Haplotype-specific PCR for NAT2 diplotyping

N-acetyltransferase 2 (NAT2) is an enzyme that acetylates many kinds of drugs, including the antituberculosis drug isoniazid. The NAT2 gene is highly diverse across populations. An individual can be classified as having a slow acetylator (SA), an intermediate acetylator (IA), or a rapid acetylator (RA) phenotype based on its two haplotypes (diplotype) of NAT2. SA individuals are at a higher risk for isoniazid-induced hepatitis, while the RA phenotype contributes to failure in tuberculosis treatment. Being able to predict individual NAT2 phenotypes is important for dose adjustment of isoniazid. NAT2 haplotypes are commonly determined via an indirect method of statistical haplotype inference from SNP genotyping. Here, we report a direct NAT2 haplotyping method using haplotype-specific PCR (HS-PCR) for the 6 most commonly found NAT2 haplotypes: NAT2*4, NAT2*5B, NAT2*6A, NAT2*7B, NAT2*12A, and NAT2*13A. Validation of this HS-PCR method via comparison with a sequencing method in 650 Thai DNA samples (107 RA, 279 IA, and 264 SA samples) showed a concordance rate for diplotype calls of 99.23% (645/650 samples). The discordant results in 5 samples were due to 3 rare NAT2 haplotypes: NAT*5C (n = 3), NAT2*7C (n = 1), and NAT2*11A (n = 1). This novel HS-PCR method allows direct NAT2 diplotyping, enabling the implementation of NAT2 acetylator phenotypes in clinical pharmacogenetic testing.

Tuberculosis (TB) is one of the most serious health problems, with more than 2 billion infected people (approximately one-third of the world's population) and an estimated 10 million new cases occurring in 2017 6 . In Thailand, more than 100,000 patients have been diagnosed with TB, and 12,000 patients die every year 7 ; hence, precise diagnosis and treatment are necessary to control TB. Anti-TB drugs are usually combined drugs that are administered for a 2-month period. The first-line anti-TB drug is composed of isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin. The most common adverse reaction to anti-TB drugs is skin rash (15.4%); hepatitis is the second most common (9.2%) adverse effect, but it is more life-threatening than skin rash and causes treatment failure 8,9 .
Patients with the SA phenotype are prone to adverse effects from drugs metabolized by NAT2. A meta-analysis that included 14 studies 10 showed that the risk of anti-TB drug-induced liver injury (AT-DILI) was higher for the SA type than for other acetylator types (OR = 4.695, 95% CI: 3.291-6.705, p < 0.001). This finding was confirmed in a study of the Thai population 11 that also explored AT-DILI (OR = 8.80, 95% CI: 4.01-19.31, p = 1.53 × 10 −8 ). Therefore, being able to determine a patient's NAT2 acetylator type would help physicians adjust the dosage of isoniazid.
NAT2 haplotypes are conventionally determined by inference from seven common SNPs located in exon 2 of the NAT2 gene. SNP genotyping methods, such as sequencing 12 , real-time polymerase chain reaction (PCR) 13 , PCR-restriction fragment length polymorphism 14,15 , allele-specific sequencing 16 , and allele-specific primer extension 17 , have complicated steps, are laborious, time-consuming and costly, and/or require sophisticated machines. These resource requirements limit the routine use of such methods in clinical applications. Furthermore, statistical inference may be error-prone and difficult for nonstatisticians to conduct. Therefore, we aimed to develop a simple and low-cost method for NAT2 diplotyping that directly provides 2 haplotypes without an inference step and can therefore be used in routine service.

Materials and methods
Frozen EDTA blood samples (n = 650) from stocks available from the Third Thailand National Health Examination Survey Program were randomly selected to represent the Thai populations of 13 health areas. The survey was approved by the Ethical Review Committee for Research in Human Subjects, Ministry of Public Health 18 , and all participants provided written informed consent. We obtained permission to use these frozen blood samples with information on sex and residential area. DNA samples were extracted using a commercial kit (QIAamp DNA blood mini kit, QIAGEN GmbH, Germany) and quantitated using a spectrophotometer (Nanodrop-100, Wilmington, DE 19810, USA).
The haplotype-specific PCR-based method (HS-PCR) for NAT2 diplotyping presented here uses 6 reaction tubes, with each tube containing a specific primer pair for one of the haplotypes most commonly found in Thai populations (NAT2*4, NAT2*5B, NAT2*6A, NAT2*7B, NAT2*12A, and NAT2*13A). These oligonucleotide primers were designed using NAT2*4 (NG_012246.1 Homo sapiens N-acetyltransferase 2 (NAT2), RefSeqGene on chromosome 8) as a reference sequence. Specific amplification of only one NAT2 haplotype was performed by using a combination of forward and reverse primers that contained a haplotype signature SNP as the last base at the 3′ end of the oligonucleotide primer, and its paired primer also contained a specific base at the 3′ end. The six variant bases of these six common NAT2 haplotypes and the last 3′ end base of each primer are provided in Table 1.
Since primers with only a single 3′ mismatched base may give false-positive results, the amplification refractory mutation system 19,20 was used to introduce an additional mismatched base at the −2 position from the 3′ end of the primer to increase the specificity of HS-PCR. A primer pair (TIMP1-Fw/TIMP1-Rv) for amplification of TIMP1, a gene located on chromosome X, was used as an internal Indicates the signature SNP for each NAT2 cluster. Fw indicates the last base at the 3′ end of the forward primer, and Rv indicates the last base at the 3′ end of the reverse primer. SNP positions were identified by designating "A" of the "ATG" start codon as the first position.
control in every reaction tube. The primer sequences used to amplify specific NAT2 haplotypes and TIMP1 and the amplified product sizes are shown in for 15 min to remove unincorporated primers and dNTPs. The sequencing reactions were performed by using 1 ml of the treated PCR products, sequencing primers (shown in Table 2), and a 1X BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). After purification (D-Pure TM Dye Terminator Removal Kit, NimaGen BV, Nimagen, NL), the sequences were read by a 3500XL genetic analyzer (Applied Biosystems). The variants in exon 2 of NAT2 were called by Variant Reporter TM Software v2 (Applied Biosystems). The NAT2 haplotype was inferred based on the most common 7 SNP positions using PHASE software 21,22 .

