Single cell chromosome rearrangements in indivuduals with reproductive failure

Abstracts

Single cell chromosome rearrangements (SCCR) are incidental findings in cell cultures. Nevertheless, some authors have implicated them in habitual abortion. Ninety individuals classified in four groups were prospectively studied: A) individuals with spontaneous abortions, malformed children or molar pregnancies (N = 36); B) azoospermic males (N = 14); C) individuals with one or more children with either de novo or inherited, constitutional, chromosome abnormalities (N = 26), and D) individuals with healthy children and no reproductive failure (control group, N = 14). Lymphocyte chromosome preparations were stained for GTG bands, and 50-100 metaphases were scored per individual. The rearrangements observed were translocations, deletions, duplications, isochromosomes, rings, fragments and markers. Thirty-four individuals (21 males, 13 females) had a range of 1-5 SCCR. Four had rearrangements only of chromosomes 7 and 14. SCCR frequency (with the exception of rearrangements involving chromosomes 7 and 14) was 0.0063, while that of rearrangements between chromosomes 7 and 14 was 0.0010. Statistical intergroup comparisons (<FONT FACE="Symbol">c</font>2 with Yates correction) did not show significant differences. Hence, the occurrence of SCCR in our sample was found to be independent of ascertainment mode and sex.

Rearranjos cromossômicos de célula única (SCCR) são achados incidentais em culturas de células. Não obstante, alguns autores os têm relacionado com o abortamento habitual. Noventa indivíduos classificados em 4 grupos foram estudados prospectivamente: A) indivíduos com abortamentos espontâneos, filhos malformados ou gravidezes molares (N = 36); B) homens azoospérmicos (N = 14); C) indivíduos com um ou mais filhos com anormalidades cromosômicas de novo ou herdadas (N = 26) e D) indivíduos sem problemas reprodutivos e com filhos saudáveis (grupo controle, N = 14). As preparações de cromossomos de linfócitos foram coradas para bandamento GTG e 50-100 metáfases foram avaliadas por indivíduo. Os rearranjos observados foram translocações, deleções, duplicações, isocromossomos, anéis, fragmentos e marcadores. Trinta e quatro indivíduos (21 homens e 13 mulheres) tiveram de 1 a 5 SCCR. Quatro tiveram rearranjos apenas dos cromossomos 7 e 14. A freqüência de SCCR (com exceção dos rearranjos envolvendo os cromossomos 7 e 14) foi de 0,0063, enquanto que para os rearranjos entre os cromossomos 7 e 14 foi 0,0010. Comparações estatísticas intergrupo (<FONT FACE="Symbol">c</font>2 com correção de Yates) não mostraram diferenças significativas. Assim, a ocorrência de SCCR em nossa amostra mostrou-se independente do modo de averiguação e do sexo.

Single cell chromosome rearrangements in indivuduals with reproductive failure

M.G. Domínguez, H. Rivera, A.I. Vásquez and A.L. Ramos

División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, y Posgrado en Genética Humana, Universidad de Guadalajara, Apdo. Postal 1-3838, Guadalajara, Jalisco, Mexico. Send correspondence to H.R. Fax: [phone] E-mail: [e-mail]

ABSTRACT

Single cell chromosome rearrangements (SCCR) are incidental findings in cell cultures. Nevertheless, some authors have implicated them in habitual abortion. Ninety individuals classified in four groups were prospectively studied: A) individuals with spontaneous abortions, malformed children or molar pregnancies (N = 36); B) azoospermic males (N = 14); C) individuals with one or more children with either de novo or inherited, constitutional, chromosome abnormalities (N = 26), and D) individuals with healthy children and no reproductive failure (control group, N = 14). Lymphocyte chromosome preparations were stained for GTG bands, and 50-100 metaphases were scored per individual. The rearrangements observed were translocations, deletions, duplications, isochromosomes, rings, fragments and markers. Thirty-four individuals (21 males, 13 females) had a range of 1-5 SCCR. Four had rearrangements only of chromosomes 7 and 14. SCCR frequency (with the exception of rearrangements involving chromosomes 7 and 14) was 0.0063, while that of rearrangements between chromosomes 7 and 14 was 0.0010. Statistical intergroup comparisons (c² with Yates correction) did not show significant differences. Hence, the occurrence of SCCR in our sample was found to be independent of ascertainment mode and sex.

INTRODUCTION

Single cell chromosome rearrangements (SCCR) are incidental findings in cell cultures (Iskra, 1985; Petersson and Mitelman, 1985, Higgins and Palmer, 1987). Chromosome participation in SCCR appears to be at random, except for inv(7)(p14q35), inv(14)(q11q32), t(7;14)(p13-15;q11) and t(7;14)(q34-36;q11), that occur only in phytohemagglutinin (PHA)-stimulated lymphocyte cultures (Herva, 1981; Dewald et al., 1986; Aurias, 1993). Some authors have suggested that SCCR can also occur in germ cells and lead to spontaneous abortions (Iskra, 1985; Higgins and Palmer, 1987).

