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Allograft steatosis is a feature of progressive familial intrahepatic cholestasis, type 1 (F1C1 disease), treated by orthotopic liver transplantation (OLTX)
HEPAFOLOGY Vol. 34, N o . 4, Pt. 2, 2 0 0 1
AASLD ABSTRACTS
209A
141
142
A POPULATION-BASED STUDY OF THE BIOCHEMICAL EXPRESSION OF THE H63D HAEMOCHROMATOSIS MUTATION. Peter A G o c h e e , L a w -
SERUM CERULOPLASMIN AND FERROXIDASE ACTIVITY ARE DECREASED IN HFE C 2 8 2 Y + / + MALE IRON-OVERLOADED PATIENTS.
rie W Powell, Q u e e n s l a n d I n s t i t u t e of M e d i c a l Research, Brisbane Australia; D i g b y J Cullen, D e p a r t m e n t o f Medicine, F r e m a n t l e Hospital, F r e m a n t l e Australia; Desire'e D u Sart, M u r d o c h C h i l d r e n s R e s e a r c h Institute, M e l b o u r n e Australia; Enrico Rossi, P a t h c e n t r e , Q u e e n Elizabeth II Medical Centre, N e d lands Australia; J o h n K O l y n y k , D e p a r t m e n t of Medicine, F r e m a n t l e Hospital, F r e m a n t l e Australia
Fabrice Laine, Svc des Maladies du Foie a n d I N S E R M U-522, U n i v H o s p Pontchaillou, R e n n e s France; M a r t i n e Ropert, Lab of Biochemistry, U n i v H o s p Pontchaillou, R e n n e s France; C a r o l i n e Le Lan, 5vc des Maladies d u Foie a n d I N S E R M U-522, U n i v H o s p Pontchaillou, R e n n e s France; Olivier Loreal, INSERM U-522, U n i v H o s p Pontchaillou, R e n n e s France; Eric Bellissant, Lab of P h a r m a c o l o g y , U n i v H o s p Pontchaillou, R e n n e s France; Michel P o u c h a r d , CPAM, R e n n e s France; A n d r e Le Treut, Lab of Biochemistry, R e n n e s France; Pierre Brissot, Svc des Maladies du Foie a n d I N S E R M U-522, U n i v H o s p Pontchaillou, R e n n e s F r a n c e
Background: The two major mutations defined within the haemochromatosis gene both predispose m tim development of iron overload. While the effects of the first mutation, C282Y, have been well characterised, the effects of the second mutation, H63D, remain unclear. We accessed a well-defined, non-blood bank population in Busselmn, Australia and determined the frequency of the H63D mutation and its influence on total body iron stores. Methods: Blood samples were obtained from 30t 1 unrelated Caucasian subjects enrolled in the Busselton population study and aged 20 to 79 years. HFE genotype was determined for each subject. Serum transferrin saturation and serum ferritin were measured and correlated with the H63D mutation in the absence of the C282Y mutation. Results: Sixty-two subjects (2.1 percent) were homozygous for the H63D mutafion and 711 (23.6 percent) were heterozygous. Serum transferrin saturation was significantly increased in males and females who were H63D homozygotes (35.1_+ 1.9 and 29.6-+ 1.6 percent, respectively) or heterozygotes (31.5-+0.5 and 26.7-+0.5 percent, respectively) compared with Dale and female subjects (28.+-+0.3 and 25.0-+0.3 percent, respectively) wild-type for both the H63D and the C282Y mutation (p<0.01). No significant difference was observed in mean serum ferrifin levels between H63D genotypes within each gender. Elevated transferrin saturation >--45 percent was observed in a greater proportion of male H63D homozygotes (15 percent) and heterozygotes (I2 percent) than male HFE wild-types (5 percent) (p<0.01). Male H63D homozygotes (9 percent) and heterozygotes (3 percent) were more likely to have both elevated transferrin saturation and elevated ferritin >- 300 ng per milliliter than male HFE wild-t3zpes (0.7 percent) (p<0.0I). Conclusions: Though a significant increase in serum transferrin satmafion was observed in iudividuals who possessed the H63D mutation, the increase observed does not reflect significantly increased body iron stores and in the absence of the c2g2Y mutation, the H63D mutation is not clinically significant. Efforts directed at population screening based on haemochromatosis genotype should therefore focus on the major mutation, C282Y.
