Liver enzymes, metabolomics and genome-wide association studies: From systems biology to the personalized medicine

Table 1 List of enzymes of the Class-I pyridoxal-phosphate-dependent aminotransferase family: Evidence from the human proteome

Protein name	Gene name	Chromosome	Number of isoforms
1-aminocyclopropane-1-carboxylate synthase-like protein 1	ACCS	11p11.2	1
Alanine aminotransferase 1	GPT1	8q24.3	1
Alanine aminotransferase 2	GPT2	16q11.2	2
Aspartate aminotransferase, cytoplasmic	GOT1	10q24.2	1
Aspartate aminotransferase, mitochondrial	GOT2	16q21	2
Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial	AADAT	4q33	2
Kynurenine--oxoglutarate transaminase 1	CCBL1	9q34.11	3
Kynurenine--oxoglutarate transaminase 3	CCBL2	1p22.2	3
Probable inactive 1-aminocyclopropane-1-carboxylate synthase-like protein 2	ACCSL	11p11.2	1
Putative aspartate aminotransferase, cytoplasmic 2	GOT1L1	8p11.23	1
Tyrosine aminotransferase	TAT	16q22.2	1

Table 2 Comparison of biological and protein function of ALT1 and ALT2: Background information and recent findings

Features and function	Glutamic-pyruvate transaminase 1 (alanine aminotransferase 1) GPT1	Glutamic pyruvate transaminase 2 (alanine aminotransferase) 2 GPT2
Gene and protein Id in data-bases	Entrez gene: 2875	Entrez gene: 84706
	Ensembl gene: ENSG00000167701	Ensemble: ENSG00000166123
	UniProtKB: P24298	UniProtKB: Q8TD30
Genomic location	Entrez gene cytogenetic band: 8q24.3	Entrez gene cytogenetic band: 16q12.1
Number of gene transcripts	7 transcripts (splice variants), 28 exons on the forward strand	5 transcripts (splice variants), 26 exons on the forward strand
Variation	GPT1 has 210 SNPs	GPT2 has 819 SNPs
Orthologues	GPT1 has 69 orthologues in Ensembl	GPT2 has 63 orthologues in Ensembl
Regulation	There are 2 regulatory elements located in the region of GPT1 gene	There are 13 regulatory elements located in the region of GPT2
	miR-122 may interact with GPT1 at multiple sites of the coding region to enhance translation[21]	GPT2 promoter has a putative ATF4 (Activating transcription factor 4 binding site[69]
	Microsomal triglyceride transfer protein inhibition augments plasma ALT/AST levels in response to endoplasmic reticulum stress[66]	GPT2 is regulated by androgens in non-hepatic tissues[70]
	GPT1, but not GPT2 promoter is induced by PPAR agonists[67]
	ALT1 catalytic activity is inhibited by the effect of glycation[68]
Protein features	Size: 496 amino acids; 54637 Da	Size: 523 amino acids; 57904 Da
	Cofactor: Pyridoxal phosphate	Cofactor: Pyridoxal phosphate
	Subunit: Homodimer	Subunit: Homodimer (By similarity)
Cellular localization in human cells1	Cytosol of hepatocytes[18]	ER and mitochondrial fraction[18]
Measurement in plasma (catalytic activity)	Represents 90% of total ALT in circulation[17,18]	Represents 10% of total ALT in circulation[17,18]
Tissue expression in humans	Evidence: WB: Liver and kidney[18]	Evidence: WB and IHQ (protein): Pancreas (islets of Langerhans), brain, adrenal gland, skeletal muscle, heart (cardiomyocytes)[18]
	Evidence: NB: GPT mRNA is moderately expressed in kidney, liver, heart, and fat[15]	Evidence: NB: mRNA is expressed at high levels in muscle, fat, kidney, and brain, and at lower levels in liver and breast[15]
Tissue expression in rodents	Evidence: NB (mRNA): Highly expressed in liver and moderately expressed in white adipose tissue (WAT), intestine, and colon[71]	Evidence: NB (mRNA): muscle, liver, and white adipose tissue (WAT), at moderate levels in brain and kidney, and at a low level in heart[71]
		Gene expression analysis suggests a sex-dependent difference in GPT2-mRNA in the liver and muscle[15]
		Hepatic and muscle ALT2 protein activity was higher in males than in females; while no sex-dependent difference was noted in the liver for ALT1, it appears 20% higher in muscle in females[15]
Biological meaning and metabolic function	ALT1 contributes to “basal” serum ALT activity, most likely associated with normal cell turnover in liver and other tissues that would release ALT1 into the circulation[15,17-19]	Generation of pyruvate for gluconeogenesis under stressful living conditions, such as starvation[18]
		ALT2 is involved in the metabolic adaptation of the cell to stress[69]
		ALT2 is associated with a liver progluconeogenic metabolic adaptive response without hepatocellular necrosis after exposure to dexamethasone[72]
		ALT2 may participate in the generation of pyruvate and glyceroneogenesis, contributing to the homeostasis of fatty acid metabolism and storage[16]
Biological meaning in human disease	NAFLD: ALT1 represented 94% of total ALT levels in circulation[19]	NAFLD: ALT2 represented 6% of total ALT levels in circulation[19]
	HCV: High levels in circulation of ALT1 (about 5-fold increase as compared to the controls)[19]	HCV: Moderate levels in circulation of ALT1 (about 2.5 fold increase as compared to the controls)[19]
	Ultra-endurance exercise: no significant changes after exercise[19]	Ultra-endurance exercise: High levels in circulation of ALT2 (about 2-fold increase as compared to the baseline conditions)[19]
Biological meaning in experimental models of disease	NAFLD (ob/ob): Compared to the normal liver of lean mice, the expression of GPT1 mRNA remained unchanged[71]	NAFLD (ob/ob): Compared to the normal liver of lean mice, the expression of GPT2 mRNA was elevated by about 2-fold, suggesting ALT2 induction during fatty liver[71]
	Both ALT1 and ALT2 increased in the liver of mice induced liver steatosis by a deficient methionine-choline diet[73]

