Mental retardation and cardiovascular malformations in NF1 microdeleted patients point to candidate genes in 17q11.2

University of Milan, Milano, Lombardy, Italy
Journal of Medical Genetics (Impact Factor: 5.64). 02/2004; 41(1):35-41. DOI: 10.1136/jmg.2003.014761
Source: PubMed
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    ABSTRACT: Neurofibromatosis type 1 (NF1) affects about one in 3,500 people in all ethnic groups. Most NF1 patients have private loss-of-function mutations scattered along the NF1 gene. Here, we present an original NF1 investigation strategy and report a comprehensive mutation analysis of 565 unrelated patients from the NF-France Network. A NF1 mutation was identified in 546 of the 565 patients, giving a mutation detection rate of 97%. The combined cDNA/DNA approach showed that a significant proportion of NF1 missense mutations (30%) were deleterious by affecting pre-mRNA splicing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements that would have been missed if only sequencing or microsatellite analysis had been performed. In four unrelated families, we identified two distinct NF1 mutations within the same family. This fortuitous association points out the need to perform an exhaustive NF1 screening in the case of molecular discordant related patients. A genotype-phenotype study was performed in patients harboring a truncating (N=368), in-frame splicing (N=36), or missense (N=35) mutation. The association analysis of these mutation types with 12 common NF1 clinical features confirmed a weak contribution of the allelic heterogeneity of the NF1 mutation to the NF1 variable expressivity. This article is protected by copyright. All rights reserved.
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    ABSTRACT: Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman's capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.
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    ABSTRACT: Cyclin-Dependent Kinase 5 Regulatory subunit 1 (CDK5R1) encodes p35, a specific activator of Cyclin-Dependent Kinase 5 (CDK5). CDK5 and p35 have a fundamental role in neuronal migration and differentiation during CNS development. Both the CDK5R1 3'-UTR's remarkable size and its conservation during evolution strongly indicate an important role in post-transcriptional regulation. We previously validated different regulatory elements in the 3'-UTR of CDK5R1, which affect transcript stability, p35 levels and cellular migration through the binding with nELAV proteins and miR-103/7 miRNAs. Interestingly, a 138bp-long region, named C2.1, was identified as the most mRNA destabilizing portion within CDK5R1 3'-UTR. This feature was maintained by a shorter region of 73bp, characterized by two poly-U stretches. UV-CL experiments showed that this region interacts with protein factors. UV-CLIP assays and pull-down experiments followed by mass spectrometry analysis demonstrated that nELAV and hnRNPA2/B1 proteins bind to the same U-rich element. These RNA-binding proteins (RBPs) were shown to oppositely control CDK5R1 mRNA stability and p35 protein content at post-trascriptional level. While nELAV proteins have a positive regulatory effect, hnRNPA2/B1 has a negative action that is responsible for the mRNA destabilizing activity both of the C2.1 region and of the full-length 3'-UTR. In co-expression experiments of hnRNPA2/B1 and nELAV RBPs we observed an overall decrease of p35 content. We also demonstrated that hnRNPA2/B1 can downregulate nELAV protein content but not viceversa. This study, by providing new insights on the combined action of different regulatory factors, contributes to clarify the complex post-transcriptional control of CDK5R1 gene expression.
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