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Genotype-phenotype correlation in APC familial and new mutation cases
Abstracts
B I O C H E M I C A L C H A R A C T E R I Z A T I O N O F A PRODUCT ENCODED BY T H E E M S 1 GENIE L O C A T E D IN T H E C H R O M O S O M E l l q 1 3 REGION AND A M P L I F I E D IN SEVERAL BREAST, HEAD AND N E C K C A R C I N O M A S . Henk van Damme., Ellen Scholtes, Herbert Brok & Ed Schuuring, Department of Pathology, University of Leiden, P.O Box 9603, 2300 RC Leiden, The Netherlands. EMS1 and CCND1 are two genes in the chromosome llq13 region, which are amplified
and overexpressed in 13 % of primary breast carcinomas and 28 % of squamons carcinomas derived from head and neck. Since these two genes are the only known genes that become independently overexpressed due to 1lq13 amplification (other genes such as FGF3 and FGF4 are not), we started to characterize the biological significance of these two genes in the development of malignancies. The gene product of CCND1 is cyclin D1, a growth factor regulated protein that is involved in the comrol of progression through the G1 phase of the cell cycle. Little is known about the role of the EMSI gene product, which appears in two forms (80 kDa and 85 kDa). The protein, from which the amino acid sequence has been resolved, consists of four distinct domains. Two of these domains, a seven-fold 34 amino acid repeat and the SH3 region, seem to be critical for its function. The SH3-domaln, located in the carboxyterminal part of the EMSI gene product, is also presem in several other proteins involved in the linkage between cytoskeletal elements and the plasmamembrane or the extracellular matrix. Moreover, studies, performed with the chicken homolog p80/85, referred to as cortactin, is tyrosine phosphorylated in chicken embryo cells transfected with ppr0 ' ~ . At the same time, cortactin is redistributed from cytoplasm to cell substratum contacts (podosomes). In addition, the repeat motif in cortactin has found to be the binding region for F-actin (Wu and Parsons, 1993). Because amplification of the llq13 region appears to be correlated with an elevated invasive behavinur of cells, we are trying to elucidate the function of the human EMS1 protein in adhesion and invasion. In two human squamous carcinoma cell lines (UMSCC2 and UMSCC8), the subcenniar localisation of the EMS1 protein was investigated by immunofluorescence. Surprisingly, in UMSCC2 cells with an 11-fold amplification of EMSI, the gene product was predominantly localized in podosomes. In UMSCC8 cells without this llq13 amplification, the EMS1 protein was only found in the cytoplasm. Presently, we are investigating whether tyrosine phosphorylation or other posttranslational modifications influence the localization of the EMS1 gene product. To further clarify the relation between the EMS1 gene product and adhesion, we investigate the biochemical properties such as phosphorylation, the presence of different isoforms and subcellular localisation in relation to the adhesive behaviour of cells transfected with EMS1 constructs.
CHARACTERIZATION OF A PRIMARY CELL CULTURE OBTAINED FROM AN ADENOMATOUS POLYP IN A PATIENT WITH FAMILIAL ADENOMATOUS POLYPOSIS (FAY). P. Grammatico (1),S. De Sanctis (1),C. Mordenti (1),L. Varesco (2),M.L. Lenti (1),C. Di Rosa (1),R. Gradini (i) and G. Del Porto (I). (I) Dipartimento di Medicina Sperimentale, Universitg "La Sapienza", c/o Osp. L. Spallanzani, V. Portuense 292 00149 Roma, Italy, (2) Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. The autors present cytogenetic, molecular, immundhisto= chemical, ultrastructural characterization of a primary :cell culture obtained from an adenomatous polyp in a patient with familial adenomatous polyposis (FAP). Single strand conformation polymorphism (SSPC) analysis of blood-derived DNA of the patient C.L.R. revealed the presence of a 5 base pair deletion (cAAAAG) at nucleotide position 3926-3930 of the APC gene confirmed also in other affected members of the family. Immunohistochemical and ultrastructural techniques were performed to a better characterization of the cell culture. Cytogenetic analysis evidentiated clonal aberrations involving chromosomes 3,8:and 9 and the results were confirmed by in situ hybridization. Moreover, as several genetic alteration occuring in colorectal tumorl = genesis are known, we characterized this cell line for the presence of somatic APC, ras and p53 mutations as well as loss of heterozygosity (LOH) and replication errors (RER).
