[Genetic differences in enzymes of folic acid metabolism in patients with lip-jaw-palate clefts and their relatives]. / Genetische Unterschiede von Enzymen des Folsäurestoffwechsels bei Patienten mit Lippen-Kiefer-Gaumen-Spalten und ihren Angehörigen.

BACKGROUND: The effectiveness of folic acid supplementation in the periconceptional period for the prevention of cleft lip/cleft lip and palate (CLP) is contradictorily discussed. Genetically determined variants of enzymes of the folic acid metabolism could be part of the key to success or failure of folate supplementation. A mutation of the methylenetetrahydrofolate reductase (MTHFR) gene is suspected to be a risk factor for CLP. METHODS: The blood samples of 66 CLP patients, their 88 relatives (without CLP), and 184 healthy controls were searched by polymerase chain reaction for mutations of MTHFR 677 C:T, MTHFR 1298 A:C and of the arylamine N-acetyltransferase (NAT1) gene [gene type NAT1 degree 4 (wild type) or not]. RESULTS: There was no significant difference in the number of MTHFR gene mutations (for 677 C:T and 1298 A:C) between the three groups (p approximately 0.3), but for the NAT1 genes (p = 0.033). The homozygote mutation was found more than twice as often in CLP patients (10.5%) and their relatives (10.6%) than in the healthy controls (4.35%). DISCUSSION: Our results provide no evidence that the above MTHFR gene mutations are a risk factor for CLP.A NAT1 gene mutation instead could be a risk factor for CLP.

Gender-specific effects of NAT2 and GSTM1 in bladder cancer.

One approach for risk assessment of cancer is the evaluation of polymorphic enzymes involved in cancer using molecular tools. Phase II enzymes are involved in the detoxification of several drugs, environmental substances and carcinogenic compounds. Here, we analyzed enzymes for their putative relevance in urinary bladder cancer. The hereditable enzyme polymorphism of arylamine N-acetyltransferase 2 (NAT2) and glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) was studied in 157 hospital-based patients and in 223 control subjects. Slow acetylation was not observed to be a significant risk factor of developing bladder cancer (OR: 1.33; 95% CI 0.85-2.09). One genotype responsible for slow acetylation (NAT2*5B/*6A) was observed significantly more frequently in bladder cancer patients compared with control subjects (OR: 1.63; 95% CI 1.03-2.58). Gender-specific effects were observed when patients were divided into subgroups. In male patients, slow acetylators were identified as carrying a significant increased risk of developing bladder cancer, in particular when the genotype NAT2*5B/*6A was combined with the GSTM1 null genotype (OR: 4.39; 95% CI 1.98-9.74). By contrast, the same genotype combination significantly protected female patients from bladder cancer (OR: 0.21; 95% CI 0.06-0.80).

Susceptibility genes: GSTM1 and GSTM3 as genetic risk factors in bladder cancer.

Glutathione S-transferase (GST, E.C. 2.5.1.18) comprises a family of isoenzymes that play a key role in the detoxification of such exogenous substrates as xenobiotics, environmental substances, and carcinogenic compounds. At least five mammalian GST gene families have been identified to be polymorphic, and mutations or deletions of these genes contribute to the predisposition for several diseases, including cancer. The gene cluster of GSTM1-GSTM5 has been reported to be localized on chromosome 1p and spans a length of nearly 100 kb. One mutation of the GSTM3 gene generates a recognition site for the transcription factor yin yang 1. As a result of this mutation, the expression of GSTM3 can be influenced. The mutated GSTM3 gene has been reported to be involved in increased susceptibility for the development of cancer, but no information is available concerning its role in bladder cancer. We have identified patients with a heterozygous GSTM3 geno- type who carry a significantly increased risk for the development of bladder cancer. Here we report that the mutation of intron 6 of GSTM3 increases the risk for bladder cancer (odds ratio: 2.31; 95% confidence interval [CI], 1.79-2.82). We developed a procedure to identify heterozygous or homozygous carriers of the GSTM1 alleles. Heterozygous carriers of the GSTM1 null genotype have a significantly elevated risk of developing bladder cancer. We calculated an odds ratio of 3.54 (95% CI, 2.99-4.11) for this genotype. These observations lead to the assumption that the lack of detoxification by glutathione conjugation predispose to bladder cancer when at least one of two alleles is affected. Furthermore, individuals presenting the homozygous wild type of GSTM1 and GSTM3 are significantly protected against bladder cancer.

