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Chromosomal studies in familial polyposis coli
Chromosomal Studies in Familial Polyposis Coli Karen Brendum Nielsen, Steffen Biilow, and Niels Tommerup
ABSTRACT: A study of five patients with familial polyposis cell did not demonstrate any structural abnormalities of prometaphase chromosomes from methotrexate synchronized peripheral blood lymphocytes. No chromosomal heritable fragile sites were observed, nor was sister chromatid exchange increased. INTRODUCTION Identification of a chromosomal aberration highly specific of familial polyposis coli (FPC) would mean a significant advance in early diagnosis of affected members of FPC families; until now, however, chromosomal studies have revealed no abnormalities. The aim of the present study was to investigate prometaphase chromosomes from FPC patients in search of minor structural aberrations and fragile sites and to examine the frequency of sister chromatid exchange. MATERIALS AND METHODS Five patients with histologically verified FPC (two male, three female) with a median age of 24 years (range, 17-26) constituted the patient population studied. All patients had diffuse colorectal polyposis with thousands of adenomas. One of the patients had multiple epidermoid cysts and the remaining four had no clinically overt extracolonic manifestations. Two were solitary FPC cases, and three belonged to two families with 17 and 34 FPC cases respectively, in six generations. Two of the latter patients were dizygotic twins. None had developed colorectal or extracolonic carcinoma, and all five patients had been treated with colectomy or proctocolectomy 3-9 years (median, 5 years) prior to the present examination. Lymphocyte cultures were obtained from samples of peripheral blood; blood cultures were set up and cultured in TC 199 medium according to the laboratory's routine method, as previously described [1]. Eleven Q-banded metaphases from each patient were examined by fluoroscence microscopy, which is suitable for demonstration of the folate-sensitive fragile sites.
From the John F. Kennedy Institute, and the Department of Surgical Gastroenterology,Bispebjerg Hospital, Copenhagen, Denmark. Address requests for reprints to Dr. Steffen Biilow, Department of Surgical Gastroenterology C, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen 0, Denmark. Received October 5, 1984; accepted November 14, 1984.
355 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 17, 355-357 (1985) 0165-4608/85/$03.30
356
K.B. Nielsen, S. Billow, and N. Tommerup Furthermore, phytohemagglutinin-stimulated lymphocytes were cultured in TC 199 medium and subjected to further treatment with methotrexate and BrdU after a modification of Yunis' method for prometaphase chromosomes [2], previously described in detail [1]. A minimum of five RBA-banded prometaphases from each patient were analyzed by fluoroscence microscopy. Finally, sister chromatid exchanges (SCE) were studied in PHA-stimulated lymphocytes, according to the method described by Latt [3]. This method is also suitable for the demonstration of BrdU-sensitive fragile sites.
RESULTS
The karyotypes of all five FPC patients were normal: 46,XX or 46,XY without any structural aberrations. Fragile sites were not observed. SCE showed values that did not differ from the baseline values of the laboratory (Table 1). DISCUSSION
FPC is an autosomal dominant disease characterized by development of hundreds or thousands of colorectal adenomas in childhood or youth. If left untreated, all affected individuals will die from colorectal carcinoma at an early age. Early diagnosis and subsequent prophylactic colectomy, however, results in a favorable prognosis [4], although in rare cases, FPC patients may suffer from extracolonic malignancy, e.g., periampullary carcinoma. Until now, the only possible method of diagnosis is proctosigmoidoscopy, which is recommended in first degree relatives of FPC at biannual intervals beginning at the age of 10 years [5]. A specific diagnostic test capable of predicting the development of colorectal adenomas would be a considerable improvement and, thus, might reduce the need for regular proctosigmoidoscopic examination of all first degree relatives to include only those who, in fact, have inherited the disease. Chromosomal studies in FPC have not yet proven successful. In 1965 a normal chromosomal constitution was found in cultures from colorectal adenomas, "normal" colorectal mucosa, and in skin fibroblasts [6]. Normal karyotypes in lymphocyte cultures from FPC patients also have been reported [7, 8]. No qualitative differences in chromosomal constitution were observed in colonic adenomas [9]. In 1980 Utsunomiya and Iwama reported an increased number of somatic cells with balanced rearrangement of chromosomes (CS-cells) in FPC patients, and concluded that these "subclinical abnormalities" in vitro may explain the occurrence of fibrous tumors in extracolonic organs [10].