171
Genotype-phenotype correlation in APC familial and new mutation cases. ~imon A. C~a.~ther,Dagan Wells, Siolmn B. SenGupta, Katta Tsioupra and Joy D.A. Delhaltty. Department of Genetics and Biometry, University College London, Wolfson House. 4 Stephenson Way, London NW1 2HE The inherited premalignant s y n d r o m e a d e n o m a t o u s polyposis coil (APC) is caused by germline mutation in the A P C gene at 5q21-22. Somatic m u t a t i o n in this g e n e is an early event in colorectal tumourigenesis. Both types of mutation are concentrated in the 5' half of exon 15 and exons 6,8,11 and 14, which together constitute 8 0 % of all mutations so far detected. W e have used single strand conformational p o l y m o r p h i s m (SSCP) and heteroduplex analysis to screen for variants in these regions of the gene in a total of 4 5 affected but unrelated individuals. Eighteen patients had no family history of the disease; of these 11 were classified as having a severe p h e n o t y p e , b a s e d on an e a r l y a g e at p r e s e n t a t i o n or c a n c e r development. This c o m p a r e s with 6 of 27 familial cases. A 5 b p deletion at codon 1309 reported to o c c u r in 10-15% of unselected A P C patients worldwide, w a s f o u n d in 5 of the 18 new mutation cases and 4 o f the 27 familial cases: all nine were classed as severe. A further 3 new mutations a n d 1 familial mutation were located
1"
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~h,..,~o~,o¢ /~ctd m~,t-a.~'oa~ pr:or Po ¢odo^ i 309.
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LINKAGE STUDIES COLORECTAL CANCER J. Wi~nen L e e u w e n I, Cornelisse Kleibeuker Khanl. 2 .
ON HEREDITARY IN T H E N E T H E R L A N D S .
NONPOLYPOSIS
I, L. S a n d k u i j l I, H. V a s e n z , C. van M. van den B r o e k I, I van LeeuwenI, F. M e n k o 2, F. N a g e n g a s t 2, A. C a t s z, J. 2, G. G r i f f i o e n 2, W. O s k a m 2 a n d P. M e e r a
iMGC-Department of Human Genetics, University, Leiden, The Netherlands; Workgroup on HNPCC, c/o the Foundation D e t e c t i o n of H e r e d i t a r y T u m o u r s .
Leiden 2 Dutch for the
In our search for the genes determining familial predisposition to hereditary nonpolyp o s i s c o l o r e c t a l c a n c e r (HNPCC), w e s c r e e n e d 277 polymorphic microsatellite markers dispersed on 22 a u t o s o m e s in 16 D u t c h f a m i l i e s w h i c h s a t i s f i e d the Amsterdam c r i t e r i a f o r HNPCC. O u r d a t a h a v e yielded significantly high positive lodscores for close linkage between COCA1 and the microsatellite marker D2S123 located in the chromosome 2p13-p16 region. T h e s e d a t a a r e in c o n f i r m a t i o n w i t h t h e r e c e n t r e p o r t b y P e l t o m ~ k i et a i . ( 1 9 9 3 ) . O u r p e a k l o d s c o r e w a s 3.57 a t 0 = 0 . 0 2 w h e n w e considered t h e a f f e c t e d s only; w h e n t h e a n a l y s i s was performed including all the family members, and using 16 p e n e t r a n c e classes, we obtained a p e a k l o d s c o r e of 4 . 2 0 a t 8 = 0 . 0 7 . Also several other markers including D2SI19, D2S147 and D2S136 known to be located in t h e vicinity of D 2 S 1 2 3 are beening screened. Based upon the results obtained from the multipoint and two-point analysis the orientation of these markers with respect to COCAI and the estimated interlocus genetic distances will be presented.
