Numerous unclassified variants (UVs) have been found in the mismatch repair genes MLH1 and MSH2 involved in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Some of these variants may have an effect on pre-mRNA splicing, either by altering degenerate positions of splice site sequences or by affecting intronic or exonic splicing regulatory sequences such as exonic splicing enhancers (ESEs). In order to determine the consequences of UVs on splicing, we used a functional assay of exon inclusion. For each variant, mutant and wild-type exons to be tested were PCR-amplified from patient genomic DNA together with approximately 150 bp of flanking sequences and were inserted into a splicing reporter minigene. After transfection into HeLa cells, the effects on splicing were evaluated by RT-PCR analysis and systematic sequencing. A total of 22 UVs out of 85 different variant alleles examined in 82 families affected splicing, including four exonic variants that affected putative splicing regulatory elements. We analyzed short stretches spanning the latter variants by cloning them into the ESE-dependent central exon of a three-exon splicing minigene and we showed in cell transfection experiments that the wild-type sequences indeed contain functional ESEs. We then used this construct to query for ESE elements in the MLH1 or MSH2 regions affected by 14 previously reported exonic splicing mutations and showed that they also contain functional ESEs. These splicing assays represent a valuable tool for the interpretation of UVs and should contribute to the optimization of the molecular diagnosis of the Lynch syndrome and of other genetic diseases.

Proteins of the GW182 family are essential components of the miRNA pathway in animal cells. Vertebrate genomes encode three GW182 paralogs (TNRC6A, TNRC6B, and TNRC6C), which may be functionally redundant. Here, we show that the N-terminal GW-repeat-containing regions of all three TNRC6s interact with the four human Argonaute proteins (AGO1-AGO4). We also show that TNRC6A, TNRC6B, and TNRC6C silence the expression of bound mRNAs. This activity is mediated by their C-terminal silencing domains, and thus, is independent of the interaction with AGO1-AGO4. Silencing by TNRC6A, TNRC6B, and TNRC6C is effected by changes in protein expression and mRNA stability that can, in part, be attributed to deadenylation. Our findings indicate that TNRC6A, TNRC6B, and TNRC6C are recruited to miRNA targets through an interaction between their N-terminal domain and an Argonaute protein; the TNRC6s then promote translational repression and/or degradation of miRNA targets through a C-terminal silencing domain.

Li-Fraumeni syndrome, resulting from p53 (TP53) germline mutations, represents one of the most devastating genetic predispositions to cancer. Recently, the MDM2 SNP309 (T-->G variation) was shown to be associated with accelerated tumour formation in p53 mutation carriers. The impact of the common p53 codon 72 polymorphism on cancer risk remains controversial. We therefore investigated the effect of these two polymorphisms in 61 French carriers of the p53 germline mutation. The mean age of tumour onset in MDMD2 SNP309 G allele carriers (19.6 years) was significantly different from that observed in patients homozygous for the T allele (29.9 years, p<0.05). For the p53 codon 72 polymorphism, the mean age of tumour onset in Arg allele carriers (21.8 years) was also different from that of Pro/Pro patients (34.4 years, p<0.05). We observed a cumulative effect of both polymorphisms because the mean ages of tumour onset in carriers of the MDM2G and p53Arg alleles (16.9 years) and those with the MDM2T/T and p53Pro/Pro genotypes (43 years) were clearly different (p<0.02). Therefore, our results confirm the impact of the MDM2 SNP309 G allele on the age of tumour onset in germline p53 mutation carriers, and suggest that this effect may be amplified by the p53 72Arg allele. Polymorphisms affecting p53 degradation therefore represent one of the rare examples of modifier genetic factors identified to date in mendelian predispositions to cancer.

The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for "unclassified variant") found in genetic screenings represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patient genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the variant constructs are compared by reverse transcription-PCR analysis and sequencing. This method represents a complementary approach to reverse transcription-PCR analyses of patient RNA, for the identification of pathogenic splicing mutations.