This page has been archived and is no longer updated
MSH2 genomic deletions are a frequent cause of HNPCC.
Author: Wijnen, J., et. al.
Keywords
Keywords for this Article
Add keywords to your Content
Save
|
Cancel
Share
|
Cancel
Revoke
|
Cancel
Rate & Certify
Rate Me...
Rate Me
!
Comment
Save
|
Cancel
Flag Inappropriate
The Content is
Objectionable
Explicit
Offensive
Inaccurate
Comment
Flag Content
|
Cancel
Delete Content
Reason
Delete
|
Cancel
Close
Full Screen
"correspondence 326 nature genetics volume 20 december 1998 our assay, both Gli3C�D ClaI, which approximates the PAP-A form, and Gli3C�D Bal#8, which approximates the PHS form, bind Smads. Our findings suggest a physiologically relevant interaction between C-termi- nally truncated Gli3 proteins and Smads. C-terminally truncated Gli proteins are nuclear repressors that may resemble endogenous processed forms (A.R.A., submitted). Thus, production of C-termi- nally truncated Gli3 mutant forms, as in PHS, may inhibit activating Gli function and may also affect the outcome of TGFb - family signalling in tissues that express Gli3. How Gli-Smad complexes act, how- ever, is not clear. Full-length or truncated forms of Gli1 or Gli3 were unable to dif- ferentially affect transcriptional activity from the BMP-inducible Vent2 enhancer 13 , the TGFb /activin-inducible Mix2 promoter 14 , or by Gal4-Smad2 from a Gal4 reporter gene 10 (data not shown). It remains possible that the Gli3-Smad complex has novel binding or transcrip- tional specificities. One role Smad-Gli3 (and possibly Smad-Gli2) complexes could have is to partially coordinate the actions of the two regulatory systems. For example, in instances where Shh and BMP signalling act antagonistically, BMP- induced dissociation of truncated Gli3- Smad complexes could induce a two-tier antagonism of the Shh pathway. On one hand, Smads would be free to bind spe- cific partners to induce BMP-responsive gene expression. On the other hand, C- terminally truncated Gli3 proteins would antagonize the activating function of Gli1 and Gli2, and thus Shh signalling. In addition, complex formation may occur in the cytoplasm where inactive Smads normally reside 9 , suggesting that depend- ing on the relative abundance of each pro- tein, Smads could render Gli3 repressors inactive by cytoplasmic sequestration until signalling occurs. The regulation of the production of C-terminally deleted forms would thus appear to be critical for determining the signalling outcome. Acknowledgements This work was supported by NIH grants (NS37352) to A.R.A. and (CA34610) to J.M., a Basil O?Connor Award from the March of Dimes and a Pew fellowship to A.R.A. and a Cancer Center grant to MSKCC. J.M. is an investigator of the Howard Hughes Medical Institute. MSH2 genomic deletions are a frequent cause of HNPCC H ereditary non-polyposis colorectal cancer (HNPCC) is a common, autosomal dominant, cancer susceptibility condition characterized by early onset col- orectal cancer 1 . HNPCC is caused by germline mutations in one of five DNA mismatch repair genes (MMR): MSH2 (ref. 2), MLH1 (refs 3,4), PMS1 (ref. 5), PMS2 (ref. 5) and MSH6 (refs 6,7). To date, more than 200 different predisposing mutations in these genes have been charac- terized in HNPCC patients, the majority of which occur in MSH2 and MLH1 (ref. 8; see also http://www.nfdht.nl/index.htm). Here, we report that genomic deletions at MSH2 also represent a frequent cause of HNPCC. In fact, these deletions comprise more than one-third of all pathogenic MSH2 mutations among Dutch HNPCC families and account for 6.5% of HNPCC defined by the Amsterdam criteria. In previous studies 9,10 , we determined the prevalence of mutations at MSH2 and MLH1 among 184 kindreds, 