ORIGINAL ARTICLE Obstructive Sleep Apnea Syndrome Affects Liver Histology and Inflammatory Cell Activation in Pediatric Nonalcoholic Fatty Liver Disease, Regardless of Obesity/Insulin Resistance Valerio Nobili1*, Renato Cutrera2*, Daniela Liccardo1, Martino Pavone2, Rita Devito3, Valentina Giorgio1, Elisabetta Verrillo2, Giuseppe Baviera4, and Giovanni Musso5 1 HepatoMetabolic Diseases Unit, 2Pneumology Unit, Sleep and Noninvasive Ventilation Laboratory, and 3Pathology Unit, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy; 4Family pediatrician, Rome, Italy; and 5Gradenigo Hospital, Turin, Italy

Abstract Rationale: Obstructive sleep apnea syndrome (OSAS) and nonalcoholic fatty liver disease (NAFLD) are frequently encountered in obese children. Whether OSAS and intermittent hypoxia are associated with liver injury in pediatric NAFLD is unknown. Objectives: To assess the relationship of OSAS with liver injury in pediatric NAFLD. Methods: Sixty-five consecutive children with biopsy-proven NAFLD (age, mean 6 SD, 11.7 6 2.1 yr; 58% boys; body mass index z score, 1.93 6 0.61) underwent a clinical-biochemical assessment and a standard polysomnography. Insulin sensitivity, circulating proinflammatory cytokines, markers of hepatocyte apoptosis (cytokeratin-18 fragments), and hepatic fibrogenesis (hyaluronic acid) were measured. Liver inflammatory infiltrate was characterized by immunohistochemistry for CD45, CD3, and CD163, surface markers of leukocytes, T cells, and activated macrophage/Kupffer cells, respectively. OSAS was defined by an apnea/hypopnea index (AHI) greater than or equal to 1 event/h, and severe OSAS was defined by an AHI greater than or equal to 5 events/h.

Nonalcoholic fatty liver disease (NAFLD) affects 3 to 10% of the general western pediatric population and 50 to 70% of obese children, and its prevalence is continuously rising along with the growing obesity epidemic (1, 2). NAFLD encompasses a histological spectrum ranging from simple steatosis to

Measurements and Main Results: Fifty-five percent of children with NAFLD had nonalcoholic steatohepatitis (NASH), and 34% had significant (stage F > 2) fibrosis. OSAS affected 60% of children with NAFLD; the presence and severity of OSAS were associated with the presence of NASH (odds ratio, 4.89; 95% confidence interval, 3.08–5.98; P = 0.0001), significant fibrosis (odds ratio, 5.91; 95% confidence interval, 3.23–7.42; P = 0.0001), and NAFLD activity score (b, 0.347; P = 0.029), independently of body mass index, abdominal adiposity, metabolic syndrome, and insulin resistance. This relationship held also in nonobese children with NAFLD. The duration of hemoglobin desaturation (SaO2 , 90%) correlated with increased intrahepatic leukocytes and activated macrophages/Kupffer cells and with circulating markers of hepatocyte apoptosis and fibrogenesis. Conclusions: In pediatric NAFLD, OSAS is associated with

biochemical, immunohistochemical, and histological features of NASH and fibrosis. The impact of hypoxemia correction on liver disease severity warrants evaluation in future trials. Keywords: sleep apnea; hypoxia; nonalcoholic fatty liver disease;

steatosis; fibrosis

nonalcoholic steatohepatitis (NASH); although the former has a benign hepatological course, NASH can progress to cirrhosis and hepatocellular carcinoma (3). Although NAFLD is generally a slowly progressive disease, children are reported with NAFLD as early as 2 years and with

NASH-related cirrhosis as early as age 8 years (4). Accordingly, early recognition of NASH in children is warranted to prevent liver disease progression (5), and recent AGA/ACG/AASLD guidelines acknowledged that those with onset of NAFLD in childhood may be most at risk

( Received in original form July 22, 2013; accepted in final form November 2, 2013 ) * These authors contributed equally as co–first authors. Author Contributions: V.N. and G.M.: conception and design of the study, analysis and/or interpretation of data; drafting and/or revision of the manuscript; approval of the final version of the manuscript. R.C., D.L., M.P., R.D., V.G., E.V., and G.B.: generation, collection, assembly, analysis, and/or interpretation of data; revision of the manuscript; approval of the final version of the manuscript. Correspondence and requests for reprints should be addressed to Giovanni Musso, M.D., Gradenigo Hospital, Corso Regina Margherita 8, 10132 Turin, Italy. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 189, Iss 1, pp 66–76, Jan 1, 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201307-1339OC on November 20, 2013 Internet address: www.atsjournals.org

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ORIGINAL ARTICLE

At a Glance Commentary Scientific Knowledge on the Subject: Obstructive sleep apnea has

been connected to the pathogenesis of liver injury and nonalcoholic steatohepatitis (NASH) in experimental nonalcoholic fatty liver disease (NAFLD). Whether obstructive sleep apnea syndrome (OSAS) is associated with the severity of NAFLD independently of coexisting overall/ abdominal obesity, insulin resistance, and metabolic syndrome is debated in humans, where most studies have been performed in bariatric surgery patients and no data in the pediatric population are available. The relative role of chronic intermittent hypoxemia and accompanying hypercarbia in the pathogenesis of liver injury in NAFLD is unclear in humans. What This Study Adds to the Field:

In children with NAFLD, the presence and severity of OSAS and of nocturnal hemoglobin desaturation are associated with the presence of NASH and with the severity of histological necroinflammation and fibrosis, independently of whole body/ abdominal adiposity, insulin resistance, and metabolic syndrome. The duration of nocturnal hypoxemia relates to increased number of activated leukocytes and macrophage/ Kupffer cells in the liver and to increased markers of hepatocyte apoptosis and fibrogenesis, regardless of systemic proinflammatory markers and cytokine levels. Children with NAFLD should be systematically screened for the presence of OSAS in clinical practice, and when OSAS is present, NASH and significant fibrosis should be ruled out. The impact of OSAS treatment and nocturnal hypoxemia correction on liver injury in pediatric NASH warrants assessment in therapeutic trials.

for severe liver-related complications later in life (6). OSAS is emerging as a highly prevalent condition in children, affecting up to 6% of the general pediatric population and up to

