Results and Discussion - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/25171/10/10_chapter...

Results and Discussion

134

© Iyer Sandhya Natarajan & Institute of Chemical Technology (ICT) Mumbai,

India, 2013

4.1 Analysis of DPYD gene

Two hundred and twenty five healthy Indian adults were analyzed for determining the

frequency of the DPYD (IVS14+1G>A) by PCR-RFLP methodology. The frequency

of the DPYD exon 14 splice-site mutation was detected to be 0.004. The gel picture

depicting different DPYD*2A genotypes is shown in Figure 4.1.1.

100 bp PCR GG GA GG

Ladder Control

Figure 4.1.1 A 2% agarose gel with DPYD*2A genotypes and 100bp ladder

Lane 1: 100bp ladder

Lane 2: Uncut PCR control- 341bp

Lane 3: GG genotype – 284bp (The 50bp fragment runs away)

Lane 4: GA genotype- 341bp + 284bp

Lane 5: GG genotype – 284bp

Since the frequency of the DPYD*2A was very low, sequencing analysis of the

DPYD exons and flanking intronic regions was undertaken as a pilot effort to

document all the variations present in the adult Indian population. Fifty samples were

analyzed by sequencing analysis of the DPYD gene to document all the variations

present in adult Indian population. A total of twenty two variations were recorded in

our study. These included sixteen known and six novel variations. The pherograms of

all the variants is as shown in Figure 4.1.2. The frequency of all the detected variants

is as shown in Table 4.1.1.

284bp

341bp


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© Iyer Sandhya Natarajan & Institute of Chemical Technology (ICT) Mumbai, India, 2013

85C>T (TT genotype)


136


IVS234-123G>C (GC genotype)


137


IVS234-81G>A (GA genotype)


138


IVS483+18G>A (GA genotype


139


1236G>A (GA genotype)


140


IVS1129-15T>C (TC genotype)


141


IVS1525-209G>A (AA genotype)


142


1627A>G (AG genotype)


143


1896T>C (TC genotype)


144


IVS1906-64A>G (AG genotype)


145


IVS1974+118A>C (AC genotype)


146




147


IVS1974+75A>G (GG genotype)


148


IVS2058+101T>C (CC genotype)


149




150


2194G>A (GA genotype)


151


IVS2300-39G>A (GA genotype)


152


IVS2443-207A>T (AT genotype)


153


2656C>T (CT genotype)


154


IVS2907+55C>T (TT genotype)


155


IVS2908-69A>G (GG genotype)


156


IVS2908-58G>C (GC genotype)

Figure 4.1.2 Pherograms of detected variations in the DPYD gene


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Table 4.1.1 Frequencies of all variations detected in DPYD gene in our study

Note: “n” represents the total number of samples typed for particular SNP. Novel

variations are recorded in bold and red font in Table 4.1.1. The data on phenotype and

global frequency is as mentioned in 1000genomes browser.


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Linkage disequilibrium analysis of the DPYD gene

Fifteen known DPYD variations having frequency of ≥ 0.01 were used for LD

analysis in our study. LD analysis was performed using software Haploview v.4.2.

Figure 4.1.3 depicts the LD plot of the DPYD gene. Strong LD was detected in a

block containing the variations 2656C>T, IVS2058+101T>C and 1896T>C at r2 1.0.

Other block with r2 value of 0.823 consisted of variants IVS2908-58G>C, IVS2908-

69A>G and IVS2300-39G>A.

Figure 4.1.3 LD plot of the DPYD gene

Comparison of allelic frequency generated in our study with data from different

population groups

DPD is the major rate limiting enzyme involved in metabolism of 5-FU. Drug

associated toxicity on treatment with 5-FU has been associated with deficiency of

DPD in several populations worldwide. However, such data on slow metabolizer


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India, 2013

status and alleles of DPD is unavailable for the Indian population. Our study is a pilot

effort to analyze the frequency of the DPYD exon 14 splice-site mutation

(IVS14+1G>A; DPYD*2A) to record factors responsible for 5-FU related toxicity.

Gene sequencing analysis of all the 23 exons and flanking intronic regions of the

DPYD gene was done to detect the presence of any other known or novel slow

metabolizer variants in the adult Indian population.

