Synergism of mutant frequencies in the mouse lymphoma cell mutagenicity assay by binary mixtures of methyl methanesulfonate and ethyl methanesulfonate

The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK-/- mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK-/- mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 micrograms/ml MMS and either 261, 392, 560 or 712 micrograms/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12, 20, 35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased -7, -6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 micrograms/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 micrograms/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration.     

Methapyrilene is a genotoxic carcinogen: studies on methapyrilene and pyrilamine in the L5178Y/TK +/- mouse lymphoma assay

Methapyrilene (MP), a sedating antihistamine, is a potent rat hepatocarcinogen which has been thought to be non-genotoxic on the basis of the negative results in a small number of short-term mutagenicity tests. The present studies show that MP is a moderately active mutagen in the L5178Y/TK +/-----TK-/- mouse lymphoma assay (MLA) in the presence of aroclor-induced rat-liver S9, and that it induces predominantly small-colony thymidine kinase-deficient (TK-/-) mutants of demonstrated chromosomal origin. 10 of 12 small colony TK-/- mutants analyzed by banded karyotype (230-band level of resolution) show aberrations to chromosome 11b, the known location of the single functional TK gene in these cells. The observed aberrations from nine of the mutants included insertions, deletions and translocations while the tenth mutant had highly rearranged, multiple copies of chromosome 11 segments. By varying the concentrations of the S9 protein and cofactors it was shown that our standard S9 composition was close to optimum for activating MP to a mutagen. The activity and stability of various lots of S9 prepared in-house or purchased from a contract laboratory revealed significant differences. The ability of 2 lots of in-house S9 to activate a standard concentration of MP increased rapidly over the first 4 weeks of liquid nitrogen storage then declined slowly over the next 16 weeks. Three separate lots of purchased S9 were essentially inactive for the first 2 weeks of liquid nitrogen storage then increased in activity thereafter; these were the only occasions in which MP was not mutagenic in our hands. The mutagenic activity of pyrilamine (PYR), a structurally related antihistamine which is far less carcinogenic in rats, but easily detected in short-term tests as being genotoxic, was also investigated in the MLA. PYR was slightly less mutagenic than MP over a comparable range of concentrations, and also induced predominantly small-colony mutants. These studies fail to adequately explain the great carcinogenic differences between these two compounds, but are consistent with the potent hepatocarcinogenicity of MP resulting through a mutagenic mechanism.     

Chromosome 11 aberrations in small colony L5178Y TK-/- mutants early in their clonal history

We have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK-/- mutagenesis of the L5178Y/TK+/-3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments we show that the major proportion (93%) of small-colony (sigma) mutants studied have chromosome 11 rearrangements (the chromosome containing the thymidine kinase gene) while large-colony (lambda) mutants do not have detectable chromosome rearrangements. In addition, we find among the chromosome abnormalities in sigma mutants a significant proportion (34%) with dicentric chromosomes involving chromosome 11. These potentially unstable chromosome rearrangements may help to explain the karyotypic instability and heterogeneity among chromosome 11 aberrations previously noted in sigma mutants when they are analyzed later in their clonal history.     

Trifluorothymidine resistance and colony size in L5178Y/TK +/- cells treated with methyl methanesulfonate

L5178Y/TK +/- cells treated with methyl methanesulfonate (MMS) were allowed to recover for 0,48,96,144, or 240 hours, and were then plated in soft-agar medium containing trifluorothymidine (TFT). Dose-dependent and consistent increases in the frequency of TFTR cells were observed after each of the 48-240-hour expression periods through the counting of predominantly large, mutant colonies. Size distributions of soft-agar colonies from either MMS-treated or control cells were bimodal in the presence, and unimodal in the absence, of TFT. An increase of small, presumptive TFTR colonies with either increasing MMS concentration or decreasing recovery time was probably a manifestation of chemical toxicity, for a similar increase in small-colony number was observed in the absence of TFT when cells were cloned immediately after MMS treatment, when no induced mutants were yet detectable. Recloning experiments with 22 small-colony-derived cell lines revealed that, with one exception, small-colony morphology was not a heritable trait. While all large- and some small-colony-derived stocks from MMS-treated cells were of the phenotypically stable TK-/- type; spontaneous small TFTR colonies generally were not, their occurrence being directly correlated with serum concentration. No aneuploidy was evident in MMS-treated cell lines several generations after isolation as small TFTR colonies. These results suggest that delayed MMS cytotoxicity in TK +/- cells can temporarily produce increased physiological resistance to TFT in some cells, giving rise to secondary populations of small-colony TFTR variants.     

