N-Acetyltransferase 2 (NAT2) Genotyping

Web Seminar: Pharmacogenetics in the Practice of Medicine

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NAT2 Genotyping

N-Acetyltransferase 2 (NAT2) plays an important role in the detoxication and/or metabolic activation of certain therapeutic drugs, occupational chemicals and carcinogens. The enzyme produced by NAT2 acts on 1% of drugs in current clinical use including isoniazid, a common tuberculosis treatment, and numerous chemicals. Approximately 50% of people in the United States are slow acetylators and 40% intermediate acetylators.

Genelex offers improved detection rates using an extended NAT2 DNA mutation panel. screens for the seven most frequent single nucleotide polymorphisms (SNPs) of NAT2 including 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, and 857G>A. The NAT2*4 allele encodes for a fully active enzyme and is traditionally considered the wild type (rapid acetylator) allele. The representative four common alleles (haplotypes) that possess signature nucleotide substitutions at positions 341, 590, 857, and 191 are designated NAT2*5, NAT2*6, NAT2*7, and NAT2*14, respectively, and several studies have shown that the members of these clusters are responsible for the slow acetylator phenotype. Analytical specificity and sensitivity for detection of these mutations are >99%.

Specimen Types

Please call Client Services at 800-523-6487 to obtain specimen kits.

• Buccal Swabs: 4 sterile Whatman OmniSwabs™.
• Blood: 5-10 cc whole blood lavender-top EDTA or Yellow-top ACD-A tubes.

• Turnaround Time: 10 -14 days turnaround

CPT Codes

NAT2 Mutation DNA Analysis (provided for your guidance only)
1 X 83891, 2 X 83892, 1 X 83900, 7 X 83914, 3 X 83901, 1 X 83909

Clinical Significance

Phenotype prevalence is approximately 50% slow acetylators and 40% intermediate acetylators in the United States. Phenotype prevalence varies greatly be area, please refer to http://www.cdc.gov/genomics/hugenet/reviews/tables/n_acet_Tables.htm#t3 for prevalence in other areas.

NAT2 is a highly polymorphic enzyme that plays a key role in drug detoxification and elimination of many commonly prescribed drugs. Genetic polymorphisms in NAT2 are common and can affect therapeutic response to drugs. The enzyme activity is expressed at highly variable levels. Three phenotypes are identified: Rapid acetylators, Intermediate acetylators, or Slow acetylators.
Detecting genetic variations in drug-metabolizing enzymes is useful for identifying individuals who may experience adverse drug reactions with conventional doses of certain medications. Individuals who possess NAT2 reduced expression variants may exhibit different pharmacokinetics (drug levels) than normal individuals. As a result, such individuals may require non-conventional doses of medications that require NAT2 activity for biotranformation or detoxification. Conversely, medications that do not require NAT2 biotranformation or detoxification may be preferentially selected for patients with potentially impaired NAT2 metabolic capacity to avoid adverse drug reactions.

Laboratory Test Interpretation

The method incorporates polymerase chain reaction (PCR) and multiplex allele specific primer extension (ASPE) using the Luminex FlexMAP™ tag sorting system on the Luminex® 100 xMAP™ platform. For each sample, genomic DNA is amplified in a single PCR reaction. To enable efficient incorporation of biotin-dCTP during the ASPE reaction, each PCR product is treated with Shrimp Alkaline Phosphatase (SAP) to inactivate any remaining nucleotides and with Exonuclease I (EXO) to degrade any primers left over from the PCR reaction. An aliquot of the treated PCR product is used in the ASPE reaction containing fourteen tagged primers. The ASPE products are then sorted by hybridization to the bead mixture containing fourteen populations of beads with anti-tags complimentary to the primer tag sequences, and incubated with Streptavidin, R-Phycoerythrin conjugate (reporter solution). Samples are read on the Luminex® 100 xMAP™ Instrument and signal is generated for each of the seven variants. The median fluorescence intensity values (MFI) are then analyzed to determine whether wild-type and/or mutant alleles for each of the variants have been detected.