78% of obese children (7–10). OSAS and obesity frequently coexist and mutually interact in promoting cardiometabolic disorders; consistently, it is difficult to separate the effects of OSAS from those of obesity, and some suggested OSAS is a manifestation of the metabolic syndrome (11). Although OSAS is an established risk factor for diabetes and cardiovascular disease, its relationship with NAFLD is poorly defined, and it is debated whether OSAS promotes liver injury independently of coexisting comorbidities, including obesity, insulin resistance, and metabolic syndrome. Although experimental evidence suggests chronic intermittent hypoxia may per se trigger liver injury, inflammation, and fibrogenesis, data connecting OSAS to NAFLD in humans are controversial and restricted to morbidly obese adults (12). Although some studies found an independent relationship between OSAS and liver histology, in other studies this relationship was no longer significant after adjusting for comorbidities (13, 14). In pediatric NAFLD, the relationship of OSAS with the presence of progressive NASH and fibrosis is unknown; the only two studies available reported a prevalence of OSAS ranging from 55 to 89% in overweight/obese children with biochemical/ultrasonographic evidence of NAFLD, without any histological data. Establishing a link between OSAS and progressive NASH/fibrosis in children would have important screening implications and prompt evaluation of the impact of OSAS treatment on liver histology, as preliminary evidence suggests OSAS improvement reduces liver enzymes in children with NAFLD (9, 15). We assessed the relationship of OSAS to liver histology in children with biopsyproven NAFLD.

Methods Patients

The study was performed at Bambino Ges`u Children’s Hospital during the period January 2011 to May 2013, after approval by the Ethics Committee of the Bambino Ges`u Children’s Hospital and Research Institute, Rome, Italy. Sixty-six consecutive children with an ultrasonographic diagnosis of NAFLD, persistently (>6 mo) elevated serum aminotransferases, and symptoms/signs suggestive of sleep apnea (see Table E1 in

Nobili, Cutrera, Liccardo, et al.: OSAS and Severity of Pediatric NAFLD

the online supplement) were prospectively seen and offered liver biopsy and polysomnography (PSG) as part of the experimental protocol, designed to evaluate the relationship of sleep disturbance with liver disease severity. The decision to perform biopsy on children with NAFLD with persistently elevated alanine aminotransferase (ALT) was based on our previously published data demonstrating a high prevalence of necroinflammatory changes and fibrosis in children with ultrasonographic steatosis and persistently elevated liver enzymes. In that study, 81% of children with normal ALT levels at the time of liver biopsy had histological fibrosis (16). Among these 66 patients, 65 children were assented, and parents consented to be in the study. Hepatitis virus infection and other competing causes of hepatic steatosis were excluded according to standard guidelines, detailed in the online supplement. Body mass index (BMI) and waist circumference and their SD score (z score) were calculated (17–19). Metabolic syndrome (MS) was defined as the presence of three or more of the following five criteria (20): abdominal obesity as defined by a waist circumference greater than or equal to the 90th percentile for age and sex (19); hypertriglyceridemia as defined by triglycerides greater than the 95th percentile for age and sex (21); low high-density lipoprotein cholesterol as defined by less than the 5th percentile for age and sex (20); elevated blood pressure as defined by systolic or diastolic blood pressure above the 95th percentile for age and sex (22); and impaired fasting glucose, impaired glucose tolerance, or type 2 diabetes mellitus as previously described (23). Laboratory assessment. Aspartate aminotransferase, ALT, g-glutamyl-transferase, total triglycerides, and total and highdensity lipoprotein cholesterol were evaluated using standard laboratory methods. Insulin was measured using a radioimmunoassay (Myria Technogenetics, Milan, Italy). All participants underwent a standard oral glucose tolerance test (OGTT), performed with 1.75 g of glucose per kilogram of body weight (up to 75 g). Insulin sensitivity. Homeostasis model assessment of insulin resistance was calculated as the product of the fasting glucose and insulin concentration divided by 22.5 (24). 67

ORIGINAL ARTICLE Glucose and insulin were measured at 0, 30, 60, 90, and 120 minutes of the OGTT, and the insulin sensitivity index was calculated using the formula: insulin sensitivity index = (10,000/square root of [fasting glucose 3 fasting insulin] 3 [mean glucose 3 mean insulin during OGTT]) (24). Proinflammatory markers and cytokines. Serum C-reactive protein (CRP)

was determined via a high-sensitivity latex agglutination method on HITACHI 911 Analyzer (Sentinel Ch., Milan). Serum tumor necrosis factor-a and IL6 were measured by sandwich ELISA (R&D System Europe Ltd, Abingdon, UK). Sensitivity and intra- and interassay coefficients of variation of each kit are detailed in the online supplement. Markers of hepatocyte apoptosis.

Circulating cytokeratin-18 (CK-18) fragment levels, a validated marker of hepatocyte apoptosis in pediatric NAFLD (25), were measured by the M30Apoptosense ELISA kit (PEVIVA) purchased from Li Starfish (Milan, Italy). Markers of extracellular matrix deposition. Serum hyaluronic acid, a marker

of extracellular matrix deposition, was measured using an enzyme-linked binding protein assay (Hyaluronan; R&D Systems, Minneapolis, MN) (detailed in the online supplement) (26). Liver histology. The clinical indication for biopsy was either to assess the presence of NASH and degree of fibrosis or other likely independent or competing liver diseases. Liver biopsy was performed after an overnight fast, using an automatic core biopsy 18-gauge needle (Biopince; Amedic, Kista, Sweden) under general anesthesia and ultrasound guidance. A Sonoline Omnia ultrasound machine (Siemens, Munich, Germany) equipped with a 5-MHz probe (5.0 C 50, Siemens) and a biopsy adaptor was used. The length of liver specimen was recorded: only samples with a length greater than or equal to 15 mm and including at least five to six complete portal tracts were considered adequate for the purpose of the study. Biopsies were routinely processed (ie, formalin-fixed and paraffin-embedded), and sections of liver tissue were stained with hematoxylin-eosin, Van Gieson, periodic acid-Schiff diastase, and Prussian blue stain. Biopsies were evaluated by a single hepatopathologist, with long-time experience in the field of liver pathology, who was blinded to clinical and laboratory data. To determine the intraobserver agreement, the pathologist 68

