Cancer Research Annual Meeting 2010 Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stella, A.
Right arrow Articles by Liu, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stella, A.
Right arrow Articles by Liu, B.
[Cancer Research 61, 7020-7024, October 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

A Nonsense Mutation in MLH1 Causes Exon Skipping in Three Unrelated HNPCC Families1

Alessandro Stella, Anja Wagner, Kazuhisa Shito, Steven M. Lipkin, Patrice Watson, Ginevra Guanti, Henry T. Lynch, Riccardo Fodde2 and Bo Liu2

University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 [A. S., K. S., B. L.]; Sez. di Genetica Medica-DIMIMP Policlinico, Universitèa di Bari, 70124 Bari, Italy [A. S., G. G.]; Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands [A. W., R. F.]; Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892 [S. M. L.]; and Department of Preventive Medicine, Creighton University School of Medicine, Omaha, Nebraska 68178 [P. W., H. T. L.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 are responsible for the majority of hereditary nonpolyposis colorectal cancer (HNPCC) families. A common mutation mechanism is to disrupt MLH1 and MSH2 mRNA splicing. The disruption creates aberrant mRNAs lacking specific coding exons (exon skipping). Here, we report a novel skipping of MLH1 exon 12 caused by an AAG to TAG nonsense mutation at codon 461 in three HNPCC families of North American origins. The nonsense codon was found in a conserved haplotype in the three unrelated families and seems to represent a founder mutation. The skipping created an aberrant MLH1 mRNA transcript lacking exon 12. The effect of the codon 461 nonsense mutation on exon 12 skipping is evident even though it was placed in a minigene construct containing entirely different coding sequences. Notably, the effect of the nonsense mutation on exon skipping is incomplete. Accordingly, a second aberrant MLH1 transcript encompassing the nonsense codon is also produced. Whereas the latter transcript is unstable, presumably because of nonsense-mediated mRNA decay, neither of the aberrant transcripts seems to affect the stability of wild-type MLH1 mRNA. This study demonstrates that the germ-line nonsense mutation at codon 461 of MLH1 disrupts normal MLH1 mRNA processing, and that exon skipping underlies pathogenesis in these HNPCC families.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
HNPCC3 is the most common inherited colorectal cancer syndrome characterized by an autosomal dominant inheritance pattern of early onset colorectal, endometrial, ovarian, urological, and upper gastrointestinal malignancies (1) . The disease is caused by germ-line mutations in MLH1 (2) , MSH2 (3) , MSH6 (4) , and PMS2 (5) . Somatic inactivation of the remaining wild-type allele leads to microsatellite instability (6) . Mutation analysis showed that the vast majority of HNPCC families are caused by a germ-line defect in MSH2 and MLH1, whereas mutations in MSH6 and PMS2 only contribute to a minority of the cases (5 , 7, 8, 9, 10, 11, 12) . Consistent with the mutation studies, tumors arising in carriers with defined germ-line MMR mutations are defective in MMR and display microsatellite instability phenotype (6 , 7 , 9 , 10) .

