Prenatal Diagnosis and Molecular Cytogenetic Characterization of an Unusual Complex Structural Rearrangement in a Pregnancy Following Intracytoplasmic Sperm Injection (ICSI)

Complex chromosomal rearrangements (CCRs) are constitutional structural rearrangements having three or more breakpoints (Gardner and Sutherland 2004). The majority of cases reported in the literature show three-way exchanges, in which three segments from three chromosomes break off, translocate, and unite. There are very few reports on exceptional CCRs involving more than three breakpoints and/or different structural rearrangements than translocations like insertions or inversions (for review see Houge et al. 2003 and Kuechler and Claussen in this issue). A de novo, apparently balanced CCR detected at prenatal diagnosis requires exact molecular cytogenetic analysis and potentially additional molecular genetic analysis to enable proper genetic counseling. We report on the prenatal diagnosis and molecular cytogenetic characterization of an exceptional CCR in a pregnancy following intracytoplasmic sperm injection (ICSI).

A 40-year-old woman was referred for prenatal diagnosis and subsequent karyotyping of amniocytes because of assisted fertilization, maternal age, and a 4-mm choroid plexus cyst. The parents had opted for ICSI after a spermiogram revealed asthenoteratozoospermia with slightly decreased sperm number and explicitly reduced sperm motility, as well as conspicuous sperm morphology. The cause of the abnormal sperm morphology and motility could not be identified, and the karyotypes of both partners were normal at a resolution level of 400 bands per haploid chromosome set in lymphocytes.

Karyotyping of GTG-banded chromosomes from cultured amnion cells at the 18th week of pregnancy revealed a structurally rearranged karyotype with a derivative chromosome 5 consisting mainly of chromosome 5p material and a derivative chromosome 15 apparently composed of nearly the whole chromosome 15 and a rearranged part of the long arm of chromosome 5, presumably an inversion (Figure 1A). This aberrant karyotype was found in all metaphases analyzed from two independent cultures, indicating that the constitutional aberrant karyotype was present in the fetus. There were no hints of mosaicism in 50 cells analyzed. Staining the nuclear organizer regions (NORs) indicated that the der(15) contained interstitial active NORs (Figure 1B). After GTG-banding the karyotype appeared to be balanced, on the basis of 400 bands per haploid genome, and hence has been described as 46,XX,der(5)t(5;15)(q11.2;p12),der(15) t(5;15)(q11.2;p12)inv(5)(q11.2q15).

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Figure 1

GTG-banding of the chromosomes involved in the rearrangement (A). Ag-NOR staining reveals an interstitial active nuclear organizer region (B). The derivative chromosome is indicated by the arrow.

Because conventional cytogenetics alone was not sufficient to elucidate the rearrangement with satisfactory accuracy, we performed additional molecular cytogenetic analyses to define the exact composition of the derivative chromosomes. An even more complex rearrangement was excluded by hybridizing chromosome-painting probes for chromosomes 5 and 15 (Qbiogene; Carlsbad, CA) following the manufacturer's instructions. No euchromatin of these two autosomes could be detected on any other chromosome, and no interruption of the hybridization signal was visible on the two derivative chromosomes (Figure 2A). Therefore, no other chromosome seemed to be involved in the rearrangement. This was confirmed by comparative genomic hybridization (CGH) performed as described elsewhere (Tönnies et al. 2001). CGH did not hint at duplications or deletions at the breakpoints or at any other chromosomal imbalances, which cannot be excluded by hybridizing whole-chromosome paints alone (Figure 2B). Hybridization and comparison of the relative positions of locus-specific yeast artificial chromosome clones on the der(15) and the normal chromosome 5 pointed toward the existence of a paracentric inversion of chromosome 5q material in the der(15) (Figure 2C).

This was confirmed by multicolor banding (MCB) (Figure 2D). Hybridization of probes specific for subcentromeric regions of chromosome 5 showed normal signals on the der(5) (Figure 2D). MCB and subcentromere-specific multicolor fluorescence in situ hybridization (subcenM-FISH) were done as described previously (Chudoba et al. 1999; Liehr et al. 2002; Starke et al. 2003). All applied molecular cytogenetic techniques supported the assumptions of the conventional cytogenetic analysis.

To exclude a uniparental disomy (UPD) due to trisomy rescue after a possible 3:1 segregation with interchange trisomy for chromosome 15, we extracted genomic DNA from the amniocytes by standard procedures and performed methylation testing on the imprinting center of the Prader-Willi syndrome (PWS)/Angelman syndrome (AS)–critical region, as described by Zeschnigk et al. (1997). Methylation-specific PCR revealed two bands for the fetal DNA and consequently excluded a UPD 15 for the PWS/AS critical region (Figure 2E).