scored the liver biopsies blindly twice, and the weighted k coefficients for different histological features were calculated. Steatosis, inflammation, hepatocyte ballooning, and fibrosis were scored using the NAFLD Clinical Research Network criteria (27). Briefly, steatosis was graded on a 4-point scale: grade 0 = steatosis involving less than 5% of hepatocytes; grade 1 = steatosis involving up to 33% of hepatocytes; grade 2 = steatosis involving 33 to 66% of hepatocytes; and grade 3 = steatosis involving more than 66% of hepatocytes. Lobular inflammation was graded on a 4-point scale: grade 0 = no foci; grade 1 = fewer than two foci per 2003 field; grade 2 = two to four foci per 2003 field; and grade 3 = more than four foci per 2003 field. Hepatocyte ballooning was graded from 0 to 2: 0 = none; 1 = few balloon cells; and 2 = many/prominent balloon cells. The stage of fibrosis was quantified using a 5-point scale: stage F0 = no fibrosis; stage F1 = perisinusoidal or periportal (1a = mild, zone 3, perisinusoidal; 1b = moderate, zone 3, perisinusoidal; 1c = portal/periportal); stage F2 = perisinusoidal and portal/periportal; stage F3 = bridging; and stage F4 = cirrhosis. Additionally, the presence of Mallory bodies and portal fibrosis were noted. Features of steatosis, lobular inflammation, and hepatocyte ballooning were combined to obtain the NAFLD activity score (NAS). As recently recommended by NASH Clinical Research Network (28), a microscopic diagnosis based on overall injury pattern (steatosis, hepatocyte ballooning, inflammation) as well as the presence of additional lesions (e.g., zonality of lesions, portal inflammation, and fibrosis) has been assigned to each case. Accordingly, biopsies were subdivided into: not-NASH and definite NASH subcategories (28). Liver immunohistochemistry. We investigated the relationship between OSAS and the nature and severity of hepatic inflammatory cell infiltration by assessing three different surface markers (CD45, CD3, and CD163) in liver tissue (detailed in the online supplement). CD45, known as common leukocyte antigen, is one of the most abundant leukocyte cell surface glycoproteins, and its expression is restricted to hematopoietic cells (29). CD3 antigen represents a specific and sensitive T-cell lineage marker, including natural killer and natural killer T cells (30). CD163

is a member of the cysteine scavenger receptor superfamily that is expressed on activated cells of monocyte/macrophage origin, including Kupffer cells (30, 31). The number of CD451, CD1631, and CD31 cells in both the portal tract and liver lobule has been recently related to the severity of liver injury in pediatric NAFLD (32). PSG study. All consecutive patients with biopsy-proven NAFLD underwent an overnight PSG using standard techniques. The patients received no dietary or lifestyle counseling or any other treatment between liver biopsy and PSG, which was performed within 3 months of the histological diagnosis of NAFLD. PSG was performed in a quiet room in the sleep laboratory of our hospital. All recordings started at the patients’ usual bedtime and continued until spontaneous awakening. No hypnotic drugs were allowed for at least 2 weeks before sleep recording. All patients were accompanied by one of their parents throughout the night. No oxygen was supplemented and no respiratory stimulants were used. The PSG montage included four EEG channels (C3-A2, C4-A1, O1-A2, and O2-A1), left and right electrooculogram, chin EMG, ECG, nasal cannula, thoracic and abdominal respiratory effort, oxygen saturation (Siesta; Compumedics, Abbottsford, Australia), and end-tidal carbon dioxide pressure were monitored simultaneously with other parameters (Capnostream; Oridion, Needham, MA). They were scored manually and interpreted according to current guidelines (2007 American Academy of Sleep Medicine Manual for the Scoring of Sleep and Associated Events: Rules, Terminology and Technical Specifications, Version 2.0) (33). Central, obstructive, and mixed apnea events were counted according to the criteria established by the American Thoracic Society (2007). An obstructive apnea was defined as the absence of airflow, with continued chest wall and abdominal movement, for a duration of at least two breaths. A central apnea was defined as the absence of airflow with the cessation of respiratory effort, lasting more than 20 seconds and associated with bradycardia and desaturation. Central apnea occurring after gross body movements or after sighs was not considered as a pathological finding. A mixed apnea was defined as an apnea that usually begins as central and ends

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 1 | January 2014

ORIGINAL ARTICLE in obstruction according to changes in the chest, abdominal, and flow traces. Hypopnea was defined as a decrease in nasal flow of at least 50%, with a corresponding decrease in SaO2 of at least 4% and/or an arousal. The apnea/hypopnea index (AHI) was defined as the number of apneas and hypopneas per hour of total sleep time (TST). Patients with an AHI greater than or equal to 1 event/h were considered to have OSAS, and subjects with an AHI greater than or equal to 5 events/h were considered to have severe OSAS, according to current guidelines (33, 34). The following parameters were also recorded: oxygen desaturation index (i.e., the number of hemoglobin desaturations [drop in SaO2 > 4% of baseline value] per hour of sleep), mean SaO2, nadir SaO2, total duration of hemoglobin desaturation (SaO2 , 90%), expressed as % TST, and mean end-tidal carbon dioxide pressure. All recordings were manually and visually scored by one of the investigators (E.V.), and the sleep parameters derived were tabulated for statistical analysis. Adenotonsillar hypertrophy and other symptoms/signs associated with OSAS. All

consecutive children with NAFLD underwent a detailed evaluation at the time of PSG to assess the prevalence of adenotonsillar hypertrophy (ATH) and of other symptoms/signs associated with OSAS, as indicated by current guidelines (34). Briefly, physical examination was performed for assessment of associated craniofacial abnormalities, middle ear effusion, and size of the tonsils. ATH was assessed and graded by a single otorhinolaryngologist, blinded to patient data, according to a standard 4-stage scale (from 0 = absent to 4 = severe) based on the percentage of oropharyngeal airway occupied by the two tonsils (35). The size of the tonsils was scored as 0 to 4. Adenoid size and nasopharyngeal airway space were assessed by lateral cephalometric X-ray (36). Statistical Analysis

Data were expressed as mean 6 SD. Differences were considered statistically significant at P , 0.05. Differences across groups were analyzed by analysis of variance and Bonferroni correction when variables were normally distributed; otherwise, the Kruskal-Wallis test, followed by the post hoc Dunn test, was used to compare nonparametric variables.

Normality was evaluated by Shapiro-Wilk test. Chi-square test or Fisher exact test were used to compare categorical variables, as appropriate. Spearman rank correlation coefficient was used to estimate the relationship between different variables. Standard multivariate logistic regression analysis was used to identify independent predictors of NASH and significant (stage F > 2) fibrosis, included as a dichotomous variable. For this analysis, quartiles of continuous variables were included. Multiple linear regression analysis was applied to identify predictors of NAS score, after log transformation of continuous variables with skewed distribution. Furthermore, stepwise regression models were built to identify the best combination of parameters to predict the outcome variables. At each step, variables were entered for P , 0.05 and removed for P . 0.1. STATISTICA 5.1 (StatSoft Italia, Padua) was used for all analyses.

