Article Text

Cytogenetic and molecular study of a jumping translocation in a baby with Dandy-Walker malformation
  1. GENEVIÈVE LEFORT*,
  2. PATRICIA BLANCHET,
  3. ANNE MARIE CHAZE*,
  4. ANNE GIRARDET,
  5. PIERRE SARDA,
  6. JACQUES DEMAILLE*,,
  7. FRANCK PELLESTOR
  1. *Cytogenetics Laboratory, CHU Montpellier, France
  2. Clinical Genetics Department, CHU Montpellier, France
  3. CNRS-UPR 1142, IGH, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5, France
  1. Dr Pellestor, Franck.Pellestor{at}igh.cnrs.fr

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Editor—Jumping translocations are rare chromosomal events in which a donor chromosome segment is translocated to various recipient chromosome sites. Jumping translocations were initially described in constitutional chromosome syndromes,1 ,2 but the majority of published cases have been observed in haematological malignancies, where their presence has been related to poor prognosis.3 ,4

Most jumping translocations involve acrocentric chromosomes and a characteristic feature is that breakpoints usually concern areas of repetitive DNA (telomeric, centromeric, or heterochromatic regions). Recipient chromosome involvement seems to be randomly distributed but with a preferential involvement of telomeric segments. This has led to the hypothesis that repetitive telomeric sequences could be implicated in the occurrence of jumping translocations, as suggested by FISH studies which have reported the presence of interstitial telomeric sequences at the junction sites of jumping translocations.5 ,6 However, the molecular basis underlying these complex chromosomal rearrangements is not well understood.

Constitutional jumping translocations are extremely rare and are usually associated with various phenotypic abnormalities. We report the finding of a jumping translocation in a baby with Dandy-Walker malformation. Dandy-Walker malformation consists of the triad (1) hypoplasia or absence of the vermis, (2) upward displacement of the falx, lateral sinuses, and torcular, and (3) a large, thin walled retrocerebellar cyst formed by the roof of the fourth ventricle. Most Dandy-Walker malformations are sporadic. The Possum database reports six autosomal dominant syndromes and 37 autosomal recessive syndromes which can be associated with Dandy-Walker malformation. However, in these disorders, Dandy-Walker malformation is always one feature of a larger spectrum of anomalies. Various cytogenetic abnormalities have been reported, but to date no consistent chromosomal aberration has been recognised.7 ,8 The present study combines molecular and cytogenetic investigations, including FISH and PRINS labelling procedures, to characterise a constitutional jumping translocation.

The patient is a baby boy, the second child of healthy, consanguineous parents (the parents are double third cousins). The family history is otherwise unremarkable. Consent was obtained from the parents for genetic studies. At the time of birth, the mother and father were 26 and 34 years old respectively. There was no history of maternal exposure to environmental hazards during pregnancy. The pregnancy was uneventful until sonographic examination at 39 weeks' gestation suggested partial agenesis of the vermis. Fetal sampling for chromosome analysis was not undertaken. The baby was delivered at 40.5 weeks' gestation; birth weight, length, and head circumference were 3260 g, 50 cm, and 34 cm, respectively. Apgar scores were 8 at one minute and 9 at five minutes. The baby was referred to our genetics service at the age of 1 month. At this time, weight, height, and head circumference were 5250 g (+3 SD), 57 cm (+2 SD), and 40.5 cm (+2 to +3 SD), respectively. He showed no facial dysmorphism; physical examination showed a small median mass of the occipital soft tissues and bilateral deep plantar furrows, but was otherwise normal. Skull xray showed an occipital bone defect. MRI showed (1) hypoplasia of the cerebellar vermis (only a part of the posterior and anterior segments are visible), (2) upward displacement of the cerebellar falx, and (3) a retrocerebellar cyst communicating with the fourth ventricle. These features led to a diagnosis of Dandy-Walker malformation. Neurological developmental milestones were normal at the age of 1 month. At the age of 5 years, neurological examination and psychomotor development were normal. The patient walked at 16 months, language skills were acquired normally, and school performance is normal for age.

