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Discussion & Conclusion



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Detection and Incidence of Cryptic Mosaicism in 41 Turner Syndrome Patients

Contact Person: Eduardo Pásaro & Rosa Fernández (pspasaro@mail.udc.es ; rossa@mail.udc.es)


In about 99% of the cases of 45,X human conception, a natural miscarriage occurs in the first stages of embryonic development (Hook and Warburton, 1983; Hassold et al., 1988). Only 1% of these fertilizations is taken successfully to term, and generally displays the characteristics of Turner's syndrome (Turner, 1938).

Both, embryonic mortality and the Turner's phenotype, are considered to be a result of monosomy of a common gene(s) of the X and Y chromosomes. For women, it is assumed that these genes are expressed in both active and inactive X chromosomes as a means of ensuring the right quantity of genetic product. It is believed that one or more of these genes is responsible for Turner's syndrome (TS) (Fisher et al., 1990).

The high percentage of foetal and embryonic miscarriage for karyotype 45,X suggests the necessity of mosaicism for survival (Held et al., 1991;1992). Natural selection does not always prevail when mosaicism is operative (Hook and Warburton, 1983; Hassold et al., 1988), although the resulting phenotype is similar. Current hypotheses argue for the existence of a feto-protective effect (Held et al., 1992) of one or more genes of the sexual chromosomes (X or Y). According to this concept, two copies of these gene(s) should be presented, either in the fetus or in extra-embryonic tissues (Kalousek et al., 1987).

The detection of mosaicism is mainly influenced by four factors: the type and number of tissues analyzes; the number of cells studied (Hook, 1977); the sensitivity of the techniques applied; and the possible selection against any of the cell lines (Procter et al., 1984; Held et al., 1992). Low percentage of mosaicism cannot be detected by conventional cytogenetic techniques because of the great number of cells that should be analyzed. The application of molecular techniques, such as FISH (Fluorescent in situ hybridization) and PCR, substantially increase the detection of low-frequency cell lines and possible structural alterations.

This poster describes a molecular investigation of 41 TS patients The application of FISH and PCR techniques has helped reevaluated 37 patients and highlight the differences between the initial diagnoses achieved by Cytogenetics and that following by application of FISH and PCR.

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Materials and Methods

Human blood lymphocytes were obtained from 41 patients with TS. Chromosome preparations were carried out according to standard techniques (Moorhead et al., 1960).

Fluorescence in situ Hybridization in blood and paraffin-embedded gonadal tissue

Analysis of the X-Y chromosomes was made by fluorescence in situ hybridization on mitotic preparations with a classical alpha-satellite chromosome-X specific probe (CEP-X, Vysis) and with CEP-Y (Vysis) probe that hybridizes to the satellite III sequence of human chromosome Y (band Yq12, locus DYZ1). We also used WCP-X and WCP-Y painting probes to identifying chromosomal structural rearrangements. The nature of the material involved in the restructuring was determined with two subchromosomal painting libraries (SCPL), viz., one (SCPL116) covering the short arm of the X chromosome, the other (SCPL102) covering the long arm of the X chromosome and the entire chromosome 16. Probes SCPL116 and SCPL102 (400ng/slide) were labeled by nick translation with biotin-16-dUTP according to the Boehringer Mannheim protocol. Hybridization, post-hybridization washes, and signal detection were performed according to Archidiacono et al., (1994).

Paraffin-embedded tissue blocks of the streak ovary were sectioned. Four-micron sections were taken and placed on slides treated with 3-aminopropyltriethoxylane (Sigma). FISH using DYZ1/DYZ3 (Oncor) probe was performed following the manufacturer's instructions (Oncor) with small variations. The paraffin was dissolved in xylene, the slides were rehydrated through graded alcohols and the sections were digested with proteinase K (1mg/ml for 30 min at 37°C). The protease digestion was stopped with glycine, and sections were then dehydrated. Denatured slides were hybridized for 16 h at 37°C in 65% formamide/ 2xSSC, 10% dextran sulfate and washed at 43°C in 50% formamide/2xSSC followed by a 2xSSC wash at 37°C. Hybridization was detected with fluorescein conjugated avidin and counter stained with propidium iodide.

PCR amplifications

DNA extraction from peripheral blood of our patient and controls was carried out using standard procedures (Sambrook et al. 1989). XES-7 and XES-2 oligonucleotide primers were used to amplify a fragment of 609-bp (Berta et al. 1990) and located within the SRY open reading frame.

