Following the epidemics of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most frequent chronic liver disease in children and adolescents in industrialized countries (1–3). NAFLD affects 3% to 10% of the paediatric population, but this figure increases to approximately 80% among obese individuals (3–6). When complicated by hepatocellular damage and inflammation, that is, in the presence of nonalcoholic steatohepatitis (NASH), NAFLD can progress to hepatic fibrosis, liver failure, and hepatocellular carcinoma (7).
Pediatric NAFLD is generally related to hyperalimentation associated with inadequate physical activity leading to a progressive increase of body mass index (BMI) and visceral adiposity. Familial, epidemiological, and twin studies suggest that inherited factors play a pivotal role in determining the susceptibility to develop NASH (8–11). Single nucleotide polymorphisms (SNPs) in genes involved in inflammation, insulin signaling, oxidative stress, and fibrogenesis have been associated with the severity of liver damage in NAFLD (12–17), but these factors explain only a minority of fibrosis variability, and several candidate genetic variants have not been replicated after initial reports (17,18). Recently, the Patatin-like phospholipase domain-containing-3 (PNPLA3), rs738409 C>G, encoding the I148 M protein variant, has been identified as a strong determinant of liver fat without affecting body mass, dyslipidemia, and insulin resistance (19). We recently showed that the rs738409 PNPLA3 SNP was strongly associated with severe steatosis, NASH, and the progression of liver fibrosis in a large series of Italian and UK patients with NAFLD (20), and a even stronger association between the PNPLA3 genotype, steatosis, and liver damage has been detected in children with NAFLD (21). Furthermore, the PNPLA3 genotype affects steatosis and the progression of liver damage in other liver diseases (22).
Other genes involved in lipid metabolism are likely involved in the pathogenesis of NAFLD. Lipin1 (LPIN1), a phospatidate phospatase highly expressed in adipose tissue, is involved in the metabolism of phospholipids and triacylglycerol, and is required for adipogenesis and the normal metabolic flux between adipose tissue and the liver, where it also acts as an inducible transcriptional coactivator to regulate fatty acid metabolism (23). LPIN1 mRNA expression in the liver and adipose tissue has been positively associated with body mass and insulin sensitivity, and LPIN1 SNPs and haplotypes that may confer variability in the protein activity have been associated with several components of the metabolic syndrome, including body mass, insulin levels, resting metabolic rate, and responsiveness to insulin sensitizers, although overall evidence is still controversial (24). Interestingly, in a recent meta-analysis conducted in 8504 subjects the LPIN1 rs13412852 T allele was associated with lower BMI and insulin levels (25), thus possibly representing a protective factor toward metabolic syndrome alterations.
Recent data from our group also indicated that hepatic LPIN1 expression is downregulated in experimental models of NAFLD (26), potentially implicating this gene in the pathogenesis of liver steatosis, but no data are available to date on the effect of LPIN1 genotype on NAFLD development and progression to advanced liver damage.
In the hypothesis that LPIN1 genotype influences the susceptibility to and progression of NAFLD, the aim of the present study was to evaluate whether LPIN1 rs13412852 SNP is associated with NASH and fibrosis in Italian pediatric and adult patients with NAFLD.
The present prospective study included 142 untreated, consecutive children and adolescents (93 boys and 56 girls) with biopsy-proven NAFLD, referred to “Bambino Gesù” Children's Hospital between May 2006 and November 2009. All of the patients were tested for secondary causes of steatosis including alcohol abuse (≥140 g/week), total parenteral nutrition, and the use of drugs known to precipitate steatosis (eg, valproate, amiodarone, prednisone). Hepatitis A, B, C, D, E, and G, cytomegalovirus, and Epstein-Barr virus infections were ruled out by appropriate tests. In all of the cases, autoimmune liver disease, metabolic liver disease, Wilson disease, and alpha-1-antitrypsin were ruled out using standard clinical and laboratory evaluation and liver biopsy features. All of the included subjects were whites of Italian descent.
Analyses were replicated in an independent cohort of 115 consecutive adult patients with NAFLD who underwent liver biopsy between January 2007 and January 2010 at the Department of Internal Medicine, Fondazione Ca’ Granda IRCCS Policlinico of Milan, who were included and described in previous series (15,20).
