Promoter Polymorphism of the CD14 Endotoxin Receptor Gene Is Associated With Biliary Atresia and Idiopathic Neonatal Cholestasis
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《小儿科》
Department of Pediatrics, Chang-Gung Memorial Hospital at Chiayi, Pu-Tz City, Chiayi Hsien, Taiwan
Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung, Taiwan
Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan
Surgery Pathology Pediatrics Chang-Gung Memorial Hospital at Kaohsiung, Kaohsiung, Taiwan
Graduate Institute of Medicine; Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan
ABSTRACT
Objective.To investigate whether single-nucleotide polymorphisms in the promoter regions of endotoxin-responsive genes CD14 and tumor necrosis factor- (TNF-) are associated with biliary atresia (BA) and idiopathic neonatal cholestasis (INC).
Methods.We obtained genomic DNA from 90 patients with established diagnosis of BA and 28 patients with INC. Forty-two adult patients with hepatitis B–related cirrhosis and 143 healthy children served as control populations. The genotypes of CD14/C(–159)T and TNF-/G(–308)A (G allele, TNF 1; A allele, TNF 2) were determined by using a restriction enzyme–based assay. Plasma soluble CD14 levels were determined in different disease stages and genotypes of BA.
Results.The frequencies of T allele and T/T homozygosity of the CD14/–159 promoter polymorphism were significantly higher in patients with BA (T allele: 61.7%; T/T genotype: 42.2%) and in patients with INC (T allele: 67.9%; T/T genotype: 53.6%) but not in control populations. Decrease of plasma soluble CD14 from the early stage of BA when the patients received a Kasai operation to the late stage of liver cirrhosis was observed in carriers of the T/T and T/C genotypes but not in carriers of the C/C genotype. The TNF-/–308 promoter polymorphisms (TNF 1 and TNF 2) were not associated with BA.
Conclusion.These findings show that the single-nucleotide polymorphism at CD14/–159 is associated with the development of BA and INC. Endotoxin susceptibility may play a role in the pathogenesis of infantile cholestasis.
Key Words: CD14 gene single nucleotide polymorphism biliary atresia neonatal cholestasis endotoxin susceptibility
Abbreviations: BA, biliary atresia TNF-, tumor necrosis factor- IL, interleukin LPS, lipopolysaccharide SNP, single-nucleotide polymorphism INC, idiopathic neonatal cholestasis sCD14, soluble CD14
Biliary atresia (BA) is characterized by inflammatory obliteration of the extrahepatic bile ducts within months of birth and by progression to liver cirrhosis despite Kasai's portoenterostomy in most patients. However, little is known about the etiopathogenesis of BA. An interaction of genetic susceptibility and undefined perinatal exposure to infectious or toxic agents has been proposed.1,2 The mutations at the human jagged 1 gene are found to be associated with enhanced production of tumor necrosis factor- (TNF-) and interleukin-8 (IL-8), contributing to disease progression in some severe cases of BA.3 A genome-wide expression analysis recently indicated genetic susceptibility to pro-TH1 immunity in patients with BA.4 These findings suggest that cytokine production is important in the pathogenesis of BA and is in part genetically determined.
Liver is prone to viral infections and exposure to enteric endotoxin via portal circulation. Lipopolysaccharide (LPS), a major component of bacterial endotoxin, can stimulate monocytes and endothelial cells to produce proinflammatory cytokines, especially TNF-, as an important innate immune response. The TNF-inducing activity is genetically dependent on endotoxin recognition with a cell-surface glycoprotein named CD14.5 Recent studies also confirm involvement of CD14 gene in antiviral immunity to control HIV, respiratory syncytial virus, and dengue virus infection.6–8 Thus, CD14 not only mediates Gram-negative infection but also confers immunity against viral infections. In addition, CD14 is known to be involved in postnatal maturation of T-helper 1 function.5,9 Previous studies have demonstrated the expression of CD14 proteins on activated Kupffer cells and liver sinusoidal endothelial cells in patients with BA.10,11 CD14 therefore is a crucial inflammatory regulator in the liver that determines innate immunity to viral and bacterial pathogens and mediates TH1 cytokine responses, ie, TNF-, IL-6, IL-8, and IL-12.12
The TNF- production induced by endotoxin/CD14 recognition is also determined by polymorphisms within the TNF- gene. The commonly studied G(–308)A promoter polymorphisms (G allele, TNF 1; and A allele, TNF 2) were shown to have a role in the development of Kawasaki disease, allograft rejection in the liver, and primary sclerosing cholangitis.13–15 Gene polymorphisms might influence transcription activity and gene expression levels. The promoter single-nucleotide polymorphism (SNP) at TNF-/–308 was related to TNF production.16 In addition, the CD14 C(–159)T promoter SNP has been shown to be functionally important in monocyte activation. Homozygous carriers of the T/T genotype have an enhanced CD14 expression than T/C and C/C genotypes.17 This CD14 promoter SNP has been associated with various inflammatory diseases, such as atherosclerosis and myocardial infarction, alcoholic liver disease, atopia, and inflammatory bowel diseases.18–22 We thus hypothesized that SNPs in the genes CD14 and TNF- might be potential risk markers for infantile cholestasis and investigated whether CD14/–159 and TNF-/–308 polymorphisms are associated with such diseases.
