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Anne De Paepe

Publications and source records attributed to Anne De Paepe.

72 records · Page 4Linked to original sources
PubMed2003

Differentiating pathogenic mutations from polymorphic alterations in the splice sites of BRCA1 and BRCA2.

About 4% of all BRCA1 and BRCA2 alterations reported to the Breast Information Core database are splice site variants. Only a limited number of them have been studied at the RNA level. By BRCA1 and BRCA2 mutation analysis of breast/ovarian cancer families, we identified two novel and eight previously reported potential splice site mutations, never characterized at the cDNA level before. RT-PCR was performed to determine whether these variants disrupted correct splicing. To ensure efficient detection of transcripts containing premature termination codons, a nonsense-mediated mRNA decay inhibitor was added to the lymphoblastoid cell lines of the patients before RNA extraction. We found that BRCA1 IVS3+3A>C, 4304G>A (in the last codon of exon 12), and IVS19+2delT and BRCA2 IVS6+1G>A, IVS23-2A>G, and IVS24+1G>A lead to aberrant transcripts in lymphocytes. Therefore, they were considered to be true pathogenic mutations, predisposing carriers to cancers of the hereditary breast/ovarian cancer syndrome. BRCA2 IVS24-16T>C is a frequent polymorphism in linkage disequilibrium, with a polymorphic stop codon in exon 27, K3326X. BRCA1 IVS2-14C>T and BRCA2 IVS9-5insT and IVS25+9A>C represent rare variants, not disrupting normal splicing in blood lymphocytes. However, some of the alterations may act differently, qualitatively and/or quantitatively, in breast or ovarian tissues. The data provided in this paper allowed more accurate risk estimation of patients and relatives carrying the mutations described herein and have facilitated genetic counseling. Furthermore, our study is important for a better understanding of splicing mechanisms and revealed new patterns of alternative splicing in BRCA1 and BRCA2.

Adult↗
PubMed2003

Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database.

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were first described in the heritable connective disorder, Marfan syndrome (MFS). FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS, called "type-1 fibrillinopathies." In 1995, in an effort to standardize the information regarding these mutations and to facilitate their mutational analysis and identification of structure/function and phenotype/genotype relationships, we created a human FBN1 mutation database, UMD-FBN1. This database gives access to a software package that provides specific routines and optimized multicriteria research and sorting tools. For each mutation, information is provided at the gene, protein, and clinical levels. This tool is now a worldwide reference and is frequently used by teams working in the field; more than 220,000 interrogations have been made to it since January 1998. The database has recently been modified to follow the guidelines on mutation databases of the HUGO Mutation Database Initiative (MDI) and the Human Genome Variation Society (HGVS), including their approved mutation nomenclature. The current update shows 559 entries, of which 421 are novel. UMD-FBN1 is accessible at www.umd.be/. We have also recently developed a FBN1 polymorphism database in order to facilitate diagnostics.

Animals↗
PubMed2003

Evidence for involvement of a tumor suppressor gene on 1p in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare soft-tissue malignancies. The genetic basis of these tumors is still poorly understood. Cytogenetic analyses predominantly revealed complex karyotypes, precluding the identification of recurrent chromosomal changes. We report loss of 1p material in a near-diploid karyotype with few or no additional structural chromosome changes in two sporadic cases of MPNST, indicating an important role of 1p loss in MPNST development. In one of these two tumors, a distal 1p deletion (1p31.2 approximately pter) was detected suggesting involvement of a tumor suppressor gene located within this distal region of 1p. Further evidence for recurrent 1p loss in MPNST was obtained by interphase fluorescence in situ hybridization, which showed loss of 1p material in 3 out of 13 tumors. These findings together with data from the literature suggest that loss of a tumor suppressor gene located within distal 1p is implicated in the pathogenesis of MPNST.