g /:
Male
gema~
Male
Female
Figure I. Mean sen!m~'atgtcrrinsaturation and serum tN'ritin tbr male ~nd ~m~lleH63D genc~pes. HH =HIREWild-type, HI) = H63D Heterozygous,DD = FI63DHomozygous. * p < 0.01
B A C K G R O U N D . A b o d y of e v i d e n c e s u g g e s t s that c e r u l o p l a s m i n , a p r o t e i n l i n k e d to copper, acts in i r o n m e t a b o l i s m , d u e to its ability to catalyze iron oxidation. T h i s ferroxidase activity p e r m i t s the c o n v e r s i o n of ferrous i o n s into ferric ions, w h i c h appears essential for iron m o v e m e n t s a n d exchanges. AIMS. T o evaluate, in C282Y + / + h e m o c h r o m a t o t i c patients, s e r u m c e r u l o p l a s m i n c o n c e n t r a t i o n a n d its ferroxidase activity, a n d to s t u d y their v a r i a t i o n s as a f u n c t i o n of i r o n status. P A T I E N T S A N D M E T H O D S . T h e p r e s e n t s t u d y investigated the s e r u m levels of c e r u l o p l a s m i n ( d e t e r m i n e d b y i m m u n o p h e l e m e t r y ) a n d its ferroxidase activity ( d e t e r m i n e d b y Erel's m e t h o d w h i c h enables to differentiate the real Cp ferroxidase activity f r o m total s e r u m o x i d a s e activity) in 53 patients w i t h C282Y + / + genetic h e m o c h r o m a t o s i s (38 iron overloaded, 15 i r o n depleted) as c o m p a r e d to age a n d s e x m a t c h e d h e a l t h y volunteers. RESULTS. l) In m e n : S e r u m levels o f c e r u l o p l a s m i n w e r e significantly decreased i n i r o n - o v e r l o a d e d p a t i e n t s ( n = 2 7 ) v e r s u s control g r o u p ( p = 0 . 0 2 ) . F u r t h e r m o r e , s e r u m ferroxidase activity w a s s t r o n g l y a n d significantly lower in this g r o u p of i r o n o v e r l o a d e d patients ( p < 0 . 0 0 1 ) . In contrast, in irondepleted h e m o c h r o m a t o t i c p a t i e n t s ( n = 1 5 ) , w h o w e r e u n d e r m a i n t e n a n c e t h e r a p y b y regular p h l e b o t o m i e s , s e r u m levels of c e r u l o p l a s m i n a n d ferroxidase activity w e r e n o t statistically different f r o m those o b s e r v e d in controls. II) In w o m e n ( n = l l ) : No differences w e r e o b s e r v e d , for b o t h p a r a m e t e r s , bet w e e n iron o v e r l o a d e d patients a n d controls. C O N C L U S I O N S . T h e s e data: i) s h o w for the first t i m e that, in i r o n - o v e r l o a d e d C 2 8 2 Y + / + h e m o c h r o m a t o t i c m a l e patients, s e r u m c e r u l o p l a s m i n a n d its ferroxidase activity are decreased, a n d that b o t h factors are corrected b y the r e m o v a l of iron excess. In contrast, no s u c h differences are o b s e r v e d in w o m e n , w h i c h could be related to the w e l l - k n o w n i n d u c i n g effect of e s t r o g e n s o n Cp synthesis; ii) s u g g e s t that, in h e m o c h r o m a t o t i c m a l e patients, iron is able to m o d u l a t e the e x p r e s s i o n of c e r u l o p l a s m i n ; iii) raise the issue of the p u t a t i v e role of d e c r e a s e d s e r u m ferroxidase activity- in the p h e n o t y p i c e x p r e s s i o n o f H F E - I g e n e t i c h e m o c h r o matosis.
