PRENATAL DIAGNOSIS, VOL. 2, 75-78 (1982)

PRENATAL DIAGNOSIS OF TRISOMY 18 MOSAICISM”

FRANK s. GRASS?, JOHN c. HISLEY, JAMES c. PARKE, JR.

Clinical Genetics Program, Departments of Pediatrics and Obstetrics-Gynecology, Charlotte Memorial Hospital and Medical Center, P.O. Box 32861,

Charlotte, North Carolina 28232, U.S.A.

AND

DAVID BUNN

I505 Doctors Circle, Wilmington, North Carolina 28401, U.S.A.

SUMMARY

Trisorny 18 mosaicism was found in multiple primary cultures of amniotic fluid cells and subsequently confirmed by chromosome analysis of several tissues derived from the aborted fetus. The overall frequency of the minority cell line was 25 per cent in the amniotic fluid cultures and 28 per cent in the fetal tissues although much intertissue variations were noted.

KEY WORDS Trisomy 18 Mosaicism Amniotic fluid

INTRODUCTION

Chromosomal mosaicism in amniotic fluid cultures has been well documented but continues to be a crucial dilemma in prenatal studies complicated by the possibility of false positive pseudomosaicism and false negative results when few cells can be examined. This problem was addressed by Bouk et al. (1979) in a study in which they identified three different manifestations of pseudomosaicism and demonstrated a technique to distinguish true mosaicism from pseudomosaicism. The cumulative experience from several centres reported recently by Hamerton et al. (1980) indicates the frequency of true amniotic fluid mosaicism is 0.26 per cent, while the frequency of pseudomosaicism is 2.4 per cent.

Bloom et al. (1974) described the first case of true mosaicism involving a 46,XX/ 47,XX,+ 13 fetus identified in amniotic fluid cultures and confirmed by fetal tissue chromosome analysis. Milunsky (1979) reviewed 82 cases of amniotic fluid mosaicism of which 14 were confirmed. Hamerton et al. (1980) cited 69 cases of mosaicism identified prenatally. Of those examined, 60.7 per cent were confirmed by chromo- some studies and 25.5 per cent displayed an abnormal phenotype. Mosaic fetuses with the following minority cell lines have been identified: 47,XXY ; 47,XY +mar; 47,XY, + 20; 47,XX,+21; 45,X; 47,XXX; 47,XX,+ 13. To our knowledge, no confirmed cases of prenatally diagnosed trisomy 18 mosaicism have been reported. We present such a case confirmed by chromosome analysis of multiple fetal tissues.

* Supported in part by Medical Service Grant C-102, ru’ational Foundation-March of Dimes. t Addressee for correspondence.

0197-3851 /82/010075-O4$01 .OO 0 1982 by John Wiley & Sons, Ltd.

Received 14 July I981 Revised I 7 September I981

Accepted I 0 October I981

76 F. S. GRASS ET AL.

CASE REPORT

A 37-year-old Gravida 111, Para 2, white female was referred for prenatal diagnostic chromosome studies because of maternal age. Her first two pregnancies were by a previous husband. The first pregnancy ended in elective abortion because of uterine infection, and the second produced a normal male. The father of this third pregnancy was 48 years old and had fathered two normal boys in a previous marriage. The family history was non-contributory. The mother had taken Butasol (Butabarbitol) 15 mg as needed two months before becoming pregnant and Lepressor (Metroprolol Tar- trate) 50 mg for hypertension during the pregnancy. Vaginal spotting occurred for one day at 8 weeks and for one day at 1 4 weeks. Ultrasonography performed at 17 menstrual weeks revealed a biparietal diameter of 2.9 cm equivalent to a 15-week gestation. Normal fetal movements were noted. Amniocentesis was performed at 17 weeks, and 20 cc of clear amniotic fluid were obtained. Two cubic centimetres were separated for alphafetoprotein determination. Amniotic fluid alphafetoprotein concentration was 8 mcg/ml.

CYTOGENETIC INVESTIGATION

The remaining 18 cc of amniotic fluid were divided into three 6 cc aliquots, and three primary cultures were established in 60 x 15 mm culture dishes (Falcon 1002) using minimum essential media supplemented with 25 per cent fetal bovine serum. Two of these cultures were harvested by trypsinization after fourteen days and the third after sixteen days.

Chromosome analysis was performed by standard Giemsa, trypsin G-banding and Q-banding. 46,XX/47,XX, + 18 mosaicism was identified in each of three primary cultures. Examination of slides from all three primary cultures was performed to rule out pseudomosaicism. The cumulative frequency of trisomy 18 cells out of a total of 130 amniotic fluid cells examined was 25 per cent (Table 1).

Tetraploid cells presumably derived from the amnion were noted from each of the three primary cultures. Five of fifteen tetraploid cells from culture A, two of five tetraploid cells from culture B and two of seven tetraploid cells from culture C pos- sessed 94 chromosomes with six No. 18 chromosomes. Thus, a total of nine (33 per cent) out of 27 tetraploid cells were hexasomic for chromosome No. 18 which caused some concern that the abnormal cell line reflected a clone in the amnion and might not reflect mosaicism in the fetus, a potential pitfall in prenatal diagnosis of mosaicism.

