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The genetics of colorectal cancer
Abstracts
R A P I D D E T E C T I O N O F A P C M U T A T I O N S IN F A P F A M I L I E S A N D C O L O R E C T A L T U M O R S BY P R O T E I N T R U N C A T I O N T E S T R.B. van der L u i j t z , P. Meera K h a n I, H . F . A . V a s e n 2, C.M.J. T o p s 1, C. v a n L e e u w e n I, C. B r e u k e l I, P.A.M. R o e s t I, J.T. den D u n n e n I, I.S.J. van L e e u w e n - C o r n e l i s s e 1,2 a n d R. F o d d e z. ZMGC-Department of Human Genetics, Leiden University, Leiden, The Netherlands. 2Foundation for the Detection of Hereditary Tumors, Leiden, The Netherlands. The adenomatous polyposis coli (APC) g e n e p l a y s an essential r o l e in c o l o r e c t a l carcinogenesis. D u e t o t h e l a r g e c o d i n g r e g i o n of A P C (8.5 kb) and the extremely heterogeneous spectrum of mutations, t h e i d e n t i f i c a t i o n of A P C m u t a t i o n s in familial adenomatous polyposis (FAP) and colorectal cancer using the currently available methods such as SSCP, RPA and DGGE is very laborious and time-consuming. In order to facilitate the direct identification of APC mutations, we developed a rapid and sensitive screening procedure, called the protein t r u n c a t i o n t e s t (PTT). T h e P T T is b a s e d on t h e i n vitro coupled transcription and translation of PCR products, and permits the detection of s t o p codons in large stretches of c o d i n g sequence. Using the PTT directed to a mutation-dense region of APC, we identified truncating germ line mutations in a b o u t 50% of t h e F A P p a t i e n t s . In o r d e r t o i n v e s t i g a t e t h e 'second hit' h y p o t h e s i s of APC, t h e s a m e s e g m e n t of A P C w a s s c r e e n e d f o r somatic mutations in m u l t i p l e a d e n o m a t o u s polyps from a single FAP patient. T h i s r e s u l t e d in t h e detection of t r u n c a t i n g APC mutations in 9 0 % of the tumor samples. Our studies indicate that the PTT is an ideal approach for the mutation a n a l y s i s of A P C in F A P a n d c o l o r e c t a l c a n c e r .
THE GENETICS OF COLORECTAL CANCER. Albert de la Chapelle, Department of Medical Genetics, University of Helsinki, 00290 Helsinki Finland That gene mutations play a fundamental role in the pathogenesis of colorectal cancer has long been realized. Our present views of these processes have evolved gradually. First somatic mutations in a number of genes established that n o r m a l cells become cancer cells t h r o u g h a multistep process. Subsequently t u m o r s u p p r e s s o r genes causing cancer susceptibility by triggering this process were identified. Recently a new class of genes was detected whose mutations confer high susceptibility to cancer by inducing mutations in other genes. These processes will be highlighted.
151
AN IMPROVED PROCEDURE TO QUICKLY ISOLATE AND SEQUENCE THE TERMINI OF DNA INSERTS OF YEAST ARTIFICIAL CHROMOSOMES. Jan M.W. Geurts, Eric F.P.M. Schoenmakers, R. Mols, Herman Van den Berghe, end Wim J.M. Van de Ven. Center for Human Genetics, University of Leuven, Herestraat 49, 3000 Leuven, Belgium. The development of Yeast Artificial Chromosome (YAC) vectors (Burke and Olson, Science 236:806-812, 1987), which has made it possible to molecularly clone large DNA segments of up to more than 1000 kbp, has provided a strong impetus to ongoing efforts of generating long range physical maps of genomes and positional cloning of genes. Such long range physical maps are often obtained from large YAC contigs, collections of overlapping YAC clones mostly isolated by a process known as chromosome walking. An important step in chromosome walking is the isolation and/or nucleotide sequence analysis of the termini of YAC inserts. Such sequence data can be used as sequence tagged sites (STS's) to screen DNA pools of YAC clones by polymerase chain reaction (PCR) and to generate probes (PCR products) to identify individual YAC clones by filter hybridization in a final round of screening. To facilitate chromosome walking, we have developed a streamlined procedure to quickly isolate and sequence termini of YAC-inserts. We have modified the protocol for vectorette-PCR of Riley eta/. (Nucleic Acids Res. 18:2887-2890, 1990) and combined it with direct solid phase fluorescent sequencing of PCR products. The modified protocol combines speed with the high success rate of vectorette-PCR. The improved procedure and its application will be presented and discussed.