A common mutation mechanism in HNPCC is the disruption of MSH2 and MLH1 splicing by exon skipping (7 , 8 , 13) . This often involves the generation of aberrant MLH1 and MSH2 mRNA transcripts detectable in cells from HNPCC patients by RT-PCR-based analysis. The transcript carrying skipped exons predicts the truncation of MLH1 or MSH2 proteins because of frameshift and premature termination. Recently, exon skipping in MLH1 and MSH2 has been shown also to occur in normal individuals without hereditary cancer susceptibility (14, 15, 16, 17) . These and other studies have suggested that MLH1 and MSH2 exon skipping can occur either as a pathogenic mutation or as a normal splice variant. Here, we have thoroughly examined the molecular genetic basis of MLH1 exon 12 skipping observed in three unrelated HNPCC families. The skipping is caused by an AAG to TAG nonsense mutation at codon 461 within exon 12. The same mutation also leads to the expression of a second aberrant transcript encompassing the stop codon and predicting the synthesis of a Mr 53,000-truncated protein. Our studies support the hypothesis that nonsense mutation has a direct cis-acting effect on pre-mRNA processing and leads to the expression of two types of aberrant mRNA transcripts. These results have important implications for the clinical molecular diagnosis of patients suspected of HNPCC who are exhibiting MLH1 exon skipping.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Samples.
The three HNPCC mutations investigated here were identified during the molecular genetic analysis of a cohort of 58 HNPCC families selected by Dr. Henry T. Lynch (Creighton University School of Medicine, Omaha, Nebraska). The clinical features of these families are strongly reminiscent of HNPCC, and 46 of 58 families comply with the modified Amsterdam criteria (1) . For the present studies, EBV-transformed lymphoblastoid cell lines were obtained from a total of 23 different individuals. Of these 23, 15 were from L1933,4 5 were from L3551,4 and 3 were from L3048.4 Of the 15 individuals from L1933, 6 had cancer at various organ sites, and those remaining were phenotypically normal individuals. RNA and DNA were purified from EBV-transformed lymphoblastoid cells as described previously (7) .

DGGE Analysis.
DGGE mutation analysis and nucleotide sequence determination were performed as described previously (18) . In short, the general strategy was to PCR-amplify each of the 16 MSH2 exons and 19 MLH1 exons of a single affected individual per family and to analyze these products by GC-clamped DGGE. Exons exhibiting altered migration patterns were sequenced to determine the molecular nature of the variation observed. When sequence variants were detected, the investigations were extended to the rest of the family to verify the segregation of the nucleotide change with the disease phenotype. In all three cases investigated here, cosegregation was observed between the codon 461 nonsense mutation and the disease phenotype.

Haplotype Analysis.
Haplotype data were derived by genotyping several affected and nonaffected individuals from the three families by PCR amplification of the following polymorphic CA repeat markers previously shown to map both proximal and distal to the MLH1 gene: cen-D3S1619-D3S1561 -D3S1277-D3S1611-MLH1 exon 8-MLH1 exon 15-D3S1298-D3S1289-tel. Primers and PCR conditions were as described previously (19 , 20) .

RT-PCR and in Vitro Coupled TnT Analysis.
cDNA was generated using random hexamers and reverse transcriptase (7) . RT-PCR amplification and sequencing of the entire coding region of MLH1 and MSH2 were performed as described previously (7) . The primer sets used for cDNA segregation analysis were 5'-GTGGACAATAATCGCTCCGTC-3' and 5'-CTCAGATATGTACTGCTTCCG-3'. Sequencing primers for the cDNA segregation analysis were 5'-TTCATTCCTGCACGAGGAGAGC-3' and 5'-TCCAGGATGTACTTTACCCAG-3'. The high specific activity radioisotope for sequencing primer labeling was purchased from ICN (150 mCi/ml). The in vitro coupled TnT assay was performed as described (7) . For cloning of RT-PCR products, BamHI and XhoI adaptors were introduced into the RT-PCR primer set used for cDNA segregation analysis. The RT-PCR products were cloned into the BamHI and XhoI digested pZero 2.0 (Invitrogen, Carlsbad, CA). The recombinant clones were screened and analyzed by DNA sequencing.

