PRESYMPTOMATIC DIAGNOSIS OF FAMILIAL ADENOMATOUS POLYPOSIS BY BRIDGING DNA MARKERS

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PRESYMPTOMATIC DIAGNOSIS OF FAMILIAL ADENOMATOUS POLYPOSIS BY BRIDGING DNA MARKERS C. M. J. TOPS1 J. TH. WIJNEN1 S. I. GRIFFIOEN2 G. J. V LEEUWEN1,3 F. C. A. DEN HARTOG JAGER4 H. F. A. VASEN2,3 F. M. NAGENGAST5 C. BREUKEL1 C. B. H. W. LAMERS2 H. M. VAN DER KLIFT1 P. MEERA KHAN1 Human Genetics Institute, Sylvius Laboratory, University of Leiden;1 Department of Gastroenterology, University Medical Centre, Leiden;2 National Polyposis Registration Centre, Foundation for the Detection of Hereditary Tumours, Leiden;3

Netherlands Cancer Institute and Department of Gastroenterology, Academic Medical Centre, Amsterdam;4 and Department of

Gastroenterology, University Hospital, Nijmegen,

The Netherlands5

Familial adenomatous polyposis (FAP) is a disorder with autosomal dominant which to colorectal inheritance, predisposes adenocarcinoma. The gene causing the disorder has been assigned to chromosome 5 by means of a polymorphic DNA marker called C11p11. An informative Dutch pedigree showed that two other linked polymorphic DNA markers, Pi227 and YN5.48, closely flank the FAP locus, one on either side. This finding will allow prenatal and presymptomatic diagnosis of FAP, with more than 99.9% reliability in the majority of families, by means of already available markers.

Summary

Introduction FAMILIAL adenomatous polyposis (FAP) is characterised the appearance of hundreds or thousands of adenomatous polyps in the colorectum. The polyps inevitably lead to adenocarcinoma if the affected part of the bowel is not removed. FAP is an autosomal dominantly inherited disorder with complete penetrance. On clinical grounds, it has traditionally been divided into two types, familial polyposis coli and Gardner syndrome.1 As well as the appearance of polyps in the large bowel, the occurrence of extracolonic lesions, such as multiple osteomas, epidermoid cysts, and desmoid tumours, was thought to distinguish Gardner syndrome from familial polyposis coli. But, the growing consensus is that Gardner syndrome is allelic to, if not the same as, familial polyposis coli at the FAP locus; gene localisation data support this opinion.2-4 The age at clinical diagnosis of FAP varies, even under continuous surveillance including regular sigmoidoscopy and confirmation by histopathology. All first-degree relatives of affected subjects have a 50% risk of carrying the disease gene. The availability and use of closely linked polymorphic markers would be helpful in prenatal and presymptomatic diagnosis, and could eventually prevent anxiety and unnecessary clinical screening in half the people at risk. Polymorphic marker studies in families with FAP showed close linkage between the FAP locus and a DNA marker called C11P11, localised by physical methods to the long arm of chromosome 5; no recombinants were found in about 50 cases.3.5 Several other DNA markers have since been reported to be closely linked to the FAP locus,w6 including Pi227 and YN5.48. The arrangement of these markers relative to the FAP locus was not known. Nevertheless, by use of these markers individually, the inheritance of the FAP gene can be followed through a family. This approach has one serious limitation-it cannot identify recombination

by

between Pi227 and the FAP gene in about 5% of cases and between YN5.48 and the FAP gene in about 1% of cases. The reliability of the diagnosis would be much greater if markers could be identified on both sides of the gene. If both polymorphisms were informative in a suitable family, recombinations which would otherwise escape detection and lead to the wrong prediction, could be identified. We report here marker information in a Dutch family which, for the first time, allowed direct localisation of a set of closely linked markers on either side of the FAP gene locus.