Initial cytogenetic investigations in the patient were performed on peripheral blood lymphocytes and then subsequently repeated on a second blood sample and skin biopsy. Chromosomes were studied according to standard procedures for RHG banding.9

Dual colour FISH labelling was performed with commercially available whole chromosome paints for chromosomes 1, 2, 5, 6, and 12, according to the suppliers' instructions (Oncor, Gaithersburg, MD). Specific painting probes were combined as follows: biotinylated chromosome 2 specific paint with chromosome 1, 5, 6, or 12 specific paint labelled with digoxigenin. Biotinylated painting was visualised using fluorescein-avidin whereas detection of digoxigenin was done with antidigoxigenin-rhodamine.

Specific labelling of subtelomeric regions 1p, 2q, 5q, 6q, and 12q was performed using subtelomeric specific probes. Probes for 2p, 5q, 6q, and 12q were direct fluorophore labelled probes provided by Vysis (Vysis Inc, Downers Grove, IL). The YAC probe 762B5, specific for the subtelomeric region 1p, was a gift from Dr Rocci (Barri, Italy). This probe was labelled by nick translation with digoxigenin-11-dUTP (Boehringer Mannheim, Meylan, France) and used under the same conditions as the purchased subtelomeric probes. The subtelomeric probes correspond to loci estimated to be within 300 kb of the end of the chromosomes.

Telomere repetitive sequences were labelled by PRINS using the telomeric consensus primer (CCTAA)7. PRINS experiments were performed as previously described.10 Fifty μl of a reaction mix composed of the telomeric consensus primer, a nucleotide mixture including fluorescein-dUTP, Taq DNA polymerase buffer, and 2 units of Taq DNA polymerase (Boehringer Mannheim) was used. PRINS reactions were performed on a programmable thermal cycler equipped with a flat plate block. The denatured slides were put on the plate block and the reaction mixes placed on the slides and overlaid with coverslips. The reaction cycle consisted of two programmed steps: 10 minutes at the specific annealing temperature of the primer (60°C) and 20 minutes at 72°C in order to allow the in situ nucleotide chain elongation. At the end of the reaction, slides were washed in 2 × SSC-0.5% Tween 20 and then directly counterstained with propidium iodide in Vectashield antifade solution (Vector Laboratories, Burlingame, CA).

Combined FISH and PRINS experiments were also performed according to the previously reported protocol.11 We used a centromeric repeat probe specific for chromosome 2 (Oncor) and the consensus telomeric primer (CCTAA)7.

Fluorescent signals generated by FISH or PRINS were visualised by using a Leitz DMRD microscope equipped with appropriate filters. Combined FISH and PRINS labelling experiments were analysed by confocal microscopy. Optical sections were sampled with a confocal Nikon microscope. Images were transferred to a Power Macintosh computer and standard software tools (Adobe Photoshop 5.0) were used for image analysis.

The parental origin of chromosome 2 was investigated by PCR analysis of two microsatellite markers, D2S337 and D2S2232, located on both sides of the breakpoint 2p12 (respectively in 2p14 and 2p11). DNA was extracted from peripheral blood of the proband and his parents. Oligonucleotide primer sets were obtained from the Genethon. PCRs were performed in a total volume of 50 μl containing 200 ng genomic DNA, 5 μl of 1 × PCR buffer (10 mmol/l Tris-HCl, pH 8.3, 50 mmol/l KCl, 1.5 mmol/l MgCl2, 0.01% (w/v) gelatin), 200 μmol/l each of dNTPs, 0.15 μl dUTP-R110, 0.2 μmol/l primers, and 1 U ofTaq DNA polymerase). After an initial denaturation (96°C for 2½ minutes), the PCR was carried out for 30 cycles consisting of 20 seconds at 94°C, 20 seconds at 55°C, 40 seconds at 72°C, followed by a final extension step of 72°C for 10 minutes. One μl of each reaction product was mixed with 2 μl of formamide, 0.5 μl of molecular weight marker, and 0.5 μl of loading buffer. The mix was denatured for three minutes at 90°C and 1 μl was loaded onto a denaturing 4% polyacrylamide gel (36 cm × 20 cm × 0.2 mm thick). The electrophoresis conditions were one hour 40 seconds at 3000 V and 51°C. The analysis was carried out with the Genescan 2.1 software (Applied Biosystems) on an ABI 377.