Genotyping of the polymorphic N-acetyltransferase (NAT2) and loss of heterozygosity in bladder cancer patients.

Acetylation is one of the major routes in metabolism and detoxification of a large number of drugs, chemicals and carcinogens. Slow acetylators are said to be more susceptible to developing bladder cancer and because of investigations about tumor risk based on phenotyping procedures, it was our aim to study the distribution of allelic constellations of the N-acetyltransferase (NAT2) by genotyping patients with bladder cancer. We analysed NAT2 gene of blood and tumor DNA from 60 patients with primary bladder cancer and DNA of blood samples from 154 healthy individuals. Using ASO-PCR/RFLP techniques we identified 70% of patients with bladder cancer (n = 42) to be slow acetylators while genotyping of controls resulted in 61% with slow acetylators (n = 94). In addition, dividing bladder cancer patients in males and females the genotype NAT2*5B/NAT2*6A occured with much higher frequencies in males (OR = 4, 95%); CI = 1.8-8.9). Furthermore, investigating bladder cancer tissues we could detect loss of heterozygosity (LOH) in slow and rapid acetylator genotypes. In eleven out of 60 tumor samples (18.3%) we observed allelic loss at the NAT2 locus while in control DNA of blood from the same patients both alleles were still detectable.

Regional fine mapping of the multiple-aberration region involved in uterine leiomyoma, lipoma, and pleomorphic adenoma of the salivary gland to 12q15.

Cytogenetic aberrations involving 12q13-15 are frequently observed in a variety of benign solid tumors. Using a chromosome walking approach combined with fluorescence in situ hybridization analysis, we were able to show that the chromosome 12 breakpoints involved in uterine leiomyoma, pleomorphic adenoma of the salivary gland, and lipoma cluster to the same chromosomal region, which we therefore designated MAR (multiple-aberration region). By comparing the G-banding pattern of prometaphase chromosomes of amniotic fluid cells and lymphocytes to the position of hybridization signals obtained with a cosmid pool encompassing this breakpoint hot spot region, MAR was assigned to 12q15. We conclude that, despite the cytogenetic breakpoint assignment to the three bands 12q13-15 in individual uterine leiomyomas, lipomas, and pleomorphic adenomas of the salivary glands in the past, most likely 12q15 is the only 12q breakpoint site in these three distinct solid tumor types.

Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry.

Melatonin, the chief hormone of the pineal gland in vertebrates, is widely distributed in the animal kingdom. Among many functions, melatonin synchronizes circadian and circannual rhythms, stimulates immune function, may increase life span, inhibits growth of cancer cells in vitro and cancer progression and promotion in vivo, and was recently shown to be a potent hydroxyl radical scavenger and antioxidant. Hydroxyl radicals are highly toxic by-products of oxygen metabolism that damage cellular DNA and other macromolecules. Herein we report that melatonin, in varying concentrations, is also found in a variety of plants. Melatonin concentrations, measured in nine different plants by radioimmunoassay, ranged from 0 to 862 pg melatonin/mg protein. The presence of melatonin was verified by gas chromatography/mass spectrometry. Our findings suggest that the consumption of plant materials that contain high levels of melatonin could alter blood melatonin levels of the indole as well as provide protection of macromolecules against oxidative damage.

Isolation and mapping of a cosmid clone containing the human NAT2 gene.