Subjects and Methods All the subjects in the study are Dutch by descent. The patients and their families are systematically reviewed and registered at the Dutch Polyposis Registration Centre, Leiden. The information recorded includes personal data, results of investigations, pathology reports, and results of treatment, as well as personal data and results of clinical screening of at-risk relatives.’ Samples of DNA for polymorphism analyses were isolated from freshly collected peripheral venous blood. The procedures for DNA isolation and Southern blot analysis were as described previously.8 The DNA marker YN5.48 detects three restriction polymorphisms recognised respectively by Taq I,4MspI, and EcoRI (Y. Nakamura, R. White, personal communication); in this study we used the MspI polymorphism. The DNA marker Pi227 recognises restriction polymorphisms with the enzymes Bell, BstXI, PstI, and MboI.6,9 Lod scores for linkage were calculated by a computer program (LINKAGE),lO modified by an age at diagnosis correction. Age-dependent penetrance classification used in these calculations was adapted from the distribution of ages at which FAP was first diagnosed in 58 Dutch call-up patients under the age of 30 years7 (H. Vasen, unpublished). The penetrance was assigned as follows: 6-0% for 0-12 years, 32-5% for 13-17 years, 66-5% for 18-22 years, 89% for 23-27 years,and 98% for 28 years and older. The 95% confidence limits (CL) were calculated by the minus 1-lod unit method." Recombination between linked genes is due to interchange of parts between homologous chromosomes (after the formation of a chiasma) during meiosis, called crossover. 12 The recombination fraction, the proportion of recombinants between two genetic loci, is a measure of the genetic distance between the loci considered for linkage analysis.12 Linkage is close when the recombination fraction is 0-02-0 06. The closest markers segregate together with the highest probability, yielding a recombination fraction close to zero. Thus, the inheritance of disease genes can be traced in families through closely linked genetic markers. 13 The occurrence of crossover at one point in the chromosome reduces the probability of another crossover in the same chromosome. This phenomenon is called chiasma interference12 and it is greater for short than for long distances in chromosomes. Within a certain minimum distance along the chromosome double crossover is not possible. Lod represents the logarithm of odds in favour of linkage, the lod score’2 being calculated as:

peak lod score of 300 means odds 1000/1in favour of linkage. The polymorphism information content (PIC)" of a genetic marker indicates its usefulness in linkage studies. For example, the YN5.48 MspI restriction fragment length polymorphism (RFLP)," with a PIC value of 0-37 in the Dutch population, has the probability of being informative for linkage with the FAP locus in 37% of the Dutch families with FAP, whereas Pi227, with an A

aggregate PIC value of 0-75, will be informative in 75% of such families.

Results Nineteen Dutch FAP pedigrees were screened for the at YN5.48. Six were informative for

MspI polymorphism

1362 LOD SCORES BETWEEN FAP LOCUS AND DNA MARKERS

&bul et; AFFECTED o UNA FFEC TED

YN5.48 AND P1227

to

*Peak combined lod score for FAP/YN5.48 linkage was estimated be 10-67 at a recombination fraction of 0.01. tUpdated with information on same and additional Dutch families. tPeak combined lod score for FAP/Pi227 linkage was estimated to be 18 09 at a recombination fraction of 005.

Pi227, YN5.48, and FAP locus segregation in part of family with an obligatory recombinant.

Arrow

between YN5.48 and the FAP locus, yielding a maximum lod score of 2-86 at a recombination fraction of 0-05 (95% CL 0-002-0-200) (see table). Linkage analysis between the FAP locus and Pi227 gave a maximum lod score of 15-29 at a recombination fraction of 0-04 (95% CL 0-01-0-11) in sixteen Dutch families for whom the polymorphism was informative. The pedigree of the single family with an obligatory recombination between the FAP locus and YN5.48 is shown in the accompanying figure. In 60 randomly ascertained (non-FAP) Dutch subjects, the frequency of the MspI alleles was B1 0-44, B2 0-56, B3 0, giving a PI04 of 0-37. The ages of the people in generation II ranged from 40 to 50 years. C 11 p 11was not informative in this family. The other markers reported by Nakamura et al4 were not included in our study.

linkage

Discussion Nakamura and colleagues found no direct evidence for the of recombination between YN5.48 and the FAP locus or between C11p11 and the FAP locus. By means of multilocus linkage analysis on data derived also from non-FAP families and by the location score method, they concluded that the most likely location of the FAP locus would be closer to YN5.48 than to Cllpll. Nevertheless, they could not determine whether the FAP locus was within or outside the region between Cllpll and YN5.48. The family shown in the figure was not informative, either with the TaqI RFLP3 or with the 4-base deletion polymorphism of Cllpll (unpublished), whereas Pi227 was completely informative for linkage and showed no recombination with the FAP locus. Since Pi227 and C11p11are located on the same side of the FAP locus,14,15 and since the recombination frequency between Pi227 or Cllplland the FAP locus is much higher than that between YN5.48 and the FAP locus, the recombinant, shown in individual 11-4 in the figure, provides the first direct evidence that the FAP locus is located between Pi227 and YN5.48. So far the FAP gene has not been isolated or identified.

occurrence

Until

its

sequence

is

available, the presymptomatic

detection of FAP gene carriers depends on family studies with linked genetic markers. The discovery that the markers Pi227 and YN5.48 lie either side of the FAP locus has raised the reliability of the laboratory diagnosis of FAP to nearly 100%. In families informative for both the markers, making an incorrect diagnosis of FAP would be possible only when a double recombination, one between the FAP locus and Pi227 and another between the FAP locus and YN5.48, could occur. When the phenomenon of chiasma interference

indicates

individual

with

recombinant;

cross

marks site ot

recombination.

