Gene expression profile suggesting immunological dysregulation in two Brazilian Bloom's syndrome cases.

BACKGROUND: Bloom syndrome (BS) is a rare autosomal recessive chromosome instability disorder. The main clinical manifestations are growth deficiency, telangiectasic facial erythema, immunodeficiency, and increased risk to develop neoplasias at early age. Cytogenetic test for sister chromatid exchanges (SCEs) is used as a diagnostic marker for BS. In addition, most patients also present mutations in the BLM gene, related to defects in the DNA repair mechanism. However, the molecular mechanism behind the pathogenicity of BS is still not completely understood. METHODS: We describe two patients confirmed with BS by SCE and molecular analysis. Also, we performed the gene expression profile by the RNA-seq methodology in mRNA transcripts for differential gene expression analysis using as a biological condition for comparison BS versus health controls. RESULTS: We detected 216 differentially expressed genes related to immunological pathways such as positive regulation and activation of B cells, immune effector process and absence of difference of DNA repair genes expression. In addition; we also observed differentially expressed genes associated with apoptosis control, such as BCL2L1, CASP7, CDKN1A, E2F2, ITPR, CD274, TNFAIP6, TNFRSF25, TNFRSF13C, and TNFRSF17. CONCLUSION: Our results suggest that the combination of altered expression of genes involved in signaling pathways of immune response and apoptosis control may contribute directly to the main characteristics observed in BS, such as recurrent infections, growth failure, and high risk of cancer. Transcriptome studies of other instability syndromes could allow a more accurate analysis of the relevant gene interactions associated with the destabilization of the genome. This is a first description of the profile of differential gene expression related to immunological aspects detected in patients with BS by RNA-seq.

Genomic instability resulting from Blm deficiency compromises development, maintenance, and function of the B cell lineage.

The RecQ family helicase BLM is critically involved in the maintenance of genomic stability, and BLM mutation causes the heritable disorder Bloom's syndrome. Affected individuals suffer from a predisposition to a multitude of cancer types and an ill-defined immunodeficiency involving low serum Ab titers. To investigate its role in B cell biology, we inactivated murine Blm specifically in B lymphocytes in vivo. Numbers of developing B lymphoid cells in the bone marrow and mature B cells in the periphery were drastically reduced upon Blm inactivation. Of the major peripheral B cell subsets, B1a cells were most prominently affected. In the sera of Blm-deficient naive mice, concentrations of all Ig isotypes were low, particularly IgG3. Specific IgG Ab responses upon immunization were poor and mutant B cells exhibited a generally reduced Ab class switch capacity in vitro. We did not find evidence for a crucial role of Blm in the mechanism of class switch recombination. However, a modest shift toward microhomology-mediated switch junction formation was observed in Blm-deficient B cells. Finally, a cohort of p53-deficient, conditional Blm knockout mice revealed an increased propensity for B cell lymphoma development. Impaired cell cycle progression and survival as well as high rates of chromosomal structural abnormalities in mutant B cell blasts were identified as the basis for the observed effects. Collectively, our data highlight the importance of BLM-dependent genome surveillance for B cell immunity by ensuring proper development and function of the various B cell subsets while counteracting lymphomagenesis.

Differential expression of Werner and Bloom syndrome genes in the peripheral blood of HIV-1 infected patients.

Human immunodeficiency virus (HIV)-induced immunodeficiency and immune-system aging share some analogies. Since Werner (WRN) and Bloom (BLM) helicases are crucial in cell repair and aging, their peripheral blood mononuclear cells (PBMC) mRNA levels were compared in HIV-1 infected patients and in normal donors. The mean levels of WRN mRNA were 3.7-fold higher in PBMCs from HIV-1 infected individuals in comparison to healthy donors, whereas BLM mRNA mean levels were slightly higher, although not significantly. WRN increase was positively correlated to CD4 and CD8 T-cell numbers, and also the percentage of naive T lymphocytes, and was observed also in T-cell subsets. Interestingly, a general trend toward increased WRN mRNA levels in individuals with lower viral load was observed, without association with patient age, time of seroconversion, and on/off antiretroviral therapy regimen. On the whole, this study shows that WRN and BLM are differentially modulated in HIV infection, as WRN--but not BLM--is significantly increased, suggesting that mechanisms different from defect or loss of helicase function, observed in WRN and BLM syndromes, may be at the basis of T-cell aging in HIV infection.

Evaluation of short stature, carbohydrate metabolism and other endocrinopathies in Bloom's syndrome.

