Skip to main content
PMC home page
Molecular Genetics & Genomic Medicine logoLink to Molecular Genetics & Genomic Medicine
. 2015 Aug 11;3(6):558–569. doi: 10.1002/mgg3.168

Genetic analysis of nonalcoholic fatty liver disease within a Caribbean–Hispanic population

Deborah Edelman 1, Harmit Kalia 2, Maria Delio 1,3, Mustafa Alani 2, Karthik Krishnamurthy 2, Mortadha Abd 2, Adam Auton 1, Tao Wang 4, Allan W Wolkoff 2,3,5, Bernice E Morrow 1,5,✉
PMCID: PMC4694126  PMID: 26740948

Abstract

We explored potential genetic risk factors implicated in nonalcoholic fatty liver disease (NAFLD) within a Caribbean–Hispanic population in New York City. A total of 316 individuals including 40 subjects with biopsy‐proven NAFLD, 24 ethnically matched non‐NAFLD controls, and a 252 ethnically mixed random sampling of Bronx County, New York were analyzed. Genotype analysis was performed to determine allelic frequencies of 74 known single‐nucleotide polymorphisms (SNPs) associated with NAFLD risk based on previous genome‐wide association study (GWAS) and candidate gene studies. Additionally, the entire coding region of PNPLA3, a gene showing the strongest association to NAFLD was subjected to Sanger sequencing. Results suggest that both rare and common DNA variations in PNPLA3 and SAMM50 may be correlated with NAFLD in this small population study, while common DNA variations in CHUK and ERLIN1, may have a protective interaction. Common SNPs in ENPP1 and ABCC2 have suggestive association with fatty liver, but with less compelling significance. In conclusion, Hispanic patients of Caribbean ancestry may have different interactions with NAFLD genetic modifiers; therefore, further investigation with a larger sample size, into this Caribbean–Hispanic population is warranted.

Keywords: ABCC2, CHUK, ENPP1, ERLIN1, fatty Liver, gene, hispanic population, liver genetics, NAFLD, NASH, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, PNPLA3, polymorphism, SAMM50.

Introduction

Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in western countries, with a prevalence of approximately 30% in the United States (Lazo et al. 2011). It is characterized by excessive fat accumulation in the liver, or hepatic steatosis. The clinical significance of steatosis is correlated with its histological severity. Nonalcoholic fatty liver disease can progress to a more severe disorder. The disease can range from simple steatosis to steatosis with inflammation (nonalcoholic steatohepatitis or NASH), fibrosis, and cirrhosis (Adams et al. 2005). A total of 2.9–20% of NAFLD patients will develop NASH dependent on the disease lead‐time (Charlton et al. 2001; Lebovics and Rubin 2011). Once NASH develops, an estimated 17–49% of cases progress to cirrhosis and, once present, patients are at risk for liver decompensation and hepatocellular carcinoma (Bugianesi et al. 2002; Ratziu et al. 2002; Argo et al. 2009). Overall, a small percentage of individuals with NAFLD progress to end‐stage cirrhosis or liver cancer. However, given the total number of cases of NAFLD, this still amounts to a large number of patients suffering from these significant morbidities.

Nonalcoholic fatty liver disease is frequently associated with several coexisting conditions including obesity, visceral fat, hyperlipidemia, and type 2 diabetes (Angulo 2002). Diabetes is not only a risk factor for NAFLD independent of obesity (Gupte et al. 2004), its presence also seems to compound the risk of histopathologic progression of steatosis among obese patients (Silverman et al. 1990). This logically follows as NAFLD is closely associated with insulin resistance, although the direction of this association has not been established (Marchesini et al. 1999; Angulo 2002; Paschos and Paletas 2009; Fabbrini et al. 2010).

In addition to environmental risk factors, NAFLD may have genetic risk factors. Specifically, there appears to be differences in risk depending on ethnic background. Based on recent population‐based studies in the United States, the prevalence of NAFLD was 30–32% overall, with rates of 39–45% among Hispanics, 30–33% among non‐Hispanic Whites, and 23–24% among non‐Hispanic Blacks (Browning et al. 2004; Smits et al. 2013). Multiple studies have indeed illustrated the lower disease burden and decreased histologic severity in the African‐American population (Giday et al. 2006; Kallwitz et al. 2009; Mohanty et al. 2009). Interestingly, although individuals of Asian ancestry tend to have lower rates of obesity, they have high rates of metabolic syndrome and NAFLD (prevalence ranging from 15 to 45%) (Farrell et al. 2013; Wong and Ahmed 2014). Asians may even demonstrate a higher disease severity than whites (Mohanty et al. 2009). A number of studies have demonstrated that Hispanics in the United States have both a higher disease burden and an increased disease severity than their counterparts of African‐American and European ancestry (Clark et al. 2003; Neuschwander‐Tetri and Caldwell 2003; Weston et al. 2005; Kallwitz et al. 2009; Sharp et al. 2009; Wagenknecht et al. 2009; Pan et al. 2011; Schneider et al. 2013). These studies investigated primarily Mexican Americans, but not Hispanics from other locations (Browning et al. 2004; Mohanty et al. 2009). This presents a complication when attempting to apply genetic and clinical principles derived from research in different Hispanic subpopulations. In the Bronx, for instance, 53.5% of the county is of Hispanic ethnicity, but over 80% are of Caribbean (Dominican and Puerto Rican) ancestry (2008–2012 American Community Survey). One study recently acknowledged the shortcoming of these NAFLD‐Hispanic investigations and analyzed rates of “suspected” NAFLD among different Hispanic populations. They discovered that rates of the condition differed between ancestries, with Hispanics of Cuban, Puerto Rican, and Dominican backgrounds with lower prevalence of NAFLD when compared with those of Mexican heritage (Kallwitz et al. 2015).

Here, we performed a new study to investigate the potential genetic risk implicated in NAFLD within a population composed of Hispanics of mostly Caribbean ancestry in Bronx County. We compared the allele frequencies of genetic variations in candidate genes found for NAFLD to those in our local population.

Materials and Methods

Human subjects and phenotype data

Blood or saliva samples were obtained from 40 patients with biopsy‐proven NAFLD, with their informed consent (IRB # 11‐06‐247E). The 12 males and 28 females are all of self‐identified Hispanic descent and are over the age of 18. Control samples (n = 24) were obtained from patients visiting various primary care, dermatology, and gastroenterology clinics at Montefiore Medical Center in the Bronx, New York. Subjects included 12 females and 12 males self‐identified as Hispanic. Patients were excluded if they had any evidence of liver disease (abnormal abdominal imaging, AST/ALT levels, or clinical history), BMI >30, or other evidence of metabolic syndrome. If patients had isolated HTN, hypercholesterolemia, or controlled DM, they were included based on clinical judgment.

Blood samples were obtained from healthy parents of children seen at the Pediatric Genetics Clinic at Montefiore Medical Center for developmental delay, autism, or multiple congenital malformations (mostly sporadic). Their DNA was used as a proxy for a random sample of our Bronx population. Their ethnic makeup was selected to be reflective of that of Bronx County, NY:−43% Hispanic, 31% White, 15.9% Black, 5% Asian. Subjects were not screened for NAFLD or other exclusion criteria. Deidentified samples were also obtained with their informed consent (IRB #1999‐201).

The Gentra Puregene Genomic DNA Purification Kit (Gentra, Minneapolis, MN) was used to purify DNA in the Molecular Cytogenetics Core, Albert Einstein College of Medicine, NY, according to standard protocols. Specimens were derived from blood (purple top EDTA tubes) or saliva (collected in Oragene OR‐250 kits). Quality of DNA was visualized by agarose gel electrophoresis and quantified by nanodrop analysis, Qubit, and/or PicoGreen.

Genotype analysis

Based on prior GWAS and candidate gene studies, we compiled a list of 87 target SNPs in 52 genes associated with the development NAFLD or disease severity. Seventy‐four SNPs (Table 1) were included in the study design. The other SNPs were rejected if there was a nearby SNP within 20 bases of the target SNP or the nearby SNP blocked the design of an extension primer. We subsequently performed SNP‐based genotyping with SNPs using Sequenom MassArray technology.

Table 1.

