Spectrum of beta-thalassemia in Jordan: identification of two novel mutations.

Two hundred and forty-four beta-thalassemia alleles were identified from 135 unrelated occasionally and periodically transfusion dependent beta- and S/beta-thalassemia patients from all regions of Jordan. Allele identification was achieved by PCR amplification of beta-globin genes, dot-blotting the amplified DNA, hybridization with allele specific synthetic probes, and direct sequencing of amplified genomic DNA. A total of 19 different mutations were detected, eight of them constituted about 86% of the Jordanian thalassemic chromosomes. These mutations were IVS1-110 (G>A) (25%), IVS2-1 (G>A) (15%), IVS2-745 (C>G) (14.2%), IVS1-1 (G>A) (10%), IVS1-6 (T>C) (8.3%), codon 37 (G>A) (6.3%), codon 39 (C>T) (4.6%), and codon 5 (-C) (3.8%). The remaining eleven mutations were rare, presented with frequencies ranging between 0.4% and 1.6%. These included two novel mutations and four others detected in Jordan for the first time. The novel mutations were the frame shift (-C) at codon 49 and the substitution (A>C) at position -29 in the TATA box. Four alleles (1.6%) remained unidentified; having no abnormalities in their beta-globin gene sequences and therefore, constituted additional defects causing beta-thalassemia in the Jordanian population. These unknown alleles are expected to be candidates for upstream or downstream mutations affecting the expression of beta-globin gene. The results provided the essential foundation for planning a national preventive program for thalassemia in Jordan and will help improving the medical services for the patients and their families by helping their clinicians and genetic counselors in evaluating their variants and designing their treatment regimens.

Mutagenicity of sodium azide and its metabolite azidoalanine in Drosophila melanogaster.

The mutagenic and toxic activities of sodium azide (NaN(3) ) and its organic metabolite L-azidoalanine [N(3)-CH(2)-CH(NH)(2)-COOH] were examined in the different stages of spermatogenesis in Drosophila melanogaster. Both azide and azidoalanine were toxic to the injected males, but azidoalanine was significantly less toxic than sodium azide. Following the injection with 0.2 microl of these compounds in the hemocoel of young adult wild-type males, the minimum concentrations of these compounds with complete toxic effects (zero survival) were 40 mM sodium azide and 160 mM azidoalanine. Sex-linked recessive lethals were scored by the Muller-5 method in three successive broods, representing sperms (brood A), spermatids (brood B), and a compiled group of meiotic and premeiotic germ cell stages (brood C). The results provide strong experimental evidence that azidoalanine is significantly (p<0.01) mutagenic to all stages of spermatogenesis in Drosophila melanogaster. Sodium azide, however, was not significantly (p>0.05) mutagenic and did not increase the rate of sex-linked recessive lethals over those produced by the control group injected with 0.45% NaCl. These results indicate the requirement of metabolic activation of azide in Drosophila as a prerequisite for its mutagenic effects.

Induction of chromosomal aberrations by the rhodium(III) complex cis-[Rh(biq)(2)Cl(2)]Cl in cultured human lymphocytes.

The genotoxicity of the rhodium(III) complex cis-[Rh(biq)(2)Cl(2)]Cl (complex R) in cultured human lymphocytes was studied using the chromosome aberrations (CAs) assay. Lymphocyte cultures were initiated from two adult healthy non-smoking male volunteers and were exposed to the complex for a duration of 3 or 20 h prior to cell collection. The reduction in mitotic indices (MI) and the induction of CAs represented the toxic and clastogenic effects of the different treatments, respectively. Complex R proved to be an intermediate toxic clastogen with a MI(50) of 1.0 microg/ml and a minimum positive dose (MPD) of 0.1 microg/ml. Like bleomycin, complex R exerted its clastogenic effects without the need for metabolic activation and induced CAs of all types in lymphocytes treated in the G(2) and late S phases and, therefore, can be considered a radiomimetic. In addition, it induced more total CAs of chromatid-type than of chromosome-type. The reduction in the frequencies of CAs following the 20 h treatment as compared with those induced following the 3 h treatments indicated that human lymphocytes in the presence of complex R can partially tolerate the lesions involved in CA production. Based on the biological effects of complex R and the similarities between its functional group and that of bleomycin, possible mechanisms for complex R genotoxicity are discussed.

The effect of trifluoperazine on the genotoxicity of bleomycin in cultured human lymphocytes.

The effects of trifluoperazine on the toxicity and mutagenicity of bleomycin were examined in cultured human lymphocytes. Lymphocyte cultures were initiated from three adult healthy non-smoking male volunteers. Cultures were exposed to the drugs for either three or twenty hours prior to cell collection. The toxic and clastogenic effects of the different treatments were represented by the reduction in the mitotic indices and the induction of chromosomal aberrations (CA) respectively. Both TFP and BLM significantly increased CA frequencies and reduced the mitotic indices (MI) following all treatments. The reduction in the mitotic indices and the increase in CA frequencies induced by the combined administration of both BLM and TFP were highly significant (p < or = 0.001), but they were not significantly different from the sum of those induced by the separate treatments with the two drugs. These combined treatments, however, potentiated the odds ratios compared to those of the separate drug treatments. Therefore, though the effect of TFP on the clastogenic and cytotoxic effects of BLM was additive, the observed potentiation of the odds ratios of the combined treatments compared to those of the separate treatments suggested a significant enhancement in the expected chemotherapeutic effects of BLM when administered with TFP.

The mutagenic and toxic effects of bleomycin and trifluoperazine in Drosophila melanogaster.

