Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 20
Filter
1.
J Clin Immunol ; 43(8): 1974-1991, 2023 11.
Article in English | MEDLINE | ID: mdl-37620742

ABSTRACT

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare and life-threatening condition characterized by recurrent localized edema. We conducted a systematic screening of SERPING1 defects in a cohort of 207 Czech patients from 85 families with C1-INH-HAE. Our workflow involved a combined strategy of sequencing extended to UTR and deep intronic regions, advanced in silico prediction tools, and mRNA-based functional assays. This approach allowed us to detect a causal variant in all families except one and to identify a total of 56 different variants, including 5 novel variants that are likely to be causal. We further investigated the functional impact of two splicing variants, namely c.550 + 3A > C and c.686-7C > G using minigene assays and RT-PCR mRNA analysis. Notably, our cohort showed a considerably higher proportion of detected splicing variants compared to other central European populations and the LOVD database. Moreover, our findings revealed a significant association between HAE type 1 missense variants and a delayed HAE onset when compared to null variants. We also observed a significant correlation between the presence of the SERPING1 variant c.-21 T > C in the trans position to causal variants and the frequency of attacks per year, disease onset, as well as Clinical severity score. Overall, our study provides new insights into the genetic landscape of C1-INH-HAE in the Czech population, including the identification of novel variants and a better understanding of genotype-phenotype correlations. Our findings also highlight the importance of comprehensive screening strategies and functional analyses in improving the C1-INH-HAE diagnosis and management.


Subject(s)
Angioedemas, Hereditary , Complement C1 Inhibitor Protein , Humans , Complement C1 Inhibitor Protein/genetics , Angioedemas, Hereditary/diagnosis , Angioedemas, Hereditary/epidemiology , Angioedemas, Hereditary/genetics , Czech Republic/epidemiology , RNA Splicing , RNA, Messenger
2.
J Clin Immunol ; 40(3): 435-446, 2020 04.
Article in English | MEDLINE | ID: mdl-31982983

ABSTRACT

PURPOSE: Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. RESULTS: Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. CONCLUSIONS: In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.


Subject(s)
Complement C1 Inhibitor Protein/genetics , DNA Mutational Analysis/methods , Exons/genetics , Hereditary Angioedema Types I and II/genetics , Introns/genetics , Mutation/genetics , RNA Splice Sites/genetics , Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Pedigree , Protein Splicing/genetics , Young Adult
4.
Oncol Rep ; 38(4): 2535-2542, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28791403

ABSTRACT

Mutations and deletions of the tumor suppressor TP53 gene are the most frequent genetic alterations detected in human tumors, though they are rather less frequent in lymphomas. However, acquisition of the TP53 mutation was demonstrated to be one of the characteristic markers in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) and prognostic value of the TP53 status has been recognized for these diseases. We present the complex analysis of the TP53 aberrations in 57 cases of MCL and 131 cases of DLBCL. The TP53 status was determined by functional analyses in yeast (FASAY) followed by cDNA and gDNA sequencing. The level of the p53 protein was assessed by immunoblotting and loss of the TP53-specific locus 17p13.3 was detected by FISH. Altogether, we detected 13 TP53 mutations among MCL cases (22.8%) and 29 TP53 mutations in 26 from 131 DLBCL cases (19.8%). The ratio of missense TP53 mutations was 76.9% in MCL and 82.8% in DLBCL. The frequency of TP53 locus deletion was rather low in both diseases, reaching 9.3% in MCL and 15.3% in DLBCL. The presence of TP53 mutation was associated with shorter overall survival (OS) and progression-free survival (PFS) in MCL. Among DLBCL cases, the TP53 mutations shortened both OS and PFS of patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) and decreased both OS and PFS of patients with secondary DLBCL disease.


