Kałużewski Bogdan, Zając Ewa, Mastalerz-Eckersdorf Anna, Płowaś Iwona, Lach Joanna, Helszer Zofia, Constantinou Maria.
1 Department of Medical Genetics
Medical University of Łódź
No 3 Sterling Street,
91-425 Łódź, Poland e-mail: majcon@kardio-sterling.lodz.pl
INTRODUCTION.
Clinicians experienced in dysmorphologic conditions often correctly arrive at the cytogenetic basis underlying the disease process, having reviewed the characteristics of the patients’ phenotype, yet they are still faced with the choice of 20-30 diferent disorders. Comparative Genomic Hybrydization (CGH or microarray CGH) may turn out to be more effective for detection of gains and losses in a single study (1, 6, 7).
MATERIAL AND METHODS:
Patients: Clinical presentations of patients, initial and final karyotype anylyses, CGH, FISH and M-FISH are showed in table 1.
Classical cytogenetic analysis.
Cytogenetic analysis was performed by use standard protocols. The resolution of the GTGbanding was in the range of 400 - 550 bands corresponding to approximately 5-10 Mbp.
CGH technique. CGH was performed according to the protocol described previously (5). The acquired CGH profile was compared to a corresponding dynamic standard reference interval based on an average of 20 control cases with a normal karyotype (Fig. 1). The resolution of presented CGH technique was in the range of ¼ - ½ band corresponding to approximately 3-5 Mb.
FISH and M-FISH.
FISH was performed using twenty commercially available probes, i.e. centromere probes: CEPY-SG
(DYZ3)/CEPX-SO(DXZ1), CEP15-SG, CEP1-SG, CEP18-SG, (VYSIS), CEPY(DYZ1)-FITC, CEP 14/22-FITC, Human
Pancentromere Probe (Q-Biogene), painting probes WCPY-SG, WCPX-SO, WCP15-SG , WCP18-SG (VYSIS), specific probes LSI SNRPN-SO/PML-SO/CEP15-SG, LSI D15S10-SO/PML-SO/CEP15-SG, LSI PML-SO/RARA-SG, LSI
SRY/CEPX(DXZ1) (VYSIS), TSPY-SG (our own), telomeric probes: Tel 1q-SO, Tel 1p-SG, Tel 12-SG (VYSIS) and
MultiVysion PGT (13q14, CEP18, 21q22, CEPX, CEPY) (VYSIS). FISH and M-FISH were performed according to the protocols, described in detail previously (4, 5).
RESULTS.
Cases with normal karyotype after routine chromosomal analysis (1 – 6). C ases 1 and 2 with mental retardation did not present chromosomal imbalances by CGH. In case 3 with some features of Prader-Willi syndrome a loss of 15q12 was detected by CGH and confirmed by FISH. Cases 4 and 5 are examples of sex reversal: true hermaphrodite XX and a male
XX respectively, previously described in detail (4, 5). CGH confirmed the presence of a sequence of Yp: from Yp11.2 to
Ypter in case 4 and from Yp11.32 to Ypter in case 5. In case 6 using CGH the deletion - del(12)(p12.1p13.1) was detected.
Cases with chromosomal aneuploidy (7 – 11). The application of CGH technique to detection of chromosomal aneuploidy did not pose any interpretational problems. Chromosomal polyploidy was not revealed in case 11 because of the fact that it was observed only in 35% of the amniocytes (3).