Cloning and DNA sequencing of the PCR products

The PCR product was ligated into the plasmid pGEM-T (Promega) and subsequently transformed into the E. coli JM109 under the conditions recommended by the manufacturer. Recombinant clones were selected as white colonies on ampicillin plates containing X-gal and IPTG.

Plasmidic DNA was obtained following the alkaline lysis method described by Sambrook et al. (1989) and used as template for sequencing reactions. Nucleotide sequencing of both strands of PCR product was done with the same primers used for PCR and the ALFexpress AutoRead Sequencing kit (Pharmacia) on the automatic laser fluorescence DNA sequencer (ALFexpress, Pharmacia).

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The application of FISH and PCR techniques allowed us to find a second cellular line in 37 out of 41 patients, about a 90%; only four patients (10%) were defined as non- mosaics. The most frequent mosaic was 45,X/46,XX (32%); the presence of isochromosomes comprised 27% and fragments 10%.The increase of the number of patients with mosaicism was due mainly to the appearance of the cell line 46,XX. (see Tabl.1).

The PCR study with primers XES-7 and XES-2, confirmed the existence of the SRY gene in patients 14 and 28. The sequencing data od the SRY gene in patient 14 showed a single mutation C-T in the SRY open reading frame that causes a conservative change from Arg (CGA) to stop codon (TGA) within the conserved binding motif of the SRY gene.

Table 1. Karyotypes of the patients studied by cytogenetic and FISH techniques.

Patients    Previous diagnoses          Final Diagnoses
   1        45,X/46,XX               45,X/46,XX (3.6%/96.4%)
   2        45,X                     45,X/46,XX (98.7%/1.3%)
   3        45,X/47,XXX              45,X/47,XXX (82.5%/17.5%)
   4        45,X                     45,X/46,XX (99.6%/0.4%)
   5        45,X                     45,X/46,XX (99.7%/0.3%)
   6        45,X                     45,X/46,XX (99.8%/0.2%)
   7        45,X                     45,X (100%)
   8        45,X                     45,X/46,XX (99.2%/0.8%)
   9        45,X/47,XXX/47,XX, del(Xp-)
                                     45,X/47,XXX/47,XX del(Xp-)(41.4%/35.5%/23.1%)
   10        45,X/46,XX              45,X/46,XX/46,X,psu dic(X)(Xp-Xq21::Xq21-Xpter)(94.7%/0.7%/4.6%)
   11        45,X                    45,X/46,X +frag(X) (99%/1%)
   12        45,X/46,X,i(Xq)         45,X/46,X,i(Xq) (81%/19%)
   13        45,X/46,XX              45,X/46,XX  (65%/35%)
   14        45,X/46,X,del(X)(q12)   45,X/46,X,idic(Ynf)(59%/41%)
   15        45,X                    45,X/46,XX  (99%/1%)
   16        45,X                    45,X (100%)
   17        45,X/46,X,del(Xq?)      45,X/46,XX/46,X +frag(X)(83.5%/0.8%/15.7%)
   18        45,X/46,XX              45,X/46,XX (5%/95%)
   19        45,X/46,X,del(X)(q11)   45,X/46,X,del(X)(q13) (25%/75%)
   20        45,X/46,X,i(Xq)/47,X,i(Xq),i(Xq)
   21         45,X/47,XXX            45,X/46,XX/47,XXX (87%/6%/7%)
   22         46,X,i(Xq)             45,X/46,X,idic(Xq)/47,X,idic(Xq),del(X)(p?)/47,X,idic(Xq),idic(Xq)(14.4%/60.6%/1.3%/23.7%)
   23         45,X                   45,X/46,XX/46,X,idic(Xq) (91%/7%/2%)
   24         45,X/46,X,i(Xq)        45,X/46,X,idic(Xq)/47,X,idic(Xq),idic(Xq)(29.6%/69.9%/0.5%) 
   25         45,X/46,X +mar(?)      45,X/46,X +frag(X) (30%/70%)
   26         45,X                   45,X (100%)
   27         45,X                   45,X/46,XX (99,7%/0,3%)
   28         45,X/46,XY             45,X/46,XY (96.5%/3.5%)
   29         45,X/46,X,idic(Xq)     45,X/46,X,idic(Xq) (83%/17%)
   30         45,X                   45,X/46,XX (94%/6%)
   31         45,X/46,XX             45,X/46,XX (85%/15%)
   32         46,X,del (X)(q13)      45,X/46,X,del(X)(q13)/47,X,del(X)(q13),del(X)(q13)(4.5%/93.8%/1.6%)
   33         45,X                   45,X/46,X,+r(X) (82%/18%)
   34         45,X/46,X,i(Xq)        45,X/46,X,i(Xq) (80%/20%)
   35         46,X, del(Xp)          45,X/46,X,del(Xpter-Xcen)(34%/66%)
   36         45,X/47,XXX            45,X/47,XXX (94%/6%)
   37         46,X,del(Xp)           46,X,del(X)(Xpter-Xp11)(100%)
   38         45,X/46,X,i(Xq)/47,X,i(Xq),i(Xq)
   39	Turner's syndrome (??)         45,X/46,XX (99.6%/0.4%)
   40         45,X                   45,X (100%)
   41         45,X/46,XX             45,X/46,X,psu dic(X)(Xp-Xq21::Xq21-Xpter) (73.3%/26.6%)