AIMS: To obtain an understanding of the etiology of proportional dwarfism and endocrinopathies of Bloom's syndrome BS. METHODS: Admission for 5-day periods to an NIH-supported Clinical Research Center of a randomly selected population of persons with BS (n = 11; mean age 11.5 years, range 9 months to 28.5 years) for clinical and genetic history-taking, physical examination, and endocrinological, gastroenterological and immunological testing. RESULTS: An oral glucose tolerance test was performed in all participants. Impaired glucose tolerance was present in 4 individuals, insulin resistance was observed in 6 individuals, and previously unrecognized diabetes was found in 1. Growth hormone provocation was normal in the 10 individuals tested. Overnight frequent GH sampling was suggestive of neurosecretory dysfunction in 3. Compensated hypothyroidism was found in 2 participants. Lipid profile abnormalities were present in 5 of 10 individuals. Low immunoglobulin concentrations (IgG and/or IgM) were seen in all tested. Intestinal absorption by D-xylose and/or fecal fat measurement was normal in all individuals tested as well. CONCLUSION: Altered carbohydrate metabolism is very common in BS, and is present from childhood. BS dwarfism is not related to growth hormone deficiency or malabsorption. The basis for the growth restriction in BS remains to be elucidated.

Expression of Werner and Bloom syndrome genes is differentially regulated by in vitro HIV-1 infection of peripheral blood mononuclear cells.

In HIV infection, continuous immune activation leads to accelerated ageing of the adaptive immune system, similar to that observed in elderly people. We investigated the expression of WRN and BLM (genes involved in disorders characterized by premature ageing, genomic instability and cancer predisposition) in peripheral blood mononuclear cells (PBMC) activated in vitro with phytohaemagglutinin (PHA) and infected with different HIV-1 strains. The steady state levels of mRNA were analysed by reverse transcription-polymerase chain reaction (RT-PCR), and protein expression was assayed using immunocytochemistry and Western blot techniques. In uninfected PBMC, PHA stimulation induced an increase in BLM mRNA and protein expression, while WRN expression remained virtually unchanged. When PBMC were infected in vitro with a lymphotropic HIV-1 strain, the level of BLM mRNA showed a peak at 24 h of infection, followed by a decline to uninfected culture levels. A similar result failed to be seen using an R5-tropic HIV-1 strain. In accordance with mRNA expression, in HIV-infected cultures PBMC were stained more frequently and more intensely by a BLM-specific antibody as compared to uninfected cultures, staining peaking at 24. Conversely, WRN expression was not modulated by HIV-1. The proportion of cells showing BLM up-regulation, established by immunocytochemical staining, was much greater than the proportion of productively infected PBMC, as established by proviral DNA measurement. This result indicates that BLM up-regulation is probably a result of an indirect bystander cell effect. Activation of the BLM gene in infected PBMC suggests that premature ageing could be a further immunopathogenetic mechanism involved in HIV-induced immunodeficiency, and points to a possible new candidate target for innovative therapeutic intervention.

Why the lupus problem remains unsolved and I am a human geneticist.

A personal account is given: 1) of my early work with lupus erythematosus including the first observation of formation of the LE cell and the experimental production in animal renal glomeruli of hematoxyphil bodies, the pathognomonic lesion of lupus; and, 2) of how discontinuation of work on lupus--by decree--diverted my life in science away from immunology into another area of human genetics, namely cytogenetics, the discovery of genetically determined genomic instability and the choice of Bloom's syndrome as an investigational model for human cancer.

MNNG-transformed Bloom syndrome B-lymphoblastoids for the detection of Hodgkin's lymphoma-associated antigen in 2D Westerns.

Twenty-four hour MNNG-exposed Bloom syndrome (BS) B-lymphoblastoid cells with the potential to form single cell colonies in soft agar and nude mouse tumour (2/6 (33%) showed a simultaneous increase in the Ras-expressing cells (using monoclonal antibody to p21 transforming protein) from 20% (at 24 h) to 85% (on day 30). In contrast, there was an absence of Ras-positive cells in MNNG-exposed fresh lymphocytes (PBMCs) from a healthy subject and a presence of only 11-18% of Ras-positive cells in normal (GA3) and unexposed BS B-lymphoblastoid cells. The Western blot analysis using sera samples from Hodgkin's lymphoma patients showed the presence of proteins of 102 and 68 kDa which in 2D Westerns were observed to be unique to BS-MNNG cells with approximate pIs of 5.3 and 5.7, respectively. It is proposed that BS-MNNG cells provide an interesting in vitro human cell model to generate unique cancer-associated antigen(s) in addition to using this system to understand the primary events associated with neoplastic transformation.

