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PubMed2001

Diagnostic strategy for analytical scanning of BRCA1 gene by fluorescence-assisted mismatch analysis using large, bifluorescently labeled amplicons.

The aim of this study was to develop a protocol for reliable, sensitive, and cost-effective mutation scanning of the BRCA1 gene, based on a modification of fluorescence-assisted mismatch analysis. The main features of this method are: (a) robust PCR amplification and strandspecific labeling of 25 large amplicons using uniform conditions and universal fluorescent primers; and (b) sensitive characterization of the position of sequence changes. The diagnostic accuracy of this method was tested by scanning the large exon 11 in 12 DNA samples with reported mutations. In a blind test, specific patterns of fluorescence profiles were obtained, and all were attributed correctly, without sequencing, to each mutation or polymorphism. Seven breast/ovarian cancer families with high probability of BRCA1-related predisposition were screened. Three truncating mutations (of which one was novel and three were missense changes, including two novel ones) were detected. The three missense mutations affect the highly conserved BRCT domain. Scanning by FAMA appears to be free of biases for particular types of sequence changes-except for exon deletions/duplications, which cannot be detected by conventional PCR-based methods-and allows substantial savings in the number of sequencing reactions and in the time invested in their interpretation. Therefore, it lends itself to screening structurally complex loci in the diagnostic context and in other fields of genetic analysis.

Base Pair Mismatch↗

PubMed2002

Biochemical characterization of the chondroitinase B active site.

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.

Animals↗

PubMed2002

Resistance profiles of cyclic and linear inhibitors of HIV-1 protease.

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

Drug Resistance, Viral↗

PubMed2004

Large-scale disruption of microtubule pathways in morphologically normal human spastin muscle.

OBJECTIVE: To investigate the molecular pathways disrupted by dominant spastin mutations in apparently unaffected skeletal muscle from patients with motor neuron disease (SPG4). METHODS: The authors studied muscle of three individuals from two unrelated families affected by spastic paraplegia caused by spastin mutations. The authors compared RNA expression profiles to 7 normal and 13 pathologic muscle U95A profiles (Duchenne dystrophy, acute quadriplegic myopathy, and spinal muscular atrophy). Data were validated with U133A arrays with seven different control specimens. mRNA and protein confirmations were done for a subset of genes. RESULTS: Both nonsense and missense mutations in the spastin gene disrupted microtubule pathways in nonpathologic tissue, including microtubule dynamics, stability, exocytosis, and endocytosis. CONCLUSIONS: Normal muscle can be used to uncover biochemical perturbation in motor neuron disease. Altered microtubule metabolism in SPG4-linked hereditary spastic paraplegia patients leads to pathology of the long descending tracks of motor neurons that likely have a stringent need for efficient microtubular transport. As many inherited neurologic conditions show a systemic biochemical defect with disease limited to neurons, our data have broader implications for biochemical pathway studies of many neurologic disorders.

Adenosine Triphosphatases↗

PubMed2000

Identification of specific BRCA1 and BRCA2 variants by DHPLC.

Denaturing high performance liquid chromatography (DHPLC) is generating increasing interest in clinical genetics as a reliable tool for the analysis of genetic alterations. In the work presented here our intentions were to optimize primer design and DHPLC analysis conditions for a qualitative detection of BRCA1 and BRCA2 variations. The BRCA1 and BRAC2 genes display a high proportion of polymorphisms. Sequencing efforts geared towards the distinction of tumor-related mutations and benign variants still remain time-consuming and expensive. DHPLC elution profiles, however, permit the correlation of a characteristic chromatographic profile with a specific sequence alteration. In this study we evaluate the sensitivity of DHPLC for the identification of unique polymorphisms, which are frequent in the Caucasian population, in lieu of sequence analysis. The complete BRCA1 gene and parts of BRCA2 were examined. In the case of BRCA1, 431 out of 432 heterozygotes were identified correctly. In addition, 18 new profiles were identified which had not been detected previously in our studies and which represented new mutations or rare polymorphisms. For BRCA2, 135 out of 137 simple sequence variants were classified correctly. In addition, six new profiles were identified which represented new mutations or rare polymorphisms.

