Hrreditas 86: 37-44
(IY77)
Cytogenetical problems in prenatal diagnosis W. SCHMID Division of Medicul Genetics, Department qf’ Pediatrics, finiversity of‘ Zurich. Switzrrlund
SCHMID, W . 1977. Cytogenetical problems in prenatal diagnosis. Sweden. ISSN 0018-0661. Received February 10, 1977
~~
Hereditus 86: 37~-44. Lund.
On the basis of 480 prenatal diagnostic karyotype studies, carried out since 1971 in Zurich. the following types of problems were considered: ( I ) The difficulty is inherent in the counselling problem itself, as an unusual chromosome anomaly in a parent. (2) True fetal mosaicism. (3) Somatic mutations during culture. (4) Growth of maternal cells. In problems of type ( I ) , i t is known in advance that the prenatal diagnosis has limitations. Problems of the types ( 2 ) to (4) can be solved to some degree by using in situ preparations and the clone-wise analysis. In ( 2 ) and ( 3 ) analysis of 1 inetaphases from 5 to 10 clones has been informative, somatic mutations usually being restricted to single cells within clones with otherwise normal karyotype. When entire clones exhibit a deviating karyotype, the proportion of abnormal to normal clones will help decide whether it is a case of ( 2 ) or (3). Also problem (4) is elucidated by the in situ method, since maternal cells. generally. differ in their growth pattern from fetal cells. If uncertainties persist, the comparison of lluorescent markers of presumed fetal cells to those of the mother is apt to resolve the problem. Somatic mutation and maternal cell growth should not be considered as a source of unavoidable pitfalls in prenatal cytogenetic diagnosis. By anticipating these events and by applying appropriate techniques. diagnostic errors can be kept at a very low level. W . Schmid. Division q / Medicul Genetics, Department of Pediutrics. Ci’niversity o/ Zurich, CH-8032 Zurich, Switzerland
Cytogenetical problems in prenatal diagnosis can be divided into four groups: ( I ) The difficulty may be inherent in the counselling problem, i.e. in an unusual kind of karyotype anomaly in a parent. (2) True fetal mosaicism, expressed in the amniotic cell culture. (3) Somatic numerical and structural chromosome mutations in cell culture. (4) Growth of maternal cells, which can be mistaken for fetal cells. Prior to setting forth my experience with these four problems I should like to present, in tabular form, the case load on which our observations were collected.
autosomal trisomies. (2) Recurrence of Down’s syndrome after a sporadic case of t(21; 21) translocation Down’s syndrome. Both parents had normal karyotypes with no mosaicism detectable in blood cultures. (3) A large omphalocele was found due to high A F P (alpha-I-fetoprotein, 110 pg/ml in 16th week of pregnancy) in the amniotic fluid of a woman with a previous 21 trisomic child.
1 , Cases studied in Zurich from 1971 to 1976
Indications
The indications for 480 prenatal diagnoses divided into major groups are shown in Table 1. Table 2 presents the results from 326 cases with primarily cytogenetic indications. By and large, these results are similar to those of other major published series. Three points are noteworthy: (1) The incidence of sex chromosome anomalies (XXY and XXX in particular) in the high maternal age group is lower than would be expected on the assumption of a similar age influence as the one observed with
Table I. Indications for 480 prenatal diagnoses (number of patients, up to Sept. 15, 1976) Karyotype studied in all cases, A F P in the last 394 cases, biochemical studies in 13 cases Patients Number High maternal age (mother 40 and over: 125) Trisomy 21 in sibs Chromosomal aberration in parent Biochemical defects MMC, anencephaly. hydrocephaly X-linked disorders Obstetrical indications Various
191 I I6 15
12 24 9
82 45
”(,
38.7 23.5 3.1 2.4 4.8 1.8 16.6 9. I
38
w. SCHMID
Hrreilitas X6 ( l Y 7 7 )
Tuhlr 2. Results in cases with primarily cytogenetic indications Total number
Indications
Abnormal Number
Details
",,
High maternal age mother 40 and over
I25
6
4.8
mother 25-39 Trisomy 21 in sibs
66 I16
I 3
I .5 2.6
I5 4
1 0
6.1
326
II
3.4
Chromosomal aberrations in parents Trisomy (except 21) in sibs Total
3 x trisomy 21 2 x trisomy 18 I x XXY I x trisomy 21 (mother 38) I x trisomy 21 (mother 27) 1 x tri-21 by t(21;21) (same as brother, parents normal) I x omphalocele I x part. trisomy 3
0
Table 3. Results in various indication groups Indications
X-chromosomal defects
No.