The first part of our study dealt with determining the frequency of the DPYD

(IVS14+1G>A) variation in 225 healthy Indian subjects. The frequency of the exon

14 splice-site mutation was detected to be very low at 0.004 in our study. Thus, it can

be concluded that though this SNP is reported to be responsible factor for 5-FU

related toxicity in different populations, it may be a mutation in the adult Indian

population. Hence in order to determine the presence of other known SNPs which

have been documented to be responsible for slow metabolizers and for 5-FU

associated toxicity or any novel mutation in Indians, gene sequencing analysis of all

the coding 23 exons and flanking intronic regions of the DPYD gene was carried out.

All the 23 exons along with the flanking intronic regions were analyzed for the DPYD

gene in fifty DNA samples using sequencing methodology. A total of twenty two

variations including six novel ones were detected. This is summarized as below:

The exon 14 skipping mutation documented to be responsible for 5-FU

associated toxicity in populations worldwide was not detected in this part of our

study. The 1679T>G (Ile560Ser) recorded to be responsible for a small

percentage of drug associated toxicity [Morel et al., 2006] was also not detected

in our study. Another non-synonymous variation 2846A>T (Asp949Val)

reported to lower DPD activity [van Kuilenburg et al., 2000; Johnson et al.,

2002] was also not detected in our study.

The synonymous variation 85C>T (Arg29Cys) known to cause unclear

consequences was detected at a frequency of 0.23 in our study.

The intronic variation IVS483+18G>A and the non-synonymous variation

1236G>A which are associated with hapB3 were detected at frequencies of 0.09


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India, 2013

and 0.07, respectively. The other two variations associated with hapB3 viz. the

680+139G>A and 959-51T>G were not detected in our study. The hapB3 is

associated with lowering the DPD activity [Amstutz et al., 2009]. Although the

1236G>A SNP associated with the hapB3 is a non-synonymous variation, its

frequency has been detected to be higher in patients who experienced 5-FU

toxicity as compared to controls [Schwab et al., 2008; Gross et al., 2008].

The 1627A>G (Ile543Val) present in exon 13 of the DPYD gene was detected

at a frequency of 0.04 in our study. Though this variation has not been

documented to lower enzyme activity, it has been detected at a frequency 0f

0.23 in patients treated with 5-FU who experienced grade 3-4 toxicity [Gross et

al., 2008]. The frequency of the 1627 A>G was detected to be 0.283 in

Japanese, 0.275 in Caucasians and 0.227 in African-Americans [Wei et al.,

1998; Maekawa et al., 2007; Ridge et al., 1998].

The 1896 T>C (Phe632Phe) silent mutation in exon14 was detected at a

frequency of 0.01 in our study. The frequency of the 1896T>C silent mutation is

reported to be present at 0.139 and 0.035 in Japanese and Caucasians,

respectively [Seck et al., 2005; Maekawa et al., 2007].

The intronic variation IVS1974+75A>G was detected at a frequency of 0.3 in

our study which is higher than 0.155 in Japanese and 0.166 in Caucasians [Seck

et al., 2005; Maekawa et al., 2007]. In a case-control study, the frequency of the

1974+75A>G SNP was as high as 16.6% in 153 control subjects with 5-FU

associated toxicity being detected in two patients [Gross et al., 2003]. In another

study, the 1974+75A>G SNP was detected in one patient who experienced

extreme grade 4 toxicity [van Kuilenburg et al., 2010]. The concentration of the

DPD enzyme activity was detected to be just 1.2 nm/mg/h in contrast to healthy

volunteers who had concentration of 6.2 nm/mg/h [van Kuilenburg et al., 2010].

The study by van Kuilenburg et al. (2010) also detected the presence of the

IVS2300-39G>A intronic variation in the same patient who experienced

extreme grade 4 toxicity on treatment with 5-FU. Our study detected this

variation at a frequency of 0.25 in healthy subjects.


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The variation IVS2907+55C>T in intron 22 was detected at a highest frequency

of 1 in our study. One explanation for this observation could be that the “T”

allele is the major allele in the Indian population.

The synonymous variation 2194G>A (Val732Ile) was detected at a frequency of

0.13 in our study. In comparison, the frequency in Japanese is reported to be

lower at 0.015 and 0.058 in Caucasians [Wei et al., 1998; Ridge et al., 1998].

