ORIGINAL ARTICLE: PANCREATOLOGY
Circulating MicroRNAs as Noninvasive Diagnostic Biomarkers of Liver Disease in Children With Cystic Fibrosis
Naomi L. Cook, Tamara N. Pereira, yPeter J. Lewindon, zRoss W. Shepherd, and Grant A. Ramm
ABSTRACT Objectives: Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. Methods: We profiled circulating serum miRNA levels in patients with CFLD (n ¼ 52), patients with CF without liver disease (CFnoLD, n ¼ 30), and non-CF pediatric controls (n ¼ 20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. Results: miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (P < 0.0001). miR-25 (P ¼ 0.0011) and miR-21 (P ¼ 0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, P ¼ 0.002) and miR-25 (AUC 0.65, P ¼ 0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, P < 0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3–F4 fibrosis (AUC 0.73, P ¼ 0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1–F4 (AUC 0.72, P ¼ 0.02).
Received March 4, 2014; accepted October 8, 2014. From the Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, the yDepartment of Gastroenterology, Royal Children’s Hospital, Brisbane, Australia, and the zDepartment of Pediatrics, Baylor College of Medicine, Houston, TX, Brisbane, Australia. Address correspondence and reprint requests to Prof Grant A. Ramm, Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, PO Royal Brisbane and Women’s Hospital, Brisbane, QLD 4029, Australia (e-mail: [email protected]). Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jpgn.org). G.A.R., P.J.L., and R.W.S are supported by the National Health and Medical Research Council of Australia (NHMRC-APP1048740). G.A.R. is also supported by an NHMRC Senior Research Fellowship (APP1061332). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000600
JPGN
Volume 60, Number 2, February 2015
Conclusions: These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis. Key Words: cystic fibrosis liver disease, hepatic fibrosis, pediatrics, reference genes, RT-qPCR, serum miRNA
(JPGN 2015;60: 247–254)
L
iver fibrosis is responsible for considerable morbidity and mortality in patients with cystic fibrosis (CF), causing clinically significant complications in up to 20% of patients and 2% to 3% of overall mortality (1,2); however, the onset and progression of fibrosis in cystic fibrosis liver disease (CFLD) are difficult to predict and monitor, and factors relating to the early pathogenesis of CFLD are incompletely understood. Current measures of liver function (eg, serum biochemistry), combined with ultrasound and clinical examination, are insensitive and nonspecific for detection of early liver disease and assessment of progressive fibrosis severity. Liver biopsy remains the criterion standard for diagnosis of hepatic fibrosis in CFLD, but is invasive and may be confounded by the focal nature of disease activity (3,4). Thus, a sensitive, specific, and noninvasive test reflecting the early pathogenesis of CFLD is required to identify individuals at risk of developing hepatic fibrosis before the advent of severe complications. Posttranscriptional regulation of gene expression is now recognized as an important contributor to disease pathogenesis, among whose mechanisms include alterations in the function and stability of translational elements within both coding and noncoding regions of messenger RNA (mRNA). A major component of this process is binding by microRNAs (miRNAs), which function via base pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation. miRNAs are noncoding endogenously transcribed RNAs that undergo a well-characterized series of processing steps that generate short single-stranded (21 nucleotides in length) RNA fragments (5). miRNAs are critical to the regulation of virtually all of the cellular processes, and increasing evidence implicates dysregulated miRNA expression in a range of human pathologies, including liver diseases (6). Because miRNAs are readily detectable in serum and other body fluids, and exhibit remarkable stability compared with mRNA and other long RNAs (7–9), cell-free miRNAs have attracted interest as potential diagnostic biomarkers. Substantial evidence implicates miRNA involvement both in maintaining normal liver function and in processes associated with liver disease. Elevated serum levels of liver-specific miR-122 have
247
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Ramm et al
JPGN
been demonstrated in viral, alcoholic, metabolic, and drug-induced liver injury (10–14). These studies suggest that serum levels of miR-122 may act as biomarkers of tissue injury. Although certain tissue miRNAs may be implicated in CF complications of the lung and intestinal tract (15,16), no study to date has characterized serum miRNAs in CFLD. In the present study, we aimed to identify a circulating miRNA signature in children with CF with diagnostic potential for the detection of early CFLD. We hypothesized that the candidate liver-specific miR-122 would be elevated in the serum of patients with CFLD compared with in that of patients with CF without liver disease (CFnoLD), and we sought to quantify circulating miRNAs, including miR-122, in patients with CFLD, patients with CFnoLD, and non-CF controls. To do this, we initially used a polymerase chain reaction (PCR) array containing 84 serum-detectable miRNAs in a subset of samples to identify candidate miRNAs, which were then validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in a larger patient cohort.