(A) GWAS – target SNPs (25). (B) Candidate gene and association studies (52). (C): SNPs excluded from study design (13)

SNP HGVS name Gene Risk allele N P‐value
(A)
rs2896019 (Kawaguchi et al. 2012) NM_025225.2:c.979+542T>G PNPLA3 G 543 7.5E‐10
rs738409 (Chambers 2011; Speliotes 2011; Kawaguchi et al. 2012) NM_025225.2:c.444C>G PNPLA3 G 543–61089 (1E‐10) – (1E‐45)
rs6006460 (Romeo et al. 2008) NM_025225.2:c.1358G>T PNPLA3 G 9229 0.0007
rs11597390 (Yuan et al. 2008) NC_000010.11:g.100101678G>A Intergenic A 7715 2.9E‐08
rs1227756 (Chalasani et al. 2010) NM_080805.3:c.364+6310G>A COL13A1 G 236 2E‐07
rs10883437 (Chambers 2011) NC_000010.11:g.100035604T>G Intergenic T 61089 4E‐09
rs2710833 (Chalasani et al. 2010) NC_000010.11:g.100035604T>G DDX60L A 236 6.3E‐07
rs887304 (Chalasani et al. 2010) NM_001144958.1:c.1118+160A>G CRACR2A A 236 7.7E‐07
rs6591182 (Chalasani et al. 2010) NM_001099409.1:c.1613T>G EHBP1L1 A 236 8.6E‐07
rs780094 (Kawaguchi et al. 2012) NM_001486.3:c.1423‐418T>C GCKR T 543 0.011
rs343062 (Chalasani et al.2010) NC_000007.14:g.35509456C>T HERPUD2 T 236 2.7E‐08
rs6834314 (Chambers 2011) NC_000004.12:g.87292656A>G HSD17B13 A 61089 3.1E‐09
rs12137855 (Speliotes 2011) NC_000001.11:g.219275036C>T LYPLAL1 C 592 4.12E‐05
rs2228603 (Speliotes 2011) NM_004386.2:c.274C>T NCAN T 592 5.29E‐05
rs6006611 (Kitamoto et al. 2013) NM_001003828.2:c.211+5145G>A PARVB G 392 1.8E‐12
rs343064 (Chalasani et al. 2010) NC_000007.14:g.35515178C>T PDGFA A 236 3E‐08
rs4240624 (Speliotes 2011) NR_040039.1:n.765+418G>A PPP1R3B A 592 3.68E‐18
rs6487679 (Chalasani et al. 2010) NC_000012.12:g.9218736C>T PZP G 236 0.000001
rs2143571 (Kawaguchi et al. 2012) NM_015380.4:c.1365‐532G>A SAMM50 A 543 6.4E‐07
rs738491 (Kawaguchi et al. 2012) NM_015380.4:c.21+2633C>T SAMM50 T 543 3.9E‐06
rs3761472 (Kawaguchi et al. 2012) NM_015380.4:c.329A>G SAMM50 G 543 1.1E‐06
rs2954021 (Chambers 2011) NC_000008.11:g.125469835A>G TRIB1 A 61089 2.3E‐13
rs2645424 (Chalasani et al. 2010) NM_001287756.1:c.201+739A>G FDFT1 A 236 6.8E‐07
rs2499604 (Chalasani et al. 2010) NC_000001.11:g.237940201C>T ZP4 A 236 0.000002
rs343062 (Chalasani et al. 2010) NC_000007.14:g.35509456C>T HERPUD2 T 236 2.70E‐08
(B)
rs8187710 (Sookoian et al. 2009) NM_000392.4:c.4544G>A ABCC2 G 109 0.035
rs17222723 (Sookoian et al. 2009) NM_000392.4:c.3563T>A ABCC2 A 109 0.037
rs2241766 (Tokushige et al. 2009) NM_004797.3:c.45T>G ADIPOQ T 70 0.0002
rs1501299 (Musso et al. 2008) NM_004797.3:c.214+62G>T ADIPOQ G 119 0.03
rs767870 (Kotronen et al. 2009) NM_024551.2:c.650+20G>A ADIPOR2 T 302 0.024
rs3772627 (Yoneda et al. 2009) NM_031850.3:c.‐1+4440A>G AGTR1 C 167 4.1E‐06
rs3772633 (Yoneda et al. 2009) NM_031850.3:c.‐85+2254T>C AGTR1 G 167 6.5E‐06
rs3772630 (Yoneda et al. 2009) NM_031850.3:c.‐1+658T>C AGTR1 G 167 0.000042
rs2276736 (Yoneda et al. 2009) NM_031850.3:c.‐1+59A>G AGTR1 C 167 0.000047
rs2854116 (Petersen et al. 2010) NM_000040.1:c.‐501C>T APOC3 C 95 0.001
rs2854117 (Petersen et al. 2010) NM_000040.1:c.‐528T>C APOC3 T 163 0.02
rs3772622 (Yoneda et al. 2009; Zain et al. 2013b) NM_031850.3:c.‐1+9939T>C ATGR1 C 144–167 0.003–1.2E‐06
rs11591741 (Feitosa et al. 2013) NM_001278.3:c.933+1251C>G CHUK C 2705 3.43E‐06
rs11597086 (Feitosa et al. 2013) NM_001278.3:c.1974+36T>G CHUK C 2705 3.07E‐05
rs6850524 (Sookoian et al. 2007) NM_004898.3:c.‐289‐5765G>C CLOCK G 136 0.00899
rs6843722 (Sookoian et al. 2007) NM_152760.2:c.1073‐31G>A CLOCK C 136 0.0229
rs4864548 (Sookoian et al. 2007) NM_004898.3:c.‐1144C>T CLOCK A 136 0.02697
rs11932595 (Sookoian et al. 2007) NM_004898.3:c.793‐1130T>C CLOCK A 136 0.0449
rs28969387 (Vazquez‐Chantada et al. 2013) NM_000773.3:c.1370A>T CYP2E1 A 69 1.56E‐07
rs1044498 (Dongiovanni et al. 2010) NM_006208.2:c.517A>C ENPP1 C 702 <0.05
rs2862954 (Feitosa et al. 2013) NM_006459.3:c.871A>G ERLIN1 C 2705 5.74E‐06
rs1260326 (Tan et al. 2013) NM_001486.3:c.1337T>C GCKR T 144 0.012
rs17883901 (Oliveira et al. 2010) NM_001498.3:c.‐594C>T GCLC T 130 0.007
rs12979860 (Petta et al. 2012) NM_001276254.2:c.151‐152G>A IL28B C 74 <0.001
rs1800795 (Carulli et al. 2009) NM_000600.3:c.‐237C>G IL6 C 114 <0.01
rs1801278 (Dongiovanni et al. 2010) NM_005544.2:c.2911G>A IRS1 C 702 <0.05
rs7324845 (Adams et al. 2013) NM_002298.4:c.1752‐1284C>T LCP1 A 13 2.96E‐06
rs6700896 (Swellam and Hamdy 2012) NM_002303.5:c.2673+1118C>T LEPR T 90 0.0001
rs1137100 (Zain et al. 2013a) NM_002303.5:c.326A>G LEPR G 144 0.003
rs1137101 (Zain et al. 2013a) NM_002303.5:c.668A>G LEPR G 144 0.013
rs13412852 (Valenti et al. 2012) NM_145693.2:c.722+1070C>T LPIN1 C 142 <0.01
rs12743824 (Adams et al. 2013) NC_000001.11:g.99317401C>A LPPR4 C 13 4.82E‐06
rs1800591 (Namikawa et al. 2004) NM_001300785.1:c.143‐7574G>T MTTP G 63 0.002
rs3816873 (Hashemi et al. 2011) NM_001300785.1:c.464T>C MTTP C 83  0.05
rs1800804 (Peng et al. 2013) NM_001300785.1:c.143‐7245T>C MTTP C 580 0.05
rs694539 (Sazci et al. 2013) NC_000011.9:g.114133419C>T NNMT A 80 0.051
rs2461823 (Sookoian et al. 2010) NM_033013.2:c.‐22‐5951T>C NR1I2 A 188 0.039
rs7946 (Song et al. 2005; Dong et al. 2007) NM_148173.1:c.523G>A PEMT A 28–107 <0.01–0.03
rs738409 (Romeo et al. 2008) NM_025225.2:c.444C>G PNPLA3 G 2111 5.9E‐10
rs2126259 (Feitosa et al. 2013) NR_040039.1:n.765+1333T>C PPP1R3B G 2705 4.81E‐08
rs1800234 (Chen et al. 2008) NM_005036.4:c.680T>C PPARA T 79 0.011
rs8192678 (Lin et al. 2013) NM_013261.3:c.1444G>A PPARGC1A A 182 0.008
rs7643645 (Sookoian et al. 2010) NM_033013.2:c.‐22‐579A>G PXR G 188 <0.0015
rs56225452 (Auinger et al. 2010) NM_012254.2:c.‐1324G>A SLC27A5 A 103 0.02
rs2229682 (Vazquez‐Chantada et al. 2013) NM_006516.2:c.588G>C SLC2A1 A 69 0.000912
rs11864146 (Adams et al. 2013) NM_001080442.2:c.1163‐58T>C SLC38A8 A 13 1.86E‐06
rs4880 (Al‐Serri et al. 2012) NM_001024466.1:c.47T>C SOD2 T 55 0.038
rs6503695 (Sookoian et al. 2008) NM_213662.1:c.129‐802A>G STAT3 C 108 0.021
rs780094 (Yang et al. 2011; Tan et al. 2013) NM_001486.3:c.1423‐418T>C GCKR T 144–436 0.013–0.0072
rs7903146 (Musso et al. 2009) NM_030756.4:c.382‐41435C>T TCF7L2 T 39 0.0001
rs11235972 (Xu et al. 2013) NM_022803.2:c.338‐96C>T UCP3 G 250 0.03
rs1800849 (Aller et al. 2010) NM_022803.2:c.‐238C>T UCP3 T 39 <0.05
(C)
rs12447924 Adams et al. 2012); NM_001286085.1:c.‐1700C>T CETP T 467 3.0E‐04
rs12597002 (Adams et al. 2012) NM_001286085.1:c.234‐894C>A CETP A 467 2.0E‐04
rs1800471 (Dixon et al. 2003) NM_000660.5:c.74G>C TGF‐β1 G 105 0.77
rs57920071 (Ludtke et al. 2005) NM_170708.3:c.1444C>T LMNA T 18 Not Given
rs3750861 (Miele et al. 2008) NM_001300.5:c.103‐27G>A KLF6 A 415 4.0E‐03
rs1554483 (Sookoian et al. 2007) NM_004898.3:c.982+247G>C CLOCK G 136 0.02697
rs9891119 (Sookoian et al. 2008) NM_213662.1:c.‐23‐7423T>G STAT3 C 108 0.02
rs1799964 (Tokushige et al. 2007) NM_000594.3:c.‐1211T>C TNF C 102 <0.01
rs1800630 (Tokushige et al. 2007) NM_000594.3:c.‐1043C>A TNF A 102 <0.01
rs1799945 (Valenti et al. 2010a,b) NM_139011.2:c.77‐2168C>G HFE G 587 <0.005
rs1800562 (Valenti et al. 2010a,b) NM_139011.2:c.77‐206G>A HFE A 587 <0.005
rs361525 (Valenti et al. 2002) NM_000594.3:c.‐418G>A TNF A 99 <0.0001
rs2290602 (Yoneda et al. 2008) NM_013261.3:c.877+171A>C PPARGC1A T 115 <0.02
Open in a new tab