Child↗
PubMed2002

Combined karyotyping, CGH and M-FISH analysis allows detailed characterization of unidentified chromosomal rearrangements in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. Cytogenetic studies have indicated that deletions and unbalanced translocations involving chromosome 1 short arm material occur in 40% of the investigated cases. Recurrent chromosomal imbalances detected by comparative genomic hybridization (CGH) analysis were loss of 3p, 10q, 13q and 17p and gains of 1q, 3q, 5p and 8q. In order to study genomic aberrations occurring in MCC in further detail, we combined karyotyping, CGH and multiplex-fluorescence in situ hybridization (M-FISH), a strategy that proved to be successful in the analysis of other malignancies. Analysis of 6 MCC cell lines and 1 MCC tumor revealed mostly near-diploid karyotypes with an average of 5 chromosomal rearrangements. The observed karyotypic changes were heterogeneous, with 3-27 breakpoints per case, leading to imbalance of the involved chromosomal regions that was confirmed by CGH. Chromosomal rearrangements involving the short arm of chromosome 1, the long arm of chromosome 3 and gain of 5p material were the most frequently observed abnormalities in our study. In keeping with previous observations, this series of MCCs showed no evidence for high-level amplification. We provid a detailed description of chromosomal translocations occurring in MCC that could be useful to direct future intensive investigation of these chromosomal regions.

Aged↗
PubMed2002

Homozygosity for a missense mutation in fibulin-5 (FBLN5) results in a severe form of cutis laxa.

Hereditary cutis laxa comprises a heterogeneous group of connective tissue disorders characterized by loose skin and variable systemic involvement. Autosomal dominant and recessive as well as X-linked forms have been described. Some dominant forms are caused by mutations in the elastine gene (ELN). The X-linked form is now classified in the group of copper transport diseases. The genetic defect underlying the autosomal recessive (AR) forms of cutis laxa is not known. The phenotypic abnormalities recently observed in a fibulin-5 knockout mouse model are reminiscent of human AR cutis laxa type I. Both share cutis laxa, lung emphysema and arterial involvement. Molecular study of the fibulin-5 (FBLN5) gene in a large consanguineous Turkish family with four patients affected by AR cutis laxa type I demonstrated the presence of a homozygous missense mutation (T998C) in the FBLN5 gene resulting in a serine-to-proline (S227P) substitution in the fourth calcium-binding epidermal growth factor-like domain of fibulin-5 protein. This amino acid substitution is predicted to have important structural and functional consequences for normal elastogenesis. As such, we provide evidence that a genetic defect in fibulin-5 (FBLN5, also known as EVEC or DANCE) is responsible for a recessive form of cutis laxa in humans.

Cutis Laxa↗
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

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.

BACKGROUND: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. RESULTS: We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. CONCLUSIONS: The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

Algorithms↗
PubMed2002

Pathological splice mutations outside the invariant AG/GT splice sites of BRCA1 exon 5 increase alternative transcript levels in the 5' end of the BRCA1 gene.

We report two novel mutations in the splice sites of BRCA1 exon 5: IVS5+3A>G, a Belgian founder mutation, and IVS3-6T>G, identified in one family with a strong family history of breast cancer. Real-time RT-PCR showed that IVS3-6T>G leads to a fivefold increase of the BRCA1-Deltaex5 (isoform with an in frame skip of exon 5) ratio to the total BRCA1 expression level. IVS5+3A>G results in a 10-fold increase of the BRCA1-Delta22ntex5 ratio (isoform with an out of frame skip of the last 22 nucleotides of exon 5) and a twofold increase of the BRCA1-Deltaex5 ratio. These altered ratios are most likely to result from increased expression of the alternative transcripts, although we cannot completely rule out a small decrease of the total BRCA1 expression level due to highly variable BRCA1 levels in cultured cell lines. In order to explore the functional significance of the isoforms, we evaluated their prevalence in normal tissues and cancer cell lines. The BRCA1-Delta22ntex5 ratio was significantly higher in an ovarian cancer cell line compared to normal ovarian tissue. Our findings suggest that revealing the defects caused by some splice mutations requires accurate quantitative methods. We hypothesize that disruption of alternative transcript ratios of BRCA1 may be a dominant mechanism affecting predisposition to hereditary breast and/or ovarian cancer.

