Molecular identification of chromosome Y sequences in Brazilian patients with Turner syndrome

The investigation of Y-specific sequences in patients with Turner Syndrome (TS) with karyotype 45,X or mosaic, has a fundamental role in the clinical management of these patients. The relationship between the presence of Y chromosome fragments and a higher risk of gonadoblastoma in TS has already been established. The aim of the study was to investigate the presence of Y-chromosome fragments in a population of 42 female Brazilian patients with TS from Mato Grosso state. Cytogenetic analysis has shown the karyotypes 45,X in 27 of them (64.3%) and mosaic in 15 (35.7%). The presence of the Y-primers SRY, DYZ3, ZFY, DYZ1, DYS1 and PABY was investigated in all patients. These markers were amplified by polymerase chain reaction (PCR) technique, using DNA genomic from peripheral blood lymphocytes. None of these patients had shown any Y-chromosome fragments when they were analysed only by the classic cytogenetic technique. The PCR analysis with the Y-specific sequences ZFY and DYZ3 were identified in two different patients (4.8%), both with karyotype 45,X. It was concluded that PCR is efficient in the investigation of hidden Y-fragments in TS patients. Therefore, this method should be included in the routine assistance of these patients.     

Screening of seven putative 45,X subjects with deoxyribonucleic acid probes to detect low-level mosaicism for Y cell lines

The frequency of monosomy X in cytogenetically abnormal abortion material (10% to 15%) suggests that viable 45,X subjects might have covert mosaicism for X or Y cell lines. The deoxyribonucleic acid samples from seven 45,X subjects with Turner syndrome were examined with three Y-specific deoxyribonucleic acid probes. Successive hybridizations with each of these three sensitive deoxyribonucleic acid probes did not reveal any Y-specific band.     

Turner syndrome in a girl with marker chromosome in karyotype]

OBJECTIVES: There are suggestion, that Turner syndrome (TS) patients with mosaic karyotype for a Y-DNA-containing cell line are at risk of Y-induced gonadoblastoma. The TS patients in whom some or all cells contain a marker chromosome of unknown origin and those in whom there is clitoromegaly or other evident virillisation should be tested by FISH or PCR techniques. DESIGN: The aim of our study to present a TS girl with mosaic karyotype and marker chromosome, which origin from X chromosome was detected by FISH method. MATERIAL AND METHODS: 5-years old girl in whom TS was established. Clinical analysis included the full dysmorphic and clinical phenotype of TS. Chromosome analysis was performed on peripheral blood samples using routine cytogenetic methods and FISH technique. RESULTS: Clinical examination of girl showed many typical signs of TS besides of normal weight and length at birth and not typical for TS patients heart defect. First routine chromosome analysis, at age of 6 month, showed only 45,X cell line, Second study revealed mosaic karyotype with marker chromosome. FISH analysis for interphase nuclei and metaphase chromosomes using X centromere probe explained origin of marker from X chromosome. The karyotype was 45,X[155]/46,X,+mar[8].fish mar(X)(DXZ1+). CONCLUSION: Presence of marker chromosome in karyotype of patient with TS may modify their phenotype and it is a indication for molecular examination by FISH technique.     

Turner综合征Y染色体物质嵌合分子遗传学研究

摘要：目的　分析Turner综合征（TS）患儿Y染色体物质及衍生物嵌合发生的情况，为TS患儿诊断后的监测提供科学建议，改善国内TS监测和保健管理的现状。方法　选取2006年2月至2007年8月在重庆医科大学附属儿童医院诊断为TS患儿30例，进行基因组DNA 的Y编码睾丸特异性蛋白基因（TSPY）、 Y染色体中心着丝粒DYZ3重复序列（DYZ3 ）和Y性别决定区域（SRY）3个Y染色体特异序列多聚酶链反应（PCR）检测，反应结果阳性的病例补充SRY探针原位荧光杂交（FISH）分析。结果　基因组DNA 的PCR结果显示，3例患儿的TSPY、 DYZ3扩增均为阳性（10.00%），其中只有1例 SRY 扩增阳性（3.33%）；3例Y染色体物质阳性病例进一步进行FISH研究，结果显示3例SRY杂交信号均为阳性。结论　运用3个Y染色体特异序列的分子遗传学研究，证实Y染色体物质嵌合在TS不少见，每一个TS患儿都应在诊断后进行Y染色体物质的分子遗传学监测。     