GWAS, genome‐wide association study; SNP, single‐nucleotide polymorphisms.

Additionally, we sequenced the PNPLA3 (OMIM # 609567) gene including SNP rs738409 (NM_025225.2:c.444C>G) in exon 3, which is the most consistently and significantly associated variation with the development of NAFLD (Anstee and Day 2013). To detect sequence variations, we amplified the nine coding exons of PNPLA3 using PCR followed by Sanger DNA sequencing. Primers were designed specifically for each exon, based on GenBank reference sequence (Table S1). PCR amplified products were purified using the AmpPure Purification System processed by Beckman Coulter BioMan and carried out according to the manufacturer's protocol (Beckman Coulter, Indianapolis, IN). Purified PCR products were sequenced on the Applied Biosystems 3730 Sequencer (Genomics Core, Einstein, NY). All sequence data were compiled using Sequencher 4.0.1 software and compared to reference sequence NM_025225.2 (Gene Codes, Ann Arbor, MI).

Statistical analysis

Allele frequencies of the variations were estimated by the gene‐counting method. Agreement with the Hardy–Weinberg equilibrium was tested by the chi‐square test. Allele frequencies between each group were also compared using the chi‐square test. PLINK software was used to evaluate all Sequenom results. All other analyses were performed in Microsoft Excel.

Results

Target variation analysis

Sequenom genotyping and PNPLA3 sequencing were used to identify allele frequencies of the target SNPs within NAFLD cases, controls, and our sample Bronx population. Additional minor allele frequencies (MAFs) were determined through 1000 Genomes Project Phase 1 report of the total population as well as subpopulation of Puerto Ricans from Puerto Rico. Complete results are included in Table S2. Nine variations are listed in Table 2 based on significant P‐values (≤0.05) across multiple avenues of comparison.

Table 2.

Selected genotyping results. (A) Comparisons relative to NAFLD cases. (B) comparisons relative to sample Bronx population

(A)
Gene SNP MAF‐NAFLD cases MAF‐controls OR P‐value MAF‐Bronx population OR P‐value MAF‐general population RR P‐value MAF‐Puerto Rican population OR P‐value
CHUK rs11597086 26% (22/15/3) 16% (15/7/0) 1.65 0.35 23% (134/75/14) 1.14 0.67 22% (694/308/90) 1.18 0.55 43% (19/25/11) 0.61 0.09
ENPP1 rs1044498 37% (19/11/9) 42% (10/8/6) 0.89 0.72 36% (105/80/42) 1.03 0.90 29% (660/224/208) 1.27 0.29 19% (37/15/3) 1.96 0.05
ERLIN1 rs2862954 29% (20/14/4) 14% (15/6/0) 2.03 0.20 26% (127/69/22) 1.12 0.70 25% (665/314/113) 1.17 0.55 48% (15/27/13) 0.60 0.07
PNPLA3 rs738409 56% (9/17/14) 19% (16/7/1) 2.95 0.003 19% (139/62/8) 3.01 4.4E‐7 28% (590/382/120) 1.97 0.0001 36% (20/30/5) 1.56 0.05
PNPLA3 rs2896019 41% (17/13/10) 17% (17/6/1) 2.48 0.04 25% (132/75/20) 1.63 0.04 28% (604/374/114) 1.50 0.06 28% (27/25/3) 1.47 0.18
SAMM50 rs3761472 38% (17/14/8) 17% (16/8/0) 2.31 0.07 21% (146/68/14) 1.83 0.02 25% (628/374/90) 1.52 0.07 21% (33/21/1) 1.83 0.06
SAMM50 rs2143571 44% (14/16/9) 25% (13/10/1) 1.74 0.14 28% (120/88/20) 1.55 0.05 32% (514/453/125) 1.35 0.14 30% (25/27/3) 1.45 0.18
ABCC2 rs17222723 10% (31/8/0) 2% (23/1/0) 4.92 0.22 7% (193/33/0) 1.40 0.52 4% (1000/91/1) 2.40 0.08 7% (47/8/0) 1.47 0.57
(B)
MAF‐Bronx population MAF‐general population OR P‐value MAF‐Puerto Rican population OR P‐value
CHUK rs11597086 23% 22% 1.04 0.79 43% 0.54 0.003
ENPP1 rs1044498 36% 29% 1.24 0.04 19% 1.90 0.02
ERLIN1 rs2862954 26% 25% 1.05 0.69 48% 0.54 1.5E‐3
PNPLA3 rs738409 19% 28% 0.66 3.5E‐3 36% 0.52 5.2E‐4
PNPLA3 rs2896019 25% 28% 0.92 0.49 28% 0.90 0.68
SAMM50 rs3761472 21% 25% 0.83 0.17 21% 1.00 0.98
SAMM50 rs2143571 28% 32% 0.87 0.22 30% 0.94 0.78
ABCC2 rs17222723 7% 4% 1.71 0.05 7% 1.04 0.94
Open in a new tab

NAFLD, nonalcoholic fatty liver disease; SNP, single‐nucleotide polymorphisms.

The SNPs in PNPLA3 and SAMM50 (OMIM # 612058) (rs738409, rs2896019; NM_025225.2:c.979+542T>G, rs3761472; NM_015380.4:c.329A>G) were previously identified in GWAS studies and generally had increased frequencies in the NAFLD population compared with our controls, the sample Bronx population, and expected population frequencies. Increased MAF of the alternative allele of SNPs rs738409 and rs2896019 in PNPLA3 was found in our Bronx population sample as compared to the MAF recorded for the Puerto Rican population. Similarly, the MAF of the alternative allele was increased significantly in disease‐related SNPs, rs1044498 ((NM_006208.2:c.517A>C) and rs17222723 (NM_000392.4:c.3563T>A), in ENPP1 (OMIM # 173335) and ABCC2 (OMIM # 601107). The MAF of the alternative allele of rs17222723 and rs1044498 were statistically significant (P < 0.05) in both the NAFLD cases and Bronx population than the expected based on population data, but were not significant when comparing NAFLD cases versus Bronx population. The SNP rs1044498 (ENPP1) was most significant (P = 0.02) when comparing the Bronx population with the Puerto Rican population.

