Pseudo dicentric chromosome (5;21): a rare example of maternal germline mosaicism: Case Report

U. Engel1,3, S.K. Bohlander1, K. Bink1, B. Hinney2, F. Laccone1 and I. Bartels1

1 Institute for Human Genetics, University of Göttingen and 2 Department of Obstetrics and Gynaecology, University of Göttingen, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Karyotyping of a malformed male newborn revealed the unbalanced karyotype of 46,XY, psudic(5;21)(q12;p13), +5 resulting in trisomy for the short arm of chromosome 5 and partial trisomy for 5q. Both parents had normal karyotypes in their peripheral blood lymphocytes. A second pregnancy ended in a miscarriage at 16 weeks gestation, sonographically 12 weeks. Karyotyping of chorionic villi from the abortus revealed the same unbalanced karyotype that had been identified in the first child. Fluorescence in-situ hybridization analysis confirmed a trisomy 5p. Microsatellite marker analysis ruled out illegitimacy and proved the maternal origin of the trisomic section of chromosome 5. Extended chromosome analysis of 60 metaphase cells from maternal skin fibroblasts and 40 metaphase cells from lymphocytes did not reveal mosaicism for psudic(5;21). These findings suggest the presence of a maternal germline mosaicism.

Key words: genetic counselling/germ line mosaicism/gonadal mosaicism/parental origin/translocation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The possibility of germ line or gonadal mosaicism is of particular concern when counselling the parents of a child with a de-novo structural chromosomal abnormality. De-novo structural chromosomal abnormalities are usually interpreted as the result of a random event during parental gametogenesis. The recurrence risk of a de-novo structural chromosomal aberration after one affected offspring is therefore estimated to be very low. Today, investigation of the presence of gonadal mosaicism, and determination of the parental origin of structural and numerical chromosome abnormalities are possible by molecular analyses.

A case is reported in which a seemingly de-novo translocation pseudodicentric chromosome recurred in a second pregnancy. The results of molecular and cytogenetic studies strongly suggest that maternal gonadal mosaicism was the cause for the increased risk of recurrence.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical report
A male infant with anal atresia, clubfeet, low set ears, mongoloid eye slant, microphthalmus, and heart disease (ventrical and atrial septum defect) was born at 35 weeks gestation to young non-consanguineous healthy parents. The baby had an unbalanced karyotype (see below) and died at age 3 weeks. The parents were chromosomally normal. Thus the cytogenetic abnormality was deemed to be de novo. One year later the woman had a spontaneous abortion at 16 gestational weeks. Karyotyping revealed the same structural abnormality as in her first child (Figure 1Go).



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Figure 1. Partial G-banded karyotype (a) and C-banded derivative chromosome (b) in the newborn.

 
Cytogenetics
Chromosome studies were performed on peripheral blood cultures (newborn, parents), skin fibroblast culture (mother) and both tissue culture and semidirect preparation of chorionic villous tissue (abortus) using standard techniques. In general G-banded chromosomes were analysed. C-banding and chromosome painting using WCP5 (Vysis Inc., Downers Grove, IL, USA) was applied in order to further characterize the derivative chromosome.

Molecular investigations
DNA was extracted from peripheral blood of both parents and from a long-term culture of chorionic villi (abortus) according to standard procedures. DNA from the first child was not available.

Eleven microsatellites from chromosome 5, 7, 9 and 21 (D5S455, D5S418, D5S260, D5S253, D5S208, D7S1870, D9S886, D9S887, D9S888, D9S889, D21S11) have been investigated. The primer sequences were deposited under the following accession identification numbers of the GDB database (www.gdb.org): 189314, 188337, 181109, 196355, 180557, 377150, 304578, 304744, 304753, 304735, 188664). The polymerase chain reaction (PCR) was carried out in a volume of 25 µl and 100 ng DNA with the HSTaq MasterMix (Qiagen, Hilden, Germany) according to the manufacturers instructions under the following conditions: denaturation/polymerase activation at 97°C for 15 min followed by 30 cycles with a denaturation at 96°C for 20 s, annealing at 55°C for 30 s and extension at 72°C for 30 s in a Primus cycler (MWG, Ebersberg, Germany). The polymorphic CAG repeats of the SCA1 gene on chromosome 6 and the CAG repeat of the MJD gene on chromosome 14 were analysed as described (Orr et al., 1993Go; Kawaguchi et al., 1994Go). All PCR was carried out with fluorescence-labelled primers. The size of the product was determined by gel electrophoresis on a ABI 373 automated sequencer by the Genscan computer programm (ABI, Weiterstadt, Germany).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytogenetic analysis
Cytogenetic analysis of the newborn and the abortus revealed an unbalanced karyotype with identical banding pattern on the derivative chromosome 21 in both samples: 46,XY,psudic (5;21)(q12;p13),+5 and 46,XX,psudic(5;21) (q12;p13),+5 respectively. One single constriction was observed at the site of the chromosome 21 centromere. The second centromere was demonstrated by C-banding (staining weak but definite) (Figure 1a,bGo). Fluorescence in-situ hybridization (FISH) with a whole chromosome 5 painting probe (WCP 5; Vysis) confirmed the origin of the chromosome 5 portion (Figure 2Go). Thus, a pseudodicentric chromosome resulting in partial trisomy for 5pter to 5q11 or 5q12 was present in the malformed child and in the aborted fetus. Parental karyotyping was performed twice on lymphocytes and revealed a normal karyotype in 40 metaphases of each parent. Additional analysis of 60 metaphases from two skin fibroblasts cultures of the mother gave a normal result without evidence of mosaicism.