Table 1
Results of scoring SCE from five patients with FPC
Patient identification 1441-82 886-82 318-82 253-82 252-82
Karyotype
Number of mitoses examined
Number of SCE
Mean SCE/cell
46,XY 46,XY 46,XX 46,XX 46,XX
15 15 15 2 15
200 136 124 19 141
13.3 9.1 8.3 9.5 9.4
62
620
10.0
Total Control range, 8-12 SCE/cell(5 controls).
Familial Polyposis Coli
35 7
The first suggestion of a possible positive chromosomal finding in FPC came in 1982 in a study by Gardner et al., who reported a consistent heteromorphism of chromosome # 2 homologs i n 17 of 17 FPC patients [11]. However, this structural abnormality could not be reproduced by Kasugawa et a1.[12] using high resolution techniques similar to those used in the present investigation; recently, F i n e m a n et al. also failed to demonstrate this abnormality and considered it to be an artifact [13]. Based on previous and present negative results, we conclude that the usefulness of chromosomal studies in the search for a diagnostic test of FPC has not been proven. Identification and chromosomal localization of FPC gene(s) possibly with the aid of DNA-sequence polymorphisms might open the door to new possibilities in gene carrier detection. Until then, proctosigmoidoscopy at regular intervals in family members that are at risk remains the only reliable diagnostic method. Supported in part by Grants 809/71 and 87/80 from the Danish Cancer Society, "Max og Anna Friedmanns Legat," "Ferdinand og Ellen Hindsgauls Fond," and "Arkitekt Holger Hjortenberg og hustru, Dagmar Hjortenbergs Fond."
REFERENCES
1. Nielsen KB, Tommerup N (1984): Cytogenetic investigations in mentally retarded and normal males from 14 families with the fragile site at Xq28. Hum Genet 66:225-229. 2. Yunis JJ (1976): High resolution of human chromosomes. Science 191:1268-1270. 3. Latt SA (1973): Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395-3399. 4. BOlow S (1984}: Unpublished survival data from the Danish Polyposis Register. 5. Billow S (1984): The Danish Polyposis Register. Description of the methods of detection and evaluation of completeness. Dis Colon Rectum 27:351-355. 6. Veale AMO (1985): Intestinal Polyposis. Cambridge University Press, Cambridge, p. 33. 7. Jones EL, Cornell WP (1965): Gardner's syndrome. Arch Surg 92:287-299. 8. Sachatello CR (1970): Familial polyposis of the colon: I. Leucocytic chromosome determinations--Report of a patient with familial polyposis of the colon and Klinefelter's syndrome. Dis Colon Rectum 13:388-371. 9. Mark J, Mitelman F, Dencker H, Norryd C, Tranberg K-G (1973}: The specificity of the chromosomal abnormalities in human colonic polyps. A cytogenetic study of multiple polyps in a case of Gardner's syndrome. Acta Pathol Microbiol Scand 81:85-90. 10. Utsunomiya J, Iwama T (1980): Adenomatosis coli in Japan. In: Colorectal Cancer: Prevention, Epidemiology and Screening, S Winawer, D. Schottenfeld, P Sherlock, eds. Raven Press, New York, pp. 83-95. 11. Gardner EJ, Rogers SW, Woodward S (1982): Numerical and structural chromosome aberrations in cultured lymphocytes and cutaneous fibroblasts of patients with multiple adenomas of the colorectum. Cancer 49:1413-1419. 12. Kasukawa T, Watanabe T, Endo A (1983}: Absence of chromosome No. 2 homologues in patients with hereditary adenomatosis of the colon and rectum. Cancer Genet Cytogenet 8:283-285. 13. Fineman RM, Morgan M, Burt RW, Gardner EJ (1984): Failure to demonstrate a chromosome 2 deletion in adenomatous colorectal polyposis patients. Cancer 53:317-318.
