Relationship of Urethral Dose and Genitourinary Toxicity Among Patients Receiving Vaginal High Dose Rate Interstitial Brachytherapy.

AIMS: Interstitial brachytherapy (ISBT) plays an important role in the management of locally advanced gynaecological malignancies. However, the relationship between urinary toxicity and dose to the urethra is not well understood. We sought to evaluate the correlation between urethral dose and the incidence of genitourinary complications among patients undergoing vaginal high dose rate ISBT. MATERIALS AND METHODS: Eighty-three patients treated with ISBT between August 2014 and April 2018 were retrospectively reviewed. CTCAE version 5.0 was used to grade toxicity. Individual treatment plans were evaluated to collect dose parameters. Urethral contours were added to the structure sets using a uniform 1 cm diameter brush and minimum doses to the hottest 0.1, 0.2 and 0.5 cm3 (D0.1cm3, D0.2cm3 and D0.5cm3) of the urethra were obtained. Total (ISBT ± external beam radiotherapy) equivalent doses in 2 Gy fractions (EQD2) received by the targets and organs at risk were calculated. Numerical counts (%) and medians (interquartile range) were used to characterise the data. Fisher's exact and the Mann-Whitney-Wilcox tests were used as appropriate. Receiver operator curve analysis was used to define the urethral threshold dose that correlated to genitourinary toxicity. RESULTS: The median age and follow-up times were 67 years (59-75) and 25 months (16-37), respectively. Patients had predominantly primary endometrial (49%) and vaginal (37%) cancer, with four (5%) patients with metastatic rectal cancer to the vagina. Twenty-four of 79 (30%) patients experienced acute genitourinary toxicity and 34 of 71 (48%) experienced late genitourinary toxicity. In both analyses, the median urethral dose was significantly higher among those with toxicity. Receiver operator curve analysis indicated that D0.1cm3, D0.2cm3 and D0.5cm3 of the urethra were associated with the development of toxicity at doses >78, >71 and >62 Gy, respectively. CONCLUSION: Urethral dose seems to predict genitourinary toxicity in ISBT of vaginal tumours. Further study with an expanded cohort and longer follow-up is warranted.

Unactivated leukocyte expression of C-reactive protein is minimal and not dependent on rs1205 genotype.

C-reactive protein (CRP), a prominent component of the innate immune system, is implicated in the pathophysiology of many conditions. CRP production primarily occurs in the liver; but contributions from other tissues is unclear. The Genotype-Tissue Expression Portal shows essentially no expression in whole blood and reports in the literature are conflicting. Multiple genomic variants influence serum levels of CRP. We measured CRP mRNA expression in leukocytes and sought to determine if rs1205 genotype influences leukocyte expression. Leukocytes were obtained from 20 women differing by genotype. Quantitative, real-time PCR (RT-qPCR) detected CRP and reference gene (GAPDH) mRNA. Leukocyte expression was calculated by the 2ΔCT method, and against a standard curve. Digital drop PCR was also used to calculate expression ratios. Student's t test and linear regression methods examined possible differences between genotypes. During 32 runs (10 replicates each), the RT-qPCR mean (SD) CRP/GAPDH ratio was 3.39 × 10-4 (SD 1.73 × 10-4) and 3.15 × 10-4 (SD 1.64 × 10-4) for TT and CC genotypes respectively, p = 0.76; and digital drop PCR results were similar. Serum CRP was not significantly different between genotypes, nor correlated with leukocyte expression. CRP is minimally expressed in unactivated leukocytes and this expression is not likely influenced by rs1205 genotype.

Results of 15 Gy HDR-BT boost plus EBRT in intermediate-risk prostate cancer: Analysis of over 500 patients.

