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Linda Hartmann,
Kornelia Neveling,
Stephanie Borkens,
Hildegard Schneider,
Marcel Freund,
Elke Grassman,
Stephan Theiss,
Angela Wawer,
Stefan Burdach,
Arleen D Auerbach,
Detlev Schindler,
Helmut Hanenberg,
Heiner Schaal
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ABSTRACT: The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.
The American Journal of Human Genetics 10/2010; 87(4):480-93. · 10.60 Impact Factor
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Fiona Vaz,
Helmut Hanenberg,
Beatrice Schuster,
Karen Barker,
Constanze Wiek,
Verena Erven,
Kornelia Neveling,
Daniela Endt,
Ian Kesterton,
Flavia Autore,
Franca Fraternali,
Marcel Freund, Linda Hartmann,
David Grimwade,
Roland G Roberts,
Heiner Schaal,
Shehla Mohammed,
Nazneen Rahman,
Detlev Schindler,
Christopher G Mathew
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ABSTRACT: Fanconi anemia (FA) is a rare chromosomal-instability disorder associated with a variety of developmental abnormalities, bone marrow failure and predisposition to leukemia and other cancers. We have identified a homozygous missense mutation in the RAD51C gene in a consanguineous family with multiple severe congenital abnormalities characteristic of FA. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-1 inhibitor camptothecin. Thus, biallelic germline mutations in a RAD51 paralog are associated with an FA-like syndrome.
Nature Genetics 05/2010; 42(5):406-9. · 35.53 Impact Factor
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Alfons Meindl,
Heide Hellebrand,
Constanze Wiek,
Verena Erven,
Barbara Wappenschmidt,
Dieter Niederacher,
Marcel Freund,
Peter Lichtner, Linda Hartmann,
Heiner Schaal, [......],
Karin Kast,
Helmut Deissler,
Christoph Engel,
Bertram Müller-Myhsok,
Kornelia Neveling,
Marion Kiechle,
Christopher G Mathew,
Detlev Schindler,
Rita K Schmutzler,
Helmut Hanenberg
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ABSTRACT: Germline mutations in a number of genes involved in the recombinational repair of DNA double-strand breaks are associated with predisposition to breast and ovarian cancer. RAD51C is essential for homologous recombination repair, and a biallelic missense mutation can cause a Fanconi anemia-like phenotype. In index cases from 1,100 German families with gynecological malignancies, we identified six monoallelic pathogenic mutations in RAD51C that confer an increased risk for breast and ovarian cancer. These include two frameshift-causing insertions, two splice-site mutations and two nonfunctional missense mutations. The mutations were found exclusively within 480 pedigrees with the occurrence of both breast and ovarian tumors (BC/OC; 1.3%) and not in 620 pedigrees with breast cancer only or in 2,912 healthy German controls. These results provide the first unambiguous evidence of highly penetrant mutations associated with human cancer in a RAD51 paralog and support the 'common disease, rare allele' hypothesis.
Nature Genetics 05/2010; 42(5):410-4. · 35.53 Impact Factor
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ABSTRACT: Pathogenic splicing alterations caused by point mutations in both splice sites and auxiliary cis-regulatory elements are increasingly recognized as an important mechanism through which gene mutations cause human disease. Unfortunately, in routine genetic diagnostic settings, splicing mutations may escape identification, due to the lack of RNA samples. Since most patients are genotyped only, any computational prediction of mutation effects on splicing can be beneficial for the human geneticist. Here, we review common techniques to identify human point mutations and delineate the molecular basis for splice site recognition. Moreover, this article provides basic insights into web-tools predicting splice sites and cis-regulatory elements and discusses their benefits for judgment of clinically identified sequence variants of disease-specific genes.
Frontiers in Bioscience 02/2008; 13:3252-72. · 3.52 Impact Factor
