PRENATAL DIAGNOSIS
Prenat Diagn 2002; 22: 645–648.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / pd.354

Prenatal detection of structural abnormalities of chromosome
18: associations with interphase ﬂuorescence in situ
hybridization (FISH) and maternal serum screening

Michael D. Graf1*, Prabhcharan Gill2, Michael Krew2 and Stuart Schwartz1
1Department of Genetics and Center for Human Genetics Laboratory, Case Western Reserve University and University
Hospitals of Cleveland, Cleveland, OH, USA
2Department of Obstetrics and Gynecology, Aultman Hospital, Canton, OH, USA

We describe two cases of prenatally ascertained isochromosome 18. Case 1 included both an isochro-
mosome 18p and an isochromosome 18q, while Case 2 involved only an isochromosome 18q. Both of these
cases were associated with a positive maternal serum triple screen trisomy 18 risk (greater than 1 in 100
risk). In addition, ﬂuorescence in situ hybridization (FISH) was performed on uncultured amniotic ﬂuid
interphase cells in both cases looking for aneuploidy for chromosomes 13, 18, 21, X and Y. The results of
the interphase analyses support the common knowledge that careful interpretation of interphase FISH
analysis is necessary and that results should be followed by full cytogenetic analysis. To our knowledge these
are the ﬁrst reported cases of structurally abnormal chromosomes 18 being associated with a positive
maternal serum triple screen for trisomy 18. Copyright # 2002 John Wiley & Sons, Ltd.

KEY WORDS: isochromosome 18; prenatal screening; cytogenetics; trisomy 18; interphase FISH

INTRODUCTION

The maternal serum triple screen is a routine prenatal
screening test performed between 15 and 25 weeks’
gestation used to estimate a pregnant woman’s risk of
having a child with trisomy 21 (Down syndrome),
trisomy 18 (Edward’s syndrome), open neural tube
defects and open abdominal wall defects (Ormond,
1997). The maternal serum triple screen is not designed
to detect structural chromosome abnormalities, or
other genetic abnormalities than those listed above. As
the maternal serum triple screen is a screening test,
false-positive results can occur; thus, a positive screen
should be followed by genetic counseling and an
amniocentesis should be offered.

When expedited results of an amniocentesis are
required, interphase ﬂuorescence in situ hybridization
(FISH) can be ordered along with routine cytogenetic
analysis. Interphase FISH results are typically avail-
able in 1–2 days in contrast to the 6–10 days that a full
cytogenetic analysis takes to complete (Weremowicz
et al., 2001). In the case of the Aneuvysion assay (Vysis
Inc., Downer’s Grove, IL, USA),
interphase FISH
uses a set of two mixtures of DNA probes speciﬁc for
either the centromere or single-site locus (SSL) of
chromosomes 13, 18, 21, X and Y (Weremowicz et al.,
2001).

This interphase FISH analysis restricted to these
probes will detect almost two-thirds of all chromo-
somal abnormalities detected at the time of amnio-
centesis and 95% of the chromosomal abnormalities
causing birth defects in newborns (Rhoads et al., 1989;

Robinson et al., 1991; Whiteman and Klinger, 1991).
Typically 50 interphase cells using each probe mix (100
cells total) are counted. To minimize excessive false-
positive results, more than 10% of the interphase cells
are required to be aneuploid to qualify as a positive
result. Should results
in the 11–89% range,
mosaicism or poor probe hybridization is suggested.
This supplemental FISH analysis is often requested in
cases of later gestational age, elevated maternal serum
triple screen risks for trisomy 18 or 21, abnormal
ultrasound ﬁndings, or parental anxiety (Cheong
Leung et al., 2001).

fall

PATIENTS AND METHODS

Sample preparation

Interphase FISH was performed using 3 cc whole
amniotic ﬂuid according to the manufacturer’s (Vysis
Inc.) instructions (Weremowicz et al., 2001). Fifty
nuclei for each set of probes were observed under the
microscope. The laboratory standard for equivocal
results for identifying aneuploidy was set at >10% of
the cells.

Six primary cultures from each amniotic ﬂuid
sample were established. Cytogenetic analyses of
G-banded chromosomes were compared using the
in situ technique. Fifteen cells were analyzed from 15
different colonies in at least two different primary
cultures for chromosome number.

Case 1

*Correspondence to: M. D. Graf, Center for Human Genetics
Laboratory, 10524 Euclid Avenue, 6th ﬂoor, Cleveland, OH 44106,
USA. E-mail: michael.graf@uhhs.com

A 20-year-old, African American woman was referred
for genetic counseling at 21.6 weeks’ gestation because

Copyright # 2002 John Wiley & Sons, Ltd.