92 of which comply with the Amsterdam criteria (AMS+) and 92 of which have a familial clustering of colorectal cancer reminiscent of HNPCC (AMS- ). Approximately one- half (41) of AMS+ families revealed a pathogenic germline mutation in MSH2 or MLH1, whereas only 6 of 92 AMS- families had a mutation in either gene. In the present study, the remaining 137 fami- lies, 51 AMS+ and 86 AMS- , were investi- gated by Southern-blot analysis of genomic DNA. We analysed NsiI, EcoRI and HindIII genomic digests with two dif- ferent MSH2 cDNA probes encompassing exons 1- 7 (5� probe) and exons 4- 16 (3� probe), respectively. Eight patients, six of which are from AMS+ families, revealed aberrant restriction fragments with these enzymes when hybridized with a 5� MSH2 probe, thus indicating the presence of genomic rearrangements. Of the four dis- tinct restriction patterns indicative of a genomic deletion in MSH2, three were identified in more than one kindred (Fig. 1). Hybridization with exon-specific probes revealed the presence of a deletion of approximately 2.1 kb encompassing exon 1 (NLB50162), a 5.4-kb deletion of exon 2 (NA17 and NA86), an exon 3 dele- tion of approximately 2.2 kb (NA64, NLB50696 and NLB51971) and a large (approximately 13 kb) deletion of exon 6 (NA33 and NLB50490; Fig. 1a?c). In four kindreds (NA17, NA33, NA64 and NLB51971) from which more family members were available, the genomic deletion co-segregated with the disease phenotype (data not shown). The dele- tions of exon 3 and 6 were confirmed by nucleotide sequencing of the shorter RT- PCR product from affected individuals (Fig. 2). No RNA was available from the carriers of the other deletions. To date, only two germline genomic deletions in MLH1 have been described, both resulting from recombination between two Alu- repeats located in introns 12, 15 and 16 (refs 11,12). Moreover, a deletion of MLH1 exons 4- 19 has also been reported in a mismatch repair-deficient cell line 13 . All the filters we employed for the South- Fang Liu 1,3 , Joan Massagu� 1 & Ariel Ruiz i Altaba 2 1 Memorial Sloan-Kettering Cancer Center, Cell Biology Program and Howard Hughes Medical Institute, New York, New York 10021, USA. 2 The Skirball Institute, Developmental Genetics Program and Department of Cell Biology, NYU School of Medicine, 540 First Avenue, New York, New York 10016, USA. 3 Present address: Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA. Correspondence should be addressed to A.R.A. (e-mail: ria@saturn.med.nyu.edu) or J.M. (e-mail: j-massague@ski.mskcc.org). 1. Lee, J. et al. Development 124, 2537?2552 (1997). 2. Sasaki H. et al. Development 124, 1313?1322 (1997). 3. Ruiz i Altaba, A. Development 125, 2203?2212 (1998). 4. Matise, M. et al. Development 125, 2759?2770 (1998). 5. Ding, G. et al. Development 125, 2533?2543 (1998). 6. Dunn, N.R. et al. Dev. Biol. 188, 235?247 (1997). 7. Kang, S. et al. Nature Genet. 15, 266?268 (1997). 8. Radhakrishna, U. et al. Nature Genet. 17, 269?271 (1997). 9. Massagu�, J. Annu. Rev. Biochem. 67, 753?791 (1998). 10. Liu, F., Pouponnot, C. & Massagu�, J. Genes Dev. 11, 3157?3167 (1997). 11. Chen, X., Rubock, M.J. & Whitman, M. Nature 383, 691?696 (1996). 12. Pavletich, N.P. & Pabo, C.O. Science 261, 1701?1707 (1993). 13. Candia, A.F. et al. Development 124, 4467?4480 (1997). 14. Huang, H.