In vitro Exon Skipping Analysis.
For in vitro exon skipping analysis, a minigene containing mutant or wild-type exon 12 genomic DNA from L1933-1 was generated using exon-trapping vector pSPL3. The genomic DNA includes a 98-bp intronic sequence upstream of the exon 12 splice acceptor site and a 110-bp intron sequence downstream of the exon 12 splice donor site. Subsequent analysis of exon skipping of the minigene system were performed in the cos-7 cell line as suggested by the manufacturer (Life Technologies, Inc., Rockville, MD). In brief, genomic DNA containing exon 12 and adjacent intronic sequences from an affected individual in family L1933 was amplified by PCR with the following primer set: 5'-GCCTCGAGGTACTGCTCCATTTGGGGACC-3' and 5'-GCGGATCCGCAGAGAGAGAA GATGCAAGTG-3'. One primer contains an XhoI linker, whereas the other one contains a BamHI linker. After the digestion with XhoI and BamHI, the PCR products were cloned into the XhoI/BamHI sites of exon trapping vector pSPL3 (Life Technologies, Inc.). The recombinant clones containing either the wild-type sequence or the AAG to TAG nonsense mutation at codon 461 were transfected into cos-7 cells using Lipofectamine-plus reagents (Life Technologies, Inc.). Total cellular RNA from the transfected cos-7 cells was purified, and cDNA was generated by reverse transcriptase reaction. Transcripts containing full-length exon 12 sequences or deletion of exon 12 sequences were detected by RT-PCR and analyzed by sequencing. The primer sets used for PCR reaction were SA2 (5'-ATCTCAGTGGTATTTGTGAGC-3') and SD6 (5'-TCTGAGTCACCTGGACAACC-3').


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
L1933, L3048, and L3551 are three unrelated HNPCC families, two of which (L1933 and L3551) comply with the Amsterdam Criteria (1) . The three families were initially analyzed as parts of the mutational studies in a cohort of 58 HNPCC kindreds. To search for germ-line mutations underlying the cancer susceptibility in these families, the entire coding region of the two most frequently mutated MMR genes, MSH2 and MLH1, was analyzed by (a) DGGE analysis of PCR-amplified genomic DNA (10 , 11 , 18) ; and (b) by sequencing of RT-PCR products prepared from affected and normal individuals from these families. The results of the mutation analysis of the entire cohort will be presented elsewhere.5 Interestingly, although no single RT-PCR alteration was detected in MSH2, an aberrant mRNA transcript carrying exon-12 skipping in MLH1 was observed in affected individuals from L1933, L3048-76, and L3551-1 but not in any of the normal individuals of the same family (Fig. 1A)Citation . The aberrant transcript lacking exon 12 cosegregates with the disease in all individuals analyzed and was confirmed by separate verification experiments.



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Analysis of the codon 461 nonsense mutation in the MLH1 gene in three HNPCC families. A, sequence analysis of MLH1. RT-PCR products were used as templates for direct sequencing of the MLH1 gene. The ddA mixes from each sequencing reaction were loaded in adjacent lanes to facilitate comparison, as were those for C, G, and T. cDNA sequences from L1933-39, L1933-1, L3048-76, L3551-1, and L3551-3 are displayed in Lanes 1–5, respectively. A skipping of exon 12 is evident in cDNA from affected L1933-1, L3048-76, and L3551-1 (Lanes 2–4; data from other members of these families is not shown here). Arrowhead, starting point at exon 12 for the wild-type or exon 13 for the mutant cDNA sequences, respectively. B, DGGE analysis of MLH1 exon 12. Lane 1, positive control (del C at codon 466); Lane 2, wild type; Lane 3, L3551-1; Lane 4, L3048-76; Lane 5, L1933-1. Genomic DNA samples were PCR-amplified with the following primers: 5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCCTAATACAGACTTTGCTACCAGGA-3' and 5'-GGTAGGCTGTACTTTTCCCA-3'. Horizontal arrows, position along the gradient gel where the homo- and heteroduplexes focus. The gradient gel used here was cast between 45 and 60% denaturing agent (80% is 6% acrylamide, 32% formamide, and 5.6 M Urea in 1x TAE buffer) and run overnight at 62°C in 1x TAE buffer. C, sequencing analysis of normal conformer shown as antisense sequences. D, sequencing analysis of mutant conformer. An AAG to TAG mutation at codon 461 within exon 12 is identified in mutant conformer as designated by an arrowhead (sequence shown as the antisense; data from other affected members of the three HNPCC families is not shown here).