METHODS
Study Population
We studied 90 children (47 boys and 43 girls) with the diagnosis of BA during the past 5 years at the Kaohsiung Chang Gung Memorial Hospital, a tertiary referral center for BA and liver transplantation in southern Taiwan (Table 1). The diagnosis was based on the surgical and histologic findings in 87 patients (84 children received a Kasai's operation, and 3 children had laparotomy and liver biopsy only). The remaining 3 patients declined an operation, and their disease was diagnosed by an abdominal ultrasound, hepatobiliary scanning, and histologic findings of the percutaneous needle biopsy liver tissue. All of the patients were followed regularly for >1 year postoperatively. Thirty (33%) patients had a jaundice-free survival for >1 year. Fifty-five (61%) children had progressive hepatic failure and were treated with liver transplantation before 5 years of age. Five patients had early liver cirrhosis and waited for liver transplantation; among them, 1 patient died of liver failure and variceal hemorrhage at 13 months of age.
All of the patients were nonrelated and belonged to the same ethnic group (Chinese). One patient had an elder brother with BA, but the latter was not included in this study. Three (3.3%) patients were of type I BA combined with small choledochal cyst of the common bile duct. Congenital anomaly occurred infrequently in the study population, including duodenal atresia, intestinal malrotation, Meckel diverticulum with internal herniation, and ectopic kidney in 1 each. No complex heart disease or situs anomaly was found in our patients. Informed consent was obtained from all of the patients' parents.
Twenty-eight patients who had a diagnosis of idiopathic neonatal cholestasis (INC) were also recruited in the study. Genetic tests for Byler disease were negative in these patients by direct DNA sequencing.23 Forty-two adult patients with chronic hepatitis B and cirrhosis were enrolled as disease control subjects, and 143 anonymous nonrelated healthy adolescent volunteers served as normal control subjects. The Human Trial and Ethic Committee at the Chang Gung Memorial Hospital approved the study protocol.
Polymorphism Analysis
Genomic DNA of healthy control children, INC, and some BA patients (n = 50) was obtained either from buffy-coat leukocytes by the standard phenol/chloroform extraction method or from the archival liver specimens (n = 40) by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Both the CD14/–159(C/T) and TNF-/–308(G/A) polymorphisms were determined by polymerase chain reaction with subsequent and respective restriction fragment length polymorphism gel electrophoresis.24–26 Briefly, we amplified the CD14 gene promoter and the TNF- promoter regions by using the 5' primer 5'-ATCATCCTTTTCCCACACC-3' and the 3' primer 5'-AACTCTTCGGCTGCCTCT-3' for CD14, and the 5' primer 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and the 3' primer 5'-TCCTCCCTGCTCCGATTCCG-3' for TNF-, respectively. The PCR product was ethanol-precipitated and then digested with either HaeIII for CD14 genotyping or NcoI for TNF- genotyping. The DNA fragments were analyzed on a 2% MetaPhor agarose gel and were visualized by ethidium bromide staining. Digestion of the CD14 amplicons yielded 2 fragments for the C allele (141 and 154 base pairs), whereas the T allele was not digested (295 base pairs). Similarly, the TNF- amplicons were either digested into 2 fragments for the G allele (87 and 20 base pairs) or not digested for the A allele (107 base pairs). All of the genotypings were determined by 2 independent readers and were repeated twice to confirm the results.