Diagnostic relevance of abortion-associated human embryonic antigen expressed on the cell surface of tumour promoter-treated Bloom syndrome cells.

We detected stable expression of human embryonic antigen associated with spontaneous abortion (HEAA) on the cell surface of a tumour promoter-treated B lymphoblastoid cell line (BS-SHY) originating from Bloom syndrome. We used indirect immunofluorescence and diluted serum from 44 patients who had recurrent spontaneous abortions. With the use of the panning procedure, we separated characteristic cells expressing strong HEAA. The BS-SHY-HEAA cells separated here would be useful for measuring serum antibody (against HEAA) produced by patients with recurrent abortions. It was also noted that aborters who received husbands' leukocyte immunization have lost this antibody, and have delivered successful pregnancies at term. Using HEAA proteins, we conducted Western blotting analysis for the amino acid sequencing (mol. wt 77 kDa). Amino acid sequencing data indicated that HEAA had 87.5% homology to the immunoglobulin (Ig) VHIII region in the framework. Recently, the protective value of high dose i.v. administration of immunoglobulin in the treatment of recurrent spontaneous abortions has been reported to be similar to that of leukocyte immunization. Therefore, the BS-SHY-HEAA cells appear to provide a valuable tool for rapid serological diagnosis and for evaluating the efficacy of immunotherapy with husbands' leukocytes in patients with recurrent spontaneous abortions.

Responses of lymphocytes to Epstein-Barr virus in patients with primary immunodeficiencies.

Several reports have demonstrated that the responses of B-cells to Epstein-Barr virus (EBV) are variable in common variable immunodeficiency (CVID). In this study in patients with selected primary immunodeficiencies, i.e., Bloom's syndrome, Wiskott-Aldrich syndrome or IgA deficiency, the responses of peripheral blood mononuclear cells (PBMCs) to EBV were investigated. In the two patients with Bloom's syndrome, PBMCs stimulated with EBV showed decreased proliferation and immunoglobulin production, suggesting a mild abnormality of B-cells. In patients with Wiskott-Aldrich syndrome, the responses were variable. In the patient with IgA deficiency, PBMCs responded normally to EBV in proliferation, whereas PBMCs responded poorly to EBV in IgA production, suggesting an abnormality only in the IgA production mechanism.

A wild-type mu s C-terminal gene is expressed in Bloom's syndrome cells.

Selective IgM deficiency is found commonly in patients with Bloom's syndrome (BS). Serum IgM concentrations were low though serum IgG and IgA concentrations were normal in both patients with BS included in the study. In a previous study the authors showed that selective IgM deficiency in BS is due to an abnormality in the maturation of surface IgM-bearing cells into IgM-secreting cells and a failure of secreted mu (mu s) mRNA synthesis. The membrane-bound mu (mu m) and mu s mRNA are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of mu s mRNA synthesis depends on the addition of poly(A) to mu s C-terminal segment. The study described here demonstrated that there was no mutation or deletion in the sequence including mu s C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of mu s C-terminal segment, and the AATAAA poly(A) signal sequence, and second GT-rich element immediately down-stream of the cleavage site in both patients.

Normal V(D)J coding junction formation in DNA ligase I deficiency syndromes.

Bloom syndrome and a clinically related syndrome represented by the cell line 46BR have been associated with reduction in DNA ligase I activity. In these syndromes, DNA ligase I deficiency severely impairs the development and function of the immune system. We undertook analysis of DNA ligase I-deficient cells to determine whether the observed immune deficiency is attributable to a perturbation in the process of V(D)J recombination. V(D)J recombination in Bloom syndrome cell lines and 46BR was examined by a transient transfection assay. No effect on the fidelity of coding and signal junction formation in DNA ligase I-deficient cells was observed. The frequency of V(D)J recombination in DNA ligase I-deficient cells was also examined using recombination substrates modified to function in human cells. Similar recombination frequencies were observed in normal and DNA ligase I-deficient cells, demonstrating that the efficiency of the V(D)J recombination process is unaffected by alterations in DNA ligase I activity. Rearranged immunoglobulin loci from Bloom syndrome cell lines and patient material were molecularly cloned by an inverse polymerase chain reaction strategy which should be applicable to a variety of human immunodeficiency syndromes and were indistinguishable from those found in normal bone marrow samples. Our data argue that the immune system defects associated with DNA ligase I deficiency do not result from perturbation of the V(D)J recombination pathway.

V(D)J recombination in ataxia telangiectasia, Bloom's syndrome, and a DNA ligase I-associated immunodeficiency disorder.