Results
Direct NAT2 diplotyping by HS-PCR A novel method for genotyping NAT2 diplotypes was developed using NAT2 HS-PCR. The detection range of this method is 5-200 ng of DNA (data not shown). For each NAT2 haplotype, the specific amplified product size can be clearly and directly observed under UV light after agarose gel electrophoresis and Et-Br staining (Fig. 1). Each NAT2-haplotype reaction tube produces a specific band when a sample is positive for that particular haplotype, except for the NAT2*13 reaction tube, which produces 2 bands of 366 bp and 641 bp from one forward primer and 2 reverse primers. The concordance rate for diplotyping between the novel HS-PCR method and the indirect sequencing method among 650 DNA samples was 99.23% (645/650). The discordant results observed for five samples were due to rare NAT2 haplotypes comprising NAT2*5C (three samples), NAT2*7C (1 sample), and NAT2*11A (1 sample), which were designated NAT2*5B, NAT2*7B, and NAT2*4 by HS-PCR, respectively. However, in all 650 samples, the interpretation of acetylator phenotypes from NAT2 diplotypes by the HS-PCR method was 100% concordant with that from the direct sequencing method.

Discussion
A novel method was developed for direct genotyping of NAT2 diplotypes using haplotype-specific primers to amplify 6 common NAT2 haplotypes (NAT2*4, *5B, *6A, *7B, *12A, and *13A) found in a non-African population, i. e, the Thai population 5 . In this method, TIMP1 amplification was used as an internal control to safeguard against false negatives from failure of the PCR experiment due to the reaction mix, thermal cycler, or DNA sample. When this 817-bp product is absent from any HS-PCR reaction tube, that sample should be interpreted as having an indeterminate result and retested. This quality-control step ensures that homozygote diplotype calls obtained using HS-PCR are not caused by the failure to amplify another haplotype.
A validation method showed that this HS-PCR technique for NAT2 diplotyping provided perfect concordance of acetylator phenotype interpretation (107 RAs, 279 IAs, and 264 SAs) with the results of the reference sequencing method.
In this study, the NAT2 haplotypes detected by Sanger sequencing and haplotype inference in 650 samples randomly selected from a nationwide Thailand population confirmed the pattern of the 6 most commonly found haplotypes (*4,*6B, and *7A at a higher frequency and *5, *12A, and *13A at a lower frequency) as being similar to those previously reported 23 in a northeastern Thai sample (n = 235 individuals). Our study found a lower frequency of *5 but a higher frequency of *13 compared with those in the study by Kulkongviriyapan et al. As of April 2016, 108 NAT2 haplotypes grouped into 20 clusters were recorded 2 . A member of a NAT2-haplotype cluster will have a cluster-signature SNP plus other SNPs. A cluster consisting of *5-*7 and *11-*14 corresponds to members with more than 1 haplotype, while the rest are rarely found. This novel HS-PCR method for NAT2 diplotyping does not cover the NAT2*14 cluster found in African populations; therefore, it cannot be used in such populations.
Since this method was developed based on the six most common NAT2 haplotypes, other uncommon haplotypes cannot be clearly determined and may be misclassified. The haplotypes that can potentially be amplified by each of the six specific NAT2-haplotype reaction tubes are shown in supplement 1. However, the misclassifications that we observed in the Thai population were mostly within the same cluster and/or shared the same acetylator phenotype, such that clinical recommendations were unchanged. Some DNA samples with rarer NAT2 haplotypes may generate unusual band patterns or show more than two haplotypes. In these cases, the DNA sample should be checked for possible cross-contamination at the DNA extraction or PCR step. If the result remains uninterpretable, the sample should be subjected to Sanger sequencing.

Conclusion
This method involves direct NAT2 diplotyping, has no risk of errors caused by statistical haplotype inference, and can be implemented in a simple molecular laboratory with a lower cost and shorter turnaround time than those for other methods. This novel HS-PCR method is the first step toward enabling the routine use of NAT2 acetylator status as an indicator in clinical practice.

Disclaimer
A patent has been filed for the primer set designed/ developed in this study in Thailand (No. 1601001130) and internationally (PCT/TH2017/000014). This study was supported by the Department of Medical Sciences, the Ministry of Public Health, Thailand, and the Japan Agency for Medical Research and Development/Japan International Cooperation Agency under the Science and Technology Research Partnership for Sustainable Development (SATREPS) project (Grant no. JP18jm0110010). We thank the Health Systems Research Institute, Thailand, and the National Health Security Office for allowing us to use frozen blood samples obtained during the Third NHES program, which was supported by the Bureau of Policy and Strategy, Ministry of Public Health, Thailand.