Received: 15 June 2001
Revised: 4 January 2002
Accepted: 7 January 2002

646

M. D. GRAF ET AL.

a maternal serum triple screen resulted in a trisomy
18 risk estimate of 1 in 14 (maternal serum alpha-
fetoprotein (MSAFP)=2.35 MoM, human chorionic
gonadotrophin (hCG)=0.35 MoM, unconjugated oes-
triol (uE3)=0.41 MoM). In addition, a fetal sonogram
revealed bilateral dilated ventricles, bilateral choroid
plexus cysts, open neural tube defect, bilateral club
feet, a ventricular septal defect, endocardial cushion
defect, Dandy Walker malformation, omphalocele,
and overlapping ﬁngers with clenched ﬁsts.

An amniocentesis was performed at 23 weeks and
interphase FISH analysis was performed in addition to
the routine amniotic ﬂuid AFP and karyotype. Amnio-
tic ﬂuid AFP was elevated at 11.84 MoM. Due to the
obvious neural tube defect and omphalocele, acetyl-
cholinesterase analysis was not requested.

Interphase FISH analysis showed two signals for
both chromosomes 13 and 21 in 49/50 cells while a
single cell showed two signals for chromosome 13 and
three signals for chromosome 21. In addition, 45
interphase cells showed three signals for chromosome
18 and one signal each for the X and Y chromosome.
The three chromosome 18 signals appeared to be
identical
in size. Five cells showed two signals for
chromosome 18 and one signal each for the X and Y
chromosome. These results suggested that the male
fetus had three copies of chromosome 18 and no
aneuploidy for chromosomes 13, 21, X or Y.

Full cytogenetic analysis on amniotic ﬂuid cells
revealed 47,XY,x18,+i(18)(q10),+i(18)(p10). In addi-
tion to one normal chromosome 18, there was a
chromosome that consisted of two long arms from
chromosome 18 and another chromosome that con-
sisted of two short arms from chromosome 18. This
results in the fetus having clinical trisomy 18 due to
the presence of two isochromosomes.

The pregnancy was terminated at 23.8 weeks and
the parents declined an autopsy. Fetal tissue was
obtained at the time of termination for conﬁrmation of
the amniocentesis results, but the sample failed to
grow in culture. Peripheral blood from the parents has
not been examined.

Case 2

A 21-year-old, gravida one, African American woman
was referred for genetic counseling at 17.3 weeks’
gestation because a maternal serum triple screen
resulted in a trisomy 18 risk estimate of 1 in 44 (MSAFP=
0.37 MoM, hCG=0.50 MoM, uE3=0.33 MoM). In
addition, a fetal sonogram revealed holoprosence-
phaly, omphalocele, and bilateral hypoplastic fore-
arms with radial deviation of hands. Interphase FISH
analysis was done in addition to the routine cytoge-
netic analysis. The family history was non-contributory.
Amniotic ﬂuid AFP was 2.14 MoM with no acetyl-
cholinesterase band detected. This ﬁnding is consist-
ent with a ventral wall defect.

Interphase FISH analysis showed two signals for
both chromosomes 13 and 21 in 49 cells while a single
cell showed two signals for chromosome 13 and three

Figure 1—Interphase FISH analysis with probes for the X, Y and
18 centromeres. This analysis showed two signals from both the 18
probes. However, one centromere signal is signiﬁcantly smaller than
the other one

signals for chromosome 21. In addition, 46 interphase
cells showed two signals for both the X chromosome
and chromosome 18 while four cells showed two sig-
nals for the X chromosome and three signals for chro-
mosome 18. These results were well within normal
limits for the analysis. However, in all cells there was a
size discrepancy for the signals speciﬁc for chromo-
some 18 as one chromosome 18 signal was signiﬁ-
cantly smaller than the other chromosome 18 signal
(Figure 1).

Full cytogenetic analysis revealed 46,XX,i(18)(q10)
(Figure 2). In addition to one normal chromosome 18,
there was a chromosome that consisted of two long
arms from chromosome 18 resulting in the fetus
having monosomy 18p and trisomy 18q. Subsequent
FISH studies on metaphase cells with a probe (D18Z1)
speciﬁc for the centromeric region of chromosome 18
showed signals on both the normal chromosome 18
and the isochromosome 18q (Figure 3). However, the
signal on the isochromosome 18q was signiﬁcantly
smaller in all cells examined.

The pregnancy was terminated at 21 weeks and an
autopsy was not available. Peripheral bloods from the
parents were requested to rule out the possibility that
the isochromosome 18 was a result of a balanced
parental rearrangement. The mother’s karyotype was
46,XX. The father was unavailable for study.

Figure 2—A partial G-banded metaphase showing both the normal
18 and the I(18)(q10)(denoted by an arrow)

Copyright # 2002 John Wiley & Sons, Ltd.