143
144
GENOMIC ANALYSIS OF RAT HEPATOCYIES UNDER CONDITIONS OF CHRONIC IRON LOADING. Edward E Cable, J o h n P Bilello, Harriet C
ALLOGRAFT STEATOSIS IS A FEATURE OF PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS, TYPE 1 ( F I C I DISEASE), TREATED BY ORTHOTOPIC LIVER TRANSPLANTATION (OLTX). G Bhagat, S J Lo-
I s o m , P e n n State College of Medicine, H e r s h e y , PA TrimethylhexanoyI-ferrocene (TMHF) is an effective iron donor to rat hepatocytes in long-term DMSO culture. Total RNA was isolated from untreated rat hepatocytes and hepatocytes treated with 20/xM TMHF for 3 weeks. Genomic analysis was performed on the RNA by lneyte Genomics (Pale Alto, CA, USA) and a total of i0,000 genes were analyzed. Following chronic iron loading of hepatocytes by TMHF, 258 transcripts were significantly increased, whereas 644 transcripts were significantly decreased. The top ten listed in order of magnitude of change are listed in the table below. Changes for several of the transcripts listed below were confirmed by an RNase protection assay and are denoted by (RPA). Those transcripts not denoted by (RPA) indicate that the change was detected in the genomic analysis, but not yet confirmed by RPA. There have not yet been any transcripts that changed in the genomic analysis that failed to yield a change when analyzed by RPA. Transcripts for the iron metabolism genes L-ferrifin (RPA) and metallothionein (RPA) were increased and transferrin (Tf, RPA) gene expression was decreased. No changes in Nramp2 (RPA) and Tf receptor-2 gene expression were observed. Gene array analysis showed that transcripts for the stress response genes heine oxygenase (RPA), heat shock protein (HSP) 27 and glutathione 5-transferase I*wme increased. Conversely, transcripts for the stress response gene HSP85, HSP70, HSP60, CuZn superoxide dismutase (SOD, RPA), glutathione synthetase and glutathione peroxidase were decreased. The divergent effects of the stress response genes following TMHF treatment suggests that chronic iron loading is not simply a prolonged version of an acute stress. Further, several other families of genes were decreased. They include genes that encode for cell cycle, extraceliular matrix, and- junctional complex proteins. Iron-mediated decreases to the junctional complex transcripts caveloin, E-cadherin, protocadherin, ~-aetinin, and junctional adhesion molecule were confirmed using an RPA. The biological significance of the decreases in these transcripts was also confirmed in iron loaded hepatocytes because these ceils were more susceptible to baculovirus-mediated gene transfer, a process whose efficiency is im,ersely proportional to the functional integrity of the junctional complexes. The data produced by Incyte Genomics yielded results that were duplicated by RNase protection, demonstrating the accuracy for this particular genomic analysis. These data also indicate that there are several specific changes that occur in iron loaded hepatocytes that can not simply be attributed to the ability of iron to catalyze oxidative reactions.
Transcripts changed bY chronic iron loading _ _ T o p 10 increased "[e_p10 decreased Rat mRNA for heart cytochrome c oxidase Rat haptoglobin rnRNA subunit Via. Rat GADD!53 mRNA Rat rnRNA for pre-alpha-inhiNtor, heavy chain 3. Rat IGF binding protein4 (dGFBP-1} mRNA Rat pcRC201 rnRNA for pre-pro-complernent £,3 Rat metallothionein-2 and metallothi0nein-1 Rat betaine homocyateine methyltransferase genes (RPA) (BHMT) Rat IgE binding protein mRNA Rat alpha-l*macrogiocaiin mRNA Rat DPP2 mRNA for dipeptidyl peptidase IL Rat mRNA for atpha(1)-inhibitor 3, variant I. i~use aspady{ protease 1 mRNA Rat calcium-independent phospholipase A2 Rat mRNA for acyI-CoA hydrotase Rat negative acute-phase protein alpha-Iinhibitor 3 Ratlus sp. mRNA for kinase ~typroteir~ 1-microgfobulin/bikunin _Phospholipase C delta Rat mRNA for transferdn (RPA) Listed in order of change, greatest first.