9
Details
Results Karyotype, AFP. biochemical
Hemophilia (5) Duchenne (3) Mucopol. Hunter ( 1 )
4 d d . 19 3 dd 1 d (biochemically abnormal)
Autosomal recessive metabolic disease
12
Metachromatic leucodystrophy (3) Mucopolysacharidoses (4) GM-I-Gangliosidosis (2) Glycogenosis I I ( I ) ADA-Deficiency ( 1 ) Fay-Sachs ( 1 )
I x abnormal 1 x abnormal
Obstetrical indications in 2nd half of pregnancy
82
Placental dysfunction (55)
1 x trisomy 21 (mother 25 y.) 1 x anencephaly with ring chrom. 13
Polyhydramnios (20) suspected malformations (7)
I x mosaic trisomy 22 (mother 48 y.) 1 x sacrococc. teratoma (high AFP) 1 x pathol. twin pregnancy (high AFP) I x anencephaly (high A F P ) 1 x anencephaly (high AFP)
Spina bifida
24
I previous anencephalic (5) 2 previous anencephalics (2) 1 previous M M C (10) 1 anencephalic + I M MC ( 1 ) 1 previous hydrocephalic (6)
All results normal in respect to A F P ( I trisomy 18, mother41)
Various
45
Mostly diverse malformations in previous sibs
All normal
Table 3 presents the results in five other indication groups. In the cases of X-chromosomal defects sex was determined by complete karyotype analysis. The biochemical studies in metabolic disease were performed by Drs. Wiesmann and Herschkowitz, Bern, Dr. Gitzelmann, Zurich, Dr. Niermeijer, Rotterdam and Dr. Hirschhorn, New York, who took over our fibroblast cultures. Prenatal genetic studies
for obstetrical diagnostic purposes, carried out in the second half of the pregnancies, are justifiable as long as the capacity of the laboratory is not impaired by this serivce to the disadvantage of more pressing cases. The percentage of abnormal results is not low as can be seen from the table. The range of action that can be taken by the obstetrician is. however, limited. In cases as severe as anencephaly a
Hrrrdirus 86 ( I Y 7 7 )
PRENATAL DIAGNOSIS
39
Fig. 1. Cover glass preparations of 2 primary amniotic fluid cell cultures. The round colonies - actually clones derived from single cells are of very unequal size. They have developed within 9 and 1 1 days, respectively.
premature birth may be induced. In cases of fetal chromosomal aberrations Caesarian sections are avoided in the interest of the mother. If the results are normal, this seems to be a stimulus for the obstetrician to d o for the pregnancy as much as is possible. 2. Technique and basic considerations in regard to methodology The techniques used in our work have been described previously (SCHMID1975). In situ analysis is important in respect to the last three of our problems to be discussed later: fetal mosaicism, somatic mutation in vitro and maternal cell growth. In primary amniotic cell cultures cellular proliferation is seen in the form of distinct colonies (Fig. I). For practical purposes it is of importance to know whether these colonies, in general, are derived from single fetal cells or from groups of fetal cells. The following observations are in favour of the single cell origin: ( I ) In cultures grown under favourable optical conditions there is evidence from direct observations. ( 2 ) The cellular morphology within colonies is strikingly uniform, whereas cellular morphology from clone to clone exhibits numerous different types (SCHMID1972; HOEHNet al. 1974). (3) Pure tetraploid colonies are found quite frequently. (4) In cases where all other colonies are normal, single colonies with aberrant karyotypes can be found. The author readily admits that a small
proportion of colonies may be derived from multiple cells - usually of the same type - but on the whole, the evidence is strongly in favour of the single cell origin. What does this mean from a practical point of view? The consequences are shown in Fig. 2 and 3 . Clone-wise analysis assures the investigator that he has studied descendants of several different fetal cells; in a subculture, he may look at a much higher number of mitoses which, however, all may be derived from the same predominant clone, i.e. from a single fetal cell. If mosaicism for two different karyotypes is present, clone-wise analysis permits a distinction between the two situations depicted in Fig. 3 . Somatic mutation involving part of a clone is harmless in contrast to true fetal mosaicism.
3. Problem 1. Cytogenetical difficulty inherent in the counselling problem Common to cases ofthis kind is the situation that it is known in advance that the answer from a prenatal study may have limitations. I would like to illustrate this problem with three examples: Case 1. - A young couple was seeking advice since the husband was found to be a carrier of a small acrocentric extra chromosome. The anomaly was discovered in a chromosome study performed because the wife had had two abortions, one in the third, the other in the first month of pregnancy. With banding techniques it was not possible to
40
H r r d t r u s XO ( I Y 7 7 )
W. SCHMID
Same material trypsinized
Primary culture
Somatic mutation
Fetal mosaic
Q . . :.' ..
Q ,
J ($
f I
15 metaphases
analyzed from 7 clones
30 metaphases analyzed. All may be derived from I big clone, i.e. the same cell
Trypsinized: In both cases the same proportion of cells may be aneuploid Fig. 3. A somatic mutation involving part of a clone (striped area) is distinguishable from true fetal mosaicism involving several independent clones. Subculturing obscures the situation.
Fig. 2. Clone-wise analysis of a primary amniotic fluid cell culture in contrast to the analysis of a subculture.
identify the origin of the satellited marker. Although it is well-known that many of these supernumerary chromosomes are harmless, this does not have to be so in every case. If, e.g., the marker is part of a chromosome 22, the different carriers in the same pedigree may exhibit a normal phenotype, the cat eye syndrome, or they may suffer from an even more severe state of malformations and mental retardation. This is one incertainty in respect to offspring, another is the possibility of secondary non-disjunction leading e.g. to a trisomy 21. After open discussions the couple insisted on having an amniocentesis. Fortunately, what emerged was a normal fetal karyotype. Cuse 2. - A pair of monozygous twins was born with the typical phenotype of cri du chat syndrome; their faces are shown in Fig. 4 at the age of 2 1/2 years. The karyotype (Fig. 5 ) presents a picture that looks like monosomy 21. Based on the clinical features of the twins this possibility is more than unlikely and we have no doubts that we are confronted with an unbalanced translocation, the long arm of a 21 being translocated to the deleted short arm of a chromosome 5. To prove this by cytological means turned out to be very difficult. The banding patterns of the two segments involved resemble each other strongly. G-banding was uninformative (Fig.
