Hypergonadotropic hypogonadism in a patient with inv ins (2;4).

We report on a 30-year-old man with azoospermia, primary hypogonadism and minor dysmorphic features who carried a balanced insertional chromosome translocation inv ins (2p24;4q28.3q31.22)de novo. Molecular cytogenetic analyses of the chromosome breakpoints revealed the localization of the breakpoint in 4q28.3 between BACs RP11-143E9 and RP11-285A15, an interval that harbours the PCDH10 gene. In 4q31.22, a breakpoint-spanning clone (RP11-6L6) was identified which contains the genes LSM6 and SLC10A7. On chromosome 2, BACs RP11-531P14 and RP11-360O18 flank the breakpoint in 2p24, a region void of known genes. In conclusion, the chromosome aberration of this patient suggests a gene locus for primary hypogonadism in 2p24, 4q28.3 or 4q31.2, and three possible candidate genes (LSM6, SLC10A7 and PCDH10) were identified by breakpoint analyses.

Small supernumerary marker chromosomes--progress towards a genotype-phenotype correlation.

Small supernumerary marker chromosomes (sSMC) are still a major problem in clinical cytogenetics as they are too small to be characterized for their chromosomal origin by traditional banding techniques, but require molecular cytogenetic techniques for their identification. Apart from the correlation of about one third of the sSMC cases with a specific clinical picture, i.e. the i(18p), der(22), i(12p) (Pallister Killian syndrome) and inv dup(22) (cat-eye) syndromes, most of the remaining sSMC have not yet been correlated with clinical syndromes. Recently, we reviewed the available >1600 sSMC cases (Liehr T, sSMC homepage: http://mti-n.mti.uni-jena.de/~huwww/MOL_ZYTO/sSMC.htm). A total of 387 cases (including the 45 new cases reported here) have been molecularly cytogenetically characterized with regard to their chromosomal origin, the presence of euchromatin, heterochromatin and satellite material. Based on analysis of these cases we present the first draft of a basic genotype-phenotype correlation for sSMC for all human chromosomes apart from the chromosomes Y, 10, 11 and 13.

Evolutionary aspects of human cancer.

Evolutionary aspects of human cancer can be dealt with at two levels--on the one hand long-term evolution involving hereditary effects between generations; and on the other hand evolutionary processes operating within the organisms between tissues, cells and cell constituents, which also comprise genetic alterations, selection and adaptation. These two levels of evolution can be designated as phylogenetic and ontogenetic evolution, respectively. Concerning phylogenetic evolution there must have been a strong selection against neoplastic diseases occurring at reproductive age and a variety of protective mechanisms against carcinogenic agents have been developed. Cancer is therefore primarily a disease of old age, which does not constitute a significant risk in natural populations for the simple reason that the life length is too short. The development of an individual comprises selection forces between cells and tissues, which are particularly striking for the multistage development of tumours. The accumulation of several genetic alterations in the same cells, as illustrated by the analysis of colorectal tumours, must require a pronounced clonal expansion between each event. Such selective growth effect has recently been demonstrated for the tumour suppressor gene p53 in brain tumours. Cancer often implies a break down of between balanced systems antagonistic forces, such as oncogenes and suppressors of oncogenes. Examples of this are provided by the genetic regulation of metastasis, involving metalloproteinase as well as the inhibitor of metalloproteinase. The immortalization of cells by transformation points to the fact that programmed cell death and the balance between suicide genes and suppressors of such suicide genes is affected.

Refinement of map position of the human GluR6 kainate receptor gene (GRIK2) and lack of association and linkage with idiopathic generalized epilepsies.

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). The pivotal function of glutamate receptors (GluRs) in excitatory neurotransmission implicates their involvement in epileptogenesis and genetic susceptibility to IGEs. A trinucleotide repeat polymorphism detected in the 3' untranslated region of the kainate-selective GluR6 receptor gene (GRIK2) on chromosome 6 makes it possible to perform linkage and association studies with this high-ranking candidate gene. The present study tested the hypothesis that allelic variants of GRIK2 contribute to the genetic susceptibility to the common IGEs. Linkage and association analyses were conducted in 63 families ascertained through IGE patients with juvenile myoclonic epilepsy, juvenile absence epilepsy, or childhood absence epilepsy. Our linkage and association results suggest that allelic variants of GRIK2 are not involved in the expression of the common familial IGEs, and radiation hybrid mapping assigns GRIK2 to the chromosomal region 6q16.3-q21. This localization excludes GRIK2 as a candidate for the putative IGE susceptibility locus "EJM1" on the short arm of chromosome 6.

