What Is Known/What Is New
What Is Known
- Obesity predisposes to nonalcoholic fatty liver disease in childhood.
- The I148M patatin-like phospholipase domain-containing 3 gene (PNPLA3) and rs58542926 transmembrane 6 superfamily member2 (TM6SF2) are the major genetic variants for NAFLD development.
- The membrane-bound O-acyltransferase domain containing protein 7 (MBOAT7) gene represents a novel genetic risk variant for nonalcoholic fatty liver disease.
- What Is New
- The MBOAT7 rs641738 variant influences alanine transaminase levels and exerts an additive effect with patatin-like phospholipase domain-containing 3 and transmembrane 6 superfamily member2 variants on nonalcoholic fatty liver disease risk in obese children.
- This is the first pediatric association between the membrane-bound O-acyltransferase domain-containing protein 7 rs641738 variant and noninvasive markers of liver fibrosis.
Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease in children (1) with increasing prevalence in line with the wide spread of childhood obesity (1). Almost 8% of the general pediatric population and 34% of obese children are affected by NAFLD (2). NAFLD is a complex disease, and a multiple-hit hypothesis has been suggested for its pathogenesis (3–5). Obesity and insulin resistance play a pivotal role in development and progression of the disease (6,7) More recently, the gut microbiota has emerged as a possible player in NAFLD progression (8,9). Moreover, the observation that not all obese subjects develop NAFLD suggests that the genetic background is involved in its pathogenesis (10). Results coming from twin studies (11,12) and the differences in the disease rates among the several ethnic groups support the relevance of the genetic involvement (13,14).
Recently, a new genetic variant that might affect the pathophysiology of NAFLD has been found in the membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7) gene. Mancina et al reported an association between the rs641738 variant and both hepatic fat deposition and liver fibrosis in a large cohort of obese adults (15). The variant leads to a C-to-T substitution linked to the 3’ untranslated region of MBOAT7 and it is associated with lower RNA expression and lower protein activity (15). The gene product acts as a lysophosphatidylinositol acyltransferase and is highly expressed in human liver, brain, and immune cells (15). It is involved in phospholipid remodeling by transferring polyunsaturated fatty acids, especially arachidonoyl-CoA, to membrane phosphatidylinositols (15). To date, evidences about the association between this variant and pediatric NAFLD are limited. In fact, only 1 study, conducted by Viitasalo et al, investigated the association between genetic variant in the MBOAT7 gene and liver enzyme serum levels in the pediatric population. The authors reported that children carrying the minor allele showed significantly higher ALT levels compared with noncarriers (16). In this work, the authors did not perform any imaging technique to assess the presence of NAFLD, moreover, the cohort included normal-weight children, whereas we aim to evaluate the association between this genetic variant and NAFLD in a cohort of obese children and adolescents that are at higher risk for the disease (16).
The aim of our study was to evaluate the association of the rs641738 variant in the MBOAT7 gene with hepatic steatosis assessed by ultrasound and biochemical markers of liver damage in a cohort of Italian obese children and adolescents. Moreover, we investigated whether there is an additional effect between this variant and the other 2 common genetic variants—I148M variant (rs738409) in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene and E167K variant (rs58542926) in the transmembrane 6 superfamily member 2 (TM6SF2)—that have been associated with liver damage in obese children.
We enrolled a total of 1002 obese (Body Mass Index [BMI] >95th percentile according to reference values) (17) children and adolescents consecutively attending our obesity clinic. The population mean age was 10.56 ± 2.97 years; the mean BMI-standard deviation score (SDS) was 2.97 ± 0.79. The ethical committee of University of Campania “Luigi Vanvitelli” approved the study. Written informed consent was obtained before any procedure.
We excluded patients taking any medication or alcohol potentially affecting liver function tests or potentially determining fatty liver. We also excluded from the study the patients affected by metabolic hepatopathy, autoimmune hepatitis, celiac disease, endocrine hepatopathy, muscular diseases, and alpha-1-antitrypsin deficiency.
