Introduction

Turner syndrome (TS) is caused by one of the most common chromosomal abnormalities of humans, its incidence being of 1:2500 live female births. The phenotypic features of TS vary widely, but usually include a low hairline, triangular facies, high arched palate, small mandible, webbed neck, broad chest with widely spaced nipples, cubitus valgus, short fourth metacarpal and/or metatarsal, multiple pigmented nevi, congenital lymphedema of hands and feet, gonadal dysgenesis, primary or secondary amenorrhea, short stature and cardiac and renal anomalies [1].

The etiology of the Turner syndrome has been associated with total or partial monosomy of the X-chromosome. About 60 % of TS patients have a 45,X karyotype, while the remaining patients have other karyotypes [2, 3]. Overall, patients with X-monosomy tend to have more severe phenotypic features and a higher morbidity, showing the importance of a chromosome analysis in TS prognosis [3].

The diagnosis of TS must take into consideration both phenotypic features and karyotype findings, i.e., chromosome analysis is necessary for a definitive diagnosis of TS. In Brazil, previous studies have demonstrated that TS is usually diagnosed late, the mean age at TS diagnosis still being higher than that reported in developed countries. Some typical characteristics of TS are already present at birth, which indicates the importance of making the information required for early TS diagnosis available to pediatricians and non-pediatricians of the newborn care team [4, 5].

TS patients who carry Y-chromosome material are at high risk of developing gonadoblastoma, a benign gonadal tumor but with a high potential of malignant transformation, the incidence of which is higher between the second and third decades of life. Prophylactic gonadectomy is currently recommended in all TS patients with Y-chromosome material [6].

For the detection of Y-chromosome sequences, cytogenetic analysis may be insufficient, as it can miss cases in which Y-chromosome material is present only in a small proportion of cells or in very small amounts, or even as part of marker chromosomes containing Y-specific regions [7]. Therefore, more reliable techniques, such as molecular analysis, should be used to improve diagnostic accuracy in TS patients.

The aim of this study was to investigate the presence of Y-chromosome sequences and to determine their frequency in Brazilian patients with TS.

Material and Methods

The study included 23 patients with TS from the outpatient Genetics clinic of the Federal University of Triângulo Mineiro (UFTM), Uberaba, State of Minas Gerais, Brazil. Karyotyping was done in the Cytogenetics Laboratory of the Discipline of Genetics of UFTM. Chromosome analysis was performed in cultured peripheral blood lymphocytes and the number of metaphases analyzed by Giemsa-Trypsin-Giemsa (GTG) banding was 100 for each patient. The karyotype distribution is presented in Table 1.

Table 1 Distribution of karyotypes in the 23 TS patients
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This study was approved by the local Research Ethics Committee (CEP/UFTM no. 927) and written informed consent was obtained from all patients and/or their parents.

Genomic DNA was extracted from lymphocytes using a phenol-chloroform method and amplified using four primer sets encompassing the Y-chromosome regions DYZ1 (Yq12), DYZ3 (Ycen), ZFY (Yp11.31) and SRY (Yp11.32). An internal control of the reaction consisting of the OPN (4q21-q25) gene was used to confirm the presence and availability of DNA in the sample. Primer sequences, annealing temperatures and expected size of the PCR products are described in Table 2.

Table 2 Primer sequences, annealing temperature and product size for Y chromosome PCR
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PCR was performed in a final reaction volume of 30 μL containing approximately 100 μg of genomic DNA, 20 pmol of each forward (F) and reverse (R) primer, 1 unit of Taq DNA polymerase, 1X PCR buffer, 1.5 mM of MgCl2, and 200 μM of each dNTP. In all PCR experiments, normal female DNA, normal male DNA and a blank reaction (with no DNA) were used as controls in all runs. The PCR products were submitted to electrophoresis on 10 % polyacrylamide gel and stained with silver nitrate.

All DNA extractions and PCR reactions were performed by a female technician, to avoid the risk of male DNA contamination.

Results

In this study, two out of the 23 patients (8.7 %) had Y-specific sequences detected by PCR analysis. These patients also had a mosaic cell line with a marker chromosome, detected by cytogenetic analysis. The cytogenetic and molecular results and clinical features of these patients are showed in the Table 3. The cytogenetic techniques did not allow the identification of the precise origin, whether X or Y, of the marker chromosomes. However, molecular analysis demonstrated that the markers were derived from a Y chromosome.

Table 3 TS patients with Y-chromosome sequences and their clinical features
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None of these patients had shown any Y-chromosome fragments when they were analyzed by classical cytogenetic techniques only. The cytogenetic analysis showed a 45,X karyotype in 9 of them (39.2 %) and mosaics in 14 (60.8 %).