is completely absent, the frequency of double recombination is equal to the product of the recombination frequencies in the respective intervals. We estimated the recombination frequency between Pi227 and the FAP locus to be 5% and that between YN5.48 and the FAP locus to be 1% (table, combined data). Since Pi227 is on one side and YN5.48 is on the other side of the FAP locus, the estimated percentage of double recombinants will be 0-05. This calculation implies that the diagnosis will be correct in 99-95% of all cases that show no recombination between the test markers. In reality, owing to chiasma interference, the likelihood of a double crossover is virtually zero within such a short genetic interval. Therefore, in these cases the reliability of diagnosis will be, in practice, 100%. In the 6 % of the cases that show a recombination between the test markers Pi227 and YN5.48 (table) the diagnosis of FAP will be possible when markers closer to the FAP locus or the FAP gene sequences are

identified. The probe Pi227 shows four restriction polymorphisms and identifies nine alleles, generating an aggregate PIC13 of 0.75,6,15,16 Also, we estimated a PIC value of 0-37 for YN5.48 at its MspI RFLP site. The product of these PIC values will give a rough estimate of the fraction of families that would be informative for diagnostic tests by linkage; for these markers it amounts to 27-75%. In fact, YN5.48 has two other restriction polymorphisms recognised by TaqI and EcoRI with PIC values 0-384 and 10-44 (Y. Nakamura and R,

White, personal communication), respectively. Moreover. the published data4 suggest that the other closely linkec DNA markers Cllpll, KK5.33, and YN5.64 are locatec centromeric to YN5.48 and that the marker YN5.61 i located telomeric to YN5.48, whereas the FAP locus map closest to YN5.48. These suggestions together with the data reported here show, for the first time, that C 11p 11, KK5.33 YN5.64, and Pi227 are located on one side, and YN5.48 anc YN5.61 on the other side of the FAP locus. Each of these markers is known to have two polymorphic sites and eact site a PIC value ranging from 0- 18 to 0.38.4,16 Therefore, Wt conclude that enough reasonably informative markers are available at present on both sides of the putative FAP locu: to make diagnosis through linkage possible for the grea majority of families. A potential source of error in the diagnosis of FAI through linked markers would be the occurrence of genetic heterogeneity. Although genetic heterogeneity has not so fa been found in about 25 families reported from fou

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countries,3,4,6,14 this possibility cannot be excluded. In a family which showed no linkage to the above markers owing such heterogeneity, an unexpected number of to recombinants might be found. Thus, the available set of linked markers will be useful also for identifying genetic

heterogeneity even in small families. These results will hasten the search for and eventual isolation of the FAP gene sequences. The information on the localisation of YN5.48 on the other side of the FAP locus from Pi227 opens a direct approach with a powerful set of DNA markers to accurate diagnosis of FAP in family members at risk. We thank Sir Walter Bodmer (Imperial Cancer Research Fund Laboratories, London), Dr N. Spurr (London), Dr G. D. Stewart (Ann Arbor, Michigan, USA), Dr Y. Nakamura (Salt Lake City, Utah, USA), and Dr R. White (Howard Hughes Medical Institute, Salt Lake City) for probes; Dr M. Dunlop (Medical Research Council Unit, Edinburgh) for helpful discussions, Dr J. G. Goedhart, Dr J. Lens, Dr G. J. D. Dorrepaal, and Dr A. L. J. van der Veer for some of the family samples; and all the members of the families, who volunteered to take part in this study. The study was supported by the Dutch Praeventiefonds.

Correspondence should be addressed to C. M. J. T., Human Genetics Institute, Sylvius Lab, University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. REFERENCES

1. Bussey HJR. Familial polyposis

coli. Baltimore: Johns Hopkins University Press, 1975. 2. Herrera L, Kakati S, Gibas S, et al. Gardner syndrome in a man with an interstitial deletion of 5q. Am J Med Genet 1986; 25: 473-76.

Preliminary Communication PRENATAL SEX DETERMINATION BY DNA AMPLIFICATION FROM MATERNAL PERIPHERAL BLOOD Y-M. D. LO1

J. S. WAINSCOAT2 M. D. G. GILLMER4

P. PATEL2 M. SAMPIETRO3 K. A. FLEMING5

University of Oxford Clinical School,1 Department of Haematology,2 Maternity Department,4 and Nuffield Department of Pathology,5 John Radcliffe Hospital, Oxford; and A. Bianchi Bonomi Hemophilia and Thrombosis Centre, University of Milan, Italy3