NAT2 Mutations Detected
NAT2 allele	Nucleotide change	Effect on Enzyme Metabolism
*4	none	Rapid (Normal)
*5A	341T>C	Slow
	481C>T
*5B	341T>C	Slow
	481C>T
	803A>G
*5C	341T>C	Slow
	803A>G
*5D	341T>C	Slow
*5E	341T>C	Slow
	590G>A
*5G	282C>T	Slow
	341T>C
	481C>T
	803A>G
*5J	282C>T	Slow
	341T>C
	590G>A
*6A	282C>T	Slow
	590G>A
*6B	590G>A	Slow
*6C	282C>T	Slow
	590G>A
	803A>G
*6E	481C>T	Slow
	590G>A
*7A	857G>A	Slow
*7B	282C>T	Slow
	857G>A
*11A	481C>T	Rapid (Normal)
*12A	803A>G	Rapid (Normal)
*12B	282C>T	Rapid (Normal)
	803A>G
*12C	481C>T	Rapid (Normal)
	803A>G
*13	282C>T	Rapid (Normal)
*14A	191G>A	Slow
*14B	191G>A	Slow
	282C>T
*14C	191G>A	Slow
	341T>C
	481C>T
	803A>G
*14D	191G>A	Slow
	282C>T
	590G>A
*14E	191G>A	Slow
	803A>G
*14F	191G>A	Slow
	341T>C
	803A>G
*14G	191G>A	Slow
	282C>T
	803A>G

Testing places individuals in one of three categories:

• Rapid acetylators (RA) represent the norm for metabolic capacity. Genotypes consistent with the RA phenotype include two active NAT2 alleles. In general rapid acetylators can be administered drugs which are substrates of the NAT2 enzyme following standard dosing practices.
• Intermediate acetylators (IA) may require lower than average drug dosages for optimal therapeutic response. Genotypes consistent with the IA phenotype are those with one active and one inactive NAT2 allele.
• Slow acetylators (SA) are at increased risk of drug-induced side effects due to diminished drug elimination or lack of therapeutic effect resulting from failure to generate the active form of the drug.. Genotypes consistent with the SA phenotype are those with no active NAT2 alleles.

Direct DNA testing will not detect all the known mutations that result in decreased or inactive NAT2. Absence of a detectable gene mutation or polymorphism does not rule out the possibility that a patient has a rare phenotype. This test does not detect polymorphisms other than those listed. Other polymorphisms in the primer binding regions can affect the testing, and ultimately, the genotyping assessments made.

Dosage Recommendations

A complicating factor in correlating NAT2 genotype with phenotype is that many drugs may reduce or increase NAT2 catalytic activity. Consequently, an individual may require a dosing decrease greater than predicted based upon genotype alone. It is important to interpret the results of testing in the context of other co-administered drugs.

NAT2 activity also is dependent upon hepatic and renal function status, as well as age. Patients also may develop toxicity if hepatic or renal function is decreased.

Starting at the lowest efficacious dose and employing therapeutic drug monitoring in Intermediate acetylators (IA) and Slow acetylators (SA) is highly recommended. Specific dosing recommendations for many medications can be found by searching for the medication name in conjunction with " NAT2 and acetylator" on PUBMED. For instance, to achieve similar isoniazid exposure a standard dose is recommended for intermediate acetylators, a 50% decrease in dose is recommended for slow acetylators and a 50% increase is recommended for rapid acetlyators.

Methodology

The method incorporates polymerase chain reaction (PCR) and multiplex allele specific primer extension (ASPE) using the Luminex FlexMAP™ tag sorting system on the Luminex® 100 xMAP™ platform. For each sample, genomic DNA is amplified in a single PCR reaction. To enable efficient incorporation of biotin-dCTP during the ASPE reaction, each PCR product is treated with Shrimp Alkaline Phosphatase (SAP) to inactivate any remaining nucleotides and with Exonuclease I (EXO) to degrade any primers left over from the PCR reaction. An aliquot of the treated PCR product is used in the ASPE reaction containing fourteen tagged primers. The ASPE products are then sorted by hybridization to the bead mixture containing fourteen populations of beads with anti-tags complimentary to the primer tag sequences, and incubated with Streptavidin, R-Phycoerythrin conjugate (reporter solution). Samples are read on the Luminex® 100 xMAP™ Instrument and signal is generated for each of the seven variants. The median fluorescence intensity values (MFI) are then analyzed to determine whether wild-type and/or mutant alleles for each of the variants have been detected.