Subject(s)
Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Mantle-Cell/drug therapy , Prognosis , Tumor Suppressor Protein p53/genetics , Adult , Aged , Aged, 80 and over , Cyclophosphamide/administration & dosage , Disease-Free Survival , Doxorubicin/administration & dosage , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Kaplan-Meier Estimate , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/pathology , Male , Middle Aged , Mutation , Prednisone/administration & dosage , Rituximab/administration & dosage , Vincristine/administration & dosage , Yeasts/genetics
5.
Clin Immunol ; 180: 33-44, 2017 07.
Article in English | MEDLINE | ID: mdl-28359783

ABSTRACT

Both variants affecting splice sites and those in splicing regulatory elements (SREs) can impair pre-mRNA splicing, eventually leading to severe diseases. Despite the availability of many prediction tools, prognosis of splicing affection is not trivial, especially when SREs are involved. Here, we present data on 92 in silico-/55 minigene-analysed variants detected in genes responsible for the primary immunodeficiencies development (namely BTK, CD40LG, IL2RG, SERPING1, STAT3, and WAS). Of 20 splicing-affecting variants, 16 affected splice site while 4 disrupted potential SRE. The presence or absence of splicing defects was confirmed in 30 of 32 blood-derived patients' RNAs. Testing prediction tools performance, splice site disruptions and creations were reliably predicted in contrast to SRE-affecting variants for which just ESRseq, ΔHZEI-scores and EX-SKIP predictions showed promising results. Next, we found an interesting pattern in cryptic splice site predictions. These results might help PID-diagnosticians and geneticists cope with potential splicing-affecting variants.


Subject(s)
Immunologic Deficiency Syndromes/genetics , RNA Splicing , Agammaglobulinaemia Tyrosine Kinase , Child , Child, Preschool , Complement C1 Inactivator Proteins/genetics , Complement C1 Inhibitor Protein , Exons , HeLa Cells , Hep G2 Cells , Humans , Infant , Interleukin Receptor Common gamma Subunit/genetics , Mutation , Protein-Tyrosine Kinases/genetics , RNA, Messenger/genetics , Recombinant Fusion Proteins/genetics , STAT3 Transcription Factor/genetics , U937 Cells , Wiskott-Aldrich Syndrome Protein/genetics
6.
J Clin Immunol ; 36(6): 610-8, 2016 08.
Article in English | MEDLINE | ID: mdl-27222152

ABSTRACT

INTRODUCTION: Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox), NCF1 (p47-phox), CYBA (p22-phox), NCF2 (p67-phox), or NCF4 (p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming. AIM: The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients. MATERIALS AND METHODS: Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis. RESULTS: The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %). CONCLUSION: In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.


Subject(s)
Granulomatous Disease, Chronic/diagnosis , Biomarkers , Child , Child, Preschool , Egypt , Female , Flow Cytometry , Genotype , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/immunology , Granulomatous Disease, Chronic/metabolism , Humans , Immunophenotyping , Infant , Male , Mutation , NADP/metabolism , NADPH Oxidases/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Risk Factors
7.
Oncol Rep ; 35(3): 1859-67, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26718964

ABSTRACT

Lung cancer is the leading cause of cancer-related deaths worldwide. The p53 tumor suppressor is a transcription factor controlling expression of its target genes in response to various stress stimuli. Mutations of the TP53 gene occur very frequently in lung carcinomas and they play an important role in both oncogenic transformation of lung epithelial cells and lung carcinoma progression. We determined the TP53 status in 42 samples of squamous cell lung carcinoma (SQCC) and 56 samples of lung adenocarcinoma (AC) by the functional analysis FASAY and its variant called split assay. Altogether, we detected 64 TP53 mutations in 63 patients and analyzed them by cDNA and gDNA sequencing. The TP53 mutations were found in 76.2% (32/42) of SQCC cases, and 55.4% (31/56) of ACs. Immunoblotting revealed the p53 protein accumulation in 18 samples (42.9%) among SQCC cases and 19 samples (33.9%) among AC cases. Using fluorescence in situ hybridization we detected loss of the TP53-specific 17p13.3 locus in 23 from 41 analyzed SQCC samples (56.1%) and in 20 from 54 analyzed AC samples (37.0%). We did not find any statistically significant differences in overall and disease-free survival in relation to TP53 status.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Squamous Cell/genetics , Lung Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Adult , Aged , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/pathology , Cell Transformation, Neoplastic/genetics , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , In Situ Hybridization, Fluorescence , Lung Neoplasms/pathology , Male , Middle Aged , Mutation , Prognosis
9.
PLoS One ; 9(2): e89570, 2014.
Article in English | MEDLINE | ID: mdl-24586880

ABSTRACT

Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.