Mini- and microsatellites.

While the faithful transmission of genetic information requires a fidelity and stability of DNA that is involved in translation into proteins, it has become evident that a large part of noncoding DNA is organized in repeated sequences, which often exhibit a pronounced instability and dynamics. This applies both to longer repeated sequences, minisatellites (about 10-100 base pairs), and microsatellites (mostly 2-4 base pairs). Although these satellite DNAs are abundantly distributed in all kinds of organisms, no clear function has been discerned for them. However, extension of trinucleotide microsatellite sequences has been associated with several severe human disorders, such as Fragile X syndrome and Huntington's disease. Rare alleles of a minisatellite sequence have been reported to be associated with the ras oncogene leading to an increased risk for several human cancers. A dynamic behavior of repeated DNA sequences also applies to telomeres, constituting the ends of the chromosomes. Repeated DNA sequences protect the chromosome ends from losing coding sequences at cell divisions. The telomeres are maintained by the enzyme telomerase. Somatic cells, however, lose telomerase function and gradually die. Cancer cells have activated telomerase and therefore they acquire immortality.

Advantages of and problems with short-term mutagenicity tests for the assessment of mutagenic and carcinogenic risk.

The Salmonella microsomal assay has become an indispensible tool for the screening of mutagens and carcinogens, particularly when a large number of samples have to be tested, as in the present context for the screening of air pollution. However, for a more definite identification of potential carcinogens, a verification of the results from bacterial tests has to be performed with a battery of other tests, including point mutations and chromosomal aberrations in eukoaryotic systems. While there is a close qualitative correlation between the mutagenic and carcinogenic property of chemicals, a corresponding quantitative correlation between the mutagenic and carcinogenic potency is not always found. One reason for this lack of quantitative correlation presumably depends on the fact that cancer is induced in two steps, of which only the initiating, but not the promoting, step constitutes a mutational event, which is reflected by mutagenicity tests. Present mutagenicity tests have concentrated on discrete major mutations, while mutations of polygenes, acting on quantitative characters, have largely been omitted. Mutational data from Drosophila indicate, however, that polygenes mutate at a considerably higher rate than major genes and that they have a comparatively strong effect in heterozygous condition. It seems of great importance to develop appropriate methods to study induced mutations of polygenic systems and to get a better understanding of the properties of these genetic systems and an evaluation of the risk connected with induced mutations in polygenes.

Chemical induction of nondisjunction in drosophila.

Tests for chemically induced nondisjunction and loss of the sex chromosomes in Drosophila were performed. Of 31 compounds tested four gave rise only to an increase of XO exceptions, indicating the induction of chromosome loss. Six compounds, all known spindle inhibitors (colchicine, organic mercury, lead, and tin compounds) gave rise to an increase both of XXY and XO or of only XXY. The effect by metalloorganic compounds of which methylmercury was studied particularly closely, follows a peculiar pattern. In females with structurally normal X chromosomes only an increase of XX gametes is obtained, while with X chromosomes heterozygous for long inversions only O gametes are increased. The data indicates that the effect of the metal compounds occurs at first meiosis and that the process is connected with a meiotic drive, giving rise to a preferential segregation of the two X chromosomes to the functioning pole. The increase only of O gametes with structurally heterozygous X chromosomes can tentatively be explained by a loss due to crossing over within the inversion. An increase of the effect of methyl mercury was obtained where the normal pairing of the X chromosomes was interfered with by means of autosomal inversions. Likewise a synergistic increase of nondisjunction was obtained when a temperature chock of 10 degrees C was applied together with treatment with methylmercury. It is concluded that chemical induction of nondisjunction can be studied in Drosophila, but the sensitivity of the test is rather low and large amount of material is required.

Some aspects on the organization of microfilaments and microtubules in relation to nondisjunction.