Anthropometrical features (weight, waist circumference, height, waist-to-height ratio [W/Hr], BMI-SDS, pubertal stage and blood pressure) were obtained as previously described (18,19). BMI-SDS was calculated using the lambda-mu-sigma (LMS) method (20). Pubertal stage according to Tanner criteria was clinically assessed (21). Pediatric NAFLD Fibrosis Index (PNFI) was calculated as previously described (22).
Biochemical Measurement and Imaging
Blood samples were obtained after overnight fasting and serum was frozen at −20 °C until analyzed.
Serum alanine transaminase (ALT), aspartate transaminase (AST), triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, and insulin were assayed as previously described (23,24). Insulin resistance was assessed using the homeostasis model assessment (HOMA) as previously described (23).
ALT greater than 40 IU/L was classified as elevated. In children with elevated liver enzymes, the presence of hepatitis B and C were excluded. We also excluded the HCV infection in the pubertal patients with hepatic steatosis and ALT levels ≤40 IU/L. None of the patient assumed alcohol, or any drug potentially determining fatty liver. If the ALT levels were persistently (≥6 months) greater than 2 times the normal (with or without hepatic steatosis) value as such as if the liver steatosis without elevated liver enzymes was persistent, we excluded the most common causes of metabolic hepatopathy (eg, Wilson disease [using ceruloplasmin]), autoimmune hepatitis (dosing anti-nuclear antibodies, anti-smooth muscle antibodies, anti-liver kidney microsomal antibody type 1 [LKM-1] and anti-liver cytosol type 1 autoantibodies), celiac disease (evaluating transglutaminase immunoglobulin A [IgA] and immunoglobulin G autoantibodies and plasmatic IgA levels), endocrine hepatopathy (evaluating thyroid-stimulating hormone, free thyroxine (Ft4), and cortisol levels), muscular diseases (evaluating creatine phosphokinase levels), and alpha-1-antitrypsin deficiency (evaluating alpha-1-antitrypsin levels).
Liver steatosis was assessed as present or absent. It was determined based on abnormally intense, high-level echoes arising from the hepatic parenchyma and liver-kidney differences in echo amplitude. The same radiologist performed ultrasound imaging of the liver to detect the presence of steatosis.
Informed consent was collected for DNA extraction. Genomic DNA was extracted from peripheral whole blood with a DNA extraction kit (Promega, Madison, WI). Patients were genotyped for MBOAT7 rs641738 polymorphism (C>T transition) by Taqman allelic discrimination assay that allows to genotype the 2 possible variants at the single- nucleotide polymorphism (SNP) site measuring the change in fluorescence of the dyes associated with the probes on ABI 7900HT real time PCR system. Predesigned assay primers and probes were purchased from Applied Biosystems (Foster City, CA).
Moreover, all the patients were both genotyped for PNPLA3 rs738409C to G variant and TM6SF2 rs58542926 as previously described (24,25).
A chi-squared test was used to assess whether the genotypes were in Hardy-Weinberg equilibrium and to compare categorical variables.
We evaluated differences among MBOAT7 genotypes for continuous variables by a general linear model. We used as covariates age, sex, pubertal stage, BMI-SDS, and W/Hr whenever it was appropriate. Not-normally distributed variables were log-transformed before the analysis, but raw means are shown.
The genotype was coded with an additive model of inheritance (ie, the genotype is coded 0,1, or 2 corresponding to the number of minor alleles carried by each subject).
The genetic risk score among MBOAT7 rs641738, PNPLA3 rs738409, and TM6SF2 rs58542926 was calculated by giving a value 0 for carrying no risk allele, 1 for carrying 1 to 2 risk alleles, 2 for carrying 3 to 4 risk alleles, and 3 for carrying 5 to 6 risk alleles for the 3 examined polymorphisms, resulting in a range of 0 to 3 for the score (16). The associations of the genetic risk score with plasma ALT and other variables were analyzed using Linear Regression, and General Linear Model adjusted for confounding factors.