B I O C H E M I C A L C H A R A C T E R I Z A T I O N O F A PRODUCT ENCODED BY T H E E M S 1 GENIE L O C A T E D IN T H E C H R O M O S O M E l l q 1 3 REGION AND A M P L I F I E D IN SEVERAL BREAST, HEAD AND N E C K C A R C I N O M A S . Henk van Damme., Ellen Scholtes, Herbert Brok & Ed Schuuring, Department of Pathology, University of Leiden, P.O Box 9603, 2300 RC Leiden, The Netherlands. EMS1 and CCND1 are two genes in the chromosome llq13 region, which are amplified
and overexpressed in 13 % of primary breast carcinomas and 28 % of squamons carcinomas derived from head and neck. Since these two genes are the only known genes that become independently overexpressed due to 1lq13 amplification (other genes such as FGF3 and FGF4 are not), we started to characterize the biological significance of these two genes in the development of malignancies. The gene product of CCND1 is cyclin D1, a growth factor regulated protein that is involved in the comrol of progression through the G1 phase of the cell cycle. Little is known about the role of the EMSI gene product, which appears in two forms (80 kDa and 85 kDa). The protein, from which the amino acid sequence has been resolved, consists of four distinct domains. Two of these domains, a seven-fold 34 amino acid repeat and the SH3 region, seem to be critical for its function. The SH3-domaln, located in the carboxyterminal part of the EMSI gene product, is also presem in several other proteins involved in the linkage between cytoskeletal elements and the plasmamembrane or the extracellular matrix. Moreover, studies, performed with the chicken homolog p80/85, referred to as cortactin, is tyrosine phosphorylated in chicken embryo cells transfected with ppr0 ' ~ . At the same time, cortactin is redistributed from cytoplasm to cell substratum contacts (podosomes). In addition, the repeat motif in cortactin has found to be the binding region for F-actin (Wu and Parsons, 1993). Because amplification of the llq13 region appears to be correlated with an elevated invasive behavinur of cells, we are trying to elucidate the function of the human EMS1 protein in adhesion and invasion. In two human squamous carcinoma cell lines (UMSCC2 and UMSCC8), the subcenniar localisation of the EMS1 protein was investigated by immunofluorescence. Surprisingly, in UMSCC2 cells with an 11-fold amplification of EMSI, the gene product was predominantly localized in podosomes. In UMSCC8 cells without this llq13 amplification, the EMS1 protein was only found in the cytoplasm. Presently, we are investigating whether tyrosine phosphorylation or other posttranslational modifications influence the localization of the EMS1 gene product. To further clarify the relation between the EMS1 gene product and adhesion, we investigate the biochemical properties such as phosphorylation, the presence of different isoforms and subcellular localisation in relation to the adhesive behaviour of cells transfected with EMS1 constructs.
CHARACTERIZATION OF A PRIMARY CELL CULTURE OBTAINED FROM AN ADENOMATOUS POLYP IN A PATIENT WITH FAMILIAL ADENOMATOUS POLYPOSIS (FAY). P. Grammatico (1),S. De Sanctis (1),C. Mordenti (1),L. Varesco (2),M.L. Lenti (1),C. Di Rosa (1),R. Gradini (i) and G. Del Porto (I). (I) Dipartimento di Medicina Sperimentale, Universitg "La Sapienza", c/o Osp. L. Spallanzani, V. Portuense 292 00149 Roma, Italy, (2) Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. The autors present cytogenetic, molecular, immundhisto= chemical, ultrastructural characterization of a primary :cell culture obtained from an adenomatous polyp in a patient with familial adenomatous polyposis (FAP). Single strand conformation polymorphism (SSPC) analysis of blood-derived DNA of the patient C.L.R. revealed the presence of a 5 base pair deletion (cAAAAG) at nucleotide position 3926-3930 of the APC gene confirmed also in other affected members of the family. Immunohistochemical and ultrastructural techniques were performed to a better characterization of the cell culture. Cytogenetic analysis evidentiated clonal aberrations involving chromosomes 3,8:and 9 and the results were confirmed by in situ hybridization. Moreover, as several genetic alteration occuring in colorectal tumorl = genesis are known, we characterized this cell line for the presence of somatic APC, ras and p53 mutations as well as loss of heterozygosity (LOH) and replication errors (RER).