One possible mechanism behind nondisjunction is a malfunctioning spindle. A defect or lack of spindle is a criterion for c-mitosis. In chemical mutagenesis research the c-mitotic effect is a well known cytological phenomemon, which can be induced by many different compounds. Pioneer work was performed during the 1940's by Ostergren and Levan, and their results and conclusions are briefly discussed. Since colchicine can induce c-mitosis and c-meiosis, by definition, and nondisjunction, a correlation between these phenomena is logical. The general importance of the spindle protein tubulin is considered and some new data from the cell biology literature on spindle formation and function as well as chromosome structure are briefly summarized. This knowledge can be used to correlate cytological and biochemical parameters among which c-mitosis and changes in sulfhydryl group metabolism after chemical treatment are the most obvious ones.

Common subtypes of idiopathic generalized epilepsies: lack of linkage to D20S19 close to candidate loci (EBN1, EEGV1) on chromosome 20.

Hereditary factors play a major role in the etiology of idiopathic generalized epilepsies (IGEs). A trait locus (EBN1) for a rare subtype of IGEs, the benign neonatal familial convulsions, and a susceptibility gene (EEGV1) for the common human low-voltage electroencephalogram have been mapped close together with D20S19 to the chromosomal region 20q13.2. Both loci are potential candidates for the susceptibility to IGE spectra with age-related onset beyond the neonatal period. The present study tested the hypothesis that a putative susceptibility locus linked to D20S19 predisposes to spectra of IGEs with age-related onset from childhood to adolescence. Linkage analyses were conducted in 60 families ascertained through IGE patients with juvenile myoclonic epilepsy, juvenile absence epilepsy or childhood absence epilepsy. Our results provide evidence against linkage of a putative susceptibility gene for four hierarchically broadened IGE spectra with D20S19 assuming tentative single-locus genetic models. The extent of an "exclusion region" (lod scores below-2) varied from 0.5 cM up to 22 cM on either side of D20S19 depending on the trait assumed. These results are contrary to the expectation that a susceptibility gene in vicinity to D20S19 confers a common major gene effect to the expression of IGE spectra with age-related onset from childhood to adolescence.

Detection of DNA alterations in human bladder tumors by DNA fingerprint analyses.

DNA fingerprint analyses were used to examine the constitutional and tumor DNA from 22 bladder tumor patients. DNA alterations, such as loss of bands, new bands, and intensity shifts were observed in 10 of the 22 patients. The most frequent DNA alteration, occurring in 80% of the patients, was a complete loss of one or several bands. Fingerprint abnormalities were present both in low-malignant superficial tumors and in high-malignant invasive tumors, but were also lacking in the latter group. Apparently no relationship exists between fingerprint abnormalities and gross chromosomal aberrations or the proportion of S-phase cells as measured by flow cytometry or development of recurrent tumors during a limited observation period. Thus, whether fingerprint aberrations express genetic alterations directly involved in the malignancy potential of bladder carcinoma remains an open question.

Studies on metabolic activation of vinyl chloride in Drosophila melanogaster after pretreatment with phenobarbital and polychlorinated biphenyls.

It is known that vinyl chloride is metabolized by the mixed function oxygenase system in the liver to reactive mutagenic and carcinogenic metabolites. This metabolic activation was studied in Drosophila melanogaster by measuring the uptake of 14C from labelled vinyl chloride in different strains and with different pretreatments with phenobarbital and polychlorinated biphenyl (PCB) Clophen A50), well known inducers of cytochrome P-450. In accordance with previously obtained data on vinyl chloride induced sex linked recessive lethals, it was shown that pretreatment with inducers increased the uptake of labelled compound up to ten times. There was, however, a marked difference in response between the five strains used. In particular, the strain Hikone, known to be resistant to insecticides, had a comparatively high initial radioactivity from vinyl chloride without any pretreatment, but it was not or insignificantly inducible with phenobarbital or PCB. Crosses between Hikone and an inducible strain indicated essentially a dominance for the Hikone genotype. Tests on inducible strains showed the same response to phenobarbital by 2 h old larvae and adult male and females. Dimethylsulphoxide (DMSO) used as a solvent decreased both the initial uptake of 14C and particularly the induction by PCB. The use of Tween 80 as an emulsifier did not have such an effect. It is emphasized that the interstrain variation in metabolic activation and inducability has to be taken into consideration in order to optimize the use of Drosophila for mutagenicity testing. This variation also opens up new possibilities of analyzing the mixed function oxygenase system biochemically and genetically.