Results Clinical and Laboratory Characteristics

Sixty-five out of 66 children assented to participate to the study. Anthropometric and laboratory data of included children are described in Table 1. In the whole NAFLD cohort, 73% of the subjects were obese, and 20% had metabolic syndrome. Patients with NASH had a higher BMI and a higher prevalence of hypertriglyceridemia and metabolic syndrome than patients without NASH. Overall, the features of children included in this study resembled those seen in the general pediatric NAFLD population seen at our Center (26). Liver Histology and Immunohistochemistry

The pathologist had good to excellent intraobserver agreement between readings, as demonstrated by a weighted k coefficient of 0.89 for steatosis, 0.72 for lobular inflammation, 0.75 for portal inflammation, 0.82 for ballooning, and 0.87 for fibrosis. Overall, the second reading of biopsy slides did not change classification of any NASH or non-NASH case. All 65 children with ultrasonographic steatosis had NAFLD, and 36 (55%) subjects had NASH on liver biopsy. Mean values of each histological parameter are reported in

Nobili, Cutrera, Liccardo, et al.: OSAS and Severity of Pediatric NAFLD

Table 2. Eleven (17%) subjects had no fibrosis, 30 (46%) subjects had stage F1 fibrosis, 22 (34%) subjects had stage F2 fibrosis, and 2 (3%) subjects had stage F3 fibrosis. No patient had cirrhosis. Consistent with previous findings (32), there was a significant positive association between the number of CD451 and CD1631 cells in both the portal tract and liver lobule with the severity of steatosis, ballooning, NAS score, and fibrosis (P , 0.01 for all). Overall, histological and immunohistochemical features resembled those seen in the general pediatric NAFLD population seen at our center (26). Impact of OSAS on NASH and metabolic parameters. In the whole NAFLD

cohort, 39 (60%) subjects had OSAS: the prevalence of OSAS was significantly higher in patients with NASH than in patients without NASH (Table 1). BMI z score, waist circumference, insulin sensitivity indices, and the percentage of obese subjects did not significantly differ between children with and without OSAS, whereas children with OSAS had a higher prevalence of metabolic syndrome than children without OSAS (Table 3). Thirty-four (87%) subjects with OSAS had NASH, as compared with 8% of subjects without OSAS (P = 0.00001) (Table 4). The presence of OSAS and BMI z score independently predicted the presence of NASH on logistic regression analysis (Table 5); in a stepwise regression model, OSAS (b = 0.521, SE[b] = 0.074), and BMI z score (b = 0.377, SE[b] = 0.072) significantly contributed to NASH (adjusted R2 = 0.61; P = 0.000001). Similarly, NAS, a quantitative index of the severity of histological steatosis and necroinflammation, was independently predicted by the severity of OSAS and of nocturnal hemoglobin desaturation, as assessed by AHI and by SaO2 less than 90% (% TST), and by BMI z score (Figure 1, Table 6). In a stepwise regression model, AHI (b = 0.481, SE[b] = 0.091), SaO2 less than 90% (b = 0.356, SE[b] = 0.083), and BMI z score (b = 0.349, SE[b] = 0.099) contributed significantly to NAS variation (adjusted R2 = 0.42; P = 0.000003). To further evaluate the additive effect of BMI and OSAS on the severity of NASH, we plotted quartiles of BMI z score and presence/absence of OSAS. At each 69

ORIGINAL ARTICLE Table 1: Demographic, Clinical, and Biochemical Features of Study Population, Grouped According to the Presence/Absence of Nonalcoholic Steatohepatitis in the Whole Population (N = 65)

Parameter

All Subjects with NAFLD (n = 65)

Subjects without NASH (n = 29)

Subjects with NASH (n = 36)

P Value, NASH vs. non-NASH

11.7 6 2.1 38 (58) 27.9 6 5.3 1.93 6 0.61 48 (73) 89.5 6 4.9 1.99 6 0.51 114 6 13 68 6 8 35 6 15 38 6 17 20 6 13 39 (60) 13 (20) 2.99 6 3.29 1.07 6 1.95 477.7 6 37.4 95.8 6 0.9 90.5 6 2.0 32 6 39 46.4 6 6.2 15.5 6 9.7 88 6 15 3.37 6 1.94 111 6 65 161 6 33 42 6 7 1.5 6 0.4 11 6 6 6.7 6 2.1 274 6 81 1,213 6 429 13 (20) 50 (77) 10 (16) 22 (34) 22 (34) 10 (15) 2 (3)

11.7 6 1.8 19 (66) 26.1 6 5.5 1.71 6 0.59 17 (58) 89.2 6 5.2 1.91 6 0.78 114 6 16 68 6 8 33 6 15 34 6 16 17 6 7 9 (14) 0 0.69 6 0.44 0.69 6 0.63 473.6 6 46.2 96.1 6 0.9 91.6 6 1.1 869 44.3 6 3.7 15.1 6 10.4 87 6 12 3.22 6 2.00 100 6 48 155 6 25 44 6 7 1.6 6 0.4 11 6 6 6.9 6 2.1 197 6 37 1,095 6 438 3 (10) 20 (72) 3 (10) 6 (21) 11 (38) 5 (17) 0

11.6 6 2.4 19 (53) 29.4 6 5.0 2.12 6 0.56 31 (86) 89.7 6 4.1 2.06 6 0.78 114 6 11 68 6 8 37 6 15 43 6 18 23 6 16 30 (83) 13 (36) 4.83 6 3.63 1.51 6 1.83 480.1 6 43.1 95.6 6 0.8 89.6 6 2.1 52 6 44 48.1 6 7.2 16.0 6 9.4 89 6 17 3.52 6 2.06 120 6 72 166 6 39 41 6 7 1.5 6 0.4 11 6 6 6.6 6 2.2 336 6 45 1,350 6 452 10 (28) 30 (83) 7 (20) 16 (44) 11 (31) 5 (14) 2 (6)

0.769 0.347 0.013 0.007 0.026 0.701 0.439 0.948 0.885 0.311 0.059 0.100 0.00001 0.001 0.000001 0.031 0.518 0.021 0.00001 0.0000001 0.009 0.713 0.543 0.736 0.209 0.224 0.237 0.568 0,634 0.667 0.0000001 0.039 0.020 0.636 0.381 0.038 0.545 0.903 0.407