Anthropometrical and Biochemical Measures
Height in meters, weight in kilograms, and BMI were calculated and converted in standard deviation score. We examined aspartate aminotransferase, alanine transaminase (ALT), and γ-glutamyl transpeptidase (GGT), as previously described (27).
Biopsies were performed in all of the children using an automatic core biopsy device (Biopince, Amedic, Sweden) with an 18-gauge needle, 150 mm long, and the ability to cut tissue with lengths up to 33 mm with extreme precision (28).
Liver biopsies were at least 18 mm in length and read by a single liver pathologist who was unaware of the patient's clinical and laboratory data. Biopsies were routinely processed (formalin-fixed, paraffin-embedded) and stained by hematoxylin and eosin, and Van Gieson stain for the assessment of fibrosis and architectural changes.
The diagnosis of NASH was based on the pathologist's overall impression, according to Kleiner et al (29). The main histological features commonly described in NAFLD including steatosis, inflammation (portal and lobular), hepatocyte ballooning, and fibrosis were scored according to the Scoring System for NAFLD recently developed by the National Institutes of Health–sponsored NASH Clinical Research Network (29). Briefly, steatosis was graded on a 4-point scale: grade 0 = steatosis involving <5% of hepatocytes; grade 1 = steatosis involving up to 33%; grade 2 = steatosis involving 33% to 66%; and grade 3 = steatosis involving >66%. Lobular inflammation was graded on a 4-point scale: grade 0 = no foci; grade 1 = <2 foci per 200× field; grade 2 = 2 to 4 foci per 200× field; grade 3 = >4 foci per 200× field. Hepatocyte ballooning was graded from 0 to 2: 0 is none, 1 is few balloon cells, 2 is many/prominent balloon cells. Stage of fibrosis was quantified in a 5-point scale: stage 0 = no fibrosis; stage 1 = perisinusoidal or periportal (1a = mild, zone 3, perisinusoidal; 1b = moderate, zone 3, perisinusoidal; 1c = portal/periportal); stage 2 = perisinusoidal and portal/periportal; stage 3 = bringing; and stage 4 = cirrhosis. The clinical and histological features of the patients included in the study are shown in Table 1.
The control group included 337 blood donors from northern Italy who were selected because of the availability of serum samples stored at −80°C, lack of clinical and biochemical evidence of liver and metabolic disease, and no alcohol abuse (<30/20 g/day in boys/girls). We excluded subjects with ALT >35/30 IU/mL in boys/girls, GGT >35 IU/mL, BMI >28, abdominal circumference >100 cm, glucose levels ≥100 mg/dL, triglycerides ≥150 mg/dL, high-density lipoprotein ≤45/55 in boys/girls, or a fatty liver index >35, a value with high specificity to rule out NAFLD in the general population (28). The clinical and demographic features of subjects included are shown in Table 1.
Informed written consent was obtained from each subject included in the study or their parents. The study protocol was approved by the institutional review board of the Ospedale Maggiore Policlinico Ca’ Granda IRCCS. Informed written consent was obtained from each patient and control subject, and the study conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
DNA was extracted from peripheral blood by the phenol-chloroform method. The success rate in extracting DNA was 100% for each study group. The LPIN1 rs13412852 and PNPLA3 rs738409 SNPs were genotyped by a 5′ nuclease Taqman assay (assay on demand, Applied Biosystems, Foster City, CA) by personnel unaware of patients’ and controls’ clinical status. Postpolymerase chain reaction allelic discrimination was carried out measuring allele-specific fluorescence on the Opticon2 detection system (MJ Research, Waltham, MA). Random samples were confirmed by direct genotyping, which provided concordant results in all of the cases, controls were included in all of the batches analyzed, and quality controls were performed to verify the reproducibility of the results. Valid genotypic data were obtained for >99% of the subjects analyzed (20).
Results are expressed as mean ± standard deviation. Mean values were compared by ANOVA or Wilcoxon, when appropriate, and frequencies by Fisher exact test for trend. The association among the LPIN1 rs13412852 SNP, steatosis severity, NASH, and fibrosis was evaluated by multivariate logistic regression analysis. Analyses were carried out with JMP 6.0 statistical analysis software (SAS Institute Inc, Cary, NC).
