Promoting genetics in non-alcoholic fatty liver disease: Combined risk score through polymorphisms and clinical variables

Table 1 Overview of the main studies testing pure genetics risk score in the assessment of non-alcoholic fatty liver disease patients

Ref.	Variables included	Risk score formula	Outcomes
Petta et al[53], 2012	IL28B rs12979860 CC and PNPLA3 rs738409 GG	IL28B rs12979860 CC + PNPLA3 rs738409 GG	Higher prevalence of moderate-severe lobular inflammation (P < 0.001) and NASH > 5 (P = 0.02)
Nobili et al[42], 2014	PNPLA3 rs738409 C>G, SOD2 rs4880 C>T, KLF6 rs3750861 G>A and LPIN1 rs13412852 C>T	1/[1+e^ (-0.804-PNPLA3 GG × 1.923 + SOD2 TT × 0.564 + LPIN1 TT × 0.551 - KLF6 AG-AA × 0.324)]	AUROC 0.75 (CI: 0.67-0.82, P < 0.0001) per NASH, cutoff 0.42%-90% sensitivity/36% specificity for NASH
Leon-Mimila et al[43], 2015	PNPLA3 rs738409, LYPLAL1 rs12137855, GCKR rs1260326 and PPP1R3B rs4240624	GRS = Sum of at-risk alleles: 0 for homozygous for the non-risk allele, 1 for heterozygous and 2 for homozygous for the risk allele	GRS ≥ 6 increased risk of NAS (OR = 2.55, P = 0.045) compared to those with GRS ≤ 5
Wang et al[40], 2016	PNPLA3 rs738409 and TM6SF2 rs58542926	Sum of at-risk alleles: 0, 1 or 2 according to the number of minor allele	OR for NAFLD increase of 1.52 per additional risk allele
Di Costanzo et al[54], 2017	PNPLA3 rs738409, GCKR rs1260326 and TM6SF2 rs58542926	Weighted sum of at-risk alleles	A 3 SNPs weighted genetic score > 0.32: five-fold increased risk of NAFLD
Krawczyk et al[38], 2017	PNPLA3 rs738409, TM6SF2 rs58542926 and MBOAT7 rs641738	Sum of at-risk alleles	Association with increasing hepatic fibrosis and steatosis; increase of serum AST activities (P < 0.0001), trends for increased ALT (P = 0.08) and GGT (P = 0.07)
Larrieta-Carrasco et al[39], 2018	PNPLA3 rs738409, PNPLA3 rs3810622, SAMM50 rs2143571, ADPOQ rs17366743, COL13A1 rs7101190 and COL13A1 rs12277556	Polygenic risk score = Sum of at-risk alleles 0 for homozygous for the non-risk allele, 1 for heterozygous and 2 for homozygous for the risk allele	9-12 vs 1-4 risk alleles: 65.8% and 48.5% higher ALT and AST levels
Vespasiani-Gentilucci et al[45], 2018	PNPLA3 rs738409, TM6SF2 rs58542926, KLF6 rs3750861	GRS: Sum of at-risk alleles PNPLA3 CC = 0, CG = 1, GG = 2; TM6SF2 CC = 0, CT and TT = 1; KLF6 CC = 0, CT and TT = 1	In healthy subjects: GRS 1-2 vs 0 increases 4-fold and GRS 3-4 vs 0 increases 20-fold the risk of non-cirrhotic NAFLD; in non-cirrhotic NAFLD: GRS 3-4 vs 0 increases 4-fold the risk of NASH cirrhosis
Di Costanzo et al[41], 2018	PNPLA3 rs738409, GCKR rs1260326, TM6SF2 rs58542926 and MBOAT7 rs641738	GRS: Sum of at-risk alleles (0-2); wGRS: Sum of at-risk alleles Beta coefficient	GRS 3 vs 2 higher in NAFLD (P = 0.001) wGRS > 0.28: three fold increased risk of NAFLD
Koo et al[44], 2018	PNPLA3 rs738409 and TM6SF2 rs58542926	Sum of at-risk alleles: PNPLA3 CC = 0, CG = 1, GG =2 TM6SF2 CC = 0, CT and TT = 1	Prevalence of NASH in NAFLD patients: 28.2% (0 allele), 41.8% (1 allele), 63.7% (2 alleles), 69.2% (3 alleles); risk of NASH: 2.04 OR per risk allele

GRS: Genetic risk score; wGRS: Weighted GRS; PNPLA3: Patatin-like phospholipase domain-containing protein 3; SOD2: Superoxide dismutase 2; KLF6: Krueppel-like factor 6; LPIN1: Lipin1; AUROC: Area under the receiver operating characteristic curve; CI: Confidence interval; NASH: Non-alcoholic steatohepatitis; GCKR: Glucokinase regulatory gene; TM6SF2: Transmembrane 6 superfamily member 2; ALT: Alanine aminotransferase; GGT: γ-glutamyl transferase; NAFLD: Non-alcoholic fatty liver disease; AST: Aspartate aminotransferase; MBOAT7: Membrane bound O-acyltransferase domain containing 7; SAMM50: Sorting and assembly machinery component 50 homolog; COL13A1: Collagen, type XIII, alpha 1.

