Cytogenetic analysis was performed with GTG-banding. Cytogenetic analysis analyses from amniotic fluid cells and parents’ peripheral blood lymphocyte cultures were prepared by routine methods. For each case at least 25 metaphases were evaluated. Structural anomalies were recorded according to the International System for Human Cytogenetic Nomenclature. Fluorescence in situ hybridization (FISH) was performed on cultured amniotic fluid cells to confirm the presence of a Ybearing cell line and determine its distribution in the fetal tissue examined. The cases were analysed by also interphase FISH technique using dual prob (Vysis, Ullinois, USA) to exhibit exist Y chromosome. Hybridization was performed in according to the manufacturer’s instructions. Dual-labeling hybridization was performed using 10Vl of the hybridization mixture containing fluorescein direct labeled chromosome Y alpha-satellite probe and rhodamine direct-labelled SRY gene probe. Molecular analysis of sex-reversed patients led to the discovery of the SRY gene (sex-determining region on Y). We performed molecular genetic analysis for Y chromosomal loci (SRY, ZFY, SY84, SY86, SY127, SY134, SY254, SY255) blood leukocytes with Y Chromosoma Deletion Kit (Dr.Zeydanlı Life Science, Ankara, Turkey).
Genomic DNA was extracted from peripheral leukocytes collected from a venous blood sample. Genomic DNA was extracted by standard methods from peripheral leukocytes of all patients3. The DNA was amplified for 30 cycles with denaturation at 94ºC for 5 min, at 94ºC for 1 min, at 54ºC 1 min, at 72ºC for 2 min and extension at 72ºC for 6 min using a PTC-100 thermal cycler (MJ Research). The PCR products were separated by electrophoresis on 2 per cent agarose gel containing ethidium bromide and photographed using Gel Doc system (Hero Lab, Germany). Product was obtained 947 bp for Y. Health male and female samples were used as positive and negative control.
A pericentric inversion of chromosome Y was detected in an unborn baby by a second-trimester amniocentesis for prenatal diagnosis because of advanced maternal age. Cytogenetic investigations of the fetal cells revealed a male karyotype with a pericentric inversion of the Y chromosome. Cytogenetic analysis from amniotic fluid cells showed 46,X,+mar (Y) inverted Y (p11.2;q11.23)? (Figure 1). Chromosome analysis of the proband’s father and other elder brother was also undertaken in order to decide whether this inversion was inherited or of de novo origin. The identical pericentric inversion was found in the phenotypically normal proband's father and a elder brother. In this case of familial pericentric inversion, the parents were assured that their unborn baby was anticipated to be normal.
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Figure 1: Pericentric inversion of the Y chromosome in this family. Left, diagram showing a normal Y (Y) from a healthy individual and the common inv(Y)(p11.2;q11.23) chromosome [inv(Y)] of the male family members. Right, high resolution G banding of a normal Y (N) and the Y chromosome of case (P). |
The existence of Y chromosome was determined by interphase FISH besides a number of metaphase analysis. It was detected SRY gene and centromeric signals in patients with 46,X,+mar(Y), inverted Y(p11.2; q11.23) karyotype (Figure 2). The formation of testes can be considered as existence of SRY (sex-determining region of Y) as a testisdetermining factor. The sequence tagged sites (STS) primers SY14, ZFY, sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc) were used for each case. In all cases the SRY gene was present. No mutations were identified in this gene in any of the patients (Figure 3).
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Figure 3: Gel photograph showing Y microdeletion. Lane 1, molecular weight marker; lane 2 and 3 negative control (female and male samples), lane 4,5,6,7,8,9,10 and 11: SY14, sY84, ZFY, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc). |
The amplified loci by PCR in each patient are indicated in the Table 1. The present report illustrates the importance of FISH and molecular techniques as a complement to cytogenetic methods for accurate identification and characterization of chromosome rearrangements in prenatal diagnosis.
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