Detection of Y chromosome sequences in a 45,X/46,XXq– patient by Southern blot analysis of PCR‐amplified DNA and fluorescent in situ hybridization (FISH)

Abstract

In some cases of gonadal dysgenesis, cytogenetic analysis seems to be discordant with the phenotype of the patients. We have applied techniques such as Southern blot analysis and fluorescent in situ hybridization (FISH) to resolve the phenotype/genotype discrepancy in a patient with ambiguous genitalia in whom the peripheral blood karyotype was 45,X. Gonadectomy at age 7 months showed the gonadal tissue to be prepubertal testis on the left side and a streak gonad on the right. The karyotype obtained from the left gonad was 45,X/46,XXq- and that from the right gonad was 45,X. Three different techniques, PCR amplification, FISH, and chromosome painting for X and Y chromosomes, confirmed the presence of Y chromosome sequences. Five different tissues were evaluated. The highest percentage of Y chromosome positive cells were detected in the left gonad, followed by the peripheral blood lymphocytes, skin fibroblasts, and buccal mucosa. No Y chromosomal material could be identified in the right gonad. Since the Xq- chromosome is present in the left gonad (testis), it is likely that the Xq- contains Y chromosomal material. Sophisticated analysis in this patient showed that she has at least 2 cell lines, one of which contains Y chromosomal material. These techniques elucidated the molecular basis of the genital ambiguity for this patient. When Y chromosome sequences are present in patients with Ullrich-Turner syndrome or gonadal dysgenesis, the risk for gonadal malignancy is significantly increased. Hence, molecular diagnostic methods to ascertain for the presence of Y chromosome sequences may expedite the evaluation of patients with ambiguous genitalia.

PCR Protocol to Detect Parental Origin and Hidden Mosaicism inSex Chromosome Aneuploidies

“…Five X polymorphic markers were amplified by PCR to determine the parental origin of the single sex chromosome: DMD49 [14], DYS II [15], DXS1283E [16], AR [17]and DXS52 [18]. To detect possible Y hidden mosaicisms, SRY [19], DYZ3 and DYZ1 [20]markers were also amplified by PCR. Internal controls were simultaneously amplified with SRY (DYS II located in Xp), DYZ1 and DYZ3 (IR5, a fragment of 275 bp located in chromosome 19) [21].…”

Identification of Y chromatin directly in gonadal tissue by fluorescence in situ hybridization (FISH): Significance for Ullrich-Turner syndrome screening in the cytogenetics laboratory

“…Most marker chromosomes remained unidentified until the advent of fluorescence in situ hybridization (FISH). Molecular analysis has revealed both X-derived and Y-derived markers [Kocova et al, 1995;Schwartz et al, 1997].…”

“…Cytogenetic analysis may fail to detect rare cells bearing a normal or structurally abnormal Y chromosome due to low-level mosaicism or particular tissue distributions [Ostrer and Clayton, 1989;Bisat et al, 1993;Kocova et al, 1995]. Detection of mosaicism depends on several factors, including 1) the number of cells examined [Hook, 1977], 2) the number and type of tissues examined, 3) culture techniques used, and 4) in vivo or in vitro selection against one of the cell lines [Procter et al, 1984].…”

“…Y material has been detected in the blood of UTS individuals by PCR-based strategies or Southern blot analysis. However, these studies did not examine the gonads or other tissues to assess the distribution of the Y chromatin [Ostrer and Clayton, 1989;Medlej et al, 1992;Nagafuchi et al, 1992;Binder et al, 1995;Coto et al, 1995;Kocova et al, 1995]. It is important to gather information regarding the correlation between detected Y chromatin in lymphocytes vs. the distribution in the gonads, since it is believed to be the presence of Y chromatin in the gonads that confers the risk for gonadoblastoma.…”

Turner Syndrome and Sex Chromosomal Mosaicism

“…This way, the application of molecular techniques substantially improves the detection of low-frequency cell lines. Here we present the results of hidden mosaicism on 192 women diagnosed as TS and we focus on the molecular study of the Y chromosome, since numerous studies have shown that 4-20% of Turner women present a Y-chromosome (Kocova et al, 1995), increasing the risk of developing gonadoblastoma (Coto et al, 1995). Although the identity of the gene (or genes) linked to gonadoblastoma has not been established yet, there is evidence indicating that these genes are located near the centromere of the Y chromosome (Salo et al, 1995;Tsuchiya et al, 1995).…”