 
The observed exon-12 deletion creates a frameshift and an early termination codon predicted to result in a truncated protein of Mr 43,000. DGGE analysis of the three HNPCC families revealed the presence of a mutant conformer (Fig. 1B)Citation . Sequencing analysis of the mutant conformer identified an AAG ->TAG nonsense mutation at codon 461 within MLH1 exon 12 (Fig. 1, C and D)Citation . Additional analysis of the three families by DGGE and DNA sequencing confirmed the presence of the variant pattern associated with the codon 461 nonsense mutation in all individuals characterized by exon-12 skipping but not in the unaffected individuals without exon-12 skipping.

The identification of the identical germ-line mutation in three apparently unrelated HNPCC families raises the issue of founder versus recurring mutation event. To address this issue, we have determined the disease haplotype around the MLH1 gene on chromosome 3. In all three cases, the mutation was associated to a conserved haplotype, although some variation was observed at the most distal marker DS31289 (data not shown). There is no obvious link among the three families by genealogic studies. It seems therefore plausible to assume that the codon 461 mutation arose only once and has spread to the three apparently unrelated HNPCC kindreds described here.

Exon skipping associated with nonsense mutation in MLH1 has been noted previously in several other HNPCC kindreds (7 , 8 , 13) . However, it is not known whether the skipping was caused by the direct effect of the nonsense codon in the mutant allele. To establish a cause-effect relationship between the codon 461 single nucleotide substitution and the observed exon-12 skipping in these HNPCC families, the effect of AAG->TAG mutation on splicing was analyzed with a minigene construct used for genomic exon trapping (Fig. 2A)Citation . DNA constructs containing either mutant or wild-type exon 12 sequences from L1933-1 were generated and introduced into cos-7 cells. mRNA from transfected cells was purified, and splicing products were analyzed by RT-PCR. Whereas wild-type exon 12 DNA produced normal splicing product, the mutant exon 12 DNA containing the AAG to TAG nonsense codon produced an aberrant splicing product with deletion of almost the entire exon 12 DNA (Fig. 2B)Citation . Sequencing analysis of the aberrant RT-PCR products showed a portion of exon 12 including the first 116 nucleotides was retained in the aberrant mRN transcript because of the activation of a cryptic splicing donor site (GTA) resided at codon 385–386. This is likely caused by the structural differences of the hybrid transcript produced from the exon-trapping construct.



View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Analysis of cis-acting effect of nonsense AAG to TAG mutation at codon 461 of exon 12 on mRNA splicing. A, structures of exon-trapping vector pSPL3 and derivatives containing either wild-type or mutant exon 12, the latter was derived from individual L1933-1. The vector-splicing donor and acceptor site is designated as SDv and SAv, respectively. SD6 and SA2 are the primer set used for RT-PCR amplification of cDNA templates generated from the transfected cos-7 cells. B, splicing products detected by RT-PCR analysis of mRNA purified from the transfected cos-7 cells. K461Stop, products detected from cells transfected with L1933-1 mutant exon 12 construct containing nonsense mutation at codon 461; Normal, product detected from cells transfected with construct containing the normal exon 12 DNA. The RT-PCR fragment representing the full-length or aberrant mRNA with exon 12 deletion is designated.

 
Interestingly, a full-length transcript was also detected by RT-PCR in cells transfected with mutant exon 12 construct (Fig. 2B)Citation . This result suggests that the effect of the nonsense mutation on exon-12 skipping is incomplete. As the result of this partial effect, two different MLH1 mRNA transcripts are derived from the same mutant MLH1 allele: an aberrant transcript lacking exon 12 and a full-length mRNA encompassing the AAG->TAG nonsense mutation. However, our initial sequencing analysis of MLH1 cDNA generated from these samples did not reveal the presence of the AAG->TAG change at codon 461. It is known that mRNA encompassing nonsense mutations is often unstable, possibly because of NMD (21) . This instability may be responsible for the difficulties we encountered in detecting the second MLH1 mRNA transcript carrying the nonsense mutation at codon 461. Previous experiments has shown that the TnT-coupled in vitro TnT system is a very sensitive assay in the detection of truncated protein products translated from an unstable transcript carrying a nonsense mutation (7) . If a second MLH1 mRNA transcript carrying the nonsense mutation at codon 461 is present in the germ-line HNPCC cells, in vitro translation of above RT-PCR products using the TnT system should reveal two truncated MLH1 proteins in addition to the wild-type one. Accordingly, in vitro translation analysis of RT-PCR products from affected carriers of the nonsense mutation at codon 461 revealed two truncated proteins (Mr 40,000 and Mr 53,000) and a full-length wild-type protein (Fig. 3A)Citation . As expected, the Mr-40,000 protein is the product of aberrant the MLH1 mRNA transcript deprived of exon 12, whereas the Mr-53,000 protein is the product of MLH1 mRNA transcript encompassing the nonsense mutation at codon 461. Furthermore, the two truncated protein bands translated from the mutant MLH1 mRNA transcripts seemed significantly weaker than the wild-type protein band, which is indicative of a decreased expression level of these truncated proteins.