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium I. Activation through conjugation with glutathion in vitro.

One of the main components in the waste products from vinyl chloride industries (EDC-tar), is ethylene dichloride (1,2-dichloroethane). This compound has been tested for mutagenicity on Salmonella typhimurium TA 1535. It is concluded that 1,2-dichloroethane gives a weak direct mutagenic effect, which is enhanced by addition of the postmitochondrial liver fraction (S-9). This activation is NADPH-independent and non microsomal. It is caused by a factor in the soluble fraction (115 000 g supernatant). This activation was further enhanced by the addition of glutathione but not by the addition of L-cysteine, N-acetyl-L-cysteine or 2-mercaptoethanol. No activation was observed when glutathione was added in the presence of a totally denaturated S-9 fraction or in the absence of this fraction. Activation of 1,2-dichloroethane was also found in the presence of glutathione and glutathione S-transferase A and C but not with glutathione S-tranferase B. A synthetic conjugate S-(2-chloroethyl)-L-cysteine gave a strong direct mutagenic effect at concentrations where no effects were seen with 1,2-dichloroethane. It is thus concluded that 1,2-dichloroethane is activated by conjugation to glutathione. Another main component in EDC-tar, 1,1,2-trichloroethane, was not mutagenic under any of our experimental conditions. For comparison 1,2-dibromoethane was also tested and gave a stronger direct mutagenic effect than 1,2-dichloroethane. Like the latter 1,2-dibromoethane was also activated by a NADPH-independent process.

Mutagenicity testing on chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene.

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.

On the mechanism of the hepatocarcinogenicity of peroxisome proliferators.

The absence of a genotoxic action in the rat of several peroxisome proliferators (PP) has been confirmed by measuring gross degradation, unscheduled DNA-synthesis (UDS), as well as by measurement of single strand breaks using alkali unwinding in absence and presence of inhibitors of DNA-repair. Similar results were obtained even after drastically lowering the glutathione content of liver. Further, after oral administration of ciprofibrate, no potentiating effect was found in vivo on the generation of micronuclei in hepatocytes by ionizing radiation. The metabolically inert PP, perfluorooctanoic acid, was found to act as a promoter of liver tumors in the rat induced by diethylnitrosamine in an initiation-selection-promotion protocol. The results are discussed in light of available information concerning the mechanism of action of PPs.

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. I. Activation of vinyl chloride, 2-aminoanthracene and benzo[a]pyrene as measured by mutagenic effects in Salmonella typhimurium.

The capacity of microsomal fractions from different Drosophila strains to activate three premutagens, 2-aminoanthracene (2-AA), vinyl chloride (VCM) and benzo[a]pyrene (BP) was investigated, using Salmonella typhimurium as the indicator organism. A significant increase in the mutation response in the Salmonella test system was obtained with all three substances in the presence of a metabolizing system (S9) from Drosophila larvae. 2-AA was converted to highly mutagenic metabolite(s) by the Drosophila S9 and the mutagenic effect was further increased after pretreatment with Aroclor 1254 (PCB) or beta-naphthoflavone (BNF). BP had only marginal mutagenic effects, causing less than a 2-fold increase in the number of mutants over the control. The data indicate that the metabolic conversion of BP is different in the Drosophila as compared to the rat liver microsomal fraction. In accordance with mutagenic data on Drosophila in vivo, vinyl chloride was a fairly weak mutagen in this Drosophila/Salmonella in vitro system.

Short-term testing--are we looking at wrong endpoints?

