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Histopathological findings in livers of patients with urea cycle disorders
Molecular Genetics and Metabolism 108 (2013) 161–165
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Histopathological findings in livers of patients with urea cycle disorders Joy Yaplito-Lee a,⁎, Chung-Wo Chow b, c, Avihu Boneh a, c a b c
Metabolic Genetics, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia Department of Anatomical Pathology, Royal Children's Hospital, Melbourne, Australia Department of Paediatrics, University of Melbourne, Australia
a r t i c l e
i n f o
Article history: Received 30 November 2012 Received in revised form 15 January 2013 Accepted 15 January 2013 Available online 23 January 2013 Keywords: Histopathology Liver Urea cycle disorders
a b s t r a c t Background: Urea cycle disorders (UCD) are caused by genetic defects in enzymes that constitute the hepatic ammonia detoxification pathway. Patients may present with variable clinical manifestations and with hyperammonaemia. Liver abnormalities have been associated with UCD, but only a few reports on the histopathological findings in the liver of UCD patients have been published. Methods: We conducted a retrospective review of liver biopsies, ex-planted livers and livers at post-mortem of patients with UCD. A single pathologist reviewed all specimens. Results: There were 18 liver samples from 13 patients with confirmed UCD: four ex-planted livers from patients with Ornithine Transcarbamylase (OTC) (n= 3) and Carbamoyl Phosphate Synthetase 1 (CPS 1) (n=1) deficiencies, eight post-mortem samples from patients with CPS 1 (n=2), OTC (n=4), Argininosuccinate Synthetase (ASS) (n=1) and Argininosuccinate Lyase (ASL) (n=1) deficiencies, and six liver biopsies, three of which came from one patient with ASL deficiency. The other three liver biopsies were from patients who subsequently received liver transplantation. Histopathological findings in samples from neonates were non-specific. Samples from three late onset OTC deficient and one ASL deficient patients showed thin fibrous septa with portal to portal bridging fibrosis and focal marked enlargement and pallor of the hepatocytes due to accumulation of glycogen particles, resembling glycogenosis and resulting in a prominent nodular pattern. Serial liver biopsies in four UCD patients with interval between samples ranging from 1 year 2 months to 17 years showed progression in fibrosis in one OTC and one ASL deficient patients. Moderate fatty changes to no progression in liver disease were noted in the two patients (OTC=1 and CPS=1). A variety of non-specific features such as fatty change, mild inflammation, cholestasis and focal necrosis were seen in the other UCD patients. Conclusions: Histopathological changes in livers from neonates with UCD are non-specific. Older patients with UCD seem to show variable hepatic fibrosis compared to those who died early. Some of these patients also show focal and superficial resemblance to a glycogen storage disorder and cirrhosis. However, progression of these changes seems to be slow. To clarify the long term consequence of these changes, more extensive periods of follow up in a larger population series is needed. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.
1. Introduction The urea cycle is the metabolic pathway responsible for ammonia detoxification. Urea cycle disorders (UCD) are caused by genetic defects in enzymes that constitute this metabolic pathway: N-Acetylglutamate Synthase (EC 2.3.1.1), Carbamoyl Phosphate Synthetase 1 (EC 6.3.4.16), Ornithine Transcarbamylase (EC 2.1.3.3), Argininosuccinate Synthetase 1 (EC 6.3.4.5), Argininosuccinate Lyase (EC 4.3.2.1) and Arginase (EC 3.5.3.1). All these enzymes are inherited in an autosomal recessive manner except for Ornithine Transcarbamylase (OTC), which is inherited in an X-linked manner. UCD disorders (except Argininaemia) lead to acute or chronic encephalopathy due to hyperammonaemic toxicity. ⁎ Corresponding author at: Metabolic Genetics, Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Melbourne, ViC, Australia 3052. Fax: +61 3 83416390. E-mail address: [email protected] (J. Yaplito-Lee).
Arginine, which is produced through the urea cycle, becomes an essential amino acid in these disorders (except in Argininaemia). Patients can present early in the neonatal period or late, up to adulthood. The liver is the only organ in which all the enzymes of the urea cycle are expressed. Acute liver failure has been reported in patients with OTC deficiency [1,2] suggesting a putative toxic effect of ammonia on the hepatocytes. However, there are only few reports describing the histopathological findings and long term changes in the liver of patients with UCD. The reported findings vary from normal histology and microvesicular steatosis, to portal/periportal fibrosis, early cirrhosis, glycogen accumulation and ultrastructural changes with no specific findings for a particular UCD disorder [3–14]. The purpose of this study was to review the hepatic histopathological findings in our cohort of patients with confirmed urea cycle disorders including ex-plants of patients who underwent transplantation, and to compare our results with previous reports.