Subject(s)
Point Mutation , Protein-Tyrosine Kinases/genetics , RNA Splice Sites , Software , Agammaglobulinaemia Tyrosine Kinase , Agammaglobulinemia/genetics , Computer Simulation , Exons , Genetic Diseases, X-Linked/genetics , HeLa Cells , Humans , Models, Genetic , Molecular Sequence Data , RNA Splicing , Sequence Analysis, DNA
10.
J Clin Immunol ; 34(2): 142-5, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24477949

ABSTRACT

Bruton's agammaglobulinemia is a rare X-linked humoral immunodeficiency manifesting with recurrent bacterial infections early in life. Klinefelter's syndrome caused by an additional X chromosome is the most common sex chromosome disorder. A previously unreported association of these two conditions is described here.


Subject(s)
Agammaglobulinemia/complications , Genetic Diseases, X-Linked/complications , Klinefelter Syndrome/complications , Abnormal Karyotype , Agammaglobulinaemia Tyrosine Kinase , Agammaglobulinemia/diagnosis , Agammaglobulinemia/drug therapy , Child , Chromosome Banding , DNA Mutational Analysis , Genetic Diseases, X-Linked/diagnosis , Genetic Diseases, X-Linked/drug therapy , Humans , Immunoglobulins, Intravenous , Klinefelter Syndrome/diagnosis , Mutation, Missense , Protein-Tyrosine Kinases/genetics , Treatment Outcome
11.
Pathol Oncol Res ; 19(3): 421-8, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23536279

ABSTRACT

Glioblastoma is the most common and the most aggressive type of brain cancer. Aberrations of the RTK/RAS/PI3K-, p53-, and RB cell signaling pathways were recognized as a core requirement for pathogenesis of glioblastoma. The p53 tumor suppressor functions as a transcription factor transactivating expression of its target genes in response to various stress stimuli. We determined the p53 status in 36 samples of glioblastoma by functional analyses FASAY and split assay. Seventeen p53 mutations were detected and further analyzed by cDNA and gDNA sequencing in 17 patients (47.2 %). Fifteen (88.2 %) of the mutations were missense mutations causing amino acid substitutions, seven of them exhibited temperature-sensitivity. Two mutations were determined as short deletions, one of them causing formation of premature termination codon in position 247. Fluorescent in situ hybridization revealed the loss of the p53-specific 17p13.3 locus in four of 33 analyzed samples (12 %). In 12 out of 30 samples (40 %), the p53 protein accumulation was shown by immunoblotting. There was high (80 %) concordance between the presence of the clonal p53 mutation and the p53 protein accumulation.


Subject(s)
Brain Neoplasms/genetics , Genes, p53 , Glioblastoma/genetics , Mutation , Aged , Brain Neoplasms/metabolism , Cell Line, Tumor , DNA Mutational Analysis , Female , Gene Deletion , Glioblastoma/metabolism , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Organ Specificity , Sequence Analysis, DNA , Temperature , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Yeasts
12.
Atherosclerosis ; 223(2): 401-8, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22698793