On the basis of literature data, PNFI cutoff was made by assigning a value 0 for subjects having PNFI 0 to 3 (indicating hepatic steatosis), 1 for PNFI 3 to 8.9 (indicating a potential early fibrosis, and 2 for PNFI ≥9 (suggesting the presence of liver fibrosis) (22).
A logistic regression was performed to calculate the odds of showing NAFLD according to the genotypes.
The IBM SPSS Statistics software, Version 24 (IBM, Armonk, NY) was used for all statistical analyses. Data were expressed as means ± SD. P values less than 0.05 were considered statistically significant.
The frequency of the different MBOAT7 rs641738 genotype distribution was in Hardy-Weinberg equilibrium (P > 0.05). The clinical and laboratory characteristics of the study population stratified according to the presence of hepatic steatosis are shown in Table 1.
A total of 290 patients were homozygous for the C allele, 218 were homozygous for the minor allele T, and 494 were heterozygous. No differences among the MBOAT7 genotypes for BMI-SDS, W/Hr, triglycerides, liver steatosis, total cholesterol, LDL-C, HDL-C ALT and AST were found (Table 2). However, systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels showed a significant association among the MBOAT7 genotypes (P = 0.04 and P = 0.007, respectively).
The carriers of the rare allele T of the MBOAT7 polymorphism showed higher serum ALT levels than noncarriers, after adjustments (T carriers means ± SD 31.39 ± 21.92; noncarriers 28.96 ± 21.71; P = 0.004). These findings were confirmed also for the carriers of the rare allele T of the MBOAT7 polymorphism with hepatic steatosis compared with noncarriers without hepatic steatosis, after adjustments (steatotic T carriers means ± SD 33.18 ± 20.35, nonsteatotic noncarriers 30.70 ± 26.37; P = 0.017; see Supplemental Table 1, Supplemental Digital Content, http://links.lww.com/MPG/B335, which showed ALT levels in MBOAT7 carriers and noncarriers in the context of presence or not of steatosis).
Subjects carrying the T allele of the MBOAT7 polymorphism showed higher PNFI values (T carriers means ± SD 7.8 ± 2.6, noncarriers 7.3 ± 2.6; P = 0.029), also after adjustment for the same covariates (P = 0.019).
Moreover, steatotic subjects carrying the T allele of the MBOAT7 polymorphism showed higher PNFI values (T carriers means ± SD 8.2 ± 2.6, noncarriers 7.5 ± 2.6; P = 0.03) than noncarriers without hepatic steatosis, also after adjustment (P = 0.04).
Moreover, we stratified the patients carrying the T allele on the basis of the PNFI cutoff points and we found a statistically significant association (P = 0.025). This association remained significant after adjusting by confounding factors (P = 0.044). The OR to present a PNFI >3 for the patients carrying the minor MBOAT7 allele was 1.3 (CI 1.03–1.63, P = 0.026).
A higher genetic risk score was associated both with higher serum ALT levels (P = 0.011; Fig. 1A) and with an increased risk to show liver steatosis (Figure 1B). A logistic regression analysis adjusted for age, sex, BMI-SDS, and pubertal stage showed that patients carrying risk alleles for the MBOAT7, PNPLA3, and TM6SF2 polymorphisms (score 3) had an OR to present liver steatosis of 2.6 (CI 1.43–4.83, P = 0.0018) compared with patients belonging to lower genetic risk score group (0–2; Table 3).
Moreover, subjects belonging to genetic risk score group 3 presented an OR to show elevated ALT levels of 3.4 (CI 1.3–5.5, P = 0.003) after adjustment for confounding factors.
No significant association between PNFI values and genetic risk score was found (Fig. 1C).
In this work, we explore for the first time in a large obese pediatric population the association of the rs641738 C>T variant in the MBOAT7 gene with markers of liver damage.