Ataxia telangiectasia (AT) and Bloom's syndrome (BS) patients are characterized by sensitivity to radiation, increased lymphoid malignancy, and frequent translocations to the antigen receptor loci. Because of these features, there has been a persistent question as to whether the V(D)J recombinase might be abnormal in cells from these patients. Such abnormalities might be due to inappropriate to inaccurate expression of components of the V(D)J recombinase or due to mutation in a component shared between V(D)J recombination and other cellular processes, such as DNA repair. Bloom's syndrome is associated with a ligation deficiency, and this activity may contribute in the end resolution steps of both site-specific and general DNA-processing reactions. In the current study, we have activated V(D)J recombination in normal, AT, and BS fibroblasts and in fibroblasts from a patient with mutations that largely abolish DNA ligase I activity. We find that the signal and coding joint formation of the V(D)J recombination reaction are entirely normal in AT, BS, and DNA ligase I mutant cells. In addition to ruling out abnormalities of the V(D)J recombinase in AT, BS, and DNA ligase I mutant cells, these studies suggest that DNA ligase I is unlikely to be required for signal or coding end joining in the V(D)J recombination reaction.

Preferential damage to IgM production by ultraviolet B in the cells of patients with Bloom's syndrome.

In most patients with Bloom's syndrome (BS), selective IgM deficiency is commonly found. We examined proliferative responses by incorporation of [3H]-thymidine and the production of immunoglobulin after ultraviolet B (UVB) irradiation in the cells of two patients with BS. With regard to the proliferative responses in peripheral blood mononuclear cells (PBMC) cultured with pokeweed mitogen (PWM), the patients' PBMC were more sensitive to UVB irradiation than controls. Although the effect of UVB irradiation in lymphoblastoid cell lines (LCL) after 0 days of culture showed no difference between one patient and controls, the patient's LCL were more sensitive to UVB than the controls after 3 and 7 days of culture. These results suggest that the proliferative responses of the patient's LCL recovered later than those of controls. IgM production was the most sensitive to UVB in the patients' PBMC and LCL. IgG and IgA production in the patients' PBMC and LCL showed the same sensitivity as controls. From our results, it is suspected that the preferential damage to IgM production by UVB is connected with the selective IgM deficiency of BS.

Characteristic mucinous ovarian cancer antigen is expressed in malignantly transformed Bloom's syndrome cells.

Using a double-antibody panning procedure, we separated a unique cancer antigen cell line (BS-SHI-4M OVC-MU) expressing a mucinous ovarian cancer (OVC) antigen from a malignantly transformed Bloom's syndrome cell line. In order to gain information concerning a mucinous OVC antigen, we tested this unique cell line in the reaction to sera from patients with various OVCs, Krukenberg (KR) tumor, and signet ring cell cancer of the stomach under immunofluorescence and Western blotting protocols and determined the mucinous OVC antigen band at M(r) 84,000. We also undertook an immune electron microscopic study to gain information concerning the antigen-antibody reaction [BS-SHI-4M OVC-MU cells-sera from patients with mucinous OVC and KR tumor] and concerning the antigenic determinant of the membrane using preembedding methods. Occasional protein A-gold particles were observed along the cell membrane of BS-SHI-4M OVC-MU cells, when treated with sera from mucinous OVC and KR tumor patients, but no labeling was observed in the cell membrane when treated with sera from normal patients and those with other cancers. Results of the immune electron microscopic study strongly support the data from the antigen-antibody reaction obtained by immunofluorescence and Western blotting analyses. The BS-SHI-4M OVC-MU cells separated here would be useful for serodiagnosis of mucinous OVC and KR tumors and for follow-up of patients after therapy.

Enhanced expression of stomach cancer antigen derived from malignantly transformed bloom syndrome cells previously labeled with bromodeoxyuridine.

Bromodeoxyuridine (BrdU) greatly enhanced expression of stomach (ST) cancer antigen (CA) that originated from a malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M), although the expression was suppressed with a decrease in sister chromatid exchange (SCE) in the presence of deoxythymidine (dT) or deoxycytidine (dC) and enhanced with an increase in SCE with deoxyguanosine (dG) or deoxyadenosine (dA). Although the exact mechanisms for enhancing CA by BrdU treatment are unknown, these findings appeared to be of special interest because of the parallelism of CA expression and SCE alterations. The finding that BrdU enhancement of the ST CA was effective not only in the immunofluorescence (IF) protocol but also in the band appearance of Western blotting would be worthwhile as a sensitive serodiagnosis of cancer. The 118-kd band obtained from proteins of ST CA cells previously labeled with BrdU was clearly more darkly stained than that from nonlabeled cells and enabled eight weak-positive ST CA to show strong-positive levels retaining complete negativity to nonmalignant sera. Some ST cancer sera (advanced cancer), which originally gave a negative reaction in the nonlabeled condition, still inhibited negative reaction even in BrdU-labeled ST CA cells, however. The inability to detect cancer antibody in our assay might be due to immunocomplexes. Acid dissociation and ultrafiltration of sera from six of seven advanced ST cancers (originally IF negative) have allowed detection of antibody responses to ST CA by Western blot assay with enhanced reactivity as compared with the negativity under native serum conditions. This technique provides a reasonable avenue for study of the mechanisms of CA expression and serodiagnosis.