According to the data on the 1000genomes browser, this variation leads to a

deleterious effect on the DPD enzyme activity. Another similar variation, the

2656C>T (Arg886Cys) was detected at a frequency of 0.01 in our study.

A recent review pointed out that the variations in DPYD gene are not uniformly

distributed and the typical hot-spots for variations lie between exons 6 and 13

and exon 2 [van Kuilenburg 2004]. In our study, highest number of variations

was recorded in exon 15 while close to 73% of variations were observed

between exon 13 and 23. Another study pointed out the presence of high

fragility (FRA1E) between introns 12 and 16 in the DPYD gene [Hormozian et

al., 2007].

Six novel variations were detected for the first time in the adult Indian

population in our study. Three of these variantions were observed in intron 15.

The IVS1974+118A>C was detected at a highest frequency of 0.1 in our study,

followed by the IVS1906-64A>G, 1940A>G and IVS2443-207A>T at 0.04.

The 2283A>G SNP in exon 18 was detected at a frequency of 0.03, while the

IVS234-81G>A was detected at 0.02 in our study.

LD analysis was also performed for fifteen known variations detected in our

study having frequency ≥0.01. Strong LD was detected in a block containing

variations 2656C>T, IVS2058+101T>C and 1896T>C at r2 1.0. This is the first

effort to detect the presence of a haplotype block in the DPYD gene for the adult

Indian population.


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4.2 Analysis of CDA gene

For the CDA 208G>A SNP, no minor allele was detected in our study. The gel picture

depicting the wild type genotype for the CDA*3 haplotype is shown in Figure 4.2.1.

The enzyme RsrII cuts the wild type allele, while the variant allele abolishes the

restriction site. The enzyme cuts the PCR product of size 845bp to generate two

fragments of sizes 454bp and 391bp respectively.

100bp GG GG GG

Ladder

Figure 4.2.1 A 2% agarose gel showing CDA*3 wild type genotype with 100bp

ladder.

Lane 1: 100 bp ladder

Lane 2, 3, 4: GG genotype- 454bp + 391bp

In order to determine if any other known slow metabolizer allele is present in the

Indian population, sequencing analysis of the coding exons and flanking intronic

regions of the CDA gene was performed in a pilot effort for the adult Indian

population. Fifty samples were analyzed by gene sequencing to document all the

variations present in the CDA gene in the adult Indian population. A total of five

variations were recorded including one novel variation. The pherograms of all the

detected CDA variants is shown in Figure 4.2.2 and the frequency of all the variants is

as listed in Table 4.2.1.

454bp

391bp


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79A>C (CC genotype)


164


IVS154+37G>A (AA genotype)


165


IVS266+242A>G (AG genotype)


166


IVS324+71T>C (TC genotype)


167


IVS325-209T>C (CC genotype)

Figure 4.2.2 Pherograms for all the detected CDA variants


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Table 4.2.1 Frequencies of all the variations in the CDA gene detected in this

study

SNP Effect Phenotype N Allelic frequency

(Our study)

Global

frequency

79A>C Lys27Gln benign 28 0.14 0.21

IVS154+37G>A Intronic Not known 27 0.13 0.139

IVS266+242A>G Intronic Not known 29 0.22 0.324

IVS324+71T>C Intronic Not known 39 0.03 0.209

IVS325-209T>C Intronic Not known 17 0.06

Note: Novel variation is depicted in bold and red font in Table 4.2.1. The data on

global frequency and phenotype is as mentioned in 1000genomes browser.

Comparison of frequency data generated in our study with data from other

population groups

CDA enzyme is majorly involved in metabolism of gemcitabine to

diflurodeoxyuracil. Genetic factors which lead to deregulation of CDA needs to be

analyzed as it is involved in detoxification of nucleotide analogues. Numerous genetic

variations in CDA responsible for gemcitabine associated toxicity have been recorded

worldwide. Our study is the one of the first few attempts to document all the

variations in CDA for the Indian population. Such data is very important from the

point of view of personalized medicine and ensuring successful chemotherapy. Our

study analyzed and documented all the variations present in the four coding exons and

flanking intronic regions of the CDA in fifty healthy adult subjects. This study was

also important as our previous effort to document the frequency of the 208G>A

(Ala70Thr) SNP in the CDA gene in 225 healthy subjects did not detect any minor

allele for this SNP.