BRCA2 Protein↗

PubMed1998

BRCA1 mutations and breast cancer in the general population: analyses in women before age 35 years and in women before age 45 years with first-degree family history.

CONTEXT: Studies of high-risk families with multiple early-onset cases of breast cancer have been useful for assessing the type and spectrum of germline mutations on the BRCA1 gene, but do not provide guidance to women with modest family history profiles. Thus, studies of women from the general population are needed to determine the BRCA1 mutation frequency in women perceived to be at high risk, and to develop profiles of those most likely to be carriers. OBJECTIVE: To characterize frequency and spectrum of germline BRCA1 mutations in 2 categories of women identified via population-based studies hypothesized to be at increased risk of carrying such mutations: those diagnosed as having breast cancer before age 35 years and those diagnosed before age 45 years who have first-degree breast cancer family history. DESIGN: Study subjects were drawn from 2 population-based case-control studies of breast cancer in young women on the basis of their family history or their age of diagnosis. Cases were younger than 35 years or were younger than 45 years with first-degree family history at the time of breast cancer diagnosis and were ascertained via a population-based cancer registry, and controls (women without breast cancer) were identified via random-digit dialing. SETTING: Three counties in western Washington State. MAIN OUTCOME MEASURE: BRCA1 germline mutations in study subjects identified in DNA from peripheral blood lymphocytes by single-strand conformation polymorphism analysis using primer pairs that span the BRCA1 coding region and intron-exon boundaries. RESULTS: Of 193 women diagnosed as having breast cancer before age 35 years, none of whom were selected on the basis of family history status, 12 (6.2%, 95% confidence interval [CI], 3.2%-10.6%) had germline BRCA1 mutations. In 208 women diagnosed before age 45 years who had first-degree breast cancer family history, 15 (7.2%, 95% CI,4.1%-11.6%) had germline mutations in BRCA1. In both groups, there were variations in mutation frequency noted by age and by family history. Mutation frequency decreased with increasing age of diagnosis. Higher proportions of mutations were seen in cases with at least 1 relative diagnosed as having breast cancer before age 45 years, in cases with greater numbers of affected relatives, and those with ovarian cancer family history. Mutation frequency did not vary by bilateral breast cancer family history. No frameshift or nonsense mutations were observed in 71 control women with a first-degree family history, although missense changes of unknown significance were seen in cases and controls. CONCLUSIONS: Women with BRCA1 germline mutations lacked a common family history profile. Also, a large proportion of the women with a first-degree breast cancer family history and women diagnosed as having breast cancer before age 35 years did not carry germline BRCA1 mutations. Hence, while early-onset disease and a strong breast cancer family history may be useful guidelines for checking BRCA1 status, these findings on women drawn from the general population suggest that it may be difficult to develop BRCA1 mutation screening criteria among women with modest family history profiles.

Adult↗

PubMed2004

Why hypocotyl extension mutants need to be characterized at the cell level: a case study of axr3-1.

Since the discovery of auxin, a debate has taken place as to whether the auxin distribution in elongating organs can account for the distinctive cell elongation profiles that have been found. In an attempt to address this important issue, the elongation profiles of cells have been compared in the hypocotyls of wild-type and auxin-hypersensitive axr3-1 Arabidopsis Columbia ecotype seedlings. Clear differences in cell elongation profiles were found in the two types of seedling, whether they were light- or dark-grown. However, it was not possible unambiguously to ascribe the cell elongation profile differences to the proposition that the axr3-1 mutation causes the hypocotyl to be hypersensitive to auxin. The possibility that the abnormal hypocotyl elongation profile of the mutant was a secondary effect, consequent on a more fundamental effect of the axr3-1 mutation, is considered. It is clear from this study that cell elongation and its control needs to be studied at the cell, and not the organ, level. To characterize a mutant as having a short, or long, hypocotyl is inadequate. To determine which factors control the timing and the magnitude of cell elongation requires the demonstration of correlations between the growth rate of cells and their content of regulating substances or their sensitivity to that substance. Studies of the cell elongation profiles of the many hypocotyl length mutants could also be a very effective means of probing the co-ordination of root and shoot elongation.

