Trisomy 22 in a liveborn infant with multiple congenital anomalies.

We report on the third confirmed case of trisomy 22 in a liveborn infant. High-resolution banding studies ruled out translocations such as the relatively common t(11;22). The infant shared many manifestations with other reported cases of trisomy 22 (e.g., growth deficiency, microcephaly, micrognathia, ear malformations, cleft palate, and congenital heart defect) and some manifestations in common with dup 22. Trisomy 22 appears to cause a severe malformation syndrome, and survival to term is rare.

Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens. I. In vivo test for compounds requiring metabolic activation.

An in vivo system has been devised in which chemical mutagen-carcinogens injected into an animal induce sister chromatid exchanges that can be observed when the animal's peripheral lymphocytes are subsequently cultured and then stained with the FPG technique. Chemicals requiring metabolic activation, as well as those that do not, produce significant increases in SCE frequency one day after exposure. The frequency then returns to control level within two weeks. This in vivo system is a highly sensitive assay for the ability of chemical agents to damage chromosomes.

Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens. II. In vitro test for compounds requiring metabolic activation.

Sister chromatid exchanges (SCE's) which are easily seen by "harlequin chromosome" techniques can be readily induced in cultured Chinese hamster ovary (CHO) cells by low concentrations of mutagen-carcinogens that do not require metabolic activation. If the cells are simultaneously treated with cyclophosphamide which does require metabolic activation before it becomes mutagenic, and an activating system consisting of an extract of rat liver containing microsomes (S-9 Mix) then numerous SCE's are induced by the compound. This indicates that the induction of sister chromatid exchanges in such cells can be used as an in vitro assay for mutagens that require activation as well as those that do not. The method, which is very simple and quick, is more sensitive than is the usual cytogenetic assay in which chromosome aberrations are assayed.

SCEs are induced by replication of BrdU-substituted DNA templates, but not by incorporation of BrdU into nascent DNA.

After 3 rounds of DNA replication in the presence of BrdU, third-division metaphase cells can be scored for the frequencies of SCEs that occurred during cycles 1 and 2, and also for the frequency of SCE during cycle 3. This procedure was used to resolve the issue of SCE induction by replication of BrdU-substituted DNA templates versus induction by BrdU incorporation into nascent DNA. It was observed that third-cycle SCE frequencies in CHO are dependent upon the amount of BrdU that was present during cycles 1 and 2 and are independent of the BrdU concentration during the third cycle. It is therefore BrdU serving as a template, rather than BrdU being incorporated, that initiates the SCE event. A model is proposed that produces reasonable fits to the observed data. It also predicts a true background or spontaneous SCE frequency of 3 per cell per cycle as previously reported by Heartlein et al. (Mutation Res., 107 (1983) (103-109). The predicted single twin ratio is higher than that reported by Wolff and Perry (Exp. Cell Res., 93 (1975) 23-30), and possible explanations for this discrepancy are discussed.

Induction of long-lived chromosome damage, as manifested by sister-chromatid exchange, in lymphocytes of animals exposed to mitomycin-C.

The cytogenetic effects of repeated vs. acute exposure to a chemical mutagen--carcinogen were determined with an in vivo system in which chemicals injected into rabbits induce sister-chromatid exchanges (SCEs). SCE induction can be monitored when the animal's peripheral lymphocytes are cultured in the presence of bromodeoxyuridine (BrdUrd) and then scored for SCE frequency. Mitomycin-C (MMC), 0.5 mg/kg, was injected intraperitoneally once a week for 8 weeks. This treatment initially induced small increases in SCE frequency within one day of injection, followed by a return to control levels within 1 week. After the 4th injection, however, the frequency failed to return to normal. After the 5th injection, however it showed a 4-fold increase over the control which was sustained for the remaining 3 weeks of treatment and for an additional 2 weeks thereafter. The frequency then dropped to twice the control value and remained at this level for more than 4 months. All of the high SCE values after the first 4 weeks were due in part to the appearance and persistence of a population of cells with high SCE frequencies. Exposure to the same total dose given as a single injection resulted in a transient elevation in the SCE frequency and a subsequent return to lower values, with no evidence of a delayed effect such as the increase observed after 4 weeks in repeatedly exposed animals. Overall, repeated exposure is at least as effective as acute exposure in eliciting long-lived SCEs in vivo.