Distribution of the LPIN1 rs13412852 SNP in Patients and Controls
The frequency distribution of LPIN1 rs13412852 SNP was significantly different between pediatric patients with NAFLD and healthy controls (7% vs 14%; P = 0.039, shown in Table 2). The difference seemed to be related to an underrepresentation of the TT genotype (homozygosity for the “protective T allele”) in patients compared than in healthy controls (odds ratio [OR] 0.58, 95% confidence interval [CI] 0.35–0.91), suggesting that it may also represent a protective factor for NAFLD development during pediatric age. The frequency distribution of LPIN1 rs13412852 SNP was not significantly different between adult patients with NAFLD and controls (Table 2).
Effect of the LPIN1 rs13412852 SNP on Metabolic Features and Liver Damage in Pediatric Patients
The clinical and histological features of pediatric patients subdivided according to LPIN1 rs13412852 SNP are shown in Table 3. The rs13412852 T allele was associated with lower triglycerides levels (P = 0.01), and there was also a nonsignificant trend for higher body mass and waist circumference in patients carrying this SNP, but not with cholesterol levels, insulin resistance, and liver enzymes. The rs13412852 T allele also was associated with lower triglycerides levels in control subjects (P = 0.005).
At liver histology, TT homozygosity was negatively associated with a major outcome of the present study, that is, the prevalence of fibrosis (P = 0.012; Fig. 1A). There was also a trend for a lower prevalence of NASH in carriers of the TT genotype (3/10, 30% vs 64/132, 49%; P = 0.3), but TT homozygosity was negatively associated with the severity of histological features associated with NAFLD, as indicated by the NAS score (P = 0.026; Fig. 1B). At multivariate analysis (shown in Table 4), homozygosity for the rs13412852 LPIN1 T allele was a predictor of the absence of histological fibrosis independent of PNPLA3 rs738409 genotype and of clinical risk factors, including age, waist circumference, the presence of hyperglycemia (IGT or diabetes), and ALT levels (OR 0.29; 95% CI 0.11–0.66; P = 0.0055).
Effect of the LPIN1 rs13412852 SNP on Metabolic Features and Liver Damage in Adult Patients
As in pediatric patients, the LPIN1 rs13412852 TT genotype was not associated with demographic features, blood pressure, and liver enzymes (Table 3), but it was associated with a significantly lower BMI (24.6 ± 2.1 vs 27.1 ± 3.5, P = 0.0001), and a trend for a lower prevalence of diabetes/IGT (2/19, 11% vs 28/96, 29%; P = 0.15) and for presentation at younger age (48.9 ± 11 vs 43.8 ± 10; P = 0.1), whereas it was not associated with triglycerides in this group (137 ± 53 vs 129 ± 66, P = 0.6).
Similar to that observed in pediatric patients, LPIN1 rs13412852 TT genotype was, albeit not significantly, associated with a lower prevalence of NASH (6/19, 31% vs 46/96, 48%; P = 0.2) NAS ≥5 (2/19, 10% vs 18/96, 19%; P = 0.5), and fibrosis (7/19, 37% vs 53/96, 55%; P = 0.2).
At logistic regression analysis considering age at biopsy, ALT levels, the presence of diabetes/IGT, PNPLA3 rs738409 genotype, BMI, and LPIN1 rs13412852 TT genotype as independent variables, LPIN1 rs13412852 TT genotype was associated with a reduced risk of histological fibrosis (OR 0.15; 95% CI 0.02–0.67; P = 0.012).
We evaluated the role of the LPIN1 rs13412852 SNP in the pathogenesis of NAFLD in children. We found that in line with previous results suggesting a protective role of the minor allele in the development of metabolic complications (24,25), homozygosity for the rs13412852 T allele was underrepresented in pediatric, but not in adult, patients with NAFLD; it was associated with less-severe dyslipidemia; children affected with this genotype had a trend for a lower prevalence of NASH and significantly less-severe liver damage; it seemed to be protected from fibrosis progression independently of PNPLA3 genotype and other clinical risk factors; and it was also confirmed at multivariate analysis in adult patients.