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↗
PubMed2002

Kallmann syndrome in a patient with congenital spherocytosis and an interstitial 8p11.2 deletion.

We describe the hitherto smallest interstitial 8p11.2 deletion in a patient with congenital spherocytosis, dysmorphic features, and growth delay in association with hypogonadotropic hypogonadism and anosmia. The latter features are characteristic for Kallmann syndrome. In contrast to the previously reported patients with 8p deletions, the present patient showed normal intelligence. Congenital spherocytosis is one of the most common hereditary hemolytic anemias. One of the three loci for congenital spherocytosis was assigned to chromosome 8p (located between 8p11.1 and 8p21) and mutations in or loss of the ankyrin-1 gene (ANK1) were identified. Molecular analysis confirmed the de novo loss of ANK1 in our patient. Kallmann syndrome, which is characterized by hypogonadotropic hypogonadism and anosmia, can be X-linked, autosomal dominant, or autosomal recessive. So far only the X-linked KAL1 gene has been identified. The present finding suggests an autosomal locus for Kallmann syndrome at 8p11.2. The simultaneous occurrence of congenital spherocytosis, Kallmann syndrome phenotype, dysmorphic features, and growth delay in this patient points to a new contiguous gene syndrome.

Abnormalities, Multiple↗
PubMed2002

Localization of the 17q breakpoint of a constitutional 1;17 translocation in a patient with neuroblastoma within a 25-kb segment located between the ACCN1 and TLK2 genes and near the distal breakpoints of two microdeletions in neurofibromatosis type 1 patients.

We have constructed a 1.4-Mb P1 artificial chromosome/bacterial artificial chromosome (PAC/BAC) contig spanning the 17q breakpoint of a constitutional translocation t(1;17)(p36.2;q11.2) in a patient with neuroblastoma. Three 17q breakpoint-overlapping cosmids were identified and sequenced. No coding sequences were found in the immediate proximity of the 17q breakpoint. The PAC/BAC contig covers the region between the proximally located ACCN1 gene and the distally located TLK2 gene and SCYA chemokine gene cluster. The observation that the 17q breakpoint region could not be detected in any of the screened yeast artificial chromosome libraries and the localization of the 17q breakpoint in the vicinity of the distal breakpoints of two microdeletions in patients with neurofibromatosis type 1 suggest that this chromosomal region is genetically unstable and prone to rearrangements.

Central Nervous System Neoplasms↗
PubMed2002

Identification of cytogenetic subclasses and recurring chromosomal aberrations in AML and MDS with complex karyotypes using M-FISH.

Complex chromosomal aberrations (CCAs) can be detected in a substantial proportion of AML and MDS patients, de novo as well as secondary or therapy-related, and are associated with an adverse prognosis. Comprehensive analysis of the chromosomal rearrangements in these complex karyotypes has been hampered by the limitations of conventional cytogenetics. As a result, our knowledge concerning the cytogenetics of these malignancies is sparse. Here we describe a multiplex-FISH (M-FISH) study of CCAs in 36 patients with AML and MDS. M-FISH generated a genome-wide analysis of chromosomal aberrations in CCAs, establishing several cytogenetic subgroups. -5/5q- was demonstrated in the majority of patients (86%). Other rearrangements (present with or without -5/5q-) included: deletion of 7q (47%), 3q rearrangements (19%), and MLL copy gain or amplification (17%). These genetic subgroups seem to display biological heterogeneity: MLL copy gain or amplification in association with 5q- was detected only in AML patients and was significantly associated with extremely short survival (median overall survival: 30 days, P = 0.0102). A partially cryptic t(4;5)(q31;q31), a balanced t(1;8)(p31;q22), and an unbalanced der(7)t(7;14)(q21;q13) were detected as possible new recurrent rearrangements in association with CCAs. Novel reciprocal translocations included t(5;11)(q33;p15)del(5)(q13q31) and t(3;6)(q26;q25). We conclude that AML and MDS with CCAs can be subdivided into molecular cytogenetic subclasses, which could reflect different clinical behavior and prognosis, and that three recurrent chromosomal aberrations are associated with karyotype complexity.