Use of human alpha-satellite deoxyribonucleic acid to detect Y-specific centromeric sequences

The Y alphoid deoxyribonucleic acid probe Y97 has proved to be specific for the human Y centromere and to define a Y-specific 5.5 kb Eco RI fragment. Three experiments were designed to evaluate the sensitivity and the specificity of this Y alphoid probe Y97. In the first experiment the centromeric Y-specific 5.5 kb Eco RI fragment was clearly seen in the mixture of 0.050 microgram of male DNA with 4.950 micrograms of female DNA (1%). In the second experiment the same dilutional study was applied to the Yq11-related probe 4B-2 for comparison purpose. In the third experiment, hybridization with the Y97 probe was performed on 20 subjects with mosaic cell lines containing a cytogenetically identifiable Y (n = 10) and a cytogenetically unidentifiable minute (n = 10) fragment. Nineteen of the 20 subjects demonstrated the Y-specific 5.5 kb Eco RI hybridization band with the centromeric Y97 probe. These experiments demonstrated the utility of the Y97 probe to consistently identify cytogenetically altered Y chromosome fragments and confirm the mapping of the alphoid repeat sequences to the centromeric region of the Y chromosome.     

Spontaneous Sexual Development and Heavy Menstrual Bleeding in 45,X Monosomy and 45,X/47,XXX Mosaic Turner Syndrome and a Review of the Literature

Turner syndrome (TS) is one of the most common sex chromosome disorders and is characterized by short stature and gonadal dysgenesis. A few patients with TS achieve normal sexual development, menarche, and even pregnancy. We encountered two cases of Turner syndrome with spontaneous sexual development and menstruation. The patients had different karyotypes, 45,X monosomy and 45,X/47,XXX mosaic TS, and presented with severe anemia due to excessive menstrual bleeding. Abnormal uterine bleeding patterns are expected in patients with primary ovarian insufficiency; however, the menstrual patterns of patients with TS have not been well described in the literature. Here, we describe these cases along with a brief review of the relevant literature.     

Pure yolk sac tumor of the ovary with mosaic 45X/46X+mar Turner's syndrome with a Y-chromosomal fragment

A pure yolk sac tumor (endodermal sinus tumor) of the dysgenetic gonad developed in a 23-year-old woman whose karyotype was mosaic 45 X/46X+mar Turner's syndrome is reported. Molecular biological studies showed that the patient's DNA contained a fragment of Y chromosome. This case seems to be extremely rare case of developing a pure yolk sac tumor in a patient with mosaic Turner syndrome with a Y-chromosomal fragment. Received: 5 September 1997 / Accepted: 9 December 1997     

Prenatal identification of a Y-chromosome deletion by Y-specific single copy DNA probes

A sex chromosome deletion was identified in the course of prenatal diagnosis for maternal age. Ultrasound pictures revealed male fetal sex and a comparison with the father's Y chromosome suggested that the altered chromosome might be a de novo deletion of the Y chromosome. DNA hybridization with five human Y-specific probes shows that, among the Y-specific sequences recognized by the probes, only two of them are absent. The normal infant, at birth, was mosaic 46, XYq-/46,XY.     

Short communication: Y chromosome microchimerism in female peripheral blood

Male DNA of recognized fetal origin can be detected in the maternal circulation many years after delivery. It is referred to as fetal microchimerism, and is thus a possible explanation for the existence of low-level Y chromosome mosaicisms. Employing the nested polymerase chain reaction (PCR) technique, Y-specific markers were investigated in 13 cases with abnormal sex chromosome and 31 normal women. Sex-determining region Y (SRY) sequences were detected in normal women with a male child, which reflects the existence of fetal progenitor cells in the maternal circulation. This was completely absent in normal women with a female child. Individuals with the Y chromosome showed amplification for Y-specific markers. Microchimerism of Y was noted in Turner phenotype cytogenetically investigated with marker chromosome, and in an individual with XX karyotype. False positive amplifications are possible in nested PCR reactions, but the same could also be true for routine PCR. However, in the absence of any identifiable factor that could contribute to the recurrence of spurious PCR amplifications, cases of therapeutic importance must be tested at least five times. In such situations, DNA from an additional tissue should also be used for nested PCR.     