Age, yr Sex, male BMI, kg/m2 BMI, z score Obese subjects* Waist circumference, cm Waist circumference, z score Systolic BP, mm Hg Diastolic BP, mm Hg AST, IU/L ALT, IU/L GGT, IU/L Subjects with OSAS Subjects with severe OSAS† AHI, events/h ODI TST, min Mean SaO2, % Nadir SaO2, % SaO2 , 90%, % TST ETpCO2, mm Hg Insulin, mU/ml Glucose, mg/dl HOMA-IR Triglyceride, mg/dl Total cholesterol, mg/dl HDL cholesterol, mg/dl C-reactive protein, mg/L IL-6, pg/ml TNF-a, pg/ml CK-18, U/L Hyaluronic acid, ng/ml Subjects with Met Sy Subjects with abdominal obesity‡ Subjects with hypertension Subjects with hypertriglyceridemia Subjects with low HDL cholesterol Subjects with IFG/diabetes Subjects with diabetes

Definition of abbreviations: AHI = apnea/hypopnea index; ALT = alanine aminotransferase; AST = aspartate aminotransferase; BMI = body mass index; BP = blood pressure; CK-18 = cytokeratin-18 fragments; ETpCO2 = mean end-tidal carbon dioxide pressure; GGT = g-glutamyl-transferase; HDL = high-density lipoprotein; HOMA-IR = homeostasis model assessment of insulin resistance; IFG = impaired fasting glucose; ISI = insulin sensitivity index; Met Sy = metabolic syndrome; NAFDL = nonalcoholic fatty liver disease; NASH = nonalcoholic steatohepatitis; ODI = oxygen desaturation index (no. of hemoglobin desaturations [drop in SaO2 > 4% of baseline value] per hour of sleep); OSAS = obstructive sleep apnea syndrome; TNF = tumor necrosis factor; TST = total sleep time. Data are reported as mean 6 SD or n (%). Differences were considered statistically significant at P , 0.05. Statistically significant differences are indicated in boldface. SaO2 , 90% is the total duration of hemoglobin desaturation, expressed as percentage of TST. *Obesity was defined as BMI > 95th percentile. † Severe OSAS was defined by an AHI > 5 events/h. ‡ Abdominal obesity was defined by a waist circumference > 90th percentile for age and sex.

quartile of BMI z score, the presence of OSAS was associated with a significantly higher NAS score (Figure 2). Impact of OSAS on hepatic fibrosis.

Children with OSAS had a higher prevalence of portal fibrosis than children without OSAS (Table 4). The prevalence of significant (stage F > 2) fibrosis was significantly higher in children with severe 70

OSAS than in children with mild OSAS or without OSAS (Table 4). On logistic regression analysis, the presence of severe OSAS and BMI z score independently predicted the presence of significant fibrosis (Table 5). Severe OSAS (b = 0.360, SE[b] = 0.137) and BMI z score (b = 0.339, SE[b] = 0.125) contributed to significant fibrosis

in a stepwise regression model (adjusted R2 = 0.26; P = 0.01). Similarly, fibrosis stage, a semiquantitative index of the severity of histological hepatic fibrosis, was independently predicted by duration of hemoglobin desaturation, expressed as SaO2 less than 90% (% TST) on multiple regression analysis (Figure 1, Table 6). In

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 1 | January 2014

ORIGINAL ARTICLE Table 2: Liver Immunohistochemistry and Histology of Study Population (N = 65), Grouped According to the Presence/Absence of Nonalcoholic Steatohepatitis

Parameter NAS score Steatosis score Lobular inflammatory score Ballooning score Fibrosis score Fibrosis stage (F) 0 1 1a 1b 1c 2 3 4 Fibrosis any stage Significant fibrosis, stage > 2 Portal fibrosis Mallory bodies CD451 cells, n* CD1631 cells, n* CD31 cells, n*

All Subjects with NAFLD (n = 65) 4.7 2.2 1.5 1.0 1.2

6 6 6 6 6

1.4 0.7 0.7 0.6 0.8

11 (17) 30 (46) 0 6 (9) 24 (37) 22 (34) 2 (3) 0 54 (83 22 (34) 44 (68) 8 (12) 124.43 6 48.22 118.42 6 34.75 19.14 6 9.72

Subjects without NASH (n = 29) 3.43 1.62 1.06 0.72 0.89

6 6 6 6 6

0.73 0.56 0.52 0.45 0.70

9 (31) 14 (48) 0 5 (17) 9 (31) 6 (21) 0 0 20 (69) 4 (14) 14 (48) 0 99.51 6 42.91 98.71 6 20.1 20.79 6 12.77

Subjects with NASH (n = 36)

P Value, NASH vs. non-NASH

6 6 6 6 6

0.00001 0.00001 0.00002 0.00001 0.001

5.69 2.61 1.78 1.31 1.50

0.86 0.50 0.43 0.54 0.70

2 (6) 16 (44) 0 1 (3) 15 (42) 16 (44) 2 (6) 0 34 (94) 18 (50) 30 (83) 8 (22) 144.81 6 43.13 134.55 6 36.18 17.77 6 6.28

0.017 0.891 0.999 0.081 0.441 0.080 0.210 0.899 0.017 0.006 0.005 0.012 0.001 0.0004 0.371

Definition of abbreviations: NAFDL = nonalcoholic fatty liver disease; NAS = nonalcoholic fatty liver disease activity score; NASH = nonalcoholic steatohepatitis. Data are reported as mean 6 SD or n (%). Differences were considered statistically significant at P , 0.05. Statistically significant differences are indicated in boldface. *Counted in 10 portal tracts and in 10 areas of lobular region at a magnification of 320 under light microscopy.

a stepwise regression model, SaO2 less than 90% (b = 0.423, SE[b] = 0.114) significantly contributed to fibrosis stage variation (adjusted R2 = 0.22; P = 0.007).

AHI between children with and without ATH. The prevalence of ATH and of other symptoms/signs associated with OSAS is reported in Table E2.

