Analysis of 30 Cases of Translocation by the Controlled Heat Denaturation

J. LEJEUNE, B. DUTRILLAUX, M.O. RETHORE, M. PRIEUR, J. COUTURIER, S. CARFENTIER, and O. RAOUL.

Modem Aspects of Cytogenetics: Constitutive Heterochromatin in Man ; Symposium Schloß Reinhartshausen / Rhein June 18th - 20th, 1972. F.K. Schattauer verlag. Stuttgart-New York.


Résumé :

Zusammenfassung :Im Rahmen einer systematischen Untersuchung von mehr als 1000 Patienten mittels verschiedener Denaturationsmethoden (kontrollierte Hitzedenaturierung, enzymatische Verdauung, Centromerfärbung) wurden 30 Fälle von Translokationen analysiert. Alle Fälle von zentrischen Fusionen blieben unberücksichtigt. Es fanden sick :2 Fälle von komplexen Strukturänderungen, die 3 oder 4 Brüche erforderlich machten,2 Fälle von reziproker Translokation zwischen dem X and einem Autosom (klinisches Motiv : Sterilität), 26 Fälle von autosomalen reziproken Translokationen (klinisches Motiv : Anomalien unter den Nachkommen). Von diesen konnten 3 mittels der klassischen Methoden nicht aufgedeckt werden.Aus diesen Befunden ergeben sich folgende Tatsachen: 28 Translokationen liegen 56 Brüche zugrunde, die 18 mal in der Zentromerregion, 20 mal in der Telomerregion und 18 mal an einer anderen Stelle des Chromatids erfolgt sind.Offensichtlich sind die Brüche nicht zuffällig verteilt, da Centromer- and Telomerregionen gegenüber dem Rest des Chromatids weitaus überwiegen.

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Introduction

The analysis of the fine structure of chromatids by new techniques allow a better determination of the type of translocations. A review of 30 cases examined in our laboratory show that centromeric and telomeric regions are more frequently involved by breakage and rejoining than other parts of the chromatids.

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Material and methods

Our technique of controlled heat denaturation has been applied successfully to more than 1.000 patients referred to the laboratory.

The patients have been examined for various reasons, mostly because of an abnormality of the patient himself or of some of his relatives, or of an abnormality of reproduction such as sterility or repeated spontaneous abortions.

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a) Controlled heat denaturation (1-3-6) ( = R-banding)

The slides are dipped for 10 to 20 min in a solution of Earle's medium at pH 6.5, heated at 87° C.

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b) Enzymatic digestion (2-4) ( = G-banding)

The slides are dipped at room temperature in a solution of 5 mg of enzyme for 100 ml of water, buffered at the optimal pH, for the enzyme used (pronase, trypsin, a-chymotrypsine, protease, etc.).

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c) Staining o f constitutive heterochromatin (= C-banding)

This technique established in the laboratory uses a 20% solution of urea in water, heated at 87° C, in which the slides are dipped for 1 to 6 min.

Giemsa staining was used for all three techniques.

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Results

In this test with more than 1.000 patients, 30 were found to be translocation carriers.

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I. Translocations undetectable by classical procedures

The hypothesis has been verified (7) that some rearrangements which do not greatly modify the length of the elements should have escaped detection by the classical procedures.

These translocations seem to occur comparatively seldom, for only three cases have been detected during the course of our experiments.

1. In one family a child having a supernumerary G-like acrocentric, was born from a carrier father (Fig. 1).

2. In another family two brothers having also a supernumerary G-like acrocentric were born from a carrier mother (9).

Although these two families are not related, so far as we know, the carrier parents exhibit rather the same translocation: The long arm of chromosome 22 is exchanged with the terminal portion of the long arm of chromosome 8, the exchanged segments being of similar length. The three children are thus essentially trisomics for the distal part of the long arm of chromosome 8 and for the short arms. and centromeric region of chromosome 22.

3. The third case concerns a sterile man exhibiting apparently a Gq- chromosome. From meiotic studies there was no evidence of a reciprocal translocation. Denaturation technique demonstrated a translocation of the distal part of the long arm of chromosome 22 and on the end portion of chromosome 11. [t(11; 22)] (Fig. 8f).