The NAT2 gene encodes for a polymorphic arylalkylamine N-acetyltransferase and thus accounts for the human N-acetylation polymorphism. By a NAT2-specific primer set we have screened a human chromosome 8-specific cosmid library. A positive cosmid clone was mapped by fluorescence in situ hybridization to 8p22. The polymerase chain reaction followed by restriction analysis of the PCR product was used to identify allele 2 to be contained in the cosmid clone.

Pleomorphic adenoma cells vary in their susceptibility to SV40 transformation depending on the initial karyotype.

Chromosomal aberrations involving 8q12 or 12q13-15 characterize two cytogenetic subgroups of salivary gland pleomorphic adenomas. As the tumors of the two groups differ in their clinical and histologic characteristics, we decided to determine their susceptibility to SV40 transformation. We transfected cell cultures from 13 adenomas with aberrations involving 8q12 and from seven adenomas with involvement of 12q13-15 using an SV40 plasmid coding for the early region of the viral genome. Whereas all cultures with aberrations of 12q13-15 showed transformed foci, only 4 of the 13 cultures with 8q12 abnormalities showed foci of transformed cells. We also observed a much higher immortalization rate in the first group (3/7 vs. 1/13). All successfully transformed tumor cell cultures showed a relatively stable karyotype in the pre-crisis stage and a high mitotic index, were T-antigen positive, and had an extended life span in vitro.

A recurrent marker chromosome involving chromosome 1 in two mammary tumors of the dog.

An apparently identical marker chromosome resulting from a chromosome 1. translocation was found in the mammary carcinomas of two bitches. Although these karyotypic aberrations were the sole clonal aberrations detected, it was not possible to unambiguously identify the material translocated to the chromosome 1 in either animal. Our observations, however, represent the first report of a recurring marker chromosome in mammary tumors of the dog and suggest that these tumors may become an interesting model for human breast cancer.

No rearrangement of c-mos in salivary gland pleomorphic adenomas with 8q12 aberrations.

The frequent occurrence of salivary gland pleomorphic adenomas characterized by clonal structural chromosome abnormalities involving 8q12 raises the question as to how the cytogenetic rearrangements correspond to molecular mechanisms of tumor development. Since the proto-oncogene c-mos maps to this breakpoint region, DNA from eight adenomas with these aberrations was isolated and checked for rearrangements of c-mos after digestion by BamHI, EcoRI and HindIII. In none of the tumors was a rearranged allele besides the germ-line fragments found.

Pleomorphic adenomas with unbalanced chromosomal abnormalities have an increased in vitro lifetime.

The in vitro lifetime of 100 salivary gland pleomorphic adenomas was investigated. On the average, they had a limited lifetime of 3.7 passages. In 86 cases, the tumors were successfully karyotyped, resulting in three major cytogenetic subgroups characterized by either an apparently normal karyotype or by rearrangements of 8q12 or 12q13-15. There was no significant difference between the in vitro lifetime of the tumors with a normal or with an aberrant karyotype. The three adenomas with the longest lifetime were characterized by an unbalanced karyotype. One of them had a deleted chromosome 6, which has also been described in malignant tumors. An increased in vitro lifetime may thus correspond to early steps of malignant transformation in vivo and can be caused by additional unbalanced chromosome aberrations. In the future, long-term cultivation can be helpful in defining chromosomal regions involved in this process.

Identification of flow-sorted chromosomes by G-banding and in situ hybridization.

The identification of flow-sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G-banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest. In particular when sorting chromosomes from human-rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin-labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.

Structural rearrangements of chromosome Nr 8 involving 8q12--a primary event in pleomorphic ademona of the parotid gland.