Frequency and Types of Chromosomal Abnormalities in Turkish Women with Amenorrhea

Study ObjectiveTo estimate the frequency and the type of chromosomal abnormalities (CA) in patients with primary (PA) and secondary amenorrhea (SA).DesignThis retrospective study was comprised of patients had been referred to our laboratory between 1990 to 2008 and designed as original article.SettingMedical Faculty of Cukurova University in Turkey.ParticipantsChromosomal analysis was carried out on 393 patients with PA and SA that were referred to Cytogenetic laboratory of Medical Biology and Genetic Department, Faculty of Medicine, Çukurova University.InterventionsLymphocyte culturing depended karyotyping.Main Outcome MeasuresStandard lymphocyte culturing procedure and karyotyping was performed to all samples.ResultsPA and SA were identified in 393 patients. The karyotype was normal in 337 cases (85.8%) and abnormal in 56 (14.2%) patients. CAs were found in 54 (13.7%) and 2 (0.5%) of women with PA and SA, respectively. Females carrying rearrangements between autosomal and sex chromosomes were detected in 2% (8/393). The numerical abnormalities of the X chromosome were detected in 39.3% (22/56) (monosomy and mosaic). Structural abnormalities of the X and the other chromosomes were detected in 25.5% (13 of 56). Structural mosaicism of X chromosome was found in 5.4% (3 of 56). Male karyotype (46, XY) was found in 33.9% (19/56). The most frequently detected abnormality were X chromosome monosomies or mosaics.ConclusionsOur study revealed that some causes of amenorrhea could be due to CAs. Therefore, cytogenetic study should be important test in the evaluation of patients with PA or SA. The most common abnormality seen is 45,X karyotype (monosomy X/Turner Syndrome) and its variants.     

Turner综合征患者标记染色体的鉴定与分析

目的 分析11例携带标记染色体的Turner综合征患者的核型,研究这类染色体的表型效应。方法 选择11例具Turner综合征表型的患者,常规核型分析均显示为携带标记染色体的嵌合体,其中6例标记染色体呈环状。患者G带核型表示为mos．45,X／46,X,＋mar或者mos．45,X／46,X,＋r．以X／Y着丝粒探针,应用荧光原位杂交（FISH）技术分析这些标记染色体起源,对其中2例较大的环状染色体,结合反向染色体涂染确定断裂位点,比较不同断裂位点的标记染色体的遗传学效应。结果11例患者所携带的标记染色体均为环状染色体,r（X）的断裂位点分别位于Xp22、Xq22、Xq24、Xq26等。结论 Turner综合征患者的标记染色体主要来源于X染色体,且表现为r（X）形式。r（X）均以嵌合型的形式存在。     

Impact of parental gonosomal mosaicism detected in peripheral blood on preimplantation embryos

This study evaluated the chromosomal condition of embryos generated by patients with an altered karyotype due to gonosomal mosaicism and the clinical outcome after preimplantation genetic diagnosis (PGD) for aneuploidy. Thirty-six patients aged 34.6 +/- 3.6 years performed 54 treatment cycles and had 295 embryos diagnosed by fluorescence in-situ hybridization (FISH). Thirty-seven per cent of the embryos were chromosomally normal and generated 19 clinical pregnancies after replacement in 39 cycles. Only one pregnancy miscarried, yielding a take-home baby rate of 33.3%. Autosomal monosomy and trisomy contributed 36.1% of total abnormalities and gonosomal aneuploidy 5.9%, similar to the results detected in patients who undergo PGD for increased maternal age. Reanalysis was performed on 114 non-transferrable embryos: 41 were found to be mosaics, which were grouped in three different types, chaotic mosaics (56%), aneuploid mosaics (29%) and diploid/haploid/polyploid mosaics (15%). The incidence of aneuploid mosaics was higher than expected compared with PGD patients of the same age and resembled the condition observed in patients of advanced maternal age. These findings suggest that constitutional carriers of sex chromosome mosaicism are predisposed to autosomal mosaicism of embryos, possibly due to errors of cell division. There is an indication that this tendency is higher in female than male carriers.     