DNA crosslinking, sister-chromatid exchange and specific-locus mutations.

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.

Further analysis of the replication bypass model for sister chromatid exchange.

The replication bypass model for sister chromatid exchange (SCE) proposed by Shafer is examined in detail. When applied through two cell cycles, the model predicts that potentially observable SCEs induced during one S phase will then be cancelled and rendered undetectable during the subsequent S phase. This aspect of replication bypass is inconsistent with the observation of twin SCEs in tetraploid cells. Furthermore, the model cannot account for the efficient induction of SCEs by non-cross-linking chemical agents.

Recovery of Pisum root meristems after mitotic-inhibitory treatments with 3H-thymidine. Inhibition of cell-cycle progression by unincorporated 3H-thymidine.

Primary root meristems of Pisum sativum recover form a 3H-thymidine-induced reduction in mitotic activity once the roots are no longer exposed to exogenous 3H-thymidine. Cells arrested in G2 during 3H-thymidine treatment apparently do not divide for at least 16 hours after treatment, whereas cells remaining in G1 and S do divide and thereby account for recovery. Recovery occurs only when meristems are no longer exposed to exogenous (i.e. unincorporated) 3H-thymidine, suggesting that cytoplasmic irradiation from unincorporated 3H-thymidine prevents cellular recovery from 3H-thymidine-induced inhibition of cell progression through the mitotic cycle. Concentrations of 14C-thymidine which result in cytoplasmic irradiation nearly equivalent to that achieved with 3H-thymidine, but much lower levels of nuclear irradiation, also prevent recovery from 3H-thymidine-induced inhibition of mitotic activity, but do not alone produced such inhibition. These results support the contention that cytoplasmic irradiation prevents recovery from the effects of nuclear irradiation. Unincorporated 3H-thymidine also prevents recovery from sucrose deprivation in stationary phase G2 cells which have not incorporated 3H-thymidine into nuclear DNA.

The interaction of Hoechst 33258 and BrdU substituted DNA in the formation of sister chromatid exchanges.

Sister chromatid exchanges (SCEs) are induced in cultured Chinese hamster cells by treatment with 5-bromodeoxyuridine (BrdU) or with Hoechst 33258 (H33258) plus BrdU. The SCE frequencies depend upon the number of H33258 molecules available per cell (or per base pair) and the number of brdU molecules available per cell, and not solely upon molarity. In addition, H33258 and BrdU act synergistically to induce SCEs. At low BrdU concentrations H 33258 induces very few SCEs. At high BrdU concentrations and similar concentrations of H33258, however, SCE frequencies are significantly increased. SCE frequencies decrease with time in successively harvested cells because of the depletion of H33258 from the medium due to DNA binding.

SCE induction is uncoupled from mutation induction in mammalian cells following exposure to ethylnitrosourea (ENU).

It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that "S-dependent" processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE-inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log-phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU-induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined.

Statistical design, analysis, and inference issues in studies using sister chromatid exchange.

While underlying biological mechanisms responsible for sister chromatid exchange (SCE) formation are not fully understood, scientists worldwide are increasingly using SCEs in the evaluation of excess risk from exposure to chemical and biological agents. SCEs are being used as endpoint measures of cell damage in many types of experimental and nonexperimental investigations. The former includes both simple and complex randomized experiments using both animals exposed in vivo and cells exposed in vitro as experimental units. The latter includes the important, yet potentially misleading, human case-control studies in which a group of humans who are or have been exposed to some agent are compared with a selected nonexposed group on SCE frequency. As more is learned about those factors which result in SCE variations, the assay techniques and study protocols can be adjusted to enhance study sensitivity and to minimize potential bias. Although research concerning sample sizes and statistical analysis methods has been conducted, more is needed. Search for other sources of intersubject variations in SCEs should continue so that such sources can be controlled in future studies, particularly the human exposure types. A number of experimental designs are presented and contrasted with their nonexperimental counterparts. Statistical methods are summarized and sample size options are given for the human and animal exposure studies and for the studies in which cells are exposed in vitro.