Cytogenetic characterization of the L5178Y TK+/-3.7.2C mouse lymphoma cell line

The cytogenetic characterization of the L5178Y TK+/-3.7.2C mouse lymphoma cell line was carried out, utilizing G-banded metaphase chromosomes, to provide a karyotypic basis for the precise delineation of induced rearrangements in TK-/- mutants. Band-pattern measurements were used to construct ideograms which represent the position, number, size and staining intensity of the chromosome bands. The TK+/-3.7.2C cell line has been shown to provide quantitation of forward mutations induced at the autosomal thymidine kinase (TK) locus in this cell line. Chromosome analysis of the TK+/-3.7.2C cell line and derived TK-/- mutants has become important in demonstrating that the TK+/-----TK-/- assay may detect and distinguish between chromosomal events and smaller, perhaps point-mutation, events in mutant colonies.     

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Clonal analysis of delayed karyotypic abnormalities and gene mutations in radiation-induced genetic instability.

Many tumors exhibit extensive chromosomal instability, but karyotypic alterations will be significant in carcinogenesis only by influencing specific oncogenes or tumor suppressor loci within the affected chromosomal segments. In this investigation, the specificity of chromosomal rearrangements attributable to radiation-induced genomic instability is detailed, and a qualitative and quantitative correspondence with mutagenesis is demonstrated. Chromosomal abnormalities preferentially occurred near the site of prior rearrangements, resulting in complex abnormalities, or near the centromere, resulting in deletion or translocation of the entire chromosome arm, but no case of an interstitial chromosomal deletion was observed. Evidence for chromosomal instability in the progeny of irradiated cells also included clonal karyotypic heterogeneity. The persistence of instability was demonstrated for at least 80 generations by elevated mutation rates at the heterozygous, autosomal marker locus tk. Among those TK- mutants that showed a loss of heterozygosity, a statistically significant increase in mutation rate was observed only for those in which the loss of heterozygosity encompasses the telomeric region. This mutational specificity corresponds with the prevalence of terminal deletions, additions, and translocations, and the absence of interstitial deletions, in karyotypic analysis. Surprisingly, the elevated rate of TK- mutations is also partially attributable to intragenic base substitutions and small deletions, and DNA sequence analysis of some of these mutations is presented. Complex chromosomal abnormalities appear to be the most significant indicators of a high rate of persistent genetic instability which correlates with increased rates of both intragenic and chromosomal-scale mutations at tk.     

Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Use of DNA purified in situ from cells embedded in agarose plugs for the molecular analysis of tk-/- mutants recovered in the L5178Y tk+/- 3.7.2C mutagen assay system

We have reported that tk-/- mutants recovered in the mouse L5178Y tk+/- 3.7.2C mutagen assay have often lost the tk+ allele. Allele loss in the tk-/- mutants is documented on Southern blots as the absence of a 6.3-kb Nco I fragment seen in both tk+/+ and tk+/- cell DNAs. For the routine screening of large- and small-colony tk-/- mutants DNAs for the absence of this genomic fragment, we have found that cells can be lysed in agarose plugs, and DNA of cells embedded in plugs can be purified, restricted with Nco I, electrophoresed, and analyzed on Southern blots without significant band distortion or diffusional loss of tk- specific fragments in the 2-7-kb range. Purification and restriction analysis of DNA in agarose plugs, originally developed to allow pulsed-field gel electrophoresis of very large DNA fragments, represents a convenient alternative to conventional DNA purification methods, allowing quantitative recovery of DNA from small numbers of cells, eliminating centrifugation, phenol extraction, and ethanol precipitation steps, and requiring smaller quantities of reagents.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

Molecular genetics of radiation-induced chromosome breaks in a gene area in Drosophila: "position" effect of gene mutation?