5). The same was true for Q-banding. The results with R-banding, using acridin orange fluorescence, were equivocal; some investigators thought they could see a difference between the two number, 5 chromosomes in some mitoses, others were less confident. To a lesser degree, the same uncertainty extends to the parental karyotypes although the same experts in R-banding considered them to be normal. The parents are young and the twins are their only children; so, sooner or later the question of prenatal diagnosis will be posed. In this respect it might be argued that all the clinically important aneuploid states resulting from a reciprocal translocation would be recognizable in spite of the inability to identify the translocated segments. This holds true for trisomy 21 and for monosomy of the short arm of chromosome 5 . It does, however, not apply to the combination of the two aneuploid states. Since patients have been described having the combination of cri du chat syndrome and trisomy 13 (LEISTIet al. 1975) the assumption that a child with trisomy 21 and cri du chat syndrome might survive is, by no means, far-fetched. We are hopeful that further improvements in banding techniques will help to solve this special and rare problem. Case 3. - The third example deals with a problem that was recognized early but, fortunately, turned out to be rare. It is the problem of families in which an occasional carrier of an apparently balanced translo-
Horrditus 86 (IY77)
PRENATAL DIAGNOSIS
Fig. 4. Two monozygous twins with the clinical picture of cri du chat syndome.
Fig. 5. G-banded karyotype of the twins with cri du chat syndrome. The translocation of a long arm of chromosome 21 to the deleted short arm of a No, 5 cannot be demonstrated cytologically with this banding technique.
41
42
Hereditus 86 (1977)
W . SCHMID
Tuhle 4 . Prenatal diagnoses carried out in 13 cases of parental translocation or inversion heterozygosity All balanced offspring was born normal Aberration
Ascertainment
Result of p.d. Outcome
a ) t(l:lO)pat. h) t(3;15)mat. c) t(13;14)pat. d) t(14;15)pat. e) inv(3)mat. f, t(3;IS)mat.. same a s h ) g) t(13;14)pat. h) t(14;21)pat. i) t(13;14)pat., same as c) k ) t(1;ll)mat. I) t(13;14)mat. m) t(14;2l)mat. n ) t(14;21)pat.
Chance Unbalanced child Unbalanced child (tri 13) Chance (in p.d.) Unbalanced child
Normal Unbalanced Balanced Balanced Balanced Balanced Normal Normal Balanced Balanced Normal Normal Balanced
Sterility in brother Abortion Abortion Chance (brother tri 21) Unbalanced child Unbalanced nephew
cation shows an abnormal phenotype while all other carriers are normal. A severely dysmorphic girl, whose features reminded of Down’s syndrome (SCHMID 1972) died at the age of two from a congenital heart defect. The karyotype showed an apparently balanced translocation involving the long arms 8 and 18. A family study revealed that several other completely normal members, including sibs, showed a cytologically identical karyotype anomaly. If a prenatal diagnosis were carried out in this family some uncertainty would always remain with respect to the outcome of a fetus with the apparently balanced translocation. Large series of prenatal studies in translocation families, however, did not run into this problem. Tuble 5 . Clones with single aberrant mitoses Types of aberrations observed in 314 cases Aberration Deletions Supernumerary centric elements Karyotypes with ”broken” chromosomes (euploid) Karyotypes containing extra acentric fragments (aneuploid) Karyotypes with translocated extra material Karyotypes with multiple (3) aberrations Total
No. Details 9 (3Cp-,Cq-,Bp-,Bq-, 3q-, I7p-,Gq-)
7 (D-like: 3, G-like: 3, other: I ) 4 (C broken at centromere 3, other I ) 5
I I
27
(FP+)
Normal Spontaneously aborted Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Neither did we find any phenotypical anomalies in our series (Table 4).
4. Problems 2 and 3. Somatic numerical and structural chromosome mutations in cell cultures versus true fetal mosaicism expressed in the amniotic cell culture So far, I have never met with what I would consider as true fetal mosaicism in any of our 480 cases. This, however, does not lead us to conclude that we could neglect this possibility. It has been observed elsewhere and confirmed postnatally (e.g. BLOOM et al. 1974). Somatic mutations, on the other hand, are seen frequently. Their true incidence can only be recognized if adequate methods for their detection are used. It is necessary to apply the in situ technique with clone-wise analysis of the karyotypes. We have collected the detailed results in a consecutive series of 314 cases. In these cases, as a routine, 15 metaphases were analyzed from between 5 to 10 different clones. Single or a few aberrant metaphases within clones were observed in 22 cases. The types of aberrations are presented in Table 5. It was soon obvious that quite a number of these aberrations had a common origin, namely the splitting of a C-group chromosome near the centromere. Based on this assumption it was possible to explain 17 of the 27 abnormal karyotypes as indicated in Fig. 6. What could be the reason for this relatively high incidence of somatic mutations in amniotic fluid cell cultures? One explanation might be that it simply is a property inherent to the prevailing cell type in these cultures. It also has been claimed that mycoplasma contamination could play a major role
PRENATAL DIAGNOSIS
43
Resulting karyot-vpes
a) C missing D-like + G-like in excess b) C missing D-like in excess c) C missing G-like in excess d) Normal karyotype + D-like e) Normal karyotype + G-like
3x 3x lx 7x 3x
Fig. 6. Breakage of a C group chromosome at the centromere. Observed abnormal karyotypes which can be explained as a consequence of such an event.
