Detection of Low Level Sex Chromosome Mosaicism

Detection of Low Level Sex Chromosome MosaicismAbstractfood turner syndrome (TS) is most parking lotly due to a 45,X chromosome defect, but is excessively seen in patients with a variety of X-chromosome ab everydayities or 45,X/46,XY photo mosaicism. The pheno caseful of TS patients is highly variable, and depends largely on the karyotype. Patients be at an increased risk of sex glandoblastoma when a Y derived chromosome or chromosome part is present. Since constitutional mosaicism is present in virtually 50% of TS patients, the denomination of minor(ip) prison kioskular ph oneness communitys is clinically important and a challenge to laboratories.Aim The design of the present playing field was the application of fluorescence in situ hybridization ( seek) hinderance to come in disordered level mosaicism for an XY or XX cell nation for TS patients with monosomy X and also to tell the character of invoke chromosome markers detected by schematic cytogenetic studies .Methods The study include 65 female patients with a clinical suspicion of TS, they were selected from the transmissible Clinic, Medical Re look for Institute, University of Alexandria. Chromosome epitome by G-banding technique was done. seek was performed using kinetochore probes for the X and Y chromosomes.Results Chromosome compend by G-banding technique revealed the fol execrableing results twenty patients (30.77%) had a 45,X karyotype mosaicism for a plump for normal or structurally abnormal X was observed in 27 (41.54%) cases, mosaicism for Y chromosome in 5 (7.69%) cases, 7 (10.77) had mosaicism involving a marker chromosome and non mosaic structural abnormalities of the X chromosome was present in 6 (9.23%) patients.To further check over the possibility of mosaicism in the 20 patients with an app arntly nonmosaic 45,X karyotype, and to put the nature of chromosome markers in the 7 patients carrying a marker, tip was performed using centromere probes for the X and Y c hromosomes. A minor XX cell line was set in 6 patients, XY mosaicism were determine in 3 cases and the 45, X result was substantiate in 11 seeks. FISH summary performed on the 7 patients with chromosome markers, identified the origin of these markers as X chromosome substantial in 3 patients, and Y-derived chromosome in 4 patients (idic Y with a double hybridization house be to double centromeric percentage).Conclusion FISH is a reusable tool around in the detection of low frequence cell lines and credit of the nature of mysterious chromosome markers that fork over important implications for the management of patients with food turner syndrome. FISH as an adjunct to karyotype synopsis provides a sensitive, specific, rapid, and informative technique to identify sex chromosome mosaicism in TS patients.Key Words Turner syndrome, monosomy X, mosaicism, 46, XY cell line, gonadoblastoma.INTRODUCTIONThe incidence of Turner syndrome (TS) is approximately one in 3,000 newborn girls and is associated with an apparently nonmosaic 45, X karyotype in many of these patients.(1) Based on chromosome compend 30%-50% are mosaic witha second X or a structurally abnormal X, andfewer than 10% of TS patients squander mosaicism with a 46, XY cell population or a Y chromosome rearrangement. The mosaic status of the remaining TS patients corpse uncertain but of clinical interest because if they do oblige cells with a Y chromosome or Y-derived fragment, they may have an increased risk of gonadoblastoma.(2)Because a 45, X karyotype usually causes fetal death, it has been postulated that all liveborn 45,X infants must be mosaic with either a Y or a second X in some cells.(3) The hypothesis of the necessity of mosaicism for survival is back up by the argument for the existence of a feto-protective effect of one or much genes on the sex chromosomes (X or Y). According to this concept, two copies of the gene(s) should be present, either in the fetus or in the extra-e mbryonic tissues.(4) Both, embryonic mortality and the Turner phenotype, are considered to be a result of monosomy of a common gene (s) of the X and Y chromosomes. It is assumed that, in women, these genes are verbalised in both active and inactive X chromosomes as a means of ensuring the right quantity of genetic product.(5)The American College of Medical genetic science recommends cytogenetic summary of 30 metaphase cells to rule out sex chromosome mosaicism.(6)This analysis can identify 10% mosaicism witha confidence level of 95% but a more sensitivelevel of detection requires analysis of many more metaphase cells, which is costly. PCR-based assays have been used to identify low-level mosaicism.(7) Fluorescence in situ hybridization (FISH) using X and Y chromosome probes has been validated (8) as a reflex test in apparently nonmosaic 45,X individuals to identify low-level mosaicism.(2,9) single of the advantages of applying the FISH technique is the possibility of studying mo saicism in both interphase nuclei and metaphases.