Acute Disease↗
PubMed2002

Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: Identification of 20 novel mutations and two novel polymorphisms in the FBN1 gene.

Mutations in the human fibrillin 1 gene (FBN1) cause the Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Knowledge about FBN1 mutations is important for early diagnosis, management, and genetic counseling. However, mutation detection in FBN1 is a challenge because the gene is very large in size ( approximately 200 kb) and the approximately 350 mutations detected so far are scattered over 65 exons. Conventional methods for large-scale detection of mutations are expensive, technically demanding, or time consuming. Recently, a high-capacity low-cost mutation detection method was introduced based on denaturing high-performance liquid chromatography (DHPLC). To assess the sensitivity and specificity of this method, we blindly screened 64 DNA samples of known FBN1 genotype exon-by-exon using exon-specific DHPLC conditions. Analysis of 682 PCR amplicons correctly identified 62 out of 64 known sequence variants. In three MFS patients of unknown FBN1 genotype, we detected two mutations and eight polymorphisms. Overall, 20 mutations and two polymorphisms are described here for the first time. Our results demonstrate 1) that DHPLC is a highly sensitive (89-99%, P = 0.05) method for FBN1 mutation detection; but 2) that chromatograms with moderate and weak pattern abnormalities also show false positive signals (in all 45-59%, P = 0.05); 3) that the difference in the chromatograms of heterozygous and homozygous amplicons is mostly independent of the type of sequence change; and 4) that DHPLC column conditions, additional base changes, and the amounts of injected PCR products influence significantly the shape of chromatograms. A strategy for FBN1 mutation screening is discussed.

Chromatography, High Pressure Liquid↗
PubMed2002

Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay.

Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for time-consuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C(T) method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.

DEAD-box RNA Helicases↗
PubMedDate not supplied

Identification of an unbalanced X-autosome translocation by array CGH in a boy with a syndromic form of chondrodysplasia punctata brachytelephalangic type.

Screening of a large series of patients with unexplained mental retardation with a 1 Mb BAC array resulted in the detection of several cryptic chromosomal imbalances. In this paper we present the findings of array CGH screening in a 14-year-old boy with the brachytelephalangic type of chondrodysplasia punctata, mental retardation and obesity. On several occasions, cytogenetic analysis of this boy revealed a normal karyotype. Subsequent screening with array CGH resulted in the detection of a distal 9p trisomy and distal Xp nullisomy caused by an unbalanced X;9 translocation: 46,Y,der(X)t(X;9)(p22.32;p23). The identification of this de novo chromosomal rearrangement not only made accurate genetic counselling possible but also explained most of the phenotypic abnormalities observed in this patient. This study confirms the power of array CGH in the detection of subtle or submicroscopic chromosomal changes.

Adolescent↗
PubMedDate not supplied

Orofacial manifestations of congenital fibrillin deficiency: pathogenesis and clinical diagnostics.

Mutations in the genes encoding fibrillin, an extracellular matrix protein involved in providing elastic properties to the connective tissues, may result in specific craniofacial and oral anomalies. A number of craniofacial (retrognathia, dolichocephaly, high palate) and dental (root deformity, pulp calcification) manifestations are considered pathognomic for the Marfan syndrome (MFS), a condition caused by congenital fibrillin-1 deficiency. Reports on similar features in congenital contractural arachnodactyly (CCA), caused by fibrillin-2 deficiency, support the hypothesis that fibrillin deficiency might result in a number of morphological anomalies by influencing tissue interaction during growth and development. Hence, clinical manifestations can be related to specific aspects of fibrillin deficiency pathogenesis, and may be adopted as diagnostic tools in the outlook for affected individuals.

Animals↗