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2. Methods A review of medical records of all patients with a confirmed UCD was done and patients who have had liver biopsies or liver transplantation were identified. A retrospective review of all biopsies, explants and livers at autopsy including histology and electron microscopy, was done by a single pathologist. The study was approved by the Institutional Ethics Committee (HREC 3215A). 3. Results A total of 13 patients (8 male; 5 female) with UCD confirmed by biochemical and/or molecular testing were identified and included in the review. Confirmation of diagnosis was based on biochemical testing (n= 6) and biochemical and molecular testing (n= 7). There were seven patients (two female) with Ornithine Transcarbamylase deficiency (OTC), three with Carbamoyl Phosphate Synthetase 1 (CPS 1) deficiency, one with Argininosuccinate Synthetase (ASS) deficiency and two with Argininosuccinate Lyase (ASL) deficiency. The age of onset of symptoms varied from one day of life to five years and the age range of UCD diagnosis was one day to five years and six months. Only five of the 13 patients were alive at the time of this review. The age range of death was two days to 11 months. The range of peak ammonia level at diagnosis was 168 umol/L to 2334 umol/L. Liver transplantation was performed when maximal conservative treatment failed to maintain metabolic stability. Liver biopsies were done for diagnostic purposes (enzymology) and for monitoring liver changes when liver transaminases in blood or liver function tests suggested hepatocellular damage. There were 18 liver samples available: 8 post-mortem samples (CPS 1 deficiency: 2, OTC deficiency: 4, ASS deficiency: 1 and ASL deficiency:1), four explanted livers (OTC deficiency:3 and CPS1 deficeincy:1) and 6 liver biopsies from two OTC, one ASL and one CPS 1 deficient patients. Three patients (OTC deficiency-2: and CPS 1 deficiency-1) had two liver samples (interval between samples: one year and two months to seven years and three months) and one ASL patient had three biopsies (interval between samples: three years and 17 years). OTC deficiency: Post-mortem liver findings in four male patients with early onset (one day to one month and seven days) showed normal liver architecture or non-specific findings such as mild focal inflammation, vacuolated liver cells, cholestasis, gross fatty change and necrosis. One female patient presented at one year of age with recurrent vomiting and protein aversion and was diagnosed at 18 months of age. Blood alkaline phosphatase (ALP) levels ranged from 255 IU/L to 460 IU/L (RR: 100–300 IU/L), alanine transaminase (ALT) ranged from 43 IU to 1000 IU/L (RR: b55 IU/L) but blood gamma glutamyl transferase (GGT) was normal. Total protein (TP) and albumin (ALB) levels were normal. Liver biopsy at 4 years and 3 months showed prominent nodularity with minimal fibrosis. The central veins were well preserved (Fig. 1). The nodules were composed of enlarged hepatocytes with pale cytoplasm and enhanced cell borders. A moderate number of halo nuclei were seen (Fig. 2). These nodules alternated with hepatocytes that showed no obvious histological abnormality. Electron microscopy of the pale enlarged hepatocytes showed uniform increase of dense masses of glycogen particles with prominent displacement of the organelles towards the cell membranes and nuclei (Fig. 3). The features showed considerable resemblance to the histology and ultrastructure of the liver in glycogen storage disease (GSD). The mitochondria and peroxisomes were normal in appearance. She underwent liver transplantation at five years and five months and the explanted liver showed more extensive fibrosis at this stage but no cirrhosis. Central veins were well preserved and conspicuous regenerative changes were not seen in hepatocytes. A liver biopsy at four years and two months from a male patient who was diagnosed at one year of age showed mild portal triaditis with minimal fibrosis. Blood ALP levels ranged from 203 IU/L to 380 IU/L (RR: 100–300 IU/L), ALT ranged from 160 IU to 717 IU/L (RR: b 55 IU/L) but GGT levels were normal. TP and ALB levels were normal. He underwent liver transplantation at five
Fig. 1. OTC deficiency: Liver biopsy of a patient at 4 years 3 months showing prominent nodularity, minimal fibrosis and well preserved central veins as shown by arrows. (Masson Trichrome; Medium Power).
years and 10 months. The explanted liver showed minimal fibrosis. Again, prominent nodules of enlarged pale hepatocytes with an increase in cytoplasmic glycogen were present. Another female patient presented at three years of age and was diagnosed at 5 years 7 months. Blood ALP level ranged from 206 IU/L to 469 IU/L (RR: 100–300 IU/L), ALT from 35 IU to 358 IU/L (RR: b 55 IU/L) but GGT levels were normal. TP and ALB levels were normal. She had liver transplantation at 8 years and 3 months. The explanted liver showed thin fibrous septa with focal portal-portal bridging (Fig. 4). Prominent nodules of enlarged pale hepatocytes with increase in glycogen were again present. Carbamoyl Phosphate Synthetase 1 (CPS) deficiency: Two patients had post-mortem biopsies collected at five days and nine months of age, respectively. There was non-specific fatty change in the livers of both patients. Another patient had liver biopsy at seven years showing mild fatty change. Blood ALT levels ranged from normal to 466 IU/L (RR: b55 IU/L) but ALP and GGT blood levels were normal. TP and ALB levels were normal. He had liver transplantation at 14 years and seven months. Histology of the explanted liver showed
Fig. 2. OTC deficiency: Liver of a patient at 4 years 3 months showing enlarged hepatocytes, pale cytoplasm, enhanced cell borders and halo nuclei resembling GSD. (Haematoxylin and Eosin; High Power).
