Download Two supernumerary marker chromosomes

Original Article
Cytogenet Cell Genet 93:182–187 (2001)
Two supernumerary marker chromosomes,
originating from chromosomes 6 and 11, in a
child with developmental delay and craniofacial
dysmorphism
B. Maurer,a T. Haaf,b K. Stout,b N. Reissmann,a C. Steinlein,a and M. Schmida
a Department
of Human Genetics, University of Würzburg, Biozentrum, Würzburg, and
for Molecular Genetics, Berlin (Germany)
b Max-Planck-Institut
Abstract. The interpretation of the significance of marker
chromosomes, which can be encountered at prenatal diagnosis,
is extremely problematic. Various factors contribute to the difficulty of clarifying the phenotypic risks of supernumerary
marker chromosomes, including differences in the size, structure, and origin of marker chromosomes, as well as the occurrence of multiple marker chromosomes of different origin in
the same proband. Research on marker chromosomes is currently in a data-accumulation phase. We report the presence of
two marker chromosomes, originating from chromosomes 6
and 11, in a child with developmental delay and craniofacial
dysmorphism and discuss the related literature.
Marker chromosomes are defined as additional or supernumerary chromosomes which are not directly equivalent to any
of the normal chromosomes. Marker chromosomes are found
in approximately 1 in 2,500 newborns (Friedrich and Nielsen,
1974; Buckton et al., 1985; Warburton, 1991) and are more
common in patients with congenital abnormalities, abnormal
sexual development, or mental impairment (Buckton et al.,
1985).
Fluorescence in situ hybridization (FISH) has proved to be
helpful in further identifying the chromosomal origin of marker
chromosomes (Callen et al., 1992; Daniel et al., 1994). However, it is still very difficult to compare clinical findings in
patients with markers of the same chromosomal origin. There
are several reasons for the difficulty to relate clinical syndromes
to the occurrence of marker chromosomes:
1. Marker chromosomes can be derived from any chromosome.
2. Even if two marker chromosomes originate from the
same chromosome, they still often differ in size and in the content of euchromatic material from either or both arms of a chromosome.
3. Structural variants of marker chromosomes, e.g., ring formation, have been described.
4. Some patients have multiple marker chromosomes of different origin.
5. Single or multiple marker chromosomes often occur in a
mosaic form.
Therefore, when a marker chromosome is detected at prenatal diagnosis, the associated phenotypic risks remain difficult to
predict for genetic counseling. Further case reports will be necessary to elucidate the true clinical significance of marker chromosomes.
We report the occurrence of two supernumerary marker
chromosomes derived from chromosomes 6 and 11 in a 5-yrold girl with craniofacial dysmorphism and developmental
delay and discuss the related literature.
Received 15 May 2001; accepted 28 May 2001.
Request reprints from Prof. Dr. M. Schmid, Department of Human Genetics,
University of Würzburg, Biozentrum, Am Hubland,
D–97074 Würzburg (Germany); telephone: +49-931-888-4077;
fax: +49-931-888-4069; e-mail: [email protected]
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Case report
The patient, a 5-yr-old girl, was born at 40 wk gestation after a pregnancy
complicated by hypertension. She is the fourth child of a 34-yr-old mother
who had given birth to three healthy children previously. Birth weight was
3,650 g, length 54 cm, and head circumference 37 cm. The Apgar index was
recorded as 7, 8, and 9 after 1, 5, and 10 min, and the pH of the umbilical
arterial blood was 7.31. Soon after the delivery, the hypotonic child developed difficulties in breathing, followed by a transient cyanosis, and had to be
transferred to an intensive pediatric care unit.
The child presented a variety of craniofacial dysmorphic features: frontal
bossing; severe hypoplasia of the middle face with a broad nasal bridge; a
down-turned mouth with thin lips; large, dysplastic ears; and an atypic furrow of the fourth left finger. A premature synostosis of the sagittal and basal
parts of the coronary sutures lead to a surgical correction (craniectomy) some
months after birth.
Postnatal sonography revealed an atrial septum defect, a persistent ductus arteriosus Botallo, and hypoplasia of the left kidney. Neurologically, nystagmus in combination with divergent strabismus was noted.
When the girl was last seen by her pediatrician at the age of 5 yr, she had
considerable motor difficulties, especially in regard to motor coordination;
however, her verbal abilities were nearly in accordance with her age. She is
now attending a special school. Further clinical data on the patient were not
available.