Familial 46,XX males coexisting with familial 46,XX true hermaphrodites in same pedigree.

Reported here is a family with which 46,XX males and 46,XX true hermaphrodites coexist. The propositus was a paternal uncle with 46,XX true hermaphroditism. One of his brothers fathered a 46,XX daughter with true hermaphroditism; a second brother fathered two 46,XX males. Both fathers have normal male karyotypes and phenotypes. No evidence for chromosomal mosaicism or any additional chromosomal abnormalities was obtained. We conclude that inheritance of the abnormality is most likely via paternal transmission of an autosomal testis-determining factor. This family provides evidence to support the hypothesis that 46,XX true hermaphrodites and 46,XX males represent alternative manifestations of the same genetic defect.

Variation in the baseline sister chromatid exchange frequency in human lymphocytes.

The utility of the sister chromatid exchange (SCE) assay for human population studies is potentially limited by the variability associated with individual baseline SCE Frequencies. This investigation identifies and quantifies the major sources of preparative and biological variation associated with the determination of baseline SCE frequencies in cultured human lymphocytes. Much of the variation in lymphocyte SCE frequencies is attributable to the amount of bromodeoxyuridine (BrdUrd) available per lymphocyte; the pooled coefficient of variation (CV) over the dose range of 10 to 160 micrometer is about 18%. Other variations in the baseline frequency result from culture-to-culture and slide-to-slide differences. The pooled coefficient of variation among donors is about 10%. The effect of cell-to-cell differences in baseline SCE frequency among donors can be minimized by increasing the number of cells scored per donor. When 20 cells are analyzed per individual the pooled cell-to-cell variation is 9% but when 40 or 80 cells are analyzed it is reduced to 6 and 4% respectively. For a single individual the cell-to-cell coefficient of variation at 100 micrometer BrdUrd is 40.8%. Under our experimental conditions, a 30% increase in SCE frequency between two cohort populations can be detected with a 95% probability at a 5% level of significance when 11 individuals per cohort are studied. For a longitudinal or in vitro dose response study of a single individual, a 50% increase in SCE frequency can be detected with a 95% probability at a 5% level of significance when 25 cells per sample are analyzed. These results indicate the feasibility of applying the SCE bioassay to humans as a measure of environmental stress.

Alphoid DNA diversity of a so-called monocentric Robertsonian fusion.

The centromeric heterochromatin of a Robertsonian translocation with t(13q14q), thought to be monocentric by conventional staining methods, was found to be dicentric using molecular techniques. The breakpoints were confined within the alphoid DNA subfamilies and fusion resulted in a compound centromere. The fragile nature of alphoid DNA sequences during Robertsonian translocation has opened new avenues in understanding other chromosomal aberrations involving centric fusion.

Centromeric alphoid DNA heteromorphisms of chromosome 21 revealed by FISH-technique.

The centromeric heterochromatin of chromosome 21 has been evaluated by the fluorescence in situ hybridization (FISH) technique. It was found that the alphoid DNA sequences of pericentromeric regions of chromosome 21 were highly heteromorphic when a centromeric specific probe was hybridized to these sequences. The variations were so extreme that they could even be arbitrarily classified into at least five sizes by comparison with the length of the short arm (p) of chromosome 18. They are negative (1); small (2); medium (3); large (4); and very large (5). We used 15 normal cases and 12 individuals with trisomy 21 (Down syndrome), and the incidences for these five classes were 3.0%, 22.7%, 59.2%, 13.6% and 1.5%, respectively. At least 3% of the chromosomes no. 21 did not show any trace of hybridization signals, which apparently escape detection at interphase level as well. Although, the variations observed in the present study are continuous, the proposed classification may yield some implications for future investigations.