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Fig. 3. OTC deficiency: Liver of a patient at 4 years and 3 months. Electron microscopy showing increase of dense masses of glycogen particles (left) with prominent displacement of the organelles towards the cell membranes and nuclei (right).
only moderate fatty changes with no conspicuous ultrastructural abnormalities. Argininosuccinate Synthetase (ASS) deficiency: A post-mortem liver biopsy from a neonate aged 10 days showed non-specific findings of slight widening of portal tracts, cholestasis and fine vacuolation of liver cells. Argininosuccinate Lyase (ASL) deficiency: The liver of one patient who died at age 10 days only showed moderate fatty changes. Another patient presented at five days of age and was diagnosed at 10 days. Blood ALT levels ranged from 169 IU/L to 332 IU/L (RR: b 55 IU/L) but ALP was normal. TP and ALB levels were normal. The first liver biopsy at 3 years of age showed only focal round cell infiltration. At 6 years of age there were focal fine fibrous septa joining adjacent portal tracts with preserved central veins. Hepatocytes were large and pale with enhanced cell borders (Fig. 5). Electron microscopy showed changes similar to those in the patient with OTC deficiency illustrated in Fig. 3. In the repeat biopsy at 23 years of age, the features were similar with little progression (Fig. 6).
4. Discussion Liver dysfunction and histopathological liver changes in patients with UCD have been reported since the late 1970s [6]. A range of findings from normal liver histology, non-specific findings i.e. inflammation, cholestasis and hepatocyte swelling, to steatosis (microvesicular and macrovesicular), portal/centrilobular fibrosis, progressive liver fibrosis, cirrhosis, abnormal glycogen accumulation and ultrastructural changes [3–5,7–14] have been reported (Table 1). There seemed to be no histological finding specific to a particular UCD disorder or established correlation of the histological findings with age of reported patients. Generally, the prevailing view in the late 1990s was that extensive hepatocellular damage, liver dysfunction and liver failure were not associated with the proximal UCD CPS 1 and OTC deficiencies [15] but, liver failure and fibrosis have been reported in patients with these deficiencies [1,2,14]. In our cohort, patients presenting in infancy had normal architecture or only showed relatively non-specific findings such as focal inflammation, vacuolated liver cells, cholestasis, fatty change and necrosis in post-mortem liver samples. These changes are probably related to the general clinical state of the patients rather than to the specific disease. Abnormal glycogen accumulation has been reported in CPS 1, OTC, ASS and ASL, deficiencies [3,8]. Our three late onset OTC deficient
N
N
Fig. 4. OTC deficiency: Liver of a patient at 8 years and 3 months. Ex-planted liver showing prominent nodules of pale enlarged hepatocytes (N) with focal thin fibrous septa. (Haematoxylin and Eosin, Medium Power).
Fig. 5. Argininosuccinate Lyase (ASL) Deficiency: Liver of a patient at 6 years of age showing focal fibrosis joining adjacent portal tracts. (Haematoxylin and Eosin, Low Power).
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Fig. 6. Repeat liver biopsy of the same ASL deficient patient at 23 years. No significant increase in fibrous tissue. (Masson Trichrome, Low Power).
patients had enlarged pale hepatocytes due to accumulation of glycogen in liver biopsies done between 4 and 8 years of age. We also observed distended and pale hepatocytes and variable focal glycogen accumulation in one ASL deficient patient. Displacement of the cytoplasmic organelles to the periphery by glycogen particles at electron microscopy resembling a glycogen storage disease was demonstrated in one of our OTC patient. Miles et al. measured the glucose 6 phosphatase, debrancher and phosphorylase enzyme activities in liver samples of their patients with UCD showing glycogen accumulation, and found normal to slightly abnormal enzyme activities but not in the range found in glycogen storage disease patients [8]. It is very likely that glycogen accumulation in the livers of these patients is a result of the carbohydrate rich diet of patients with UCD [9]. Indeed, all our patients were on a strict dietary management at the time of apparent glycogen excess. In addition, the amino acid composition of the diets of patients with UCD could be a possible mechanism since certain amino acids such as leucine are important stimulants of insulin secretion and glycogen synthesis [8]. Variable liver fibrosis, predominantly portal, has been reported in CPS 1, OTC, ASS, and ASL deficient patients [3,9,13,14]. In our cohort of patients with late onset OTC deficiency, fibrosis was mainly in the
form of thin fibrous septa with focal portal to portal bridging; central veins were well preserved. Although there was prominent focal nodularity with a superficial resemblance to cirrhosis, this was due to focal marked enlargement of the hepatocytes from glycogen accumulation as shown in Figs. 1, 2, 4. Conspicuous regenerative changes were not seen in the nodules and cirrhosis was not established. Cirrhosis has been reported in ASS and ASL deficiencies [5,7]. Liver findings of our 23 year old ASL deficient patient showed no cirrhosis but only fibrosis that did not progress significantly in a 17 year follow-up. The underlying mechanism of liver fibrosis in UCD is not well understood. Hyperammonaemia could be a contributing factor but additional factors are suggested. The long term toxic effect of elevated citrulline seen in ASS and ASL deficiencies could be a contributing factor [9,12]. Liver fibrosis may also be a result of the reparative process following