Impact of OSAS on Intrahepatic Inflammatory Infiltrate and on Circulating Markers of Hepatocyte Apoptosis and Fibrogenesis

Impact of OSAS on Liver Disease in Nonobese Children

On multiple regression analysis, SaO2 less than 90% independently predicted serum CK-18 fragments (b = 0.398, P = 0.002) and hyaluronic acid (b = 0.551, P = 0.0005) and the number of CD451 cells (b = 0.358, P = 0.017) and of CD1631 cells (b = 0.435, P = 0.002) in the blood and liver, respectively, of patients with NAFLD (Figures 1A–1D; Table E1). ATH and other symptoms/signs associated with OSAS. ATH was present in

15% of children and was stage 3 in 8% of children. There was no significant difference in the prevalence and severity of ATH between children with and without OSAS (P = 0.701), either in the whole cohort or in nonobese or obese subgroups (P = 0.812). Furthermore, there was no difference in

It has been suggested that OSAS can trigger liver injury and NASH only when superimposing on background obesity (37); furthermore, in children, two distinct phenotypes of OSAS have been proposed to exist, one in the absence of obesity and often associated with marked lymphadenoid hypertrophy (type I) and the other associated with obesity (type II), with distinct comorbidities and cardiometabolic risk (38). On this basis we examined the impact of OSAS on liver disease and cardiometabolic profile separately in obese and nonobese children with NAFLD (Tables E3 and E4). Among nonobese children with NAFLD (n = 18), 39% of subjects had OSAS. Despite similar BMI z score and waist circumference, children with OSAS had a higher prevalence of NASH, higher

Nobili, Cutrera, Liccardo, et al.: OSAS and Severity of Pediatric NAFLD

serum CRP, and more severe insulin resistance than nonobese children without OSAS (Table E2). The presence of OSAS was associated with the presence of NASH independently of CRP and insulin resistance (odds ratio, 5.21; 95% confidence interval, 3.71–7.92; P = 0.009).

Discussion Main findings of our study are the following: 1. OSAS is a frequent finding in pediatric NAFLD, affecting 68% of obese and 44% of nonobese subjects in a sample of consecutive children with biopsy-proven NAFLD. 2. In children with NAFLD, the presence and severity of OSAS is associated with the severity of liver histology, independently of whole-body/ abdominal obesity, metabolic syndrome, and insulin resistance. 3. In pediatric NAFLD, the duration of nocturnal hypoxia correlates with increased liver infiltration by leukocytes and activated macrophage/Kupffer cells 71

ORIGINAL ARTICLE Table 3: Main Demographic, Clinical, and Biochemical Features of Study Population, Grouped According to the Presence and Severity of Obstructive Sleep Apnea Syndrome (N = 65)

Parameter Age, yr Sex, male BMI, kg/m2 BMI, z score Obese subjects Waist circumference, cm Waist circumference, z score Systolic BP, mm Hg Diastolic BP, mm Hg AST, IU/L ALT, IU/L GGT, IU/L AHI, events/h ODI TST, min Mean SaO2, % Nadir SaO2, % SaO2 , 90%, % TST ETpCO2, mm Hg Insulin, mU/ml Glucose, mg/dl HOMA-IR ISI Triglyceride, mg/dl Total cholesterol, mg/dl HDL cholesterol, mg/dl C-reactive protein, mg/L IL-6, pg/ml TNF-a, pg/ml CK-18, U/L Hyaluronic acid, ng/ml Subjects with Met Sy Subjects with abdominal obesity Subjects with hypertension Subjects with hypertriglyceridemia Subjects with low HDL cholesterol Subjects with IFG/diabetes Subjects with diabetes

Subjects without OSAS (n = 26)

Subjects with OSAS (n = 39)

P Value, OSAS vs. non-OSAS

Mild OSAS (n = 26)

Severe OSAS (n = 13)

P Value, Mild vs. Severe OSAS

11.6 6 2.0 16 (62) 26.4 6 5.8 1.80 6 0.64 15 (58) 88.7 6 5.5 1.97 6 0.82 113 6 16 68 6 8 33 6 15 35 6 16 18 6 7 0.51 6 0.27 0.59 6 0.58 471.8 6 44.1 96.4 6 0.9 91.8 6 1.2 668 44.1 6 3.5 16.0 6 10.6 88 6 15 3.45 6 2.01 3.68 6 2.50 103 6 49 157 6 25 44 6 7 1.6 6 0.4 11.1 6 4.5 6.8 6 2.0 212 6 56 1,089 6 427 3 (12) 19 (73) 5 (19) 6 (23 9 (35) 4 (15) 1 (4)

11.8 6 2.0 22 (56) 28.3 6 5.2 1.99 6 0.61 34 (87) 90.0 6 4.3 2.00 6 0.76 114 6 11 68 6 8 36 6 15 40 6 18 22 6 15 4.43 6 2.89 1.51 6 1.83 482.1 6 47.3 95.5 6 0.9 89.8 6 2.0 47 6 43 47.8 6 6.9 15.7 6 9.4 89 6 17 3.47 6 2.00 3.16 6 1.42 118 6 73 165 6 39 41 6 7 1.5 6 0.4 11 6 6 2.2 6 6.6 311 6 71 1,205 6 482 10 (26) 31 (79) 5 (13) 16 (41) 13 (33) 6 (15) 1 (3)

0.598 0.686 0.069 0.072 0.081 0.288 0.897 0.948 0.885 0.812 0.281 0.276 0.0000001 0.020 0.113 0.0006 0.000006 0.0000005 0.006 0.988 0.488 0.712 0.336 0.233 0.269 0.251 0.568 0,634 0.667 0.0000008 0.255 0.028 0.813 0.781 0.214 0.739 0.991 0.713

11.8 6 2.0 15 (58) 28.1 6 5.1 1.95 6 0.58 22 (85) 89.9 6 4.5 1.95 6 0.67 112 6 9 67 6 8 32 6 13 37 6 16 22 6 18 1.96 6 0.41* 1.01 6 0.60† 490.2 6 46.9 95.7 6 0.7‡ 90.3 6 1.8† 22 6 18* 45.5 6 3.8 13.1 6 8.7 84 6 17 2.72 6 1.83 3.38 6 1.25 122 6 70 164 6 39 40 6 6‡ 1.5 6 0.4 10.9 6 4.9 6.9 6 2.1 294 6 59 1,004 6 439 7 (27)‡ 21 (81) 2 (8) 12 (46) 10 (38) 3 (12) 0

11.9 6 2.7 7 (54) 30.1 6 5.3 2.10 6 0.62 12 (92) 89.9 6 4.9 2.11 6 0.89 117 6 13 71 6 7 45 6 22 47 6 17 19 6 13 8.71 6 2.5* 2.44 6 1.10* 479.4 6 41.4 95.1 6 0.9† 88.8 6 1.9* 91 6 37* 51.7 6 9.3 16.1 6 9.1 93 6 17 3.70 6 2.11 3.01 6 0.99 117 6 71 152 6 33 40 6 7‡ 1.5 6 0.5 13.3 6 4.8 6.9 6 2.3 338 6 47 1,558 6 461† 3 (23)‡ 10 (77) 3 (23) 4 (31) 3 (23) 3 (23) 1 (8)