britto, J H Lel~owitch, N e w Y o r k P r e s b y t e r i a n Hospital, N e w York, NY; B Bourke, M M c D e r m o t t , C h i l d r e n ' s Research Centre, O u r Lady's Hospital, D u b lin Ireland; B C P o r t m a n n , S S S t r a u m i e k s , R J T h o m p s o n , I n s t of L i v e r Studies, King's Coil Hosp, L o n d o n U n i t e d K i n g d o m ; B B D a h m s , R a i n b o w Babies Children's Hospital, Cleveland, O H ; R Jaffe, S A Kocoshis, Children's H o s p i t a l of P i t t s b u r g h , P i t t s b u r g h , PA; L N Bull, U n i v of California, San Francisco, CA; A S Knisely, I n s t of Liver Studies, King's Coil H o s p , L o n d o n U n i t e d K i n g d o m Background: Progressive intrahepatic cholestasis, type 1 (PFIC-1), due to mutations in FIC1, a gene expressed in small bowel, liver, and elsewhere, is characterized by intrahepatic cholestasis, hepatic fibrosis, and malabsor-ption with diarrhea. While cholestasis resolves with OLTX, diarrhea may continue. Steatosis is not a feature of the native liver in PFIC-1. Aim: To study histological alterations in hepatic allograft biopsies performed to assess abnormally elevated serum concentrations of liver-enzyme activity post-OLTX in children with PFIC-I and known FIC1 mutations (n = 3, 1 M, 2 F) and in children with clinically diagnosed PFIC-1 (n = 5, 3 M, 2 F). These last children were members of 2 different kindreds, both consanguine. Results of microsatellite haplotype analysis in the 2 children in one kindred were consistent with linkage of their disorder to FIC1 ; no FIC1 mutation has yet been identified. In the 3 children in the other kindred, genetic analysis has not begun. All these 5 children had liver disease with conjugated hyperbilirubinemia, low serum gamma-glutamyl transpeptidase activity, and bland intracanalicnlar cholestasis, with coarsely granular canalicular bile on ultrastructural study. Post-OLTX diarrhea refractory to treatment was present in all 8 patients. Methods: Patient age at OLTX ranged from 1 to 14 years. Two patients' alIografts came from living related donors (FIC1 status not known); the others were cadaveric. Post-OLTX intervaIs to biopsy for allograft dysfunction ranged from 7 days to 8 years. Findings on light microscopy were correlated with post-OLTX histories, clinical symptoms, liver-enzyme activities, and immunosuppressive regimens. Results: Macrovesicular steatosis variably mixed with small-droplet fat was seen in all 8 children, but not within the first 4 weeks post-OLTX. Panlobular (zones 1-3) steatosis was marked in 7 and moderate in 1. Fat was first detected between 4 and 5 weeks post-OLTX. In one patient, steatosis decreased in severity from moderate to mild and shifted in distribution from panlobular to pericentral ( . . . . 3) bet . . . . . . . . . ths 7 and 8 post-OLTX. In another.i c ~,ithehr, , wc' oni ejectiom, denovopost-OLTX"autoimmune'hepatitis, and cirrhosis, fat had disappeared 8 years post-OLTX. Mild portal-tract fibrosis was seen in 2 patients (1 and 9 months post-OLTX), portal-tract and perivenular fibrosis in 1 (4 months postOLTX), and cirrhosis in 1, as described above. Steatohepatitis was seen in none. Episodes of mild acute rejection were histologically documented in 4 patients, with progression to chronic rejection in 1, but no association appeared to exist between these, corticosteroid use, and the time of appearance, lobular distribution, and droplet size of fat. No patient was obese or diabetic. Conclusions: Macrovesicular steatosis of the allograft liver can be seen in children with PFIC-1 treated by OLTX. (This is not invariably the case; Hart J, Nguyen V: personal communications ) We suggest that such steatosis is due to disease in native bowel caused by malfunction of the FIC1 gene product, with disordered enterohepatic interaction. Perturbations of lipid transport and metabolism in PFIC-1 patients after OLTX remain to be studied, and possible complications of hepatic steatosis in such patients await prospective evaluation.