recurrent liver cell necrosis [14]. Importantly, hepatocellular carcinomas have been identified in adults with OTC and ASL deficiencies in spite of the absence of cirrhotic changes [15]. These observations raise the possibility of UCD as an independent risk factor for primary liver cancer due to the chronic liver damage by accumulating toxic metabolites in these disorders [15]. Given the small number of patients in most series, it is difficult to ascertain a correlation between age and severity of histopathological findings in patients with UCD. Badizadegan et al. have not been able to establish a relationship between age and degree of fibrosis in their cohort of 10 patients (age range: 4 days–14 years)[3]. Nevertheless, progressive liver findings have been observed in patients with UCD. Our CPS 1 deficient patient showed only moderate progression from mild fatty change 7 years and 3 months after the initial liver biopsy. LaBrecque et al. reported that the histology of the liver of an OTC deficient patient was normal at age 21 months but, repeat biopsy 6 months later showed microvesicular fat, portal fibrosis and mild piecemeal necrosis [6]. In our series, one OTC deficient patient showed more extensive fibrosis at age 5 years and 5 months, 14 months after the first liver sample, but another did not show significant changes 20 months after the first biopsy. The only OTC patient who had marked portal to portal bridging fibrosis in the series of Badizadegan et al. was the oldest, at 14 years [3]. Mori et al. reported mild periportal fibrosis and abnormal glycogen accumulation at 1 year of age on a liver biopsy of a ASL deficient patient [9]. A repeat liver biopsy two years later showed progressive liver fibrosis in periportal and central areas [9]. In our series,
Table 1 Hepatic Histological Findings in Urea Cycle Disorders. Urea Cycle Disorder [reference]
CPS 1 deficiency [3,6,8,14]
OTC deficiency [2–4,6,8,10,11]
ASS deficiency [5,8,12]
ASL deficiency [7–9,13]
Published patients
Our patients
LM*
EM**
LM
EM
Normal Focal/diffuse distended hepatocytes microvesicular steatosis Diffuse fibrosis Normal/non-specific changes
Increase in glycogen Normal to variable changes in mitochondria
Non-specific changes Moderate fatty changes
No changes
Increase in glycogen
Normal/non-specific changes
Macro and micro-vesicular steatosis Focal/diffuse distended hepatocytes Variable fibrosis and necrosis Variable fibrosis Early cirrhosis Focal necrosis, cholestasis Diffuse swelling of hepatocytes Diffuse swelling of hepatocytes
Normal to variable changes in mitochondria and peroxisomes
Nodularity with enlarged pale hepatocytes variable fibrosis
Increase in glycogen Normal mitochondria and peroxisomes
Increase in glycogen Normal to variable changes in mitochondria
Non-specific changes Cholestasis
Increase in glycogen
Moderate fatty changes
Cholestasis Variable fibrosis Micronodular cirrhosis
Normal mitochondria
Enlarged pale hepatocytes Fibrosis
*LM = light microscopy; **EM = electron microscopy.
Increase in glycogen
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one ASL deficient patient did not show significant changes 3 years and 17 years after the first liver sample, respectively. As far as we know, this is the longest follow-up of histological findings in a patient with UCD. Finally, LaBrecque et al. reported normal liver histology and microvesicular steatosis and diffuse glycogen deposits in a 48 year old and 42 year old OTC deficient patients, respectively [6]. Taken together, it seems that the progression in liver pathological findings is overall slow in patients with UCD. However, it will require more reports of liver histology from patients with UCD to establish whether a correlation exists between age and histopathological findings. Conclusion In our cohort, older patients with UCD showed variable liver fibrosis compared to those who died early. Some of these patients also showed interesting histological liver changes with focal and superficial resemblance to a glycogen storage disorder (GSD) and cirrhosis. However, progression of these changes seemed to be slow. The long term consequence of these changes is unclear. Longer term follow up in a larger population series is needed in order to clarify this. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments This study was supported by the Victorian Government's Operational Infrastructure Support Program. An earlier version of this work was presented at the SSIEM Annual Symposium, Geneva, 2011 (J. Inherit. Metab. Dis, 34 (supp 3):S89, Abstract P-040, 2011).
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References [1] A. Mustafa, J.T. Clarke, Ornithine transcarbamoylase deficiency presenting with acute liver failure, J. Inherit. Metab. Dis. 29 (2006) 586. [2] U. Teufel, J. Weitz, C. Flechtenmacher, V. Prietsch, J. Schmidt, G.F. Hoffmann, S. Kolker, G. Engelmann, High urgency liver transplantation in ornithine transcarbamylase deficiency presenting with acute liver failure, Pediatr. Transplant. 15 (2011) E110–E115. [3] K. Badizadegan, A.R. Perez-Atayde, Focal glycogenosis of the liver in disorders of ureagenesis: its occurrence and diagnostic significance, Hepatology 26 (1997) 365–373. [4] S. Capistrano-Estrada, D.L. Marsden, W.L. Nyhan, R.O. Newbury, H.F. Krous, M. Tuchman, Histopathological findings in a male with late-onset ornithine transcarbamylase deficiency, Pediatr. Pathol. 14 (1994) 235–243. [5] S. Gucer, E. Asan, P. Atilla, A. Tokatli, M. Caglar, Early cirrhosis in a patient with type I citrullinaemia (CTLN1), J. Inherit. Metab. Dis. 27 (2004) 541–542. [6] D.R. LaBrecque, P.S. Latham, C.A. Riely, Y.E. Hsia, G. Klatskin, Heritable urea cycle enzyme deficiency-liver disease in 16 patients, J. Pediatr. 