Common SNP variations in an intron of CHUK (OMIM # 600664) (rs11597086; (NM_001278.3:c.1974+36T>G)) and exon of ERLIN1 (OMIM # 611604) (rs2862954; (NM_001100626.1:c.871A>G)) occurred less frequently in both the NAFLD cases and the sample Bronx population than would be expected in a random Puerto Rican population. The intronic CHUK, SNP variant, rs11591741, also occurred less frequently in our Bronx population than would be expected in a random Puerto Rican population (31 vs. 43%, P = 0.03); however, it occurred more often than would be expected in the general population (31 vs. 23%, P = 0.004).

PNPLA3 sequencing

Sixteen common and six rare DNA variations were uncovered upon sequencing of PNPLA3 (Table 3). Two SNPs, rs738409 and rs6006460 (NM_025225.2:c.1358G>T), have known associations with NAFLD genes from previous GWAS studies and were also included in our Sequenom assay; three other SNPs are associated with hepatic steatosis as suggested in the literature (rs738408; NM_025225.2:c.447C>T (Speliotes 2011), rs4823173; NM_025225.2:c.487‐28G>A (DiStefano et al. 2014), rs139051; NM_025225.2:c.421‐28A>G (Peng et al. 2012)). The remaining 17 SNP variations have no known disease or phenotypic associations based on literature review or Online Mendelian Inheritance in Man (OMIM). Six variants with a MAF of ≤1% in the general, Hispanic, and Puerto Rican populations were discovered and included in Table 3. Of note, four of these variations occurred more often in our NAFLD cases than in controls (rs144730517; NM_025225.2:c.1184C>G, rs192841252; NM_025225.2:c.420+23C>T, rs41278871; NM_025225.2:c.*393G>C, rs144821153 NM_025225.2:c.‐40C>G).

Table 3.

PNPLA3 sequencing results (all P‐values are for relative comparisons to NAFLD cases)

SNP NAFLD allele frequency n = 40 Control allele frequency n = 24 P OR Minor allele Chromosome position MAF n = 2184 P MAF (HIS) n = 362 P MAF (PUR) n = 110 P
rs738409 56% 19% 0.003 3.00 G 43,928,847 28.4% 1.18E‐04 45% 0.18 36% 0.03
rs738408 55% 19% 0.004 2.93 T 43,928,850 28.4% 2.35E‐04 45% 0.23 36% 0.04
rs2294919 10% 30% 0.040 0.33 T 43,946,445 26.1% 0.02 19% 0.16 19% 0.19
rs4823173 41% 19% 0.063 2.20 A 43,932,850 25.7% 0.03 39% 0.78 28% 0.12
rs139047 35% 14% 0.086 2.45 A 43,927,194 34.2% 0.92 25% 0.17 25% 0.23
rs2294918 20% 39% 0.112 0.52 A 43,946,236 23.0% 0.65 24% 0.57 31% 0.18
rs139051 54% 38% 0.208 1.43 A 43,928,796 47.8% 0.46 55% 0.88 45% 0.34
rs34879941 25% 8% 0.215 3.00 T 43,936,998 27.3% 0.75 40% 0.06 28% 0.72
rs41278873 10% 2% 0.246 4.60 C 43,946,811 3.0% 0.01 6% 0.33 9% 0.85
rs144730517 3% 0% 0.353 2.56a G 43,944,762 0.1% 6.94E‐05 0% 0.00 0% 0.10
rs115043594 0% 2% 0.445 0.29 A 43,946,514 0.0% NA NA NA NA NA
rs192841252 4% 0% 0.614 2.15 T 43,927,190 0.1% 1.85E‐08 1% 0.14 1% 0.25
rs2294917 16% 21% 0.652 0.77 C 43,946,106 35.2% 0.01 28% 0.11 29% 0.11
rs2076213 14% 10% 0.696 1.32 G 43,927,042 7.6% 0.15 17% 0.60 13% 0.90
rs41278871 3% 0% 0.743 1.68 C 43,946,775 0.6% 0.13 0% 0.00 0% 0.10
rs2072906 23% 18% 0.758 1.24 G 43,937,292 27.5% 0.48 40% 0.03 28% 0.50
rs2076212 14% 17% 0.760 0.83 T 43,927,090 11.0% 0.58 13% 0.89 15% 0.87
rs6006461 1% 0% 0.793 1.56 T 43,946,433 0.2% 0.78 0% NA 0% NA
rs76510336 3% 2% 0.797 1.55 G 43,946,295 1.4% 0.56 0% 0.00 1% 0.49
rs6006460 1% 2% 0.898 0.78 T 43,946,294 3.1% 0.26 0% NA 0% NA
rs144821153 1% 0% 0.908 1.22 G 43,923,872 0.4% 0.41 1% 0.88 1% 0.85
rs34179073 3% 2% 0.915 1.20 T 43,932,952 4.4% 0.56 7% 0.28 6% 0.39
Open in a new tab

NAFLD, nonalcoholic fatty liver disease; MAF, minor allele frequency; SNP, single‐nucleotide polymorphisms.

a

Using zero‐cell correction.

Discussion

NAFLD is a highly prevalent disease that is the most common cause of liver disease worldwide. With its connection to obesity and metabolic syndrome, NAFLD and its more severe form, NASH, are projected to continually increase as a public health burden. Although NAFLD occurs with increased incidence and severity in the Mexican Hispanic population, the principal aim of this study was to explore previously discovered NAFLD variants in our largely Caribbean–Hispanic population. The NAFLD patients seen at Montefiore Medical Center are of mostly Dominican or Puerto Rican ancestry, populations highly affected with fatty liver disease and to our knowledge never studied in this context. We performed liver biopsies as part of the clinical evaluation of these patients. Additionally, our analysis benefits from the inclusion of previously discovered and/or validated genes and variants related to fatty liver disease by providing more power. This may enhance any implications made in the context of our limited number of samples. Indeed, the greatest weakness in our study is the relatively small number of patients when examining a common disease. Therefore, the purpose of our discussion is to provide a framework for future analyses. All results of our experiments are suggestive, but not intended to be conclusive.

As discussed previously, NAFLD is a complex disease with a multifactorial etiology associated with diet, obesity, and a variety of comorbidities related to insulin resistance (Marchesini et al. 1999). Although environmental factors are important in determining risk for the disease, evidence from familial and twin studies support the assumption that genetics provide an important modulator of NAFLD development and disease progression (Brouwers et al. 2006; Makkonen et al. 2009; Schwimmer et al. 2009). The most studied and replicated variant is rs738409 (NM_025225.2:c.444C>G) in PNPLA3. This common missense variant has demonstrated increased risk for the development of NAFLD independent of diabetes or obesity and is also significantly associated with degree of histological severity (Rotman et al. 2010; Speliotes et al. 2010; Valenti et al. 2010a,b). The strongest effect of the variant appears to exist within the Hispanic population versus African‐American or European‐American individuals (Romeo et al. 2008). The overall strength of the association between this polymorphism and NAFLD has recently been confirmed in a genetic meta‐analysis (Sookoian and Pirola 2011).

Although the missense SNP, rs738409, has been repeatedly evaluated and tested, to our knowledge the entire coding region of PNPLA3 had never been sequenced in the Hispanic population with respect to NAFLD. Our study demonstrated that the minor, G allele is more common in our Caribbean–Hispanic patients with NAFLD than in any other group analyzed. This included ethnically matched controls (OR 2.95, P = 0.003), the sample Bronx population (OR 3.01, P = 4.4E‐7), the general population (OR 1.97, P = 0.0001), and even the average Puerto Rican population (OR 1.56, P = 0.05).