(SCHNEIDER et al. 1974). If mycoplasma, however, really should be such a frequent contaminant either in many different lots of commercial fetal calf serum or in amniotic fluid samples, I d o not see what, realistically, could be done to remedy this situation. As we have seen, clone-wise analysis of the cultures is a feasible solution to the problems posed by somatic mutations. Cases containing single clones in which all the cells had the identical aberration occurred much more rarely than clones with one or a few aberrant cells. In our consecutive series of 314 cases we observed two such instances. One involved a large deletion of the short arm of a chromosome I , the other monosomy for chromosome 22. In cases like these, clone-wise analysis is helpful, again, to arrive at a judgement whether the possibility of fetal mosaicism is likely or not. If one out of ten clones exhibits a karyotype not known from a viable malformation syndrome, this is not worth worrying the mother by telling her about it. In more serious cases of mosaic findings, e.g. if trisomy 21 would be found in a clone, one certainly ought to repeat amniocentesis before abortion is considered.
no means always maternal as we know from cases where the fetus is male. In two cases the maternal cells observed in our study grew in colonies which practically were indistinguishable from fetal clones. In both cases only a single colony was involved. The minimal safeguard against confusing fetal and maternal karyotypes consists in studying a sufficient number of different clones, if possible six to ten. Helpful, at least in cases of male fetuses, is the study of Y chromatin in the direct preparations of native cells. The way to obtain absolute assurance in cases of female fetuses consists in comparing the fluorescent markers of mother and presumed fetus. HAUGEet al. (1975) demonstrated in 50 cases that all the fetuses differed from their mothers in two or more - up to nine - markers. An interesting example illustrating this feature is shown in Fig. 8. In this case the obstetrician (Prof. W. E. Schreiner) succeeded in puncturing the two different amniotic fluid sacs in a twin pregnancy. A dye was injected into the first sac and by obtaining clear fluid from the second puncture the operator was
5. Problem 4. Maternal cell growth Maternal cell growth is a problem which cannot be dismissed lightly. I know several colleagues who have changed their technique from trypsinisation to in situ analysis after having made one or two wrong diagnoses of fetal sex. In our study we were able to document maternal cell growth, in between fetal clones, in seven cases and probably missed a similar number in which the fetus was of female sex. In five cases only few maternal cells were discovered and these were growing loosely in between typical fetal clones in a fashion sketched in Fig. 7. Cells growing in this way are by
possible maternal cell growth Fig. 7. In the majority of cases maternal cell growth was observed in the form of loose colonies in between clones of fetal cells. There are exceptions t o this rule: maternal cell colonies resembling fetal clones do occur.
44
w. scmm
13
Hereditas 86 ( I Y 7 7 )
21
15
14
22
Fig. 8. Fluorescent markers in a set of non-identical female twins, A and B,diagnosed prenatally, and in their mother, M. The large fluorescent marker o n a No. 15 is present only in twin B and in the mother; the two differ, however, in other markers.
sure of having reached the other sac. The twins were female but non-identical as was obvious from the fluorescent markers. In conclusion, prenatal diagnosis definitely poses some special cytogenetical problems. Among these, somatic mutation and maternal cell growth should, however, not be considered as a source of unavoidable pitfalls. By anticipating these events and by applying the necessary high technical standards most instances can be recognized in time and diagnostic errors can be kept at a very low level. Literature cited BLOOM,A. D., SCHMICKEL. R., BARR,M . and BIJRDI.A . R. 1974. Prenatal detection of autosomal mosaicism. J. Pediar. 84: 732-133 HAUGE.M . , POULSEN.H., HAI.BERG, A. and MIKKELSEN, M. ~
1975. The value of fluorescence markers in the distinction between maternal and fetal chromosomes. Ifutn. Genet. 26: 187-191 HOEHN,H., BRYANT,E. M . . KARP.L. E. and MARTIN,G. M 1974. Cultivated cells from diagnostic amniocentesis in second trimester pregnancies. 1. Clonal morphology and growth potential. Pediat. Res. 8: 746-754 LEISTI. J . , KABACK,M . M . and RIMOIN. D. L. 1975. I . Cri-duchat and trisomy 13 syndromes in an infant with an unbalanced chromosomal translocation. ~ - Birth D+cts.Original Article Series. I I ( 5 ) :3 17 -3 I9 SCHMIU,W. 1972. Die prlnatale Diagnose von Chromosomenanomalien. ~~-Triangel 1 1 : 91--102 SCHMIII,W. 1975. A technique for in situ karyotyping of primary amniotic fluid cell cultures. - Hum. Genet. 30. 325---330 SCHNEIDER, E. L., STANBRIDCE, E. J . , EPSTEIN.C. J . , GOLRUS, M . , ABBO-HALBASCH, G. and RODGERS,G. 1974. Mycoplasma contamination of cultured amniotic fluid cells: Potential hazard to prenatal chromosomal diagnosis. - Science 184: 477 -479 ~~
~
(IY77)
Cytogenetical problems in prenatal diagnosis W. SCHMID Division of Medicul Genetics, Department qf’ Pediatrics, finiversity of‘ Zurich. Switzrrlund
SCHMID, W . 1977. Cytogenetical problems in prenatal diagnosis. Sweden. ISSN 0018-0661. Received February 10, 1977
~~
Hereditus 86: 37~-44. Lund.