Arabidopsis↗

PubMed2002

The Ewing's sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in primary human fibroblasts.

Ewing's sarcoma is associated with a fusion between the EWS and FLI1 genes, forming an EWS/FLI fusion protein. We developed a system for the identification of cooperative mutations in this tumor through expression of EWS/FLI in primary human fibroblasts. Gene expression profiling demonstrated that this system recapitulates many features of Ewing's sarcoma. EWS/FLI-expressing cells underwent growth arrest, suggesting that growth arrest-abrogating collaborative mutations may be required for tumorigenesis. Expression profiling identified transcriptional upregulation of p53, and the growth arrest was rescued by inhibition of p53. These data support a role for p53 as a tumor suppressor in Ewing's sarcoma and demonstrate the use of transcriptional profiling of model systems in the identification of cooperating mutations in human cancer.

Anti-Bacterial Agents↗

PubMed2026

Whole genome and exome sequencing of pancreatic neuroendocrine tumour to investigate PRRT response.

Patients with pancreatic neuroendocrine tumours (PNETs) often have similar baseline clinical characteristics, including grade and molecular imaging phenotype, yet have highly variable responses to peptide receptor radionuclide therapy (PRRT). To identify genomic alterations and mutational patterns associated with PRRT treatment response and acquired somatic changes following PRRT exposure, whole genome or exome sequencing was applied to 40 PNET samples from 32 patients, including eight paired pre- or post-PRRT samples. The genomic profile of tumours reflected the known mutational landscape of PNET with MEN1 (34%), ATRX/DAXX (47%) alterations and a recurrent pattern of aneuploidy (38%) detected. A recurrent PSIP1::TBL1X fusion of unknown function was also identified in four tumours. The disease control rate following PRRT using RECIST1.1 and molecular imaging criteria was 88% (28/32). No mutational features were found to be statistically associated with progression-free survival. There was no significant increase in tumour mutational burden in the post-PRRT tumours, nor recurrent emergent mutational changes in cancer driver genes to explain progression to higher-grade disease, when observed. However, a small indel signature (ID8) previously associated with DNA damage repair by non-homologous end joining (NHEJ) was higher in PRRT-exposed compared with PRRT-naive samples (23.8 vs 4.8%, respectively; P < 0.001). Thus, comprehensive DNA analysis of pancreatic NETs did not identify biomarkers predictive of PRRT response nor evidence for high-level PRRT-induced genomic instability or hypermutation, yet mutation signature analysis supports NHEJ as being important for DNA repair and survival of neuroendocrine cells following exposure to beta-particle radiation.

Humans↗

PubMed2006

Prothrombin gene 20209 C >T along with the first description of a homozygous polymorphism at the 3' downstream region +4 C >T in the Turkish population.

Mutation screening studies related with thrombosis revealed that prothrombin 20210A allele of the prothrombin gene leads to an increase in plasma prothrombin levels. The G20210A mutation screening has become a routine analysis in patients with thrombosis. For the detection of the mutation, several molecular techniques have been defined. Real-time polymerase chain reaction is an easy and feasible way to monitor this mutation according to the melting point analysis. Here we report a different pattern in the LightCycler melting point analysis of 2 patients with thrombosis. DNA sequencing of the prothrombin gene showed that these patients with an atypical melting point profile carry a 20209 C >T mutation along with a novel homozygous polymorphism at the 3' downstream region, +4 C >T.

Adolescent↗

PubMed1999

The calcium-binding sites of heparinase I from Flavobacterium heparinum are essential for enzymatic activity.

In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R. (1999) J. Biol. Chem. 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif. In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More specifically, we identified critical residues (viz. Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.

Amino Acid Sequence↗

PubMed2003

Detection of cystic fibrosis mutations by peptide mass signature genotyping.