METHODS Patients The present study was approved by the human ethics committees of QIMR Berghofer Medical Research Institute and the Royal Children’s Hospital, Brisbane, and informed consent was obtained from parents or guardians. Sera from 102 children were processed as described in the following text. Patients with CFLD (n ¼ 52) were initially selected by the presence of at least 2 of the following: hepatomegaly splenomegaly; persistent elevation of serum alanine aminotransferase (ALT >1 upper limit normal) longer than 6 months; abnormal ultrasound scan with abnormal echogenicity or nodular edge suggestive of cirrhosis, with subsequent dual-pass liver biopsies to assess CFLD and stage fibrosis, as previously reported (17). Liver histology was staged for hepatic fibrosis by blinded Scheuer scoring system (18). The highest fibrosis staging was used if fibrosis stages between the 2 biopsies were discordant. Age- and sex-matched children with CF but no signs of liver disease by clinical, biochemical, and ultrasonographic means were assigned to the CFnoLD group (n ¼ 30). Children attending the CF clinic underwent regular clinical examination, blood testing, and hepatobiliary ultrasound scanning. Patients were included in the CFnoLD group if they had normal clinical, biochemical, and ultrasonographic examination in the 2 years before study serum sampling. A non-CF, non–liver disease pediatric control group comprised 20 age- and sex-matched children who were recruited while attending the hospital for minor plastic procedures.
RNA Extraction and Reverse Transcription RNA was extracted from 200 mL serum using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) or the miRCURY RNA Isolation Kit for Biofluids (Exiqon, Vedbaek, Denmark), according to the manufacturer’s protocol. RNA was reverse transcribed using the miScript II RT Kit (Qiagen) or the miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon). RNA isolation and reverse transcription are described in detail in the supplementary Methods, http://links.lww.com/MPG/A394.
miRNA PCR Array and RT-qPCR Validation We first conducted a PCR array (Human Serum & Plasma miRNA PCR Array; Qiagen, cat. no. MIHS-106Z) in sera from a subset of 32 children, including 12 with CFLD, 10 with CFnoLD, and 10 controls, to assess the levels of 84 miRNAs detectable in
248
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serum. Candidate miRNAs with the potential to distinguish liver disease in CF were identified from the PCR array screen for subsequent RT-qPCR validation using Exiqon reagents in the entire cohort of 102 patients. PCR amplification is described in detail in the supplementary Methods, http://links.lww.com/MPG/A394. Sequences of candidate miRNAs are found in supplemental Table 1, http://links.lww.com/MPG/A391.
Reference Gene Validation Reference gene validation was considered to be a critical prerequisite to RT-qPCR data normalization. The geNorm (19) tool in qbaseþ was used to rank the detectable miRNAs from the PCR array in order of stability and to identify an appropriate panel of reference genes for data normalization (supplemental Fig. 1, http:// links.lww.com/MPG/A392). geNorm assigned an expression stability measure (M) to each miRNA, with a recommended cutoff value of 1.5. Next, pairwise variation (V) analysis determined the optimal number of reference genes required for accurate data normalization, with a cutoff value of 0.15, below which additional reference genes are not required. geNorm determined that miR-93 and miR-19b comprised an appropriate set of reference genes for our experimental conditions; therefore, these reference genes were used to normalize all of the RT-qPCR data in the present study.
Data Analysis Quantification cycle (Cq) values were determined using instrument software and input into qbaseþ version 2.5 software (Biogazelle, Ghent, Belgium) (20) to calculate normalized relative quantities of each miRNA in the Qiagen PCR array and thus to identify candidate miRNAs that would be validated in the subsequent RT-qPCR experiments. The geNorm tool (19) was used for reference gene validation. In the present study, we have adhered to the Minimum Information for Publication of Quantitative RealTime PCR Experiments guidelines (21) in conjunction with recent recommendations for standardized reporting in cell-free miRNA studies (22), in which information was available. More details regarding data analysis are included in the supplementary Methods, http://links.lww.com/MPG/A394.