Mutations in PNPLA3 provide a possible genetic basis for the underlying mechanisms in the genesis of NAFLD. The PNPLA3 gene is located on chromosome 22q13.3 and encodes for a membrane‐bound protein mediating triacylglycerol hydrolysis in adipocytes. Its expression is highest in human liver tissue (Huang et al. 2010) and is induced during feeding and insulin resistance by fatty acids and other regulators of lipogenesis (Huang et al. 2010; Dongiovanni et al. 2013). The rs738409 missense variation specifically is assumed to promote triglyceride accumulation through relative inhibition of triglyceride hydrolysis (He et al. 2010). An understanding of the theoretical pathophysiology behind NAFLD illustrates how PNPLA3's function is consistent with a theoretical role in the progression of the disease.

Although the exact pathogenesis of NAFLD is still under debate, there are a number of mechanisms that are clearly involved. These include insulin resistance, free fatty acid flux, endoplasmic reticulum stress, oxidative stress, and inflammation (Yoon and Cha 2014). Simplistically, steatosis in the liver develops when supply of fatty acids to the liver exceed the demand in requirements for mitochondrial oxidation and synthesis of phospholipids, triglycerides, and cholesterol (Lall et al. 2008). Triglyceride accumulation results from either lipid uptake in the liver or de novo synthesis in the setting of excess carbohydrates (Kawano and Cohen 2013). Insulin resistance has repeatedly been implicated as an important cause of lipid accumulation in the liver (Marchesini et al. 1999; Sanyal et al. 2001; Chitturi et al. 2002; Pagano et al. 2002). Once the liver is overrun with lipid accumulation, the mitochondria attempt to remove the fatty acids through oxidation. However, this process can inadvertently cause oxidative stress and mitochondrial dysfunction through excessive production of reactive oxygen species (ROS) (Rolo et al. 2012; Yoon and Cha 2014). This hepatocellular injury is further exacerbated by the secretion of inflammatory cytokines (e.g., TNF‐α, IL‐6, and NO) that are induced through the presence of adipose tissue in the liver and basal insulin resistance (Carter‐Kent et al. 2008; Hijona et al. 2010; Odegaard and Chawla 2011; Yoon and Cha 2014). This proinflammatory state and resultant fibrosis/necrosis define the histopathological spectrum of NAFLD.

Many of the genes identified in our study as having variations with multiple significant results have functions that fit in with the pathogenesis of NAFLD and may logically influence disease susceptibility or progression. These particularly include SAMM50, ENPP1, and ABCC2. ENPP1's function can be directly linked to hepatic steatosis as it codes for a membrane glycoprotein functioning to inhibit insulin signaling. The rs1044498, SNP variation causes a gain‐of‐function mutation that causes overexpression in peripheral insulin target tissues and is associated with human insulin resistance (Prudente and Trischitta 2006). If SAMM50 is involved in NAFLD, it is likely related to its role in mitochondrial function. SAMM50 encodes a component of the SAM assembly complex, which helps integrate β‐barrel proteins into the outer mitochondrial membrane. Any mitochondrial dysfunction and resultant decrease in removal of reactive oxygen species (ROS) as a result of mutations in SAMM50 is consistent with biochemical rationale for the importance of SAMM50 in NAFLD (Kitamoto et al. 2013). ABCC2 has a function that is more removed from a direct correlation with NAFLD. It encodes a protein expressed in the apical area of the hepatocyte and functions in biliary transport, likely critical to the elimination of conjugates of many toxins from hepatocytes into bile (Nies and Keppler 2007) and likely predisposing liver toward injury from excessive adipose tissue (Sookoian et al. 2009).

There is one gene cluster, ERLIN1‐CHUK‐CWF19L1, which revealed associations with SNPs that appear to confer protection in our study. The relation of these genes to NAFLD is theoretically logical as ERLIN1 comprises a component of lipid rafts (Browman et al. 2006) and CHUK proteins modulate NF‐kB activation of several genes involved in insulin resistance (Yuan et al. 2008). For the SNPs rs11597086 and rs2862954, the rate of polymorphism in our sample Bronx population was lower than in the average Puerto Rican population (OR 0.54, P = 0.003; OR 0.54, P = 1.5E‐3). In our study, these variations also occur less frequently in NAFLD cases than would be expected in a random Puerto Rican population (OR 0.61, P = 0.09, OR 0.60, P = 0.07). The fact that these polymorphisms only seem to confer protection when compared with the Puerto Rican community suggest that there is some other genetic modifier in the Hispanic subpopulation that interacts with these genes to protect patients from the development of hepatic steatosis. This phenomenon is interesting and warrants further investigation.

In conclusion, this study implies significant interactions of variants from PNPLA3, ERLIN1‐ CHUK, SAMM50, ENPP1, and ABCC2 with NAFLD in Hispanics from a majority Caribbean ancestry. An interaction of these genes with NAFLD is pathophysiologically plausible and the extent of impact of these variations in NAFLD generally and in sub‐Hispanic populations deserves further analysis.

Conflict of Interest

None declared.

Supporting information

Table S1. PNPLA3 primers.

Table S2. Full sequenom results (Target SNPs).

Click here for additional data file. (17.9KB, docx)