A very rare complex chromosomal aberration was diagnosed after chromosome analysis of cultured amniocytes. Conventional and molecular cytogenetic analysis showed a balanced karyotype [46,XX,der(5)t(5;15)(q11.2;p12),der(15)t(5;15)(q11.2;p12)inv(5)(q11.2q15)]. The risk for phenotypic abnormalities associated with a de novo reciprocal two-breakpoint translocation is estimated to be 6%, 3% above the background risk of about 3% for malformations and/or defects in all pregnancies (Warburton 1991). The risk estimation in Warburton's study is slightly higher for balanced two-breakpoint inversions (9.4%) but probably merely due to a smaller case number. De novo, apparently balanced CCRs have presumably a high risk for mental retardation and malformations. The few postnatal studies suggest that the risk for abnormal phenotypes increases with a greater number of breakpoints (Madan et al. 1997). In our case, the exact molecular cytogenetic characterization of the rearrangement did not point toward any imbalances. The rearrangement involves only three breakpoints that were defined by cytogenetic analysis: the breakpoint at 5q11.2 is involved in two events, the inversion and the translocation. Furthermore, the breakpoint in chromosome 15 localizes in the short arm of the chromosome, a region containing no relevant single-copy genes, but genes coding for rRNA. Thus, the risk for fetal malformations and developmental or mental retardation should not exceed the 9.4% given for the usual two-breakpoints inversion, although it concerns a complex rearrangement. Non-acrocentric chromosomes with interstitial NORs derived by translocations or insertions from acrocentric chromosomes are rare forms of chromosomal rearrangements without phenotypic effect, if there is no loss of euchromatic material from the non-acrocentric chromosome (Willatt et al. 2001).

Accurate definition of the chromosomal breakpoints was essential to enable proper genetic counseling. After genetic counseling and additional inconspicuous ultrasound examination, the parents decided to continue with the pregnancy; meanwhile, a healthy girl was born, with a birth weight of 3.005 g and a birth length of 49 cm.

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Figure 2

Chromosome painting of chromosomes 5 (red) and 15 (green) did not point toward an involvement of additional chromosomes in the rearrangement (A). CGH analysis revealed no small deletions or duplications at the translocation and inversion breakpoints (B) or for any other chromosome (data not shown). The relative positions of locus-specific probes on the der(15) pointed toward an inversion including 5q12~13 (C): YAC 962f04, localizing to chromosomal bands 5q12~13 (green), and YAC 753f05, localizing on 5q34 (red). YACs were obtained through the Positional Cloning Centre at the Max-Planck-Institute of Molecular Genetics (Berlin, Germany). Multicolor banding confirmed that a paracentric inversion of 5q material had occurred in the derivative chromosome 15, and hybridization of probes specific for subcentromeric regions of chromosome 5 showed normal signals on the der(5) (D). Methylation-specific PCR for the SNRPN locus excluded an UPD 15. Samples 1–3, controls; sample 4, our patient; samples 5–6, AS; samples 7–8, PWS (E).

The chromosomal constitution will have considerable implications for the girl in the event of of a possible wish for having her own children in the future. If the rearrangement does not cause a general meiotic arrest, two segregant gametes may be produced by the translocation heterozygote that could lead to viable, severely affected offspring. Tertiary trisomy including the der(5) following 3:1 segregation results in trisomy for the whole short arm of chromosome 5, which is associated with severe malformations and mental retardation. Over 40 cases with partial trisomy of 5p have been reported to date (Grosso et al. 2002). The corresponding tertiary monosomy would result in a constellation with a cri-du-chat syndrome, a well-described partial monosomy resulting from deletion of the short arm of chromosome 5. A trisomy rescue after interchange trisomy for chromosome 15 would cause a PWS due to maternal UPD 15 in prospective offspring of the girl, while monosomy rescue of the paternal chromosome 15 following the corresponding interchange monosomy of chromosome 5 would result in an Angelman syndrome. Recombination products following crossing over in a meiotic inversion loop would lead to dicentric recombinant chromosomes or acentric fragments. These are lethal constellations in early embryonic development and therefore do not enhance the risk for abnormal offspring.

The findings could also have implications for further pregnancies of the couple itself, if one of the partners had a gonadal mosaicism. However, the specified unbalanced segregation products could be easily identified by karyotyping fetal cells in further pregnancies. Exclusion of a UPD 15 would require molecular genetic analysis of fetal DNA.

Although most familial complex rearrangements are transmitted through the mother, paternal transmission has been described in a few cases (Grasshoff et al. 2003; Gardner and Sutherland 2004). Infertility in male carriers of complex translocations is well known and the abnormal spermiogram could possibly be due to gonadal mosaicism for the rearrangement. A potential gonadal mosaicism—if existing in the father—could be verified by analyzing segregation patterns in sperm analysis using fluorescence in situ hybridization.