PURPOSE/OBJECTIVE: To report biochemical control associated with single fraction 15 Gy high-dose-rate brachytherapy (HDR-BT) boost followed by external beam radiation (EBRT) in patients with intermediate-risk prostate cancer. MATERIALS AND METHODS: A retrospective chart review of all patients with intermediate-risk disease treated with a real-time ultrasound-based 15 Gy HDR-BT boost followed by EBRT between 2009 and 2016 at a single quaternary cancer center was performed. Freedom from biochemical failure (FFBF), cumulative incidence of androgen deprivation therapy use for biochemical or clinical failure post-treatment (CI of ADT) and metastasis-free survival (MFS) outcomes were measured. RESULTS: 518 patients met the inclusion criteria for this study. Median age at HDR-BT was 67 years (IQR 61-72). 506 (98%) had complete pathologic information available. Of these, 146 (28%) had favorable (FIR) and 360 (69%) had unfavorable (UIR) intermediate-risk disease. 83 (16%) received short course hormones with EBRT + HDR. Median overall follow-up was 5.2 years. FFBF was 91 (88-94)% at 5 years. Five-year FFBF was 94 (89-99)% and 89 (85-94)% in FIR and UIR patients, respectively (p = 0.045). CI of ADT was 4 (2-6)% at 5 years. Five-year CI of ADT was 1 (0-3)% and 5 (2-8)% in FIR and UIR patients, respectively (p = 0.085). MFS was 97 (95-98)% at 5 years. Five-year MFS was 100 (N/A-100)% and 95 (92-98)% in FIR and UIR patients, respectively (p = 0.020). CONCLUSION: In this large cohort of intermediate-risk prostate cancer patients, 15 Gy HDR-BT boost plus EBRT results in durable biochemical control and low rates of ADT use for biochemical failure.

Absolute percentage of biopsied tissue positive for Gleason pattern 4 disease (APP4) appears predictive of disease control after high dose rate brachytherapy and external beam radiotherapy in intermediate risk prostate cancer.

BACKGROUND AND PURPOSE: To identify if, in intermediate risk prostate cancer (IR-PCa), the absolute percentage of biopsied tissue positive for pattern 4 disease (APP4) may be a predictor of outcome. MATERIALS AND METHODS: 411 patients with IR-PCa were retrospectively reviewed. APP4 was calculated based on biopsy reports. Multivariable competing risk analysis was then performed on optimized APP4 cutpoints to predict for biochemical failure (BF), androgen deprivation use for BF (ADT-BF) and development of metastases (MD). RESULTS: Median follow-up for the cohort was 5.2 (Inter Quartile Range: 2.9-6.6) years. Median baseline PSA was 7.3 (5.3-9.8) ng/mL. 234 (56.9%) patients had T1 and 177 (43.1%) had T2 disease. Median APP4 was 2.00 (0.75-7.50)%. 38 (9.3%) patients experienced BF. The optimal cutpoint of APP4 for BF was >3.3% with an area under the curve (AUC) of 0.66. 17 (4.1%) received ADT-BF. The ADT-BF cutpoint was >6.6% with an AUC of 0.72. Eight (2.0%) developed MD. The MD cutpoint was >17.5% with an AUC of 0.86. Using APP4 >3.3 vs ≤ 3.3, log-transformed baseline PSA ln(PSA) (HR 2.5, 1.1-6.1; p = 0.037) and APP4 (HR 2.3, 1.1-4.7; p = 0.031) predicted for BF. Using APP4 >6.6 vs ≤ 6.6, ln(PSA) (HR 4.2, 1.4-12.4; p = 0.010) and APP4 (HR 3.7, 1.4-10.0; p = 0.009) were predictive of ADT-BF. APP4 >17.5 vs ≤ 17.5 alone was predictive of MD (HR 25.7, 4.9-135.3; p < 0.001). CONCLUSION: APP4 cutpoints of >3.3%, >6.6% and >17.5% were strongly associated with increased risk of BF, ADT-BF and developing MD respectively. These findings may inform future practice when treating IR-PCa but require external validation.

Rates of cannabis use in patients with cancer.