References

  1. 2008. –2012 American Community Survey . U.S. Census Bureau.
  2. Adams, L. A. , Sanderson S., Lindor K. D., and Angulo P.. 2005. The histological course of nonalcoholic fatty liver disease: a longitudinal study of 103 patients with sequential liver biopsies. J. Hepatol. 42:132–138. [DOI] [PubMed] [Google Scholar]
  3. Adams, L. A. , Marsh J. A., Ayonrinde O. T., Olynyk J. K., Ang W. Q., Beilin L. J., et al. 2012. Cholesteryl ester transfer protein gene polymorphisms increase the risk of fatty liver in females independent of adiposity. J. Gastroenterol. Hepatol. 27:1520–1527. [DOI] [PubMed] [Google Scholar]
  4. Adams, L. A. , White S. W., Marsh J. A., Lye S. J., Connor K. L., Maganga R., et al. 2013. Association between liver‐specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease. Hepatology 57:590–600. [DOI] [PubMed] [Google Scholar]
  5. Aller, R. , De Luis D. A., Izaola O., Gonzalez Sagrado M., Conde R., Alvarez T., et al. 2010. Role of ‐55CT polymorphism of UCP3 gene on non alcoholic fatty liver disease and insulin resistance in patients with obesity. Nutr. Hosp. 25:572–576. [PubMed] [Google Scholar]
  6. Al‐Serri, A. , Anstee Q. M., Valenti L., Nobili V., Leathart J. B., Dongiovanni P., et al. 2012. The SOD2 C47T polymorphism influences NAFLD fibrosis severity: evidence from case‐control and intra‐familial allele association studies. J. Hepatol. 56:448–454. [DOI] [PubMed] [Google Scholar]
  7. Angulo, P. 2002. Nonalcoholic fatty liver disease. N. Engl. J. Med. 346:1221–1231. [DOI] [PubMed] [Google Scholar]
  8. Anstee, Q. M. , and Day C. P.. 2013. The genetics of NAFLD. Nat. Rev. Gastroenterol. Hepatol. 10:645–655. [DOI] [PubMed] [Google Scholar]
  9. Argo, C. K. , Northup P. G., Al‐Osaimi A. M., and Caldwell S. H.. 2009. Systematic review of risk factors for fibrosis progression in non‐alcoholic steatohepatitis. J. Hepatol. 51:371–379. [DOI] [PubMed] [Google Scholar]
  10. Auinger, A. , Valenti L., Pfeuffer M., Helwig U., Herrmann J., Fracanzani A. L., et al. 2010. A promoter polymorphism in the liver‐specific fatty acid transport protein 5 is associated with features of the metabolic syndrome and steatosis. Horm. Metab. Res. 42:854–859. [DOI] [PubMed] [Google Scholar]
  11. Brouwers, M. C. , Cantor R. M., Kono N., Yoon J. L., van der Kallen C. J., Bilderbeek‐Beckers M. A., et al. 2006. Heritability and genetic loci of fatty liver in familial combined hyperlipidemia. J. Lipid Res. 47:2799–2807. [DOI] [PubMed] [Google Scholar]
  12. Browman, D. T. , Resek M. E., Zajchowski L. D., and Robbins S. M.. 2006. Erlin‐1 and erlin‐2 are novel members of the prohibitin family of proteins that define lipid‐raft‐like domains of the ER. J. Cell Sci. 119(Pt 15):3149–3160. [DOI] [PubMed] [Google Scholar]
  13. Browning, J. D. , Szczepaniak L. S., Dobbins R., Nuremberg P., Horton J. D., Cohen J. C., et al. 2004. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 40:1387–1395. [DOI] [PubMed] [Google Scholar]
  14. Bugianesi, E. , Leone N., Vanni E., Marchesini G., Brunello F., Carucci P., et al. 2002. Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology 123:134–140. [DOI] [PubMed] [Google Scholar]
  15. Carter‐Kent, C. , Zein N. N., and Feldstein A. E.. 2008. Cytokines in the pathogenesis of fatty liver and disease progression to steatohepatitis: implications for treatment. Am. J. Gastroenterol. 103:1036–1042. [DOI] [PubMed] [Google Scholar]
  16. Carulli, L. , Canedi I., Rondinella S., Lombardini S., Ganazzi D., Fargion S., et al. 2009. Genetic polymorphisms in non‐alcoholic fatty liver disease: interleukin‐6‐174G/C polymorphism is associated with non‐alcoholic steatohepatitis. Dig. Liver Dis. 41:823–828. [DOI] [PubMed] [Google Scholar]
  17. Chalasani, N. , Guo X., Loomba R., Goodarzi M. O., Haritunians T., Kwon S., et al. 2010. Genome‐wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease. Gastroenterology 139:1567–1576, 1576.e1561–1566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Chambers, J. C. 2011. Genome‐wide association study identifies loci influencing concentrations of liver enzymes in plasma. Nat. Genet. 43:1131–1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Charlton, M. , Kasparova P., Weston S., Lindor K., Maor‐Kendler Y., Wiesner R. H., et al. 2001. Frequency of nonalcoholic steatohepatitis as a cause of advanced liver disease. Liver Transpl. 7:608–614. [DOI] [PubMed] [Google Scholar]
  20. Chen, S. , Li Y., Li S., and Yu C.. 2008. A Val227Ala substitution in the peroxisome proliferator activated receptor alpha (PPAR alpha) gene associated with non‐alcoholic fatty liver disease and decreased waist circumference and waist‐to‐hip ratio. J. Gastroenterol. Hepatol. 23:1415–1418. [DOI] [PubMed] [Google Scholar]
  21. Chitturi, S. , Abeygunasekera S., Farrell G. C., Holmes‐Walker J., Hui J. M., Fung C., et al. 2002. NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 35:373–379. [DOI] [PubMed] [Google Scholar]
  22. Clark, J. M. , Brancati F. L., and Diehl A. M.. 2003. The prevalence and etiology of elevated aminotransferase levels in the United States. Am. J. Gastroenterol. 98:960–967. [DOI] [PubMed] [Google Scholar]
  23. DiStefano, J. K. , Kingsley C., Craig Wood G., Chu X., Argyropoulos G., Still C. D., et al. 2014. Genome‐wide analysis of hepatic lipid content in extreme obesity. Acta Diabetol. 52:373–382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Dixon, J. B. , Bhathal P. S., Jonsson J. R., Dixon A. F., Powell E. E., and O'Brien P. E.. 2003. Pro‐fibrotic polymorphisms predictive of advanced liver fibrosis in the severely obese. J. Hepatol. 39:967–971. [DOI] [PubMed] [Google Scholar]
  25. Dong, H. , Wang J., Li C., Hirose A., Nozaki Y., Takahashi M., et al. 2007. The phosphatidylethanolamine N‐methyltransferase gene V175M single nucleotide polymorphism confers the susceptibility to NASH in Japanese population. J. Hepatol. 46:915–920. [DOI] [PubMed] [Google Scholar]
  26. Dongiovanni, P. , Valenti L., Rametta R., Daly A. K., Nobili V., Mozzi E., et al. 2010. Genetic variants regulating insulin receptor signalling are associated with the severity of liver damage in patients with non‐alcoholic fatty liver disease. Gut 59:267–273. [DOI] [PubMed] [Google Scholar]
  27. Dongiovanni, P. , Anstee Q. M., and Valenti L.. 2013. Genetic predisposition in NAFLD and NASH: impact on severity of liver disease and response to treatment. Curr. Pharm. Des. 19:5219–5238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fabbrini, E. , Sullivan S., and Klein S.. 2010. Obesity and nonalcoholic fatty liver disease: biochemical, metabolic, and clinical implications. Hepatology 51:679–689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Farrell, G. C. , Wong V. W., and Chitturi S.. 2013. NAFLD in Asia–as common and important as in the West. Nat. Rev. Gastroenterol. Hepatol. 10:307–318. [DOI] [PubMed] [Google Scholar]
  30. Feitosa, M. F. , Wojczynski M. K., North K. E., Zhang Q., Province M. A., Carr J. J., et al. 2013. The ERLIN1‐CHUK‐CWF19L1 gene cluster influences liver fat deposition and hepatic inflammation in the NHLBI Family Heart Study. Atherosclerosis 228:175–180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Giday, S. A. , Ashiny Z., Naab T., Smoot D., and Banks A.. 2006. Frequency of nonalcoholic fatty liver disease and degree of hepatic steatosis in African‐American patients. J. Natl Med. Assoc. 98:1613–1615. [PMC free article] [PubMed] [Google Scholar]
  32. Gupte, P. , Amarapurkar D., Agal S., Baijal R., Kulshrestha P., Pramanik S., et al. 2004. Non‐alcoholic steatohepatitis in type 2 diabetes mellitus. J. Gastroenterol. Hepatol. 19:854–858. [DOI] [PubMed] [Google Scholar]
  33. Hashemi, M. , Hoseini H., Yaghmaei P., Moazeni‐Roodi A., Bahari A., Hashemzehi N., et al. 2011. Association of polymorphisms in glutamate‐cysteine ligase catalytic subunit and microsomal triglyceride transfer protein genes with nonalcoholic fatty liver disease. DNA Cell Biol. 30:569–575. [DOI] [PubMed] [Google Scholar]
  34. He, S. , McPhaul C., Li J. Z., Garuti R., Kinch L., Grishin N. V., et al. 2010. A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis. J. Biol. Chem. 285:6706–6715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hijona, E. , Hijona L., Arenas J. I., and Bujanda L.. 2010. Inflammatory mediators of hepatic steatosis. Mediators Inflamm. 2010:837419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Huang, Y. , He S., Li J. Z., Seo Y.‐K., Osborne T. F., Cohen J. C., et al. 2010. A feed‐forward loop amplifies nutritional regulation of PNPLA3. Proc. Natl Acad. Sci. 107:7892–7897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kallwitz, E. R. , Guzman G., TenCate V., Vitello J., Layden‐Almer J., Berkes J., et al. 2009. The histologic spectrum of liver disease in African‐American, non‐Hispanic white, and Hispanic obesity surgery patients. Am. J. Gastroenterol. 104:64–69. [DOI] [PubMed] [Google Scholar]