On the sample of 43 gamma-ray and neutron-induced inversion or translocation exchanges with the vestigial (vg) phenotype, the molecular cytogenetic analysis of distribution of exchange breakpoints on the molecular map of Drosophila vg region (subsection 49D3-4 on the polytene chromosome 2R) was performed using hybridisation in situ technique. Simultaneously, PCR-assay of DNA alterations in all exons and introns (except for intron 4) of the vg gene for 18 mutants with exchange breakpoints outside of the gene was carried out. The results obtained by these molecular genetic techniques have shown that 1) radiation-induced breaks under chromosome exchanges with the vg phenotype were regularly located inside of the vg gene (19 cases out of 43 studied ones or 44.2%) passing through the large introns; 2) breakpoints were frequently flanked by deletions of the gene as whole (3 exchanges) or of its major part (3 exchanges); 3) many of the breaks (18/43 or 41.8%) are situated outside (distal or proximal) of the gene although such mutants have got the vg phenotype; 4) 2/3 (12/18 or 66.7%) vg mutants with the breakpoint outside of gene show the intragenic DNA lesions (microdeletions, microinversions) occurring obviously independently and simultaneously with the neighbor chromosome breaks; 5) only each third vg mutant with break outside of the gene (6/18 or 33.3%) have the unchanged gene subregions under study and presents obviously the result of "position effect" which appear to manifest itself for a distance of 2-30 kb (more near and farther locations of the proximal and distal breakpoints, respectively, relative to the vg gene). Our findings showing regular induction of the multiple genetic lesions (chromosome breaks and mutations of the adjacent genes) on the both ends of chromosome exchange induced by single track produced by gamma-rays or neutrons were discussed as a scientific basis for the conceptually new approaches to the assessment of both genetic damage numbers in the cell genome with chromosome exchange (the multiple genetic lesions) and radiation genetic risk (our molecular genetic approach showing the need for an increase of risk levels at least on a factor of 3 for the heritable chromosome alterations detected by the ordinary cytogenetic monitoring).     

Stability of tk-/- mutants of L5178Y mouse lymphoma cells in Fischer's medium

The phenotypic stability of over 2000 large- and small-colony trifluorothymidine-resistant (TFTres) variants of L5178Y/tk(+/-)-3.7.2C cells has been examined. All except 4 of 488 spontaneously arising small-colony variants analyzed (0.8%) retained the TFTres phenotype when rechallenged with TFT after growth for several generations in its absence. All of 558 spontaneous large-colony variants, and 440 small-colony or 487 large-colony variants arising from 13 different mutagens showed similar stability. These results attest to the completeness of TFT selection in the mouse-lymphoma assay when used at 1 microgram/ml in Fischer's medium supplemented with heat-inactivated serum and, together with previous cytogenetic and molecular studies, justify considering essentially all such TFTres variants as stable mutants. The implications of these results for those versions of the mouse lymphoma assay that fail to optimize the recovery and scoring of small-colony mutants is discussed.     

Induction of multilocus lesions by UVC-radiation in mouse L5178Y lymphoblasts

The survival, the mutant frequency and the nature of the DNA alteration responsible for the inactivation of the thymidine kinase (tk) locus were investigated in 5 strains of mouse L5178Y lymphoblasts exposed to UVC radiation. The nature of the DNA alteration was investigated in independent TK-/- mutants using Southern blot analysis. The concomitant loss of galactokinase (GK) activity in homogenates of individual TK-/- mutants was taken as an indication that the lesion inactivating the tk allele extended to the neighboring galactokinase (gk) allele. The survival of strains LY-R16 and LY-R83 was decreased to a greater extent than that of strains LY-S1, LY-SR1, and LY-3.7.2C, reflecting a deficiency in excision repair in strains derived from LY-R cells. The TK-/- mutant frequency of strain LY-R83, which is monosomic for chromosome 11 and thus hemizygous for the tk and gk genes, was only 50% of the mutant frequency of strain LY-R16 which is heterozygous for the tk gene. Moreover, a greatly reduced percentage of individual spontaneous and UVC-induced TK-/- mutants of strain LY-R83 showed loss of GK activity in comparison to the other strains. This result indicates that UVC irradiation induces intergenic mutations and that such mutants are poorly recovered in the hemizygous strain. Strain LY-3.7.2C appears to have only one active galactokinase (gk) allele, and very few TK-/- mutants of this strain showed loss of GK activity, possibly because this strain, although heterozyogous for the tk gene, is hemizygous in the region of the gk gene. Strains LY-R16 and LY-S1 are deficient in the repair of UVC- and X-radiation-induced damage, respectively, and the percentage of TK-/- mutants with intergenic mutations was higher for strain LY-R16 after UVC-radiation and for strain LY-S1 after X-radiation. These results indicate that unrepaired DNA lesions lead to an increase in intergenic mutations.     

Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium

L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants.     

The mutagenicity of 2-amino-N6-hydroxyadenine to L5178Y tk +/- 3.7.2C mouse lymphoma cells: measurement of mutations to ouabain, 6-thioguanine and trifluorothymidine resistance, and the induction of micronuclei

2-Amino-N6-hydroxyadenine (AHA) was tested in the mouse lymphoma L5178Y tk +/- assay using the microtitre cloning technique over concentrations from 0.005 micrograms/ml-1 (100% viability) to 6 micrograms/ml (10% viability) as measured by cloning efficiency immediately after treatment. At low, non-toxic concentrations (0.005-0.25 micrograms/ml) a dose-related linear increase in the frequency of ouabain-resistant mutants was seen, in addition to an increase in 6-thioguanine- and trifluorothymidine-resistant mutants. No consistent induction of micronucleated cells was observed in this concentration range. Toxic concentrations (20-90% kill) induced a dose-related increase in micronuclei, while the frequency of ouabain-resistant mutants fell (although it was still highly significantly above the control value). These results suggest that the mechanism of action of AHA depends on the concentration, with point mutations being induced at low, non-toxic doses and detectable chromosome breakage occurring only at higher doses. Both large-colony and small-colony trifluorothymidine-resistant mutants were induced at all concentrations. The utility of using multiple genetic end-points in one cell line and the importance of dose range selection for risk assessment and an understanding of the mode of action of test substances is underlined.     

The nature of radiation-induced mutations at the white locus of Drosophila melanogaster

X-Ray- and neutron-induced mutations at the white locus of Drosophila melanogaster were used to study the nature of radiation-induced genetic damage. Genetic analysis showed the presence of multi-locus deficiencies in 15 out of 31 X-ray mutants and in 26 out of 35 mutants induced by neutrons. The DNA from 11 X-ray and 4 neutron mutants, which were not multi-locus deficiencies, was analyzed by Southern blot-hybridization. Deletions were observed in 2 X-ray and 1 neutron mutant. In combination with cytogenetic techniques, chromosomal rearrangements affecting the white locus (translocations, inversions, etc.) were identified in 3 X-ray and in 2 neutron mutants. A hot-spot for translocation breakpoints was identified in the left arm of the third chromosome. 5 X-ray mutants, which apparently did not contain large deletions, were subjected to further analysis by the nuclease S1 protection method, after cloning of the white gene. In 4 mutants a small deletion could indeed be detected in this way. Thus it seems that by far the main part of X-ray- and neutron-induced white mutants have arisen through large changes in the white gene, especially deletions.     

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10(6) survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.     

A Genetic and Molecular Analysis of the 46c Chromosomal Region Surrounding the Fmrfamide Neuropeptide Gene in Drosophila Melanogaster

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes.     

A case of prenatal detection of a de novo unbalanced complex chromosomal rearrangement involving four chromosomes

Complex chromosomal rearrangements are rarely observed prenatally. Genetic counceling of CCR carriers is complicated, especially in cases of de novo origin of the rearrangement. Here we present a new case of a de novo CCR involving four chromosomes observed in amniotic fluid cells of the fetus at 17 weeks of gestation. The rearrangement was characterized as an apparently balanced four-way trans-location t(1;11;7;13)(~p21;~q13.5;~q32;~q22)dn by conventional cytogenetic studies. However, array-based comparative genomic hybridization revealed 5 submicroscopic heterozygous interstitial deletions on chromosome 1, 11, 7, 13 with a total loss of 21.1 Mb of genetic material in regions close to those, designated as breakpoints by conventional cytogenetic analysis. The described case clearly illustrates that high-resolution molecular genetic analysis should be combined with conventional cytogenetic techniques to exclude subtle chromosomal abnormalities in CCR cases detected prenatally.