To date, in the complex puzzle of the pathophysiology of pediatric NAFLD, SNP in the PNPLA3 and TM6SF2 have been recognized as the major common genetic determinants (14,24–29). The I148M variant in the PNPLA3 gene is strongly associated with NAFLD in both adults (30–33) and children (25–27). This nonsynonymous variant has been shown to increase the lipogenic activity and to reduce the triglyceride hydrolysis of the gene product (also known as adiponutrin) (34–36). Moreover, it has been reported that the effect is driven by adiposity (25,37). The TM6SF2 variant has been associated with higher hepatic triglyceride content with higher serum levels of ALT and lower plasma levels of liver-derived triglyceride-rich lipoproteins (14). The TM6SF2 protein promotes the VLDL secretion (38) and the E167K genetic variant leads to a substitution of a glutamate to a lysine that enhances degradation of the TM6SF2 protein and subsequently triglyceride hepatic accumulation (38).
Recently, the MBOAT7 gene involvement in the NAFLD susceptibility has been studied, but its mechanism remains to be elucidated (15,39–41).
MBOAT7 is a protein involved in the remodeling of phosphatidylinositols with arachidonic acid in the Lands cycle (40). The MBOAT7 rs641738 variant has been associated not only with the risk of NAFLD, inflammation, and fibrosis but also with NAFLD progression (15,40).
In fact, data from adult population showed that T carriers of the MBOAT7 polymorphism presented greater risk for developing NAFLD, likely because of reduced enzymatic activity leading to increased liver fat accumulation (15). This risk variant has also been related in adults to histologic liver damage, particularly to significant fibrosis increasing with number of MBOAT7 variant alleles (42).
Examining the effect of MBOAT7 rs641738 variant in NAFLD pathophysiology in childhood, Viitasalo et al have reported that healthy children carrying the minor (T) allele showed higher alanine aminotransferase (ALT) and C-reactive protein (CRP) serum levels compared with noncarriers (16). In line with these findings, we demonstrated, in a pediatric obese population, that the MBOAT7 rare allele is associated with higher ALT levels.
The same authors have also evaluated the association of a genetic risk score combining information from the same variants mentioned above and demonstrated higher serum ALT levels in children carrying the risk alleles for the MBOAT7, PNPLA3, and TM6SF2 polymorphisms (16).
Similarly, we showed that obese subjects carrying the risk alleles for the 3 examined variants involved in NAFLD pathogenesis (PNPLA3, TM6F2, and MBOAT7) had higher risk to show both NAFLD and elevated ALT levels compared with patients belonging to lower genetic risk score group (0–2).
Consistent with available data, this result suggests that these 3 risk variants exert an additive effect both on serum ALT levels and on the liver steatosis risk, involving a potential role in clinical practice of this combined genetic risk score in improving screening for increased risk of NAFLD in children.
Interestingly, we also demonstrated, for the first time in literature, an association between the rs641738 variant in the MBOAT7 gene and a fibrosis index score in childhood. This indicates, in line with the first findings indicating a correlation between minor MBOAT7 allele and histological evidence of liver fibrosis in adulthood that MBOAT7 gene is mainly linked to increased fibrosis, whereas the PNPLA3 and TM6SF2 polymorphisms are mostly related to steatosis (15,42).However, recent data regarding the exact role of these SNPs in adult NAFLD severity are conflicting (41) and they have not yet been replicated in pediatric population.
We also found lower SBP-DS levels among patients with MBOAT7 CT genotype and lower DBP-DS levels among patients with MBOAT7 TT genotype. This could suggest, as such as TM6SF2 polymorphism, that the MBOAT7 T allele while influencing ALT levels and PNFI could reduce important cardiovascular risk factor as blood pressure levels in the same patients.
The strength of this study is represented by the large cohort of obese children examined and carefully evaluated for the anthropometric profile.
We are aware, however, that this study has some limitations. The major weakness is the evaluation of the liver steatosis and fibrosis with hepatic ultrasound and PNFI, respectively, and without liver biopsy. In fact, the diagnostic gold standard both for NAFLD and hepatic fibrosis diagnosis is currently represented by liver biopsy, but this procedure represents an ethical and economic issue in pediatric population (43). Moreover, further limitations are represented by the lack of other noninvasive diagnostic test such as magnetic resonance imaging (MRI)/resonance spectroscopy for steatosis and transient elastometry for fibrosis (44).
