Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.     

Chromosomal damage induced by furocoumarins and UVA in hamster and human cells including cells from patients with ataxia telangiectasia and xeroderma pigmentosum

The comparative photosensitizing effects to near-UV irradiation (UVA) of several naturally occurring furocoumarins, 5-methoxypsoralen (5MOP), psoralen, 8-methoxypsoralen (8MOP) and angelicin in producing chromosome damage in vitro in cells derived from hamster, normal human, ataxia telangiectasia (AT) and xeroderma pigmentosum (XP) patients were studied. In Chinese hamster cells, lethality was greatest with psoralen and least with angelicin; 8MOP and 5MOP were intermediate. 8MOP and 5MOP produced sister-chromatid exchanges with almost equal efficiency and to a larger extent by far than angelicin. In all human cell lines studied 8MOP and 5MOP were similarly effective in the production of sister-chromatid exchanges and chromosomal aberrations. AT and XP cells responded with higher frequencies of sister-chromatid exchanges as well as chromosomal aberrations than normal human cells to 5MOP, 8MOP and angelicin. Evidence is presented which suggests that cell death in Chinese hamster cells following angelicin photosensitization is not clearly related to the production of sister-chromatid exchanges. AT cells were unexpectedly more sensitive to angelicin than normal cells. The presence of 5MOP in some sun-tan preparations is not acceptable in view of the present evidence of its biological activity.     

High frequency of spontaneous translocations revealed by FISH in cells from patients with the cancer-prone syndromes ataxia telangiectasia and Nijmegen breakage syndrome.

The application of fluorescence in situ hybridization (FISH) using whole-chromosome paints (WCPs) is proving to be a very powerful technique for revealing chromosomal instability that, for the most part, has gone undetected by conventional cytogenetic analysis. We have analyzed the frequency of translocations in lymphocytes and lymphoblastoid cell lines from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) homozygotes and heterozygotes using a three-color chromosome-painting technique (WCP 1, 2, 4). With this assay we were able to detect an increased frequency of spontaneous translocations in AT homozygotes (median, 18.47 +/- 10.82 translocations per 1,000 metaphase cells; 10 patients) and AT heterozygotes (median, 7.87 +/- 3.15 translocations per 1,000 cells; 7 patients), in comparison to controls (median, 2.26 +/- 1.75 translocations per 1,000 cells; 10 controls). Analysis of NBS homozygotes (median, 19.05 +/- 11.27 translocations per 1,000 cells; 5 patients) and NBS heterozygotes (median, 6.93 +/- 3.04 translocations per 1,000 cells; 6 patients) also showed an increased frequency of translocations in these patients compared to controls. The presence of such hitherto undetected chromosomal aberrations corroborate previous findings of spontaneous chromosomal instability in AT and NBS patients, as manifested by an increased rate of open breaks and rearrangements involving chromosomes 7 and 14. Moreover, we show that the degree of genomic instability in AT and NBS patients is even higher than previously established and that some AT and NBS heterozygotes evidence spontaneous chromosomal instability as well. These increased levels of nonspecific translocations could be an important risk factor for the development of malignancies in homozygotes and heterozygotes for ATM or NBS1 gene mutations.     

Cells from patients with ataxia telangiectasia are abnormally sensitive to the cytotoxic effect of a tumor promoter, phorbol-12-myristate-13-acetate

Fibroblast strains from 6 patients with ataxia telangiectasia (A-T) were found to be markedly hypersensitive to the cytotoxic action of the tumor promoter phorbol-12-myristate-13-acetate (PMA), their D37 values being 5 times lower than those of two normal controls. Two A-T heterozygous strains were slightly hypersensitive to PMA, while a third one showed normal sensitivity. It is concluded that the DNA lesion which is critical in A-T cells is an important component of the damage caused by PMA-induced free radicals and may play a role both in the tumor-promoting activity of PMA and the cancer proneness of A-T patients.     

UV mutation spectra in cell lines from patients with Cockayne's syndrome and ataxia telangiectasia, using the shuttle vector pZ189.

We have used the SV40-based shuttle vector pZ189 to determine ultraviolet mutation spectra in SV40-transformed cell lines from two patients with Cockayne's syndrome (CS) and ataxia telangiectasia (AT). The shuttle vector was UV-irradiated, transfected into the cells and recovered two days later, after many rounds of replication had occurred. Plasmid DNA was used to transform indicator bacteria in which plasmids containing a mutation in the supF gene resulted in white colonies. Mutant plasmids were analysed both by agarose gels and by DNA sequencing. In contrast to published spectra for xeroderma pigmentosum cells, the types of mutation induced by UV mutation in the CS and AT cell lines were similar to each other and to published spectra for normal cell lines. There were however, some differences in the sequence distribution of the mutations.     

