CHROMOSOMAL MOSAICISM IN THE PATIENTS WITH TURNER'S SYNDROME
Vyatkina S1., Nagornaya I1,3., Loginova J1,3., Lyazina L2., Vasileva I2., Prozorova M2., Verlinskaya D2., Kuznetzova T 1 Baranov V.S. 1
*Corresponding Author: Professor Dr. Vladislav S. Baranov, Laboratory of Prenatal Diagnosis of Inherited Disorders, 1The D. O. Ott Institute of Obstetrics & Gynecology, the Russian Academy of Medical Sciences, Mendeleevskaya l., 3, Saint Petersburg, Russia fax +7 (812) 328 04 87; e-mail: svetavyach@mail.ru 2 Saint Petersburg Centre for Medical Genetics, Tobolskaya ul., 5, Saint Petersburg, Russia fax +7 (812) 542 67 76, e-mail: gkdmgenc@zdrav.spb.ru 3Russian-Finnish Medical Centre "AVA-Peter", Nevskii pr., 22/24, Saint Petersburg, Russia fax: +7 (812) 314 51 19, e-mail: avapeter@bcltele.com
page: 17

RESULTS AND DISCUSSION

 

Karyotyping of PHA-stimulated   lymphocytes   has  proved  45,X karyotype  in 41 of total 88 patients studied,  karyotype 46,XX - in 1 case and  46,X,der (X) - in 4 cases. Mosaic karyotypes with admixture of  45 cell lines with normal or abnormal ?-, Y- or marker chromosomes were revealed in 42 cases. FISH analysis with X-specific DNA-probes proved the existance of additional cell lines with aberrant X-chromosomes in 5 out of  41 patients with 45,X.  PCR analysis of DNA samples from 34 patients with monosomy X revealed the presence of Y specific DNA sequences in 2  cases.
Taking into account that the rate of chromosomal mosaicism depends on the  number of the scored cells, on the  methods applied  (Hook, 1977; Binder et al., 1995), as well as on the type of tissues we tried to study  the cells  of different germ-line origin.
The  combined  FISH analysis of   lymphocytes and buccal mucosal cells was achieved in 69 cases. Karyotype 45,X  was ascertained in  29 cases, karyotype 46, X,der(X)  in  both  tissues studied was recorded in one case. In 4 patients (10%) mosaic karyotypes (45,X/46X,der(X)) has been proven        in peripheral blood lymphocytes in one case and  in   buccal mucosa cells in 3 cases (tab. 1, ? 39 and ?? 1, 11, 22 accordingly).  Chromosomal mosaicism in both tissues was revealed in 35 from 39 patients (90 %).
Obvious quantitative differences of mosaicism in  cell lines of different tissues  have been  detected in overwhelming majority of mosaics (tab. 1). The preponderence  of one cell line  in two tissues was found in 28 out of 35 cases (80%) studied.  In 7 patients the cell clone, minor in one tissue, was  dominant in  other  tissue (?? 4, 6, 9, 10, 20, 28, 33, tab. 1). The portion of cells with aberrant X-chromosome in "cryptic" mosaics was 5-8 % in blood and 12-22 % - in buccal mucosa (?? 1, 15, 17, 23, 25, tab. 1).
 Cryptic mosaicism involving Y-chromosome  was found in 6 % and  X-chromosome – in 15% patients with a pure  X monosomy detected by standard cytogenetic analysis. Karyotype 45, X in both tissues  has been assessed in the majority  of patients.  According to availble data   the frequency of "cryptic" mosaicism varies from 0 % to 61 % for Y-chromosome (Lorda-Sanchez et al., 1992; Coto et al., 1995; Yorifuji et al., 1997) and from 2,4 % to 90 % for X-chromosome (Yorifuji et al., 1997; Nazarenko et al., 1999; Fernandez-Garcia et al., 2000).
Substantial fluctuation in the rate of mosaicism could be attributed to the  sample’s volume   as well as  to the  sensitivity of the techniques applied. Owing to  its high sensitivity and specificity, PCR  technique  readily  reveals   chromosomal mosaicism attributed  to extra genetic material  but it does not permit estimation of a cell line ratio. The quantitative characteristic of cell lines in mosaics might be achieved by means of  FISH analysis of interphase nuclei. Meanwhile spontaneous aneuloidy and hybridization efficiency might  substantially  influence   the results.   5% cut-off value for mosaicism both  in peripheral blood, and in buccal mucosa was chosen and this value roughly corresponded to the  background  level of spontaneous sex chromosomes aneuploidy   (Eastmond, Pinkel, 1991). However, quantitative variations of X-chromosome aneuploidy in different tissues can vary (2 %-4,04 % for non-cultivated and cultivated lymphocytes respectively and 1,79 % for native buccal mucosal cells) (Nazarenko, etc., 1997; Jacobs et al., 1997). It should be menthioned  that the number of marker loci detected by specific DNA probes might remain constant in spite of structural chromosomal abnormalities and the relevent cell lines with abnormal karyotype could not be detected by  FISH analysis. Thus the results of FISH analysis in interphase nuclei could not  precisely correspond  to metaphase studies.