MATERIAL AND METHODS

Ninety individuals classified into four groups were prospectively studied to determine the frequency and type of SCCR in lymphocyte cultures: A) individuals with spontaneous abortions, malformed children or molar pregnancies (N = 36); B) azoospermic males (N = 14); C) individuals with one or more children with either de novo or inherited, constitutional, chromosome abnormalities (N = 26), and D) individuals with healthy children and no reproductive failure (control group, N = 14).

Chromosome preparations were stained for GTG bands, and 50-100 metaphases were scored per individual. The rearrangements observed were translocations, deletions, duplications, isochromosomes, rings, fragments and markers. Rearrangements were recorded only when identified by two observers, and were described according to the International System for Human Cytogenetic Nomenclature (ISCN, 1995).

RESULTS

In 56 individuals (25, 6, 17 and 8 from groups A-D, respectively) no SCCR were observed. The remaining 34 subjects (21M:13F; 11, eight, nine and six from groups A-D, respectively) had 1-5 SCCR, although four of them presented only PHA-induced rearrangements: two inv(7) (p14q35), one t(7;14)(p13;q11) and one t(7;14)(q36;q11) (Tables I-^IV). There were 44 SCCR unrelated to PHA: 21 deletions, including three in which the deleted segment appeared as either an isoacentric or an acentric fragment, 12 markers, eight balanced translocations, two rings, and one chromosome 22 with additional material on its long arm. Therefore, 36/44 SCCR were unbalanced. The frequency of SCCR (excluding PHA-related rearrangements) was 0.0063, while that of PHA-related rearrangements was 0.0010. Although no mosaicism was detected, two contiguous cells with del(X)(q24) were found in a female control, who had 5/100 cells with SCCR without PHA treatment (^{Table IV}). Statistical analysis (c² with Yates correction) did not show significant differences between the groups, including the comparison of the whole study population (N = 76) versus the control group (P = 0.91); likewise, SCCR occurrence was independent of the individual's sex (P = 0.55).

Table I
- Single cell chromosome rearrangements in individuals with spontaneous abortions, malformed children or molar pregnancies (group A, N = 36).

Constitutional karyotypes	SCCR	Metaphases analyzed
46,XY	46,XY,del(13)(q21)	60
	46,XY,del(2)(q13)
	47,XY,+mar
46,XY	46,XY,del(6)(q24)	50
46,XX	46,XX,del(17)(p11)	72
	47,XX,+mar
46,XX	46,XX,dic(15;22)(p1;p1), ace(15;22)(p1;p1)	100
	47,XX,+mar*
46,XX	46,XX,del(9)(q22)	63
46,XY	46,XY,del(3)(p11)	54
46,XY	46,XY,del(1)(p21)	100
46,XY	46,XY,del(3)(p25)	69
46,XX	47,XX,+mar	100
46,XX	45,X,-X,inv(7)(p14q35)	100
	47,XX,+mar
46,XX	46,del(X)(q22),ace(X) (q22®Xqter)	100

* = Isoacentric fragment of unknown origin.

Constitutional karyotypes SCCR Metaphases analyzed 46,XY 46,XY,del(8)(q11) 53 47,XY,+mar 46,XY 46,XY,inv(7)(p14q35) 53 46,XY 46,XY,del(19)(q13) 65 47,XY,+mar 46,XY 46,XY,del(3)(p14) 66 46,Y, del(X)(q21) 46,XY 46,XY,del(9)(q11) 50 46,XY 46,XY,t(1;2)(p10;q10) 80 46,XY 46,XY,inv(7)(p14q35) 100 46,XY 45,XY,t(1;7)(q12;q36),-21 100

Constitutional karyotypes	SCCR	Metaphases analyzed
46,XY	46,XY,del(8)(q11)	53
	47,XY,+mar
46,XY	46,XY,inv(7)(p14q35)	53
46,XY	46,XY,del(19)(q13)	65
	47,XY,+mar
46,XY	46,XY,del(3)(p14)	66
	46,Y, del(X)(q21)
46,XY	46,XY,del(9)(q11)	50
46,XY	46,XY,t(1;2)(p10;q10)	80
46,XY	46,XY,inv(7)(p14q35)	100
46,XY	45,XY,t(1;7)(q12;q36),-21	100

Table II - Single cell chromosome rearrangements in azoospermic males (group B, N = 14).

Table III - Single cell chromosome rearrangements in individuals with one or more children with either de novo or inherited constitutional chromosome abnormalities (group C, N = 26). Constitutional karyotypes SCCR Metaphases analyzed 46,XY 46,XY,del(21)(q22) 65 46,XX 46,XX,t(7;14)(p13;q11) 73 47,X,del(X)(q11), +iace(X)(q11®qter)* 46,XY 46,XY,t(7;14)(q36;q11) 60 46,XY 46,XY,t(7;22)(p15;q13) 62 46,XY 46,XY,del(4)(q16) 100 46,XY,add(22)(q13) 46,XX 47,XX,del(1)(q11) 100 42,XX,t(1;2)(p11;q21), -6,-7,-12,-18 46,XX,t(2;4)(q37;q35) 46,XX,t(19;20)(p10;q10) 100 46,XX,r(2)(p25q37) 46,XY 46,XY,-22,+mar 100 46,XY,t(7;14)(q34;q11) 46,XX,t(4;20)(q31;q11.2) 46,XX,t(9;19)(q12;p13.3) 70

* = Isoacentric fragment.