AASLD ABSTRACTS
209A
141
142
A POPULATION-BASED STUDY OF THE BIOCHEMICAL EXPRESSION OF THE H63D HAEMOCHROMATOSIS MUTATION. Peter A G o c h e e , L a w -
SERUM CERULOPLASMIN AND FERROXIDASE ACTIVITY ARE DECREASED IN HFE C 2 8 2 Y + / + MALE IRON-OVERLOADED PATIENTS.
rie W Powell, Q u e e n s l a n d I n s t i t u t e of M e d i c a l Research, Brisbane Australia; D i g b y J Cullen, D e p a r t m e n t o f Medicine, F r e m a n t l e Hospital, F r e m a n t l e Australia; Desire'e D u Sart, M u r d o c h C h i l d r e n s R e s e a r c h Institute, M e l b o u r n e Australia; Enrico Rossi, P a t h c e n t r e , Q u e e n Elizabeth II Medical Centre, N e d lands Australia; J o h n K O l y n y k , D e p a r t m e n t of Medicine, F r e m a n t l e Hospital, F r e m a n t l e Australia
Fabrice Laine, Svc des Maladies du Foie a n d I N S E R M U-522, U n i v H o s p Pontchaillou, R e n n e s France; M a r t i n e Ropert, Lab of Biochemistry, U n i v H o s p Pontchaillou, R e n n e s France; C a r o l i n e Le Lan, 5vc des Maladies d u Foie a n d I N S E R M U-522, U n i v H o s p Pontchaillou, R e n n e s France; Olivier Loreal, INSERM U-522, U n i v H o s p Pontchaillou, R e n n e s France; Eric Bellissant, Lab of P h a r m a c o l o g y , U n i v H o s p Pontchaillou, R e n n e s France; Michel P o u c h a r d , CPAM, R e n n e s France; A n d r e Le Treut, Lab of Biochemistry, R e n n e s France; Pierre Brissot, Svc des Maladies du Foie a n d I N S E R M U-522, U n i v H o s p Pontchaillou, R e n n e s F r a n c e
Background: The two major mutations defined within the haemochromatosis gene both predispose m tim development of iron overload. While the effects of the first mutation, C282Y, have been well characterised, the effects of the second mutation, H63D, remain unclear. We accessed a well-defined, non-blood bank population in Busselmn, Australia and determined the frequency of the H63D mutation and its influence on total body iron stores. Methods: Blood samples were obtained from 30t 1 unrelated Caucasian subjects enrolled in the Busselton population study and aged 20 to 79 years. HFE genotype was determined for each subject. Serum transferrin saturation and serum ferritin were measured and correlated with the H63D mutation in the absence of the C282Y mutation. Results: Sixty-two subjects (2.1 percent) were homozygous for the H63D mutafion and 711 (23.6 percent) were heterozygous. Serum transferrin saturation was significantly increased in males and females who were H63D homozygotes (35.1_+ 1.9 and 29.6-+ 1.6 percent, respectively) or heterozygotes (31.5-+0.5 and 26.7-+0.5 percent, respectively) compared with Dale and female subjects (28.+-+0.3 and 25.0-+0.3 percent, respectively) wild-type for both the H63D and the C282Y mutation (p<0.01). No significant difference was observed in mean serum ferrifin levels between H63D genotypes within each gender. Elevated transferrin saturation >--45 percent was observed in a greater proportion of male H63D homozygotes (15 percent) and heterozygotes (I2 percent) than male HFE wild-types (5 percent) (p<0.01). Male H63D homozygotes (9 percent) and heterozygotes (3 percent) were more likely to have both elevated transferrin saturation and elevated ferritin >- 300 ng per milliliter than male HFE wild-t3zpes (0.7 percent) (p<0.0I). Conclusions: Though a significant increase in serum transferrin satmafion was observed in iudividuals who possessed the H63D mutation, the increase observed does not reflect significantly increased body iron stores and in the absence of the c2g2Y mutation, the H63D mutation is not clinically significant. Efforts directed at population screening based on haemochromatosis genotype should therefore focus on the major mutation, C282Y.