BACKGROUND: The diversity of genetic mutations and polymorphisms calls for the development of practical detection methods capable of assessing more than one patient/one nucleotide position per analysis. METHODS: We developed a new method, based on peptide mass signature genotyping (PMSG), for the detection of DNA mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Exons of the gene were amplified, cloned, and expressed in Escherichia coli as peptide fusions, in natural as well as unnatural reading frames. Peptide analytes were purified by immobilized metal affinity chromatography and analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry. Synthetic and natural DNA samples with the 25 mutations recommended for CFTR carrier screening (Grody et al. Genet Med 2001;3:149-54) were assessed using the PMSG test for the CFTR gene. RESULTS: Peptide analytes ranged from 6278 to 17 454 Da and varied 30-fold in expression; highly expressing peptides were observed by electron microscopy to accumulate as inclusion bodies. Peptides were reliably recovered from whole-cell lysates by a simple purification method. CFTR mutations caused detectable changes in resulting mass spectrometric profiles, which were >95% reliably detected in blinded testing of replicate synthetic heterozygous DNA samples. Mutation detection was possible with both sample pooling and multiplexing. The PMSG CFTR test was used to determine compound heterozygous mutations in DNA samples from cystic fibrosis patients, which were confirmed by direct DNA sequencing. CONCLUSIONS: The PMSG test of the CFTR gene demonstrates unique capabilities for determining the sequence status of a DNA target by sensitively monitoring the mass of peptides, natural or unnatural, generated from that target.

Cloning, Molecular↗

PubMed2005

Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation.

Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway.

Animals↗

PubMed2026

KRAS Expression Complements Genomic Profiling in Identifying Therapeutic Vulnerability in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) remains a major therapeutic challenge. Although alterations in the RAS pathway occur in over 50% of tumors, only a limited proportion are clinically actionable. We investigated whether KRAS expression complements genomic profiling for patient stratification and therapeutic vulnerability in GC. METHODS: Comprehensive genomic profiling was performed in 19 Taiwanese GC patients and compared with TCGA-STAD data (n = 434). KRAS mRNA expression and overall survival were evaluated by meta-analysis of 13 independent cohorts (n = 2,521). Protein-level validation was performed by immunohistochemistry in an independent cohort (n = 121). Functional KRAS dependency and response to combined MEK/SHP2 inhibition were assessed in eight GC cell lines. RESULTS: KRAS amplification was entirely contained within the KRAS-high population, whereas most KRAS-high tumors lacked detectable amplification. High KRAS expression was associated with poorer overall survival (HR 1.23, p = 0.001) and remained an independent prognostic factor after multivariable adjustment (adjusted HR 1.24, p = 0.003). Protein-level analysis showed a concordant trend. KRAS expression correlated strongly with functional dependency (R2 = 0.88, p = 0.005), was enriched in MSI and CIN subtypes, and identified cell lines with enhanced sensitivity to combined MEK/SHP2 inhibition. CONCLUSIONS: KRAS expression complements genomic profiling by identifying biologically relevant KRAS-dependent GCs beyond mutation or amplification alone. Integrating expression-based stratification with genomic profiling may improve patient selection for RAS pathway-directed combination therapies.

Biomarker↗

PubMed2005

The EGFR mutation and its correlation with response of gefitinib in previously treated Chinese patients with advanced non-small-cell lung cancer.

BACKGROUND: The aim of the study was to evaluate the efficacy of gefitinib and the epidermal growth factor receptor (EGFR) mutation to gefitinib response in a series of Chinese patients with pretreated advanced non-small-cell lung cancer (NSCLC). METHODS: A total of 98 patients who had failed at least one platinum-based regimen received gefitinib 250 mg once daily. The mutation analysis of the EGFR kinase domain was performed for 30 patients using paraffin-embedded tumor tissue. RESULTS: The response rate was 31.6% and the disease control rate was 67.3%. Objective response was correlated with adenocarcinoma, female gender and non-smokers. Median progress free survival (PFS) was 7.0 months, median overall survival (OS) was 12.0 months and 1-year survival was 53.1%. The median PFS and OS were improved among patients with adenocarcinoma, gefitinib responders and non-smokers. Active gene mutation was detected in 12 patients. Mutation rates were higher among gefitinib responders, non-smokers, patients with adenocarcinoma and female patients. OS was longer for patients with gene mutation than for patients without mutation. CONCLUSION: Gefitinib demonstrated significant antitumor activity with a favorable toxicity profile for pretreated Chinese patients with advanced NSCLC. The active mutation of the EGFR kinase domain was strongly associated with response to gefitinib and prolonged overall survival.