-C. et al. EMBO J. 14, 5965?5973 (1995). � 1998 Nature America Inc. ? http://genetics.nature.com � 1998 Nature America Inc. ? http://genetics.nature.com correspondence nature genetics volume 20 december 1998 327 ern analysis of MSH2 were also hybridized with MLH1 cDNA probes to assay the presence of deletions in the chromosome 3p locus; no aberrant restriction pattern indicative of a genomic rearrangement at the MLH1 locus was found with the restriction endonucleases used here. Little is known about the number or location of Alu-repeats in MSH2, however, MSH2 deletions encompassing exon 1, exons 1- 6 and exons 4- 8 have been detected 13- 15 . The presence of recurring deletions in several HNPCC families could be indicative of a founder mutation or of the independent occurrence of the same rearrangement due to the presence of recombinogenic sequences such as Alu repeats. Haplotype analysis of the kin- dreds sharing the same deletions failed to show evidence of a founder effect (data not shown). These observations indicate that the deletions reported here arose independently through a common recom- bination event. In the present study, the HNPCC series comprises 86 Dutch and 51 Norwegian families. The eight genomic deletions were found exclusively in kindreds of Dutch origin; thus, genomic deletions may show inter-ethnic differences and could be prevalent in the Dutch population, as was previously reported for BRCA1 (ref. 16). We had already reported 19 MSH2 (16 in AMS+) and 28 MLH1 (25 in AMS+) pathogenic mutations by DGGE (refs 9,11) in our set of 92 HNPCC and 92 HNPCC-like families. Therefore, MSH2 accounts for 24% of HNPCC defined by the Amsterdam criteria, 6.5% (6/92) due to genomic deletions and 17% (16/92) to point mutations. Moreover, the eight genomic deletions comprise 30% (8/27) of all mutations detected in MSH2, and 36% (8/22) of those found among Dutch HNPCC families. Our results indicate that genomic dele- tions at MSH2 are a frequent cause of dis- ease among HNPCC patients. These findings have implications for the improvement of HNPCC mutation screening protocols, as deletions will often escape detection by currently employed methods such as SSCP, DGGE, heterodu- plex-analysis and direct sequencing of genomic DNA. RNA-based mutation detection technologies such as RT-PCR and PTT may also fail to detect such rearrangements due to complete deletion of the gene, or because of the instability of Fig. 2 Nucleotide sequence analysis of deleted MSH2 transcripts. RT-PCR of exons 1- 7 from patients NA64 and NLB50490 yielded the expected 1.3-kb fragment in addition to two shorter products of approximately 1.0 and 1.15 kb, respectively. Sequence analysis of the latter fragments revealed the loss of exon 3 (top) and 6 (bottom) sequences. Fig. 1 Mapping of MSH2 deletions responsible for HNPCC. a, NsiI digests from patients NA64, NLB 50696 and NLB 51971 hybridized with the 5? MSH2 probe revealed the constitutional 10.6-kb band encompassing exons 2- 6 and an aberrant 8.4-kb fragment. Additional hybridizations with exon- specific probes indicated an approximately 2.2-kb deletion encompassing exon 3 (p2, p3 and p4). b, EcoRI digests from kindreds NA33 and NLB50490 hybridized with the 5? MSH2 probe showed an additional 5.7-kb band as well as the constitutional 9.2-kb band. Additional analyses with BamHI and exon-specific probes (data not shown) revealed that the 9.2-kb band resulted from the comigration of two EcoRI fragments encompassing exons 4- 6 and exon 7. EcoRI digests hybridized with exon-specific probes were indica- tive of an approximately 13-kb deletion encom- passing exon 6 (p5, p6 and p7). c, In NA17 and NA86, HindIII digestion and hybridization with the 5? probe revealed a 10.6-kb fragment encompassing exons 1- 2 and an aberrant 8.0-kb band. Hybridization with exon-specific probes (p2 and p3) and addi- tional NsiI and BamHI digests (data not shown) confirmed the presence of an exon 2 deletion of approximately 5.4 kb. Hybridization of HindIII digests from NLB50162 with an exon 2-specific probe showed an aberrant 8.5-kb band. The same band was not recognized by the exon 