PRENATAL DIAGNOSIS, VOL. 15 843-848 (1995)

IiDENTIFICATION OF A CASE OF MATERNAL UNIPARENTAL DISOMY OF CHROMOSOME 10 ASSOCIATED WITH CONFINED PLACENTAL

MOSAICISM CARRIE JONES*, CAROL BOOTH*, DEBRA RITA*, LYDIA JAZMINES*, RHONDA SPIRO*, BRIAN M~CULLOCH*,

CHRISTOPHER McCASKILL AND LISA G. SHAFFERt

*Department of Genetics, Lutheran General Hospital, Park Ridge, Illinois, U.S.A.; ?Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, U. S. A.

Received I February 1995 Revised 15 May 1995 Accepted I June 1995

SUMMARY We report a case of maternal uniparental disomy of chromosome 10 discovered after chorionic villus sampling

(CVS). Direct preparations revealed mosaic msomy 10, while cultured CVS cells, as well as amniotic fluid cells, showed only a normal 46,XY complement. DNA analysis using microsatellite markers showed both chromosomes 10 to have been inherited from the mother. The pregnancy was complicated by polyhydramnios. A phenotypically normal male infant of appropriate size was delivered by Caesarean section at 41 weeks’ gestation. Since only the direct preparations showed trisomy 10, this case illustrates the importance of CVS direct preparations in the detection of pregnancies at risk of uniparental disomy (UPD). Although the increased frequency of confined placental mosaicism (CPM) diagnosed when direct preparations are performed has been viewed negatively, identification of both CPM and UPD may have biological and clinical sigdicance for a pregnancy. Even though only a single case of maternal disomy 10 is reported here, the apparently normal phenotype provides evidence that there are no major imprinted loci on chromosome 10 that affect in uzero growth and development. However, other potential effects such as mental retardation will require long-term follow-up of this as well as additional cases.

KEY WORDS: uniparental disomy; confined placental mosaicism; trisomy 10; direct CVS preparations

INTRODUCTION

Since the advent of chorionic villus sampling (CVS), approximately 2 per cent of pregnancies have been shown to have disparity between placen- tal and fetal chromosome constitutions (Hall, 1990; Kalousek et aL, 1991). In a subset of these cases, euploidaneuploid mosaicism is present in the placenta, but not in the fetus. This phenom- enon has been termed confined placental mo- saicism (CPM). Pregnancies characterized by CPM may result in phenotypically normal infants. How- ever, a number of problems have been reported to

Addressee for correspondence: Came Jones, Department of Genetics, Lutheran General Hospital, 1875 Dempster Suite 340, Park Ridge, IL, USA.

0 1995 by John Wiley & Sons, Ltd. CCC 0 197-385 1/95/090843-O5

occur in pregnancies with CPM, including intra- uterine growth retardation (IUGR) and fetal death (Johnson et al., 1990; Kennerknecht and Terinde, 1990). Most cases of CPM probably originate as trisomic conceptuses. It has been hypothesized that an aneuploid embryo may be ‘rescued’ from demise by the loss of the extra chromosome early post-conception. Because two chromosomes origi- nate from one parent and one from the other parent, there is a theoretical 1 in 3 chance that the lost chromosome was the contribution from the parent donating only one chromosome and the two remaining chromosomes came from the other parent, representing uniparental disomy (UPD). UPD may produce an abnormal phenotype if the chromosomes involved carry imprinted genes [e.g., chromosome 15 and Prader-Willi syndrome

844 C. 30NES ET’ AL.

(Nicholls et al. , 1991)] or if a mutant recessive gene is homozygous due to UPD [e.g., chromosome 7 and cystic fibrosis (Spence et aL, 1988; Voss et aL, 1989)]. We report a case investigated for UPD due to the finding of trisomy 10 mosaicism on the C V S direct preparation.

MATERIALS AND METHODS Clinical report

The mother was a 41-year-old gravida 3, para 0020 who had a CV S at 10 weeks’ gestation for advanced maternal age. Her first two pregnancies had been terminated electively in the &st trimes- ters. The pregnancy was complicated by polyhy- dramnios beginning at 26 weeks’ gestation with an amniotic fluid volume (AFV) index of 30-58 (nor- mal 8-24). At 33 weeks’ gestation, the AFV index was 33-9; however, by 39 weeks it had decreased to 25.74. The mother never required amniocentesis for removal of amniotic fluid. A detailed ultra- sound examination showed no evidence of fetal anomalies. The infant was delivered at 41 weeks’ gestation by Caesarean section because of an abnormal fetal heart rate tracing. The apparently normal infant had Apgar scores of 7 at 1 min and 9 at 5 min. His birth weight was 3.1 kg (50 per cent for 41 weeks). A complete physical examhation at birth revealed no anomalies or dyssnorphic fea- tures. He was re-evaluated at 8 months. At that time, his weight was 8-1 kg (25 per cent), height 6 9 3 m (25 per cent), and head circumference 46.5 cm (80 per cent). A Denever Developmental Assessment showed him to be age-appropriate for gross motor, h e motor, social, and language skills.