Statistical Analysis Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA) or SPSS Statistics version 19 (IBM, Armonk, NY), with P < 0.05 considered significant. Clinical characteristics of the CFLD and CFnoLD groups shown in Table 1 were analyzed using Student t test or Mann-Whitney U test. Normalized RT-qPCR data were subjected to log base 10 transformations and analyzed using 1-way analysis of variance (ANOVA) with Bonferroni post hoc test to compare all of the groups. Correlations between miRNA levels and serum aminotransferase levels were determined using Spearman rank correlations. Receiver operating characteristic (ROC) analyses and logistic regression were carried out to assess the ability of miRNAs, both individually and in combination, to distinguish between patient groups. To optimize both the positive predictive value (PPV) and negative predictive value (NPV) and provide the most clinically useful biomarker test derived from the ROC analysis to the clinician treating children suspected as having CFLD with abnormal routine tests, data are presented using maximum specificity for the test. Sample size evaluation for a clinical study is almost always a matter of compromise between the available resources and the various objectives. Our patients were chosen out of expedience, www.jpgn.org
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 60, Number 2, February 2015
Noninvasive Diagnostic Biomarkers of CF Liver Disease
TABLE 1. Demographic and clinical characteristics of the CFLD and CFnoLD groups
Boys, n (%) Girls, n (%) Age Mean (median) SD Range Alanine aminotransferase, U/L Mean (median) SD Range Aspartate aminotransferase, U/L Mean (median) SD Range g-Glutamyl transpeptidase, U/L Mean (median) SD Range Alkaline phosphatase, U/L Mean (median) SD Range Ultrasound Normal Abnormal Not available Scheuer fibrosis score (n) 0 (no fibrosis) 1 2 3 4 (cirrhosis)
CFLD (n ¼ 52)
CFnoLD (n ¼ 30)
P
29 (56) 23 (44)
16 (53) 14 (47)
NS
10.9 (10.9) 4.6 2.3–17.6
12.2 (12.0) 3.1 6.2–17.0
0.132
53 (42) 33 19–174
23 (20) 11 6–50
Circulating MicroRNAs as Noninvasive Diagnostic Biomarkers of Liver Disease in Children With Cystic Fibrosis
Naomi L. Cook, Tamara N. Pereira, yPeter J. Lewindon, zRoss W. Shepherd, and Grant A. Ramm
ABSTRACT Objectives: Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. Methods: We profiled circulating serum miRNA levels in patients with CFLD (n ¼ 52), patients with CF without liver disease (CFnoLD, n ¼ 30), and non-CF pediatric controls (n ¼ 20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. Results: miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (P < 0.0001). miR-25 (P ¼ 0.0011) and miR-21 (P ¼ 0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, P ¼ 0.002) and miR-25 (AUC 0.65, P ¼ 0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, P < 0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3–F4 fibrosis (AUC 0.73, P ¼ 0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1–F4 (AUC 0.72, P ¼ 0.02).