View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Analysis of two aberrant MLH1 mRNA transcripts. A, in vitro coupled TnT analysis of RT-PCR products generated from a normal control (N) and affected individuals L1933-1, L3048-76, and L3551-1. To enhance the detection of mutant mRNA, cDNA sequences encoding codons 1–546 were PCR-amplified and translated by TnT. The two truncated proteins of Mr 53,000 and Mr 40,000 are evident in the RT-PCR products generated from affected individuals L1933-1, L3048-76, and L3551-1 and designated with arrowheads (results from other affected individuals of the three HNPCC families are not shown here). wt, full-length wild-type protein of Mr 63,000 representing open reading frame 1–546. B, sequence analysis of cloned RT-PCR products containing the exon-12 skipping in MLH1 mRNA transcripts. The ddA mixes from each sequencing reaction were loaded in adjacent lanes to facilitate comparison, as were those for C, G, and T. DNA clones representing the aberrant transcripts from L1933-1, L3048-76, and L3551-1 are displayed in Lanes 2–4, whereas DNA clones representing the wild-type transcript are displayed in Lanes 1 and 5, respectively. Exon-12 skipping is shown in Lanes 2–4. Arrow, the beginning of exon 12 sequences from the wild-type cDNA clones (Lanes 1 and 4) and of exon 13 sequences from the mutant cDNA clones (Lanes 2–4). C, sequencing analysis of cloned RT-PCR products containing nonsense mutation at codon 461. Arrowhead, the A to T mutation.

 
To provide direct evidence for the existence of the second aberrant transcript arising from the mutant MLH1 gene, cDNA generated from one affected member from each of the three HNPCC families were amplified by RT-PCR, and the products were cloned and analyzed. Analysis each of these clones by sequencing identified three types of DNA sequences representing each of the MLH1 mRNA transcripts as we have hypothesized above (Fig. 3, B and C)Citation . Finally, extended exposure of the sequencing gel revealed a faint band representing A to T changes at the codon 461 of exon 12 in cDNA from the affected individual of these families (data not shown).


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The results presented here demonstrate that MLH1 exon-12 skipping identified in three unrelated HNPCC families is caused by the cis-acting effect of an AAG->TAG nonsense mutation. The exon 12 skipping is associated with the affected HNPCC members carrying the nonsense mutation at codon 461 but was not observed in any of the family members who tested negative for the mutation. Our data suggest that exon skipping is directly associated with the affected individuals in these HNPCC families. Several studies have documented the presence of similar nonsense mutations within skipped exons, thus suggesting that it may represent a common mechanism of gene inactivation (21, 22, 23, 24) . Although the exact biochemical mechanism of exon skipping caused by the nonsense mutation is not completely understood, two models have been proposed to explain nonsense-associated altered splicing (22 , 23) . One suggests that exon skipping is caused by a nonsense mutation as the result of its effect on RNA secondary structure. According to the second hypothesis, nonsense codons could be recognized by the "nuclear scanning" machinery during the processing of nascent RNA transcripts. The latter process seems to be mediated by specific exonic sequences, the so-called ESEs and exonic splicing silencers, which are recognized and bound by specific splicing factors containing RNA recognition motifs and an Arg/Ser-rich region. ESEs are short (6–8 nt) degenerate sequence motifs that are usually, though not necessarily, purine-rich (24) . A recent study showed that BRCA1 exon-18 skipping is caused by the disruption of an ESE by a G->T nonsense mutation (25) . At present, it is not clear whether the nonsense mutation at codon 461 of the MLH1 gene will affect the binding capacity of a specific ESE or exonic splicing silencer. However, codon 461 of the MLH1 gene is located within a purine-rich region 29 bp upstream of the splice donor site of exon 12. It is therefore possible that the AAG->TAG mutation within exon 12 disrupts the purine-rich ESE and results in structural changes of pre-mRNA during splicing as proposed previously (26) .