94 (1979) 580–587. [7] M. Marble, R.R. McGoey, E. Mannick, B. Keats, S.S. Ng, S. Deputy, H. Gereighty, E. Schmidt-Sommerfeld, Living related liver transplant in a patient with argininosuccinic aciduria and cirrhosis: metabolic follow-up, J. Pediatr. Gastroenterol. Nutr. 46 (2008) 453–456. [8] L. Miles, J.E. Heubi, K.E. Bove, Hepatocyte glycogen accumulation in patients undergoing dietary management of urea cycle defects mimics storage disease, J. Pediatr. Gastroenterol. Nutr. 40 (2005) 471–476. [9] T. Mori, K. Nagai, M. Mori, M. Nagao, M. Imamura, M. Iijima, K. Kobayashi, Progressive liver fibrosis in late-onset argininosuccinate lyase deficiency, Pediatr. Dev. Pathol. 5 (2002) 597–601. [10] J.M. Shapiro, F. Schaffner, H.H. Tallan, G.E. Gaull, Mitochondrial abnormalities of liver in primary ornithine transcarbamylase deficiency, Pediatr. Res. 14 (1980) 735–739. [11] H.H. Tallan, F. Schaffner, S.L. Taffet, K. Schneidman, G.E. Gaull, Ornithine carbamoyltransferase deficiency in an adult male patient: significance of hepatic ultrastructure in clinical diagnosis, Pediatrics 71 (1983) 224–232. [12] S.A. Zamora, A. Pinto, R.B. Scott, H.G. Parsons, Mitochondrial abnormalities of liver in two children with citrullinaemia, J. Inherit. Metab. Dis. 20 (1997) 509–516. [13] A. Zimmermann, C. Bachmann, R. Baumgartner, Severe liver fibrosis in argininosuccinic aciduria, Arch. Pathol. Lab. Med. 110 (1986) 136–140. [14] A. Zimmermann, C. Moll, C. Bachmann, Liver fibrosis in carbamoylphosphate synthetase deficiency, Pediatr. Pathol. 7 (1987) 191–200. [15] J.M. Wilson, O.A. Shchelochkov, R.C. Gallagher, M.L. Batshaw, Hepatocellular carcinoma in a research subject with ornithine transcarbamylase deficiency, Mol. Genet. Metab. 105 (2012) 263–265.
Contents lists available at SciVerse ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Histopathological findings in livers of patients with urea cycle disorders Joy Yaplito-Lee a,⁎, Chung-Wo Chow b, c, Avihu Boneh a, c a b c
Metabolic Genetics, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia Department of Anatomical Pathology, Royal Children's Hospital, Melbourne, Australia Department of Paediatrics, University of Melbourne, Australia
a r t i c l e
i n f o
Article history: Received 30 November 2012 Received in revised form 15 January 2013 Accepted 15 January 2013 Available online 23 January 2013 Keywords: Histopathology Liver Urea cycle disorders
a b s t r a c t Background: Urea cycle disorders (UCD) are caused by genetic defects in enzymes that constitute the hepatic ammonia detoxification pathway. Patients may present with variable clinical manifestations and with hyperammonaemia. Liver abnormalities have been associated with UCD, but only a few reports on the histopathological findings in the liver of UCD patients have been published. Methods: We conducted a retrospective review of liver biopsies, ex-planted livers and livers at post-mortem of patients with UCD. A single pathologist reviewed all specimens. Results: There were 18 liver samples from 13 patients with confirmed UCD: four ex-planted livers from patients with Ornithine Transcarbamylase (OTC) (n= 3) and Carbamoyl Phosphate Synthetase 1 (CPS 1) (n=1) deficiencies, eight post-mortem samples from patients with CPS 1 (n=2), OTC (n=4), Argininosuccinate Synthetase (ASS) (n=1) and Argininosuccinate Lyase (ASL) (n=1) deficiencies, and six liver biopsies, three of which came from one patient with ASL deficiency. The other three liver biopsies were from patients who subsequently received liver transplantation. Histopathological findings in samples from neonates were non-specific. Samples from three late onset OTC deficient and one ASL deficient patients showed thin fibrous septa with portal to portal bridging fibrosis and focal marked enlargement and pallor of the hepatocytes due to accumulation of glycogen particles, resembling glycogenosis and resulting in a prominent nodular pattern. Serial liver biopsies in four UCD patients with interval between samples ranging from 1 year 2 months to 17 years showed progression in fibrosis in one OTC and one ASL deficient patients. Moderate fatty changes to no progression in liver disease were noted in the two patients (OTC=1 and CPS=1). A variety of non-specific features such as fatty change, mild inflammation, cholestasis and focal necrosis were seen in the other UCD patients. Conclusions: Histopathological changes in livers from neonates with UCD are non-specific. Older patients with UCD seem to show variable hepatic fibrosis compared to those who died early. Some of these patients also show focal and superficial resemblance to a glycogen storage disorder and cirrhosis. However, progression of these changes seems to be slow. To clarify the long term consequence of these changes, more extensive periods of follow up in a larger population series is needed. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.
1. Introduction The urea cycle is the metabolic pathway responsible for ammonia detoxification. Urea cycle disorders (UCD) are caused by genetic defects in enzymes that constitute this metabolic pathway: N-Acetylglutamate Synthase (EC 2.3.1.1), Carbamoyl Phosphate Synthetase 1 (EC 6.3.4.16), Ornithine Transcarbamylase (EC 2.1.3.3), Argininosuccinate Synthetase 1 (EC 6.3.4.5), Argininosuccinate Lyase (EC 4.3.2.1) and Arginase (EC 3.5.3.1). All these enzymes are inherited in an autosomal recessive manner except for Ornithine Transcarbamylase (OTC), which is inherited in an X-linked manner. UCD disorders (except Argininaemia) lead to acute or chronic encephalopathy due to hyperammonaemic toxicity. ⁎ Corresponding author at: Metabolic Genetics, Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Melbourne, ViC, Australia 3052. Fax: +61 3 83416390. E-mail address: [email protected] (J. Yaplito-Lee).
