|Ahead of print publication
Clinical spectrum and Cytogenetic characterization of patients with Turner Syndrome – Twin case report
Sriambika Kumar, Rema Devi
Department of Anatomy, Division of Human Genetics, Pondicherry Institute of Medical Sciences, Puducherry, India
|Date of Submission||20-Sep-2021|
|Date of Decision||27-Apr-2022|
|Date of Acceptance||27-Apr-2022|
|Date of Web Publication||17-Sep-2022|
Department of Anatomy, Division of Human Genetics, Pondicherry Institute of Medical Sciences, Ganapathychettikulam, Puducherry - 605 014
Source of Support: None, Conflict of Interest: None
Diagnosis of Turner syndrome (TS) is usually made in mid-childhood, where 50% of the patients have 45, X karyotype in peripheral lymphocytes, which results from haploinsufficiency of the genes that commonly escape X-inactivation. 30%–40% have mosaicism of different forms, like 45,X/46,X,dic(Xp)/46,X,idic(Xq) and less commonly 45,X/46,XY. The goal of this case report is to analyze the cytogenetic and molecular characterization of two cases with dicentric X chromosomal abnormalities with varying degrees of mosaicism, demonstrating shared clinical features of TS. Combined conventional cytogenetic analysis, centromere banding, and fluorescence in situ hybridization (FISH) was done for the patients who presented with short stature and irregular menstrual cycles. Chromosome studies showed two cell lines: one with a single copy of X chromosome (45,X) and the other with a structural variation in X chromosome (isodicentric X chromosome), which is described as a Turner variant. C-banding also revealed the presence of two centromeres. Metaphase FISH with centromere probes for X revealed two mosaic cell lines: one with 45,X and a second one showing isodicentric X chromosome. The accurate diagnosis and characterization of a genomic imbalance in patients with sex chromosome disorders are essential for evaluating phenotype–karyotype correlations, genetic counseling, and having a clinical follow-up.
Keywords: Fluorescence in situ hybridization, isodicentric, mosaic, short stature, Turner variant
|How to cite this URL:|
Kumar S, Devi R. Clinical spectrum and Cytogenetic characterization of patients with Turner Syndrome – Twin case report. J Curr Res Sci Med [Epub ahead of print] [cited 2022 Oct 4]. Available from: https://www.jcrsmed.org/preprintarticle.asp?id=356216
| Introduction|| |
Turner syndrome (TS) is the most common genetic abnormality, with a prevalence of 1 in 2500 live births., The chromosomal abnormalities in TS are quite variable. Short stature, ovarian dysgenesis, broad webbed neck, shield-shaped chest, and congenital cardiac or renal anomalies are the common clinical features in TS patients. Diagnosis is usually made in mid-childhood, where approximately 45% of the patients have 45, X karyotype in peripheral lymphocytes, which results from haploinsufficiency of the genes that commonly escape X-inactivation. 30%–40% have mosaicism of different forms, like 45, X/46, X, idic(Xp)/46, X, idic(Xq) and less commonly 45, X/46, XY. 10%–20% have structural abnormalities in the X chromosome involving deletion of some or all of Xp/Xq. TS patients with dicentric X-chromosome is not a common chromosomal abnormality.
The dicentric chromosome is a structurally abnormal chromosome with two centromeres. It is formed by an end-to-end fusion of chromatids after a break, with subsequent loss of the acentric fragment, followed by the inactivation of one centromere. However, the mechanisms of centromere inactivation are poorly understood. In any case, clinical manifestation depends on breakpoint location, chromosomal segment involved in the duplication, type of structural abnormality, level of mosaicism, and chromosome inactivation pattern.
The cytogenetic and molecular characterization of two cases of dicentric X chromosomes with various degrees of mosaicism, as well as their clinical presentation, is given in the accompanying case report.
| Case Report|| |
The reported subjects were referred for chromosomal analysis to the Division of Human Genetics, Pondicherry Institute of Medical Sciences, Pondicherry, with the primary complaints of either short stature or irregular menstrual cycles. A detailed history and written informed consent were taken before karyotyping.
