Project:Alcohol Flush Genotyping

Overview

The main pathway of ethanol metabolism in humans consists of two critical reactions that occur in the liver. First the ethanol is oxidised to acetaldehyde (a toxic substance) by alcohol dehydrogenases (ALDHs). Second, the acetaldehyde is further oxidised to acetate (ethanoic acid) by aldehyde dehydrogenases (ALDHs).

Several SNP based alleles exist for many of the ADH and ALDH genes, each of which alters the kinetic activity of the enzymes encoded by each gene. Among these alleles, the ALDH2*2 allele of the of the ALDH2 is commonly found in people of Chinese, Japanese, and Korean descent and is associated with alcohol flush.

Genetics

ALDH2

ALDH2 is a ~43 kb gene on chromosome 12 consisting of 13 exons and encodes the ALDH2 enzyme found in the mitochondria, it has two alleles ALDH2*1 and ALDH2*2. The rs671 SNP (FASTA), in which a guanine in the ancestral ALDH2*1 is substituted for an adenine in ALDH2*2 is responsible for these two alleles. The G -> A substitution in ALDH2*2 causes the amino acid lysine to be expressed instead of glutamate and causes an inactive ALDH2 enzyme to be expressed. ALDH2*2 is considered nearly dominant as heterozygous individuals have almost no detectable ALDH2 activity in the liver and people who carry two copies of the ALDH2*2 allele have no detectable activity.

The inactivity of the ALDH2 enzyme in individuals with at least one ALDH2*2 allele causes acetaldehyde levels in the blood to increase after the imbibing of alcohol to a level high enough to cause a flushing reaction and possibly nausea or increased heart rate.

ALDH2*2 is relatively common in people of Chinese, Japanese, and Korean descent but is essentially absent in people of European or African descent and is strongly associated with a drastic reduction in the likelihood of an individual becoming alcohol dependent.

Other Relevant Genes

ADH Genes

A small region of chromosome 4 contains all seven ADH genes found in humans, ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6, and ADH7. The various ADH enzymes that are encoded by these genes are dimers. The ADH1A, ADH1B, and ADH1C genes are closely related to each other but each produces a different subunit that form either homodimers or heterodimers (to give a total of 9 possible ADH1 enzymes).

2 SNPs in the ADH1B gene give rise to three commonly found alleles: ADH1B*1, ADH1B*2 (common in Asians), ADH1B*3 (found mainly in people of African descent). The ADH1B*2 allele results in an enzyme with a 70- to 80-fold higher turnover rate than that produced by the ADH1B*1 allele and is associated with a lower risk of alcoholism.

SNP genotyping

Tetra-primer ARMS-PCR

Requires the design of 2 primer pairs. See this PDF

rs671 FASTA sequence

FASTA		
>gnl|dbSNP|rs671|allelePos=501|totalLen=1001|taxid=9606|snpclass=1|alleles='A/G'|mol=Genomic|build=138

AGGCATAGTG GCACATACTT GTTATCTTAA CTACTTGGGA GGCTGAGGCA GGAGGATCAC
TGAAGACCAG GAGTTGGAGA CCAGCCTGGG TAACATAATC AGACCCTGTC TCTTAAAAAA
AAATTTATTG CCAGGCGTGG TTGCACGTGC TGGTAGTCCA GCTACTCAGG AAGCTGAGGC
AGGAGAATCT CTTGAACCCC AGATGTGGAG GTTGCAACGA GCCAAGATCA TGCCATGGCA
ACTCCAGCCT GGGCAACAGA GAAAGATTCT ATCTCAAAAA AAAAAATTTT TTTTTAAGTT
AAAAATAAAA TAAAGACTTT GGGGCAATAC AGGGGGTCCT GGGAGTGTAA CCCATAACCC
CCAAGAGTGA TTTCTGCAAT CTCGTTTCAA ATTACAGGGT CAACTGCTAT GATGTGTTTG
GAGCCCAGTC ACCCTTTGGT GGCTACAAGA TGTCGGGGAG TGGCCGGGAG TTGGGCGAGT
ACGGGCTGCA GGCATACACT
R
AAGTGAAAAC TGTGAGTGTG GGACCTGCTG GGGGCTCAGG GCCTGTTGGG GCTTGAGGGT
CTGCTGGTGG CTCGGAGCCT GCTGGGGGAT TGGGGTCTGT TGGGGGCTCG GGGCCTGCCA
GAGGTTCAGG ACCTGCCGGG GACTCAGGGC CTGCTGGAAG TTCAGGACCT GCTGGGGATC
AGGGCCTGCC AGGGATTTAG GGTCTGCTGG GCGGGCCACC TTTTGGCCTC TCCCTCATGC
TTGAGGCCAT CAGTGTTTCC TACTAATTTC CCATTTTAAG CCTGAGAAGT GACAAGAGAG
GGTAAAGACC CAGCCTCTGC TCTGTCCCAT GAGAAATACT GAGGGACGTG CCCCCATCAG
GCCTATGCGG TCATTTGCTG GGCTTCGTTA TACGCCAAGG CCTGTAGGCC TGAGAAGAGG
GAGAGACTTC AGGGGGCGGA GCGGAGAGGA AAAGCTTCTA GTAAGAATCT TTTCAGATTT
TCACCAGGCG CGGTGGCTTT

Tetra-primers

Suggested primers for tetra=primer PCR from tetra-primer designer:

Forward inner primer (A allele):                Melting temperature
474 GGCGAGTACGGGCTGCAGGCATACAATA 501                    74

Reverse inner primer (G allele):
530 AGCAGGTCCCACACTCACAGTTTTCACGTC 501                  74

Forward outer primer (5' - 3'):
238 GCAACTCCAGCCTGGGCAACAGAGAAA 264                     74

Reverse outer primer (5' - 3'):
669 GTCCTGAACTTCCAGCAGGCCCTGAGTC 642                    74

Product size for A allele: 197
Product size for G allele: 293
Product size of two outer primers: 432

Links

Ensembl rs671 summary

NCBI dbSNP rs671

An efficient procedure for genotyping single nucleotide polymorphisms

Tetra-primer designer