Plasma Soluble CD14 Levels
Plasma samples were collected from 59 children with BA at different disease stages: 19 at Kasai operation, 25 at liver transplantation with liver cirrhosis, and 15 at outpatient clinic with long-term jaundice-free survival >1 year. All samples were aliquotted and immediately stored at –80°C until tested. The levels of soluble CD14 (sCD14) were determined in duplicate by using a commercially available enzyme-linked immunosorbent assay kit (Quantikine; R&D Systems, Minneapolis, MN).
Statistical Analysis
Allele and genotype frequencies were estimated by the gene-counting method. With 2 x 2 or 3 x 2 contingency tables, we calculated odds ratios together with 95% confidence intervals by the 2 test. The Hardy-Weinberg equilibrium was tested for each SNP using the 2 test. Student's t test for independent samples was used to determine the differences of sCD14 levels between groups. All statistic tests were performed using the SPSS software for Windows, Version 10, (SPSS Inc Chicago, IL). A given two-sided P < .05 was considered to be statistically significant.
RESULTS
CD14 Promoter Polymorphism and Risk for Infantile Cholestasis
All of the observed genotypic frequencies were in Hardy-Weinberg equilibrium. The frequency of the T allele at position –159 of the CD14 promoter gene was significantly higher in patients with BA and INC than that in control populations. Table 1 shows that gender, pathologic types of BA, incidence of associated anomaly, and clinical outcomes were not statistically different between the genotype subgroups of the CD14 and the TNF- promoter polymorphisms. The genotypic distributions of the CD14 promoter polymorphisms in patients with BA and INC versus control subjects are shown in Table 2. Comparing the genotypic distribution between patients and control subjects yielded P = 0.013 (x22 = 8.63). The T/T genotype of the CD14 SNP was higher in patients with BA (42.2%; P = .004) and INC (53.6%; P = .002) but not in adult hepatitis B–related cirrhosis, by using a recessive model (comparing T/T genotype vs T/C + C/C genotypes). This result indicates that the T/T genotype confers an increase risk for developing BA and INC. Considering possible damage to DNA resulting in false polymorphisms, we compared the source of DNA and frequencies of CD14 polymorphisms in 90 BA patients. Both the T/T genotype frequencies (48% in the buffy-coat group; 35% in the archival liver group) and T allele frequencies (63% in the buffy-coat group; 60% in the archival liver group) showed no significant difference between the buffy-coat group and the archival liver group. The T/T genotype and T allele in both groups were higher than in healthy control subjects (24% of T/T and 52% of T allele frequencies). TNF- G(–308)A polymorphisms, however, were not associated with BA (Table 2).
Plasma sCD14 Levels in BA
To examine whether the CD14 promoter polymorphism is associated with variations in protein expression, we measured plasma sCD14 levels in 59 patients. Individuals with the T/T and T/C genotypes of the CD14 promoter polymorphism showed a profound decrease of plasma sCD14 levels at the late stage of BA (mean ± SD: 1222.1 ± 356.8 and 949.6 ± 448.1 ng/mL, respectively), compared with the early stage at Kasai operation (1867.9 ± 566.6 and 2036.1 ± 793.7 ng/mL, respectively; P = .009 and .006, respectively). Patients with the C/C homozygote, however, had a similar level of plasma sCD14 between the early and the cirrhotic stages of BA (1420.4 ± 559.1 vs 1605.8 ± 656.1 ng/mL; P = .63; Fig 1).
We also noted that the plasma sCD14 levels might serve as a predictive factor for development of liver cirrhosis in BA patients (Fig 2). Patients with liver cirrhosis that required liver transplantation had a significant decrease of plasma sCD14, compared with those in the early stage when receiving a Kasai operation (1205.2 ± 524.3 vs 1785.6 ± 624.3 ng/mL; P = .002). Patients with long-term jaundice-free survival had a higher level of plasma sCD14 than that at Kasai operation (2596.1 ± 862.2 vs 1785.6 ± 624.3 ng/mL; P = .003). Thirteen (86.7%) of 15 patients with long-term survival had a plasma sCD14 level >2000 ng/mL. However, 22 (88%) of 25 patients in the transplantation group had plasma sCD14 levels <2000 ng/mL (Fig 2).
DISCUSSION
Our results indicated that the T/T homozygote at position –159 for the CD14 promoter polymorphism is associated with the development of BA and INC in our study population. In addition, the plasma sCD14 level might serve as a biological marker that is significantly decreased in patients with the T/T and T/C genotypes when disease progresses to liver cirrhosis, in contrast to nonsignificant change in patients with the C/C genotype. Such findings suggest a potential role of CD14-mediated innate immune response in the pathogenesis of infantile cholestasis syndromes.