¹Cellular localization differs among species. ER: Endoplasmic reticulum; WB: Western blotting; IHQ: Immunohistochemistry; NB: Northern blot analysis; HCV: Hepatitis C virus; NAFLD: Non-alcoholic fatty liver disease.

Table 3 Evidence from genome-wide association studies on the heritability of circulating levels of alanine-aminotransferase and aspartate-aminotransferase

Ref.	Number of participants/study design	GWAS strategy (genotyping)	Number of variants	Phenotype	Identified locus
Chambers et al[36]	n = 61089	Affymetrix, Illumina and perlegen sciences arrays	About 2.6 million directly genotyped or imputed autosomal SNPs	Plasma levels of ALT	HSD17B13, MAPK10, TRIB1, CPN1, PNPLA3, SAMM50
	Population-based
	Adults
Yuan et al[74]	Initial study n = 7715	Affymetrix	-	Plasma levels of ALT	CHUK, PNPLA3, SAMM50, CPN1
	Replication n = 704
	Population-based
	Adults
Park et al[75]	n = 532	Illumina HumanOmni1-Quad BeadChip	747076 SNPs	Plasma levels of ALT	ST6GALNAC3, MMADHC, CCDC102B, RGS5, BRD7, GALNT13, SIRPA, CD93, SLC39A11, ADAMTS9, CELF2
	Population-based Children			Plasma levels of AST	CYB5APS, CELF2, GOT1, ST6GALNAC3, ADAMTS9, THSD7B, EIF4A1P1, ROBO1, THSD7B
Shen et al[76]	n = 866	Affymetrix GeneChip Human Mapping 500 K Array set	500568 SNPs	Plasma levels of AST	GOT1
	Population-based
	adults

GWAS: Genome-wide association studies; SNPs: Single nucleotide polymorphisms; ALT: Alanine-aminotransferase; AST: Aspartate-aminotransferase.

