We identified four haplotypes, NAT2*4, NAT2*5B, NAT2*6A, and NAT2*7B, in the 31 donors (Table 1). The wild-type NAT2*4 haplotype was present in 79.0% of the examined subjects, whereas the remaining three haplotypes were variants. There was no significant difference (Pearson χ2 test) in the distributions of haplotypes between our data and the data from a study that assessed 200 healthy Japanese volunteers [25]. As exceptions, the NAT2*11 and NAT2*13 haplotypes, which are present in 0.25% and 1.25% of the Japanese population, respectively, were not detected in the cell donors enrolled in our study.
Table 1 Distribution of NAT2 haplotypes in the cell donors and the Japanese population
Allele
|
Nucleotide change(s)
|
Amino acid change(s)
|
Type
|
Freq. (%)
detected
|
Freq. (%)
literaturea
|
P valueb
|
NAT2*4
|
—
|
—
|
Rapid
|
79.0
|
69.5
|
0.125
|
NAT2*5B
|
T341C,C481T, A803G
|
Ile114Thr, Lys268Arg
|
Slow
|
1.6
|
0.5
|
0.317
|
NAT2*6A
|
C282T, G590A
|
Arg197Gln
|
Slow
|
9.7
|
19.8
|
0.056
|
NAT2*7B
|
C282T, G857A
|
Gly286Glu
|
Slow
|
9.7
|
8.8
|
0.811
|
a Allele frequency in the literature, the data were from healthy volunteers aged 20–60 years; b calculated for haplotype frequencies.
Next, genotyping was performed to assess the acetylator status based on the inferred NAT2 haplotypes. We identified four genotypes, NAT2*4/*4, NAT2*4/*5B, NAT2*4/*6A, and NAT2*4/*7B, at frequencies of 58.0%, 3.2%, 19.4%, and 19.4%, respectively (Table 2 Panel A). The NAT2*4/*4 alleles of rapid/rapid homozygotes were classified as a rapid acetylator phenotype, whereas rapid/slow heterozygotes, i.e., with NAT2*4/*5B, NAT2*4/*6A, and NAT2*4/*7B alleles, were classified as an intermediate acetylator phenotype. However, none of the individuals were slow/slow homozygotes, i.e., a slow acetylator phenotype. The results of the statistical analysis showed that no single genotype was positively correlated with the efficacy of NTP treatment (Pearson χ2 test). Subsequently, we analyzed the differences between phenotypes regarding the response to NTP treatment. The phenotype frequencies of NAT2 in the donors were 58.0% and 42.0% for rapid (NAT2*4/*4) and intermediate (NAT2*4/*5B, NAT2*4/*6A, and NAT2*4/*7B combined) acetylators, respectively.
Table 2 NAT2 genotype frequencies and response to NTP treatment
Panel A
Genotype
|
N
|
%b
|
Phenotype
|
NTP+c N
|
NTP+ % d
|
OR (95% CI)
|
P value
|
*4/*4
|
18
|
58.0
|
Rap.
|
7
|
38.9
|
0.19 (0.04–0.95)
|
0.036
|
*4/*5B
|
1
|
3.2
|
Int.
|
1
|
100
|
–
|
–
|
*4/*6A
|
6
|
19.4
|
Int.
|
4
|
66.7
|
1.9 (0.28–11.98)
|
0.517
|
*4/*7B
|
6
|
19.4
|
Int.
|
5
|
83.3
|
5.4 (0.55–53.27)
|
0.118
|
*4/*5B,*6A,*7B a
|
13
|
42.0
|
Int.
|
10
|
76.9
|
5.2 (1.06–26.0)
|
0.036
|
Panel B
|
|
|
|
|
|
|
|
Genotype in genders
|
N
|
%b
|
Phenotype
|
NTP+c N
|
NTP+
%d
|
OR (95% CI)
|
P value
|
Male *4/*4
|
9
|
29.0
|
Rap.
|
2
|
22.2
|
0.1 (0.01–0.65)
|
0.012
|
*4/*5B,*6A,*7Ba
|
10
|
32.3
|
Int.
|
8
|
80.0
|
14.0 (1.54–127.2)
|
0.012
|
Female *4/*4
|
9
|
29.0
|
Rap.
|
5
|
55.5
|
0.6 (0.04–0.97)
|
0.735
|
*4/*5B,*6A,*7Ba
|
3
|
9.7
|
Int.
|
2
|
66.7
|
1.6 (0.10–24.7)
|
0.735
|
a Total of the variants; b frequency in the total sample; c responders to NTP treatment; d frequency of responders in each group. Rap.: rapid; Int.: intermediate.