CD14 plays a key role in the innate immunity through recognition of bacterial endotoxin, involved in an inflammatory cascade in favor of the development of Th1 cells from nave CD4-positive T cells.27 A biologically relevant finding is that the T/T homozygote increases expression levels of the CD14 gene in monocytes.17 The T/T homozygote had a significantly higher sCD14 level but a lower level of serum immunoglobulin E than that of carriers with either the T/C or the C/C genotypes.17,27,28 The findings are different from the results of this study. The present data demonstrate that circulating sCD14, an important mediator in neutralization of LPS, decreased among patients with progressive disease and liver cirrhosis, especially in carriers of T/T and T/C genotypes. The difference may lie in the different population and different disease entities between this study and those reported earlier. In fact, our results suggest that the ability to neutralize LPS by circulating sCD14 is deficient in patients with liver cirrhosis but is preserved in long-term survivors. The C(–159)T genetic variant in the CD14 gene promoter influences the response of infants' hepatobiliary system to endotoxin or to some unknown viral agents in favor of Th1-cytokine production. The latter may in turn activate liver Kupffer cells and aggravate portal inflammation, leading to the pathologic features that are characteristic of BA.
It is noteworthy that infants are prone to postnatal gut-derived bacterial or endotoxin translocation and that most patients with BA develop acholic stool and biliary obstruction gradually after a period of normal feeding. Studies of the role of endotoxin in BA are still lacking. Goldberg et al29 reported a close relationship between hyperbilirubinemia in neonates without underlying hemolytic disorders and serum levels of endotoxin. Furthermore, endotoxin can be found in great quantities in human bile. Administration of probiotics to infants with BA might decrease bacterial translocation and serum endotoxin levels.30
In addition to bacteria, viral stimuli may mediate a Th1-polarized proinflammatory cytokine reaction through a CD14-dependent pathway.9 The viral agents, including cytomegalovirus, human papilloma virus, herpesvirus, rotavirus, and retrovirus, have been implicated as the causative agent for the development of BA for many years. Although some have been identified in a limited number of patients with BA, none is universally isolated or identified in every case.31–39 From the experience of the infectious animal model of BA, there is an inherent immunologic gap that is required for virally induced BA to occur.40 As CD14 also mediates infection caused by viral agents, the implication of CD14 promoter polymorphisms in the virus-associated pathogenesis of BA needs additional clarification.
Various associations of the CD14 promoter polymorphisms and inflammatory diseases are conflicting in previous reports. In Taiwan, BA is a more common disease of liver in infants than in Western countries, with an estimated incidence up to 1 in 3500 newborn infants. Host genetic susceptibility and environmental triggering factors are important in multifactorial diseases, such as BA. Additional studies might identify these risk populations and provide prevention from development of BA and neonatal cholestasis syndrome. In conclusion, the present data delineate a novel association between the CD14 promoter SNP and the development of BA and INC. Our findings support the notion that BA is an inflammatory disorder of biliary tracts in infants and suggest a potential role of CD14-dependent endotoxin response in triggering Th1-differentiation of infants' livers.
ACKNOWLEDGMENTS
This study was supported by Chang Gung Memorial Grant CMRPG8006 (to F.-C.H.) and NSC92-2314-B182-076 (to J.-H.C.).
We thank Drs Chie-Song Hsieh, Shin-Ye Lee, Chih-Chi Wang, and Yaw-Sen Chen for providing the samples used in this study and Ming-Huei Chou, Chia-Lin Wu, and Ching-Ting Cheng for technical assistance.
FOOTNOTES
Accepted Nov 23, 2004.
No conflict of interest declared.