On the basis of 480 prenatal diagnostic karyotype studies, carried out since 1971 in Zurich. the following types of problems were considered: ( I ) The difficulty is inherent in the counselling problem itself, as an unusual chromosome anomaly in a parent. (2) True fetal mosaicism. (3) Somatic mutations during culture. (4) Growth of maternal cells. In problems of type ( I ) , i t is known in advance that the prenatal diagnosis has limitations. Problems of the types ( 2 ) to (4) can be solved to some degree by using in situ preparations and the clone-wise analysis. In ( 2 ) and ( 3 ) analysis of 1 inetaphases from 5 to 10 clones has been informative, somatic mutations usually being restricted to single cells within clones with otherwise normal karyotype. When entire clones exhibit a deviating karyotype, the proportion of abnormal to normal clones will help decide whether it is a case of ( 2 ) or (3). Also problem (4) is elucidated by the in situ method, since maternal cells. generally. differ in their growth pattern from fetal cells. If uncertainties persist, the comparison of lluorescent markers of presumed fetal cells to those of the mother is apt to resolve the problem. Somatic mutation and maternal cell growth should not be considered as a source of unavoidable pitfalls in prenatal cytogenetic diagnosis. By anticipating these events and by applying appropriate techniques. diagnostic errors can be kept at a very low level. W . Schmid. Division q / Medicul Genetics, Department of Pediutrics. Ci’niversity o/ Zurich, CH-8032 Zurich, Switzerland
Cytogenetical problems in prenatal diagnosis can be divided into four groups: ( I ) The difficulty may be inherent in the counselling problem, i.e. in an unusual kind of karyotype anomaly in a parent. (2) True fetal mosaicism, expressed in the amniotic cell culture. (3) Somatic numerical and structural chromosome mutations in cell culture. (4) Growth of maternal cells, which can be mistaken for fetal cells. Prior to setting forth my experience with these four problems I should like to present, in tabular form, the case load on which our observations were collected.
autosomal trisomies. (2) Recurrence of Down’s syndrome after a sporadic case of t(21; 21) translocation Down’s syndrome. Both parents had normal karyotypes with no mosaicism detectable in blood cultures. (3) A large omphalocele was found due to high A F P (alpha-I-fetoprotein, 110 pg/ml in 16th week of pregnancy) in the amniotic fluid of a woman with a previous 21 trisomic child.
1 , Cases studied in Zurich from 1971 to 1976
Indications
The indications for 480 prenatal diagnoses divided into major groups are shown in Table 1. Table 2 presents the results from 326 cases with primarily cytogenetic indications. By and large, these results are similar to those of other major published series. Three points are noteworthy: (1) The incidence of sex chromosome anomalies (XXY and XXX in particular) in the high maternal age group is lower than would be expected on the assumption of a similar age influence as the one observed with
Table I. Indications for 480 prenatal diagnoses (number of patients, up to Sept. 15, 1976) Karyotype studied in all cases, A F P in the last 394 cases, biochemical studies in 13 cases Patients Number High maternal age (mother 40 and over: 125) Trisomy 21 in sibs Chromosomal aberration in parent Biochemical defects MMC, anencephaly. hydrocephaly X-linked disorders Obstetrical indications Various
191 I I6 15
12 24 9
82 45
”(,
38.7 23.5 3.1 2.4 4.8 1.8 16.6 9. I
38
w. SCHMID
Hrreilitas X6 ( l Y 7 7 )
Tuhlr 2. Results in cases with primarily cytogenetic indications Total number
Indications
Abnormal Number
Details
",,
High maternal age mother 40 and over
I25
6
4.8
mother 25-39 Trisomy 21 in sibs
66 I16
I 3
I .5 2.6
I5 4
1 0
6.1
326
II
3.4
Chromosomal aberrations in parents Trisomy (except 21) in sibs Total
3 x trisomy 21 2 x trisomy 18 I x XXY I x trisomy 21 (mother 38) I x trisomy 21 (mother 27) 1 x tri-21 by t(21;21) (same as brother, parents normal) I x omphalocele I x part. trisomy 3
0
Table 3. Results in various indication groups Indications
X-chromosomal defects
No.