Background: A comprehensive assessment of cannabis use by patients with cancer has not previously been reported. In this study, we aimed to characterize patient perspectives about cannabis and its use. Methods: An anonymous survey about cannabis use was offered to patients 18 years of age and older attending 2 comprehensive and 2 community cancer centres, comprising an entire provincial health care jurisdiction in Canada (ethics id: hreba-17011). Results: Of 3138 surveys distributed, 2040 surveys were returned (65%), with 1987 being sufficiently complete for analysis (response rate: 63%). Of the respondents, 812 (41%) were less than 60 years of age; 45% identified as male, and 55% as female; and 44% had completed college or higher education.Of respondents overall, 43% reported any lifetime cannabis use. That finding was independent of age, sex, education level, and cancer histology. Cannabis was acquired through friends (80%), regulated medical dispensaries (10%), and other means (6%). Of patients with any use, 81% had used dried leaves.Of the 356 patients who reported cannabis use within the 6 months preceding the survey (18% of respondents with sufficiently complete surveys), 36% were new users. Their reasons for use included cancer-related pain (46%), nausea (34%), other cancer symptoms (31%), and non-cancer-related reasons (56%). Conclusions: The survey demonstrated that prior cannabis use was widespread among patients with cancer (43%). One in eight respondents identified at least 1 cancer-related symptom for which they were using cannabis.

The "VH1-like" dual-specificity protein tyrosine phosphatases.

The number of dual-specificity protein tyrosine phosphatases has grown considerably in the last few years, and thus it would be helpful to organize these novel enzymes. The simple term "VH1-like" or "dual-specificity" phosphatase does not adequately reflect the different subclasses within this new and important phosphatase subfamily. In this article, we review the salient features of dual-specificity phosphatases and propose a novel classification scheme of these enzymes based on their structural motifs. Classification of dual-specificity phosphatases based on their motifs should serve as a useful organizational framework for bringing together this now large subgroup of phosphatases. Moreover, this classification scheme may hold predictive value, since some of these motifs may hold the key to new, undiscovered functional properties.

Characterization of the PEST family protein tyrosine phosphatase BDP1.

Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues.

Slow acetylation in mice is caused by a labile and catalytically impaired mutant N-acetyltransferase (NAT2 9).

Three N-acetyltransferase genes (NAT*) were detected in inbred parental and congenic mice. Direct sequencing of NAT2* and liver cytosolic N-acetylation activity determinations with NAT2-specific (p-aminobenzoic acid) and NAT2-selective (2-aminofluorene) substrates have established that the acetylator congenic A.B6 and B6.A mice are genotypically and phenotypically identical to the parental B6 ("wild-type"; rapid acetylator) and A (mutant; slow acetylator) mice, respectively, from which they originated. The apparent KM for p-aminobenzoic acid and thermal inactivation rates determined with liver cytosol from the mutant (A and B6.A) mice were 3-fold and one order of magnitude higher than the corresponding values with liver cytosol from the wild-type (B6 and A.B6) strains. Northern blotting and immunoblotting revealed hepatic NAT2 mRNA and protein bands of equal size and intensity, regardless of the NAT2* genotype or phenotype of the animals. Incubation of liver cytosol from mutant A and B6.A mice at 37 degrees C for 6 hr resulted in virtual cessation of p-aminobenzoate N-acetylation activity, whereas the steady-state level of immunoreactive NAT2 remained unchanged. The results indicate that the amino acid change (N99I) in mutant NAT2* from slow acetylator mice does not hinder the synthesis of hepatic NAT2 protein, but, rather, leads to production of a conformationally modified NAT2 molecule that resists degradation by tissue proteases but is labile and catalytically impaired.

hVH-5: a protein tyrosine phosphatase abundant in brain that inactivates mitogen-activated protein kinase.