The results of this study also demonstrate that the cis-acting effect of a nonsense mutation on the exon skipping is incomplete. This notion is in contrast with the effect caused by mutations at the most conserved positions in consensus donor or acceptor splice sites. As the result of this incomplete cis-acting effect, two MLH1 mRNA transcripts are produced from the mutant MLH1 allele encompassing the AAG->TAG nonsense mutation at codon 461. However, the presence of the nonsense mutation clearly affects mRNA stability. NMD has been documented in several human diseases (22 , 23 , 26) . Our current studies provide additional evidence supporting this notion. It is not clear whether NMD observed in our HNPCC families is attributable to the position effect of the mutation or to other mechanisms (22) .

The expression of two types of MLH1 transcripts from the same MLH1 mutant allele in HNPCC is of great interest. A single mutation encoding two different but equally nonfunctional products has not been observed previously in MSH2 or MLH1. On the basis of these studies, we hypothesize that the two aberrant transcripts are produced from the posttranscriptional processing of mutant pre-mRNA. Cells from HNPCC carriers of the codon 461 nonsense mutation express a wild-type and a mutant MLH1 hnRNA transcript. During mRNA processing, the cis-acting effect of the nonsense codon interferes with normal splicing and causes skipping of exon 12. Because the cis-acting effect of the nonsense codon is not complete, the full-length mRNA was also observed, though in significantly lower levels because of nonsense-mediated mRNA decay. It is likely that a similar mechanism should exist in other HNPCCs with germ-line nonsense mutation in MLH1 or MSH2.


   ACKNOWLEDGMENTS
 
We thank Ann Harty, Barbara Franklin, Janet Lynch, and Alicia Barrows for clinical coordination and sample collection and Juul Wijnen for his assistance in the mutation and haplotype analysis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the V-Foundation for Cancer Research, funds from the Nebraska Department of Health and Human Services, and NIH Grants CA74684-03 and CA81357-01. A. S. was the recipient of a Luisa Santunione Fellowship and was supported by the Associazione Italiana Ricerca sul Cancro and Ministero dell’Universitèa e Ricerca Scientifica e Tecnologica Cofin98 of Italy. A. W. is supported by a fellowship from the Dutch Cancer Society (KWF). Back

2 To whom requests for reprints should addressed, at Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands (R. F.) or University of Pittsburgh Cancer Institute, E1047BST, 200 Lothrop Street, Pittsburgh, PA 15213 (B. L.). Back

3 The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal cancer; MMR, DNA mismatch repair; RT-PCR, reverse transcription-PCR; DGGE, denaturing gradient gel electrophoresis; TnT, transcription and translation; NMD, nonsense-mediated mRNA decay; ESE, exonic splicing enhancer. Back