In one patient, a previous cytogenetic analysis performed in another laboratory had reported a plain 45,X karyotype (25 metaphases analyzed). The authors' evaluation, done in 100 peripheral blood lymphocyte metaphases revealed a 45,X[90]/46,X,mar[10] karyotype. These diverging findings may be explained by improved identification techniques and changes in clinical screening practices. In authors' laboratory, the diagnosis of TS is based on the chromosome analysis of 100 peripheral blood lymphocyte metaphases. In most of the studies, the diagnosis of TS is based on 30 analyzed cells only, and therefore, they may not detect low-grade mosaicism.

Discussion

In the index study, two patients that showed marker chromosomes on conventional karyotype analysis actually carried hidden Y-chromosome material, detectable by DNA analysis of blood cells. In fact, about half of the unidentifiable marker chromosomes that occur in TS at an estimated frequency of 3 % are Y-chromosome-derived [8].

The prevalence of Y-chromosome sequences in the present sample was 8.7 %, which is in agreement with previous studies [9–11]. A study done in Italy investigated 171 TS patients for sequences SRY and DYZ3 showed a prevalence of 8.2 % [9]. FISH analysis of buccal smears revealed five patients (7.9 %) with Y-chromosome material [11]. In another, recently published study on 32 Mexican patients with TS, the prevalence of Y-chromosome sequences was 9.4 % (3/32) [10].

According to the literature, TS patients with marker chromosomes identified by cytogenetic analysis have demonstrated a frequency of Y-chromosome sequences ranging from 6.9 % to 60 % (Table 4). The present results are also consistent with other studies conducted in Brazilian patients that reported the presence of Y-chromosome material in 4.8 % [18], 6.8 % [19], and 10.4 %, respectively [14]. According to the literature, the frequency of hidden Y-chromosome material ranges between 0 % and 61.1 % [10]. Several factors contribute to these different findings, such as: patient selection, number of metaphases analyzed in the cytogenetic study, sample size, methodology, and the Y-chromosome sequence markers used [9–19].

Table 4 TS patients with positive PCR results for Y-chromosome segments and carrying marker chromosomes identified by cytogenetic analysis, described in previous studies
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It has traditionally been recommended that a search for Y-chromosome fragments in TS should only be performed under two circumstances: when there are signs of virilization and/or when there is a marker chromosome not identified by classical cytogenetics [20, 21]. Thus, patients with TS and a marker chromosome detected by conventional cytogenetic analysis may benefit from molecular screening analysis to detect the presence of Y-chromosome material. PCR is a rapid and low-cost technique that is more sensitive than karyotyping for detecting hidden Y-chromosome material.

The present study showed that one patient with TS, initially reported to have a 45,X karyotype, actually carried a mosaic cell line. Another, recently published study showed that: 1) all 45,X individuals with Turner syndrome are cryptic mosaics; 2) absence of the X chromosome in 45,X embryos is caused primarily by mitotic factors; and 3) the placenta is a strong candidate for the location of the rescue line in apparently non-mosaic 45,X individuals [22]. In addition, in routine laboratory diagnosis, the classical cytogenetic analysis is performed in thirty metaphases, which allows detection of 10 % mosaicism with a 95 % confidence interval [23–25]. If the hypothesis that all patients with TS carry mosaicism is true, it is likely that cell lines that are present in blood at frequencies below 10 % go undetected by this method. Molecular studies present the advantages of not requiring cell culture, can be done with a rather small amount of material, and increase the sensitivity of the cytogenetic methods [26]. In the present study, molecular investigation revealed that 8.7 % patients (2/23) carried Y-chromosome sequences. These sequences are localized on the Y centromere region and short arm Y chromosome, but not amplification was observed to Yq.

Moreover, the clinical guideline for TS advises testing for Y-chromosome material in any TS patient (or fetus) with a marker chromosome (a sex chromosome fragment of unknown origin, i.e., X vs. Y), which can be achieved by DNA studies or FISH [24]. Therefore, based on the index results and data from the literature, the authors recommend that a search of hidden Y-chromosome mosaicism should be carried out by molecular techniques in all TS patients, regardless of the karyotype found.

Conclusions

Thus, it was found that 8.7 % of the patients with TS studied had Y-chromosome sequences detected by the PCR method, with the presence of marker chromosomes found in the cytogenetic analysis. So, it is indicated that in patients suspected of TS, at least 100 metaphases should be analyzed cytogenetically, in order to allow the detection of low-grade mosaicism. Moreover, a molecular investigation for Y-chromosome sequences should be done routinely in patients with a cytogenetic diagnosis of TS, regardless of their karyotype.