Arginine, which is produced through the urea cycle, becomes an essential amino acid in these disorders (except in Argininaemia). Patients can present early in the neonatal period or late, up to adulthood. The liver is the only organ in which all the enzymes of the urea cycle are expressed. Acute liver failure has been reported in patients with OTC deficiency [1,2] suggesting a putative toxic effect of ammonia on the hepatocytes. However, there are only few reports describing the histopathological findings and long term changes in the liver of patients with UCD. The reported findings vary from normal histology and microvesicular steatosis, to portal/periportal fibrosis, early cirrhosis, glycogen accumulation and ultrastructural changes with no specific findings for a particular UCD disorder [3–14]. The purpose of this study was to review the hepatic histopathological findings in our cohort of patients with confirmed urea cycle disorders including ex-plants of patients who underwent transplantation, and to compare our results with previous reports.
1096-7192/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymgme.2013.01.006
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2. Methods A review of medical records of all patients with a confirmed UCD was done and patients who have had liver biopsies or liver transplantation were identified. A retrospective review of all biopsies, explants and livers at autopsy including histology and electron microscopy, was done by a single pathologist. The study was approved by the Institutional Ethics Committee (HREC 3215A). 3. Results A total of 13 patients (8 male; 5 female) with UCD confirmed by biochemical and/or molecular testing were identified and included in the review. Confirmation of diagnosis was based on biochemical testing (n= 6) and biochemical and molecular testing (n= 7). There were seven patients (two female) with Ornithine Transcarbamylase deficiency (OTC), three with Carbamoyl Phosphate Synthetase 1 (CPS 1) deficiency, one with Argininosuccinate Synthetase (ASS) deficiency and two with Argininosuccinate Lyase (ASL) deficiency. The age of onset of symptoms varied from one day of life to five years and the age range of UCD diagnosis was one day to five years and six months. Only five of the 13 patients were alive at the time of this review. The age range of death was two days to 11 months. The range of peak ammonia level at diagnosis was 168 umol/L to 2334 umol/L. Liver transplantation was performed when maximal conservative treatment failed to maintain metabolic stability. Liver biopsies were done for diagnostic purposes (enzymology) and for monitoring liver changes when liver transaminases in blood or liver function tests suggested hepatocellular damage. There were 18 liver samples available: 8 post-mortem samples (CPS 1 deficiency: 2, OTC deficiency: 4, ASS deficiency: 1 and ASL deficiency:1), four explanted livers (OTC deficiency:3 and CPS1 deficeincy:1) and 6 liver biopsies from two OTC, one ASL and one CPS 1 deficient patients. Three patients (OTC deficiency-2: and CPS 1 deficiency-1) had two liver samples (interval between samples: one year and two months to seven years and three months) and one ASL patient had three biopsies (interval between samples: three years and 17 years). OTC deficiency: Post-mortem liver findings in four male patients with early onset (one day to one month and seven days) showed normal liver architecture or non-specific findings such as mild focal inflammation, vacuolated liver cells, cholestasis, gross fatty change and necrosis. One female patient presented at one year of age with recurrent vomiting and protein aversion and was diagnosed at 18 months of age. Blood alkaline phosphatase (ALP) levels ranged from 255 IU/L to 460 IU/L (RR: 100–300 IU/L), alanine transaminase (ALT) ranged from 43 IU to 1000 IU/L (RR: b55 IU/L) but blood gamma glutamyl transferase (GGT) was normal. Total protein (TP) and albumin (ALB) levels were normal. Liver biopsy at 4 years and 3 months showed prominent nodularity with minimal fibrosis. The central veins were well preserved (Fig. 1). The nodules were composed of enlarged hepatocytes with pale cytoplasm and enhanced cell borders. A moderate number of halo nuclei were seen (Fig. 2). These nodules alternated with hepatocytes that showed no obvious histological abnormality. Electron microscopy of the pale enlarged hepatocytes showed uniform increase of dense masses of glycogen particles with prominent displacement of the organelles towards the cell membranes and nuclei (Fig. 3). The features showed considerable resemblance to the histology and ultrastructure of the liver in glycogen storage disease (GSD). The mitochondria and peroxisomes were normal in appearance. She underwent liver transplantation at five years and five months and the explanted liver showed more extensive fibrosis at this stage but no cirrhosis. Central veins were well preserved and conspicuous regenerative changes were not seen in hepatocytes. A liver biopsy at four years and two months from a male patient who was diagnosed at one year of age showed mild portal triaditis with minimal fibrosis. Blood ALP levels ranged from 203 IU/L to 380 IU/L (RR: 100–300 IU/L), ALT ranged from 160 IU to 717 IU/L (RR: b 55 IU/L) but GGT levels were normal. TP and ALB levels were normal. He underwent liver transplantation at five
Fig. 1. OTC deficiency: Liver biopsy of a patient at 4 years 3 months showing prominent nodularity, minimal fibrosis and well preserved central veins as shown by arrows. (Masson Trichrome; Medium Power).