The lack of both longitudinal data and age-matched control population are also recognized as limitation.
As future perspective, considering that there is an impressive amount of data implicating gut microbiota in the development of NAFLD (5,8,9), it could be interesting to investigate the interplay between MBOAT7 genotypes and gut microbiota on NAFLD development.
In conclusion, we investigated in childhood obesity the role of the rs641738 variant in the MBOAT7 gene on ALT levels and the combined effect of the MBOAT7, PNPLA3, and TM6SF2 variants both on ALT serum levels and liver steatosis risk. We also provided the first pediatric evidence of the association of the MBOAT7 polymorphism with noninvasive markers of liver fibrosis.
Nevertheless, further studies are needed to confirm these effects and to elucidate the pathophysiological mechanism of the MBOAT7 gene.
1. Welsh JA, Karpen S, Vos MB. Increasing prevalence of nonalcoholic fatty liver disease among United States adolescents, 1988-1994 to 2007-2010. J Pediatr
2. Anderson EL, Howe LD, Jones HE, et al. The prevalence of non-alcoholic fatty liver disease in children and adolescents: a systematic review and meta-analysis. PLoS One
3. Buzzetti E, Pinzani M, Tsochatzis EA. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism
4. Guercio Nuzio S, Di Stasi M, Pierri L, et al. Multiple gut-liver axis abnormalities in children with obesity with and without hepatic involvement. Pediatr Obes
5. Troisi J, Pierri L, Landolfi A, et al. Urinary metabolomics in pediatric obesity and NAFLD identifies metabolic pathways/metabolites related to dietary habits and gut-liver axis perturbations. Nutrients
2017; 9:pii: E485.
6. Marzuillo P, Del Giudice EM, Santoro N. Pediatric non-alcoholic fatty liver disease: new insights and future directions. World J Hepatol
7. Di Sessa A, Umano GR, Miraglia Del Giudice E. The association between non-alcoholic fatty liver disease and cardiovascular risk in children. Children (Basel)
2017; 4: pii: E57.
8. Poeta M, Pierri L, Vajro P. Gut-liver axis derangement in non-alcoholic fatty liver disease. Children (Basel)
2017; 4: pii: E66.
9. Clemente MG, Mandato C, Poeta M, et al. Pediatric non-alcoholic fatty liver disease: recent solutions, unresolved issues, and future research directions. World J Gastroenterol
10. D’Adamo E, Santoro N, Caprio S. Metabolic syndrome in pediatrics: old concepts revised, new concepts discussed. Curr Probl Pediatr Adolesc Health Care
11. Makkonen J, Pietilainen KH, Rissanen A, et al. Genetic factors contribute to variation in serum alanine aminotransferase activity independent of obesity and alcohol: a study in monozygotic and dizygotic twins. J Hepatol
12. Loomba R, Schork N, Chen CH, et al. Genetics of NAFLD in Twins Consortium. Heritability of hepatic fibrosis and steatosis
based on a prospective twin study. Gastroenterology
13. Marzuillo P, Miraglia del Giudice E, Santoro N. Pediatric fatty liver disease: role of ethnicity and genetics. World J Gastroenterol
14. Marzuillo P, Grandone A, Perrone L, et al. Understanding the pathophysiological mechanisms in the pediatric non-alcoholic fatty liver disease: the role of genetics. World J Hepatol
15. Mancina RM, Dongiovanni P, Petta S, et al. The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of european descent. Gastroenterology
16. Viitasalo A, Eloranta AM, Atalay M, et al. Association of MBOAT7 gene variant with plasma ALT levels in children: the PANIC study. Pediatr Res
17. Cacciari E, Milani S, Balsamo A, et al. Italian cross-sectional growth charts for height, weight and BMI (2 to 20 yr). J Endocrinol Invest
18. Santoro N, Del Giudice EM, Grandone A, et al. Y2 receptor gene variants reduce the risk of hypertension in obese children and adolescents. J Hypertens