Table 1. Marker chromosome derived from chromosome 6 by YAC
hybridization and determination of the presence or absence of specific chromosome bands
Chromosome preparation and banding
Metaphase chromosomes were prepared in the standard way from
human peripheral blood. R-, C-, and G-banding were performed according to
the techniques of Dutrillaux and Lejeune (1971), Sumner (1972), and Seabright (1973). One hundred metaphase spreads were analyzed in the patient
and in each of her parents.
thiocyanate (FITC)-conjugated avidin (Vector) and digoxigenated probes by
Cy3-conjugated anti-digoxin antibody (Dianova). Chromosomes and cell
nuclei were counterstained with 1 Ìg/ml 4),6-diamidino-2-phenylindole
(DAPI) in 2 × SSC for 5 min. The slides were mounted in 90 % glycerol,
100 mM Tris-HCl (pH 8.0) and 2.3 % 1,4-diazobicyclo-2,2,2-octane.
Images were taken with a Zeiss epifluorescence microscope equipped
with a thermoelectronically cooled charge-coupled device camera (Photometrics CH250), which was controlled by an Apple Macintosh computer.
Vysis imaging software was used to capture grayscale images, to superimpose
these into a color image, and to convert the DAPI image into a G-banded
metaphase spread for identification of the chromosomes.
DNA probes
Chromosome-specific ·-satellite subsets have been described for the
majority of human chromosomes and are widely used for marker-chromosome identification (Haaf et al., 1992; Haaf, 2000). Clone pLC11A is specific
for chromosome 11 (Wevrick and Willard, 1989). A set of oligonucleotide
primers directed to a conserved region of the human ·-satellite consensus
sequence (Haaf and Willard, 1998) was used to amplify, by PCR, chromosome 6-specific ·-satellite fragments from genomic DNA of a monochromosomal somatic cell hybrid.
To FISH map the ring chromosome 6 in fine detail, we selected regionspecific non-chimeric clones (see Table 1) from a standard set of, up to now,
more than 3,000 cytogenetically and genetically anchored CEPH YACs (for
more information, see the Molecular Cytogenetics and Positional Cloning
Center internet site at http://www.mpimg-berlin-dahlem.mpg.de).
Spectral karyotyping (SKY)
Optimally aged slides (3–15 d at room temperature) were hybridized
with a SKY probe mixture supplied by Applied Spectral Imaging. The probe
mixture contains 57 uniquely labeled chromosome-specific probes, which
are combinatorially labeled with three fluorochromes and two haptens (SpectrumGreen, SpectrumOrange, and TexasRed for direct labeling, as well as
biotin-16-dUTP and digoxigenin-11-dUTP for indirect labeling; Macville et
al., 1999). After hybridization, biotin was detected with avidin-Cy5 and digoxigenin with mouse anti-digoxigenin, followed by sheep anti-mouse custom-conjugated to Cy5.5. Metaphase preparations were counterstained with
DAPI (150 ng/ml) in 2 × SSC and covered with antifade solution (Vectashield mounting medium; Vector Laboratories). Image acquisition was
achieved with the SpectraCube system (Applied Spectral Imaging) and analyzed with SKY view imaging software (Garini et al., 1996).
Materials and methods
FISH
Standard protocols for FISH were followed (Haaf, 2000). Briefly, the
slides were treated with 100 Ìg/ml RNase A in 2 × SSC (pH 7.0) at 37 ° C for
30 min and with 0.01 % pepsin in 10 mM HCl at 37 ° C for 10 min. After
refixing the preparations for 10 min in 1 × PBS, 50 mM MgCl2, 1 % formaldehyde, they were dehydrated in an ethanol series (70 %, 80 %, and 100 %).
Slides were denatured for 1 min at 90 ° C in 70 % formamide, 2 × SSC (pH
7.0) and again dehydrated in an alcohol series.
Probes were labeled by standard nick-translation procedures with biotin16-dUTP or digoxigenin-11-dUTP (Boehringer Mannheim). Either 2 ng/Ìl
of fluorescent-labeled ·-satellite DNA or 10 ng/Ìl of YAC DNA was coprecipitated with 100 ng/Ìl human Cot-1 (GIBCO BRL) competitor DNA (for
single-copy probes) and 500 ng/Ìl salmon sperm carrier DNA and then redissolved in 50 % formamide, 20 % dextran sulfate, and 2 × SSC. After 10 min
denaturation at 80 ° C, 30 Ìl of hybridization mixture was applied to each
slide and sealed under a cover slip. Slides were left to hybridize in a moist
chamber at 37 ° C for 1 d. Slides were washed 3 × 5 min in 50 % formamide,
2 × SSC at 42 ° C, followed by a 5-min wash in 0.1 × SSC at 65 ° C. Some
hybridizations with ·-satellite probes were carried out under higher-stringency conditions, using 65 % formamide in the hybridization mixture and posthybridization washings. Biotinylated probes were detected by fluorescein iso-
Results
Cytogenetic analysis of peripheral lymphocytes from the
proband showed a complex karyotype involving two marker chromosomes. The mosaic karyotype consisted of four
cell lines: 46,XX/47,XX,+mar1/47,XX,+mar2/48,XX,+mar1,
+mar2 in 36 %, 26 %, 22 % and 16 % of the cells analyzed. Both
parents had normal karyotypes.