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Discussion and Conclusion

The application of FISH and PCR techniques has helped reevaluated 41 TS patients and highlight the differences between the initial diagnoses achieved by Cytogenetics and that following by application of specific probes. Initially, 15 TS patients were defined as non-mosaics. As a result of FISH study this number was reduced to four patients. In these cases the analysis of a higher number of metaphases would be need for the detection of the second cell line. Using Hook tables (1977) of mosaicism exclusion, we could exclude up to 2% of mosaicism.

For patient group mentioned above, the percentage of the second cell line (46,XX) fluctuated between 1% and 2% (Tab.1). The detection a such low mosaicism by cytogenetics techniques would be an arduous task because it should be necessary analyzed a lot of metaphases. The use of specific probes by FISH facilitates this work substantially.

The presence of a pseudodicentric X chromosome (psu dic(X)(Xp-Xq21::Xq21-Xpter)for the short arm in patients 10 and 41, is another example of the alterations detected by FISH which had been unnoticed by G-banding. The application of FISH with SCPL116 and SCPL102 probes, showed that the derivated X chromosome in these patients were a pseudo-dicentric isochromosome containing two copies of the short arm of the X chromosome, two centromeres and two copies of the very proximal region of the long arm and the inactivation center. One of the two centromeres was nonconstricted and therefore inactive.

The complex mosaicism in other patients is characterized by isodicentric X chromosomes idic(Xq). These chromosomes correspond to dicentric isochromosomes constituted by two copies of the q arm and two centromeres located very close to each other. Using G-banding techniques these chromosomes have been previously defined as monocentric isochromosomes. Due to the great instability of dicentric chromosomes, an accumulation of idic(Xq) chromosomes is a result of mitotic nondisjunction. The same principle of such instability gives rise to ruptures that produce dicentric chromosomes lacking one of their arms. In these patients, the dicentric fragments present variation of length and structure and a tendency to form rings that also vary in size.

The study of the nature of the fragments derived from Y chromosome in females with gonadal dysgenesis is important since the risk of developing gonadal cancer is increased by 30% (Wolman et al., 1985; Page,1994). In the group of TS patients only two had a chromosome derived from Y (patients 14 and 28). Patient 14 had been initially diagnosed as mosaic form 45,X/46,X +der(?) using conventional cytogenetic techniques (G-banding). However, FISH and PCR analyses allowed us to redefine the der(?) chromosome as a pseudodicentric nonfluorescent Y-chromosome psu dic(Y)(pter-q11.2::q11.2-pter). This dicentric chromosome undergoes a complex restructuring characterized by two copies of the short arm, two centromeres and two copies of the region most proximal to the long arm. The heterochromatic region that fluoresces brightly with 4', 6-diamino-2-phenylindole (DAPI) was not observed.

A high level of mosaicism for the psu dic(Y) cell line (41%) was observed in peripheral blood lymphocytes. The dicentric nature of the Y-chromosome became apparent in FISH studies in the paraffin-embedded streak ovary sections and in blood. Molecular analysis of the SRY open reading frame using PCR with XES-7, XES-2 primers (Berta et al. 1990) confirmed the presence of the SRY gene in patient 14. These amplified DNAs were cloned for DNA sequencing.

DNA sequencing for the PCR clones revealed an apparent single base change in our TS patient's SRY gene. It was detected as a mutation C to T in the open reading frame at position 594 that causes a conservative change from CGA (Arg) to TGA (Stop codon) at codon 198.