Our study documented five variations in the CDA gene for the first time in the adult

Indian population. These include four known variations and one novel intronic

variation. These are summarized below:


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India, 2013

The deleterious 208G>A SNP documented to be responsible for gemcitabine

associated toxicity majorly in Japanese population was not detected in our

study.

The non-synonymous variation 79A>C (Lyn27Gln) was recorded in our study

at a frequency of 0.14. This polymorphism has also been projected as a

promising biomarker for increasing treatment efficiency and preventing toxic

side effects [Okazaki et al., 2010]. The frequency of the variant allele is as high

as 36.5% in Europeans and only 3.5% in Africans [Wong et al., 2009]. This

difference clearly indicates the need for testing on the basis of ethnicity. In a

study carried out in AML children treated with Ara-C based therapy, the post-

induction treatment mortality was significantly higher in children with the

minor allele [Bhatla et al., 2009]. This data emphasizes the fact that carriers of

CDA*2 haplotype are at an increased risk of mortality on treatment with Ara-C

based therapy. Another study has also reported lower deamination of

gemcitabine due to presence of the minor allele in codon 27 of the CDA gene

[Gilbert et al., 2006]. A recent study on Chinese cancer patients, detected high

incidence of severe neutropenia in patients treated with gemcitabine harbouring

the 79C allele [Xu et al., 2012b]. A recent Indian study by Abraham et al.

(2012) analysed the mRNA expression and variations of the CDA gene in 100

patients with AML on treatment with Ara-C and 36 controls. This study

detected the 79A>C SNP to be significantly associated with Ara-C toxicity.

The intronic variation IVS154+37G>A was detected at frequency of 0.13 in our

study. In a study carried out in Japanese cancer patients treated with

gemcitabine, the frequency of this intronic variation was recorded at 0.175 and

was found to increase gemcitabine clearance [Sugiyama et al., 2010].

The other intronic variation detected in our study were the IVS266+242A>G in

intron 2 at a higher frequency of 0.22 and the IVS324+71T>C in intron 3 at a

frequency of 0.03.


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A novel intronic variation IVS325-209T>C was detected for the first time in the

adult Indian population at a frequency of 0.06 and the variant was detected in

the homozygous variant form.

4.3 Analysis of NAT2 gene

A total of 181 individuals were genotyped by gene sequencing of the NAT2 in our

study. Frequency of slow, intermediate as well as fast acetylators was calculated.

Assignment of haplotypes to all the samples was done using PHASE v.2.1.1 software

and frequency of different NAT2 haplotypes detected in our study was calculated.

Pherogram of all the detected NAT2 variants is as shown in Figure 4.3.1 (a, b).

Figure 4.3.1 (a) Pherogram of 282C>T and 341T>C SNPs of the NAT2 gene


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Figure 4.3.1 (b) Pherogram of 481C>T, 590G>A and 803A>G SNPs of the NAT2

gene


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The gel picture depicting PCR-RFLP analysis for the 481C>T SNP is shown in

Figures 4.3.2 (I) and 4.3.3 (II).

100bp CT CT

Ladder

Figure 4.3.2 (I) A 2% agarose gel showing the heterozygous genotype for the

481C>T variant along with 100bp ladder


Lane 2 and 3: CT genotype- 896bp + 480bp + 416bp

TT CC CT CC

Figure 4.3.3 (II) A 2% agarose gel showing different 481C>T genotypes

Lane 1: TT genotype – 896bp

Lane 2: CC genotype – 480bp + 416bp

Lane 3: CT genotype- 896bp + 480bp + 416bp

Lane 4: CC genotype- 480bp + 416bp

896bp

480bp

416bp

896bp

480bp

416bp


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The restriction enzyme DdeI cuts the PCR product of 896 bp to generate fragments of

345bp, 278bp, 153bp and 120bp, respectively in case of wild type allele. In case of

variant, allele fragments of 345bp, 278bp, 153bp, 97bp and 23bp, respectively, are

generated. Figure 4.3.4 depicts the different genotypes for the 803A>G SNP of the

NAT2 gene.

AA AG AA AA

Figure 4.3.4 A 10% native PAGE showing the different genotypes for the

803A>G variant

Lane 1, 3 and 5: AA genotype- 345bp + 278bp + 153bp + 120bp

Lane 2: AG genotype- 345bp + 278bp + 153bp + 97bp (23bp fragment runs away)

For the 341T>C i.e the TT genotype showed the presence of two bands in the wild

type PCR tube, the 428bp band of the control and 314bp band of the wild type allele.