Evaluation of a culture system for enrichment of CD34+ hematopoietic progenitor cells present in maternal blood

BACKGROUND: Clonogenic expansion of fetal cells in maternal blood is one approach to overcome the very low number of target cells available for prenatal genetic analysis. However, efficient methods of enrichment, culturing conditions and subsequent analysis of fetal cells are lacking. Optimization of this technique requires more detailed evaluation of the composition and distribution of fetal cells that cross the placenta into the maternal circulation. Previous studies by others have shown that fetal blood is rich in CD34+ progenitor cells capable of expansion in cultures supplemented with hematopoeitic growth factors. Moreover, CD34+ fetal cells have been recovered from maternal blood following enrichment. OBJECTIVE: In this study, we examine the type and frequency of hematopoietic progenitor cells detected in maternal (n = 13) and non-pregnant control (n = 4) peripheral blood specimens. METHODS: A methylcellulose-based culture system was used to perform colony assays on CD34+-enriched or non-enriched cells. Overall, a total of 2,249 colonies were scored for colony type among the 17 samples. To determine whether fetal cells were present and expanded, all colonies present in each of the 10 confirmed male-cases (n = 1,525 colonies) were examined either by PCR or FISH. RESULTS: With CD34+-enriched maternal samples, we observed a significantly higher number of burst-forming unit-erythroid (BFU-E) and a reduced number of colony-forming unit-granulocyte, macrophage (CFU-GM) colonies compared to the non-enriched samples. Of 1,067 colonies analyzed by PCR for the amelogenin locus on X and Y, none were found positive for the 250-bp Y-specific sequences. Of 458 colonies tested by FISH for presence of X and Y probe signals, no XY-male cells were detected. CONCLUSION: We conclude that hematopoiesis is enhanced during pregnancy, but the number of fetal progenitor cells is either very low or fail to expand using the enrichment techniques and culturing conditions described in this study. Further development of methods is warranted before considering this approach for prenatal diagnosis.     

Molecular and cytogenetic characterization of extra-structurally abnormal chromosomes (ESACs) found prenatally: outcome and follow-up

A 40-year-old woman underwent amniocentesis at 15.3 weeks of gestation. Chromosome analysis performed using QFQ, DA-DAPI and CBG banding revealed two de novo extra-chromosomal markers (ESACs) in 11 of the 16 colonies analysed. Fluorescence in situ hybridization (FISH) showed that both chromosomes came from the Yq11.22.1 region of the Y chromosome. PCR analysis of genes and STS localized on the Y chromosome excluded the Yp presence specifically of the SRY gene, and most of the euchromatic region of Yq. After extensive genetic counselling and considering both laboratory and second-level ultrasound data, the couple decided to continue the pregnancy. At 37.4 weeks of gestational age, a girl weighing 2750 g was born with an Apgar score of 9/10. A blood sample taken from the umbilical cord showed three cellular lines: mos47,XX, +mar1 ish.der (Y)(wcpY+) [21%]/48,XX, +mar1 ish.der (Y)(wcpY+), +mar2 ish.der (Y)(wcpY+) [41%]/46,XX [38%]. One year after birth, the baby was developing normally and had normal psychomotorial activity.     

Reliability of gender determination using the polymerase chain reaction (PCR) for single cells

Contamination with extraneous DNA sequences is a frequent problem when performing PCR analysis of single cells. This report describes our experience with eliminating contaminating DNA sequences from PCR reagents for the purposes of gender identification. We have used amplification of Y-specific sequences to identify the gender of single human amniocytes. Female cells consistently showed no Y-specific bands but only 80% of male cells showed the expected intense Y-specific band. This phenomenon could lead to incorrect gender identification of single cells. We developed a technique of simultaneous amplification of X- and Y-specific sequences to prevent misdiagnosis because of failed PCR, which allows accurate preimplantation gender determination for women at risk for conceiving children with X-linked genetic discoses. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment     

Review: Sex Chromosome Evolution and the Expression of Sex-Specific Genes in the Placenta