Characterisation of mutations in 77 patients with X-linked myotubular myopathy,including a family with a very mild phenotype

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.     

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.     

Characterization of a B-lymphocyte t(2;14) (p11;q32) translocation from an ataxia telangiectasia patient conferring a proliferative advantage on cells in vitro

Patients with the recessively inherited disorder ataxia telangiectasia (AT) are particularly prone to the development of both B-cell and T-cell tumours. Specific translocations involving T-cell gene rearrangements and an unknown locus 3' of IGH have been described in AT T-cell clone and tumour cells. We describe here a t(2;14)(p11;q32) translocation which was observed in nonmalignant short-term-cultured B lymphocytes from an AT patient. In vivo, the clone of cells grew from 1% to 6% of the total cell population over a period of 2 yr. Clonal translocations may therefore be associated with AT B cells, as well as AT T cells. B lymphocytes were transformed with Epstein-Barr virus, and the t(2;14) translocation cell was cellularly cloned. Using Southern filter analysis and in situ hybridisation to define more clearly the positions of the breakpoints, we show that the translocation at 14q32 involves a deletion within the IGH chain gene of at least J1, J2, DQ52, and sequences 1.5 kb 5' of DQ52 and that the breakpoint is either adjacent to the non-deleted JH sequences or upstream of these sequences, within the D or V regions, but proximal to all members of the VHII family of genes. The breakpoint at 2p11 is outside and proximal to IGK with respect to the centromere in an unknown gene. Sub-lines with an initially low proportion of translocation cells eventually became monoclonal in vitro for these cells. This suggests they have a growth advantage in vitro.     

Expression of lamin A mutated in the carboxyl-terminal tail generates an aberrant nuclear phenotype similar to that observed in cells from patients with Dunnigan-type partial lipodystrophy and Emery-Dreifuss muscular dystrophy

Autosomal dominantly inherited missense mutations in lamins A and C cause familial partial lipodystrophy of the Dunnigan-type (FPLD), and myopathies including Emery-Dreifuss muscular dystrophy (EDMD). While mutations responsible for FPLD are restricted to the carboxyl-terminal tails, those responsible for EDMD are spread throughout the molecules. We observed here the same structural abnormalities in the nuclear envelope and chromatin of fibroblasts from patients with FPLD and EDMD, harboring missense mutations at codons 482 and 453, respectively. Similar nuclear alterations were generated in fibroblasts, myoblasts, and preadipocytes mouse cell lines overexpressing lamin A harboring either of these two mutations. A large variation in sensitivity to lamin A overexpression was observed among the three cell lines, which was correlated with their variable endogenous content in A-type lamins and emerin. The occurrence of nuclear abnormalities was reduced when lamin B1 was coexpressed with mutant lamin A, emphasizing the functional interaction of the two types of lamins. Transfected cells therefore develop similar phenotypes when expressing lamins mutated in the carboxyl-terminal tail at sites responsible for FPLD or EDMD.     

The characteristics of the phenotype and lymphocyte function in patients with a mucosal lesion of the large intestine

The results of studies of functional activity of lymphocyte subpopulations: theophylline-sensitive (thphs-1) and theophylline-resistant (thphr-1), separated from peripheral blood of healthy people and patients with nonspecific ulcerative colitis (NUC) are represented in this paper. The evaluation of the separated subpopulations was performed by local xenogeneic graft-VS-host reaction, study of distribution of nonspecific esterase in cells and determination of relative amount of Fc gamma-R positive lymphocytes among thphs-1 and thphr-1. It was established that lymphocytes of patients with NUC had an alternative form of action on development of xenogeneic graft-VS-host reaction in contrast with cells of donors, that is inhibited the reaction. Among thphr-1 patients with NUC the amount of EC gamma-R cells and cells with diffuse distribution of enzyme with small degree activity was positively increased.     