19. Santoro N, Amato A, Grandone A, et al. Predicting metabolic syndrome in obese children and adolescents: look, measure and ask. Obes Facts
20. Cole TJ. The LMS method for constructing normalized growth standards. Eur J Clin Nutr
21. Tanner JM, Whitehouse RH. Clinical longitudinal standards for height, weight, height velocity, weight velocity, and stages of puberty. Arch Dis Child
22. Nobili V, Alisi A, Vania A, et al. The pediatric NAFLD fibrosis index: a predictor of liver fibrosis in children with non-alcoholic fatty liver disease. BMC Med
23. Marzuillo P, Grandone A, Conte M, et al. Novel association between a nonsynonymous variant (R270H) of the G-protein-coupled receptor 120 and liver injury in children and adolescents with obesity. J Pediatr Gastroenterol Nutr
24. Grandone A, Cozzolino D, Marzuillo P, et al. TM6SF2 Glu167Lys polymorphism is associated with low levels of LDL-cholesterol and increased liver injury in obese children. Pediatr Obes
25. Giudice EM, Grandone A, Cirillo G, et al. The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat. PLoS One
26. Santoro N, Kursawe R, D’Adamo E, et al. A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents. Hepatology
27. Romeo S, Sentinelli F, Cambuli VM, et al. The 148M allele of the PNPLA3 gene is associated with indices of liver damage early in life. J Hepatol
28. Viitasalo A, Pihlajamaki J, Lindi V, et al. Associations of I148M variant in PNPLA3 gene with plasma ALT levels during 2-year follow-up in normal weight and overweight children: the PANIC Study. Pediatr Obes
29. Goffredo M, Caprio S, Feldstein AE, et al. Role of TM6SF2 rs58542926 in the pathogenesis of nonalcoholic pediatric fatty liver disease: a multiethnic study. Hepatology
30. Romeo S, Kozlitina J, Xing C, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet
31. Sookoian S, Pirola CJ. Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology
32. Hyysalo J, Stojkovic I, Kotronen A, et al. Genetic variation in PNPLA3 but not APOC3 influences liver fat in non-alcoholic fatty liver disease. J Gastroenterol Hepatol
33. Dongiovanni P, Donati B, Fares R, et al. PNPLA3 I148M polymorphism and progressive liver disease. World J Gastroenterol
34. Wilson PA, Gardner SD, Lambie NM, et al. Characterization of the human patatin-like phospholipase family. J Lipid Res
35. Huang Y, Cohen JC, Hobbs HH. Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease. J Biol Chem
36. Li JZ, Huang Y, Karaman R, et al. Chronic overexpression of PNPLA3I148M in mouse liver causes hepatic steatosis
. J Clin Invest
37. Marzuillo P, Grandone A, Perrone L, et al. Weight loss allows the dissection of the interaction between abdominal fat and PNPLA3 (adiponutrin) in the liver damage of obese children. J Hepatol
38. Kozlitina J, Smagris E, Stender S, et al. Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease. Nat Genet
39. Krawczyk M, Rau M, Schattenberg JM, et al. NAFLD Clinical Study Group. Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study. J Lipid Res
40. Eslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: Clinical impact. J Hepatol
41. Koo BK, Joo SK, Kim D, et al. Additive effects of PNPLA3 and TM6SF2 on the histological severity of non-alcoholic fatty liver disease. J Gastroenterol Hepatol
2017; [Epub ahead of print].
42. Luukkonen PK, Zhou Y, Hyotylainen T, et al. The MBOAT7 variant rs641738 alters hepatic phosphatidylinositols and increases severity of non-alcoholic fatty liver disease in humans. J Hepatol
43. Marzuillo P, Grandone A, Perrone L, et al. Controversy in the diagnosis of pediatric non-alcoholic fatty liver disease. World J Gastroenterol
44. Vajro P, Lenta S, Socha P, et al. Diagnosis of nonalcoholic fatty liver disease in children and adolescents: position paper of the ESPGHAN Hepatology Committee. J Pediatr Gastroenterol Nutr