Drug Metabolism Guide

This list is not all inclusive and is for your guidance only. A more comprehensive Cytochrome list of substrates, inhibitors, and inducers can be found here.

Substrates Metabolized through NAT2
Substrates refers to drugs that are either activated or deactivated by the pathway.

caspofungin
caffeine
dapsone
hydralazine
isoniazid
nitrazepam
procainamide
retigabine
sulfadiazine
zonisamide
sulfonamides such as:
sulfapyridine
sulfamethazine
sulfadiazine
sulfasalazine
carcinogenic aromatic and heterocyclic amines
benzidine
beta-naphthylamine

Inhibitors of NAT2
Inhibitors refers to drugs that reduce the ability of the pathway to process drugs.
Co-administration will decrease the rate of metabolism of drugs through the metabolic pathway listed, increasing the possibility of toxicity.

acetaminophen

Inducers of NAT2
Inducers refers to drugs that increase the activity of a pathway.
Co-administration increases the rate of excretion for drugs metabolized through the pathway indicated, reducing the drug's effectiveness.

retinoic acid

References

1) http://www.cdc.gov/genomics/population/file/print/genvar/NAT2.pdf
2) Brans R, Laizane D, Khan A, Blomeke B. N-acetyltransferase 2 genotyping: an accurate and feasible approach for simultaneous detection of the most common NAT2 alleles. Clin Chem. 2004 Jul;50(7):1264-6.
3) Kinzig-Schippers M, Tomalik-Scharte D, Jetter A, Scheidel B, Jakob V, Rodamer M, Cascorbi I, Doroshyenko O, Sorgel F, Fuhr U. Should we use N-acetyltransferase type 2 genotyping to personalize isoniazid doses? Antimicrob Agents Chemother. 2005 May;49(5):1733-8.
4) Furet Y, Bechtel Y, Le Guellec C, Bechtel PR, Autret-Leca E, Paintaud G. Clinical relevance of N-acetyltransferase type 2 (NAT2) genetic polymorphism. Therapie. 2002 Sep-Oct;57(5):427-31.
5) Tanaka, et al. Adverse effects of sulfasalazine in patients with rheumatoid arthritis are associated with diplotype configuration at the N-acetyltransferase 2 gene. J Rheumatol. 2002 Dec;29(12):2492-9.
6) Blum, et al. Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell Biol. 1990 Apr;9(3):193-203.
7) Bell, et al. Genotype/phenotype discordance for human arylamine N-acetyltransferase (NAT2) reveals a new slow-acetylator allele common in African-Americans. Carcinogenesis. 1993 Aug;14(8):1689-92.
8) Deguchi, et al. Correlation between acetylator phenotypes and genotypes of polymorphic arylamine N-acetyltransferase in human liver. J Biol Chem. 1990 Aug 5;265(22):12757-60.
9) Vatsis, et al. Diverse point mutations in the human gene for polymorphic N-acetyltransferase. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6333-7.
10) Blum, et al. Molecular mechanism of slow acetylation of drugs and carcinogens in humans. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5237-41.
11) Hickman, et al. N-acetyltransferase polymorphism. Comparison of phenotype and genotype in humans. Biochem Pharmacol. 1991 Aug 8;42(5):1007-14.
12) Cascorbi, et al. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated Caucasian individuals: correlation with phenotypic activity. Am J Hum Genet. 1995 Sep;57(3):581-92.
13) Brockton N, Little J, Sharp L, Cotton SC. N-Acetyltransferase Polymorphisms and Colorectal Cancer. Am J Epidemiol. 2000 May 1;151(9):846-61.