years and 10 months. The explanted liver showed minimal fibrosis. Again, prominent nodules of enlarged pale hepatocytes with an increase in cytoplasmic glycogen were present. Another female patient presented at three years of age and was diagnosed at 5 years 7 months. Blood ALP level ranged from 206 IU/L to 469 IU/L (RR: 100–300 IU/L), ALT from 35 IU to 358 IU/L (RR: b 55 IU/L) but GGT levels were normal. TP and ALB levels were normal. She had liver transplantation at 8 years and 3 months. The explanted liver showed thin fibrous septa with focal portal-portal bridging (Fig. 4). Prominent nodules of enlarged pale hepatocytes with increase in glycogen were again present. Carbamoyl Phosphate Synthetase 1 (CPS) deficiency: Two patients had post-mortem biopsies collected at five days and nine months of age, respectively. There was non-specific fatty change in the livers of both patients. Another patient had liver biopsy at seven years showing mild fatty change. Blood ALT levels ranged from normal to 466 IU/L (RR: b55 IU/L) but ALP and GGT blood levels were normal. TP and ALB levels were normal. He had liver transplantation at 14 years and seven months. Histology of the explanted liver showed
Fig. 2. OTC deficiency: Liver of a patient at 4 years 3 months showing enlarged hepatocytes, pale cytoplasm, enhanced cell borders and halo nuclei resembling GSD. (Haematoxylin and Eosin; High Power).
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Fig. 3. OTC deficiency: Liver of a patient at 4 years and 3 months. Electron microscopy showing increase of dense masses of glycogen particles (left) with prominent displacement of the organelles towards the cell membranes and nuclei (right).
only moderate fatty changes with no conspicuous ultrastructural abnormalities. Argininosuccinate Synthetase (ASS) deficiency: A post-mortem liver biopsy from a neonate aged 10 days showed non-specific findings of slight widening of portal tracts, cholestasis and fine vacuolation of liver cells. Argininosuccinate Lyase (ASL) deficiency: The liver of one patient who died at age 10 days only showed moderate fatty changes. Another patient presented at five days of age and was diagnosed at 10 days. Blood ALT levels ranged from 169 IU/L to 332 IU/L (RR: b 55 IU/L) but ALP was normal. TP and ALB levels were normal. The first liver biopsy at 3 years of age showed only focal round cell infiltration. At 6 years of age there were focal fine fibrous septa joining adjacent portal tracts with preserved central veins. Hepatocytes were large and pale with enhanced cell borders (Fig. 5). Electron microscopy showed changes similar to those in the patient with OTC deficiency illustrated in Fig. 3. In the repeat biopsy at 23 years of age, the features were similar with little progression (Fig. 6).
4. Discussion Liver dysfunction and histopathological liver changes in patients with UCD have been reported since the late 1970s [6]. A range of findings from normal liver histology, non-specific findings i.e. inflammation, cholestasis and hepatocyte swelling, to steatosis (microvesicular and macrovesicular), portal/centrilobular fibrosis, progressive liver fibrosis, cirrhosis, abnormal glycogen accumulation and ultrastructural changes [3–5,7–14] have been reported (Table 1). There seemed to be no histological finding specific to a particular UCD disorder or established correlation of the histological findings with age of reported patients. Generally, the prevailing view in the late 1990s was that extensive hepatocellular damage, liver dysfunction and liver failure were not associated with the proximal UCD CPS 1 and OTC deficiencies [15] but, liver failure and fibrosis have been reported in patients with these deficiencies [1,2,14]. In our cohort, patients presenting in infancy had normal architecture or only showed relatively non-specific findings such as focal inflammation, vacuolated liver cells, cholestasis, fatty change and necrosis in post-mortem liver samples. These changes are probably related to the general clinical state of the patients rather than to the specific disease. Abnormal glycogen accumulation has been reported in CPS 1, OTC, ASS and ASL, deficiencies [3,8]. Our three late onset OTC deficient
N
N
Fig. 4. OTC deficiency: Liver of a patient at 8 years and 3 months. Ex-planted liver showing prominent nodules of pale enlarged hepatocytes (N) with focal thin fibrous septa. (Haematoxylin and Eosin, Medium Power).
Fig. 5. Argininosuccinate Lyase (ASL) Deficiency: Liver of a patient at 6 years of age showing focal fibrosis joining adjacent portal tracts. (Haematoxylin and Eosin, Low Power).
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Fig. 6. Repeat liver biopsy of the same ASL deficient patient at 23 years. No significant increase in fibrous tissue. (Masson Trichrome, Low Power).