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LeVan TD, Bloom JW, Bailey TJ, Karp CL, Halonen M, Martinez FD, Vercelli D. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity. J Immunol. 2001;167 :5838 –5844
Hubacek JA, Rothe G, Pit'ha J, Skodova Z, Stanek V, Poledne R, Schmitz G. C(-260)->T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation. 1999;99 :3218 –3220
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Tyler KL, Sokol RJ, Oberhaus SM, et al. Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts. Hepatology. 1998;27 :1475 –1482
Steele MI, Marshall CM, Lloyd RE, Randolph VE. Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. Hepatology. 1995;21 :697 –702
Riepenhoff-Talty M, Gouvea V, Evans MJ, et al. Detection of group C rotavirus in infants with extrahepatic biliary atresia. J Infect Dis. 1996;174 :8 –15
Bobo L, Ojeh C, Chiu D, Machado A, Colombani P, Schwarz K. Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia. Pediatr Res. 1997;41 :229 –234
Mason AL, Xu L, Guo L, Munoz S, et al. Detection of retroviral antibodies in primary biliary cirrhosis and other idiopathic biliary disorders. Lancet. 1998;351 :1620 –1624
Czech-Schmidt G, Verhagen W, Szavay P, Leonhardt J, Petersen C. Immunological gap in the infectious animal model for biliary atresia. J Surg Res. 2001;101 :62 –67(Hsiang-Hung Shih, MD, Tsu)
Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung, Taiwan
Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan
Surgery Pathology Pediatrics Chang-Gung Memorial Hospital at Kaohsiung, Kaohsiung, Taiwan
Graduate Institute of Medicine; Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan
ABSTRACT
Objective.To investigate whether single-nucleotide polymorphisms in the promoter regions of endotoxin-responsive genes CD14 and tumor necrosis factor- (TNF-) are associated with biliary atresia (BA) and idiopathic neonatal cholestasis (INC).
Methods.We obtained genomic DNA from 90 patients with established diagnosis of BA and 28 patients with INC. Forty-two adult patients with hepatitis B–related cirrhosis and 143 healthy children served as control populations. The genotypes of CD14/C(–159)T and TNF-/G(–308)A (G allele, TNF 1; A allele, TNF 2) were determined by using a restriction enzyme–based assay. Plasma soluble CD14 levels were determined in different disease stages and genotypes of BA.
Results.The frequencies of T allele and T/T homozygosity of the CD14/–159 promoter polymorphism were significantly higher in patients with BA (T allele: 61.7%; T/T genotype: 42.2%) and in patients with INC (T allele: 67.9%; T/T genotype: 53.6%) but not in control populations. Decrease of plasma soluble CD14 from the early stage of BA when the patients received a Kasai operation to the late stage of liver cirrhosis was observed in carriers of the T/T and T/C genotypes but not in carriers of the C/C genotype. The TNF-/–308 promoter polymorphisms (TNF 1 and TNF 2) were not associated with BA.
Conclusion.These findings show that the single-nucleotide polymorphism at CD14/–159 is associated with the development of BA and INC. Endotoxin susceptibility may play a role in the pathogenesis of infantile cholestasis.
Key Words: CD14 gene single nucleotide polymorphism biliary atresia neonatal cholestasis endotoxin susceptibility
Abbreviations: BA, biliary atresia TNF-, tumor necrosis factor- IL, interleukin LPS, lipopolysaccharide SNP, single-nucleotide polymorphism INC, idiopathic neonatal cholestasis sCD14, soluble CD14
Biliary atresia (BA) is characterized by inflammatory obliteration of the extrahepatic bile ducts within months of birth and by progression to liver cirrhosis despite Kasai's portoenterostomy in most patients. However, little is known about the etiopathogenesis of BA. An interaction of genetic susceptibility and undefined perinatal exposure to infectious or toxic agents has been proposed.1,2 The mutations at the human jagged 1 gene are found to be associated with enhanced production of tumor necrosis factor- (TNF-) and interleukin-8 (IL-8), contributing to disease progression in some severe cases of BA.3 A genome-wide expression analysis recently indicated genetic susceptibility to pro-TH1 immunity in patients with BA.4 These findings suggest that cytokine production is important in the pathogenesis of BA and is in part genetically determined.