A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH-5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen-activated protein kinase. The catalytic region of hVH-5 was expressed as a fusion protein and was shown to hydrolyze p-nitrophenylphosphate and inactivate mitogen-activated protein kinase, thus proving that hVH-5 possessed phosphatase activity. A unique proline-rich region distinguished hVH-5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH-5 from related phosphatases was that hVH-5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH-5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH-5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate-early gene, suggesting a possible role in the signal transduction cascade.

Chromosomal localization of four human VH1-like protein-tyrosine phosphatases.

Four human protein-tyrosine phosphatase (PTPase) genes of the VH1-like subclass were cloned by low-stringency screening of a genomic library. These genes were localized to their respective chromosomes by G-banding and fluorescence in situ hybridization. The genes were localized to unique regions of different chromosomes: CL100, a stress-induced PTPase, to 5q35; PAC-1, a mitogen-induced nuclear PTPase, to 2q11;hVH-3 to 10q25; and hVH-4 to 10q11.

Isolation and characterization of a human dual specificity protein-tyrosine phosphatase gene.

Vaccinia phosphatase VH-1 and its mammalian counterparts, including protein-tyrosine phosphatases (PTPase) CL100 and VHR, constitute a novel subfamily of protein-tyrosine phosphatases that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates. The expression of human VH-1-like PTPase CL100 is rapidly inducible by mitogen stimulation and oxidative stress, suggesting that this gene is transcriptionally regulated. In order to study the mechanism underlying this transcriptional regulation, we isolated the first human gene of this subfamily, the CL100 gene, and characterized its promoter. The gene consists of four exons intervened by three short introns 400-500 base pairs in length. Analysis of the protein sequence encoded by each exon revealed that there is a second region of similarity between CL100 protein and cdc25 in addition to the PTPase catalytic domain. Promoter analysis of the CL100 gene indicates that an 800-base pair region flanking the transcriptional initiation site is sufficient to confer a transcriptional response to serum and 12-O-tetradecanoylphorbol-13-acetate stimulation. The CL100 gene is expressed in numerous tissues, including nonmitotic cells in the brain. Within the brain, CL100 mRNA is localized in discrete neuronal populations, suggesting that this PTPase is likely to play a key role in neurotransmission as well as in mitotic signaling. Finally, although extracellular signal-regulated kinase has recently been shown to act as substrate for CL100 in vitro, we find no clear correspondence between the distribution of extracellular signal-regulated kinase and CL100 mRNA in the brain. The potential significance of a second cdc25 homology domain of CL100 is discussed.

A novel receptor-type protein tyrosine phosphatase is expressed during neurogenesis in the olfactory neuroepithelium.

Tyrosine phosphorylation plays a central role in the control of neuronal cell development and function. Yet, few neuronal protein tyrosine phosphatases (PTPs) have been identified. We examined rat olfactory neuroepithelium for expression of novel PTPs potentially important in neuronal development and regeneration. Using the polymerase chain reaction with degenerate DNA oligomers directed to the conserved tyrosine phosphatase domain, we identified 6 novel tyrosine phosphatases. One of these, PTP NE-3, is a receptor-type PTP expressed selectively in both rat brain and olfactory neuroepithelium. In the olfactory neuroepithelium, PTP NE-3 expression is restricted to neurons and describes a novel pattern of expression with a high level in the immature neurons and a lower level in mature olfactory sensory neurons.

A protein phosphatase related to the vaccinia virus VH1 is encoded in the genomes of several orthopoxviruses and a baculovirus.

The vaccinia virus VH1 gene product is a dual specificity protein phosphatase with activity against both phosphoserine- and phosphotyrosine-containing substrates. We investigated the potential presence of VH1 analogs in other viruses. Hybridization and sequence data indicated that a phosphatase related to the VH1 phosphatase is highly conserved in the genomes of smallpox variola virus and other orthopoxviruses. The open reading frames from the raccoonpox virus and the smallpox variola virus Bangladesh major strain genomes encoding the VH1 analogs were sequenced and found to be highly conserved with the vaccinia virus VH1. An open reading frame from the baculovirus Autographa californica has sequence similarity to the VH1 phosphatase. The viral proteins appear to be structurally related to the cell cycle control protein p80cdc25. A recombinant phosphatase expressed from the baculovirus gene was found to share with the VH1 phosphatase the ability to hydrolyze substrates that contained both phosphoserine and phosphotyrosine.