Table 1. Molecular-cytogenetic characteristic of mosaic karyotypes

#

Lab. index

  blood lymphocytes (metaphase plate n=100)

  blood lymphocytes (interphase nucleus n=500)

 buccal mucosal cells
(interphase nucleus n=500)

1

760

45,?

DXZ1?1[482], DXZ1?2[18]

DXZ1?1[438], DXZ1?2[61]

2

93

45,?[88]/47,???[5]/46,??[7]

DXZ1?1[409], DXZ1?3[44], DXZ1?2[47]

DXZ1?1[375], DXZ1?3[44], DXZ1?2[81]

3

15

45,?[91]/46,??[9]

DXZ1?1[471], DXZ1?2[29]

DXZ1?1[427], DXZ1?2[73]

4

319

45,?[40]/46,??[60]

DXZ1?1[248], DXZ1?2[252]

DXZ1?1[376], DXZ1?2[124]

5

419

45,X[71]/46,XX[29]

DXZ1?1[328], DXZ1?2[172]

DXZ1?1[349], DXZ1?2[151]

6

632

45,X[41]/46,XX[59]

DXZ1?1[222], DXZ1?2[278]

DXZ1?1[266], DXZ1?2[234]

7

422

45,X[88]/46,XX[12]

DXZ1?1[449], DXZ1?2[51]

DXZ1?1[413], DXZ1?2[87]

8

1161

45,X[8]/46,XX[92]

DXZ1?1[33], DXZ1?2[467]

DXZ1?1[40], DXZ1?2[460]

9

1323

45,X[61]/46,XX[39]

DXZ1?1[321], DXZ1?2[179]

DXZ1?1[115], DXZ1?2[385]

10

1062

45,?[89]/47,???[6]/46,??[5]

DXZ1?1[426], DXZ1?3[42], DXZ1?2[32]

DXZ1?3[443], DXZ1?1[23], DXZ1?2[34]

11

874

46,??

DXZ1?2

DXZ1?1[72], DXZ1?2[428]

12

43

45,?[85]/46,?,r(?)[15]

DXZ1?1[442], DXZ1?2[58]

DXZ1?1[400], DXZ1?2[100]

13

28

46,?,r(?)[67]/45,?[33]

DXZ1?2[369], DXZ1?1[131]

DXZ1?2[363], DXZ1?1[137]

14

4

46,?,r(?)[64]/45,?[36]

DXZ1?2[344], DXZ1?1[156]

DXZ1?1[362], DXZ1?2[138]

15

82

45,X[92]/46,X,r(X)[8]

DXZ1?1[471], DXZ1?2[29]

DXZ1?1[395], DXZ1?2[105]

16

107

45,X[87]/46,X,r(X)[13]

DXZ1?1[411], DXZ1?2[89]

DXZ1?2[267], DXZ1?1[233]

17

194

45,X[95]/46,X,r(X)[5]

DXZ1?1[474], DXZ1?2[26]

DXZ1?1[414], DXZ1?2[86]

18

1529

45,X[60]/46,X,r(X)[35]/47,X,r(X), +r(X)[5]

DXZ1?1[321], DXZ1?2[154], DXZ1?3[25]

DXZ1?2[304], DXZ1?1[196]

19

33

45,?[80]/46,?,r(?)[10]/46,?,idic(X)(q22)[5]/47,?,r(?),+r(?)[5]

DXZ1?1[390], DXZ1?2[48], DXZ1?3[62]

DXZ1?1[428], DXZ1?2[72]

20

680

46,X,der(X)[51]/46,X,r(X)[29]/45,X [15]/46,X,dicr(X)[5]

DXZ1?2[405], DXZ1?1[69], DXZ1?3[26]

DXZ1?1[285], DXZ1?2[189], DXZ1?3[26]

21

220

45,?[81]/46,X,i(X)(q10)[19]

DXZ1?1[401], DXZ1?2[99]

DXZ1?1[405], DXZ1?2[95]

22

914

46,X,i(X)(q10)

DXZ1?2[490], DXZ1?1[10],

DXZ1?2[473], DXZ1?1[27],

23

85

45,X[92]/46,X,idic(X)(p11)[8]

DXZ1?1[466], DXZ1?3[34]

(DXZ1?1[417], DXZ1?3[83]

24

901

45,X[67]/46,X,idic(X)(p11)[33]

DXZ1?1[335], DXZ1?3[165]

DXZ1?1[337], DXZ1?3[163]

25

558

45,?[95]/46,X,idic(X)(q22)[5]

DXZ1?1[469], DXZ1?3[31]

DXZ1?1[392], DXZ1?3[108]

26

581

45,X[70]/46,X,dup(X)(p21p22)[25]/46,XX[5]

  • DXZ1?1[362], DXZ1?2[138]
  • DXZ1?1[448], DXZ1?2[52]

27

151

45,X[79]/46,X,der(X)[21]

DXZ1?1[403], DXZ1?2[97]

DXZ1?1[326], DXZ1?2[174]

28

313

46,X,der(X)[62]/45,?[33]/46,X, dicder(X)[5]

DXZ1?2[318], DXZ1?1[154], DXZ1?3[28]