Liver is prone to viral infections and exposure to enteric endotoxin via portal circulation. Lipopolysaccharide (LPS), a major component of bacterial endotoxin, can stimulate monocytes and endothelial cells to produce proinflammatory cytokines, especially TNF-, as an important innate immune response. The TNF-inducing activity is genetically dependent on endotoxin recognition with a cell-surface glycoprotein named CD14.5 Recent studies also confirm involvement of CD14 gene in antiviral immunity to control HIV, respiratory syncytial virus, and dengue virus infection.6–8 Thus, CD14 not only mediates Gram-negative infection but also confers immunity against viral infections. In addition, CD14 is known to be involved in postnatal maturation of T-helper 1 function.5,9 Previous studies have demonstrated the expression of CD14 proteins on activated Kupffer cells and liver sinusoidal endothelial cells in patients with BA.10,11 CD14 therefore is a crucial inflammatory regulator in the liver that determines innate immunity to viral and bacterial pathogens and mediates TH1 cytokine responses, ie, TNF-, IL-6, IL-8, and IL-12.12
The TNF- production induced by endotoxin/CD14 recognition is also determined by polymorphisms within the TNF- gene. The commonly studied G(–308)A promoter polymorphisms (G allele, TNF 1; and A allele, TNF 2) were shown to have a role in the development of Kawasaki disease, allograft rejection in the liver, and primary sclerosing cholangitis.13–15 Gene polymorphisms might influence transcription activity and gene expression levels. The promoter single-nucleotide polymorphism (SNP) at TNF-/–308 was related to TNF production.16 In addition, the CD14 C(–159)T promoter SNP has been shown to be functionally important in monocyte activation. Homozygous carriers of the T/T genotype have an enhanced CD14 expression than T/C and C/C genotypes.17 This CD14 promoter SNP has been associated with various inflammatory diseases, such as atherosclerosis and myocardial infarction, alcoholic liver disease, atopia, and inflammatory bowel diseases.18–22 We thus hypothesized that SNPs in the genes CD14 and TNF- might be potential risk markers for infantile cholestasis and investigated whether CD14/–159 and TNF-/–308 polymorphisms are associated with such diseases.
METHODS
Study Population
We studied 90 children (47 boys and 43 girls) with the diagnosis of BA during the past 5 years at the Kaohsiung Chang Gung Memorial Hospital, a tertiary referral center for BA and liver transplantation in southern Taiwan (Table 1). The diagnosis was based on the surgical and histologic findings in 87 patients (84 children received a Kasai's operation, and 3 children had laparotomy and liver biopsy only). The remaining 3 patients declined an operation, and their disease was diagnosed by an abdominal ultrasound, hepatobiliary scanning, and histologic findings of the percutaneous needle biopsy liver tissue. All of the patients were followed regularly for >1 year postoperatively. Thirty (33%) patients had a jaundice-free survival for >1 year. Fifty-five (61%) children had progressive hepatic failure and were treated with liver transplantation before 5 years of age. Five patients had early liver cirrhosis and waited for liver transplantation; among them, 1 patient died of liver failure and variceal hemorrhage at 13 months of age.
All of the patients were nonrelated and belonged to the same ethnic group (Chinese). One patient had an elder brother with BA, but the latter was not included in this study. Three (3.3%) patients were of type I BA combined with small choledochal cyst of the common bile duct. Congenital anomaly occurred infrequently in the study population, including duodenal atresia, intestinal malrotation, Meckel diverticulum with internal herniation, and ectopic kidney in 1 each. No complex heart disease or situs anomaly was found in our patients. Informed consent was obtained from all of the patients' parents.
Twenty-eight patients who had a diagnosis of idiopathic neonatal cholestasis (INC) were also recruited in the study. Genetic tests for Byler disease were negative in these patients by direct DNA sequencing.23 Forty-two adult patients with chronic hepatitis B and cirrhosis were enrolled as disease control subjects, and 143 anonymous nonrelated healthy adolescent volunteers served as normal control subjects. The Human Trial and Ethic Committee at the Chang Gung Memorial Hospital approved the study protocol.
Polymorphism Analysis
Genomic DNA of healthy control children, INC, and some BA patients (n = 50) was obtained either from buffy-coat leukocytes by the standard phenol/chloroform extraction method or from the archival liver specimens (n = 40) by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Both the CD14/–159(C/T) and TNF-/–308(G/A) polymorphisms were determined by polymerase chain reaction with subsequent and respective restriction fragment length polymorphism gel electrophoresis.24–26 Briefly, we amplified the CD14 gene promoter and the TNF- promoter regions by using the 5' primer 5'-ATCATCCTTTTCCCACACC-3' and the 3' primer 5'-AACTCTTCGGCTGCCTCT-3' for CD14, and the 5' primer 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and the 3' primer 5'-TCCTCCCTGCTCCGATTCCG-3' for TNF-, respectively. The PCR product was ethanol-precipitated and then digested with either HaeIII for CD14 genotyping or NcoI for TNF- genotyping. The DNA fragments were analyzed on a 2% MetaPhor agarose gel and were visualized by ethidium bromide staining. Digestion of the CD14 amplicons yielded 2 fragments for the C allele (141 and 154 base pairs), whereas the T allele was not digested (295 base pairs). Similarly, the TNF- amplicons were either digested into 2 fragments for the G allele (87 and 20 base pairs) or not digested for the A allele (107 base pairs). All of the genotypings were determined by 2 independent readers and were repeated twice to confirm the results.