ABSTRACT

BACKGROUND: Familial hypercholesterolemia (FH), a major risk for coronary heart disease, is predominantly associated with mutations in the genes encoding the low-density lipoprotein receptor (LDLR) and its ligand apolipoprotein B (APOB). RESULTS: In this study, we characterize the spectrum of mutations causing FH in 2239 Czech probands suspected to have FH. In this set, we found 265 patients (11.8%) with the APOB mutation p.(Arg3527Gln) and 535 patients (23.9%) with a LDLR mutation. In 535 probands carrying the LDLR mutation, 127 unique allelic variants were detected: 70.1% of these variants were DNA substitutions, 16.5% small DNA rearrangements, and 13.4% large DNA rearrangements. Fifty five variants were novel, not described in other FH populations. For lipid profile analyses, FH probands were divided into groups [patients with the LDLR mutation (LDLR+), with the APOB mutation (APOB+), and without a detected mutation (LDLR-/APOB-)], and each group into subgroups according to gender. The statistical analysis of lipid profiles was performed in 1722 probands adjusted for age in which biochemical data were obtained without FH treatment (480 LDLR+ patients, 222 APOB+ patients, and 1020 LDLR-/APOB- patients). Significant gradients in i) total cholesterol (LDLR+ patients > APOB+ patients = LDLR-/APOB- patients) ii) LDL cholesterol (LDLR+ patients > APOB+ patients = LDLR-/APOB- patients in men and LDLR+patients > APOB+ patients >LDLR-/APOB- patients in women), iii) triglycerides (LDLR-/APOB- patients > LDLR+ patients > APOB+ patients), and iv) HDL cholesterol (APOB+ patients > LDLR-/APOB- patients = LDLR+ patients) were shown. CONCLUSION: Our study presents a large set of Czech patients with FH diagnosis in which DNA diagnostics was performed and which allowed statistical analysis of clinical and biochemical data.


Subject(s)
Hyperlipoproteinemia Type II/genetics , Mutation , Receptors, LDL/genetics , Adolescent , Adult , Apolipoproteins B/genetics , Biomarkers/blood , Chi-Square Distribution , Child , Czech Republic/epidemiology , DNA Mutational Analysis , Female , Gene Frequency , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Hyperlipoproteinemia Type II/blood , Hyperlipoproteinemia Type II/diagnosis , Hyperlipoproteinemia Type II/epidemiology , Linear Models , Lipids/blood , Male , Middle Aged , Phenotype , Risk Assessment , Risk Factors , Young Adult
13.
Int J Oncol ; 41(3): 1157-63, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22710932

ABSTRACT

The p53 protein is a sequence-specific transcription factor controlling the expression of multiple genes and protecting cells from oncogenic transformation. In many tumors, the p53 protein is completely or partially inactivated by mutations in the p53 gene. We analyzed the transactivating activity of nine human temperature-dependent (td) p53 mutants in yeast cells. Mutations in seven of them were localized in the ß-sandwich-coding region of the p53 gene, eight p53 mutants were temperature-sensitive and the R283C mutant was cold-sensitive. Patterns of their transactivation abilities towards three different responsive elements, the extent of their temperature dependency as well as discriminativity, were considerably variable. Similarly, their capacity to become reactivated by amifostine varied from complete resistance to high sensitivity. Transactivation abilities and temperature dependency of six p53 td mutants were determined in transiently-transfected H1299 human cells and revealed substantial concordance between the activity patterns of the p53 mutants in yeast and human cells. We concluded that the td p53 mutants do not comprise a uniform group, therefore, the behavior of each mutant has to be tested individually.


Subject(s)
Genes, p53 , Transcriptional Activation , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Yeasts/genetics , Amifostine/pharmacology , Humans , Mutation , Radiation-Protective Agents/pharmacology , Temperature , Transformation, Genetic
14.
Pediatrics ; 129(2): e523-8, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22271700

ABSTRACT

SH2D1A gene defects are the cause of X-linked lymphoproliferative disorder (XLP-1), a rare condition characterized by severe immune dysregulation. We present a patient lacking the typical symptoms of XLP-1, but experiencing a severe unusual skin condition encompassing features of dermatosclerosis and vesiculobullous skin disease. A maternal cousin of the patient was diagnosed with XLP-1 and found to carry a deletion of the SH2D1A gene. SH2D1A deletion was also identified in our patient, which offered a possible explanation for his skin symptoms. Subsequent analysis showed that the deletion in both cousins was identical and involved the whole SH2D1A gene and a part of the adjacent ODZ1 gene. High phenotypic variability of XLP-1 observed in this family prompted us to analyze the genotype-phenotype correlation of 2 different-sized deletions involving SH2D1A and ODZ1 in 5 patients from 2 families, and we report the clinical and laboratory data on these individuals. Our findings illustrate the wide clinical variability of XLP-1, both inter- and intrafamilial, which may complicate the diagnosis of this condition. The comparison of phenotypes of our patients argues against a strong involvement of the ODZ1 gene in the skin disorder and other symptoms observed in our index patient. His hitherto not described severe skin condition extends the phenotypic range of XLP-1.