Summary

The polymerase chain reaction was used to amplify a Y-specific repeat sequence in peripheral blood DNA samples from 19 pregnant women who had a gestational age of 9 to 41 weeks. Y-specific sequences were amplified from all 12 women who bore a male fetus but in none of 7 women who bore a female fetus. With stringent precautions against contamination, this technique may assist prenatal diagnosis of sex-linked genetic

disorders. INTRODUCTION

FETAL cells in peripheral maternal blood are, in theory, an alternative source of tissue to specimens obtained by invasive techniques such as amniocentesis and chorionic villus biopsy. In 1969, by examination of metaphase chromosomes, Walknowska et all found cells with a probable male karyotype in the peripheral circulation of pregnant women who later gave birth to boys. This method, however, is time-consuming and such cells may also be

3. Bodmer WF, Baily CJ, Bodmer J, et al. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 1987; 328: 614-16. 4. Nakamura Y, Lathrop M, Leppert M, et al. Localization of the genetic defect in familial adenomatous polyposis within a small region of chromosome 5 Am J Hum Genet 1988; 43: 638-44. 5. Leppert M, Dobbs M, Scambler P, et al. The gene for familial polyposis coli maps to the long arm of chromosome 5. Science 1987; 238: 1411-13. 6. Meera Khan P, Tops CMJ, Broek Mvd, et al. Close linkage of a highly polymorphic marker (D5S37) to familial adenomatous polyposis (FAP) and confirmation of FAP localization on chromosome 5q21-22. Hum Genet 1988; 79: 183-85. 7. Vasen HFA, Griffioen G, Offerhaus GJA, et al. The value of screening and central registration of families with familial adenomatous polyposis. Dis Colon Rectum (in

press). MH, Wapenaar MC, Goor N, et al. Isolation of probes detecting restriction fragment length polymorphisms from X chromosome-specific libraries: potential use for diagnosis of Duchenne muscular dystrophy. Hum Genet 1985; 70: 148-56. 9. Stewart GD, Bruns GAP, Wasmuth JJ, Kurnitt DM. An anonymous DNA segment (Pi227) maps to the long arm of chromosome 5 and identifies a BstX I polymorphism (D5S37). Nucl Acid Res 1987; 15: 3939. 10. Lathrop GM, Lalouel JM. Easy calculations of Iodscores and genetic risks on small computers. Am JHum Genet 1984; 36: 460-65. 11. Conneally PM, Edwards JH, Kidd KK, et al. Report of the committee on methods of linkage and reporting. 8th International Workshops on Human Gene Mapping. Cytogenet Cell Genet 1985; 40: 356-59. 12. Ott J. In: Analysis of human genetic linkage. Baltimore: Johns Hopkins University Press, 1985. 13. Botstein D, White LW, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am JHum Genet 1980; 8. Hofker

32: 314-31.

Dunlop MG, Steel CM, Wyllie AH, et al. Linkage ana1ysis in familial adenomatous polyposis: Order of C11p11 (D5S71) and Pi227 (D5S37) loci at the apc gene. Genomics 1989; 5: 350-53. 15. Tops CMJ, Breukel C, Wijnen JTh, et al. Linkage relationships between APC, D5S37 (Probe pi227) and D5S71 (probe C11p11) in the Dutch polyposis families. Cytogenet Cell Genet (in press). 16. Meera Khan P, Tops CMJ, Breukel C, et al. Closely linked DNA markers for preclinical prediction of familial adenomatous polyposis. In: Herrera L, ed. Familial adenomatous polyposis. New York: Alan R Liss, 1989: 389-92. 14.

found in the blood of normal women who are not pregnant.2 Quinacrine staining has also been used to detect cells that contain Y bodies in the maternal circulation, as a guide to fetal sex,3,4 but drawbacks of this technique include non-uniform criteria for Y-body detection between laboratories,5 the occurrence of false positives,3,4and failure of others to reproduce the results.6 Herzenberg et aF used flow cytometry to detect cells of probable fetal origin, by immunogenetic and cytogenic criteria, from peripheral maternal blood. By a combination of flow cytometry and monoclonal antibody technology, Covone et al found cells that react with the monoclonal antibody H315 in the peripheral blood of pregnant women.8 H315 identifies a glycoprotein expressed on the surface of human syncytiotrophoblasts,9 but most H315-positive cells in the maternal circulation do not contain Y-chromosome-derived DNA when the fetus is male, and H315-negative cells can adsorb H315 antigen in vitro10-observations that cast doubt on the meaning of earlier results. An air-culture technique has also been developed to enrich for fetal cells in cultures of maternal blood." The polymerase chain reaction (PCR)12,13 offers an alternative way to detect the occasional fetal cell amongst numerous maternal cells in maternal venous blood. However, a previous attempt to detect circulating male fetal cells by PCR was unsuccessful. 14 We describe use of PCR to detect male fetal cells from maternal peripheral circulation -a relatively non-invasive method for sex determination. We believe this report to be the first to describe molecular genetic analysis of the fetus by use of maternal blood. PATIENTS AND METHODS

7-10 ml blood were taken from the antecubital veins of 19 pregnant women at various stages of gestation. Early pregnancy