9
Details
Results Karyotype, AFP. biochemical
Hemophilia (5) Duchenne (3) Mucopol. Hunter ( 1 )
4 d d . 19 3 dd 1 d (biochemically abnormal)
Autosomal recessive metabolic disease
12
Metachromatic leucodystrophy (3) Mucopolysacharidoses (4) GM-I-Gangliosidosis (2) Glycogenosis I I ( I ) ADA-Deficiency ( 1 ) Fay-Sachs ( 1 )
I x abnormal 1 x abnormal
Obstetrical indications in 2nd half of pregnancy
82
Placental dysfunction (55)
1 x trisomy 21 (mother 25 y.) 1 x anencephaly with ring chrom. 13
Polyhydramnios (20) suspected malformations (7)
I x mosaic trisomy 22 (mother 48 y.) 1 x sacrococc. teratoma (high AFP) 1 x pathol. twin pregnancy (high AFP) I x anencephaly (high A F P ) 1 x anencephaly (high AFP)
Spina bifida
24
I previous anencephalic (5) 2 previous anencephalics (2) 1 previous M M C (10) 1 anencephalic + I M MC ( 1 ) 1 previous hydrocephalic (6)
All results normal in respect to A F P ( I trisomy 18, mother41)
Various
45
Mostly diverse malformations in previous sibs
All normal
Table 3 presents the results in five other indication groups. In the cases of X-chromosomal defects sex was determined by complete karyotype analysis. The biochemical studies in metabolic disease were performed by Drs. Wiesmann and Herschkowitz, Bern, Dr. Gitzelmann, Zurich, Dr. Niermeijer, Rotterdam and Dr. Hirschhorn, New York, who took over our fibroblast cultures. Prenatal genetic studies
for obstetrical diagnostic purposes, carried out in the second half of the pregnancies, are justifiable as long as the capacity of the laboratory is not impaired by this serivce to the disadvantage of more pressing cases. The percentage of abnormal results is not low as can be seen from the table. The range of action that can be taken by the obstetrician is. however, limited. In cases as severe as anencephaly a
Hrrrdirus 86 ( I Y 7 7 )
PRENATAL DIAGNOSIS
39
Fig. 1. Cover glass preparations of 2 primary amniotic fluid cell cultures. The round colonies - actually clones derived from single cells are of very unequal size. They have developed within 9 and 1 1 days, respectively.
premature birth may be induced. In cases of fetal chromosomal aberrations Caesarian sections are avoided in the interest of the mother. If the results are normal, this seems to be a stimulus for the obstetrician to d o for the pregnancy as much as is possible. 2. Technique and basic considerations in regard to methodology The techniques used in our work have been described previously (SCHMID1975). In situ analysis is important in respect to the last three of our problems to be discussed later: fetal mosaicism, somatic mutation in vitro and maternal cell growth. In primary amniotic cell cultures cellular proliferation is seen in the form of distinct colonies (Fig. I). For practical purposes it is of importance to know whether these colonies, in general, are derived from single fetal cells or from groups of fetal cells. The following observations are in favour of the single cell origin: ( I ) In cultures grown under favourable optical conditions there is evidence from direct observations. ( 2 ) The cellular morphology within colonies is strikingly uniform, whereas cellular morphology from clone to clone exhibits numerous different types (SCHMID1972; HOEHNet al. 1974). (3) Pure tetraploid colonies are found quite frequently. (4) In cases where all other colonies are normal, single colonies with aberrant karyotypes can be found. The author readily admits that a small
proportion of colonies may be derived from multiple cells - usually of the same type - but on the whole, the evidence is strongly in favour of the single cell origin. What does this mean from a practical point of view? The consequences are shown in Fig. 2 and 3 . Clone-wise analysis assures the investigator that he has studied descendants of several different fetal cells; in a subculture, he may look at a much higher number of mitoses which, however, all may be derived from the same predominant clone, i.e. from a single fetal cell. If mosaicism for two different karyotypes is present, clone-wise analysis permits a distinction between the two situations depicted in Fig. 3 . Somatic mutation involving part of a clone is harmless in contrast to true fetal mosaicism.
3. Problem 1. Cytogenetical difficulty inherent in the counselling problem Common to cases ofthis kind is the situation that it is known in advance that the answer from a prenatal study may have limitations. I would like to illustrate this problem with three examples: Case 1. - A young couple was seeking advice since the husband was found to be a carrier of a small acrocentric extra chromosome. The anomaly was discovered in a chromosome study performed because the wife had had two abortions, one in the third, the other in the first month of pregnancy. With banding techniques it was not possible to
40
H r r d t r u s XO ( I Y 7 7 )
W. SCHMID
Same material trypsinized
Primary culture
Somatic mutation
Fetal mosaic
Q . . :.' ..
Q ,
J ($
f I
15 metaphases
analyzed from 7 clones
30 metaphases analyzed. All may be derived from I big clone, i.e. the same cell
Trypsinized: In both cases the same proportion of cells may be aneuploid Fig. 3. A somatic mutation involving part of a clone (striped area) is distinguishable from true fetal mosaicism involving several independent clones. Subculturing obscures the situation.
Fig. 2. Clone-wise analysis of a primary amniotic fluid cell culture in contrast to the analysis of a subculture.
identify the origin of the satellited marker. Although it is well-known that many of these supernumerary chromosomes are harmless, this does not have to be so in every case. If, e.g., the marker is part of a chromosome 22, the different carriers in the same pedigree may exhibit a normal phenotype, the cat eye syndrome, or they may suffer from an even more severe state of malformations and mental retardation. This is one incertainty in respect to offspring, another is the possibility of secondary non-disjunction leading e.g. to a trisomy 21. After open discussions the couple insisted on having an amniocentesis. Fortunately, what emerged was a normal fetal karyotype. Cuse 2. - A pair of monozygous twins was born with the typical phenotype of cri du chat syndrome; their faces are shown in Fig. 4 at the age of 2 1/2 years. The karyotype (Fig. 5 ) presents a picture that looks like monosomy 21. Based on the clinical features of the twins this possibility is more than unlikely and we have no doubts that we are confronted with an unbalanced translocation, the long arm of a 21 being translocated to the deleted short arm of a chromosome 5. To prove this by cytological means turned out to be very difficult. The banding patterns of the two segments involved resemble each other strongly. G-banding was uninformative (Fig.