(5)The conclude of the present study was to show the value of FISH analysis to identify low level sex chromosome mosaicism in Turner syndrome with nonmosaic monosomy X, and also to identify the nature of marker chromosomes detected by conventional cytogenetic studies.METHODSThe study included 65 patients with clinical features suggestive of TS, they were selected from the out patient clinic, sympathetic race Genetics Department, Medical Research Institute, University of Alexandria over a period of 4 years. Chromosomes were prepared according to standard techniques for culturing lymphocytes from peripheral blood, and the preparation was treated with trypsin to gain G-banding.(10) A minimum of 30 metaphases were analysed, and 3 were photographed for each patient.FISH analysis using the classic alpha-satellite probes for the X DXZ1 and Y DYZ3 centromeres (CEP-X and CEP-Y, Vysis Inc., spectrum green hybridizes to the centromere of hum an chromosome X, and spectrum orange hybridizes to the centromere of human chromosome Y) was performed in cases with nonmosaic 45, X karyotype to detect low level sex chromosome mosaicism and also in cases with chromosome markers to identify the nature of these markers. The protocol followed was that provided by the manufacturer. The normal cutoff was determined to be 1.0% for a second X planetary house and 0.6% for a Y signal in analysis of 500 interphase cells. (11) Whenever interphase FISH analysis revealed evidence of a second cell population, a search was undertaken using FISH for metaphase cells to confirm its presence and date the structure of the sex chromosomes in that population. FISH was performed in the Genetic Center, Genetic Counseling Society, Alexandria.Statistical analysisData were presented in the form of frequency and percentages.RESULTSBased on G-banded chromosome analysis of a minimum of 30 metaphase cells for the 65 patients included in this study, 45,X kary otype was found in 20 cases (30.77%), unhomogeneous mosaic complements was detected in 39 (60%), and non mosaic structural abnormalities of the X chromosome in 6 (9.23%) (table I). Mosaicism detected were as follow 8 (12.31%) with numerical mosaicism involvingthe X chromosome, 19 (29.23%) with structural mosaicism of the X chromosome, 5 (7.69%) with Y chromosome mosaicism, and 7 (10.77%) with mosaicism involving a marker chromosome, the level of mosaicism ranged from 8% to 86%.FISH analysis, using centromere probes for the X and Y chromosomes, identified mosaicism with a second X chromosome in 6 of the 20 patients with an apparently non-mosaic 45,X karyotype. The level of mosaicism detected ranged from 3.8% to 8.2%. Mosaicism with a Y chromosome was detectedin 3 patients, the level of mosaicism ranged from 2.4% to 7.2% (table II) (figure1). FISH modify the appellative of mosaicism from 60% (39/65) to about 73.85% (48/65).FISH analysis performed on the 7 patients with chromosome markers, identified the nature of these markers as X chromosome material in 3 patients, and Y-derived chromosome in 4 patients (idic Y with a double hybridization signal like to double centromeric region) (table III).FISH highlighted the differences between the initial diagnosis, based on G-banding, and the final diagnosis, determined by specific probes for theX and Y chromosomes. FISH analysis detectedmore Y-chromosomal material than karyotyping (18.46% (12/65) vs. 7.69% (5/65), respectively), and also detected more X-chromosomal mosaicism among the TS patients (55.38% (36/65) vs. 41.54% (27/65), respectively).Clinical, ultrasound and laparoscopic examination of gonads in patients with Y chromosome material revealed normal females with zygomorphic rudimentary streak gonads in 9 patients and females with clitoromegaly, unilateral streak gonads, and contralateral intraabdominal testis in 3 patients.Interphase cells showing one green signal of the X chromosomeInterphase cell showing 2 green signals for X chromosomeInterphase cell with one green signal for X chromosome and one red signal for Y chromosomeMetaphase cell with one green signal for X chromosome and one red signal for Y chromosomeDISCUSSIONAn estimated 1 in 50 conceptuses is affected with TS. However, only 1% of TS conceptuses survive to birth. It has been observed that there is a higher ratio of mosaic karyotypes to monosomy X in live births compared to aborted fetuses. This determination has led to speculation that most if not all patients born with TS must have mosaicism.(3)Phenotypic expression in TS patients largely depends on the karyotype, and identification ofsex chromosome mosaicism plays a key role in clinical management. Patients with put down mosaicism for a 46, XX or duplication of thelong arm have a moderate phenotype. Mental retardation is seen more frequently in patients with a elflike ring chromosome and deletion of the X-inactivation center (XIST).(12) Patients with a Y or Y-deri ved chromosome identified by routineG-banding analysis may have as high as a 30%risk of exploitation gonadoblastoma, althoughmost reports suggest an incidence of 7%-10%. Therefore, identification of low levelY chromosome mosaicism is also clinically important.(13)Out of the 65 patients included in the present study, 45, X karyotype was found in 20 cases (30.77%), various mosaic complements wasdetected in 39 (60%), and non mosaic structural abnormalities of the X chromosome in 6 (9.23%). Previous studies reported constitutional mosaicism in approximately 50% of TS patients based on chromosome analysis.(14) The detection of mosaicism is mainly influenced by the type and number of tissues analysed, the number of cells studied, and the sensitivity of the techniques applied.