Cytogenetic analysis Metaphase cells were prepared from the cyto-

trophoblast and mesenchyme from the CVS to obtain a direct preparation and cultured prepar- ation, respectively, according to standard pro- cedures. Amniotic fluid cells, placental tissues, cord biopsy, and cord blood were cultured and metaphase chromosomes were obtained and analysed according to standard procedures.

DNA analysis

Genomic DNA was extracted from peripheral blood from the parents and cultured chononic villi

from the fetus by standard methods. Three di- nucleotide repeat polymorphic markers specific for chromosome 10 (DlOS245, D10S179, D10S169) were used to identify the chromosome 10 origins in CVS. Additionally, markers from chromosome 7 [intron 17 of elastin (Foster et aL, 1993) and D7S476] and chromosome 13 (D13S121 and D13S125) (Bowcock et aL, 1993) were used to confirm paternity. The primer sequence infor- mation was obtained through the Genome Data Base for markers D10S245, D10S179, DlOS169, and D7S476, and primers for markers D10S169, D7S476, intron 17 of elastin, D13S121, and D13S125 were synthesized by the sequencing core within the Department of Molecular and Human Genetics at Baylor College of Medicine. For markers D10S245 and DlOS179, primers were purchased through Research Genetics Inc. (Huntsville, AL). The precise location of the markers on chromosome 10 is not known. The molecular analyses were performed individually for each locus, using previously published methods (Shaffer et al., 1993). DNA was extracted from cultured amniotic fluid cells and analysed as described above for the CVS. At birth, DNA was extracted from cord blood, tissue obtained from the cord, fetal membranes, and placenta. DNA analysis was performed on these additional tissues as described above.

RESULTS

Direct analysis of the CVS sample showed mosaicism for trisomy 10, with three cells being normal (46,XY) and two Ceus showing 47,XY, +lo. In the analysis of 40 cells from the cultured villi, only normal chromosome complements were observed (46,XY). Since a complete dichotomy can theoretically occur in the chromosome consti- tution between the placenta and fetus, we were concerned that the placenta (CVS) could show uniparental disomy while the fetus was biparental. Therefore, amniotic fluid was drawn for chromo- some and DNA analyses at 17 weeks’ gestation. The cytogenetic analysis of the amniotic fluid showed a normal male karyotype in each of the 16 cells examined.

Mter delivery, the placenta was analysed cytoge- netically for the presence of trisomy 10. In cultured samples derived from villi obtained from multiple sites, 10 of 60 cells analysed (- 17 per cent) dem- onstrated trkorny 10. Eighty cultured cells derived

MATERNAL UNIPARENTAL DISOMY OF CHROMOSOME 10 845

Table I-Cytogenetic studies

Tissue 47,XY, + 10 (%) -

CVS (direct) CVS (cultured) Amniocytes Placenta villi Placental membrane Umbilical cord Cord blood

2 (40%) 0 0

0 0 0

10 (17%)

3 40 16 50 80 20 16

from amniotic membranes and 20 from the umbili- cal cord showed only 46,XY compliments with no trisomic cells seen (Table r).

The DNA analysis using the chromosome 10 markers revealed inheritance of both maternal alleles resulting in heterodisomy for all markers, and failure of inheritance of any paternal chromo- some 10 alleles for all tissues examined from the fetus and placenta (Fig. 1). No evidence for tri- somy 10 (inheritance of a paternal allele) was found in the vi l lus samples derived from the placenta. The failure to detect a paternally contributed allele is most likely due to selection for diploid cells during extensive culturing needed for the DNA extraction. The results using chromo- some 7 and 13 markers were consistent with cor- rect paternity for all loci tested. Therefore, the fetus had maternal disomy for chromosome 10. Since for at least these three markers heterodisomy was found, this case most likely occurred from a maternal meiosis I non-disjunction resulting in a trisomic 10 conceptus and subsequent early post-zygotic loss of the paternal chromosome 10 resulting in maternal WD.

DISCUSSION

The concept of UPD was first described in 1980 (Engel, 1980) and subsequently, several cases of UPD involving various chromosomes were reported (Engel, 1993). A number of mechanisms have been hypothesized to result in UPD (Engel, 1993). The current case appears to represent ‘rescue’ from a trisomy 10. Initially, the gamete of one parent had two copies of chromosome 10 and the gamete of the other parent had one copy of chromosome 10, resulting in trisomy 10 at fertiliz- ation. In the zygote, one of the three chromosomes

Fig. 1-Molecular results for a case of maternal disomy 10. For each marker, the DNA samples are shown in the lanes as indicated. For the chromosome 10 markers, all tissues derived from the fetus demonstrated inheritance of both maternal alleles and failure of inheritance of any paternal allele, consist- ent with maternal disomy 10. For the elastin polymorphism located on chromosome 7, all tissues derived from the fetus show normal inheritance of one matemal allele and one pater- nal allele, consistent with biparental inheritance and correct paternity

10 was lost. In two-thirds of such cases, normal biparental disomy would be predicted for cells developing into the embryo. In one-third of such cases, however, UPD is predicted to occur (Cassidy et d., 1992; Purvis-Smith et aL, 1992). This is believed to be the most common mech- anism by which UPD occurs and presumably occurred in this case resulting in maternal UPD 10. Furthermore, since the mother was heterozygous at all three markers tested for chromosome 10 and

846 C. JONES ET AL.

the infant inherited both of these maternal alleles for each locus, the infant has heterodisomy at these loci. Isodisomy, resulting from recombination, cannot be excluded at other loci on chromosome 10. Therefore, although the infant is apparently normal at age 8 months, homozygous recessive genes, inherited due to isodisomy, cannot be excluded.