5). The same was true for Q-banding. The results with R-banding, using acridin orange fluorescence, were equivocal; some investigators thought they could see a difference between the two number, 5 chromosomes in some mitoses, others were less confident. To a lesser degree, the same uncertainty extends to the parental karyotypes although the same experts in R-banding considered them to be normal. The parents are young and the twins are their only children; so, sooner or later the question of prenatal diagnosis will be posed. In this respect it might be argued that all the clinically important aneuploid states resulting from a reciprocal translocation would be recognizable in spite of the inability to identify the translocated segments. This holds true for trisomy 21 and for monosomy of the short arm of chromosome 5 . It does, however, not apply to the combination of the two aneuploid states. Since patients have been described having the combination of cri du chat syndrome and trisomy 13 (LEISTIet al. 1975) the assumption that a child with trisomy 21 and cri du chat syndrome might survive is, by no means, far-fetched. We are hopeful that further improvements in banding techniques will help to solve this special and rare problem. Case 3. - The third example deals with a problem that was recognized early but, fortunately, turned out to be rare. It is the problem of families in which an occasional carrier of an apparently balanced translo-
Horrditus 86 (IY77)
PRENATAL DIAGNOSIS
Fig. 4. Two monozygous twins with the clinical picture of cri du chat syndome.
Fig. 5. G-banded karyotype of the twins with cri du chat syndrome. The translocation of a long arm of chromosome 21 to the deleted short arm of a No, 5 cannot be demonstrated cytologically with this banding technique.
41
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W . SCHMID
Tuhle 4 . Prenatal diagnoses carried out in 13 cases of parental translocation or inversion heterozygosity All balanced offspring was born normal Aberration
Ascertainment
Result of p.d. Outcome
a ) t(l:lO)pat. h) t(3;15)mat. c) t(13;14)pat. d) t(14;15)pat. e) inv(3)mat. f, t(3;IS)mat.. same a s h ) g) t(13;14)pat. h) t(14;21)pat. i) t(13;14)pat., same as c) k ) t(1;ll)mat. I) t(13;14)mat. m) t(14;2l)mat. n ) t(14;21)pat.
Chance Unbalanced child Unbalanced child (tri 13) Chance (in p.d.) Unbalanced child
Normal Unbalanced Balanced Balanced Balanced Balanced Normal Normal Balanced Balanced Normal Normal Balanced
Sterility in brother Abortion Abortion Chance (brother tri 21) Unbalanced child Unbalanced nephew
cation shows an abnormal phenotype while all other carriers are normal. A severely dysmorphic girl, whose features reminded of Down’s syndrome (SCHMID 1972) died at the age of two from a congenital heart defect. The karyotype showed an apparently balanced translocation involving the long arms 8 and 18. A family study revealed that several other completely normal members, including sibs, showed a cytologically identical karyotype anomaly. If a prenatal diagnosis were carried out in this family some uncertainty would always remain with respect to the outcome of a fetus with the apparently balanced translocation. Large series of prenatal studies in translocation families, however, did not run into this problem. Tuble 5 . Clones with single aberrant mitoses Types of aberrations observed in 314 cases Aberration Deletions Supernumerary centric elements Karyotypes with ”broken” chromosomes (euploid) Karyotypes containing extra acentric fragments (aneuploid) Karyotypes with translocated extra material Karyotypes with multiple (3) aberrations Total
No. Details 9 (3Cp-,Cq-,Bp-,Bq-, 3q-, I7p-,Gq-)
7 (D-like: 3, G-like: 3, other: I ) 4 (C broken at centromere 3, other I ) 5
I I
27
(FP+)
Normal Spontaneously aborted Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal
Neither did we find any phenotypical anomalies in our series (Table 4).
4. Problems 2 and 3. Somatic numerical and structural chromosome mutations in cell cultures versus true fetal mosaicism expressed in the amniotic cell culture So far, I have never met with what I would consider as true fetal mosaicism in any of our 480 cases. This, however, does not lead us to conclude that we could neglect this possibility. It has been observed elsewhere and confirmed postnatally (e.g. BLOOM et al. 1974). Somatic mutations, on the other hand, are seen frequently. Their true incidence can only be recognized if adequate methods for their detection are used. It is necessary to apply the in situ technique with clone-wise analysis of the karyotypes. We have collected the detailed results in a consecutive series of 314 cases. In these cases, as a routine, 15 metaphases were analyzed from between 5 to 10 different clones. Single or a few aberrant metaphases within clones were observed in 22 cases. The types of aberrations are presented in Table 5. It was soon obvious that quite a number of these aberrations had a common origin, namely the splitting of a C-group chromosome near the centromere. Based on this assumption it was possible to explain 17 of the 27 abnormal karyotypes as indicated in Fig. 6. What could be the reason for this relatively high incidence of somatic mutations in amniotic fluid cell cultures? One explanation might be that it simply is a property inherent to the prevailing cell type in these cultures. It also has been claimed that mycoplasma contamination could play a major role
PRENATAL DIAGNOSIS
43
Resulting karyot-vpes
a) C missing D-like + G-like in excess b) C missing D-like in excess c) C missing G-like in excess d) Normal karyotype + D-like e) Normal karyotype + G-like
3x 3x lx 7x 3x
Fig. 6. Breakage of a C group chromosome at the centromere. Observed abnormal karyotypes which can be explained as a consequence of such an event.