4 Family identification number. Back

5 Wagner et al., manuscript in preparation. Back

Received 4/27/01. Accepted 8/15/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Vasen H. F., Watson P., Mecklin J. P., Lynch H. T. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology, 116: 1453-1456, 1999.[Medline]
  2. Bronner C. E., Baker S. M., Morrison P. T., Warren G., Smith L. G., Lescoe M. K., Kane M., Earabino C., Lipford J., Lindblom A., et al Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature (Lond.), 368: 258-261, 1994.[Medline]
  3. Fishel R., Lescoe M. K., Rao M. R., Copeland N. G., Jenkins N. A., Garber J., Kane M., Kolodner R. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell, 77: 167 1994.[Medline]
  4. Miyaki M., Konishi M., Tanaka K., Kikuchi-Yanoshita R., Muraoka M., Yasuno M., Igari T., Koike M., Chiba M., Mori T. Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nat Genet., 17: 271-272, 1997.[Medline]
  5. Nicolaides N. C., Papadopoulos N., Liu B., Wei Y. F., Carter K. C., Ruben S. M., Rosen C. A., Haseltine W. A., Fleischmann R. D., Fraser C. M., et al Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature (Lond.), 371: 75-80, 1994.[Medline]
  6. Boland C. R., Thibodeau S. N., Hamilton S. R., Sidransky D., Eshleman J. R., Burt R. W., Meltzer S. J., Rodriguez-Bigas M. A., Fodde R., Ranzani G. N., Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res., 58: 5248-5257, 1998.[Abstract/Free Full Text]
  7. Liu B., Parsons R., Papadopoulos N., Nicolaides N. C., Lynch H. T., Watson P., Jass J. R., Dunlop M., Wyllie A., Peltomaki P., de la Chapelle A., Hamilton S. R., Vogelstein B., Kinzler K. W. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat. Med., 2: 169-174, 1996.[Medline]
  8. Nystrom-Lahti M., Wu Y., Moisio A-L., Hofstra R. M. W., Osinga J., Mecklin J. P., Jarvinen H. J., Leisti J., Buys C. H. C. M., de la Chapelle, Peltomaki P. DNA mismatch repair gene mutations in 55 kindreds with verified or putative hereditary non-polyposis colorectal cancer. Hum. Mol. Genet., 5: 763-769, 1996.[Abstract/Free Full Text]
  9. Peltomaki P., Vasen H. F. Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology, 13: 1146-1158, 1997.
  10. Wijnen J. T., Vasen H. F., Khan P. M., Zwinderman A. H., van der Klift H., Mulder A., Tops C., Moller P., Fodde R. Clinical findings with implications for genetic testing in families with clustering of colorectal cancer. N. Engl. J. Med., 339: 511-518, 1998.[Abstract/Free Full Text]
  11. Wijnen J., de Leeuw W., Vasen H., van der Klift H., Moller P., Stormorken A., Meijers-Heijboer H., Lindhout D., Menko F., Vossen S., Moslein G., Tops C., Brocker-Vriends A., Wu Y., Hofstra R., Sijmons R., Cornelisse C., Morreau H., Fodde R. Familial endometrial cancer in female carriers of MSH6 germline mutations. Nat Genet., 23: 142-144, 1999.[Medline]
  12. Wu Y., Berends M. J., Mensink R. G., Kempinga C., Sijmons R. H., van Der Zee A. G., Hollema H., Kleibeuker J. H., Buys C. H., Hofstra R. M. Association of hereditary nonpolyposis colorectal cancer-related tumors displaying low microsatellite instability with MSH6 germline mutations. Am. J. Hum. Genet., 65: 1291-1298, 1999.[Medline]
  13. Kohonen-Corish M., Ross V. L., Doe W. F., Kool D. A., Edkins E., Faragher I., Wijnen J., Khan P. M., Macrae F., St John D. J. RNA-based mutation screening in hereditary nonpolyposis colorectal cancer. Am. J. Hum. Genet., 59: 818-824, 1996.[Medline]
  14. Mori Y., Shiwaku H., Fukushige S., Wakatsuki S., Sato M., Nukiwa T., Horii A. Alternative splicing of hMSH2 in normal human tissues. Hum. Genet., 99: 590-595, 1997.[Medline]