DXZ1?1[467], DXZ1?2[33]

29

1442

46,X,der(X)[53]/45,?[42]/47,X,der (X),+der(X)[5]

DXZ1?2[269], DXZ1?1[204], DXZ1?3[27]

DXZ1?2[295], DXZ1?1[205]

30

166

46,X,del(X)(qter-p11.2:)[95]/45,?[5]

DXZ1?2[469], DXZ1?1[31]

DXZ1?2[430], DXZ1?1[70]

31

880

45,X[54]/46,X,del(X)(qter-p11.2:) [46]

DXZ1?1[253], DXZ1?2[247]

DXZ1?1[374], DXZ1?2[126]

32

156

46,?,del(X)(pter-q23:)[77]/45,X[23]

DXZ1?2[391], DXZ1?1[109]

DXZ1?2[434], DXZ1?1[66]

33

1314

45,?[57]/46,?Y[43]

DYZ3?0[290], DYZ3?1[210]

DYZ3?1[282], DYZ3?0[218]

34

748

47,XYY[28]/45,X[14]/46,XY[58]

DYZ3?2[144], DYZ3?0[63], DYZ3?1[290]

DYZ3?2[98], DYZ3?0[183], DYZ3?3[25], DYZ3?1[194]

35

654

46,?,dic(Y)[86]/45,?[14]

DYZ3?2[441], DYZ3?0[59]

DYZ3?2[303], DYZ3?0[197]

36

19

46,?,r(Y)[7]/45,?[6]/46,?,dicr(Y) [5]/46,?Y[82]

DYZ3?1[442], DYZ3?0[27], DYZ3?0[31]

DYZ3?1[328], DYZ3?0[172]

37

1065

46,X,dicder(Y)[66]/45,X[34]

DYZ3?2[333], DYZ3?0 [167]

DYZ3?2[305], DYZ3?0 [173], DYZ3?4[22]

38

1445

45,X[70]/46,X,dicder(Y)[30]

DYZ3?0[362], DYZ3?2[138]

DYZ3?0[402], DYZ3?2[98]

39

1511

45,X[73]/46,X,der(Y)[27]

DYZ3?0[371], DYZ3?1[129]

DYZ3?0

Meanwhile,    limited tissue mosaicism (tab. 2)  revealed by different methods could be proved in only about 20 % of cases. Therefore, the hypothesis that  the patients possessing  45,X karyotype in their lymphocytes,  should have  additional cell lines with normal or aberrant sex chromosomes  (Hook, Warburton, 1983) is fair only for a part of patients.  True chromosomal mosaicism  in all tissues, irrespective of their germinal origin  is typical for the most  of TS cases. Domination of the same cell line  in  the tissues of   different embryonic origin is encountered  in more than 70 % cases

Table 2. Cell lines distribution   in different tissues  of Turners syndrome patients  .

Authors

Number of cases

Analyzed
tissue

Research method

Character of mosaicism

Character of cell line domination

True

limited

In two tissues

In one tissue

Mashkova M.V., 1974

3

lymphocytes, skin fibroblasts

cytogenetic

3

0

2

1

Held et al., 1992

56

lymphocytes,  skin fibroblasts

cytogenetic

32

24

24

8

Reddy, Sulcova, 1998

5

lymphocytes, ovarian tissue

fish

5

0

4

1

Nazarenko et al.,1999

13

lymphocytes, buccal mucosal cells

fish

9

4

2

7

Hanson et al., 2001

35

lymphocytes, buccal mucosal cells

fish

32

3

-

-

Hanson et al., 2002

11

lymphocytes, buccal mucosal cells

fish

11

0

8

3

Fernandez et al., 2000

2

lymphocytes, ovarian tissue

fish

2

0

-

-

Osipova et al., 1998

2

leukocytes, ovarian tissue

PCR

1

1

-

-

Mendes et al., 1999

2

leukocytes, ovarian tissue

PCR

2

0

-

-

Own data

39

lymphocytes,
buccal mucosal cells

fish

35

4

28

 

7

summary

168

132 (79%)

36 (21%)

68 (72%)

27 (28%)

Presence of Y-chromosome derivatives might be crucial for the health prognosis  of X0 patients because of high risk of malignant gonadoblastoma development. Our results substantiate  the following  algorithm  of mosaicism detection in   patients with clinical features  of TS (fig. 1). Molecular detection of Y chromosomes or its fragments with DNA probes for PCR reaction in  DNA samples from peripheric blood  should be taken as the 1st step. In the absense of Y-chromosome issue in blood,   DNA analysis of the  buccal mucosa cells might be suggested at the 2nd  step. Positive results of DNA tests substantiate further thorough  FISH analysis with Y-specific DNA-probes in lymphocytes and buccal mucosa cells for more precise determination of der(Y) structure at the 3rd step. However, not all patients with Y-chromosome  derivatives reveal any masculinization traits.   FISH-analysis of blood lymphocytes and  buccal cells with X and  Y-chromosome specific DNA probes  should be  recommended for  all  the rests of  XO patients .

Figure.1. Algorythm  of laboratory genetic  tests in  TS patients.




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