ABSTRACT: A study of five patients with familial polyposis cell did not demonstrate any structural abnormalities of prometaphase chromosomes from methotrexate synchronized peripheral blood lymphocytes. No chromosomal heritable fragile sites were observed, nor was sister chromatid exchange increased. INTRODUCTION Identification of a chromosomal aberration highly specific of familial polyposis coli (FPC) would mean a significant advance in early diagnosis of affected members of FPC families; until now, however, chromosomal studies have revealed no abnormalities. The aim of the present study was to investigate prometaphase chromosomes from FPC patients in search of minor structural aberrations and fragile sites and to examine the frequency of sister chromatid exchange. MATERIALS AND METHODS Five patients with histologically verified FPC (two male, three female) with a median age of 24 years (range, 17-26) constituted the patient population studied. All patients had diffuse colorectal polyposis with thousands of adenomas. One of the patients had multiple epidermoid cysts and the remaining four had no clinically overt extracolonic manifestations. Two were solitary FPC cases, and three belonged to two families with 17 and 34 FPC cases respectively, in six generations. Two of the latter patients were dizygotic twins. None had developed colorectal or extracolonic carcinoma, and all five patients had been treated with colectomy or proctocolectomy 3-9 years (median, 5 years) prior to the present examination. Lymphocyte cultures were obtained from samples of peripheral blood; blood cultures were set up and cultured in TC 199 medium according to the laboratory's routine method, as previously described [1]. Eleven Q-banded metaphases from each patient were examined by fluoroscence microscopy, which is suitable for demonstration of the folate-sensitive fragile sites.
From the John F. Kennedy Institute, and the Department of Surgical Gastroenterology,Bispebjerg Hospital, Copenhagen, Denmark. Address requests for reprints to Dr. Steffen Biilow, Department of Surgical Gastroenterology C, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen 0, Denmark. Received October 5, 1984; accepted November 14, 1984.
355 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 17, 355-357 (1985) 0165-4608/85/$03.30
356
K.B. Nielsen, S. Billow, and N. Tommerup Furthermore, phytohemagglutinin-stimulated lymphocytes were cultured in TC 199 medium and subjected to further treatment with methotrexate and BrdU after a modification of Yunis' method for prometaphase chromosomes [2], previously described in detail [1]. A minimum of five RBA-banded prometaphases from each patient were analyzed by fluoroscence microscopy. Finally, sister chromatid exchanges (SCE) were studied in PHA-stimulated lymphocytes, according to the method described by Latt [3]. This method is also suitable for the demonstration of BrdU-sensitive fragile sites.
RESULTS
The karyotypes of all five FPC patients were normal: 46,XX or 46,XY without any structural aberrations. Fragile sites were not observed. SCE showed values that did not differ from the baseline values of the laboratory (Table 1). DISCUSSION
FPC is an autosomal dominant disease characterized by development of hundreds or thousands of colorectal adenomas in childhood or youth. If left untreated, all affected individuals will die from colorectal carcinoma at an early age. Early diagnosis and subsequent prophylactic colectomy, however, results in a favorable prognosis [4], although in rare cases, FPC patients may suffer from extracolonic malignancy, e.g., periampullary carcinoma. Until now, the only possible method of diagnosis is proctosigmoidoscopy, which is recommended in first degree relatives of FPC at biannual intervals beginning at the age of 10 years [5]. A specific diagnostic test capable of predicting the development of colorectal adenomas would be a considerable improvement and, thus, might reduce the need for regular proctosigmoidoscopic examination of all first degree relatives to include only those who, in fact, have inherited the disease. Chromosomal studies in FPC have not yet proven successful. In 1965 a normal chromosomal constitution was found in cultures from colorectal adenomas, "normal" colorectal mucosa, and in skin fibroblasts [6]. Normal karyotypes in lymphocyte cultures from FPC patients also have been reported [7, 8]. No qualitative differences in chromosomal constitution were observed in colonic adenomas [9]. In 1980 Utsunomiya and Iwama reported an increased number of somatic cells with balanced rearrangement of chromosomes (CS-cells) in FPC patients, and concluded that these "subclinical abnormalities" in vitro may explain the occurrence of fibrous tumors in extracolonic organs [10].