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Figure 2. Fluorescence in-situ hybridization (FISH) with a whole chromosome 5 painting probe (WCP5) confirmed the origin of the chromosome 5 portion.

 
Molecular investigations
Microsatellite analysis confirmed the presumed paternity with a probalitity >99.9%. Three microsatellites (D5S455, D5S418, D5S208) identified a trisomy of the fetus. The polymorphic marker D5S455 showed two different maternal alleles and one paternal allele, which is consistent with a maternal origin of the derivative chromosome 5 (Figure 3Go).



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Figure 3. Three microsatellites of chromosome 5 (D5S455, D5S418, D5S208) identified a trisomy of the fetus. The polymorphic marker D5S455 showed two different maternal alleles and one paternal allele which is consistent with a maternal origin of the derivative chromosome 5. Red line = maternal genotype, blue line = fetal genotype, green line = paternal genotype. The arrows indicate the maternal alleles and the arrowheads the paternal ones.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cases of male or female germline mosaicism were first identified in several X-linked diseases, including Duchenne muscular dystrophy (Bakker et al., 1987Go, 1989Go; Passos-Bueno et al., 1992Go; Melis et al., 1993Go; Bunyan et al., 1994Go), fragile X-syndrome (Prior et al., 1995Go), ornithine transcarbamylase deficiency (Maddalena et al., 1988Go), Wiskott-Aldrich syndrome (Arveiler et al., 1990Go), severe combined immunodeficiency (SCID) (Puck et al., 1995Go), X-linked hydrocephalus (Jouet et al., 1996Go), myotubular myopathy (Vincent et al., 1998Go), Coffin-Lowry syndrome (Jacquot et al., 1998Go) and others. It is becoming increasingly clear that germline mosaicism for new mutations is not limited to X-linked recessive disorders.

Byers et al. reported data on recurrence risk of autosomal dominant osteogenesis imperfecta (Byers et al., 1988Go). The authors postulated that the 6% empirical recurrence risk of the disorder is best explained by gonadal mosaicism in one of the parents. In Duchenne muscular dystrophy the proportion of germline mosaicism was estimated at 15% (Passos-Bueno et al., 1992Go). Germline mosaicism has been demonstrated also for interstitial deletions in chromosome 22q11 (Hatchwell et al. 1998Go), Smith-Magenis syndrome 17p11.2 (Zori et al. 1993Go), Ehlers-Danlos syndrome type IV (Milewicz et al., 1993Go), and Williams-Beuren-syndrome (Kara-Mostefa et al. 1999Go).

Only a few reports exist in the literature about gonadal mosaicism for chromosome rearrangements. In 1990 Sachs et al. detected mosaicism in two couples with three and four pregnancies in which trisomy 21 was found (Sachs et al., 1990Go). In one couple ovarian biopsy showed 44 and 47% trisomic cells in the right and left ovary respectively. Gross et al. reported on a female patient with mosaicism for two different Robertsonian translocations, both involving chromosome 21 (Gross et al., 1996Go). The patient had cells with an unbalanced karyotype with an i(21q) and balanced cell with der(21;22)(q11;q11). In skin fibroblasts only balanced karyotypes were observed. In lymphocytes and in one-third of the cells in the ovarian biopsy the unbalanced karyotype with the i(21q) was observed. The patient was phenotypically normal but two of her children inherited the i(21q) and had Down's syndrome. Yu et al. reported on maternal gonadal mosaicism for a Robertsonian translocation (21;21) (Yu et al., 1998Go). Molecular cytogenetic detection of confined gonadal mosaicism in a conceptus with trisomy 16 placental mosaicism has also been reported (Stavropoulos et al., 1998Go).

One problem in genetic counselling of couples with suspected gonadal mosaicism for chromosomal aberration is the question of recurrence risk. While analysis of spermatozoa by means of multicolour FISH allows the assessment of risk for the offspring in cases with paternal gonadal mosaicism (Mercier and Bresson, 1997Go), maternal gonadal mosaicism is not readily amenable to diagnostic procedures.

Due to the high risk of Down's syndrome pregnancies in the couples reported by Yu et al. and Conn et al., the couples elected to pursue preimplantation genetic diagnosis (PGD) (Yu et al., 1998Go; Conn et al., 1999Go). Because of the elevated recurrence risk in further pregnancies for the same chromosomal aberration, our patients might well ask for new diagnostic techniques such as PGD or polar body analysis.

Due to the present legal situation, PGD in Germany is not possible. However, we could offer polar body analysis, chorionic villous sampling or amniocentesis for prenatal diagnosis in further pregnancies. Ovarian biopsy and subsequent chromosomal analysis does not provide the necessary information for risk estimation. When doing a chromosomal analysis on ovarian tissue only the cells from the stromal tissue are analysed. These cells are not more closely related to the germ cells than, for example, lymphocytes. The usefulness of ovarian biopsy in estimating the recurrence risk for such abnormalities is very questionable.

Because germline mosaicism can never be effectively excluded, the possibility of germ line mosaicism should always be borne in mind.


    Acknowledgments
 
We thank Mrs A.Winkler for preparing the manuscript and Professor W.Engel for discussion and advice.


    Notes
 
3 To whom correspondence should be addressed at: Institut für Humangenetik, Universität Göttingen, Heinrich-Düker-Weg 12, D-37073 Göttingen, Germany. E-mail: wengel{at}gwdg.de Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 28, 2000; accepted on October 5, 2000.





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