The first patient was a 23-year-old female who presented with irregular menstrual cycles. She had attained menarche at the age of 17 years and was said to have experienced only withdrawal bleeding after that. On examination, she was found to have short stature and no other Turner stigmata. The second patient was a 19-year-old female, who was short, and her primary complaint was irregular menstrual cycles. She had attained menarche at the age of 14 years. On examination, she was found to have short stature and hyperpigmented patches on the lips. No other features suggestive of Turner syndrome were noticed. Biochemical reports with regard to sex hormonal assay were found to be abnormal in both the patients. [Table 1] summarizes the clinical aspects of both occurrences.
Karyotyping was done following the conventional cytogenetic methods. 72-h peripheral lymphocyte culture was set up as per the modified laboratory standardized protocol. The culture was arrested at the metaphase stage using colcemid, harvested by hypotonic treatment, fixed and washed with Carnoy's solution, and cast on clean prechilled slides. Chromosomal analysis was performed by G-banding using trypsin and Giemsa. Fifty metaphase spreads were captured. In both the cases, the chromosome study revealed two cell lines, with one major cell line showing a single copy of the X chromosome and a second cell line showing a structural variation in the X chromosome (isodicentric X chromosome) reported as a Turner variant [Figure 1].
|Figure 1: The karyotype images of two cases showing the presence of two cell lines and dicentric X chromosome. Row A shows karyotype of both the cases with 45, X cell line. Row B shows karyotype of a second cell line with dicentric X chromosome|
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Centromere banding is said to preferentially stain constitutive heterochromatin regions which mainly includes repetitive DNA sequences and satellite DNA. The darkly staining C-bands are generally located at the centromeres of the chromosomes and on the distal region of the q arm of the Y chromosome. The significance of C-banding is to look for chromosome polymorphism near centromeres.
Three-day-old aged slides were dipped in 0.2 N HCl for 15 min before being incubated for 8–10 min in a saturated solution of barium hydroxide at 60°C. To remove excess barium hydroxide, the slides were washed in distilled water for 30 s before being quickly dipped in 0.1 N HCl. They were then re-incubated for 1 h in a 60°C water bath in a coplin jar with 2× sodium citrate and sodium chloride salt solution (SSC). Finally, the slide was rinsed in distilled water before being stained in a 2% Giemsa working solution for 5 min and then washed in running tap water. Centromere banding in both the cases revealed the presence of two centromeres.
Fluorescence in situ hybridization
Using a SpectrumGreen tagged probe for the centromeric region of chromosome X, fluorescence in situ hybridization (FISH) was done to rule out a complex structural rearrangement.
As instructed in the kit, a conventional protocol was followed (MetaSystems). The slides with metaphase chromosomes were prepared, 3 μl of centromere probe was applied, and the slides were subsequently sealed with rubber cement and coverslips. The metaphase chromosomes and the probes were subjected to denaturation and hybridization at 72°C for 2 min and 37°C for 20 h. The coverslip was removed after 20 h of hybridization, and the slides were placed in 0.4 × SSC at 72°C for 2 min, followed by a wash in 2 × SSC for 30 s, the third wash in distilled water, and air drying. The slides were counterstained with DAPI – 4',6 – diamidino- 2-phenylindole (3 μl/slide), covered with a coverslip, and examined under a fluorescence microscope for appropriate signals.
With the help of software, 200 interphases and 20 metaphases were taken under a fluorescent microscope. FISH analysis revealed two signals with an X-specific centromeric probe. Interphase FISH with an X centromere probe revealed three signals at 68% and one signal at 32% of the interphase cells in patient 1, whereas three signals at 77.5% and one signal at 22.5% of interphase cells in patient 2. Three signals indicate the dicentromeric nature of the X chromosome, and this analysis confirmed the mosaic condition with two cell lines: one with a normal cell line and a second cell line showing isodicentric X chromosome [Figure 2].
|Figure 2: FISH image showing three green signals: one on chromosome X and two on dicentric (x) chromosome and two blue signals (control). FISH: Fluorescence in situ hybridization|
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The values of follicular-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone values in patients are summarized in [Table 2] with references to ranges in different age groups.