0.916 0.912 0.111 0.160 0.294 0.813 0.552 0.291 0.578 0.073 0.179 0.613 0.000001 0.0001 0.229 0.028 0.021 0.000003 0.024 0.682 0.501 0.284 0.341 0.514 0.312 0.244 0.748 0.213 0.713 0.009 0.004 0.562 0.693 0.297 0.313 0.469 0.119 0.401

Data are reported as mean 6 SD or n (%). Differences were considered statistically significant at P , 0.05. Statistically significant differences are indicated in boldface. For definition of abbreviations, see Table 1. *P , 0.0001 vs. subjects without OSAS. † P , 0.01 vs. subjects without OSAS. ‡ P , 0.05 vs. subjects without OSAS.

and with markers of hepatocyte apoptosis. OSAS is characterized by episodes of chronic intermittent hypoxia and sleep fragmentation, which increase sympathetic activity and promote oxidative stress, proinflammatory cytokine production, endothelial dysfunction, and metabolic dysregulation (11, 12). These mechanisms provide the pathophysiological basis for the increased cardiometabolic risk of OSAS observed in adults, which was 72

reduced by effective OSAS treatment (11, 34, 39–41). OSAS is also increasingly recognized in the pediatric population, where it is associated with a host of adverse conditions, including cognitive and behavioral abnormalities, growth retardation, hypertension, cor pulmonale, glucose intolerance, and metabolic syndrome, most of which improve after OSAS treatment (34, 42–45). On this basis, the International Diabetes Federation recommended that adults with OSAS should be routinely

screened for cardiometabolic disorders and that the possibility of OSAS should be considered in all patients with diabetes and the metabolic syndrome (46); the recent American Academy of Pediatrics guidelines recognized that childhood OSAS may jeopardize long-term cardiometabolic health (34). Growing experimental evidence connects OSAS to the pathogenesis and progression of NAFLD, as well (12); in cellular and animal models, chronic intermittent hypoxia promoted hepatic triglyceride accumulation, necroinflammation, and fibrosis (12).

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ORIGINAL ARTICLE Table 4: Liver Immunohistochemistry and Histology Of Study Population (N = 65), Grouped According to the Presence and Severity of Obstructive Sleep Apnea Syndrome

Parameter Subjects with NASH NAS score Steatosis score Lobular inflammatory score Ballooning score Fibrosis stage Subjects with fibrosis stage (F) 0 1 1a 1b 1c 2 3 4 Fibrosis any stage Significant (F > 2) fibrosis Portal fibrosis Mallory bodies CD451 cells, nx CD1631 cells, nx CD31 cells, nx

Subjects without OSAS (n = 26)

Subjects with OSAS (n = 39)

P Value, OSAS vs. non-OSAS

2 (8) 3.53 6 0.95 1.65 6 0.52 1.07 6 0.56 0.80 6 0.45 1.1 6 0.6

34 (87) 5.44 6 1.09 2.51 6 0.57 1.72 6 0.47 1.21 6 0.59 1.3 6 0.7

0.00001 0.00001 0.00001 0.00002 0.013 0.171

5 (19) 14 (54) 0 5 (19) 9 (35) 7 (27) 0 0 21 (81) 7 (27) 13 (50) 0 96.65 6 41.22 102.42 6 29.06 21.02 6 13.48

6 (15) 16 (41) 0 1 (3) 15 (38) 15 (38) 2 (5) 0 33 (85) 17 (44) 31 (79) 8 (21) 142.94 6 44.05 129.09 6 34.64 17.88 6 6.13

0.811 0.412 0.999 0.034 0.798 0.512 0.492 0.999 0.734 0.218 0.026 0.037 0.001 0.009 0.393

Mild OSAS (n = 26)

Severe OSAS (n = 13)

21 (81)* 5.15 6 1.01* 2.30 6 0.43* 1.46 6 0.45† 1.09 6 0.37† 1.1 6 0.6

13 (100)* 5.71 6 0.82* 2.69 6 0.49* 1.72 6 0.48* 1.42 6 0.39* 1.6 6 0.7‡

6 (23) 0 14 (54 2 (15)‡ 0 0 1 (4) 0 11 (42) 3 (23) 6 (23) 9 (69)‡ 0 2 (15) 0 0 20 (77) 13 (100) 6 (23) 11 (85)‡ 20 (77) 11 (87) 2 (8) 6 (46) 140.65 6 40.20 178.33 6 38.17 125.86 6 34.10 172.85 6 25.84 21.06 6 13.89 17.49 6 6.16

P Value, Mild vs. Severe OSAS 0.229 0.030 0.045 0.187 0.041 0.037 0.158 0.048 0.999 0.959 0.304 0.015 0.199 0.999 0.148 0.0001 0.888 0.017 0.009 0.0002 0.301

Definition of abbreviations: NAFDL = nonalcoholic fatty liver disease; NAS = nonalcoholic fatty liver disease activity score; NASH = nonalcoholic steatohepatitis; OSAS = obstructive sleep apnea syndrome. Data are reported as mean 6 SD or n (%). Differences were considered statistically significant at P , 0.05. Statistically significant differences are indicated in boldface. *P , 0.0001 vs. subjects without OSAS. † P , 0.01 vs. subjects without OSAS. ‡ P , 0.05 vs. subjects without OSAS. x Counted in 10 portal tracts and in 10 areas of lobular region at a magnification of 320 under light microscopy.