The variant CC genotype showed the presence of the 428bp band of control and a

314bp band in the variant PCR tube. Heterozygous genotype TC showed the presence

of both the bands- the 428bp of the control and 314bp in both the PCR tubes

containing the wild type as well as the variant allele primer. The PCR-ARMS results

of the 341T>C SNP is shown in Figure 4.3.5.

345bp

278bp

153bp

120bp 97bp


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India, 2013

100bp

Ladder TC PCR Failure TC

Figure 4.3.5 A 2% agarose gel showing the different genotypes for 341T>C along

with 100bp ladder


Lane 2 and 3: TC genotype- 428bp + 314bp (in both wild type and variant allele well)

Lane 4 and 5: PCR failure (Shows control gene is important to prevent reporting of

false positive results)

Lane 6 and 7: TC genotype- 428bp + 314bp (in both the wild type and variant allele

well

The frequency of different NAT2 acetylators detected in our study is shown in Table

4.3.1 and the list of all the detected NAT2 haplotypes in our study is shown in Table

4.3.2.

Table 4.3.1 Frequency of overall NAT2 acetylators detected in our study

Acetylator status N Frequency

Slow 117 64.64%

Intermediate 47 25.97%

Rapid 17 9.39%

Total 181 100%

428bp

314bp


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Table 4.3.2 Frequency of different NAT2 haplotypes detected in our study

Predicted

acetylator status

Haplotypes n Frequency

(%)

Phase probability

Rapid

*4/*4 9 4.97 0.998

*4/*12A 1 0.55 0.999

*4/*12C 2 1.1 0.977

*4/*13A 5 2.76 0.997

Intermediate

*4/*5B 14 7.73 0.98

*4/*5C 1 0.55 0.961

*4/*5D 1 0.55 0.999

*4/*6A 22 12.15 0.987

*4/*6B 8 4.42 0.995

*4/*6C 2 1.1 ~0.524

*5B/*12C 1 0.55 0.997

*5B/*13A 3 1.66 0.979

*6A/*11A 2 1.1 ~0.946

*6A/*12C 2 1.1 0.973

*6A/*13A 1 0.55 0.995

*6B/*12C 1 0.55 0.979

*6C/*13A 1 0.55 0.758

Slow

*5A/*5B 2 1.1 ~0.94

*5A/*6A 1 0.55 0.968

*5B/*5B 14 7.73 0.997

*5B/*5C 6 3.31 ~0.998

*5B/*5D 8 4.42 0.962

*5B/*5J 1 0.55 0.963

*5B/*6A 45 24.86 ~0.993

*5C/*5D 1 0.55 0.999

*5C/*5J 1 0.55 0.62

*5C/*6A 3 1.66 0.95


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*5D/*6A 1 0.55 0.927

*6A/*6A 18 9.94 0.995

*6B/*6B 1 0.55 0.997

*6C/*6C 1 0.55 0.994

*14A/*14C 1 0.55 0.493

*14F/*14F 1 0.55 0.997

Total 181

Linkage disequilibrium analysis of the NAT2 gene

Six known variants in the NAT2 gene having frequency ≥0.01 were subjected to

linkage disequilibrium analysis using Haploview v.4.2 software. LD was detected in

four blocks viz. (i) 282C>T and 590G>A at r2 0.772, (ii) 481C>T and 803G>A at r

2

0.72, (iii) 341T>C and 481C>T at r2 0.608, and (iv) 341T>C and 803G>A at r

2 0.698.

Figure 4.3.6 depicts the LD plot for the NAT2 gene in our study.

Figure 4.3.6 LD plot of NAT2

Comparison of frequency data generated in our study with data from other

population groups

NAT2 is the major enzyme involved in metabolism of INH used in treatment of TB

and other environmental carcinogens. The entire NAT2 gene is intronless and hence

sequencing of a single PCR amplicons of size 896bp is enough to record all the

variations present in the gene. In NAT2 gene, presence of many variations and their


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India, 2013

combinations give rise to different haplotypes. Till date more than 66 different alleles

have been recorded in different populations and documented by the NAT2

nomenclature committee. Our study involved documenting all the variations present

in the NAT2 gene in 181 healthy adult Indian subjects. Though many studies have

been carried out in different regions of the country in specific regional groups of the

Indian population for the NAT2 gene, our study to the best of our knowledge is the

first to be carried out in a mixed cohort of different regional groups. The results of

these are summarized below:

Thirty three different haplotypes were recorded in our study in the Indian

population. For the NAT2 gene, genotype was recorded for 24 loci of the

known 27 loci. The combination of all the genotypes at the 24 loci recorded in

our study led to the detection of the 33 haplotypes.