Alveolar macrophage function in rats with severe protein calorie malnutrition. Arachidonic acid metabolism, cytokine release, and antimicrobial activity

To investigate the effects of protein calorie malnutrition (PCM) on alveolar macrophage function, we measured antimicrobial activity, IL-1 and TNF production, and arachidonic acid metabolism in alveolar macrophages of infant rats with moderate and severe PCM. Groups of weanling male rats were fed a diet containing 0.8% protein (PCM) or 24% protein (control). A third group (pair fed) was fed limited amounts of the control diet that matched the mean daily dietary intake of the PCM group. After 4 wk on the diets, alveolar macrophages from all three groups functioned similarly with respect to surface adherence, phagocytosis and killing of Listeria monocytogenes, release of hydrogen peroxide and superoxide anion, and production of IL-1 and TNF. In contrast, Listeria-stimulated alveolar macrophages from the PCM group exhibited a marked shift in arachidonic acid metabolism, with impaired production of leukotriene B4 and enhanced release of thromboxane B2 and PGE2. The membrane arachidonic acid content and the uptake of [3H]arachidonate by alveolar macrophages did not differ among the three groups. The shift toward the cyclooxygenase pathway was not seen after 2 wk of dietary restriction and was reversed if PCM animals were fed the control diet for 1 wk. Thus, PCM does not affect the antimicrobial activity or cytokine production of alveolar macrophages, but causes alterations in arachidonic acid metabolism that may interfere with the modulatory functions of alveolar macrophages.     

Response to DNA-damaging agents in cultured cells from patients with X-linked duchenne muscular dystrophy phenotype: male DMD,female DMD,possible carriers

Using blood cultures the response to gamma () radiation was examined in a male DMD and his mother, in a female DMD and her mother and in a normal control. In a series of experiments chromosome aberrations were determined after 3 separate -irradiation dose levels: 0, 150, 300 rads. The DMD patients showed a response to ionising radiations different from control, in fact the percentage of aberrations was lower than the control. In this preliminary study a slight difference between normal and possible carriers was also found.     

Evaluation of acetylator phenotype,renal function and serum sulfadiazine levels in patients with paracoccidioidomycosis treated with cotrimazine (a combination of sulfadiazine and trimethoprim)

The authors evaluated the relationships among renal function, acetylator phenotype and serum sulfadiazine levels in 22 patients with paracoccidioidomycosis treated with 1 tablet of cotrimazine (a combination of 820 mg sulfadiazine and 180 mg trimethoprim) administered orally every 12 hours.Fifteen patients (68.18%) presented free sulfadiazine levels above 50 g/ml, 6(27.28%) presented serum levels above 40 g/ml, and 1(4.54%), levels lower than 40 g/ml, this being the patient in which treatment failed. The highest free sulfadiazine levels were obtained in slow acetylator patients with reduced renal function. One patient with neuroparacoccidioidomycosis presented free sulfadiazine levels in cerebrospinal fluid corresponding to 55% of the serum levels.Finally, the authors consider cotrimazine to be an important therapeutic alternative for neuroparacoccidioidomycosis and conclude that administration every 12 hours can provide therapeutic sulfadiazine levels. They also suggest that when the sulfadiazine-trimethoprim combination is used, the therapeutic levels of sulfadiazine should be above 40 g/ml.     

GLUT4 and UBC9 protein expression is reduced in muscle from type 2 diabetic patients with severe insulin resistance

Aims

Subgroups of patients with type 2 diabetes mellitus demand large insulin doses to maintain euglycemia. These patients are characterized by severe skeletal muscle insulin resistance and the underlying pathology remains unclear. The purpose of this study was to examine protein expression of the principal glucose transporter, GLUT4, and associated proteins in skeletal muscle from type 2 diabetic patients characterized by severe insulin resistance.

Methods

Seven type 2 diabetic patients with severe insulin resistance (mean insulin dose 195 IU/day) were compared with seven age matched type 2 diabetic patients who did not require insulin treatment, and with an age matched healthy control group. Protein expression of GLUT4 and associated proteins was assessed in muscle and fat biopsies using standard western blotting techniques.

Results

GLUT4 protein expression was significantly reduced by ∼30 pct in skeletal muscle tissue from severely insulin resistant type 2 diabetic subjects, compared with both healthy controls and type 2 diabetic subjects that did not require insulin treatment. In fat tissue, GLUT4 protein expression was reduced in both diabetic groups. In skeletal muscle, the reduced GLUT4 expression in severe insulin resistance was associated with decreased ubiquitin-conjugating enzyme 9 (UBC9) expression while expression of GLUT1, TBC1D1 and AS160 was not significantly different among type 2 diabetic patients and matched controls.

Conclusions

Type 2 diabetic patients with severe insulin resistance have reduced expression of GLUT4 in skeletal muscle compared to patients treated with oral antidiabetic drugs alone. GLUT4 protein levels may therefore play a role in the pathology behind type 2 diabetes mellitus among subgroups of patients, and this may explain the heterogeneous response to insulin treatment. This new finding contributes to the understanding of the underlying mechanisms for the development of extreme insulin resistance.