(SCHNEIDER et al. 1974). If mycoplasma, however, really should be such a frequent contaminant either in many different lots of commercial fetal calf serum or in amniotic fluid samples, I d o not see what, realistically, could be done to remedy this situation. As we have seen, clone-wise analysis of the cultures is a feasible solution to the problems posed by somatic mutations. Cases containing single clones in which all the cells had the identical aberration occurred much more rarely than clones with one or a few aberrant cells. In our consecutive series of 314 cases we observed two such instances. One involved a large deletion of the short arm of a chromosome I , the other monosomy for chromosome 22. In cases like these, clone-wise analysis is helpful, again, to arrive at a judgement whether the possibility of fetal mosaicism is likely or not. If one out of ten clones exhibits a karyotype not known from a viable malformation syndrome, this is not worth worrying the mother by telling her about it. In more serious cases of mosaic findings, e.g. if trisomy 21 would be found in a clone, one certainly ought to repeat amniocentesis before abortion is considered.
no means always maternal as we know from cases where the fetus is male. In two cases the maternal cells observed in our study grew in colonies which practically were indistinguishable from fetal clones. In both cases only a single colony was involved. The minimal safeguard against confusing fetal and maternal karyotypes consists in studying a sufficient number of different clones, if possible six to ten. Helpful, at least in cases of male fetuses, is the study of Y chromatin in the direct preparations of native cells. The way to obtain absolute assurance in cases of female fetuses consists in comparing the fluorescent markers of mother and presumed fetus. HAUGEet al. (1975) demonstrated in 50 cases that all the fetuses differed from their mothers in two or more - up to nine - markers. An interesting example illustrating this feature is shown in Fig. 8. In this case the obstetrician (Prof. W. E. Schreiner) succeeded in puncturing the two different amniotic fluid sacs in a twin pregnancy. A dye was injected into the first sac and by obtaining clear fluid from the second puncture the operator was
5. Problem 4. Maternal cell growth Maternal cell growth is a problem which cannot be dismissed lightly. I know several colleagues who have changed their technique from trypsinisation to in situ analysis after having made one or two wrong diagnoses of fetal sex. In our study we were able to document maternal cell growth, in between fetal clones, in seven cases and probably missed a similar number in which the fetus was of female sex. In five cases only few maternal cells were discovered and these were growing loosely in between typical fetal clones in a fashion sketched in Fig. 7. Cells growing in this way are by
possible maternal cell growth Fig. 7. In the majority of cases maternal cell growth was observed in the form of loose colonies in between clones of fetal cells. There are exceptions t o this rule: maternal cell colonies resembling fetal clones do occur.
44
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Hereditas 86 ( I Y 7 7 )
21
15
14
22
Fig. 8. Fluorescent markers in a set of non-identical female twins, A and B,diagnosed prenatally, and in their mother, M. The large fluorescent marker o n a No. 15 is present only in twin B and in the mother; the two differ, however, in other markers.
sure of having reached the other sac. The twins were female but non-identical as was obvious from the fluorescent markers. In conclusion, prenatal diagnosis definitely poses some special cytogenetical problems. Among these, somatic mutation and maternal cell growth should, however, not be considered as a source of unavoidable pitfalls. By anticipating these events and by applying the necessary high technical standards most instances can be recognized in time and diagnostic errors can be kept at a very low level. Literature cited BLOOM,A. D., SCHMICKEL. R., BARR,M . and BIJRDI.A . R. 1974. Prenatal detection of autosomal mosaicism. J. Pediar. 84: 732-133 HAUGE.M . , POULSEN.H., HAI.BERG, A. and MIKKELSEN, M. ~
1975. The value of fluorescence markers in the distinction between maternal and fetal chromosomes. Ifutn. Genet. 26: 187-191 HOEHN,H., BRYANT,E. M . . KARP.L. E. and MARTIN,G. M 1974. Cultivated cells from diagnostic amniocentesis in second trimester pregnancies. 1. Clonal morphology and growth potential. Pediat. Res. 8: 746-754 LEISTI. J . , KABACK,M . M . and RIMOIN. D. L. 1975. I . Cri-duchat and trisomy 13 syndromes in an infant with an unbalanced chromosomal translocation. ~ - Birth D+cts.Original Article Series. I I ( 5 ) :3 17 -3 I9 SCHMIU,W. 1972. Die prlnatale Diagnose von Chromosomenanomalien. ~~-Triangel 1 1 : 91--102 SCHMIII,W. 1975. A technique for in situ karyotyping of primary amniotic fluid cell cultures. - Hum. Genet. 30. 325---330 SCHNEIDER, E. L., STANBRIDCE, E. J . , EPSTEIN.C. J . , GOLRUS, M . , ABBO-HALBASCH, G. and RODGERS,G. 1974. Mycoplasma contamination of cultured amniotic fluid cells: Potential hazard to prenatal chromosomal diagnosis. - Science 184: 477 -479 ~~
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