Subject(s)
Chromosome Deletion , Chromosomes, Human, X/genetics , Intracellular Signaling Peptides and Proteins/genetics , Lymphoproliferative Disorders/genetics , Scleroderma, Localized/genetics , Skin Diseases, Vesiculobullous/genetics , Adolescent , Adult , Anemia, Aplastic/diagnosis , Anemia, Aplastic/genetics , Anemia, Aplastic/therapy , Child , Child, Preschool , Comorbidity , Exons/genetics , Histiocytic Sarcoma/diagnosis , Histiocytic Sarcoma/therapy , Humans , Infectious Mononucleosis/diagnosis , Infectious Mononucleosis/therapy , Longitudinal Studies , Lymphoproliferative Disorders/diagnosis , Lymphoproliferative Disorders/therapy , Male , Membrane Proteins/genetics , Nerve Tissue Proteins , Peripheral Blood Stem Cell Transplantation , Phenotype , Scleroderma, Localized/diagnosis , Signaling Lymphocytic Activation Molecule Associated Protein , Skin Diseases, Vesiculobullous/diagnosis , Skin Diseases, Vesiculobullous/therapy , Tenascin
15.
Clin Biochem ; 45(1-2): 96-100, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22119890

ABSTRACT

OBJECTIVES: The aim of this study was to determine whether the atherogenic index of plasma (AIP=log[triglycerides/HDL-cholesterol]) differs in heterozygous familial hypercholesterolemia (FH) patients with and without a history of cardiovascular disease (CVD). DESIGN AND METHODS: A total of 555 FH patients with known mutations in the LDL receptor or the apolipoprotein B gene, of whom 53 had a history of CVD (CVD+ group), were retrospectively analyzed. RESULTS: Compared to patients without CVD (CVD- group), CVD+ patients showed significantly higher fasting LDL-cholesterol, triglycerides and AIP as well as lower HDL-cholesterol. After both adjustment for age and diabetes and using analysis based on age and sex matched groups, only the increase in triglycerides and AIP in the CVD+ vs. the CVD- group remained significant. CONCLUSION: The results of the present study indicate that AIP, which reflects the presence of atherogenic small LDL and small HDL particles, may be connected to the risk of CVD in FH patients.


Subject(s)
Cardiovascular Diseases/blood , Cardiovascular Diseases/diagnosis , Cholesterol, HDL/blood , Hyperlipoproteinemia Type II/blood , Mutation , Triglycerides/blood , Adult , Age Factors , Atherosclerosis/diagnosis , Cohort Studies , Female , Heterozygote , Humans , Lipoproteins/chemistry , Male , Middle Aged , Particle Size , Retrospective Studies , Sex Factors
16.
Int J Oncol ; 39(6): 1413-20, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21874232

ABSTRACT

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma in adults. There are specific alterations that appear repeatedly in DLBCL cases and play a role in lymphomagenesis or progression of the disease. Some aberrations were used as prognostic markers in the pre-rituximab era. Addition of rituximab to the classical anthracycline-based chemotherapy significantly increased the survival rate in DLBCL. Only few prognostic factors have been re-evaluated for patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). We performed complex analysis of the p53 tumor suppressor in collection of 75 DLBCL cases. Fifty-four patients were de novo cases, twenty-one cases developed into DLBCL by transformation from less aggressive disease. We determined functional status by analysis of separated alleles in yeast (FASAY) and analyzed the p53 mutations by cDNA sequencing. We assessed the level of the p53 protein by immunoblot analysis. We used FISH to analyze loss of the p53 and ATM (ataxia telangiectasia mutated) gene deletions. We detected 16 p53 mutations (21.3%) including the mutation activating non-sense-mediated RNA decay pathway. Deletion of the p53 allele was more common in cases with p53 mutation. Mutations and/or deletions of p53 had statistically significant negative impact on progression-free survival and tended to decrease also overall survival in 46 de novo DLBCL patients treated with R-CHOP. p53 aberrations are negative predictors for survival of DLBCL patients treated with R-CHOP.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclophosphamide/therapeutic use , Doxorubicin/therapeutic use , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/mortality , Prednisone/therapeutic use , Tumor Suppressor Protein p53/genetics , Vincristine/therapeutic use , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Murine-Derived/administration & dosage , Base Sequence , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 17/genetics , Cyclophosphamide/administration & dosage , Doxorubicin/administration & dosage , Female , Gene Expression Regulation, Neoplastic , Genetic Loci , Humans , Kaplan-Meier Estimate , Lymphoma, Large B-Cell, Diffuse/diagnosis , Lymphoma, Large B-Cell, Diffuse/genetics , Male , Middle Aged , Mutation/genetics , Prednisone/administration & dosage , Prognosis , Rituximab , Treatment Outcome , Tumor Suppressor Protein p53/metabolism , Vincristine/administration & dosage , Young Adult
17.
Atherosclerosis ; 216(1): 139-45, 2011 May.
Article in English | MEDLINE | ID: mdl-21310417