The mutagenic and toxic effects of trifluoperazine and bleomycin on Drosophila were investigated in the progenies of males injected with 0.2 microliter of bleomycin and/or trifluoperazine. The Muller-5 method was used to study the induction of complete- and mosaic-sex-linked recessive lethals induced by 0.1 microgram/ml bleomycin and/or 0.1 mM trifluoperazine in the five successive broods, mainly representing the different stages of spermatogenesis. Trifluoperazine increased the induction rate of sex-linked recessive mutations above the spontaneous rates of the control, but these increases were not statistically significant at the 5% level27 in any of the five different broods. Contrary to trifluoperazine, bleomycin significantly (5% level)27 increased the induction rate of the complete sex-linked recessive lethals over those of the control in the meiotic and premeiotic broods C and D, and the meiotic brood E. As with the separate treatment with bleomycin, the frequencies of the complete sex-linked recessive lethals induced by the simultaneous combination treatment of 0.1 microgram/ml bleomycin and 0.1 mM trifluoperazine were significantly higher than those of the control at the 5%27 level, only in the meiotic and premeiotic broods, but they were not significantly higher than those induced by bleomycin treatment alone19. Treatments with 0.1 mM trifluoperazine enhanced the toxicity, sterility and the number of mutated clusters induced by 0.1 mM bleomycin but did not significantly increase the rates of induced lethals over the additive effects of both drugs in the meiotic and premeiotic stages, suggesting no potentiation effects for trifluoperazine over those of bleomycin in Drosophila. Higher concentrations of the two drugs could not be used due to their high toxicity and sterility effects.

Studies on fetal hemoglobin and gamma globin gene triplication in newborns in Jordan.

Quantitation of fetal hemoglobin (Hb F) and quantitation of it's gamma chain composition is important for the identification of different hemoglobinopathies. This is the first study done on the Jordanian newborns to test the hematological data and the gamma globin chain variants. A total of 52 randomly selected healthy Jordanian newborns were examined. The quantitation of the G gamma and A gamma chains combined with gene mapping using XmnI digestion, were used in the identification of one case of G gamma triplication among the studied samples. A family study of this case showed that adults carrying one copy of this G gamma triplication (13Kb XmnI fragment) had normal levels of HbF (< 1%) and high levels of G gamma (> 80%) while no homozygotes were detected. The remaining 51 newborns had normal frequency values of G gamma and A gamma chains. The frequency of the A gamma T chain among the 52 samples was 0.22. No abnormal alpha or beta chain variants were detected except for one case of HbS.

Induction of mosaic sex-linked recessive lethals in the different germ cell stages of Drosophila melanogaster by bleomycin.

The mutagenicity of bleomycin was studied in the different stages of spermatogenesis in Drosophila melanogaster. Following the injection of 2 microliters of 0.1 micrograms/ml of the chemical into young wild-type males, complete and mosaic sex-linked recessive lethals were scored by the Muller-5 method in five successive broods, mainly representing the different stages of spermatogenesis. The delayed mutagenic effect of the chemical was measured by the proportion of mosaic progeny produced. The results showed that bleomycin significantly increased the proportions of both complete and mosaic lethals in the broods representing the meiotic and pre-meiotic stages, but did not show any significant increase in these proportions in the broods representing the sperms and spermatids. The sizes of the mutated areas in the F1 gonads represented by the proportions of lethal-bearing females in F2 mosaic cultures were small, indicating that the genetic instabilities induced by bleomycin were transformed into actual mutations in later zygotic divisions. The significant divisions. The significant production of mosaic progeny in the F4 generation of the treated males showed that the mosaic F1 females produced by bleomycin were able to produce further mosaic progeny and suggested that bleomycin-induced instabilities can be transmitted as such for many future generations.

Molecular characterization of beta-thalassemia in north Jordan.

We have studied the beta-thalassemia mutations in 91 chromosomes of 43 patients with beta-thalassemia major and five with Hb S-beta-thalassemia, aged 6 months to 24 years. Many are blood transfusion-dependent and are being treated at the major hospital, the Princess Basma Hospital, in Irbid, Jordan. As many as 13 different mutations have been identified; three Mediterranean mutations [IVS-I-110 (G-->A), IVS-II-I (G-->A), and IVS-II-745 (C-->G)] were present in 54% of the chromosomes tested, while six other Mediterranean alleles were found in 24% of the chromosomes, for a total of 78% of Mediterranean origin. Sixteen chromosomes carried mutations which were observed in Arabian, Southeast Asian/Indian, and Iranian/Egyptian or Black populations; four beta-thalassemia mutations remained unidentified.

Production of mosaic lethals in different germ cell stages of drosophila by N-methyl-N'-nitro-N-nitrosoguanidine.

The delayed effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in Drosophila melanogaster by the proportion of mosaic progeny produced after this treatment. Following injection of the chemical into wild type males, complete and mosaic sex-linked recessive lethals were scored by the Muller-5 method, in five successive broods representing the different stages of spermatogenesis. All broods showed significant increase over the control in the frequencies of complete lethals with gradual decrease in mutation rate from the post-meiotic stages to the pre-meiotic ones. In the case of mosaic lethals, too, the post-meiotic stages were generally more sensitive; but the increase over the control was significant only for the mature spermatozoa. The extension of the experiment to F4 generation showed that a mosaic F1 female may produce further mosaic progeny. The production of lethal mutations in successive generations after treatment with MNNG supports the view that chemically induced instabilities can be transmitted as such over several generations.