These data, together with previous studies implicating deregulation of LPIN1 in the mechanism of liver fat accumulation in experimental models (26), suggest that LPIN1 is involved in the pathogenesis of fatty liver and in the progression of liver damage in NASH, and that genotyping for the LPIN1 rs13412852 SNP may contribute, with PNPLA3 mutations and clinical risk factors, to refine the risk of progressive liver disease (identified by the presence of liver fibrosis) in obese children with fatty liver. It could be hypothesized that the mechanism involves either a decreased flux of free fatty acids to the liver because of the reduced lipolysis associated with the protective allele, or directly reduced lipogenesis in the liver.
As opposed to previous studies (23–25), we did not detect any association between rs13412852 and either BMI or insulin resistance in children, but it should be mentioned that the design of the study was not adequate to test an effect of LPIN1 genotype on adiposity, and that rs13412852 TT genotype was strongly associated with lower BMI in adults with NAFLD. Furthermore, rs13412852 TT genotype was negatively associated with protection from NAFLD in pediatric age, but not in adult patients. This finding may be related to an age-specific effect of LPIN1 rs13412852 TT on lipid metabolism, as suggested by the differential association with serum lipids and body mass, which could lead to a more marked effect during development, or to the fact that this genetic variant protects from the most severe forms of the disease, but the effect on mild uncomplicated steatosis is more limited. Indeed, pediatric patients in this series had a higher prevalence of fibrosis and NASH.
We could replicate at multivariate analysis the association between LPIN1 rs13412852 TT genotype and protection from progression of liver damage to fibrosis in adults with histological NAFLD, suggesting that the advantageous metabolic effect of this genetic variant persists after the developmental age, although the association was only significant after correction for confounding factors at multivariate analysis, and therefore requires confirmation in independent cohorts and is likely less stronger than in pediatric patients. We did not observe a significantly lower prevalence of NASH in adults with the protective LPIN1 variant, however, possibly because of the relatively limited number of patients evaluated. Additional studies are required to define whether the effect of LPIN1 genotype on hepatic fibrogenesis is independent of NASH and its features, including the severity of steatosis, parenchymal necroinflammation, and hepatocellular damage.
Therefore, the phenotype associated with LPIN1 rs13412852 TT genotype appeared to be dependent on age because it was associated with susceptibility to NAFLD, serum triglycerides, NAS score, and the presence of liver fibrosis in children, whereas in adults it was associated with BMI and likely with liver fibrosis. These differences may be related to specific and different roles of LPIN1 on adipose tissue biology during development and adult age and to an even more important role of genetic factors in the pathogenesis of NASH in children as compared with adults, as already observed for the PNPLA3 I148 M variant (21).
The strengths of the present study include the careful clinical, metabolic, and genetic characterization of cases and controls, but the study also has limitations. The limitations include the relative low number of patients carrying the protective genotype included in the present study and the lack of evaluation of the distribution of LPIN1 genotype in healthy children without NAFLD. In addition, validation of the association between LPIN1 genotype and NASH risk should be performed in future studies in children at risk.
Furthermore, we evaluated the effect of a single SNP in the LPIN1 gene, which was chosen based on a meta-analysis of previously published studies; however, this genetic polymorphism does not encode for a protein variant, and thus may influence the clinical phenotype either because it is in linkage disequilibrium with another coding variant or because it influences, directly or indirectly, the expression of LPIN1 in the liver or in the adipose tissue. Indeed, LPIN1 mRNA levels in these tissues were previously correlated with the severity of metabolic abnormalities and insulin resistance. Additional studies are required to clarify this issue. In addition, despite its remarkable protective effect, given the relatively low prevalence, it is unlikely that the LPIN1 rs13412852 TT genotype explains a major proportion of NAFLD variability in the population, but it still can improve our ability to predict progressive disease and represent an attractive new therapeutic target for NAFLD.
In conclusion, LPIN1 rs13412852 SNP is associated with fibrosis progression in pediatric and adult patients with histological NAFLD. Further studies are warranted to confirm and evaluate the clinical relevance of these findings.
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Keywords:Copyright 2012 by ESPGHAN and NASPGHAN
dyslipidemia; lipin1; liver fibrosis; nonalcoholic fatty liver disease; steatosis