171
Genotype-phenotype correlation in APC familial and new mutation cases. ~imon A. C~a.~ther,Dagan Wells, Siolmn B. SenGupta, Katta Tsioupra and Joy D.A. Delhaltty. Department of Genetics and Biometry, University College London, Wolfson House. 4 Stephenson Way, London NW1 2HE The inherited premalignant s y n d r o m e a d e n o m a t o u s polyposis coil (APC) is caused by germline mutation in the A P C gene at 5q21-22. Somatic m u t a t i o n in this g e n e is an early event in colorectal tumourigenesis. Both types of mutation are concentrated in the 5' half of exon 15 and exons 6,8,11 and 14, which together constitute 8 0 % of all mutations so far detected. W e have used single strand conformational p o l y m o r p h i s m (SSCP) and heteroduplex analysis to screen for variants in these regions of the gene in a total of 4 5 affected but unrelated individuals. Eighteen patients had no family history of the disease; of these 11 were classified as having a severe p h e n o t y p e , b a s e d on an e a r l y a g e at p r e s e n t a t i o n or c a n c e r development. This c o m p a r e s with 6 of 27 familial cases. A 5 b p deletion at codon 1309 reported to o c c u r in 10-15% of unselected A P C patients worldwide, w a s f o u n d in 5 of the 18 new mutation cases and 4 o f the 27 familial cases: all nine were classed as severe. A further 3 new mutations a n d 1 familial mutation were located
1"
-'2
"
~h,..,~o~,o¢ /~ctd m~,t-a.~'oa~ pr:or Po ¢odo^ i 309.
--
LINKAGE STUDIES COLORECTAL CANCER J. Wi~nen L e e u w e n I, Cornelisse Kleibeuker Khanl. 2 .
ON HEREDITARY IN T H E N E T H E R L A N D S .
NONPOLYPOSIS
I, L. S a n d k u i j l I, H. V a s e n z , C. van M. van den B r o e k I, I van LeeuwenI, F. M e n k o 2, F. N a g e n g a s t 2, A. C a t s z, J. 2, G. G r i f f i o e n 2, W. O s k a m 2 a n d P. M e e r a
iMGC-Department of Human Genetics, University, Leiden, The Netherlands; Workgroup on HNPCC, c/o the Foundation D e t e c t i o n of H e r e d i t a r y T u m o u r s .
Leiden 2 Dutch for the
In our search for the genes determining familial predisposition to hereditary nonpolyp o s i s c o l o r e c t a l c a n c e r (HNPCC), w e s c r e e n e d 277 polymorphic microsatellite markers dispersed on 22 a u t o s o m e s in 16 D u t c h f a m i l i e s w h i c h s a t i s f i e d the Amsterdam c r i t e r i a f o r HNPCC. O u r d a t a h a v e yielded significantly high positive lodscores for close linkage between COCA1 and the microsatellite marker D2S123 located in the chromosome 2p13-p16 region. T h e s e d a t a a r e in c o n f i r m a t i o n w i t h t h e r e c e n t r e p o r t b y P e l t o m ~ k i et a i . ( 1 9 9 3 ) . O u r p e a k l o d s c o r e w a s 3.57 a t 0 = 0 . 0 2 w h e n w e considered t h e a f f e c t e d s only; w h e n t h e a n a l y s i s was performed including all the family members, and using 16 p e n e t r a n c e classes, we obtained a p e a k l o d s c o r e of 4 . 2 0 a t 8 = 0 . 0 7 . Also several other markers including D2SI19, D2S147 and D2S136 known to be located in t h e vicinity of D 2 S 1 2 3 are beening screened. Based upon the results obtained from the multipoint and two-point analysis the orientation of these markers with respect to COCAI and the estimated interlocus genetic distances will be presented.