Table 2 Overview of the main studies testing combined genetic/clinical risk score in the assessment of non-alcoholic fatty liver disease patients

Ref.	Variables included	Risk score formula	Outcomes
Kotronen et al[46], 2009	Clinical and laboratory data (metabolic syndrome, type 2 diabetes, insulin, AST, AST/ALT ratio and PNPLA3 rs738409 GG)	NAFLD liver fat score: [-2.89 + 1.18 × metabolic syndrome (yes = 1/no = 0) + 0.45 × type 2 diabetes (yes = 2/no = 0) + 0.15 × fS-insulin (mU/L) + 0.04 × fS-AST (U/L) - 0.94 × AST/ALT] and PNPLA3 rs738409 GG	Independent predictor of NAFLD with AUROC of 0.872 ± 0.02 (95%CI: 0.84-0.91); Addition of rs738409 to the score improved the accuracy of the prediction by only < 1%.
Francque et al[47], 2012	ALT, fasting levels of C-peptide, ultrasound steatosis scores and PNPLA3 rs738409 genotypes	ND	Predictor of NASH with AUROC of 0.8 Rs738409 correlated with development of NASH but did not add value
Guichelaar et al[55], 2013	PNPLA3 rs738409 G allele, CK-18 > 145 IU/ L, Glucose > 100 mg/dL, C-reactive protein > 0.8 mg/dL	Sum of risk factors	82% probability of NASH (all four risk factors) vs 9% in their absence
Hyssalo et al[56], 2014	PNPLA3 genotype, AST, fasting insulin	NASH score: -3.05 + 0.562 × PNPLA3 genotype (CC = 1/GC = 2/GG = 3) - 0.0092 × fS-insulin (mU/L) + 0.0023 × AST (IU/L) + 0.0019 × (fS-insulin × AST)	Finnish cohort NPV 86%, PPV 53% for NASH Italian cohort NPV 74%, PPV 65% for NASH
Zhou et al[48], 2016	Glutamate, isoleucine, glycine, lysophosphatidylcholine 16:0, phosphoethanolamine 40:6, AST, and fasting insulin and	NASH ClinLipMet score: -8.167 + 0.954 × PNPLA3 genotype (CC = 1/GC = 2/GG = 3) + 0.0451 × AST (IU/L) + 0.0667 × fS12 insulin (mU/L) - 3.151× log10(LysoPC(16:0)) (μmol/L) + 2.617 × log10(PE(40:6)) (μmol/L) + 2.357 × 13 log10(Glu) (μmol/L) + 7.813 × log10(Ile) (μmol/L) - 6.102 × log10(Gly) (μmol/L)	Identified patients with NASH with an AUROC of 0.866 (95%CI: 0.820-0.913)
Donati et al[28], 2017	PNPLA3rs738409 genotypes PNPLA3 rs738409, TM6SF2 rs58542926 and MBOAT7 rs641738, age, sex, obesity, type 2 diabetes, severe fibrosis	HCC risk score: 1/{1 + e-[(-12.588 + (0.162 × age) + (0.404 × sex: 1 if male, -1 if female) + (0.259 × obesity: 1 present, -1absent) + (0.587 × T2DM: 1 present,-1 absent) + (1.299 × severe fibrosis: 1 yes, -1 no) + (0.442 × number of risk alleles)]}	Identified patients with HCC with an AUROC of 0.96 ± 0.04 (96% sensitivity, 89% specificity)

PNPLA3: Patatin-like phospholipase domain-containing protein 3; AUROC: Area under the receiver operating characteristic curve; CI: Confidence interval; NASH: Non-alcoholic steatohepatitis; TM6SF2: Transmembrane 6 superfamily member 2; ALT: Alanine aminotransferase; GGT: γ-glutamyl transferase; NAFLD: Non-alcoholic fatty liver disease; AST: Aspartate aminotransferase; MBOAT7: Membrane bound O-acyltransferase domain containing 7; HCC: Hepatocellular carcinoma; NPV: Negative predictive value; PPV: Positive predictive value.

Table 3 Primary and secondary objectives for which combined genetic/clinical scores are expected in the field of non-alcoholic fatty liver disease

Primary objectives	Prediction of hard outcomes (cirrhosis decompensation, hepatocellular carcinoma, liver transplantation)
	Prediction of fibrosis progression
Secondary objectives	Complementary instruments for the diagnosis of NASH and/or advanced fibrosis
	Prediction of response to lifestyle interventions
	Prediction of response to pharmacologic interventions

NASH: Non-alcoholic steatohepatitis.