  38. Kallwitz, E. R. , Daviglus M. L., Allison M. A., Emory K. T., Zhao L., Kuniholm M. H., et al. 2015. Prevalence of suspected non‐alcoholic fatty liver disease in hispanic/latino individuals differs by heritage. Clin. Gastroenterol. Hepatol. 13:569–576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kawaguchi, T. , Sumida Y., Umemura A., Matsuo K., Takahashi M., Takamura T., et al. 2012. Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non‐alcoholic fatty liver disease in Japanese. PLoS ONE 7:e38322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kawano, Y. , and Cohen D. E.. 2013. Mechanisms of hepatic triglyceride accumulation in non‐alcoholic fatty liver disease. J. Gastroenterol. 48:434–441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kitamoto, T. , Kitamoto A., Yoneda M., Hyogo H., Ochi H., Nakamura T., et al. 2013. Genome‐wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan. Hum. Genet. 132:783–792. [DOI] [PubMed] [Google Scholar]
  42. Kotronen, A. , Yki‐Järvinen H., Aminoff A., Bergholm R., Pietiläinen K. H., Westerbacka J., et al. 2009. Genetic variation in the ADIPOR2 gene is associated with liver fat content and its surrogate markers in three independent cohorts. Eur. J. Endocrinol. 160:593–602. [DOI] [PubMed] [Google Scholar]
  43. Lall, C. G. , Aisen A. M., Bansal N., and Sandrasegaran K.. 2008. Nonalcoholic fatty liver disease. Am. J. Roentgenol. 190:993–1002. [DOI] [PubMed] [Google Scholar]
  44. Lazo, M. , Hernaez R., Bonekamp S., Kamel I. R., Brancati F. L., Guallar E., et al. 2011. Non‐alcoholic fatty liver disease and mortality among US adults: prospective cohort study. BMJ 343:d6891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Lebovics, E. , and Rubin J.. 2011. Non‐alcoholic fatty liver disease (NAFLD): why you should care, when you should worry, what you should do. Diabetes Metab. Res. Rev. 27:419–424. [DOI] [PubMed] [Google Scholar]
  46. Lin, Y. C. , Chang P. F., Chang M. H., and Ni Y. H.. 2013. A common variant in the peroxisome proliferator‐activated receptor‐gamma coactivator‐1alpha gene is associated with nonalcoholic fatty liver disease in obese children. Am. J. Clin. Nutr. 97:326–331. [DOI] [PubMed] [Google Scholar]
  47. Ludtke, A. , Genschel J., Brabant G., Bauditz J., Taupitz M., Koch M., et al. 2005. Hepatic steatosis in dunnigan‐type familial partial lipodystrophy. Am. J. Gastroenterol. 100:2218–2224. [DOI] [PubMed] [Google Scholar]
  48. Makkonen, J. , Pietilainen K. H., Rissanen A., Kaprio J., and Yki‐Jarvinen H.. 2009. Genetic factors contribute to variation in serum alanine aminotransferase activity independent of obesity and alcohol: a study in monozygotic and dizygotic twins. J. Hepatol. 50:1035–1042. [DOI] [PubMed] [Google Scholar]
  49. Marchesini, G. , Brizi M., Morselli‐Labate A. M., Bianchi G., Bugianesi E., McCullough A. J., et al. 1999. Association of nonalcoholic fatty liver disease with insulin resistance. Am. J. Med. 107:450–455. [DOI] [PubMed] [Google Scholar]
  50. Miele, L. , Beale G., Patman G., Nobili V., Leathart J., Grieco A., et al. 2008. The Kruppel‐like factor 6 genotype is associated with fibrosis in nonalcoholic fatty liver disease. Gastroenterology 135:282–291.e281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Mohanty, S. R. , Troy T. N., Huo D., O'Brien B. L., Jensen D. M., and Hart J.. 2009. Influence of ethnicity on histological differences in non‐alcoholic fatty liver disease. J. Hepatol. 50:797–804. [DOI] [PubMed] [Google Scholar]
  52. Musso, G. , Gambino R., De Michieli F., Durazzo M., Pagano G., and Cassader M.. 2008. Adiponectin gene polymorphisms modulate acute adiponectin response to dietary fat: possible pathogenetic role in NASH. Hepatology 47:1167–1177. [DOI] [PubMed] [Google Scholar]
  53. Musso, G. , Gambino R., Pacini G., Pagano G., Durazzo M., and Cassader M.. 2009. Transcription factor 7–like 2 polymorphism modulates glucose and lipid homeostasis, adipokine profile, and hepatocyte apoptosis in NASH. Hepatology 49:426–435. [DOI] [PubMed] [Google Scholar]
  54. Namikawa, C. , Shu‐Ping Z., Vyselaar J. R., Nozaki Y., Nemoto Y., Ono M., et al. 2004. Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non‐alcoholic steatohepatitis. J. Hepatol. 40:781–786. [DOI] [PubMed] [Google Scholar]
  55. Neuschwander‐Tetri, B. A. , and Caldwell S. H.. 2003. Nonalcoholic steatohepatitis: summary of an AASLD single topic conference. Hepatology 37:1202–1219. [DOI] [PubMed] [Google Scholar]
  56. Nies, A. T. , and Keppler D.. 2007. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Archiv. 453:643–659. [DOI] [PubMed] [Google Scholar]
  57. Odegaard, J. I. , and Chawla A.. 2011. Alternative macrophage activation and metabolism. Ann. Rev. Pathol. 6:275–297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Oliveira, C. P. M. S. , Stefano J. T., Cavaleiro A. M., Zanella Fortes M. A. H., Vieira S. M., Rodrigues Lima V. M., et al. 2010. Association of polymorphisms of glutamate‐cystein ligase and microsomal triglyceride transfer protein genes in non‐alcoholic fatty liver disease. J. Gastroenterol. Hepatol. 25:357–361. [DOI] [PubMed] [Google Scholar]
  59. Pagano, G. , Pacini G., Musso G., Gambino R., Mecca F., Depetris N., et al. 2002. Nonalcoholic steatohepatitis, insulin resistance, and metabolic syndrome: further evidence for an etiologic association. Hepatology 35:367–372. [DOI] [PubMed] [Google Scholar]
  60. Pan, J. J. , Qu H. Q., Rentfro A., McCormick J. B., Fisher‐Hoch S. P., and Fallon M. B.. 2011. Prevalence of metabolic syndrome and risks of abnormal serum alanine aminotransferase in Hispanics: a population‐based study. PLoS ONE 6:e21515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Paschos, P. , and Paletas K.. 2009. Non alcoholic fatty liver disease and metabolic syndrome. Hippokratia 13:9–19. [PMC free article] [PubMed] [Google Scholar]
  62. Peng, X. E. , Wu Y. L., Lin S. W., Lu Q. Q., Hu Z. J., and Lin X.. 2012. Genetic variants in PNPLA3 and risk of non‐alcoholic fatty liver disease in a Han Chinese population. PLoS ONE 7:e50256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Peng, X. E. , Wu Y. L., Lu Q. Q., Hu Z. J., and Lin X.. 2013. MTTP polymorphisms and susceptibility to non‐alcoholic fatty liver disease in a Han Chinese population. Liver Int. 34:118–128. [DOI] [PubMed] [Google Scholar]
  64. Petersen, K. F. , Dufour S., Hariri A., Nelson‐Williams C., Foo J. N., Zhang X.‐M., et al. 2010. Apolipoprotein C3 gene variants in nonalcoholic fatty liver disease. N. Engl. J. Med. 362:1082–1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Petta, S. , Grimaudo S., Camma C., Cabibi D., Di Marco V., Licata G., et al. 2012. IL28B and PNPLA3 polymorphisms affect histological liver damage in patients with non‐alcoholic fatty liver disease. J. Hepatol. 56:1356–1362. [DOI] [PubMed] [Google Scholar]
  66. Prudente, S. , and Trischitta V.. 2006. The pleiotropic effect of the ENPP1 (PC‐1) gene on insulin resistance, obesity, and type 2 diabetes. J. Clin. Endocrinol. Metab. 91:4767–4768. [DOI] [PubMed] [Google Scholar]
  67. Ratziu, V. , Bonyhay L., Di Martino V., Charlotte F., Cavallaro L., Sayegh‐Tainturier M. H., et al. 2002. Survival, liver failure, and hepatocellular carcinoma in obesity‐related cryptogenic cirrhosis. Hepatology 35:1485–1493. [DOI] [PubMed] [Google Scholar]
  68. Rolo, A. P. , Teodoro J. S., and Palmeira C. M.. 2012. Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. Free Radic. Biol. Med. 52:59–69. [DOI] [PubMed] [Google Scholar]
  69. Romeo, S. , Kozlitina J., Xing C., Pertsemlidis A., Cox D., Pennacchio L. A., et al. 2008. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat. Genet. 40:1461–1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Rotman, Y. , Koh C., Zmuda J. M., Kleiner D. E., and Liang T. J.. 2010. The association of genetic variability in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) with histological severity of nonalcoholic fatty liver disease. Hepatology 52:894–903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Sanyal, A. J. , Campbell‐Sargent C., Mirshahi F., Rizzo W. B., Contos M. J., Sterling R. K., et al. 2001. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 120:1183–1192. [DOI] [PubMed] [Google Scholar]