1-specific probe, thus indicating the presence of a 2.1-kb deletion encompassing exon 1 (p1 and p2). d, Schematic representation of the deletion types found in MSH2. Deletions are depicted as solid bars under the corresponding region of the 5? genomic structure of MSH2; dashed lines indicate that the deletion breakpoints have not been charac- terized. Note that the location of the intronic restriction sites (N, NsiI; E, EcoRI; H, HindIII) is approximate, as it has been inferred from Southern analysis. a b c d � 1998 Nature America Inc. ? http://genetics.nature.com � 1998 Nature America Inc. ? http://genetics.nature.com correspondence 328 nature genetics volume 20 december 1998 the altered mRNA. Therefore, a thorough mutation analysis of MSH2 should include the examination of its genomic structure by Southern analysis. Acknowledgements The authors are grateful to M. de Almeida for technical assistance and the following clinicians for contributing their HNPCC families: F. Nagengast, A. Br�cker-Vriends, G. Griffioen, A. Cats and J. Kleibeuker. This work has been supported in part by the Dutch Cancer Society and Praeventiefonds (project no. PRF 28-1383-1). Juul Wijnen 1 , Heleen van der Klift 1 , Hans Vasen 2 , P. Meera Khan 1 , Fred Menko 3 , Carli Tops 4 , Hanne Meijers Heijboer 5 , Dick Lindhout 5 , Allelic variation in ABCR associated with Stargardt disease but not age-related macular degeneration A ge-related macular degeneration (AMD) is a potentially blinding dis- ease that has been estimated to affect as many as 30% of people over age 65 (refs 1,2). Clinical manifestations of AMD include deposition of debris within and beneath the retinal pigment epithelium (RPE), atrophy of the RPE and haemor- rhage and exudation beneath the retina from aberrant choroidal blood vessels. The latter complication (sometimes referred to as ?wet? AMD) occurs in approximately 10% of eyes with AMD overall, but is present in approximately 90% of eyes that have become legally blind from this condition 3 . There is no evidence at this time that patients with this compli- cation have a pathophysiologically distinct form of macular degeneration. Recently, mutations in a gene (ABCR) encoding an ATP-binding transmembrane transporter protein have been associated with Stargardt disease 4?6 , an autosomal recessive retinal disease that, similar to AMD, affects the central retina (macula). Mutations in ABCR were later reported to be associated with up to 16% of AMD (ref. 7), although the methodology used in this study was controversial (http://www. sciencemag.org/cgi/content/full/279/5354/ 1107a). To further investigate the role of ABCR in Stargardt disease and AMD, we studied three populations: (i) 215 individ- uals with a clinical diagnosis of Stargardt disease; (ii) 182 patients with AMD diag- nosed at the University of Iowa; and (iii) 96 unrelated subjects also from Iowa. The latter group was chosen to represent an ethnically matched sample of the popula- tion which would be expected to develop the population rate of AMD. Despite the loss of power, we decided not to choose an elderly, ?AMD free? control group, as this would require the subjective differentia- tion between the retinal changes involved in normal ageing and early AMD, a dis- tinction which remains poorly under- stood. Sixty percent of the AMD group had, by the time of the study, developed a choroidal neovascular membrane in at least one eye. This rate of choroidal neo- vascularization is typical for a retina spe- cialty clinic of a tertiary care hospital, and reflects the fact that the more severely affected AMD patients in a population are more likely to be cared for in such a venue than their less affected family members. Our study was