Chromosome studies on the initial blood sample and control blood and fibroblast cultures showed four different cell lines: 46,XY,t(1;2)(p36;p12)/46,XY,t(2;5)(p12;q35)/46,XY,t(2;6)(p12;q27)/46,XY,t(2;12)(p12;q24). A summary of the cytogenetic data is given in table 1 and partial ideograms and RHG banded karyotypes are presented in fig 1. This constitutional mosaicism was consistent with a jumping translocation involving almost the entire short arm of chromosome 2 (donor chromosome) alternately transferred to the telomeric regions of four recipient chromosomes (chromosomes 1, 5, 6, and 12). The predominant cell line in both initial and control samples was t(1;2)(p36;p12) (table 1). The parents' chromosomes were normal with RHG banding. Derivative chromosomes resulting from the jumping translocation were confirmed in metaphase spreads by the use of whole chromosome painting probes (fig 2). No normal or unbalanced chromosome complements were found. However, dual colour whole chromosome painting did not show evidence of translocation of chromosomal material from recipient chromosomes 1, 5, 6, and 12 onto another chromosome. Specific subtelomeric FISH labelling (fig 3) showed that these sequences were not translocated between chromosome 2 and the four recipient chromosomes, whereas consensus PRINS labelling of telomeric repetitive sequences clearly indicated the presence of interstitial telomeric sequences on the recipient chromosomes (at the junction sites of the translocation) (fig 4), and at both ends of the derived chromosome 2 (fig 5).

Table 1

Distribution of different cell lines in patient tissues

Figure 1

Partial idiograms and R banded karyotypes of the chromosome pairs involved in the jumping translocation.

Figure 2

Examples of dual colour painting of donor chromosome 2 (in red) and the four recipient chromosomes (in green): (A) translocation t(1;2)(p36;p12), (B) translocation t(2;5)(p12;q35), (C) translocation t(2;6)(p12;q27), (D) translocation t(2;12)(p12;q24).

Figure 3

Results of combined in situ hybridisation of the subtelomeric specific probes for region 2p, labelled in green, and the four regions 1p (A), 5q (B), 6q (C), and 12q (D), labelled in red. In each case, lack of labelling of the der(2) can be seen.  

Figure 4

Detection of telomeric sequences by the PRINS technique showing the presence of interstitial telomeric sequences (arrow) at the junction of chromosome arm 2p12-pter and the recipient chromosomes 1 (A) and 6 (B).

Figure 5

Confocal analysis of combined FISH and PRINS labelling on the derivative chromosome 2. (A) The centromere of the derivative chromosome 2 was identified with a rhodamine labelled centromeric specific probe. (B) Telomeric repetitive sequences were detected on both ends of the broken chromosome 2 (arrows) by PRINS with a telomeric consensus primer and fluorescein-dUTP. (C) The combined picture shows the overlapping of the two signals close to the 2p12 breakpoint.  

The results of microsatellite marker analysis of chromosome 2 are given in fig 6. Both paternal and maternal alleles were present in the proband for each locus. This heterozygosity was not in favour of uniparental disomy for chromosome 2.

Figure 6

Results of PCR analysis of chromosome 2 microsatellite markers D2S337 and D2S2232 on DNA extracted from peripheral blood of the proband and his parents. For each locus, the two parental alleles were found in the proband's DNA (A and B).  

The proband is the product of a consanguineous union. He shows signs of Dandy-Walker malformation and normal neurological evaluation at the age of 1 month. Repeat chromosome studies in blood and in fibroblasts, as well as chromosome painting experiments, show de novo mosaicism for a jumping translocation in which chromosome 2p is alternately transferred to various recipient chromosomes (1, 5, 6, and 12). There is no evident relationship between the phenotype and the chromosome abnormality in our patient. Subtelomeric rearrangement does not seem to contribute to the phenotype; however, mosaicism cannot be totally excluded and it is also possible that the translocation breakpoint on chromosome 2 has disrupted the sequences of one (or more) genes implicated in Dandy-Walker syndrome. As shown in a recent systematic study, cryptic deletions may be an important cause of disease in patients with apparently balanced chromosome rearrangements.12 The possibility of uniparental disomy of chromosome 2, which might be related to the malformation of the proband, has been minimised by the microsatellite analysis.