|Table 2: The hormonal values of the patients with reference ranges in different age groups|
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In both the cases, FSH and LH values were found to be in the postmenopausal range, suggesting a premature ovarian cessation of activity.
| Discussion|| |
As a result of genetic abnormalities, the affected people, families, and the community bear a significant health and financial burden. Since environmental diseases have been successfully controlled, genetically determined diseases are becoming increasingly significant. As a result, finding a genetic component to the condition is critical for diagnosis, followed by risk assessment and genetic counseling.
In females, menstrual problems and infertility are the most common indications for genetic referral. Amenorrhea affects 20% of women with infertility. Amenorrhea has a wide range of reasons, including endocrine diseases, genetic or anatomical abnormalities, psychological issues, and environmental factors.
Puberty and stature are significant turning points in a person's life characterized by rapid changes in physical size, shape, and composition. Pure monosomy X, structural abnormalities of the X chromosome, or mosaicism of 45, X are all cytogenetically associated with TS. Short stature, pubertal delay, and gonadal dysgenesis are all evident in cell lines with the 46, XX or 46, XY complement. Over the last 30 years, cytogenetic abnormalities were present in 95% of the affected individuals who had short stature and primary amenorrhea.
Genes involved in somatic development can be found on the X chromosome's short arm (Xp). The short stature phenotype is caused by a haploinsufficiency of the short-stature homeobox gene, which encodes for a transcription factor involved in skeletal development and is found at Xp22.33; Yp11.3., In both the patients, the chromosomal study showed monosomy X and mosaic chromosome complement, an isodicentric X chromosome involving the critical region Xp22. This could account for the short stature seen in both the patients.
An end-to-end fusion of chromatids after a break, with subsequent loss of an acentric fragment, followed by inactivation of one centromere, and the remaining acentric fragments would be lost in ongoing cell division, is one probable reason for dicentric X chromosome formation. Therefore, deletion involving the short arm of the X chromosome results in short stature and typical skeletal changes in TS.
On the other hand, deletions involving the long arm of the X chromosome that affect the crucial area Xq13-q26 result in ovarian failure. The percentage of chromosomal abnormalities varies from 15.9% to 63.3% in patients with primary amenorrhea., The long arm (Xq) deletions infer primary or secondary ovarian failure. This is attributable to the loss of the premature ovarian failure gene, which is positioned in the Xq region, as seen in the current cases, where both the patients exhibited chromosomal defects in this area.,
Gonadal dysgenesis is a diagnostic feature of TS. Because of gonadal dysgenesis, gonadotropin levels are high in most girls with TS. In the present case, FSH and LH were measured, which were in the postmenopausal range.
Yu et al. reported a case where the patient presenting with normal stature, primary amenorrhea, short neck, and hypoplastic gonads had one X chromosome containing complete duplication of Xp and a partial deletion of Xq. Despite having a 45, X cell line, they believed that cells with a second defective X chromosome had a favorable effect on the phenotypic.
Petković et al. expounded that evaluation of clinical features associated with gain and loss of the X chromosomal segment is inconsistent. The phenotypic diversity is most likely owing to mosaicism, chromosomal inactivation patterns, location effects, or the patient's age.
A sex chromosomal anomaly should be suspected in those patients presenting with primary or secondary amenorrhea, especially when they have some features of TS. Identifying such chromosomal abnormalities at an early stage helps in hormone replacement, surgery, and appropriate genetic counseling. If mosaic, menstrual irregularities may be successfully managed, with a chance of maintaining fertility.,
| Conclusion|| |
The accurate diagnosis and characterization of a genomic imbalance in patients with sex chromosome disorders play a long way in evaluating phenotype–karyotype correlations, genetic counseling, and clinical follow-up. In addition, genetic counseling should address the risk of the use of hormonal replacement therapy, the possibility of infertility, and premature menopause in patients presenting with mosaic TS.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
We thank the clinical departments for referring the patients.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]