designed to compare the three groups with respect to the propor- tion of non-conservative nucleotide Table 1 ? Distribution of ABCR sequence variants among three study groups Controls AMD Stargardt Total (n=96) (n=182) (n=215) non-conservative changes 26 59 204 289 conservative missense changes 12 20 92 124 synonymous codon changes 96 177 316 589 intronic changes 49 82 188 319 total 183 338 800 1321 51 Primer pairs 4 were used for SSCP analysis of the entire coding sequence as well as all exon-intron boundaries of the 50 exons of ABCR in 493 individuals. Amplimers showing a band shift were reamplified and sequenced using an ABI 373 automated sequencer. Non-conservative variants were defined as those that would be expected to cause a change in the charge, polarity, or number of amino acids of ABCR. Table 2 ? Individuals harbouring non-conservative ABCR variants Controls AMD Stargardt (n=96) (n=182) (n=215) all non-conservative changes 26 57 137 (P=0.49) (P<0.0001) rare non-conservative changes 2 3 82 (P=1.0) (P<0.0001) The number of AMD and Stargardt patients harbouring one or more non-conservative ABCR variants was compared with the number of control subjects with such changes. P-values were calculated with Fisher?s exact test. The 52 ?rare variants? were all present in less than 1% of controls. The 3 common variants (Asn1868Ile, Arg943Gln and Ser2255Ile) were all present in more than 4% of all 3 groups. P�l M�ller 6 & Riccardo Fodde 1* 1 MGC-Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. 2 The Foundation for the Detection of Hereditary Tumors, and Department of Gastroenterology, Leiden University Medical Center, Leiden, The Netherlands. 3 Department of Clinical Genetics, Free University Hospital, Amsterdam, The Netherlands. 4 Clinical Genetic Center Leiden, Leiden University Medical Center, Leiden, The Netherlands. 5 MGC-Department of Clinical Genetics, Erasmus University Rotterdam, Rotterdam, The Netherlands. 6 The Norwegian Radium Hospital, Oslo, Norway. Correspondence should be addressed to R.F. (e-mail: fodde@ruly46.medfac.leidenuniv.nl). 1. Lynch, H.T. et al. Gastroenterology 104, 1535- 1549 (1993). 2. Leach, F.S. et al. Cell 75, 1215- 1225 (1993). 3. Papadopoulos, N. et al. Science 263, 1625- 1629 (1994). 4. Bronner, C.E. et al. Nature 368, 258- 261 (1994). 5. Nicolaides, N.C. et al. Nature 371, 75- 80 (1994). 6. Akiyama, Y. et al. Cancer Res. 57, 3920- 3923 (1997). 7. Miyaki, M. et al. Nature Genet. 17, 271- 272 (1997). 8. Peltom�ki, P. et al. Gastroenterology 113, 1146- 1158 (1997). 9. Wijnen, J.T. et al. Am. J. Hum. Genet. 61, 329- 335 (1997). 10. Wijnen, J.T. et al. N. Engl. J. Med. 339, 511- 518 (1998). 11. Nystr�m-Lahti, M. et al. Nature Med. 1, 1203- 1206 (1995). 12. Mauillon, J.L. et al. Cancer Res. 56, 5728- 5733 (1996). 13. Boyer, J.C. et al. Cancer Res. 55, 6063- 6070 (1995). 14. Liu, B. et al. Nature Genet. 9, 48- 55 (1995). 15. Papadopoulos, N. et al. Nature Genet. 11, 99- 101 (1995). 16. Petrij-Bosch, A. et al. Nature Genet. 17, 341- 345 (1997). � 1998 Nature America Inc. ? http://genetics.nature.com � 1998 Nature America Inc. ? http://genetics.nature.com "
Add Content to Group
|
Bookmark
|
Keywords
|
Flag Inappropriate
share
Close
Digg
Facebook
MySpace
Google+
Comments
Close
Please Post Your Comment
*
The Comment you have entered exceeds the maximum length.
Submit
|
Cancel
*
Required
Comments
Please Post Your Comment
No comments yet.
Save Note
Note
View
Public
Private
Friends & Groups
Friends
Groups
Save
|
Cancel
|
Delete
Please provide your notes.
Next
|
Prev
|
Close
|
Edit
|
Delete
Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
Loading ...
Scitable Chat
Register
|
Sign In
Visual Browse
Close
Comments
CloseComments
Please Post Your Comment