Plasma Soluble CD14 Levels
Plasma samples were collected from 59 children with BA at different disease stages: 19 at Kasai operation, 25 at liver transplantation with liver cirrhosis, and 15 at outpatient clinic with long-term jaundice-free survival >1 year. All samples were aliquotted and immediately stored at –80°C until tested. The levels of soluble CD14 (sCD14) were determined in duplicate by using a commercially available enzyme-linked immunosorbent assay kit (Quantikine; R&D Systems, Minneapolis, MN).
Statistical Analysis
Allele and genotype frequencies were estimated by the gene-counting method. With 2 x 2 or 3 x 2 contingency tables, we calculated odds ratios together with 95% confidence intervals by the 2 test. The Hardy-Weinberg equilibrium was tested for each SNP using the 2 test. Student's t test for independent samples was used to determine the differences of sCD14 levels between groups. All statistic tests were performed using the SPSS software for Windows, Version 10, (SPSS Inc Chicago, IL). A given two-sided P < .05 was considered to be statistically significant.
RESULTS
CD14 Promoter Polymorphism and Risk for Infantile Cholestasis
All of the observed genotypic frequencies were in Hardy-Weinberg equilibrium. The frequency of the T allele at position –159 of the CD14 promoter gene was significantly higher in patients with BA and INC than that in control populations. Table 1 shows that gender, pathologic types of BA, incidence of associated anomaly, and clinical outcomes were not statistically different between the genotype subgroups of the CD14 and the TNF- promoter polymorphisms. The genotypic distributions of the CD14 promoter polymorphisms in patients with BA and INC versus control subjects are shown in Table 2. Comparing the genotypic distribution between patients and control subjects yielded P = 0.013 (x22 = 8.63). The T/T genotype of the CD14 SNP was higher in patients with BA (42.2%; P = .004) and INC (53.6%; P = .002) but not in adult hepatitis B–related cirrhosis, by using a recessive model (comparing T/T genotype vs T/C + C/C genotypes). This result indicates that the T/T genotype confers an increase risk for developing BA and INC. Considering possible damage to DNA resulting in false polymorphisms, we compared the source of DNA and frequencies of CD14 polymorphisms in 90 BA patients. Both the T/T genotype frequencies (48% in the buffy-coat group; 35% in the archival liver group) and T allele frequencies (63% in the buffy-coat group; 60% in the archival liver group) showed no significant difference between the buffy-coat group and the archival liver group. The T/T genotype and T allele in both groups were higher than in healthy control subjects (24% of T/T and 52% of T allele frequencies). TNF- G(–308)A polymorphisms, however, were not associated with BA (Table 2).
Plasma sCD14 Levels in BA
To examine whether the CD14 promoter polymorphism is associated with variations in protein expression, we measured plasma sCD14 levels in 59 patients. Individuals with the T/T and T/C genotypes of the CD14 promoter polymorphism showed a profound decrease of plasma sCD14 levels at the late stage of BA (mean ± SD: 1222.1 ± 356.8 and 949.6 ± 448.1 ng/mL, respectively), compared with the early stage at Kasai operation (1867.9 ± 566.6 and 2036.1 ± 793.7 ng/mL, respectively; P = .009 and .006, respectively). Patients with the C/C homozygote, however, had a similar level of plasma sCD14 between the early and the cirrhotic stages of BA (1420.4 ± 559.1 vs 1605.8 ± 656.1 ng/mL; P = .63; Fig 1).
We also noted that the plasma sCD14 levels might serve as a predictive factor for development of liver cirrhosis in BA patients (Fig 2). Patients with liver cirrhosis that required liver transplantation had a significant decrease of plasma sCD14, compared with those in the early stage when receiving a Kasai operation (1205.2 ± 524.3 vs 1785.6 ± 624.3 ng/mL; P = .002). Patients with long-term jaundice-free survival had a higher level of plasma sCD14 than that at Kasai operation (2596.1 ± 862.2 vs 1785.6 ± 624.3 ng/mL; P = .003). Thirteen (86.7%) of 15 patients with long-term survival had a plasma sCD14 level >2000 ng/mL. However, 22 (88%) of 25 patients in the transplantation group had plasma sCD14 levels <2000 ng/mL (Fig 2).