Fig. 1. - Partial karyotype of two cells of a patient carrying a t(8 ; 22). The translocation is detectable only after denaturation (below).

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II. Complex translocations

Of the 30 cases, two cases have been demonstrated as complex rearrangements, which are due to more than two break points.

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1. Insertion:

In one family an apparently reciprocal translocation had been detected by the classical procedure, the carrier mother having a t(3p-; Cq+) translocation.

Denaturation showed (11) that a part of the short arm of the 3 is in fact inserted in the long arm of chromosome 7 (Fig. 2).

A rearrangement of this kind needs three break points, two in the donor and one in the recipient chromosome.


Fig. 2. Three brews accident: A segment of the short arm of chromosome 3 is inserted in the long arm of chromosome 7.

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2. Complex duplication:

A child with typical trisomy 21 had previously been proven, by classical procedures (8), to be a carrier of an abnormally long G chromosome.

This element was interpreted as a tandem translocation between two chromosomes 21, the whole arm of one of them being translocated on the telomeric portion of the other. Because of a further case of free trisomy 21 in a relative of the child, the eventuality of an " aneusomie de recombinaison " was discussed.

The analysis of the band pattern after controlled heat denaturation as well as after enzymatic digestion, shows that both chromosomes 21 are fused together by their telomeric region.

At the free end of the element the chromaticism tend to keep close to each other and show a fuzzy region which could be the remanants of satellites stalks (Fig. 3b and 3d). Nevertheless this rearranged chromosome is definitely not a true dicentric as confirmed by the centromeric staining (Fig. 3 c).

From this aspect the element can be designed, using the Paris nomenclature (10) as

t (21; 21) (21 pter ? 21 qter : : ? 21 q11 : : 21 p 11 ? 21 pter) (Fig. 4).

A new analysis of the karyotypes of the parents did not demonstrate a rearrangement of the chromosomes 21 which look normal. This negative result does not permit to propose a simple model of aneusomy of recombination to explain this strange chromosomal aberration.

If the accident really occurred de novo in the affected child, 4 break points are needed to explain it. It cannot be excluded that a particularity of the centromeric region of one of the chromosomes 21, undetectable with the present techniques, preexisted in one of the parents.


Fig. 3. Partial caryotype of 8 cells with a complex translocation t(21:21) : a) after controlled denaturation, b) after enzymatic digestion, c) after urea treatment, d) after conventional staining.


Fig. 4. Interpretation of the rearrangement t(21; 21) with a four breaks hypothesis.

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III. Translocations X-autosome

Among the 30 cases of translocations here discussed, 4 of them were ascertained for sterility. From the 4, two cases showed a reciprocal autosomal translocation and two showed an X-autosomal translocation.

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1. Translocation: t (6; X)

An amenorrheic woman carries a t (6 q+ ; Xq-) translocation. Analysis of band patterns after denaturation shows that break points occurred at the end of the long arm of 6 (6q27) and in band q 21 of the X. One third of this clear band remained on the Xq- and 2/3 were transferred to the 6q+ (Fig. 5).

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2. Translocation: t (1 ; X )

The translocation is observed in an azoospermic man (Fig. 6 c) and is also found in his mother (Fig. 6a) and his aunt (Fig. 6b). This person is also sterile. The sister of the proband does not carry the translocation but one of her chromosome I has an elongated secondary constriction (Fig. 6d). The same feature is found in the normal chromosome I of the mother and in the 1q- of the proband. It follows that a crossing over took place between the secondary constriction and the break point in the maternal meiosis (5).

The study of the meiosis of the proband (Fig. 7) reveals in many cells a very long element consisting of the rearranged chromosome 1 associated by the short arms to the normal one, itself associated by long arms to the segment translocated to the X ; this latter being itself associated by its short arms to the Y chromosome (Figs. 5 and 6).


Fig. 5. Translocation t(6;X) in an amenorrheic woman.


Fig. 6. Translocation t(1;X): a) The proband's mother. b) The proband's aunt (sterile). c) Proband (sterile). d) Proband's sister (non-sterile).


