Biomedical subjects

Ina Vandenbroucke

Publications and source records attributed to Ina Vandenbroucke.

6 recordsLinked to original sources

PubMed2004

Neurofibromin is actively transported to the nucleus.

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors 'shuttle' between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28-49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.

Active Transport, Cell Nucleus↗

PubMed2004

Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1.

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.

Alleles↗

PubMed2003

Genetic and clinical mosaicism in a patient with neurofibromatosis type 1.

Patients with typical features of neurofibromatosis type 1 (NF1) limited to a specific body segment are usually referred to as having "segmental NF1", which is generally assumed to be the result of somatic mosaicism for a NF1 mutation. Mosaicism has also been demonstrated at the molecular level in some sporadic cases with phenotypically classic NF1. In the present report, we describe a patient with NF1 disease manifestations throughout the whole body, but leaving a few sharply delineated segments of the skin unaffected, suggestive of revertant mosaicism. A large intragenic deletion was found by mutation analysis using long-range RT-PCR. The intra-exonic breakpoints were characterized in exon 13 and exon 28, resulting in a deletion of 99,571 bp at the genomic level. The presence of two genetically distinct cell populations, confirming mosaicism for this NF1 mutation, was shown by analysis of several tissues. Revertant mosaicism was excluded by demonstrating heterozygosity for markers residing in the deletion region. The findings in this patient demonstrate two things: (1) although the entire body is affected, mosaicism can still be suspected at clinical examination and proven by DNA analysis and skin biopsies; (2) long-range RT-PCR is a feasible method for demonstrating large intragenic deletions in NF1.

Adult↗

PubMed2003

Quantitative real time polymerase chain reaction for measurement of human interleukin-5 receptor alpha spliced isoforms mRNA.

BACKGROUND: Expression of human Interleukin-5 receptor alpha (hIL-5Ralpha) is controlled by alternative splicing, which generates two different transcripts encoding a membrane-anchored and a soluble form of the receptor, respectively. Although the study of the expression and regulation of hIL-5Ralpha is of crucial importance in the field of immunological processing, methods and techniques until now described lack sufficient sensitivity for detection of small differences in the expression of these isoforms. The aim of this study was to develop a reliable and sensitive real-time quantitative PCR assay to analyse the expression level of each isoform. METHODS: For the quantitative real-time PCR assay, two standard curves specific for each splice variant were constructed. PCR amplifications were performed on CDNA from peripheral blood, eosinophilic chronic rhinosinusitis and normal nasal tissue using a common forward and two specific reverse primers, in combination with SYBR Green I as the detection format. RESULTS AND CONCLUSION: We have developed an accurate and reliable assay for quantification of interleukin-5 receptor alpha mRNA isoforms over a broad dynamic range of input molecules. Importantly, excess of one isoform did not influence accurate quantification of the other isoform. Quantification of hIL-5Ralpha variants in human samples demonstrated an overexpression of both membrane-anchored and soluble encoding variants in eosinophilic chronic rhinosinusitis tissue and peripheral blood in patients with eosinophilic chronic rhinosinusitis compared to healthy subjects. The implementation of this assay will allow a better understanding of the regulatory mechanisms of the hIL-5Ralpha gene and hence its role in the pathogenesis of chronic inflammatory diseases.

Alternative Splicing↗

PubMed2002

Quantification of NF1 transcripts reveals novel highly expressed splice variants.

Previously, we have shown that the NF1 gene gives rise to multiple novel splice variants. In the present study, nine NF1 variants were quantified by real-time PCR in various human tissues. Some of these variants were expressed at low to moderate low levels and possible implications of these findings are discussed. Interestingly, two variants (NF1-DeltaE4b and NF1-DeltaE43) were shown to be highly expressed in specific tissues. NF1-DeltaE43 lacks a nuclear targeting sequence and might be functionally different from full-length NF1. These novel NF1 splice variants might expand our understanding of the role of neurofibromin.

Alternative Splicing↗

PubMed2002

Complex splicing pattern generates great diversity in human NF1 transcripts.

BACKGROUND: Mutation analysis of the neurofibromatosis type 1 (NF1) gene has shown that about 30% of NF1 patients carry a splice mutation resulting in the production of one or several shortened transcripts. Some of these transcripts were also found in fresh lymphocytes of healthy individuals, albeit typically at a very low level. Starting from this initial observation, we were interested to gain further insight into the complex nature of NF1 mRNA processing. RESULTS: We have used a RT-PCR plasmid library based method to identify novel NF1 splice variants. Several transcripts were observed with specific insertions/deletions and a survey was made. This large group of variants detected in one single gene allows to perform a comparative analysis of the factors involved in splice regulation. Exons that are prone to skipping were systematically analysed for 5' and 3' splice site strength, branch point strength and secondary structure. CONCLUSION: Our study revealed a complex splicing pattern, generating a great diversity in NF1 transcripts. We found that, on average, exons that are spliced out in part of the mRNA have significantly weaker acceptor sites. Some variants identified in this study could have distinct roles and might expand our knowledge of neurofibromin.

Journal Article↗