DISCUSSION
Our results indicated that the T/T homozygote at position –159 for the CD14 promoter polymorphism is associated with the development of BA and INC in our study population. In addition, the plasma sCD14 level might serve as a biological marker that is significantly decreased in patients with the T/T and T/C genotypes when disease progresses to liver cirrhosis, in contrast to nonsignificant change in patients with the C/C genotype. Such findings suggest a potential role of CD14-mediated innate immune response in the pathogenesis of infantile cholestasis syndromes.
CD14 plays a key role in the innate immunity through recognition of bacterial endotoxin, involved in an inflammatory cascade in favor of the development of Th1 cells from nave CD4-positive T cells.27 A biologically relevant finding is that the T/T homozygote increases expression levels of the CD14 gene in monocytes.17 The T/T homozygote had a significantly higher sCD14 level but a lower level of serum immunoglobulin E than that of carriers with either the T/C or the C/C genotypes.17,27,28 The findings are different from the results of this study. The present data demonstrate that circulating sCD14, an important mediator in neutralization of LPS, decreased among patients with progressive disease and liver cirrhosis, especially in carriers of T/T and T/C genotypes. The difference may lie in the different population and different disease entities between this study and those reported earlier. In fact, our results suggest that the ability to neutralize LPS by circulating sCD14 is deficient in patients with liver cirrhosis but is preserved in long-term survivors. The C(–159)T genetic variant in the CD14 gene promoter influences the response of infants' hepatobiliary system to endotoxin or to some unknown viral agents in favor of Th1-cytokine production. The latter may in turn activate liver Kupffer cells and aggravate portal inflammation, leading to the pathologic features that are characteristic of BA.
It is noteworthy that infants are prone to postnatal gut-derived bacterial or endotoxin translocation and that most patients with BA develop acholic stool and biliary obstruction gradually after a period of normal feeding. Studies of the role of endotoxin in BA are still lacking. Goldberg et al29 reported a close relationship between hyperbilirubinemia in neonates without underlying hemolytic disorders and serum levels of endotoxin. Furthermore, endotoxin can be found in great quantities in human bile. Administration of probiotics to infants with BA might decrease bacterial translocation and serum endotoxin levels.30
In addition to bacteria, viral stimuli may mediate a Th1-polarized proinflammatory cytokine reaction through a CD14-dependent pathway.9 The viral agents, including cytomegalovirus, human papilloma virus, herpesvirus, rotavirus, and retrovirus, have been implicated as the causative agent for the development of BA for many years. Although some have been identified in a limited number of patients with BA, none is universally isolated or identified in every case.31–39 From the experience of the infectious animal model of BA, there is an inherent immunologic gap that is required for virally induced BA to occur.40 As CD14 also mediates infection caused by viral agents, the implication of CD14 promoter polymorphisms in the virus-associated pathogenesis of BA needs additional clarification.
Various associations of the CD14 promoter polymorphisms and inflammatory diseases are conflicting in previous reports. In Taiwan, BA is a more common disease of liver in infants than in Western countries, with an estimated incidence up to 1 in 3500 newborn infants. Host genetic susceptibility and environmental triggering factors are important in multifactorial diseases, such as BA. Additional studies might identify these risk populations and provide prevention from development of BA and neonatal cholestasis syndrome. In conclusion, the present data delineate a novel association between the CD14 promoter SNP and the development of BA and INC. Our findings support the notion that BA is an inflammatory disorder of biliary tracts in infants and suggest a potential role of CD14-dependent endotoxin response in triggering Th1-differentiation of infants' livers.
ACKNOWLEDGMENTS
This study was supported by Chang Gung Memorial Grant CMRPG8006 (to F.-C.H.) and NSC92-2314-B182-076 (to J.-H.C.).
We thank Drs Chie-Song Hsieh, Shin-Ye Lee, Chih-Chi Wang, and Yaw-Sen Chen for providing the samples used in this study and Ming-Huei Chou, Chia-Lin Wu, and Ching-Ting Cheng for technical assistance.
FOOTNOTES
Accepted Nov 23, 2004.
No conflict of interest declared.
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