Fig. 7. Primary metaphase and interpretation of the tetravalent observed in the proband's meiosis.

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IV. Location of the break points

If we limit the analysis to the 28 instances of reciprocal translocations, we can divide the 56 break points according to their location :

in the centromeric region,

in the telomeric region,

or somewhere else in the chromatid (see Table 1 and Fig. 8, a, b, c, d, e, f).

The analysis shows that out of these 56 break points,

18 occur in a centromeric region,

20 occur in a telomeric region,

and only 18 occur in another part of a chromatid.

No case of translocation has been recorded in which both breaks are in the intermediate region.

Although any comparison of lengths of these regions is very difficult, it appears that the break points tend to lie in either end of a chromatid and occur rather unfrequently in the middle part of the chromatids.

The particular mechanism of " centric fusion " has often been used to explain the high frequency of translocation between acrocentrics.

It is possible that a comparable tendency exists also in metacentric chromosomes, their centromeric and telomeric regions being more prone to chromosome exchanges than the rest of the chromatid.

Table 1. Classification of 28 cases of reciprocal translocation according to the location of the break points.
Location of the break pointnumber of cases
1telomeric arm15
1arm
2centromeric regions5
1centromeric region5
1telomeric region
1centromeric region3
1arm
2arms0
2telomeric regions0*
Total28


Fig. 8. Various types of translocations: a) t(5 ; 12) between two centromeric regions; b) t(5 ; 7) between a telomeric region and a centromeric region; c) t(10 ; 11) between a telomeric region and a chromatid; d) t(9 ; 22) between two centromeric regions; e) t(8; 22) between a chromatid and a centromeric region; f) t(11 ; 22) probably between a telomeric region and a chromatid.


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References

(1) CARPENTIER, S., B. DUTRILLAUX, J. LEJEUNE: Effet du milieu ionique sur la denaturation thermique ménagée des chromosomes humains. Ann. Génét. 15: 3 (1972).

(2) COUTURIER, J.: Etude d'une technique de marquage des chromosomes humains par action d'enzymes protéolytiques. Thèse Médecine, Paris 1972.

(3) DUTRILLAUX, B., J. LEJEUNE: Sur une nouvelle technique d'analyse du caryotype humain. R. C. Acad. Sc. Paris, 272: 2638 (1971).

(4) DUTRILLAUX, B., J. DE GROUCHY, C. FINAZ, J. LEJEUNE: Mise en evidence de la structure fine des chromosomes humains par digestion enzymatique (pronase en particulier). R. C. Acad. Sc. Paris 273: 587 (1971).

(5) DUTRILLAUX, B., J. COUTURIER, J. ROTMAN, J. SALAT, J. LEJEUNE: Stérilité et translocation familiale t(1q-;Xq+). R. C. Acad. Sc. Paris 274: 3324 (1972).

(6) LAURENT, C., B. DUTRILLAUX, P. BINDER: Application de la méthode de dénaturation ménagée : dénaturation de préparations antérieurement utilisées. Ann. de Génét. 15: 3 (1972).

(7) LEJEUNE, J.: Autosomal disorders. Pediatrics 32: 326 (1963).

(8) LEJEUNE, J., R. BERGER, O. R. VIDAL, M. O. RETHORÉ: : Un cas de translocation G-G en tandem. Ann. Génét. 8: 60 (1965).

(9) LEJEUNE, J., M. U. RETHORÉ, B. DUTRILLAUX, J. MARTIN: Trisomie de la partie distale du bras long du chromosome 8 par translocation familiale 8-22. Exp. Cell Res. 74: 293 (1972).

(10) Paris Conference: Standardization in Human Genetics. Birth Defects. The National Foundation. March of Dimes, vol. VIII, n° 7 (1972).

(11) RETHORÉ M. O., J. LEJEUNE, S. CARPENTIER, M. PRIEUR, B. DUTRILLAUX, P. SERINGE, A. ROSSIER, J. C. JOB: : Trisomie pour la partie distale du bras court du chromosome 3 chez 3 germains. Premier exemple d'insertion chromosomique; ins (7:3) (q31; p21p26). Ann. Génét. 15: 3 (1972).