Sex chromosomes have a disproportionate influence on health and disease. Both the X and Y are atypical in gene content and activity, as a result of their unique evolutionary trajectory. The X and Y chromosomes originated in a pair of autosomes, and differentiated as the Y chromosome degenerated progressively. The Y contains few active genes and is composed largely of repetitive DNA sequences. Most Y genes have copies on the X from which they evolved; this includes even the sex-determining gene SRY as well as several genes required for spermatogenesis. The X contains a disproportionate number of genes that affect reproduction and brain function (or both). It is also subject to inactivation in females, so that females are mosaics composed of patches of tissue that express only the genes on either the maternally or the paternally derived X chromosome. Several widely expressed genes on the Y chromosome code for male-specific proteins that provoke an immune reaction in females; this HY antigen has a measurable effect on maternal-fetal incompatibility. Imprinted paternal X inactivation in rodent extraembryonic tissues would be expected to mitigate the effect of foreign paternal antigens; however, paternal inactivation seems not to occur in the human placenta.     

Rapid determination of fetal sex by deoxyribonucleic acid amplification of Y chromosome-specific sequences

Rapid fetal sex tests use either dot blot hybridization to Y chromosome-specific (male) repeat sequences or polymerase chain reaction deoxyribonucleic acid amplification of these Y-specific sequences. We have performed 35 fetal sex determinations, 16 by dot blot alone, 13 by polymerase chain reaction alone, and 6 by dot blot and polymerase chain reaction on samples of fetal blood, amniotic fluid, and chorionic villi. All results have been confirmed by karyotyping. Dot blots have given false-positive "male" results three times. In contrast, the polymerase chain reaction has correctly determined fetal sex in every case, even when the dot blot was in error. The Y-specific polymerase chain reaction has been applied to fetal deoxyribonucleic DNA with a chromosome 15; Y translocation to identify the origin of the translocated material. Thus the polymerase chain reaction appears to be a reliable method to rapidly determine fetal sex that also can be used diagnostically to identify translocated Y-chromosomal material.     

Prenatal application of fluorescent in situ hybridization (FISH) for identification of a mosaic Y-chromosome marker, idic(Yp).

An amniocentesis was performed at 13.3 weeks' gestation for advanced maternal age. A mosaic sex chromosome pattern was found: of 50 cells examined, 34 had a 45,X karyotype. In 14 cells with a modal number of 46, a recognizable Y was substituted by a small non-fluorescent marker. C-banding identified the marker as an isodicentric in 12 cells. In two cells, the non-fluorescent marker appeared to be monocentric and looked like a non-fluorescent del (Yq), but could have been an isodicentric Y with inactivation of one of the centromeres. Two cells with a modal number of 47 showed two copies of the monocentric marker. Fluorescent in situ hybridization with an alpha satellite Y-specific centromeric probe confirmed the Y-chromosome origin of the markers and allowed for more accurate prenatal diagnostic information.     

Reproductive outcomes after preimplantation genetic testing in mosaic Turner syndrome: a retrospective cohort study of 100 cycles

PurposeThe purpose of this study is to explore the reproductive outcomes of women with Turner syndrome (TS) in preimplantation genetic testing (PGT) cycles.MethodsA retrospective study of 100 controlled ovarian stimulating cycles, 68 TS (sixty-four mosaic Turner syndrome (MTS) and four pure Turner syndrome (PTS)) women underwent PGT was conducted from 2013 to 2018.ResultsEmbryo X chromosome abnormal rates of TS women were significantly higher than women with normal karyotype (7.04 vs 1.61%, P<0.01). Cumulative live birth rates (CLBR) after PGT-NGS treatment were lower in TS than control (31.15 vs 45.59%, P<0.05). Clinical pregnancy rates per transfer (CPR), miscarriage rates (MR) and live birth rates per transfer (LBR) remained comparable between TS and control group. Reproductive outcomes (X chromosome abnormal rates, CPR, MR, LBR and CLBR) among low (<10%), medium (10–50%) and high (>50%) level 45,X mosaicism groups were not statistically different.ConclusionsTo avoid high risk of embryo X chromosome abnormalities, prenatal or preimplantation genetic testing should be recommended to mosaic or pure TS patients.