Analysis of t(Dq Dq) translocations after heat denaturation

Dutrillaux B. (*), Rethoré Marie-Odile (**) and Lejeune, J.

Chromosome Information Service N°14, 1973.


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Introduction:

After heat treatment under specific conditions (1) the staining pattern allows a recognition of each chromosome pair. The analysis is especially suitable when translocation occur, between pairs not detectable by standard techniques: e.g. between elements of the D group.

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Material and Methods:

Cultures of peripheral blood and hypotonic treatment are performed in the routine manner. The fixation of the cells is then achieved in two steps :

a) Thirty five minutes in Carnoy fixative (ethanol 6 vol., acetic acid 1 vol., and chloroform 3 vol.).

b) After centrifugation and removal of the first fixative, the cells are gently resuspended in Carnoy solution (ethanol 3 vol. and acetic acid 1 vol.) for 15 minutes.

After spontaneous air drying during several days of the chromosomes spreads, the slides are dipped for ten minutes in a batch of phosphate buffer pH 0.5 of 20 mM molarity, and maintained at 87 centigrade degrees of temperature.

After tap water rinsing, the slides are stained for ten minutes with Giemsa diluted a 4 per cent. The staining of the chromosomes is then rather faint and the analysis under the microscope requires phase contrast.

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Results :

very chromosome pair can be identified by the specific sequence of dark and faint bands. In general, the outlook of the chromosomes is not modified and the dark band observed on the telomeres allows always a correct measure of the chromatid lengths.

With our techniques the pattern of a given pair is strictly the contrary, the "countertype", of the sequences observed with fluorescence by U.V. of chromosomes colored by quinacrine mustard (2). Every dark band with our technique corresponds to a non fluorescent band with Q.M. staining; conversely the faint bands with our technique correspond to brilliantly fluorescent ones with Q.M. technique.

For these complementarities are extremely striking, we have followed the scheme of classification of Caspersson and Zech (2), in establishing the final classification of the caryotype.

t(Dq Dq) Translocation analysis: Three unrelated women ascertained because of repeated spontaneous miscarriages (Fig. 2) have been found carriers of a t(Dq Dq) translocation.

By the detection of the dark band claw to centromere, typical of 13 and the terminal one, typical of 14, the first and the thrid instances (I.P.N° 8831 and 10207) can be demonstrated as being of the t(13q 14q) type (Fig. 3, a and c).

In these families, a research for the translocation exhibited a normal segregation on 2 generations. Thus the genealogical data are in accordance with the cytological findings, because a transmissible t(Dq Dq) is necessarily made of two non homologous chromosomes.

In the second case (I.P. N° 9531), the pattern revealed a t(13q 13q) translocation (Fig. 3, b). This accident was indeed de novo and cannot be transmitted.

Considering the great difference of prognosis between a t(13q 14q) with a rather slight load of malsegregation, around: 5 % (3), and a t(13q 13q) which can only lead to 13 trisomy or to miscarriage by monosomy, the practical interest of this detection is evident.

Especially, advantage should he taken of the new technique for every translocation arisen de novo.


Fig. 1 - Normal female caryotype.


Fig. 2. Pedigrees of the 3 families.


Fig. 3. a) Partial caryotype showing a t(13q 14q) (I.P.N° 8831), b) Partial caryotype showing a t(13q 13q) (I.P.N° 9531). c) Partial caryotype showing a t(13q 14q) (I.P.N° 10207).


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References

1. Dutrillaux B, and Lejeune J. - 1971 Sur une nouvelle technique d'analyse du caryotype humain. C.R. Acad. Sci. Paris, 272: 2638-2640.

2. Caspersson T., Zech L., Johansson C. - 1979 Analysis of human metaphase chromosome set by aid of DNA binding florescent agents. Exptl. Cell. Res. 62: 490-492.

3. Dutrillaux B, Lejeune J. - 1970 Etude de la descendance des individus porteurs dune translocation t(Dq Dq). Ann. Genet, 13; 11-18.

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Notes

(*) Chargé de Recherches au C.N.R.S.

(**) Maître de Recherches à l'INSERM.