g /:
Male
gema~
Male
Female
Figure I. Mean sen!m~'atgtcrrinsaturation and serum tN'ritin tbr male ~nd ~m~lleH63D genc~pes. HH =HIREWild-type, HI) = H63D Heterozygous,DD = FI63DHomozygous. * p < 0.01
B A C K G R O U N D . A b o d y of e v i d e n c e s u g g e s t s that c e r u l o p l a s m i n , a p r o t e i n l i n k e d to copper, acts in i r o n m e t a b o l i s m , d u e to its ability to catalyze iron oxidation. T h i s ferroxidase activity p e r m i t s the c o n v e r s i o n of ferrous i o n s into ferric ions, w h i c h appears essential for iron m o v e m e n t s a n d exchanges. AIMS. T o evaluate, in C282Y + / + h e m o c h r o m a t o t i c patients, s e r u m c e r u l o p l a s m i n c o n c e n t r a t i o n a n d its ferroxidase activity, a n d to s t u d y their v a r i a t i o n s as a f u n c t i o n of i r o n status. P A T I E N T S A N D M E T H O D S . T h e p r e s e n t s t u d y investigated the s e r u m levels of c e r u l o p l a s m i n ( d e t e r m i n e d b y i m m u n o p h e l e m e t r y ) a n d its ferroxidase activity ( d e t e r m i n e d b y Erel's m e t h o d w h i c h enables to differentiate the real Cp ferroxidase activity f r o m total s e r u m o x i d a s e activity) in 53 patients w i t h C282Y + / + genetic h e m o c h r o m a t o s i s (38 iron overloaded, 15 i r o n depleted) as c o m p a r e d to age a n d s e x m a t c h e d h e a l t h y volunteers. RESULTS. l) In m e n : S e r u m levels o f c e r u l o p l a s m i n w e r e significantly decreased i n i r o n - o v e r l o a d e d p a t i e n t s ( n = 2 7 ) v e r s u s control g r o u p ( p = 0 . 0 2 ) . F u r t h e r m o r e , s e r u m ferroxidase activity w a s s t r o n g l y a n d significantly lower in this g r o u p of i r o n o v e r l o a d e d patients ( p < 0 . 0 0 1 ) . In contrast, in irondepleted h e m o c h r o m a t o t i c p a t i e n t s ( n = 1 5 ) , w h o w e r e u n d e r m a i n t e n a n c e t h e r a p y b y regular p h l e b o t o m i e s , s e r u m levels of c e r u l o p l a s m i n a n d ferroxidase activity w e r e n o t statistically different f r o m those o b s e r v e d in controls. II) In w o m e n ( n = l l ) : No differences w e r e o b s e r v e d , for b o t h p a r a m e t e r s , bet w e e n iron o v e r l o a d e d patients a n d controls. C O N C L U S I O N S . T h e s e data: i) s h o w for the first t i m e that, in i r o n - o v e r l o a d e d C 2 8 2 Y + / + h e m o c h r o m a t o t i c m a l e patients, s e r u m c e r u l o p l a s m i n a n d its ferroxidase activity are decreased, a n d that b o t h factors are corrected b y the r e m o v a l of iron excess. In contrast, no s u c h differences are o b s e r v e d in w o m e n , w h i c h could be related to the w e l l - k n o w n i n d u c i n g effect of e s t r o g e n s o n Cp synthesis; ii) s u g g e s t that, in h e m o c h r o m a t o t i c m a l e patients, iron is able to m o d u l a t e the e x p r e s s i o n of c e r u l o p l a s m i n ; iii) raise the issue of the p u t a t i v e role of d e c r e a s e d s e r u m ferroxidase activity- in the p h e n o t y p i c e x p r e s s i o n o f H F E - I g e n e t i c h e m o c h r o matosis.