Hybridization experiments with chromosome-specific ·satellites demonstrated that the microchromosomes are derived from chromosomes 6 (mar 1) and 11 (mar 2). The larger
marker chromosome contains the centromeric region of chromosome 6. In several metaphase cells the marker chromosome
6 showed ring formation and a dicentric structure. YACs
938d08 from the distal short arm of chromosome 6, as well as
YACs 819a10, 694h11, 840g08, and 803e10 from the proximal
Cytogenet Cell Genet 93:182–187 (2001)
183
Fig. 1. (a–g) Marker chromosomes 6
and 11 as well as selected chromosome pairs
1, 6, 7, 9, 11 and 16 of the patient showing
C-banding (a–c)), R-banding (d–f), and Gbanding (g). The additional chromosome
pairs were chosen to allow size comparison
with the marker chromosomes, as well as for
the demonstration of the C-banding quality.
Note the ring structure of mar 6 in a), d, f,
and g. (h) Metaphase cell of the patient after
FISH analysis with ·-satellite fragments
specific for chromosome 6 and clone
pLC11A specific for chromosome 11. (i)
Selected chromosome pairs 6 and 11 showing FISH signals. (k) SKY analysis of chromosomes 6, 11, mar 6, and mar 11.
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Cytogenet Cell Genet 93:182–187 (2001)
end of the short arm were FISH-mapped to both the normal
chromosomes 6 and the ring(6). In contrast, YACs 810b02 and
758c05 from the region 6p23 → p22. as well as YACs from the
long arm, were not present on the ring(6) (see Table 1). Marker
chromosome 11 was not analyzed further, as it consists of constitutive heterochromatin. Karyotype details and marker chromosomes, as well as the FISH and SKY results, are shown in
Fig. 1. Additionally, chromosome pairs 1, 7, 9, and 16 are
shown to compare their size with that of the two marker chromosomes and to demonstrate the quality of the C-banding.
Discussion
Although the identification of marker chromosomes has
improved with the application of modern techniques, such as
FISH, the interpretation of the clinical significance of supernumerary chromosome fragments still remains highly problematic, especially when these are encountered at prenatal diagnosis.
A chromosomal analysis of both parents is necessary in order to
understand the mutant origin of the marker chromosomes.
When inherited from a normal parent, the marker chromosome
is less likely to cause phenotypic anomalies in the child than
when it is appears de novo. Buckton et al. (1985) found that one
of seven probands with a familial marker chromosome was
mentally retarded, compared to six of eight probands with de
novo marker chromosomes.
Various attempts have been made to classify marker chromosomes according to different chromosomal origin and size
in order to better predict the phenotypic risk, especially of de
novo marker chromosomes (Soudek et al., 1973; Friedrich and
Nielsen, 1974; Soudek and Sroka, 1977; Nishi et al., 1982;
Steinbach et al., 1983; Buckton et al., 1985; Djalali, 1990; Callen et al., 1991, 1992; Raimondi et al., 1991; Plattner et al.,
1993; Daniel et al., 1994; Blennow et al., 1995). Evaluation of
this literature reveals an association of some karyotypes involving marker chromosomes with either a low or a high risk of
phenotypic abnormalities. Small marker chromosomes with a
low proportion of euchromatin, e.g., those derived from metaor submetacentric chromosomes 14 or 15, belong to the lowrisk group, whereas markers identified as isochromosomes for
18p, ring chromosomes derived from various autosomes, and
satellited markers from chromosome 22 harbor a high risk of
phenotypic abnormalities (Condron et al., 1974; Buckton et al.,
1985; Wisniewski and Doherty, 1985; McDermid et al., 1986;
Callen et al., 1992; Webb, 1994).