Consistent with these data, OSAS was connected to an increased NAFLD prevalence and severity in morbidly obese adults, and two randomized trials documented an improvement in transaminases and ultrasonographic steatosis with OSAS treatment (9, 10, 14, 15, 47). However, the relationship of OSAS to the severity of liver histology in pediatric NAFLD was unknown to date, as only two studies documented an increased prevalence of NAFLD, as estimated by transaminase elevation or ultrasound, in overweight/obese children referred for suspected sleep-disordered breathing at two sleep medicine centers (9, 10). Our study revealed a high prevalence of OSAS in consecutive children with biopsyproven NAFLD, approaching 68% of obese and 44% of nonobese subjects. The presence of OSAS was associated with the presence of NASH and of significant fibrosis, and the severity of sleep apnea and nocturnal hypoxemia correlated with NAS score and fibrosis stage, independently

of overall/abdominal obesity, metabolic syndrome, and insulin resistance (Tables 5, 6, and E1). Furthermore, the duration of

nocturnal hemoglobin desaturation independently correlated with the number of liver-infiltrating leukocytes and activated

Table 5: Logistic Regression Analysis of the Predictors of Nonalcoholic Steatohepatitis and of Significant (Stage F > 2) Fibrosis in the Whole Nonalcoholic Fatty Liver Disease Population (N = 65) Factor Predictors of NASH Presence of metabolic syndrome Presence of OSAS BMI z score Hypertriglyceridemia Insulin sensitivity index Predictors of significant (stage F > 2) fibrosis Presence of metabolic syndrome Presence of severe OSAS BMI z score Abdominal obesity NASH

OR

95% CI

P Value

3.13 4.89 1.78 1.41 0.99

0.88–6.33 3.08–5.98 1.11–6.82 0.62–3.89 0.45–2.38

0.065 0.0001 0.037 0.383 0.691

1.21 5.91 2.51 0.41 1.66

0.24–6.49 3.23–7.42 1.22–5.86 0.09–1.43 0.71–4.49

0.712 0.0001 0.030 0.270 0.182

Definition of abbreviations: BMI = body mass index; CI = confidence interval; NASH = nonalcoholic steatohepatitis; OR = odds ratio; OSAS = obstructive sleep apnea syndrome. OSAS was entered as a dichotomous variable (present/absent). Severe OSAS was defined by an AHI > 5 events/h. Abdominal obesity was defined by a waist circumference > 90th percentile for age and sex. Statistically significant associations are indicated in boldface.

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ORIGINAL ARTICLE

Figure 1. Correlation of total duration of hemoglobin desaturation on polysomnogram, expressed as SaO2 , 90% (% total sleep time [TST]), with serum cytokeratin-18 (CK18) fragments, a marker of hepatocyte apoptosis (A); with serum hyaluronic acid, a marker of fibrogenesis (B); with the number of liver CD451 and CD1631 cells (C, D); with histological nonalcoholic fatty liver disease activity score (E); and with fibrosis stage (F). SaO2 , 90% had a skewed distribution and was log-transformed.

Kupffer cells/macrophages, which are believed to play a key role in the pathogenesis of liver injury in NAFLD (48).

These novel immunohistochemical findings and the association of SaO2 less than 90% (% TST) w ith markers of hepatocyte

Table 6: Multiple Regression Analysis of the Predictors of the Severity of Nonalcoholic Steatohepatitis, as Estimated by Nonalcoholic Fatty Liver Disease Activity Score and of Fibrosis Stage, in the Whole Nonalcoholic Fatty Liver Disease Population (N = 65) Parameter Predictors of NAS BMI, z score Metabolic syndrome, n-criteria HDL cholesterol Insulin sensitivity index AHI, events/h ODI, events/h Nadir SaO2, % Mean SaO2, % SaO2 , 90%, % ETpCO2, mm Hg Predictors of fibrosis stage BMI, z score Waist circumference Metabolic syndrome, n-criteria NAS AHI, events/h Nadir SaO2, % Mean SaO2, % SaO2 , 90%, % ETpCO2, mm Hg

b

SE(b)

P Value

0.311 0.095 20.021 20.105 0.347 0.071 20.002 20.097 0.320 0.099

0.104 0.116 0.104 0.111 0.160 0.126 0.170 0.148 0.127 0.121

0.004 0.417 0.841 0.345 0.029 0.575 0.989 0.516 0.014 0.417

0.126 0.017 0.050 0.074 0.132 20.022 20.055 0.615 0.164

0.121 0.111 0.113 0.147 0.177 0.178 0.154 0.142 0.126

0.300 0.880 0.653 0.618 0.461 0.902 0.724 0.0007 0.197

Definition of abbreviations: AHI = apnea/hypopnea index (number of apnea/hypopneas per hour of sleep); BMI = body mass index; ETpCO2 = mean end-tidal carbon dioxide pressure; HDL = high-density lipoprotein; NAS = nonalcoholic fatty liver disease activity score; ODI = oxygen desaturation index (number of hemoglobin desaturations, i.e., in SaO2 > 4% of baseline value per hour of sleep). Different polysomnographic parameters were entered as continuous variables. AHI, insulin sensitivity index, ODI, nadir SaO2, mean SaO2, SaO2 , 90%, and ETpCO2 were log-transformed due to skewed distribution. Statistically significant associations are indicated in boldface. SaO2 , 90%: total duration of hemoglobin desaturation, expressed as percentage of total sleep time.

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apoptosis and fibrogenesis (Table E1) suggest prolonged nocturnal hypoxemia may directly trigger necroinflammation in the liver, independently of activation of extrahepatic and systemic inflammation, as suggested by similar circulating proinflammatory markers/cytokine levels between children with and without NASH. Consistently, mounting experimental data demonstrate chronic intermittent hypoxia directly activates hypoxia inducible factor-1a and hypoxia inducible factor-2a, two key transcription factors regulating the expression of genes involved in hepatocyte de novo lipogenesis and free fatty acid oxidation and in Kupffer and hepatic stellate cell activation, eventually promoting hepatic steatosis, necroinflammation, and fibrogenesis (11, 12, 49–53). These findings may have relevant clinical and research implications. Children with NAFLD should be systematically screened for the presence of OSAS, as this condition increases the risk of having progressive NASH and fibrosis, independently of obesity. Future research will have to define the optimal screening strategy for OSAS in these patients, as our data demonstrate that no specific OSASrelated symptom seems accurate enough (Table E1), and screening questionnaires for OSAS have modest sensitivity and specificity, which may be even lower in NAFLD population, where fatigue,

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ORIGINAL ARTICLE

Figure 2. Impact of the presence of obstructive sleep apnea syndrome (OSAS) on nonalcoholic fatty liver disease activity score (NAS) within the same quartile of body mass index (BMI) z score. Data are expressed as mean 6 SD. Stars indicate P , 0.05 versus OSAS within the same quartile of BMI z score. Crosses indicate P , 0.01 versus OSAS within the same quartile of BMI z score.

troubled sleeping, and daytime sleepiness are frequent even in the absence of OSAS (54). Conversely, children with ultrasonographic NAFLD and newly diagnosed OSAS should be investigated for the presence of progressive NASH and

fibrosis, which demand experimental treatment and tight monitoring. Our study has some limitations. First, its cross-sectional nature prevents any definitive causal inference between OSAS and liver injury. Second, only a single

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American Journal of Respiratory and Critical Care Medicine Volume 189 Number 1 | January 2014