ABSTRACT

OBJECTIVE: Familial hypercholesterolemia (FH) is an inborn disorder of lipid metabolism characterised by elevated plasma concentrations of low-density lipoprotein cholesterol and total cholesterol. This imbalance results in accelerated atherosclerosis and premature coronary heart disease. The early identification and treatment of FH patients is extremely important because it leads to significant reduction of both coronary morbidity and mortality. FH is transmitted in an autosomal dominant manner and associated predominantly with mutations in the genes encoding the low-density lipoprotein receptor (LDLR) and its ligand apolipoprotein B (APOB). To date, more than 1000 sequence variants have been described in the LDLR gene. In marked contrast to LDLR, only one APOB mutation is prevalent in Europe. METHODS AND RESULTS: The aim of this study was, on the basis of data obtained by the molecular genetic analysis of 1945 Czech FH probands, to propose, generate, and validate a new diagnostic tool, an APEX (Arrayed Primer EXtension)-based genotyping DNA microarray called the FH chip. The FH chip contains the APOB mutation p.Arg3527Gln, all 89 LDLR point mutations and small DNA rearrangements detected in Czech FH patients, and 78 mutations frequent in other European and Asian FH populations. The validation phase revealed the sensitivity and specificity of this platform, 100% and 99.1%, respectively. CONCLUSIONS: This FH chip is a rapid, reproducible, specific, and cost-effective tool for genotyping, and in combination with MLPA (multiple ligation-dependent probe amplification) represents a reliable molecular genetic protocol for the large-scale screening of FH mutations in the Czech population.


Subject(s)
Apolipoproteins B/genetics , DNA Mutational Analysis , Gene Expression Profiling/methods , Genetic Testing/methods , Hyperlipoproteinemia Type II/diagnosis , Mass Screening/methods , Mutation , Oligonucleotide Array Sequence Analysis , Receptors, LDL/genetics , Czech Republic/epidemiology , Genetic Predisposition to Disease , Humans , Hyperlipoproteinemia Type II/epidemiology , Hyperlipoproteinemia Type II/genetics , Predictive Value of Tests , Reproducibility of Results , Sensitivity and Specificity
18.
Int J Oncol ; 36(3): 699-706, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20126990

ABSTRACT

Mantle cell lymphoma (MCL) is typified by translocation t(11;14)(q13;q32) causing upregulation of cyclin D1 and deregulation of cell cycle. The cyclin D1 activation plays a critical role in MCL pathogenesis but additional oncogenic events, such as aberrations of the ARF/MDM2/p53 pathway are also necessary for progression of the disease. We analyzed the p53 tumor suppressor in tumor tissue of 33 patients with MCL. The p53 status was determined by functional analyses in yeast (FASAY) and by cDNA sequencing. The level of the p53 protein was assessed by immunohistochemistry and immunoblotting. Loss of the p53-specific locus 17p13.3 was detected by FISH. Mutations in the p53 gene were detected in nine samples and they included eight missense mutations and one short deletion causing frame shift and premature stop codon formation in position 169. This mutation was associated with mRNA decay as revealed by sequencing of the p53 gDNA. All eight missense mutations were manifested by accumulation of the p53 protein in nuclei of tumor cells and three of them exhibited loss of the p53-specific locus 17p13.3. The p53 mutations were shown to be a negative prognostic marker in MCL.