Adenocarcinoma↗

PubMed2002

RNA expression profiling as prognostic tool in renal patients: toward nephrogenomics.

Damage to the kidney generally elicits tissue repair mechanisms, but these processes themselves conversely may result in the progression of chronic renal disease. In a majority of patients chronic renal insufficiency progresses to a common histological end point, marked by the presence of a vast amount of scar tissue, that is, glomerulosclerosis and interstitial fibrosis. These lesions are the result of an excessive production of extracellular matrix (ECM) components. Studies on RNA expression in experimental kidney disease have shown that renal mRNA levels for ECM components and cytokines can function as prognostic tools. This suggests that mRNA levels potentially predict outcome and reaction to therapy in patients with renal diseases. Timely detection of molecular alterations could allow early therapeutic intervention that slows down or even prevents the development of sclerotic and fibrotic lesions. This review first provides a short introduction on mechanisms of initiation and progression of renal disease. Molecular techniques are available to identify renal RNA sequences potentially involved in disease progression. We discuss several molecular techniques that are being used in kidney research for quantitation and detection of mRNA. This is followed by a brief overview of investigation in experimental renal diseases, which reveal that alterations in tissue ECM mRNA levels precede histological damage and can function as predictors of clinical outcome. In particular, studies in human kidney biopsies that evaluate the prognostic value of mRNA levels with respect to renal function are examined, paying special attention to the pitfalls that potentially are encountered when interpreting the results of such studies. Then, we elaborate on ways of optimal exploitation of mRNA quantification as a prognostic tool. The potential and limitations of microarray technology in the search for genes specifically involved in progression of renal disease are reviewed, including RNA expression profiling and large-scale DNA mutation screening. Finally, the future utilities of microarray in nephrology and renal pathology are discussed.

Gene Expression Profiling↗

PubMed2006

Type 2N von Willebrand disease due to compound heterozygosity for R854Q and a novel R763G mutation at the cleavage site of von Willebrand factor propeptide.

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factorVIII (FVIII) and is caused by mutations in the D' or D3 domain of mature VWF. We now report a French patient with an atypical 2N VWD phenotype associating FVIII deficiency with plasmaVWF unable to bind FVIII (undetectableVWF:FVIIIB) but with an abnormal multimeric profile. This patient is heterozygous for both the frequent R854Q type 2NVWD mutation and a novel R763G mutation at the cleavage site between VWF propeptide and mature VWF. Four children of the patient displayed moderately decreased VWF:FVIIIB of plasma VWF and were heterozygous for either the R763G or the R854Q mutation. Children with the R763G mutation displayed the same abnormal multimeric profile as their father. Recombinant VWF (rVWF) expression studies performed in COS-7 cells showed that the R763G mutation subtly affects its multimeric profile and dramatically impairs its FVIII binding function. Furthermore, the characteristics of hybrid G763/Q854 rVWF resulting from cotransfection experiments were in agreement with the type 2N VWD diagnosis of the patient. We conclude that R763G is a new type 2N VWD mutation located in the VWF propeptide which alters the proteolytic processing of VWF and consequently its binding to FVIII.

Aged↗

PubMed2004

Novel molecular aspects of prostate carcinogenesis.

Prostate adenocarcinoma is one of the most frequently diagnosed forms of cancer in the male population of the Western world. The pivotal role of androgen and its receptor in this disease has been abundantly documented and indeed, chemical castration and treatment with antiandrogens are now standard therapies. However, relapse is often observed after 18-24 months, due to the remarkable ability of prostate tumour cells to adapt to low or undetectable androgen levels. Amplification and mutations of the androgen receptor (AR) gene have been described as well as alterations in cofactor expression and crosstalk with other signalling pathways. Another recent line of research focused on the re-programming of gene expression taking place in prostate tumours. Global expression profiling of normal and cancerous prostate tissues led to the identification of tumour-distinctive patterns. Validation studies are currently underway to identify novel drug targets as well as diagnostic and outcome prediction markers.

Animals↗