A number of inherited diseases have been de- scribed to result from UPD, including cystic fibrosis (maternal WD7) (Spence et al., 1988; Voss et al., 1989), thalassaemia major (UPD16) (Beldjord et al., 1992), and rod monochromacy (maternal UPD14) (Pentao et al., 1992). In those cases, copies of mu- tant recessive genes were present on both chromo- somes inherited from the single parent. Genomic imprinting is revealed merely in the presence of UPD, but recessive diseases may occur only if the parent is a carrier. Examples of the effects of im- printing include hader-Willi syndrome resulting from maternal UPD for chromosome 15 and Angelman syndrome resulting from paternal UPD 15 (Nicholls et al , 1991; Malcolm et al., 1991). The present case has provided evidence that maternal UPD for chromosome 10 does not produce any major imprinting effect on in utero growth and de- velopment. However, long-term follow-up and ad- ditional cases will be required to assess any milder imprinting effects. It may be diEicult to sort out effects due to imprinting from those attributable to homozygous inheritance of a mutant recessive gene.

A number of conditions predispose to aneu- ploidy and therefore could predispose to UPD. These include both advancing maternal age and malsegregation of a balanced translocation. CPM combined with UPD for chromosome 16 has been shown to be associated with intra- uterine growth retardation (IUGR) (Kalousek et al., 1993). In contrast, UPD for chromosomes 15, 21, and 22 has not been associated with IUGR. While the evidence for no association of IUGR with UPDl5 is extensive, the number of UPD cases for chromosomes 21 and 22 is small (Schinzel et aL, 1994; Bloudin et aL, 1993). Poly- hydramnios, noted near the end of the preg- nancy, was the only complication in the current case. However, this had resolved by 39 weeks’ gestation. In addition, the fetus was of appropriate size at birth and therefore did not exhibit IUGR.

While ‘false-positive’ cases of autosomal triso- mies have been described from both direct and

cultured CVS, mosaicism involving rare aneu- ploidies is often not confirmed by amniocentesis or in the liveborn (Verp et al., 1989). In one study, 13 cases of mosaicism were found at CVS but not confirmed at amniocentesis. In all six placentae available for analysis after delivery, investigators confirmed the presence of CPM (Miny et al., 1989). While such results were once considered examples of tissue culture artifact or pseudo- mosaicism, many cases now appear to represent true mosaicism which may be confined to the placenta. The presence of aneuploidies in cultured CVS material is conlinned in the fetus or infant in 5-25 per cent of cases (Ledbetter et al., 1990). Awareness of the potential for CPM means that any aneuploidy discovered at CVS by either direct or culture methods warrants further evaluation.

A number of cytogenetic laboratories have stopped performing direct analysis on CVS because the duplication of procedures increases the cost and may produce results which are dficult to interpret. Relying on only direct analyses produces the potential of terminating a chromosomally nor- mal fetus. In the present case, the direct analysis of CVS indicated the CPM and the possibility of UPD being present in the fetus. While neither cultured CVS cells nor amniotic fluid cells appeared abnormal, the presence of CPM was later confirmed by cultures of several placenta biopsies after the birth of the infant. It has been suggested that CPM may be uncovered more often in CVS direct preparations than in the cultured cells (Ledbetter er aL, 1992). One study found mosaicism twice as frequently in the direct preparations as compared to the cultured cells (Vejerslev and Mekkelsen, 1989). Therefore, al- though long-term cultures are clinically more reliable as a true indication of the fetus, direct analysis of CVS material provides valuable infor- mation and could be performed in conjunction with cultured cells if material is available. This would help to identify those pregnancies which may be at risk for UPD. This case also raises an issue about appropriate management fe.g., molecular analysis) in cases of CPM for un- covering UPD prospectively. Clearly, the risks for CPM of chromosome 15 may be great for Prader- Willi syndrome (Cassidy et al., 1992; purvis-Smith et al., 1992). However, the effects of UPD for many other chromosomes remain unclear and the ‘appropriate’ case management is currently under debate.

MATERNAL. UNIPARENTAL. DISOMY OF CHROMOSOME 10 847

ACKNOWLEDGEMENTS

We gratefully acknowledge the critical evalu- ation and helpful discussions of this manuscript by Drs D. Ledbetter (NIH) and S. Elias (Baylor College of Medicine). This research was supported in part by a Basil O’Connor Starter Scholar Research grant (No. 5-FY93-0958) from the March of Dimes Birth Defects Foundation (L.G.S.).

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