Subject(s)
Gene Expression Regulation, Neoplastic , Genes, p53 , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Cyclin D1/biosynthesis , Female , Humans , Male , Middle Aged , Mutation , Prognosis , Translocation, Genetic
19.
Oncol Rep ; 20(4): 773-8, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18813817

ABSTRACT

Burkitt's lymphomas (BL) are aggressive rapidly growing tumors typified by a high c-myc expression resulting from t(8;14)(q24;q32), t(2;8)(p12;q24) or t(8;22)(q24;q11) translocations. Alterations of the p53 tumor suppressor are also relatively frequent in BL. Several approaches have been adopted for detection of the p53 aberrations such as immunohistochemical analyses, immunoblotting, DNA sequencing, fluorescence in situ hybridization (FISH), and functional assays. We used these methods to characterize the p53 mutation in tumor cells of a 53-year-old male suffering from Burkitt's lymphoma. By immunohistochemical analyses, we detected high levels of the p53 protein in the tumor tissue. Immunoblotting showed a higher molecular weight of the p53 protein overexpressed in the tumor tissues than that of the standard p53 protein. Similarly, the molecular weight of the PCR product prepared by amplification of the tumor p53 cDNA was higher than that of the standard p53 cDNA. Functional analyses of separated alleles in yeast evidently revealed that the tumor p53 protein was transcriptionally non-functional. The yeast colonies expressing this p53 variant possessed a unique phenotype in that they were red with many white spots on their surface. Sequencing of the tumor cDNA revealed a duplication of the 30 bp region of the p53 gene (g.12155_12184dup30) leading to a repeat of 10 amino acids (Pro-77 to Ala-86) in the p53 protein. Further analyses showed that the mutation was unstable in yeast cells. The FISH analyses did not confer loss of the p53-specific locus 17p13.


Subject(s)
Burkitt Lymphoma/genetics , Gene Duplication , Genes, p53 , Mutation , DNA, Complementary/chemistry , Humans , In Situ Hybridization, Fluorescence , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/analysis
20.
Oncol Rep ; 14(4): 901-7, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16142349

ABSTRACT

p53 tumor suppressor is a sequence-specific DNA-binding protein that controls the expression of many genes in response to diverse stress stimuli. p53 gene is often mutated in human cancer and in cancer cell lines. Several methods are available for identification of p53 mutations, including functional analysis of separated alleles in yeast (FASAY). FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. We analyzed the p53 status of 26 human cell lines of different tissue origin using the functional assay in yeast. Wild-type p53 was found in six cell lines and various p53 aberrations in the remaining twenty. FASAY detected temperature-sensitive p53 mutations in breast cancer cell line, BT474, and leiomyosarcoma cell line, SK-LMS-1, and two independent p53 point mutations in SK-UT-1 and SK-LMS-1 leiomyosarcoma cell lines. In addition, the assay revealed that a recombination occurred between the two mutated p53 alleles producing a stable ratio of p53 wild-type alleles (11.7 or 2.7% respectively). In the case of acute myeloid leukemia cell line, ML-1, we detected both wild-type and heterozygous p53 status, depending on the source of the cell line. In Hs913T, HL60 and Saos-2 cell lines, FASAY failed to assess p53 status due to a large deletion/rearrangement of the p53 gene. In acute lymphoid leukemia HPB cell line, we disclosed unknown non-sense mutation in codon 124 of the p53 gene.


Subject(s)
Genes, p53/genetics , Genetic Techniques , Mutation , Tumor Suppressor Protein p53/physiology , Alleles , Cell Line, Tumor , Codon , Codon, Nonsense , DNA/metabolism , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/metabolism , HL-60 Cells , Heterozygote , Humans , Immunoblotting , Leukemia, Myeloid, Acute/metabolism , Plasmids/metabolism , Point Mutation , Saccharomyces cerevisiae/metabolism , Sequence Analysis, DNA , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL
...