Received March 4, 2014; accepted October 8, 2014. From the Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, the yDepartment of Gastroenterology, Royal Children’s Hospital, Brisbane, Australia, and the zDepartment of Pediatrics, Baylor College of Medicine, Houston, TX, Brisbane, Australia. Address correspondence and reprint requests to Prof Grant A. Ramm, Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, PO Royal Brisbane and Women’s Hospital, Brisbane, QLD 4029, Australia (e-mail: [email protected]). Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jpgn.org). G.A.R., P.J.L., and R.W.S are supported by the National Health and Medical Research Council of Australia (NHMRC-APP1048740). G.A.R. is also supported by an NHMRC Senior Research Fellowship (APP1061332). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000600
JPGN
Volume 60, Number 2, February 2015
Conclusions: These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis. Key Words: cystic fibrosis liver disease, hepatic fibrosis, pediatrics, reference genes, RT-qPCR, serum miRNA
(JPGN 2015;60: 247–254)
L
iver fibrosis is responsible for considerable morbidity and mortality in patients with cystic fibrosis (CF), causing clinically significant complications in up to 20% of patients and 2% to 3% of overall mortality (1,2); however, the onset and progression of fibrosis in cystic fibrosis liver disease (CFLD) are difficult to predict and monitor, and factors relating to the early pathogenesis of CFLD are incompletely understood. Current measures of liver function (eg, serum biochemistry), combined with ultrasound and clinical examination, are insensitive and nonspecific for detection of early liver disease and assessment of progressive fibrosis severity. Liver biopsy remains the criterion standard for diagnosis of hepatic fibrosis in CFLD, but is invasive and may be confounded by the focal nature of disease activity (3,4). Thus, a sensitive, specific, and noninvasive test reflecting the early pathogenesis of CFLD is required to identify individuals at risk of developing hepatic fibrosis before the advent of severe complications. Posttranscriptional regulation of gene expression is now recognized as an important contributor to disease pathogenesis, among whose mechanisms include alterations in the function and stability of translational elements within both coding and noncoding regions of messenger RNA (mRNA). A major component of this process is binding by microRNAs (miRNAs), which function via base pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation. miRNAs are noncoding endogenously transcribed RNAs that undergo a well-characterized series of processing steps that generate short single-stranded (21 nucleotides in length) RNA fragments (5). miRNAs are critical to the regulation of virtually all of the cellular processes, and increasing evidence implicates dysregulated miRNA expression in a range of human pathologies, including liver diseases (6). Because miRNAs are readily detectable in serum and other body fluids, and exhibit remarkable stability compared with mRNA and other long RNAs (7–9), cell-free miRNAs have attracted interest as potential diagnostic biomarkers. Substantial evidence implicates miRNA involvement both in maintaining normal liver function and in processes associated with liver disease. Elevated serum levels of liver-specific miR-122 have
247
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Ramm et al
JPGN
been demonstrated in viral, alcoholic, metabolic, and drug-induced liver injury (10–14). These studies suggest that serum levels of miR-122 may act as biomarkers of tissue injury. Although certain tissue miRNAs may be implicated in CF complications of the lung and intestinal tract (15,16), no study to date has characterized serum miRNAs in CFLD. In the present study, we aimed to identify a circulating miRNA signature in children with CF with diagnostic potential for the detection of early CFLD. We hypothesized that the candidate liver-specific miR-122 would be elevated in the serum of patients with CFLD compared with in that of patients with CF without liver disease (CFnoLD), and we sought to quantify circulating miRNAs, including miR-122, in patients with CFLD, patients with CFnoLD, and non-CF controls. To do this, we initially used a polymerase chain reaction (PCR) array containing 84 serum-detectable miRNAs in a subset of samples to identify candidate miRNAs, which were then validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in a larger patient cohort.
METHODS Patients The present study was approved by the human ethics committees of QIMR Berghofer Medical Research Institute and the Royal Children’s Hospital, Brisbane, and informed consent was obtained from parents or guardians. Sera from 102 children were processed as described in the following text. Patients with CFLD (n ¼ 52) were initially selected by the presence of at least 2 of the following: hepatomegaly splenomegaly; persistent elevation of serum alanine aminotransferase (ALT >1 upper limit normal) longer than 6 months; abnormal ultrasound scan with abnormal echogenicity or nodular edge suggestive of cirrhosis, with subsequent dual-pass liver biopsies to assess CFLD and stage fibrosis, as previously reported (17). Liver histology was staged for hepatic fibrosis by blinded Scheuer scoring system (18). The highest fibrosis staging was used if fibrosis stages between the 2 biopsies were discordant. Age- and sex-matched children with CF but no signs of liver disease by clinical, biochemical, and ultrasonographic means were assigned to the CFnoLD group (n ¼ 30). Children attending the CF clinic underwent regular clinical examination, blood testing, and hepatobiliary ultrasound scanning. Patients were included in the CFnoLD group if they had normal clinical, biochemical, and ultrasonographic examination in the 2 years before study serum sampling. A non-CF, non–liver disease pediatric control group comprised 20 age- and sex-matched children who were recruited while attending the hospital for minor plastic procedures.