Prenat Diagn 2002; 22: 645–648.

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647

The present cases are also interesting in regards to
the results of the interphase FISH tests performed
looking for aneuploidy of chromosomes 13, 18, 21, X
and Y. Although in Case 2 the results indicated there
was no aneuploidy for any of those chromosomes, one
chromosome 18 signal was smaller than the other
chromosome 18 signal. On two other occasions, inter-
phase studies in this laboratory revealed altered size of
the 18 signals, both resulting in cytogenetically normal
fetuses, suggesting that there may have been reduced
amounts of chromosome 18 alpha satellite in these two
cases. In the present case, however, there was a strong
suspicion that this fetus may have an abnormality
involving chromosome 18 because of the maternal
serum triple screen results and ultrasound ﬁndings.
Therefore, the laboratory was concerned about the
size discrepancy of these signals since it seemed logical
that the discrepant signals could be the result of a
structural abnormality of one chromosome 18. There-
fore, the results of the interphase FISH test were
described to the referring physician as being within
normal limits, but the laboratory suggested to inter-
pret the normal interphase FISH results with caution
until a full cytogenetic analysis was available. FISH
analysis on metaphase cells using a probe (D18Z1)
speciﬁc for the centromere of chromosome 18 showed
a smaller sized signal
for the isochromosome as
compared to the normal chromosome 18, suggesting
the centromeres of these two chromosomes to be
different.

In Case 1, the interphase FISH results were not
unusual, but they still point out the importance of
carefully interpreting such results. The interphase
FISH results showed three chromosome 18 signals,
and although it would be easy to conclude that the
fetus had trisomy 18 which, while phenotypically
correct, would not be cytogenetically accurate. Full
cytogenetic analysis indicated that the fetus had one
normal chromosome 18, one isochromosome 18p
and one isochromosome 18q. Although a trisomy 18
phenotype would be expected, it would be important
to explain the difference between trisomy 18 and the
actual cytogenetic ﬁndings of this case. In the present
case it is possible, although unlikely, that a parent
could carry a structural abnormality of chromosome
18 that could lead to the occurrence of isochromo-
somes, which would increase the recurrence risk. This
conﬁrms the importance of following interphase FISH
results with full cytogenetic analysis.

Additional reports are needed to determine whether
there is a relationship between the maternal serum
triple screen and structural abnormalities of chromo-
some 18, such as isochromosome 18q. In addition,
although the interphase FISH analysis used in pre-
natal cases is not designed to detect structural chromo-
somal abnormalities, perhaps it is possible to detect
certain structural abnormalities of chromosome 18
and 21. Finally,
interphase FISH probe signal size
discrepancy needs to be interpreted with caution,
especially if using probes from regions of the genome
containing repetitive DNA.

Figure 3—A metaphase showing hybridisation to both the normal
and isochromosome 18. The isochromosome 18 demonstrates a
reduced signal with the alpha-satellite chromosome 18 probe
(D18Z1)

DISCUSSION

As the critical region for trisomy 18 has been demon-
strated to be localized to the long arm of chromosome
18 (Turleau and deGrouchy, 1977), individuals with a
normal chromosome 18 and an isochromosome 18q
(Case 2) will typically have a phenotype similar to that
of trisomy 18, but may also have features of 18p
monosomy (Wurster-Hill et al., 1991; Bohosioan-Sell
et al., 1994).

There have been only two other cases involving both
an isochromosome 18p and an isochromosome 18q
reported in the literature (Muller et al., 1972; Larson
et al., 1978). It is assumed and has been shown in these
cases that the phenotype of these individuals mimics
that of individuals with three number 18 chromo-
somes.

The two cases presented here are interesting for two
reasons. First, these cases are, to our knowledge, the
ﬁrst reported cases involving structural abnormalities
of chromosome 18 associated with a positive maternal
serum triple screen risk for trisomy 18. Second, Case 2
raises the role of atypical interphase FISH results in
possibly indicating the presence of a chromosomal
rearrangement and both cases conﬁrm the need for
caution when making interpretations of interphase
FISH results.

Although there has been no reported association
between an isochromosome 18 and a trisomy 18 risk
by maternal serum triple screen, there is a report of a
fetus with Down syndrome caused by an isochromo-
some 21, as well as a report of several undetermined
D;G and G;G translocations, having positive results
for Down syndrome by maternal serum triple screen
(Cheng et al., 1993; Meyers et al., 1997). However, as
the critical region for trisomy 18 is located in the long
arm, it can be speculated that an isochromosome 18q
would probably cause the same maternal serum triple
screen pattern as full trisomy 18 since the phenotypes
are similar.

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