Short-term testing has been performed and interpreted on the basis of correlation between these tests and animal carcinogenicity. This empirical approach has been the only feasible one, due to a lack of knowledge of the actual genetic endpoints of relevance in carcinogenicity. However, the rapidly growing information on genetic alterations actually involved in carcinogenicity and in particular activation of oncogenes, provides facts of basic importance for the strategy of short-term testing. The presently used sets of short-term tests focus on standard genetic endpoints, mainly point mutations and chromosomal aberrations. Little attention has been paid in that connection to other endpoints, which have been shown or suspected to play an important role in carcinogenicity. These endpoints include gene amplification, transpositions, hypomethylation, polygene mutations and recombinogenic effects. Furthermore, indirect effects, for instance via radical generation and an imbalance of the nucleotide pool, may be of great significance for the carcinogenic and cocarcinogenic effects of many chemicals. Modern genetic and molecular technology has opened entirely new prospects for identifying genetic alterations in tumours and in its turn these prospects should be taken advantage of in order to build up more sophisticated batteries of assays, adapted to the genetic endpoints actually demonstrated to be involved in cancer induction. Development of new assay systems in accordance with the elucidation of genetic alterations in carcinogenicity will probably constitute one of the most important areas in genetic toxicology in the future. From a regulatory point of view the prerequisite for a development in this direction will be a flexibility of the handling of questions concerning short-term testing also at a bureaucratic level.

Deployment of short-term assays for the detection of carcinogens; genetic and molecular considerations.

The deployment of short-term assays for the detection of carcinogens inevitably has to be based on the genetic alterations actually involved in carcinogenesis. This paper gives an overview of oncogene activation and other mutagenic events connected with cancer induction. It is emphasized that there are indications of DNA alterations in carcinogenicity, which are not in accordance with "conventional" mutations and mutation frequencies, as measured by short-term assays of point mutations, chromosome aberrations and numerical chromosome changes. This discrepancy between DNA alterations in carcinogenicity and the endpoints of short-term assays in current use include transpositions, insertion mutations, polygene mutations, gene amplifications and DNA methylations. Furthermore, tumourigenicity may imply an induction of a genetic instability, followed by a cascade of genetic alterations. The evaluation of short-term assays for carcinogenesis mostly involves two correlations that is, between mutation and animal cancer data on the one hand and between animal cancer data and human carcinogenicity on the other. It should be stressed that animal bioassays for cancer in general imply tests specifically for the property of chemicals to function as complete carcinogens, which may be a rather poor reflection of the actual situation in human populations. The primary aim of short-term mutagenicity assays is to provide evidence as to whether a compound can be expected to cause mutations in humans, and such evidence has to be considered seriously even against a background of negative cancer data. For the evaluation of data from short-term assays the massive amount of empirical data from different assays should be used and new computer systems in that direction can be expected to provide improved predictions of carcinogenicity.

The nature of spontaneous mutations.

The induction of mutations is often expressed in relation to spontaneous mutations and designated for instance by the doubling-dose concept. A problem in that context is the fact that the mutational spectrum for spontaneous and induced mutations is not the same and it can furthermore vary considerably between loci. This is illustrated by molecular characterization of spontaneous and ionizing-radiation-induced mutations in mammalian cells. Furthermore changes of the genetic machinery are not limited to those endpoints usually measured in mutational assays, that is, base substitutions, frameshifts, classical chromosomal aberrations and numerical alterations of chromosomes. Additional alterations, of which far less is known, include insertion mutations, recombinogenic events and disproportionate replication of DNA giving rise to gene amplifications, and changes of gene expression through methylation of cytosine. There are reasons to believe that these endpoints are of importance in the development of tumors. Amplification of oncogenes is a well-known phenomenon in tumorigenicity and lately mutations by the insertion of mobile DNA elements have been demonstrated in cancer cells. Often these alterations are induced by stress and they constitute a manifestation of the dynamics and instability of DNA and the genetic system revealed in recent years. It is of interest in that context that the flow of genetic information does not only occur in one direction that is, DNA-RNA-protein, but also from RNA to DNA through reverse transcription and possibly even from the protein end of the sequence.

Mutagenicity research and testing in Sweden.

A survey is given of Swedish legislation for control of chemicals in the environment. Although no direct legal requirements for mutagenicity testing of chemicals exist at present in Sweden, such requirements can be enforced within the existing laws. Testing and research in chemical mutagenicity are especially performed at the Environmental Toxicology Unit of the Wallenberg laboratory, University of Stockholm. An outline is given of the organization of the unit, which is based on an interdisciplinary cooperation, among divisions of organic and analytical chemistry, cellular toxicology, and genetics. As examples of projects under joint investigation results on polychlorinated biphenyl (PCB) and on vinyl chloride are briefly described.