Table 1
Results of scoring SCE from five patients with FPC
Patient identification 1441-82 886-82 318-82 253-82 252-82
Karyotype
Number of mitoses examined
Number of SCE
Mean SCE/cell
46,XY 46,XY 46,XX 46,XX 46,XX
15 15 15 2 15
200 136 124 19 141
13.3 9.1 8.3 9.5 9.4
62
620
10.0
Total Control range, 8-12 SCE/cell(5 controls).
Familial Polyposis Coli
35 7
The first suggestion of a possible positive chromosomal finding in FPC came in 1982 in a study by Gardner et al., who reported a consistent heteromorphism of chromosome # 2 homologs i n 17 of 17 FPC patients [11]. However, this structural abnormality could not be reproduced by Kasugawa et a1.[12] using high resolution techniques similar to those used in the present investigation; recently, F i n e m a n et al. also failed to demonstrate this abnormality and considered it to be an artifact [13]. Based on previous and present negative results, we conclude that the usefulness of chromosomal studies in the search for a diagnostic test of FPC has not been proven. Identification and chromosomal localization of FPC gene(s) possibly with the aid of DNA-sequence polymorphisms might open the door to new possibilities in gene carrier detection. Until then, proctosigmoidoscopy at regular intervals in family members that are at risk remains the only reliable diagnostic method. Supported in part by Grants 809/71 and 87/80 from the Danish Cancer Society, "Max og Anna Friedmanns Legat," "Ferdinand og Ellen Hindsgauls Fond," and "Arkitekt Holger Hjortenberg og hustru, Dagmar Hjortenbergs Fond."
REFERENCES
1. Nielsen KB, Tommerup N (1984): Cytogenetic investigations in mentally retarded and normal males from 14 families with the fragile site at Xq28. Hum Genet 66:225-229. 2. Yunis JJ (1976): High resolution of human chromosomes. Science 191:1268-1270. 3. Latt SA (1973): Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395-3399. 4. BOlow S (1984}: Unpublished survival data from the Danish Polyposis Register. 5. Billow S (1984): The Danish Polyposis Register. Description of the methods of detection and evaluation of completeness. Dis Colon Rectum 27:351-355. 6. Veale AMO (1985): Intestinal Polyposis. Cambridge University Press, Cambridge, p. 33. 7. Jones EL, Cornell WP (1965): Gardner's syndrome. Arch Surg 92:287-299. 8. Sachatello CR (1970): Familial polyposis of the colon: I. Leucocytic chromosome determinations--Report of a patient with familial polyposis of the colon and Klinefelter's syndrome. Dis Colon Rectum 13:388-371. 9. Mark J, Mitelman F, Dencker H, Norryd C, Tranberg K-G (1973}: The specificity of the chromosomal abnormalities in human colonic polyps. A cytogenetic study of multiple polyps in a case of Gardner's syndrome. Acta Pathol Microbiol Scand 81:85-90. 10. Utsunomiya J, Iwama T (1980): Adenomatosis coli in Japan. In: Colorectal Cancer: Prevention, Epidemiology and Screening, S Winawer, D. Schottenfeld, P Sherlock, eds. Raven Press, New York, pp. 83-95. 11. Gardner EJ, Rogers SW, Woodward S (1982): Numerical and structural chromosome aberrations in cultured lymphocytes and cutaneous fibroblasts of patients with multiple adenomas of the colorectum. Cancer 49:1413-1419. 12. Kasukawa T, Watanabe T, Endo A (1983}: Absence of chromosome No. 2 homologues in patients with hereditary adenomatosis of the colon and rectum. Cancer Genet Cytogenet 8:283-285. 13. Fineman RM, Morgan M, Burt RW, Gardner EJ (1984): Failure to demonstrate a chromosome 2 deletion in adenomatous colorectal polyposis patients. Cancer 53:317-318.