Polymorphic N-acetylation of 2-aminofluorene by cell-free colon extracts from inbred mice.

The increased risk of rapid acetylator humans for the development of colorectal cancer has created interest in experimental animal models to study the relationship of N-acetyltransferase phenotype to colon cancer. Colon cytosols from inbred mouse lines were assayed for the ability to N-acetylate 2-aminofluorene to determine if the mouse model of the N-acetyltransferase polymorphism could be used to study this relationship. The results indicate that the colon acetylcoenzyme A: 2-aminofluorene-N-acetyltransferase activity parallels that of the liver. Colon activity from slow acetylator (A and B6.A) mouse lines is significantly lower than that of rapid acetylator (B6, B6.D, and A.B6) lines. p-Aminobenzoic acid N-acetyltransferase activity also differed between colon cytosols from rapid and slow acetylator strains. Isoniazid acetylation in colon and in liver did not differ between phenotypes. Northern blot analysis demonstrated the presence of mRNA for both NAT-1 and NAT-2 in mouse colon as well as in mouse liver. These results indicate that the N-acetyltransferase polymorphism is expressed in mouse colon when 2-aminofluorene or p-aminobenzoic acid is used as substrate and therefore the mouse may be a model for study of the effect of acetylator phenotype on development of colorectal cancer in humans.

Metabolic, molecular genetic and toxicological aspects of the acetylation polymorphism in inbred mice.

Over the past 10 years, much fascinating information has been obtained concerning the biochemistry, genetics, toxicological implications and molecular genetics of the N-acetylation polymorphism in mice. Using C57BL/6J (B6) mice as representative of rapid acetylation and A/J (A) mice as representing slow acetylation, it has been shown that the polymorphism observed in N-acetyltransferase (NAT) activity in liver also occurs in kidney, bladder, blood, and other tissues. The development of congenic acetylator mouse lines derived from B6 and A, have provided the necessary tools to study the role of the acetylation polymorphism, on either the B6 or A genetic background, free of nearly all other genetic differences between these strains. Eliminating genes which modify and complicate the differences due to the acetylator genes make the congenic lines very useful in toxicology studies, particularly those involving carcinogenesis. The molecular genetic basis of the acetylator polymorphism in B6 and A mice involves two Nat genes. Nat-1 encodes a protein termed NAT1 which is identical in rapid and slow acetylator strains. Nat-2, however, differs between rapid and slow strains by a single nucleotide change in the coding region. The corresponding NAT2 proteins differ by a single change at amino acid 99: an hydrophilic asparagine in rapid acetylator NAT2 to an hydrophobic isoleucine in NAT2 from slow acetylators. The mechanistic basis for the differences between rapid and slow acetylation in mice appears to be that NAT2 from the rapid B6 strain is 15-fold more stable at 37 degrees C and is transcribed/translated with a maximal efficiency twice that of the enzyme from slow acetylator A mice. Results discussed in this review indicate that mice provide an excellent system for studying the N-acetyltransferase polymorphism and also are useful for modelling several aspects of the human N-acetyltransferase polymorphism.

Cloned mouse N-acetyltransferases: enzymatic properties of expressed Nat-1 and Nat-2 gene products.