At present, little is known about the origin of jumping translocations. In three other reports of constitutional jumping translocations, interstitial telomeric sequences have been observed.5 ,13 ,14 Interstitial telomeric sequences thus seem to be a common feature of constitutional jumping translocations occurring at telomeric recipient sites. The presence of interstitial telomeric sequences was not systematically observed in cases of jumping translocations associated with haematological disorders.15Nevertheless, failure to observe interstitial telomeric sequences may be because of an inherent limitation of in situ labelling procedures for the detection of submicroscopic telomeric sequences.

The results of our classical cytogenetic investigations and whole chromosome painting suggested a simple transposition with fusion between the 2p12 segment and the telomeres of recipient chromosomes rather than a reciprocal exchange, since recipient chromosomes appeared cytogenetically intact. The observed subtelomeric labelling was in accord with this interpretation since no translocation of subtelomeric sequences was observed between chromosome 2 and the four recipient chromosomes. Nevertheless, this does not rule out the possiblity of translocations occcurring distal to subtelomeric sequences since the subtelomeric probes are usually located between 40 and 300 kb from the telomeres.

PRINS labelling showed interstitial telomeric sequences at the telomeric breakpoint of the recipient chromosomes and at the broken end of chromosome 2. These data may suggest that the telomeric repeat sequences of the recipient chromosomes were partially deleted and translocated onto the proximal end of the broken chromosome 2, which would be in favour of a translocation event. However, for the jumping translocation we report, this event would have to occur four times which seems unlikely.

An alternative explanation for the presence of telomeric sequences at the proximal end of der(2) is the de novo formation of telomeric sequence at the end of the der(2). This has been shown to occur through healing of broken chromosomal ends by seeding of telomeric sequences and requires the expression of telomerase in the cell.16The stabilisation of broken chromosomes could also result from the recognition by telomerase of internal telomere-like sequences proximal to the breakpoint.17 In many eukaryotes, the ability to heal broken chromosome ends by telomeric addition is a well documented phenomenon and appears to be controlled by developmental factors.18 This phenomenon occurs early in embryonic development, shortly after zygote formation when telomerase activity is still efficient.19

This information contributes to an understanding of when our proband's rearrangement occurred. Mosaicism was found in blood and skin cultures of the patient. For both tissues, there is one largely predominant cell line, involving chromosomes 1 and 2. No chromosomally normal cells were found. It seems likely that chromosome 2 is prone to breakage. Breakage could have occurred in four (or more) different cell lineage precursors and the broken chromosome 2 fragment translocated to the end of different chromosomes.

The presence of interstitial telomeric sequences is considered to represent a form of chromosome instability and, consequently, these sequences could play a role in generating the observed mosaicism.20 Although the telomeric repeat nucleotide sequences are similar in telomeres and intrachromosomal areas, their different locations (terminal or interstitial) could contribute to differential organisation of the chromatin in the two domains, as shown by the variable effect of exonuclease on terminal and interstitial telomeric sequences.21 In jumping translocations, the presence of interstitial telomeric sequences on a receptor chromosome makes this chromosome unstable and makes the transferred fragment susceptible to “jumping” from one telomere to another. An alternative suggestion is that preferential involvement of telomeric regions in jumping translocations could result from the stabilisation of chromosome breakage by a telomeric capture process. Our report provides further evidence that the jumping process and the presence of interstitial telomeric sequences could be related. The preferential involvement of interstitial telomere-like sequences in chromosome breakage, rearrangement, and instability could be the result of quantitative or qualitative differences in the constitution of proteins associated with telomeric repeat sequences according to their location. It would be interesting to determine whether specific telomeric proteins such as TRF1 and TRF2 are present in interstitial telomere sequence regions.22

The elucidation of the underlying mechanism and the role of telomeres in the jumping translocation process will require studies combining cytogenetic and molecular technologies. In the future, the understanding of jumping translocation processes will greatly benefit by the molecular cloning of breakpoints as well as the search for specific genes or sequences involved in the occurrence of these rare chromosomal events.

Acknowledgments

We wish to thank Dr Mariano Rocci for providing the YAC probe 762B5. This work was supported by the Association Française Contre les Myopathies (AFM).

References

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