Table 4 Summary of the variants associated with alanine-aminotransferase and aspartate-aminotransferase levels in population-based genome-wide association studies: Biological function and variants characteristics

Variant ID	Variant features	Significant P value for GWAS association	Gene or nearest gene	Reported biological function of the associated locus
ALT
rs6834314	Intergenic	3.1 × 10-9	HSD17B13/MAPK10	Oxidoreductase involved in the metabolism of steroid hormones, prostaglandins, retinoids, lipids and xenobiotics
				A member of the MAP kinase family
rs2954021	Intron variant	5.3 × 10-9	TRIB1	Involved in protein amino acid phosphorylation and controlling mitogen-activated protein kinase cascades. Potent negative regulator of MAPK pathways influencing apoptosis. Regulates hepatic lipogenesis and very low density lipoprotein production
rs10883437	Intergenic	4.0 × 10-9	CPN1	A plasma metallo-protease that cleaves basic amino acids from the C terminal of peptides and proteins
rs11597390	intergenic	2.9 × 10-8
rs738409	Missense	1.2 × 10-45	PNPLA3	Acylglycerol O-acyltransferase and triacylglycerol lipase that mediates triacylglycerol hydrolysis
	p.Ile148Met
rs2281135	Intron variant	8.2 × 10-12
rs3761472	Missense	3.7 × 10-29	SAMM50	Component of the sorting and assembly machinery (SAM) of the mitochondrial outer membrane
	p.Asp110Gly
rs2143571	Intron variant	9.4 × 10-7
rs11597086	Non coding exon variant	3.6 × 10-7	CHUK	Member of the serine/threonine protein kinase family; a component of a cytokine-activated protein complex that is an inhibitor of the essential transcription factor NF-kappa-B complex
rs11591741	Intron variant	4.5 × 10-7
rs4949718	Intron variant	1.87 × 10-7	ST6GALNAC3	Transfer sialic acids from CMP-sialic acid to terminal positions of carbohydrate groups in glycoproteins and glycolipids
rs17801127	Intergenic	2.37 × 10-7	MMADHC	Mitochondrial protein that is involved in an early step of vitamin B12 metabolism
rs1539893	Intron variant	3.40 × 10-6	CCDC102BRGS5	Unknown Member of the regulators of G protein signaling (RGS) family
rs12035879	Intron variant	3.97 × 10-6
rs9941219	Intergenic	4.06 × 10-6	BRD7	Member of the bromodomain-containing protein family
rs731660	Intergenic
rs12621256	Intron variant	4.36 × 10-6	GALNT13	Member of the glycosyltransferase 2 family; catalyzes the initial reaction in oligosaccharide biosynthesis; neurons cell biogenesis
rs6035126	Intergenic	4.94 × 10-6	SIRPA	Receptor-type transmembrane glycoproteins involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes
rs13433286	Intergenic
rs844917	Intergenic	5.64 × 10-6	CD93	Cell-surface glycoprotein and type I membrane protein
rs844914	Intergenic	5.98 × 10-6
rs903107	Intron variant	6.11 × 10-6	SLC39A11	Mediates zinc uptake
rs80311637	Missense p.Val653Met	7.18 × 10-6	ADAMTS9	Disintegrin and metalloproteinase with thrombospondin motifs
rs596406	Intron variant	9.18 × 10-6	CELF2	RNA-binding protein implicated in the regulation of several post-transcriptional events
AST
rs11597390	Intergenic	0.0009	CHUK	Explained previously
rs2281135	Intron variant	5.7 × 10-6	PNPLA3	Explained previously
rs862946	Intergenic	2.41 × 10-7	CYB5AP5	Pseudogene
rs596406	Intron variant	3.69 × 10-7	CELF2	Explained previously
rs76850691	Missense p.Gln349Glu	8.55 × 10-7	GOT1	Biosynthesis of L-glutamate from L-aspartate or L-cysteine
rs17109512	Intergenic	2.80 × 10-14
rs4949718	Intron variant	1.49 × 10-6	ST6GALNAC3	Explained previously
rs80311637	Missense p.Val1597Met	1.85 × 10-6	ADAMTS9	Explained previously
rs892877	Intron variant	3.75 × 10-6	THSD7B	Unknown
rs984295	Intron variant	5.86 × 10-6
rs457603	Intergenic	4.57 × 10-6	EIF4A1P1	Pseudogene
rs452621	Intergenic
rs7617400	Intron variant	6.16 × 10-6	ROBO1	Neuronal development
rs11924965	Intron variant
rs7644918	Intron variant