Regarding with the efficacy of NTP, 38.9% and 76.9% of the individuals with rapid and intermediate acetylator phenotypes, respectively, were responders to NTP treatment (aggrecan upregulation, ³1.1-fold). The OR of the comparison of the intermediate acetylator phenotype between responders and nonresponders was 5.2 (95% CI, 1.06–26.0, P = 0.036, Pearson χ2 test), which suggests that the intermediate acetylator phenotype was significantly correlated with the efficacy of the NTP treatment. The gender-specific statistics show that the frequency of responders among male and female intermediate acetylator individuals was 80.0% and 66.7%, respectively (Table 2 Panel B). In male donors, the OR of the comparison of the intermediate acetylator phenotype between responders and nonresponders was 14.0 (95% CI, 1.54–127.2, P = 0.012, Pearson χ2 test). In contrast, in female donors, the frequency of responders was not significantly correlated with the NAT2 phenotype.
Phenotype and age-related changes in the expression of the aggrecan mRNA after NTP treatment
The quantitative data pertaining to the changes in the expression levels of the aggrecan mRNA after NTP treatment compared with each control were plotted against the NAT2 phenotypes (Fig. 2a). As shown in the box-whisker plots, there was a large variance in the rapid acetylator phenotype; consequently, no significant effect of the NTP treatment was detected compared with the control (mean ± SD, 1.12 ± 0.56, P = 0.187, Student’s t-test), while there was a significant increase in the intermediate acetylator phenotype (mean ± SD, 1.15 ± 0.16, P = 0.002, Student’s t-test). Although the mean values of each phenotype were similar (1.12 and 1.15, respectively), there was a statistically significant difference in the distributions of data between the rapid and immediate acetylator phenotypes (P = 2.69E–05, F-test). The change in the expression of the aggrecan mRNA after NTP administration is plotted against donor’s age in Fig. 2b. There was a weakly negative correlation with age in all data (r = –0.532, P = 0.0021, Student’s t-distribution test). Regarding the rapid acetylator phenotype, the same tendency was found at higher significance (r = –0.683, P = 0.002, Student’s t-distribution test). In contrast, no significant correlation was found for the immediate acetylator phenotype.
Male donors classified as having an intermediate acetylator phenotype exhibited a favorable response to NTP treatment. Gender-specific analyses are shown in Fig. 3a, b. An age-related correlation was not observed in the male donors, while a significantly negative correlation was observed in the female donors (r = –0.773, N = 12, P = 0.006, Student’s t-distribution test). In Fig. 3c, data from the male donors are plotted according to NAT2 phenotype. As shown in the box-whisker plots, there was a large variance in the rapid acetylator phenotype; consequently, no significant effect of the NTP treatment was detected compared with the control (mean ± SD, 1.00 ± 0.48, N = 9, P = 0.98, Student’s t-test), while there was a significant increase in the intermediate acetylator phenotype (mean ± SD, 1.19 ± 0.13, N = 10, P = 0.001, Student’s t-test). Moreover, there was a statistically significant difference in the distributions of the data between the rapid and immediate acetylator phenotypes (P = 2.8E–04, F-test). These results suggest that male donors classified as having an intermediate acetylator phenotype are favorable responders to NTP treatment.
Reconfirmation of efficacy of NTP on expression of the CSGALNACT1 mRNA
To confirm the efficacy of NTP that we reported previously [7], the changes in the relative expression of the CSGALNACT1 mRNA were examined (Fig. 3d). Ten samples were impartially selected according to the results of NAT2 phenotype (rapid:immediate = 5:5), responsiveness (responder:nonresponder = 5:5), and gender (female:male = 4:6) presented above. Quantitative PCR showed that NTP treatment significantly increased the expression of the CSGALNACT1 mRNA in NP cells compared with the control (mean ± SD, 1.28 ± 0.37, N = 10, P = 0.013, Student’s t-test).