Table IV - Single cell chromosome rearrangements in individuals with healthy children and no reproductive failure (group D, N = 14).

Constitutional

karyotypes

SCCR

Metaphases

analyzed

46,XX

45,XX,dic(2;6)(p13;q25)

100

46,XX

46,XX,del(9)(q11)

100

46,XY

46,XY,t(7;14)(p13;q11)

100

46,XY

47,XY,+mar

100

46,XY

46,XY,r(21)(p11q22)

100

46,XX

46,XX,-16,+mar

100

48,X,del(X)(q22), +ace(X)(q22®qter),+mar

46,X,del(X)(q24)

46,X,del(X)(q24)

47,XX,+mar

DISCUSSION

Our data differ from retrospective reports (Iskra, 1985; Higgins and Palmer, 1987) in which a significantly greater number of single cell structural abnormalities had been found in couples with multiple spontaneous abortions than in control individuals referred for other reasons (Higgins and Palmer, 1987) or in couples having children with a normal karyotype (Iskra, 1985). Although this discrepancy may be due to our small sample (N = 36), our study was prospective, with a large number of metaphases analyzed per individual; in addition, our control subjects (N = 14) were parents with healthy children and no reproductive failure. Incidentally, the largest number of SCCR was observed in a female control.

The unbalance implicit in 80% of the SCCR observed in this study, especially the deletions, is likely to be lethal; therefore, these rearrangements would hardly be compatible with gamete or zygote viability (Sonta et al., 1991). Moreover, the potential unbalances for 7/8 translocations are so great that they might also determine inviability.

Higgins and Palmer (1987) suggested that SCCR indicate parental mosaicism. Gardner et al. (1994) argued that an unbalanced rearrangement ascertained in a fetus or child can be secondary to a balanced rearrangement present in parental germ cells and occasionally detected in somatic cells. However, we found no correlation between SCCR observed in the parents and constitutional abnormality of their children (group C).

Only 18 out of 108 breakpoints were repeated in the SCCR found in previous reports (Iskra, 1985; Petersson and Mitelman, 1985; Higgins and Palmer, 1987); likewise, among the 42 non-PHA-related breakpoints found in this study, five occurred on two occasions, and 10 had been documented in the above mentioned reports. Such lack of specificity of breakpoints indicates that these abnormalities were mostly secondary to culture conditions; for example, the two translocations of chromosome 7 with an autosome other than 14 affected bands 7p15 and 7q36, which are implicated in PHA-related rearrangements (Herva, 1981; Dewald et al., 1986).

In this study there were 42 breakpoints and 37 bands involved in 31 SCCR unrelated to PHA (translocations, deletions, and rings). Among these bands, 10 have common fragile sites (Sutherland and Ledbetter, 1989). Therefore, a cause-effect relationship between common fragile sites and SCCR appears unlikely. Moreover, increased occurrence of common fragile sites in couples with spontaneous abortions but without SCCR (Schlegelberger et al., 1989; Turleau et al., 1979) needs to be confirmed. In conclusion, in our sample SCCR occurred independently of reproductive problems or sex.

ACKNOWLEDGMENTS

We thank R. Troyo for statistical advice and R.M. González, A. Mayorquin, H. Gómez and M. Aguilar for technical assistance. We also appreciate the valuable criticisms of the Technical Editor and referees of the Brazilian Journal of Genetics.

RESUMO

Rearranjos cromossômicos de célula única (SCCR) são achados incidentais em culturas de células. Não obstante, alguns autores os têm relacionado com o abortamento habitual. Noventa indivíduos classificados em 4 grupos foram estudados prospectivamente: A) indivíduos com abortamentos espontâneos, filhos malformados ou gravidezes molares (N = 36); B) homens azoospérmicos (N = 14); C) indivíduos com um ou mais filhos com anormalidades cromosômicas de novo ou herdadas (N = 26) e D) indivíduos sem problemas reprodutivos e com filhos saudáveis (grupo controle, N = 14). As preparações de cromossomos de linfócitos foram coradas para bandamento GTG e 50-100 metáfases foram avaliadas por indivíduo. Os rearranjos observados foram translocações, deleções, duplicações, isocromossomos, anéis, fragmentos e marcadores. Trinta e quatro indivíduos (21 homens e 13 mulheres) tiveram de 1 a 5 SCCR. Quatro tiveram rearranjos apenas dos cromossomos 7 e 14. A freqüência de SCCR (com exceção dos rearranjos envolvendo os cromossomos 7 e 14) foi de 0,0063, enquanto que para os rearranjos entre os cromossomos 7 e 14 foi 0,0010. Comparações estatísticas intergrupo (qui-quadrado com correção de Yates) não mostraram diferenças significativas. Assim, a ocorrência de SCCR em nossa amostra mostrou-se independente do modo de averiguação e do sexo.

(Received July 5, 1997)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  05 July 1997