patients had enlarged pale hepatocytes due to accumulation of glycogen in liver biopsies done between 4 and 8 years of age. We also observed distended and pale hepatocytes and variable focal glycogen accumulation in one ASL deficient patient. Displacement of the cytoplasmic organelles to the periphery by glycogen particles at electron microscopy resembling a glycogen storage disease was demonstrated in one of our OTC patient. Miles et al. measured the glucose 6 phosphatase, debrancher and phosphorylase enzyme activities in liver samples of their patients with UCD showing glycogen accumulation, and found normal to slightly abnormal enzyme activities but not in the range found in glycogen storage disease patients [8]. It is very likely that glycogen accumulation in the livers of these patients is a result of the carbohydrate rich diet of patients with UCD [9]. Indeed, all our patients were on a strict dietary management at the time of apparent glycogen excess. In addition, the amino acid composition of the diets of patients with UCD could be a possible mechanism since certain amino acids such as leucine are important stimulants of insulin secretion and glycogen synthesis [8]. Variable liver fibrosis, predominantly portal, has been reported in CPS 1, OTC, ASS, and ASL deficient patients [3,9,13,14]. In our cohort of patients with late onset OTC deficiency, fibrosis was mainly in the
form of thin fibrous septa with focal portal to portal bridging; central veins were well preserved. Although there was prominent focal nodularity with a superficial resemblance to cirrhosis, this was due to focal marked enlargement of the hepatocytes from glycogen accumulation as shown in Figs. 1, 2, 4. Conspicuous regenerative changes were not seen in the nodules and cirrhosis was not established. Cirrhosis has been reported in ASS and ASL deficiencies [5,7]. Liver findings of our 23 year old ASL deficient patient showed no cirrhosis but only fibrosis that did not progress significantly in a 17 year follow-up. The underlying mechanism of liver fibrosis in UCD is not well understood. Hyperammonaemia could be a contributing factor but additional factors are suggested. The long term toxic effect of elevated citrulline seen in ASS and ASL deficiencies could be a contributing factor [9,12]. Liver fibrosis may also be a result of the reparative process following recurrent liver cell necrosis [14]. Importantly, hepatocellular carcinomas have been identified in adults with OTC and ASL deficiencies in spite of the absence of cirrhotic changes [15]. These observations raise the possibility of UCD as an independent risk factor for primary liver cancer due to the chronic liver damage by accumulating toxic metabolites in these disorders [15]. Given the small number of patients in most series, it is difficult to ascertain a correlation between age and severity of histopathological findings in patients with UCD. Badizadegan et al. have not been able to establish a relationship between age and degree of fibrosis in their cohort of 10 patients (age range: 4 days–14 years)[3]. Nevertheless, progressive liver findings have been observed in patients with UCD. Our CPS 1 deficient patient showed only moderate progression from mild fatty change 7 years and 3 months after the initial liver biopsy. LaBrecque et al. reported that the histology of the liver of an OTC deficient patient was normal at age 21 months but, repeat biopsy 6 months later showed microvesicular fat, portal fibrosis and mild piecemeal necrosis [6]. In our series, one OTC deficient patient showed more extensive fibrosis at age 5 years and 5 months, 14 months after the first liver sample, but another did not show significant changes 20 months after the first biopsy. The only OTC patient who had marked portal to portal bridging fibrosis in the series of Badizadegan et al. was the oldest, at 14 years [3]. Mori et al. reported mild periportal fibrosis and abnormal glycogen accumulation at 1 year of age on a liver biopsy of a ASL deficient patient [9]. A repeat liver biopsy two years later showed progressive liver fibrosis in periportal and central areas [9]. In our series,
Table 1 Hepatic Histological Findings in Urea Cycle Disorders. Urea Cycle Disorder [reference]
CPS 1 deficiency [3,6,8,14]
OTC deficiency [2–4,6,8,10,11]
ASS deficiency [5,8,12]
ASL deficiency [7–9,13]
Published patients
Our patients
LM*
EM**
LM
EM
Normal Focal/diffuse distended hepatocytes microvesicular steatosis Diffuse fibrosis Normal/non-specific changes
Increase in glycogen Normal to variable changes in mitochondria
Non-specific changes Moderate fatty changes
No changes
Increase in glycogen
Normal/non-specific changes
Macro and micro-vesicular steatosis Focal/diffuse distended hepatocytes Variable fibrosis and necrosis Variable fibrosis Early cirrhosis Focal necrosis, cholestasis Diffuse swelling of hepatocytes Diffuse swelling of hepatocytes
Normal to variable changes in mitochondria and peroxisomes
Nodularity with enlarged pale hepatocytes variable fibrosis
Increase in glycogen Normal mitochondria and peroxisomes
Increase in glycogen Normal to variable changes in mitochondria
Non-specific changes Cholestasis
Increase in glycogen
Moderate fatty changes
Cholestasis Variable fibrosis Micronodular cirrhosis
Normal mitochondria
Enlarged pale hepatocytes Fibrosis
*LM = light microscopy; **EM = electron microscopy.
Increase in glycogen
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one ASL deficient patient did not show significant changes 3 years and 17 years after the first liver sample, respectively. As far as we know, this is the longest follow-up of histological findings in a patient with UCD. Finally, LaBrecque et al. reported normal liver histology and microvesicular steatosis and diffuse glycogen deposits in a 48 year old and 42 year old OTC deficient patients, respectively [6]. Taken together, it seems that the progression in liver pathological findings is overall slow in patients with UCD. However, it will require more reports of liver histology from patients with UCD to establish whether a correlation exists between age and histopathological findings. Conclusion In our cohort, older patients with UCD showed variable liver fibrosis compared to those who died early. Some of these patients also showed interesting histological liver changes with focal and superficial resemblance to a glycogen storage disorder (GSD) and cirrhosis. However, progression of these changes seemed to be slow. The long term consequence of these changes is unclear. Longer term follow up in a larger population series is needed in order to clarify this. Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments This study was supported by the Victorian Government's Operational Infrastructure Support Program. An earlier version of this work was presented at the SSIEM Annual Symposium, Geneva, 2011 (J. Inherit. Metab. Dis, 34 (supp 3):S89, Abstract P-040, 2011).
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