N-Acetylation plays an important role in the metabolism of a wide variety of hydrazine drugs and arylamine drugs and carcinogens. Humans have genetically determined differences in their N-acetyltransferase activities and are phenotypically classified as rapid or slow acetylators. Mice have a similar genetic polymorphism in N-acetyltransferase activity and have been used as models of the human polymorphism in many studies of the toxicology and carcinogenicity of arylamines. Recently, two N-acetyltransferase genes, Nat-1 and Nat-2, were cloned from rapid (C57BL/6J) and slow (A/J) acetylator mouse strains. The genomic clone encoding NAT-1 is identical in rapid and slow acetylator mouse strains, whereas the clone encoding NAT-2 differs between rapid and slow strains by a single base pair, which changes the encoded amino acid from Asn99 in the rapid acetylator strain to Ile99 in the slow acetylator strain. In this report, the N-acetylation polymorphism in mice was investigated by transiently expressing the cloned N-acetyltransferase genes in COS-1 cells. The intronless coding regions of Nat-1 and Nat-2 showed different substrate specificities; isoniazid was a preferred substrate for NAT-1, whereas p-aminobenzoic acid was preferred for NAT-2(99asn) and NAT-2(99ile). All three enzymes acetylated 2-aminofluorene, but none of them acetylated sulfamethazine. Kinetic constants determined for the expressed enzymes with 2-aminofluorene and p-aminobenzoic acid indicated that Km values were not significantly different between the enzymes, although the Vmax value of NAT-2(99asn) was consistently 2-3-fold higher than that of NAT-1 or NAT-2(99ile). Nat-1 and Nat-2 encoded mRNAs of approximately 1.4 kilobases in livers of rapid and slow acetylators. Nat-2 mRNA was more abundant in liver than Nat-1 mRNA. The abundance of Nat-2 mRNA and Nat-1 mRNA was equivalent in both rapid and slow acetylator mouse strain livers. Incubation of transfected COS-1 cell cytosols at 37 degrees showed that the time for decline of NAT activity to 50% of its initial value was 45 hr for NAT-1, 60 hr for NAT-2(99asn), and 4 hr for NAT-2(99ile). This 15-fold difference in the heat stability of the rapid and slow isoforms of NAT activity was also observed in cytosols from rapid and slow acetylator livers. Comparison of the rates of translation of the rapid and slow isoforms of NAT-2 in an in vitro system showed that NAT-2(99asn) was translated at approximately twice the rate of NAT-2(99ile).(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular genetic basis of rapid and slow acetylation in mice.

The molecular genetic basis of N-acetylation polymorphism has been investigated in inbred mouse models of the human acetylation polymorphism. Two genomic clones, Nat1 and Nat2, were isolated from a C57BL/6J (B6) mouse (rapid acetylator) genomic library. The Nat1 and Nat2 genes both have intronless coding regions of 870 nucleotides and display greater than 47% deduced amino acid similarity with human, rabbit, and chicken N-acetyltransferases. Amplification of Nat1 and Nat2 from A/J (A) mouse (slow acetylator) genomic DNA by the polymerase chain reaction and subsequent sequencing revealed that Nat1 was identical in B6 and A mice, whereas Nat2 contained a single nucleotide change from adenine in B6 to thymine in A mice. This nucleotide substitution changes the deduced amino acid at position 99 from asparagine in B6 to isoleucine in A mice. Hydropathy analysis revealed that this amino acid change alters the hydropathy of the flanking peptide segment in NAT2 from hydrophilic in the B6 mouse to hydrophobic in the A mouse. The amino acid change occurs in a region of the gene where no polymorphism has yet been reported in human or rabbit NAT2 and may represent an important structural domain for N-acetyltransferase activity. Nat1 and Nat2 have the same 5' to 3' orientation in the B6 mouse; the two genes are separated by approximately 9 kilobases, with Nat1 located 5' of Nat2.

Diverse point mutations in the human gene for polymorphic N-acetyltransferase.

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variations. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants. One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (codon 94) and a separate G----A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions: Thr for Ile (codon 114) and Arg for Lys (codon 268). Heterozygosity was detected in three NAT2 samples: two were heterozygous for the rapid and one of the allelic variants, and the third was a compound heterozygote of both mutant alleles. The results show conclusively that the genetically variant NAT is encoded by NAT2.