RNA Extraction and Reverse Transcription RNA was extracted from 200 mL serum using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) or the miRCURY RNA Isolation Kit for Biofluids (Exiqon, Vedbaek, Denmark), according to the manufacturer’s protocol. RNA was reverse transcribed using the miScript II RT Kit (Qiagen) or the miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon). RNA isolation and reverse transcription are described in detail in the supplementary Methods, http://links.lww.com/MPG/A394.
miRNA PCR Array and RT-qPCR Validation We first conducted a PCR array (Human Serum & Plasma miRNA PCR Array; Qiagen, cat. no. MIHS-106Z) in sera from a subset of 32 children, including 12 with CFLD, 10 with CFnoLD, and 10 controls, to assess the levels of 84 miRNAs detectable in
248
Volume 60, Number 2, February 2015
serum. Candidate miRNAs with the potential to distinguish liver disease in CF were identified from the PCR array screen for subsequent RT-qPCR validation using Exiqon reagents in the entire cohort of 102 patients. PCR amplification is described in detail in the supplementary Methods, http://links.lww.com/MPG/A394. Sequences of candidate miRNAs are found in supplemental Table 1, http://links.lww.com/MPG/A391.
Reference Gene Validation Reference gene validation was considered to be a critical prerequisite to RT-qPCR data normalization. The geNorm (19) tool in qbaseþ was used to rank the detectable miRNAs from the PCR array in order of stability and to identify an appropriate panel of reference genes for data normalization (supplemental Fig. 1, http:// links.lww.com/MPG/A392). geNorm assigned an expression stability measure (M) to each miRNA, with a recommended cutoff value of 1.5. Next, pairwise variation (V) analysis determined the optimal number of reference genes required for accurate data normalization, with a cutoff value of 0.15, below which additional reference genes are not required. geNorm determined that miR-93 and miR-19b comprised an appropriate set of reference genes for our experimental conditions; therefore, these reference genes were used to normalize all of the RT-qPCR data in the present study.
Data Analysis Quantification cycle (Cq) values were determined using instrument software and input into qbaseþ version 2.5 software (Biogazelle, Ghent, Belgium) (20) to calculate normalized relative quantities of each miRNA in the Qiagen PCR array and thus to identify candidate miRNAs that would be validated in the subsequent RT-qPCR experiments. The geNorm tool (19) was used for reference gene validation. In the present study, we have adhered to the Minimum Information for Publication of Quantitative RealTime PCR Experiments guidelines (21) in conjunction with recent recommendations for standardized reporting in cell-free miRNA studies (22), in which information was available. More details regarding data analysis are included in the supplementary Methods, http://links.lww.com/MPG/A394.
Statistical Analysis Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA) or SPSS Statistics version 19 (IBM, Armonk, NY), with P < 0.05 considered significant. Clinical characteristics of the CFLD and CFnoLD groups shown in Table 1 were analyzed using Student t test or Mann-Whitney U test. Normalized RT-qPCR data were subjected to log base 10 transformations and analyzed using 1-way analysis of variance (ANOVA) with Bonferroni post hoc test to compare all of the groups. Correlations between miRNA levels and serum aminotransferase levels were determined using Spearman rank correlations. Receiver operating characteristic (ROC) analyses and logistic regression were carried out to assess the ability of miRNAs, both individually and in combination, to distinguish between patient groups. To optimize both the positive predictive value (PPV) and negative predictive value (NPV) and provide the most clinically useful biomarker test derived from the ROC analysis to the clinician treating children suspected as having CFLD with abnormal routine tests, data are presented using maximum specificity for the test. Sample size evaluation for a clinical study is almost always a matter of compromise between the available resources and the various objectives. Our patients were chosen out of expedience, www.jpgn.org
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 60, Number 2, February 2015
Noninvasive Diagnostic Biomarkers of CF Liver Disease
TABLE 1. Demographic and clinical characteristics of the CFLD and CFnoLD groups
Boys, n (%) Girls, n (%) Age Mean (median) SD Range Alanine aminotransferase, U/L Mean (median) SD Range Aspartate aminotransferase, U/L Mean (median) SD Range g-Glutamyl transpeptidase, U/L Mean (median) SD Range Alkaline phosphatase, U/L Mean (median) SD Range Ultrasound Normal Abnormal Not available Scheuer fibrosis score (n) 0 (no fibrosis) 1 2 3 4 (cirrhosis)
CFLD (n ¼ 52)
CFnoLD (n ¼ 30)
P
29 (56) 23 (44)
16 (53) 14 (47)
NS
10.9 (10.9) 4.6 2.3–17.6
12.2 (12.0) 3.1 6.2–17.0
0.132
53 (42) 33 19–174
23 (20) 11 6–50