Haplotype assignment was done using PHASE v .2.1.1 software freely

available online. Most haplotypes were recorded at a probability of >0.9 by

the software. The frequency of slow acetylators was detected to be highest at

64.64%, followed by intermediate and rapid acetylators at 25.97% and 9.39%,

respectively.

Sixteen slow acetylator’s diplotypes were recorded in our study. The

NAT2*5B/*6A documented as a major responsible factor for slow acetylation

in populations worldwide was recorded at the highest frequency of 24.86% in

our study. The NAT2*6A/*6A was detected at a frequency of 9.94%, while

the *5B/*5B diplotype was recorded at 7.73%. The frequency of the NAT2*5

allele is highest in Arabs at 55%, followed by Egyptians at 50% and Iranians

at 32% [Bakayev et al., 2004; Woolhouse et al., 1997; Hamdy et al., 2003].

The NAT2*5B is the most common allele in the Caucasians occurring in 40-

46% of the population, while it is very rare in Koreans at 1.5% [Lee et al.,

2002; Lin et al., 1993]. In a study carried out in Maharashtrian Indian

population from the western part of the country, the frequency of slow

acetylators was recorded at 55% [Singh et al., 2009], while in North Indians it

was recorded to be 55.71% [Arif et al., 2007]. In South Indians, the frequency

is higher at 74% [Anitha and Banerjee, 2003]. These previous studies have


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India, 2013

been carried out in pure native regional groups with genotyping carried out

only at specific loci for the NAT2 gene.

Combination of a slow and rapid allele gives rise to intermediate acetylation.

A total of thirteen intermediate acetylators diplotypes were detected in our

study. The frequency of the NAT2*4/*6A was recorded at a highest of

12.15%, followed by *4/*5B at 7.73% and *4/*6B at 4.42%. In populations

worldwide, the frequency of intermediate acetylators is highest in Iranians at

48.9%, followed by Koreans at 46.9% and the Sub-Saharan Africans at 45.4%

[Bakayev et al., 2004; Lee et al., 2002; Patin et al., 2006b]. In Indian studies,

the frequency of intermediate acetylators was detected at 32% in the Western

Maharashtrian Indians, while in North Indians no intermediate acetylators

have been detected [Singh et al., 2009; Kukongviriyapan et al., 2003]. In

South Indians, the frequency detected was 23.5% [Anitha and Banerjee, 2003].

The NAT2*4 allele is a wild type haplotype and denotes the absence of any

variation in the NAT2 gene. Four different rapid acetylators diplotype were

recorded in our study. The frequency of the *4/*4 was recorded at a highest of

4.97% followed by *4/*13A at 2.76%. Globally, the frequency of rapid

acetylators is higher as compared to the Indian population. In Iranians, the

frequency is 18.2%, while for Koreans and the Sub-Saharan Africans it is

reported to be 42.8% and 14.9%, respectively [Bakayev et al., 2004; Lee et al.,

2002; Patin et al., 2006b]. In Indians, the frequency of rapid acetylators has

been detected at 13% in the Western Indian population, while it is just 2% in

South Indians [Anitha and Banerjee, 2003; Singh et al., 2009]. The frequency

of rapid acetylators has been detected to be higher at 44.29% in the Indian

population [Arif et al., 2007].

Our study is one of the few attempts to document all the variations in the

NAT2 gene in a mixed cohort of adult Indians. No novel variations or

haplotypes were detected in our study.

Sequencing of the NAT2 gene is important for full proof assignment of haplotypes.

The Indian population is ethnically different from other populations worldwide and


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also within the country different regional groups exist. This difference is highlighted

by the finding of different frequencies of NAT2 acetylators in the Indian population.

Our study is the first pilot effort to document an LD plot of different variants of the

NAT2 gene.

Results and Discussion - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/25171/10/10_chapter...

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