Y

normal reacting controls (Figs. 1 and 2; Ref. 11 and unpublished data). The aim was to demonstrate that a difference in radiosensitivity which was already known between these individuals is also detectable using the 24-color FISH technique. Thus the increased radiosensitivity of NBS and AT patients can serve as a positive control in a predictive assay.

In the following, the criteria used in our own studies are specified to demonstrate that a reliable evaluation requires an experienced cytogeneticist. After the 24-color FISH procedure including hybridization, posthybridiza-tion washes, and detection, at least 100 metaphases per irradiation dose and patient/proband have to be acquired. This can be performed filter-based (m-FISH)[17] or spectracube-based (SKY).[18] All captured metaphases have to be karyotyped. For detailed evaluation of each metaphase, it is not enough to rely only on the pseudocolor functions of the used software, but for unambiguous results, one has to check the different fluorochrome channels or hybridization profiles. Then, aberration types and involved chromosomes have to be registered in detail.

All occurring aberrations were classified into reciprocal and nonreciprocal translocations, ring chromosomes, acentric fragments, dicentric fragments, inversions, insertions, and complex rearrangements. Translocations, insertions, and complex rearrangements are visible because of a color change along a rearranged chromosome. Dicen-trics and ring chromosomes can easily be identified using the inverted DAPI picture. Complex chromosomal rearrangements (CCR) consist, per definition, of at least two chromosomes with three or more breaks.[20]

For further evaluation, the frequency of break events constituting the observed aberrations was estimated as the minimal number of breaks considered to be necessary for producing the aberrations in each metaphase. The total number of break events in each patient/control and irradiation dose was summed up and divided by the number of metaphases analyzed to obtain the average rate of breaks per mitosis (B/M). The radiosensitivity of lymphocytes was expressed as number of radiation-induced B/M and CCR/M after each irradiation dose, subtracted by the 0.0-Gy control value to correct the

controls NBS-het AT-het NBS-hm AT-hm

Fig. 2 For the graphic depicted here, four normal controls, two Nijmegen breakage syndrome (NBS), and two ataxia telangiectasia (AT) heterozygotes (NBS-het, AT-het) plus one NBS and one AT homozygote (NBS-hm, AT-hm) were included. The upper diagram shows the average number of breaks per mitosis, while the lower one presents the average number of CCR occurring in each mitosis for each of the five groups. Three different in vitro irradiation doses were analyzed per patient. The applied doses were 0.0, 0.7, and 2.0 Gy; in the AT-hm patient, instead of 2.0 Gy, 1.4 Gy was used. A clear differentiation was possible in both diagrams between normal controls and the homozygote patients, as well as between controls and AT-het. (View this art in color at www.dekker.com.)

controls NBS-het AT-het NBS-hm AT-hm

Fig. 2 For the graphic depicted here, four normal controls, two Nijmegen breakage syndrome (NBS), and two ataxia telangiectasia (AT) heterozygotes (NBS-het, AT-het) plus one NBS and one AT homozygote (NBS-hm, AT-hm) were included. The upper diagram shows the average number of breaks per mitosis, while the lower one presents the average number of CCR occurring in each mitosis for each of the five groups. Three different in vitro irradiation doses were analyzed per patient. The applied doses were 0.0, 0.7, and 2.0 Gy; in the AT-hm patient, instead of 2.0 Gy, 1.4 Gy was used. A clear differentiation was possible in both diagrams between normal controls and the homozygote patients, as well as between controls and AT-het. (View this art in color at www.dekker.com.)

these approaches—the multicolor banding (MCB) technique (or mBAND)[22]—was also used for the analysis of X-ray-induced aberrations.[23'24] However, only a probe set for chromosome 5 was applied, demonstrating that intrachromosomal aberrations are present in a considerable portion in radiation-induced changes.

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