143
144
GENOMIC ANALYSIS OF RAT HEPATOCYIES UNDER CONDITIONS OF CHRONIC IRON LOADING. Edward E Cable, J o h n P Bilello, Harriet C
ALLOGRAFT STEATOSIS IS A FEATURE OF PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS, TYPE 1 ( F I C I DISEASE), TREATED BY ORTHOTOPIC LIVER TRANSPLANTATION (OLTX). G Bhagat, S J Lo-
I s o m , P e n n State College of Medicine, H e r s h e y , PA TrimethylhexanoyI-ferrocene (TMHF) is an effective iron donor to rat hepatocytes in long-term DMSO culture. Total RNA was isolated from untreated rat hepatocytes and hepatocytes treated with 20/xM TMHF for 3 weeks. Genomic analysis was performed on the RNA by lneyte Genomics (Pale Alto, CA, USA) and a total of i0,000 genes were analyzed. Following chronic iron loading of hepatocytes by TMHF, 258 transcripts were significantly increased, whereas 644 transcripts were significantly decreased. The top ten listed in order of magnitude of change are listed in the table below. Changes for several of the transcripts listed below were confirmed by an RNase protection assay and are denoted by (RPA). Those transcripts not denoted by (RPA) indicate that the change was detected in the genomic analysis, but not yet confirmed by RPA. There have not yet been any transcripts that changed in the genomic analysis that failed to yield a change when analyzed by RPA. Transcripts for the iron metabolism genes L-ferrifin (RPA) and metallothionein (RPA) were increased and transferrin (Tf, RPA) gene expression was decreased. No changes in Nramp2 (RPA) and Tf receptor-2 gene expression were observed. Gene array analysis showed that transcripts for the stress response genes heine oxygenase (RPA), heat shock protein (HSP) 27 and glutathione 5-transferase I*wme increased. Conversely, transcripts for the stress response gene HSP85, HSP70, HSP60, CuZn superoxide dismutase (SOD, RPA), glutathione synthetase and glutathione peroxidase were decreased. The divergent effects of the stress response genes following TMHF treatment suggests that chronic iron loading is not simply a prolonged version of an acute stress. Further, several other families of genes were decreased. They include genes that encode for cell cycle, extraceliular matrix, and- junctional complex proteins. Iron-mediated decreases to the junctional complex transcripts caveloin, E-cadherin, protocadherin, ~-aetinin, and junctional adhesion molecule were confirmed using an RPA. The biological significance of the decreases in these transcripts was also confirmed in iron loaded hepatocytes because these ceils were more susceptible to baculovirus-mediated gene transfer, a process whose efficiency is im,ersely proportional to the functional integrity of the junctional complexes. The data produced by Incyte Genomics yielded results that were duplicated by RNase protection, demonstrating the accuracy for this particular genomic analysis. These data also indicate that there are several specific changes that occur in iron loaded hepatocytes that can not simply be attributed to the ability of iron to catalyze oxidative reactions.
Transcripts changed bY chronic iron loading _ _ T o p 10 increased "[e_p10 decreased Rat mRNA for heart cytochrome c oxidase Rat haptoglobin rnRNA subunit Via. Rat GADD!53 mRNA Rat rnRNA for pre-alpha-inhiNtor, heavy chain 3. Rat IGF binding protein4 (dGFBP-1} mRNA Rat pcRC201 rnRNA for pre-pro-complernent £,3 Rat metallothionein-2 and metallothi0nein-1 Rat betaine homocyateine methyltransferase genes (RPA) (BHMT) Rat IgE binding protein mRNA Rat alpha-l*macrogiocaiin mRNA Rat DPP2 mRNA for dipeptidyl peptidase IL Rat mRNA for atpha(1)-inhibitor 3, variant I. i~use aspady{ protease 1 mRNA Rat calcium-independent phospholipase A2 Rat mRNA for acyI-CoA hydrotase Rat negative acute-phase protein alpha-Iinhibitor 3 Ratlus sp. mRNA for kinase ~typroteir~ 1-microgfobulin/bikunin _Phospholipase C delta Rat mRNA for transferdn (RPA) Listed in order of change, greatest first.