The parents were counselled concerning these res ults and elected to terminate the pregnancy. The fetus was aborted by prostaglandin suppository at 20 weeks gestation and weighed 275 g. Its crown-to-rump length was 163 cm. The face was somewhat

Table 1. Number (and percentage) normal and trisomy 18 cells in amniotic fluid cell cultures

Primary culture 46,XX 47,xx, + 18 Total

A 19 (76 %) 6 (24%) 25 B 49 (84%) 13 (16%) 62

Total 97 (75%) 33 (25%) 130 C 29 (67%) 14 (33%) 43

TRISOMY 18 MOSAICISM 77

triangular with a broad cranium and micrognathia. The ears were marginally low set. Examination of the brain revealed cerebellar hypoplasia. No other features suggestive of trisomy 18 were noted externally or internally.

Fetal blood, skin, and amnion were obtained and cultured. The amniotic fluid findings were confirmed in all tissues examined (Table 2). The cumulative frequency of the trisomy 18 cell line out of a total of 134 fetal cells examined was 28 per cent; however, a wide range of frequency in the minority cell line was noted among the three tissues.

Table 2. Number (and percentage) of normal and trisomy 18 cells in fetal tissues

Tissue 46,XX 47,XX, + 18 Total

Blood 31 (52%) 29 (48%) 60 Skin 49 (98%) 1(2% ) 50 Membrane 17 (71 %) 7 (29%) 24 Total 97 (72%) 37 (28%) 134

DISCUSSION

To our knowledge, this is the first reported case of trisomy 18 mosaicism detected in amniotic fluid cultures and confirmed in fetal tissue. Prenatal diagnosis of mosaicism is often tenuous because of the possibility of pseudomosaicism in which a minority cell line develops in tissue culture and does not reflect the chromosomal status of the fetus (Peakman et al., 1979; BouC et al., 1979). The data from the prenatal studies in this case met two major criteria for true mosaicism: (1) cells with the same abnormality were found in all three primary cultures, and (2) the karyotypic abnormality has been associated with a documented syndrome.

The importance of the first of these criteria cannot be over-emphasized because it rules out pseudomosaicism. Of 82 cases of mosaicism in amniotic fluid cultures sited by Milunsky (1979), only 14 were confirmed. In 11 of the 82 cases, chromosomal examination of fetuses, aborted because of mosaic amniotic fluid cultures, revealed normal karyotypes.

Observation of tetraploid cells with six chromosome No. 18’s caused concern that the trisomy 18 cell line could have arisen in the amnion only and not reflect fetal mosaicism. Confirmatory fetal tissue studies revealed trisomy 18 in cells from cultured amnion as in the fetus proper. The non-disjunctional event producing mosaicism apparently occurred before separation of embryonic disc and the amnion. Clearly, observation of an abnormal cell line in known amnion tissue cannot rule out true fetal mosaicism.

Regarding the second of the two criteria, trisomy 18 mosaicism has been described in infants and children. The phenotypes and mean survival times tended to be more variable than in complete trisomy 18, the severity of the clinical picture depending upon the proportion of trisomy 18 cells and upon the tissue in which the abnormal cell line was found (Hamerton, 1971). In the present case, the percentage of abnormal cells varied from 2 per cent in the skin to 48 per cent in the fetal blood. Similar tissue variability in children mosaic for trisomy 18 was reported by Rao et a1 (1978) and Schuler et al. (1972).

78 F. S. GRASS ET AL.

Cerebellar hypoplasia, triangular face, and low set ears were the only features associated with trisomy 18 that were noted in the fetus in the present case. This was not surprising since a milder clinical picture would be expected in mosaics. The prenatally diagnosed trisomy 13 mosaic reported by Bloom et al. (1974) showed no obvious stigmata of trisomy 13 and examination of fetal lymphocytes and fibroblasts revealed trjsomy 13 in 18 per cent and 48 per cent respectively of the cells examined. Variability in phenotype, likelihood of a fetus with no grossly obvious malformation, and the basis for the diagnosis of true mosaicism versus pseudomosaicism should be discussed in detail with the parents.

ACKNOWLEDGEMENTS

We wish to thank Mrs. Jane Deal for her work in the cytological aspects of the report and Mrs. Freida Hinson for her help in preparation of the manuscript.

REFERENCES

Bloom, A.D., Schmickel, R., Barr, M., Bardi, A.R. (1974). Prenatal detection of autosomal mosaicism, J . Pediatrics, 84, 732-733.

BouC, J., Nicolas, H., Bairchard, F., BouC, A. (1979). Le clonage des cellules du liquide amniotique, aide dans l’interpretation des mosaiques chromosomiques en diagnostic prenatale, Ann. Genet. Paris, 22, 3-9.

Hamerton, J.L. (1971). Human Cytogenetics: Clinical Genetics, Vol. 11, New York: Academic Press.

Hamerton, J.L., Boue, A., Cohen, M.M., De la Chapelle, A., Hsu, L.Y., Lindsten, J., Mik- kelsen, M., Robinson, A., Stengel-Rutkowski, D., Webb, T., Willey, A., Worton, R. (1980). Chromosome disease, Prenatal Diagnosis, Special Issue, 1 1-21.

Milunsky, A. (1979). Genetic Disorders and the Fetus. New York: Plenum Press. Peakman, D.C., Moreton, M.F., Corn, B.J., Robinson, A. (1979). Chromosomal mosaicism

in amniotic fluid cell cultures, Am. J. oJ Hum. Genet., 31, 149-155. Rao, K. W., Buchanan, P.D., Aylsworth, A S . (1 978). Asymmetric clinical and cytogenetic

findings in a Cyear-old girl with trisomy 18 mosaicism, Birth Defects: Orig. Art. Series,

Schuler, D., Ferenczi, I., Gorgenyi, A., Dubes, M., Pekete, G., Ruzicska, R. (1972). Uni- lateral congenital malformation with chromosome aberration, Hum. Hered., 22, 198-203.

14(6C), 349-365.