  15. Charbonnier F., Martin C., Scotte M., Sibert L., Moreau V., Frebourg T. Alternative splicing of MLH1 messenger RNA in human normal cells. Cancer Res., 55: 1839-1841, 1995.[Abstract/Free Full Text]
  16. Genuardi M., Viel A., Bonora D., Capozzi E., Bellacosa A., Leonardi F., Valle R., Ventura A., Pedroni M., Boiocchi M., Neri G. Characterization of MLH1 and MSH2 alternative splicing and its relevance to molecular testing of colorectal cancer susceptibility. Hum. Genet., 102: 15-20, 1998.[Medline]
  17. Palmirotta R., Veri M. C., Curia M. C., Aceto G., D’Amico F., Esposito D. L., Arcuri P., Mariani-Costantini R., Messerini L., Mori S., Cama A., Battista P. Transcripts with splicings of exons 15 and 16 of the hMLH1 gene in normal lymphocytes: implications in RNA-based mutation screening of hereditary non-polyposis colorectal cancer. Eur. J. Cancer, 34: 927-930, 1998.
  18. Wijnen J., Khan P. M., Vasen H., Menko F., van der Klift H., van den Broek M., van Leeuwen-Cornelisse I., Nagengast F., Meijers-Heijboer E. J., Lindhout D., Griffioen G., Cats A., Kleibeuker J., Varesco L., Bertario L., Bisgaard M. L., Mohr J., Kolodner R., Fodde R. Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15–16. Am. J. Hum. Genet., 58: 300-307, 1996.[Medline]
  19. Gyapay G., Morissette J., Vignal A., Dib C., Fizames C., Millasseau P., Marc S., Bernardi G., Lathrop M., Weissenbach J. The 1992–94 Genethon human genetic linkage map. Nat. Genet., 7: 246-339, 1994.[Medline]
  20. Hutter P., Couturier A., Rey-Berthod C. Two common forms of the human MLH1 gene may be associated with functional differences. J. Med. Genet., 37: 776-781, 2000.[Free Full Text]
  21. Hentze M. W., Kulozik A. E. A perfect message: RNA surveillance and nonsense-mediated decay. Cell, 96: 307-310, 1999.[Medline]
  22. Valentine C. R. The association of nonsense codons with exon skipping. Mutat. Res., 411: 87-117, 1998.[Medline]
  23. Nystrom-Lahti M., Holmberg M., Fidalgo P., Salovaara R., de la Chapelle A., Jiricny J., Peltomaki P. Missense and nonsense mutations in codon 659 of MLH1 cause aberrant splicing of messenger RNA in HNPCC kindreds. Genes Chromosomes Cancer, 26: 372-375, 1999.[Medline]
  24. Blencowe B. J. Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem Sci., 25: 106-110, 2000.[Medline]
  25. Liu H. X., Cartegni L., Zhang M. Q., Krainer A. R. A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet., 27: 55-58, 2001.[Medline]
  26. Maquat L. E. The power of point mutations. Nat. Genet., 27: 5-6, 2001.[Medline]



This article has been cited by other articles:


Home page
 J. Med. Genet.Home page
S McVety, L Li, P H Gordon, G Chong, and W D Foulkes
Disruption of an exon splicing enhancer in exon 3 of MLH1 is the cause of HNPCC in a Quebec family
J. Med. Genet., February 1, 2006; 43(2): 153 - 156.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
E Renkonen, H Lohi, H J Jarvinen, J-P Mecklin, and P Peltomaki
Novel splicing associations of hereditary colon cancer related DNA mismatch repair gene mutations
J. Med. Genet., July 1, 2004; 41(7): e95 - e95.
[Full Text] [PDF]

Home page
 J. Med. Genet.Home page
P Lastella, N Resta, I Miccolis, A Quagliarella, G Guanti, and A Stella
Site directed mutagenesis of hMLH1 exonic splicing enhancers does not correlate with splicing disruption
J. Med. Genet., June 1, 2004; 41(6): e72 - e72.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stella, A.
Right arrow Articles by Liu, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stella, A.
Right arrow Articles by Liu, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online