Reduced secreted mu mRNA synthesis in selective IgM deficiency of Bloom's syndrome.

Serum IgM concentrations were low although serum IgG and IgA concentrations were normal in both our patients with Bloom's syndrome. Although the percentages of surface IgM-bearing cells were not reduced, the numbers of IgM-secreting cells were markedly reduced. The membrane-bound mu (microns) and secreted mu (microseconds) mRNAs are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of microseconds mRNA synthesis depends on the addition of poly(A) to microseconds C-terminal segment. In both patients, mu mRNA was well detected but microseconds C-terminal mRNA was scarcely detected, suggesting that microns mRNA was well transcribed but microseconds mRNA was not. There was, at least, no mutation or deletion in the microseconds C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of microseconds C-terminal segment and the AATAAA poly(A) signal sequence in both patients. Our results suggest that selective IgM deficiency in Bloom's syndrome is due to an abnormality in the maturation of surface IgM-bearing B cells into IgM-secreting cells and a failure of microseconds mRNA synthesis. Moreover, reduced microseconds mRNA synthesis may be due to the defect on developmental regulation of the site at which poly(A) is added to transcripts of the mu gene.

Evidence for abnormally regulated alternative RNA processing of mu chain gene in B-lymphoblastoid cells from Bloom's syndrome.

Selective IgM deficiency is commonly found in patients with Bloom's syndrome. In this study, mu mRNA synthesis was investigated in B-lymphoblastoid cells transformed by Epstein-Barr virus (LCL) from a patient with Bloom's syndrome who showed selective IgM deficiency. LCL established from the patient with Bloom's syndrome well expressed IgM molecules in their surface, but scarcely produced secreted IgM, compared with healthy controls. The JH hybridization patterns of digested DNA of LCL from the patient with Bloom's syndrome showed the rearrangement of VDJ as well as those of control LCLs. The mu mRNA was well detected, but mu s C-terminal mRNA was poorly detected compared with control LCLs, indicating that secreted mu mRNA was poorly transcribed though membrane-bound mu mRNA was well transcribed. These results suggest that alternative RNA processing of mu chain gene is abnormally regulated in LCL from patients with Bloom's syndrome.

Long-term study of the immunodeficiency of Bloom's syndrome.

The immune state was evaluated over a 10-year period in two individuals with Bloom's syndrome. In both patients, serum concentrations of IgM were markedly low. Mildly decreased serum concentrations of IgG and IgA increased significantly with age, whereas the IgM levels remained low. From assessments of B-cell and T-cell functions in pokeweed mitogen-induced immunoglobulin production, the IgM deficiencies were thought to result from B-cell dysfunction. T-cell function appeared intact. Moreover, although the percentages of surface IgM-bearing cells were not reduced, the numbers of IgM-secreting cells were reduced. These findings suggest that the IgM deficiency is due to an abnormality in the maturation of surface IgM-bearing B cells into IgM-secreting cells.

Host cell reactivation of sunlamp-exposed adenovirus in fibroblasts from patients with Bloom's syndrome, ataxia telangiectasia, and Huntington's disease.

In this study, a sensitive host cell reactivation (HCR) technique was used to examine the repair capacity for DNA damaged by sunlamp exposure in fibroblast strains derived from 5 normal individuals and 8 patients representing three different diseases associated with DNA repair deficiencies. Adenovirus type 2 (Ad 2) was exposed to radiation from a GE 275 W sunlamp and subsequently used to infect fibroblast monolayers. At 48 hr after infection, cells were scored for the presence of viral structural antigens (Vag) using indirect immunofluorescent staining. Previous reports using this technique showed a substantial reduction in the HCR of sunlamp-exposed Ad 2 for infection of excision repair deficient fibroblasts from patients with xeroderma pigmentosum. In contrast, the HCR of Vag synthesis for sunlamp-exposed Ad 2 was in the normal range for the three ataxia telangiectasia, three Bloom's syndrome, and two Huntington's disease fibroblasts strains.