericellular fibrosis (arrow), and mild macro- and microvesicular steatosis (hematoxylin and eosin staining 40 magnification). CV, central vein.
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