Cases with marker chromosome (12 – 19). In case 12 with small chromosome marker (NOR+, DAPI+) the FISH technique confirmed that the marker was i(15p) and the CGH did not detected any chromosomal imbalance (2). Cases 13 – 14 were patients with some features of Turner’s syndrome and a mosaic, small ring chromosome. FISH detected that the marker chromosome originated from X chromosome in case 13 and Y chromosome in case 14. CGH established the chromosomal breakpoints: from Xp21.3 to Xq13.3 in case 13 and from Yq10 to Yq11.23 in case 14. In case 15 with defective sexual differentiation the CGH technique revealed the total loss of Yq. Ring chromosome (NOR-, DAPI-, CBG+) was found in case
16 with like-UPD(14) Syndrome. CGH did not detect of any chromosomal imbalance. Case 17 was a female with a r(18) and del(18). CGH confirmed the loss of the chromosomal region 18p11.32 and telomeric region - 18q. FISH technique with probes specific for telomeres of 18p/18q detected the absence of 18p telomere in the short arm of del(18) and the presence of
18q telomere in both arm of del(18). Cases 18 and 19 were patients with analphoid marker chromosome. CGH technique allowed for the finding that the marker chromosome originated from chromosome 1 (1q21-q22) in case 18 and chromosome
15 (15q21-15q24) in case 19.
Cases with other chromosomal aberrations (20 – 24). Case 20 with multiple congenital abnormalities presented with the following karyotype - 45,XX,der(9;14)(p22;q22). CGH showed a partial monosomy of 9p. CGH technique did not reveal any chromosomal imbalances in three cases of familial balanced translocation (cases 21, 22 and 23). The microdeletionsegments of 3q25 and 18q21 were identified in case 24 with balanced t(3;18).
DISCUSSION.
A total of 24 clinical cases (8 prenatal and 16 postnatal) was analyzed by CGH. The chromosomal imbalance was detected in
16 of 24 cases. The CGH allowed for clarification of chromosome aberration in 8 cases and for confirmation of chromosome aberrations in 6 cases. In 2 cases the CGH allowed for identification of subtle chromosome rearrangation.
CGH turned out to be a more informative technique than specific FISH. Unlike FISH, the CGH allows for the identification of chromosomal material of unknown origin in a single study. CGH enabled the elimination of the stage of molecular probe selection (cases: 4, 5, 6, 13,14, 16, 19, 24). CGH facilitated predicting the clinical consequences associated with the revealed imbalances. Positive results of CGH point to the fact that additional chromosomal material may contain unique DNA posing
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the potential threat of revealing a dose effect (6). Chromosomal imbalances observed in cases 3, 4, 6, 17 and 19 might have been described only with the use of CGH analysis based on dynamic standard reference intervals. Three out of four patients suspected of subtelomeric rearrangements came out with negative results, i.e. no chromosomal imbalances were found, in
CGH (cases 1, 2 and 6). These results do not exclude any possible microdeletions or microduplications within the subtelomeric regions. A separate problematic issue is the correlation between the results obtained in CGH analysis and the patients phenotypes. Phenotype dependent on „gain or loss” can be differentiated and not always correlating with the established imbalance. Classical cytogenetics lay foundations for the claim that chromosomal imbalances comprising euchromatic regions are associated with abnormal phenotype whereas those limited to heterochromatic regions may remain without any clinical consequence. However, certain cases studied by our team, i.e. cases of neocentric marker clinically presented with uncharacteristic very mild phenotypes. It took place despite the fact that additional chromosomal material contained mainly euchromatic regions: bands 15q22, 15q23 and the part of 15q24 and bands 1p21.1, 1p22.1 and 1p22.3 in cases 18 and 19 respectively. On the other hand, in cases 12 and 16 heterochromatic marker chromosomes were found, i(15p) and r(q14) respectively. They were accompanied by tangible phenotypic abnormalities pointing to UPD as their underlying cause. Balanced chromosomal translocations occurring de novo can be associated with critical microdeletions
(case 24). Such deletions may exert stronger influence on patients phenotypes than more gene disruptions (6).
Summing up, in our studies the CGH was especially useful for clarification of chromosome aberrations and identification of origin of chromosome markers. The presented results confirm the attractiveness of the CGH for these laboratories, which, for economical reasons, are not able to use the microarrayCGH.
REFERENCES.
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Constantinou M, Kaluzewski B, Helszer Z, et al. 2003. Prenatal detection of maternal UPD15 in a new case with i(15p) by
Timing Replication Test (TRT) and methylation analysis. J Appl Genet, 44(2):209-18.
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TSPY positive true hermaphrodite with oligozoospermia. J Med Genet, 37(10):E28.
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