The occurrence of more than one marker chromosome further complicates the karyotype-phenotype correlation. Only
nine cases of two or more marker chromosomes have been
reported in the literature (Van Dyke et al., 1977; Callen et al.,
1991; Plattner et al., 1993; Daniel et al., 1994; Aalfs et al.,
1996) (see Table 2). In this case report we describe a girl presenting craniofacial dysmorphic features and developmental
delay associated with a karyotype involving two marker chromosomes derived from chromosomes 6 and 11 (see Table 2,
last row).
The involvement of a marker chromosome originating from
chromosome 6 has been found in two patients with more than
one additional chromosome fragment before. Callen et al.
(1991) described a patient with two marker chromosomes
derived from chromosome 6 (ring formation) and the X chromosome afflicted with dysmorphic features, microcephaly, delayed development, and seizures. The other proband, identified
by Aalfs et al. (1996), had a mosaic karyotype with two marker
chromosomes derived from chromosomes 6 and 9 and ring formation. The clinical findings of mild developmental delay and
mild dysmorphic features are inconsistent with the observation
of Plattner et al. (1993), that patients with multiple markers are
likely to present rather severe and multiple physical abnormalities. Possibly, when two marker chromosomes are associated
with only a mild phenotype in a patient, one of the marker
chromosomes might predominantly contain phenotypically silent heterochromatic domains. Also, in our patient the second
marker chromosome originating from chromosome 11 might
have only a minor phenotypic influence, as the additional chromosome material seems to consist predominantly of constitutive heterochromatin. The phenotype in our patient might
therefore mainly result from the additional chromosome 6
material. In several metaphase spreads the marker chromosome 6 shows a ring formation and dicentric structure which
originated from a sister chromatid exchange during DNA replication of the monocentric ring chromosome. Compared to other patients with only one additional chromosome 6 fragment,
there is little phenotypic similarity (James et al., 1995; Crolla et
al., 1998).
Interestingly, in one case reported in the literature two similar familial marker chromosomes lead to very different clinical
abnormalities (see Table 2, case 6). These phenotypic differences in a mother and child can only be explained by the higher
frequency of one mosaic cell line with an additional chromosome 12 fragment and/or the low percentage of cell lines containing both marker chromosomes in the child. This example
illustrates the difficulty of assessing phenotypic risk even in the
presence of morphologically similar additional chromosomal
material. At the same time, it also hints at a possible dosage
effect of markers found in mosaic karyotypes. The incidence of
marker chromosome mosaicisms involving only one additional
chromosome fragment is about 20 % to 30 % (Buckton et al.,
1985). The occurrence of two or more marker chromosomes,
however, always seems to be associated with mosaic cell lines.
The presence of mosaicism of cell lines involving marker chromosomes could be explained by two hypotheses: the postzygotic formation of these chromosome fragments in a certain proportion of cells or by a postzygotic instability resulting in a loss
of primarily non-mosaic marker chromosomes in only some
cells, with a subsequently arising mosaicism (Callen et al.,
1991). It could be argued that a postzygotic instability, as suggested by the second hypothesis, is more frequently found in
cells with more than one marker chromosome and therefore
leads to a higher percentage of mosaic cell lines in these cases.
Marker chromosomes in individuals with several additional
chromosome fragments most probably originate independently.
Moreover, several cases of uniparental disomy (UPD) in
association with marker chromosomes have been reported (Robinson et al., 1993a; Cheng et al., 1994; James et al., 1995). The
Cytogenet Cell Genet 93:182–187 (2001)
185
Table 2. Summary of cytogenetic results and clinical data on probands with two or more marker chromosomes
presence of marker chromosomes is thought to interfere with
normal chromosome disjunction during meiosis, resulting in an
increased risk of aneuploidy. The increased incidence of aneuploidy has been shown to be associated with an increased risk of
UPD as a means of aneuploidy correction (Engel, 1980, 1993;
Robinson et al., 1993b). This presence of concomitant UPD of
the normal chromosome homologs of probands with marker
chromosomes would further complicate the karyotype-phenotype correlation. So far, the parental origin of the normal homologs of the chromosome from which the marker chromosomes
are derived has only been determined in very few patients,
excluding our own patient. The identification of additional probands with supernumerary marker chromosomes will hopefully
make both the clarification of the association of UPD and marker chromosomes and the assessment of the phenotypic risks in
probands with one or more marker chromosomes possible.
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Cytogenet Cell Genet 93:182–187 (2001)
Acknowledgements
We would like to thank Prof. Dr. U. Töllner and Dr. N. Bier for providing us with clinical information about the girl described in this publication.
We are grateful to A. Nieschlag for language editing.
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