britto, J H Lel~owitch, N e w Y o r k P r e s b y t e r i a n Hospital, N e w York, NY; B Bourke, M M c D e r m o t t , C h i l d r e n ' s Research Centre, O u r Lady's Hospital, D u b lin Ireland; B C P o r t m a n n , S S S t r a u m i e k s , R J T h o m p s o n , I n s t of L i v e r Studies, King's Coil Hosp, L o n d o n U n i t e d K i n g d o m ; B B D a h m s , R a i n b o w Babies Children's Hospital, Cleveland, O H ; R Jaffe, S A Kocoshis, Children's H o s p i t a l of P i t t s b u r g h , P i t t s b u r g h , PA; L N Bull, U n i v of California, San Francisco, CA; A S Knisely, I n s t of Liver Studies, King's Coil H o s p , L o n d o n U n i t e d K i n g d o m Background: Progressive intrahepatic cholestasis, type 1 (PFIC-1), due to mutations in FIC1, a gene expressed in small bowel, liver, and elsewhere, is characterized by intrahepatic cholestasis, hepatic fibrosis, and malabsor-ption with diarrhea. While cholestasis resolves with OLTX, diarrhea may continue. Steatosis is not a feature of the native liver in PFIC-1. Aim: To study histological alterations in hepatic allograft biopsies performed to assess abnormally elevated serum concentrations of liver-enzyme activity post-OLTX in children with PFIC-I and known FIC1 mutations (n = 3, 1 M, 2 F) and in children with clinically diagnosed PFIC-1 (n = 5, 3 M, 2 F). These last children were members of 2 different kindreds, both consanguine. Results of microsatellite haplotype analysis in the 2 children in one kindred were consistent with linkage of their disorder to FIC1 ; no FIC1 mutation has yet been identified. In the 3 children in the other kindred, genetic analysis has not begun. All these 5 children had liver disease with conjugated hyperbilirubinemia, low serum gamma-glutamyl transpeptidase activity, and bland intracanalicnlar cholestasis, with coarsely granular canalicular bile on ultrastructural study. Post-OLTX diarrhea refractory to treatment was present in all 8 patients. Methods: Patient age at OLTX ranged from 1 to 14 years. Two patients' alIografts came from living related donors (FIC1 status not known); the others were cadaveric. Post-OLTX intervaIs to biopsy for allograft dysfunction ranged from 7 days to 8 years. Findings on light microscopy were correlated with post-OLTX histories, clinical symptoms, liver-enzyme activities, and immunosuppressive regimens. Results: Macrovesicular steatosis variably mixed with small-droplet fat was seen in all 8 children, but not within the first 4 weeks post-OLTX. Panlobular (zones 1-3) steatosis was marked in 7 and moderate in 1. Fat was first detected between 4 and 5 weeks post-OLTX. In one patient, steatosis decreased in severity from moderate to mild and shifted in distribution from panlobular to pericentral ( . . . . 3) bet . . . . . . . . . ths 7 and 8 post-OLTX. In another.i c ~,ithehr, , wc' oni ejectiom, denovopost-OLTX"autoimmune'hepatitis, and cirrhosis, fat had disappeared 8 years post-OLTX. Mild portal-tract fibrosis was seen in 2 patients (1 and 9 months post-OLTX), portal-tract and perivenular fibrosis in 1 (4 months postOLTX), and cirrhosis in 1, as described above. Steatohepatitis was seen in none. Episodes of mild acute rejection were histologically documented in 4 patients, with progression to chronic rejection in 1, but no association appeared to exist between these, corticosteroid use, and the time of appearance, lobular distribution, and droplet size of fat. No patient was obese or diabetic. Conclusions: Macrovesicular steatosis of the allograft liver can be seen in children with PFIC-1 treated by OLTX. (This is not invariably the case; Hart J, Nguyen V: personal communications ) We suggest that such steatosis is due to disease in native bowel caused by malfunction of the FIC1 gene product, with disordered enterohepatic interaction. Perturbations of lipid transport and metabolism in PFIC-1 patients after OLTX remain to be studied, and possible complications of hepatic steatosis in such patients await prospective evaluation.