IMPRINTING IN DEVELOPMENT AND DISEASE. W. Reik, R. Feil, J. Walter and N.D. Allen, Laboratory of Developmental Genetics and Imprinting, AFRC Babraham Institute, Eabrabam, cambridge CE2 4AT, UK. Epigenetic modifications can be introduced into the mammalian genome at various stages of development. Parental imprinting is presumably initiated in gametes or at fertilisation. We are studying the mouse insulin like growth factor 2 (Igf2) gene, whose maternally inherited allele is largely repressed in the foetus. In mice carrying only maternally inherited alleles, this deficiency of Igf2 can lead to growth retardation. We find that the repressed copy of Igf2 is potentially active for transcription: DNase I hypersensitive sites are retained, there is no CpG island methylation, and primary transcripts are d e t e c t e d at low but significant levels. Parent specific methylation differences are detected in a region several kb upstream of the first exon and in an intronic region. These allelic methylations are mosaic in nature and arise in the early embryo (together with allelic methylation of the neigbbouring H19 gene} on differentiation of stem cells. Allelic methylation for both genes adopts a paternal type pattern in choroid plexus, a brain tissue in which Igf2 is biallelically expressed. An understanding of imprinting and epiganetic programming at the molecular level will require identification of genes that control these processes, namely modifier genes. We have set up a genetic screen in which a m a j o r dominant m o d i f i e r locus segregates, with the aim of identifying and c l o n i n g the gena. This backcross is now being characteriaed with a genome-spanning panel of microsatellite markers and two loci have b e e n d e t e c t e d that show specific associations with the phenotypes. The major gene has been located to a region on proximal chromosome 17. Characterisation of modifiers will be important in the understanding of imprinting and epigenetic inheritance and will also provide insight into the m a n y genetic disorders that are influenced by variant modifier alleles.
R A P I D D E T E C T I O N O F A P C M U T A T I O N S IN F A P F A M I L I E S A N D C O L O R E C T A L T U M O R S BY P R O T E I N T R U N C A T I O N T E S T R.B. van der L u i j t z , P. Meera K h a n I, H . F . A . V a s e n 2, C.M.J. T o p s 1, C. v a n L e e u w e n I, C. B r e u k e l I, P.A.M. R o e s t I, J.T. den D u n n e n I, I.S.J. van L e e u w e n - C o r n e l i s s e 1,2 a n d R. F o d d e z. ZMGC-Department of Human Genetics, Leiden University, Leiden, The Netherlands. 2Foundation for the Detection of Hereditary Tumors, Leiden, The Netherlands. The adenomatous polyposis coli (APC) g e n e p l a y s an essential r o l e in c o l o r e c t a l carcinogenesis. D u e t o t h e l a r g e c o d i n g r e g i o n of A P C (8.5 kb) and the extremely heterogeneous spectrum of mutations, t h e i d e n t i f i c a t i o n of A P C m u t a t i o n s in familial adenomatous polyposis (FAP) and colorectal cancer using the currently available methods such as SSCP, RPA and DGGE is very laborious and time-consuming. In order to facilitate the direct identification of APC mutations, we developed a rapid and sensitive screening procedure, called the protein t r u n c a t i o n t e s t (PTT). T h e P T T is b a s e d on t h e i n vitro coupled transcription and translation of PCR products, and permits the detection of s t o p codons in large stretches of c o d i n g sequence. Using the PTT directed to a mutation-dense region of APC, we identified truncating germ line mutations in a b o u t 50% of t h e F A P p a t i e n t s . In o r d e r t o i n v e s t i g a t e t h e 'second hit' h y p o t h e s i s of APC, t h e s a m e s e g m e n t of A P C w a s s c r e e n e d f o r somatic mutations in m u l t i p l e a d e n o m a t o u s polyps from a single FAP patient. T h i s r e s u l t e d in t h e detection of t r u n c a t i n g APC mutations in 9 0 % of the tumor samples. Our studies indicate that the PTT is an ideal approach for the mutation a n a l y s i s of A P C in F A P a n d c o l o r e c t a l c a n c e r .