Reddy et al. (1996) reports that unequal distribution of the two cell lines (45,X and 46,X+ der(Y)) in various tissues causes different phenotypes. The 45,X cell line is apparently, in some cases, more influential in sex determination and sexual development than is the presence of the Y chromosome material (Hsu 1994). Clinically, most patients with dicentric Y-chromosomes present short stature or various disorders of sexual development. The great majority of reported patients are chromosomal mosaics with two or more cell lines, usually including a cell line lacking the dicentric Y. The literature suggests that sexual development in such patients depends on two factors: the structure of the dicentric Y chromosome and the level and distribution of mosaicism present (Cathy et al. 1995).

In conclusion, on the bases on the FISH and PCR data obtained, we could detect:

(1) an increase of the number of mosaic individuals.

(2) a higher complexity of mosaicism due to the appearance of new cellular lines.

(3) alterations at a centromeric level.

(4) the origin of chromosomic fragment.

(5) a new mutation in the SRY open reading frame.

(6) our data of mosaicism support the hypothesis of "the necessity of mosaicism for survival", and thus, a mitotic origin of this syndrome.

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  1. Berta, P et al (1990) Genetic evidence equating SRY and the testis-determining factor. Nature,348:448-450
  2. Cathy, M et al (1995) Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature. Hum Genet, 96:119-129
  3. Fisher, EMC et al (1990) Homologous ribosomal protein genes on the human X and Y chromosome: escape from X inactivation and possible implications for Turner syndrome. Cell, 61:1205-1218
  4. Hassold, TJ et al (1988) Cytogenetic and molecular analysis of sex-chromosome monosomy. Am J Hum Genet, 42:534-541
  5. Held, KR et al (1991) Hypothesis: 45,X Turner Syndrome does not exist. All surviving patients have sex-chromosomal mosaicism. Elsevier Science Publishers B.V. (Biomedical Division). Turner Syndrome: Growth promoting therapies. M.B. Ranke, R.G. Rosenfeld (eds). pp 3-8
  6. Held, KR et al (1992) Mosaicism in 45,X Turner syndrome: does survival in early pregnancy depend on the presence of two sex chromosomes?. Hum Genet, 88:288-294
  7. Hook, EB (1977) Exclusion of chromosome mosaicism: tables of 90%, 95%, and 99% confidence limits and comments on use. Am J Hum Genet, 29:94-97
  8. Hook, EB; Warburton, D (1983) The distribution of chromosomal genotypes associated with Turner's syndrome: livebirth prevalence and evidence for dismissed fetal mortality and severity in genotypes associated with structural X abnormalities or mosaicism. Hum Genet, 64:24-27
  9. Hsu, LYF (1994) Phenotype/karyotype correlations of the Y chromosome aneuploidy with emphasis on structural aberrations in postnatally diagnosed cases. Am J Med Genet, 53:108-140
  10. Kalousek, DK et al (1987) Confined chromosomal mosaicism in prenatal diagnosis. Hum Genet, 77:163-167
  11. Moorhead, PS et al (1960) Chromosome preparations of leucocytes from human peripheral blood. Exp Cell Res, 20:613
  12. Page, DC (1994) Y chromosome sequences in Turner's syndrome and risk of gonadoblastoma or virilization. The Lancet, 343:240
  13. Procter, SE et al (1984) Problems of detecting mosaicism in skin. A case of trisomy 8 mosaicism illustrating the advantages of in situ tissue culture. Clin Genet, 25:273-277
  14. Reddy, KS et al (1996) An infant with a mosaic 45,X/46,X, psu dic(Y)(pter-q11.2::q11.2-pter) karyotype and mixed gonadal dysgenesis studied for extent of mosaicism in the gonads. Am J Med Genet, 66:441-444
  15. Sambrook, J et al (1989) Molecular Cloning. 2nd ed. New York: Cold Spring Harbor Laboratory Press.
  16. Turner, HH (1938) A syndrome of infantilism, congenital webbed neck, and cubitus valgus. Endocrinology, 23:566
  17. Wolman, SR et al (1985) The Y chromosome in female phenotype. In: Sandberg AA (ed) The Y chromosome, part A: Basic characteristics of the Y chromosome. Liss, New York, pp 79-505

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Fernández, R.; Costoya, S.; García, S.; Pásaro, E.; (1998). Detection and Incidence of Cryptic Mosaicism in 41 Turner Syndrome Patients. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/medicine/fernandez0514/index.html
© 1998 Author(s) Hold Copyright