(4,5,15)FISH analysis of the 20 patients with 45, X karyotype included in the present study detected mosaicism in 9 patients 6 had an XX cell line, and3 had Y chromosome material. Therefore, FISH improved the identi fication of mosaicism from 60% to about 73.85%. Van Dyke and Wiktor (11) reported that FISH analysis improves the identification of mosaicism from 55% to 67% in patients with nonmosaic 45,X karyotype.They concluded that the identification of a cell population with a second X chromosome is able to exclude, with a high degree of confidence, the presence of a Y-bearing cell population in that patient. Other investigators compared the results of lymphocyte G-banded karyotype with the use of interphase X/Y FISH analysis. They detected more Y-chromosomal matrial by FISH than karyotyping (in 15% vs. 11% of the women, respectively) and also detected more X-chromosomal mosaicism among the TS women (in 70% vs. 45% of the women respectively). They suggested the use of X/Y interphase FISH as a complement to karyotyping in order to regain a more complete knowledge of the chromosome constitution of each individuals with TS.(16)The Y-chromosomal material in TS individualsis often present in the form of small marker chromosomes, which are difficult to positively identify by routine karyotyping. Furthermore, small markers are frequently missed altogether usingthis technique, especially if limit numbers of metaphases are evaluated.(17) In the presentstudy, 7 (10.77%) patients were detected with mosaicism involving a marker chromosome, FISH analysis identified the origin of these markers as X chromosome material in 3 patients, and Y-derived chromosome in 4 patients, the nature of the Y chromosome was defined as isodicentric withtwo centromeres. Approximately 20% of mosaic patients with TS have a sex marker chromosome.(14) The use of fluorescence in situ hybridization(FISH) analysis has been well documented asbeing effective in detecting and identifying sex chromosome markers.(18,19,20)In the present study, final diagnosis followed G-banding and FISH analysis identified Y-bearing cell population in approximately 18% of TS patients. criticism of the literature suggested that 6-12% of patients with TS had 45,X/46,XY cell lines withor without structurally abnormal Y.(21) This variation is a reflection of the numbers of patients studied,the technique used, and the strategies employedby different investigators to search for small populations of Y containing cells.Virilization with clitoromegaly was found in3 cases in the present study with Y cell line mosaicism. It is believed that virilization in patients with TS is due to the presence of Y cell line within the gonad even if the Y cell line is not identified in peripheral blood. Therefore, virilization is an indication for detailed studies looking for the presence of Y mosaicism. (22)Early detection of Y-derived material in the genome of TS individuals is of great importance because of the sexual relation high risk (10-30%) of developing gonadal tumors (i.e., gonadoblastoma or dysgerminoma).(23,24) Gonadoblastoma is a forerunner tumor which may undergo malignant transformation into one of the baneful ger m cell tumors (dysgerminoma, embryonal carcinoma, endodermal sinus tumor, chorioepithelioma or yolk liberation tumor).(21) It has been suggested that a locus (GBY) predisposing to the development of this tumor is located in the pericentromeric region of Yp.(25) Although the natural history of gonadoblastoma in prepubertal patients is unknown, this tumor can be evident even in the first decade of life in streak gonads with Y mosaicism and may be bilateral. Therefore, prophylactic gonadectomy shouldbe recommended in patients with TS and Y chromosome mosaicism, because fertility is not an issue, surgical morbidity is minor, and the potential for malignant transformation is unknown.(26)If a patient declines gonadectomy, monitoring for germ cell neoplasm is the only option. However,it is unclear whether methods in common usetoday (vaginal ultrasound, biochemical markers, proteomics, etc.), even with compliant patients,are able to identify germ cell neoplasms at early enough stages to i mprove the natural history of the disease. It is possible that a predictable and specific marker of malignant potential may be identified in the future. Until then, physicians will need to be continually updated on these important issues as they relate to the clinical management of patients with Turner syndrome.(21)In conclusion, FISH for the X and Y centromere probes is a useful adjunct to conventional cytogenetic studies in patients with apparently nonmosaic monosomy X. This additional assay improved the identification of mosaicism from 60% to about 73.85%. FISH method provides a sensitive, specific, rapid, and informative means of identifying low level X and Y mosaicism in TS patients, and can be employed on the same blood sample that is used for the conventional cytogenetic studies. FISH helped in identifying the nature of the unknown markers which has an important implication in the development of gonadal tumors. Metaphase FISH, and interphase FISH should complement and valida te each other in the detection of covert Y and identification of rearranged X vs Y chromosomes.