THE GENETICS OF COLORECTAL CANCER. Albert de la Chapelle, Department of Medical Genetics, University of Helsinki, 00290 Helsinki Finland That gene mutations play a fundamental role in the pathogenesis of colorectal cancer has long been realized. Our present views of these processes have evolved gradually. First somatic mutations in a number of genes established that n o r m a l cells become cancer cells t h r o u g h a multistep process. Subsequently t u m o r s u p p r e s s o r genes causing cancer susceptibility by triggering this process were identified. Recently a new class of genes was detected whose mutations confer high susceptibility to cancer by inducing mutations in other genes. These processes will be highlighted.
151
AN IMPROVED PROCEDURE TO QUICKLY ISOLATE AND SEQUENCE THE TERMINI OF DNA INSERTS OF YEAST ARTIFICIAL CHROMOSOMES. Jan M.W. Geurts, Eric F.P.M. Schoenmakers, R. Mols, Herman Van den Berghe, end Wim J.M. Van de Ven. Center for Human Genetics, University of Leuven, Herestraat 49, 3000 Leuven, Belgium. The development of Yeast Artificial Chromosome (YAC) vectors (Burke and Olson, Science 236:806-812, 1987), which has made it possible to molecularly clone large DNA segments of up to more than 1000 kbp, has provided a strong impetus to ongoing efforts of generating long range physical maps of genomes and positional cloning of genes. Such long range physical maps are often obtained from large YAC contigs, collections of overlapping YAC clones mostly isolated by a process known as chromosome walking. An important step in chromosome walking is the isolation and/or nucleotide sequence analysis of the termini of YAC inserts. Such sequence data can be used as sequence tagged sites (STS's) to screen DNA pools of YAC clones by polymerase chain reaction (PCR) and to generate probes (PCR products) to identify individual YAC clones by filter hybridization in a final round of screening. To facilitate chromosome walking, we have developed a streamlined procedure to quickly isolate and sequence termini of YAC-inserts. We have modified the protocol for vectorette-PCR of Riley eta/. (Nucleic Acids Res. 18:2887-2890, 1990) and combined it with direct solid phase fluorescent sequencing of PCR products. The modified protocol combines speed with the high success rate of vectorette-PCR. The improved procedure and its application will be presented and discussed.
IMPRINTING IN DEVELOPMENT AND DISEASE. W. Reik, R. Feil, J. Walter and N.D. Allen, Laboratory of Developmental Genetics and Imprinting, AFRC Babraham Institute, Eabrabam, cambridge CE2 4AT, UK. Epigenetic modifications can be introduced into the mammalian genome at various stages of development. Parental imprinting is presumably initiated in gametes or at fertilisation. We are studying the mouse insulin like growth factor 2 (Igf2) gene, whose maternally inherited allele is largely repressed in the foetus. In mice carrying only maternally inherited alleles, this deficiency of Igf2 can lead to growth retardation. We find that the repressed copy of Igf2 is potentially active for transcription: DNase I hypersensitive sites are retained, there is no CpG island methylation, and primary transcripts are d e t e c t e d at low but significant levels. Parent specific methylation differences are detected in a region several kb upstream of the first exon and in an intronic region. These allelic methylations are mosaic in nature and arise in the early embryo (together with allelic methylation of the neigbbouring H19 gene} on differentiation of stem cells. Allelic methylation for both genes adopts a paternal type pattern in choroid plexus, a brain tissue in which Igf2 is biallelically expressed. An understanding of imprinting and epiganetic programming at the molecular level will require identification of genes that control these processes, namely modifier genes. We have set up a genetic screen in which a m a j o r dominant m o d i f i e r locus segregates, with the aim of identifying and c l o n i n g the gena. This backcross is now being characteriaed with a genome-spanning panel of microsatellite markers and two loci have b e e n d e t e c t e d that show specific associations with the phenotypes. The major gene has been located to a region on proximal chromosome 17. Characterisation of modifiers will be important in the understanding of imprinting and epigenetic inheritance and will also provide insight into the m a n y genetic disorders that are influenced by variant modifier alleles.