  72. Sazci, A. , Ozel M. D., Ergul E., and Aygun C.. 2013. Association of nicotinamide‐N‐methyltransferase gene rs694539 variant with patients with nonalcoholic steatohepatitis. Genet. Test. Mol. Biomarkers 17:849–853. [DOI] [PubMed] [Google Scholar]
  73. Schneider, A. L. C. , Lazo M., Selvin E., and Clark J. M.. 2013. Racial differences in nonalcoholic fatty liver disease in the U.S. population. Obesity 22:292–299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Schwimmer, J. B. , Celedon M. A., Lavine J. E., Salem R., Campbell N., Schork N. J., et al. 2009. Heritability of nonalcoholic fatty liver disease. Gastroenterology 136:1585–1592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Sharp, D. B. , Santos L. A., and Cruz M. L.. 2009. Fatty liver in adolescents on the U.S.‐Mexico border. J. Am. Acad. Nurse Pract. 21:225–230. [DOI] [PubMed] [Google Scholar]
  76. Silverman, J. F. , O'Brien K. F., Long S., Leggett N., Khazanie P. G., Pories W. J., et al. 1990. Liver pathology in morbidly obese patients with and without diabetes. Am. J. Gastroenterol. 85:1349–1355. [PubMed] [Google Scholar]
  77. Smits, M. M. , Ioannou G. N., Boyko E. J., and Utzschneider K. M.. 2013. Non‐alcoholic fatty liver disease as an independent manifestation of the metabolic syndrome: results of a US national survey in three ethnic groups. J. Gastroenterol. Hepatol. 28:664–670. [DOI] [PubMed] [Google Scholar]
  78. Song, J. , da Costa K. A., Fischer L. M., Kohlmeier M., Kwock L., Wang S., et al. 2005. Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD). FASEB J. 19:1266–1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Sookoian, S. , and Pirola C. J.. 2011. 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 53:1883–1894. [DOI] [PubMed] [Google Scholar]
  80. Sookoian, S. , Castano G., Gemma C., Gianotti T. F., and Pirola C. J.. 2007. Common genetic variations in CLOCK transcription factor are associated with nonalcoholic fatty liver disease. World J. Gastroenterol. 13:4242–4248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Sookoian, S. , Castaño G., Gianotti T. F., Gemma C., Rosselli M. S., and Pirola C. J.. 2008. Genetic variants in STAT3 are associated with nonalcoholic fatty liver disease. Cytokine 44:201–206. [DOI] [PubMed] [Google Scholar]
  82. Sookoian, S. , Castano G., Gianotti T. F., Gemma C., and C. J. Pirola . 2009. Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease. J. Nutr. Biochem. 20:765–770. [DOI] [PubMed] [Google Scholar]
  83. Sookoian, S. , Castano G. O., Burgueno A. L., Gianotti T. F., Rosselli M. S., and Pirola C. J.. 2010. The nuclear receptor PXR gene variants are associated with liver injury in nonalcoholic fatty liver disease. Pharmacogenet. Genomics 20:1–8. [DOI] [PubMed] [Google Scholar]
  84. Speliotes, E. K. 2011. Genome‐wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits. PLoS Genet. 7:e1001324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. Speliotes, E. K. , Butler J. L., Palmer C. D., Voight B. F., and Hirschhorn J. N.. 2010. PNPLA3 variants specifically confer increased risk for histologic nonalcoholic fatty liver disease but not metabolic disease. Hepatology 52:904–912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Swellam, M. , and Hamdy N.. 2012. Association of nonalcoholic fatty liver disease with a single nucleotide polymorphism on the gene encoding leptin receptor. IUBMB Life 64:180–186. [DOI] [PubMed] [Google Scholar]
  87. Tan, H. L. , Zain S. M., Mohamed R., Rampal S., Chin K. F., Basu R. C., et al. 2013. Association of glucokinase regulatory gene polymorphisms with risk and severity of non‐alcoholic fatty liver disease: an interaction study with adiponutrin gene. J. Gastroenterol. 49:1056–1064. [DOI] [PubMed] [Google Scholar]
  88. Tokushige, K. , Takakura M., Tsuchiya‐Matsushita N., Taniai M., Hashimoto E., and Shiratori K.. 2007. Influence of TNF gene polymorphisms in Japanese patients with NASH and simple steatosis. J. Hepatol. 46:1104–1110. [DOI] [PubMed] [Google Scholar]
  89. Tokushige, K. , Hashimoto E., Noto H., Yatsuji S., Taniai M., Torii N., et al. 2009. Influence of adiponectin gene polymorphisms in Japanese patients with non‐alcoholic fatty liver disease. J. Gastroenterol. 44:976–982. [DOI] [PubMed] [Google Scholar]
  90. Valenti, L. , Fracanzani A. L., Dongiovanni P., Santorelli G., Branchi A., Taioli E., et al. 2002. Tumor necrosis factor α promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease. Gastroenterology 122:274–280. [DOI] [PubMed] [Google Scholar]
  91. Valenti, L. , Al‐Serri A., Daly A. K., Galmozzi E., Rametta R., Dongiovanni P., et al. 2010a. Homozygosity for the patatin‐like phospholipase‐3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease. Hepatology 51:1209–1217. [DOI] [PubMed] [Google Scholar]
  92. Valenti, L. , Fracanzani A. L., Bugianesi E., Dongiovanni P., Galmozzi E., Vanni E., et al. 2010b. HFE genotype, parenchymal iron accumulation, and liver fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology 138:905–912. [DOI] [PubMed] [Google Scholar]
  93. Valenti, L. , Motta B. M., Alisi A., Sartorelli R., Buonaiuto G., Dongiovanni P., et al. 2012. LPIN1 rs13412852 polymorphism in pediatric nonalcoholic fatty liver disease. J. Pediatr. Gastroenterol. Nutr. 54:588–593. [DOI] [PubMed] [Google Scholar]
  94. Vazquez‐Chantada, M. , Gonzalez‐Lahera A., Martinez‐Arranz I., Garcia‐Monzon C., Regueiro M. M., Garcia‐Rodriguez J. L., et al. 2013. Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease. Hepatology 57:505–514. [DOI] [PubMed] [Google Scholar]
  95. Wagenknecht, L. E. , Scherzinger A. L., Stamm E. R., Hanley A. J. G., Norris J. M., Chen Y.‐D. I., et al. 2009. Correlates and heritability of nonalcoholic fatty liver disease in a minority cohort. Obesity 17:1240–1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Weston, S. R. , Leyden W., Murphy R., Bass N. M., Bell B. P., Manos M. M., et al. 2005. Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology 41:372–379. [DOI] [PubMed] [Google Scholar]
  97. Wong, R. J. , and Ahmed A.. 2014. Obesity and non‐alcoholic fatty liver disease: disparate associations among Asian populations. World J. Hepatol. 6:263–273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  98. Xu, Y. P. , Liang L., Wang C. L., Fu J. F., Liu P. N., Lv L. Q., et al. 2013. Association between UCP3 gene polymorphisms and nonalcoholic fatty liver disease in Chinese children. World J. Gastroenterol. 19:5897–5903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  99. Yang, Z. , Wen J., Tao X., Lu B., Du Y., Wang M., et al. 2011. Genetic variation in the GCKR gene is associated with non‐alcoholic fatty liver disease in Chinese people. Mol. Biol. Rep. 38:1145–1150. [DOI] [PubMed] [Google Scholar]
  100. Yoneda, M. , Hotta K., Nozaki Y., Endo H., Uchiyama T., Mawatari H., et al. 2008. Association between PPARGC1A polymorphisms and the occurrence of nonalcoholic fatty liver disease (NAFLD). BMC Gastroenterol. 8:27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  101. Yoneda, M. , Hotta K., Nozaki Y., Endo H., Uchiyama T., Mawatari H., et al. 2009. Association between angiotensin II type 1 receptor polymorphisms and the occurrence of nonalcoholic fatty liver disease. Liver Int. 29:1078–1085. [DOI] [PubMed] [Google Scholar]
  102. Yoon, H.‐J. , and Cha B. S.. 2014. Pathogenesis and therapeutic approaches for non‐alcoholic fatty liver disease. World J. Hepatol. 6:800–811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  103. Yuan, X. , Waterworth D., Perry J. R., Lim N., Song K., Chambers J. C., et al. 2008. Population‐based genome‐wide association studies reveal six loci influencing plasma levels of liver enzymes. Am. J. Hum. Genet. 83:520–528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  104. Zain, S. M. , Mohamed Z., Mahadeva S., Cheah P. L., Rampal S., Chin K. F., et al. 2013a. Impact of leptin receptor gene variants on risk of non‐alcoholic fatty liver disease and its interaction with adiponutrin gene. J. Gastroenterol. Hepatol. 28:873–879. [DOI] [PubMed] [Google Scholar]
  105. Zain, S. M. , Mohamed Z., Mahadeva S., Rampal S., Basu R. C., Cheah P. L., et al. 2013b. Susceptibility and gene interaction study of the angiotensin II type 1 receptor (AGTR1) gene polymorphisms with non‐alcoholic fatty liver disease in a multi‐ethnic population. PLoS ONE 8:e58538. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. PNPLA3 primers.

Table S2. Full sequenom results (Target SNPs).

Click here for additional data file. (17.9KB, docx)

Articles from Molecular Genetics & Genomic Medicine are provided here courtesy of Blackwell Publishing

RESOURCES