HSD17B13: Hydroxysteroid (17-beta) dehydrogenase 13; MAPK10: Mitogen-activated protein kinase 10; TRIB1: Tribbles pseudokinase 1; CPN1: Carboxypeptidase N, polypeptide 1; PNPLA3: Patatin-like phospholipase domain containing 3; SAMM50: Sorting and assembly machinery component; CHUK: Conserved helix-loop-helix ubiquitous kinase; ST6GALNAC3: ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 3; MMADHC: Methylmalonic aciduria (cobalamin deficiency) cblD type, with homocystinuria; CCDC102B: Coiled-Coil Domain Containing 102B; RGS5: Regulator of G-protein signaling 5; BRD7: Bromodomain containing 7; GALNT13: Polypeptide N-acetylgalactosaminyltransferase 13; SIRPA: Signal-regulatory protein alpha; CD93: CD93 molecule; SLC39A11: Solute carrier family 39, member 11; ADAMTS9: ADAM metallopeptidase with thrombospondin type 1 motif, 9; CYB5AP5: Cytochrome b5 type A (microsomal) pseudogene 5; CELF2: CUGBP, Elav-like family member 2; GOT1: Glutamic-oxaloacetic transaminase 1, soluble; THSD7B: thrombospondin, type I, domain containing 7B; EIF4A1P1: Eukaryotic translation initiation factor 4A1 pseudogene 1; ROBO1: Roundabout, axon guidance receptor, homolog 1. Biological function was extracted from Gene Atlas (http://genatlas.medecine.univ-paris5.fr).

Table 5 PNPLA3-rs738409 metabolite trait associations from the genome-wide association studies catalog

Variant ID	LD with rs738409	Metabolite ratio	P value for association
rs12483959	0.657	Cholesterol/gamma-glutamyltyrosine	7.76 × 10-6
rs2076211	0.657	Cholesterol/gamma-glutamyltyrosine	1.19 × 10-5
rs2076211	0.657	Aspartylphenylalanine/docosapentaenoate (n3 DPA; 22:5n3)	5.26 × 10-6
rs2294922	0.657	Docosapentaenoate (n3 DPA; 22:5n3)/eicosapentaenoate (EPA; 20:5n3)	4.64 × 10-7
rs2073081	0.568	3-methoxytyrosine/gamma-glutamylthreonine	2.14 × 10-5
rs2281135	0.609	Glycocholate/levulinate (4-oxovalerate)	3.26 × 10-5
rs1010023	0.609	Docosapentaenoate (n3 DPA; 22:5n3)/phenylacetylglutamine	1.43 × 10-5
rs926633	0.609	Docosapentaenoate (n3 DPA; 22:5n3)/myristate (14:0)	1.08 × 10-5
rs2896019	0.607	Aspartylphenylalanine/docosapentaenoate (n3 DPA; 22:5n3)	1.31 × 10-5

Information was retrieved from the GWAS server at the freely accessible URL: http://metabolomics.helmholtz-muenchen.de. This server combines data on large GWAS and non-targeted and metabolome-wide panel of small molecules, using blood samples and phenotype data from 2824 individuals of two major population-based European cohorts: the German KORA study and the British TWINs UK study. LD: Linkage disequilibrium that refers to a non-random association in the occurrence of alleles at two loci was assessed by R² (the square of the correlation coefficient between the presence or absence of a particular allele at the first locus and the other representing the presence or absence of a particular allele at the second locus). GWAS: Genome-wide association studies.