Nine pleomorphic adenomas of the human parotid gland were investigated. Within this series the group of cases having clonal aberrations of chromosome Nr 8 predominates. The occurrence of cases with trisomy-8 and cases with structural rearrangements involving a breakpoint in 8q11-8q13 allows a further subdivision of this group of tumors. Our special interest in this paper is devoted to the latter group. The hypothesis is proposed that in these cases the chromosomal rearrangement is the primary event in tumorigenesis, leading to activation of a so far unknown oncogene located most likely at 8q12. The translocations to different recipient chromosomes may serve as sign posts to transcriptionally active chromosomal domains in the salivary gland.

Rearrangements of chromosome region 12q13----q15 in pleomorphic adenomas of the human salivary gland (PSA).

Nine of 40 pleomorphic salivary gland adenomas (PSAs) showed clonal aberrations of chromosome 12, with a breakpoint at 12q13----q15. The cytogenetic findings in these cases and those of nine additional cases reported in the literature suggest that this type of aberration is a primary change directly involved in the genesis of PSA.

Inhibition of day time, but not isoproterenol-stimulated pineal N-acetyltransferase activity by an unidentified pineal compound.

Day time rat pineal N-acetyltransferase (P-NAT) activity is higher when homogenate used for incubation is diluted. Samples containing homogenate equivalent to 1/80 pineal gland had highest enzyme activity when compared with samples that contained homogenate equivalent to 1/40 and 1/20 pineal gland, respectively. Thus P-NAT activity is inhibited by a compound present in the pineal gland of the rat. This inhibition was not found in pineal glands of animals that had been injected with isoproterenol. Furthermore day time P-NAT activity is inhibited by excess of substrate, whereas even at 10 mmol/l of substrate isoproterenol stimulated P-NAT activity is not. It is suggested that these two phenomena are involved in the physiologic regulation of the circadian changes of P-NAT activity.

A new banding pattern of human chromosomes by in situ nick translation using ECO RI and biotin-dUTP.

Here we present first results of an attempt to replace the nicking activity of DNAase I by ECO RI for in situ nick translation of human chromosomes. For the detection of nick translated sites we used biotinylated dUTP. The procedure resulted in a distinct banding pattern of the chromosomes which does not seem to correspond to that reached by any known banding technique.

Interindividual differences of corneal sensitivity. Genetic aspects.

By means of an electronic optical esthesiometer corneal sensitivity was examined in 91 volunteers of different age groups. Additionally, the anesthetic duration of the local anesthetic benoxinate was investigated. Corneal sensitivity decreases with advancing age. Comparing male and female subjects, we can suppose that there are age and sex specific differences of corneal sensitivity. There might be additional genetic factors. There are great interindividual differences in the anesthetic duration of benoxinate. It can be assumed that benoxinate is metabolized by pseudocholinesterase. One possible explanation for the great differences in the anesthetic duration of benoxinate can be seen in the genetically determined variants of pseudocholinesterase.

The pipette-method: its application to cytogenetic studies of tumor cells cloned in semisolid media.

A simple method is herein described which allows easy cytogenetic analyses of human tumor cells grown in semisolid media. The quality of the metaphases is relatively good, comparable with that reached by using conventional cytogenetic methods. Therefore, the demonstration of G-, Q-, C-bands, and nucleolus organizing regions (NOR) is possible without any problems. Its particular advantage, however, lies in the fact that single colonies can be removed under sterile conditions at different stages of cell culturing.

Further evidence for nonrandom chromosome changes in carcinoma cells--a report of 28 cases.

A cytogenetic study was made of pleural and ascitic effusions from 28 carcinoma patients. Gross chromosome abnormalities were observed in each case. A selection against heteroploid cells occurred generally in long-term cell cultures. Although no further evidence for the existence of primary specific chromosome abnormalities was found in this study, we postulate three types of chromosome abnormalities in carcinoma cells: (a) primary, specific chromosome changes; (b) secondary, but nonrandom, chromosome changes; and (c) random chromosome changes. We feel that it may be a feature of the secondary changes to cause high mitotic instability, which leads to further karyotype variability, new changes of type b and c, and an increased potential for malignancy.