Detection of Low Level Sex Chromosome Mosaicism

Detection of Low Level Sex Chromosome Mosaicism Abstract Turner syndrome (TS) is most commonly due to a 45,X chromosome defect, but is also seen in patients with a variety of X-chromosome abnormalities or 45,X/46,XY mosaicism. The phenotype of TS patients is highly variable, and depends largely on the karyotype. Patients are at an increased risk of gonadoblastoma when a Y derived chromosome or chromosome fragment is present. Since constitutional mosaicism is present in approximately 50% of TS patients, the identification of minor cell populations is clinically important and a challenge to laboratories. Aim: The purpose of the present study was the application of fluorescence in situ hybridization (FISH) assay to identify low level mosaicism for an XY or XX cell population for TS patients with monosomy X and also to identify the nature of sex chromosome markers detected by conventional cytogenetic studies. Methods: The study included 65 female patients with a clinical suspicion of TS, they were selected from the Genetic Clinic, Medical Research Institute, University of Alexandria. Chromosome analysis by G-banding technique was done. FISH was performed using centromere probes for the X and Y chromosomes. Results: Chromosome analysis by G-banding technique revealed the following results: twenty patients (30.77%) had a 45,X karyotype; mosaicism for a second 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 investigate the possibility of mosaicism in the 20 patients with an apparently nonmosaic 45,X karyotype, and to identify the nature of chromosome markers in the 7 patients carrying a marker, FISH was performed using centromere probes for the X and Y chromosomes. A minor XX cell line was identified in 6 patients, XY mosaicism were identified in 3 cases and the 45, X result was confirmed in 11 samples. FISH analysis performed on the 7 patients with chromosome markers, identified the origin of these markers as X chromosome material in 3 patients, and Y-derived chro mosome in 4 patients (idic Y with a double hybridization signal corresponding to double centromeric region). Conclusion: FISH is a useful tool in the detection of low frequency cell lines and identification of the nature of unknown chromosome markers that have important implications for the management of patients with Turner syndrome. FISH as an adjunct to karyotype analysis 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. INTRODUCTION The 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 analysis 30%-50% are mosaic with  a second X or a structurally abnormal X, and  fewer than 10% of TS patients have mosaicism with a 46, XY cell population or a Y chromosome rearrangement. The mosaic status of the remaining TS patients remains uncertain but of clinical interest because if they do have 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 supported by the argument for the existence of a feto-protective effect of one or more 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-embryonic 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 expressed in both active and inactive X chromosomes as a means of ensuring the right quantity of genetic product.(5) The American College of Medical Genetics recommends cytogenetic analysis of 30 metaphase cells to rule out sex chromosome mosaicism.(6) This analysis can identify 10% mosaicism with  a confidence level of 95% but a more sensitive  level 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) One of the advantages of applying the FISH technique is the possibility of studying mosaicism in both interphase nuclei and metaphases.(5) The purpose 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. METHODS The study included 65 patients with clinical features suggestive of TS, they were selected from the out patient clinic, Human 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 obtain 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 human 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 signal 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 examine the structure of the sex chromosomes in that population. FISH was performed in the Genetic Center, Genetic Counseling Society, Alexandria. Statistical analysis: Data were presented in the form of frequency and percentages. RESULTS Based on G-banded chromosome analysis of a minimum of 30 metaphase cells for the 65 patients included in this study, 45,X karyotype was found in 20 cases (30.77%), various 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 involving  the 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 detected  in 3 patients, the level of mosaicism ranged from 2.4% to 7.2% (table II) (figure1). FISH improved the identification 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 corresponding 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 the  X and Y chromosomes. FISH analysis detected  more 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 bilateral 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 chromosome Interphase cell showing 2 green signals for X chromosome Interphase cell with one green signal for X chromosome and one red signal for Y chromosome Metaphase cell with one green signal for X chromosome and one red signal for Y chromosome DISCUSSION An 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 finding 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 of  sex chromosome mosaicism plays a key role in clinical management. Patients with documented mosaicism for a 46, XX or duplication of the  long arm have a moderate phenotype. Mental retardation is seen more frequently in patients with a small ring chromosome and deletion of the X-inactivation center (XIST).(12) Patients with a Y or Y-derived chromosome identified by routine  G-banding analysis may have as high as a 30%  risk of developing gonadoblastoma, although  most reports suggest an incidence of 7%-  10%. Therefore, identification of low level  Y 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 was  detected 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, and  3 had Y chromosome material. Therefore, FISH improved the identification 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 sufficient 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 interpha se FISH as a complement to karyotyping in order to obtain a more complete knowledge of the chromosome constitution of each individuals with TS.(16) The Y-chromosomal material in TS individuals  is 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 using  this technique, especially if limited numbers of metaphases are evaluated.(17) In the present  study, 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 with  two 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 as  being 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. Review of the literature suggested that 6-12% of patients with TS had 45,X/46,XY cell lines with  or without structurally abnormal Y.(21) This variation is a reflection of the numbers of patients studied,  the technique used, and the strategies employed  by different investigators to search for small populations of Y containing cells. Virilization with clitoromegaly was found in  3 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 relative high risk (10-30%) of developing gonadal tumors (i.e., gonadoblastoma or dysgerminoma).(23,24) Gonadoblastoma is a precursor tumor which may undergo malignant transformation into one of the virulent germ cell neoplasms (dysgerminoma, embryonal carcinoma, endodermal sinus tumor, chorioepithelioma or yolk sac 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 should  be 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 use  today (vaginal ultrasound, biochemical markers, proteomics, etc.), even with compliant patients,  are able to identify germ cell neoplasms at early enough stages to improve 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 validate each other in the detection of covert Y and identification of rearranged X vs Y chromosomes.

Rubber Company

Atlas Tire and Rubber Company 1) What were Atlas’ major challenges prior to the promotion of Walter Harrison as CEO? The main challenges faced by Atlas’ were competitive and financial challenges. As for the first time from 1905 the company was facing losses for consecutive 2 years in row as the company was facing a severe downturn. This was all because of the new challenges, which the company was facing due to their growth and expansion policiesThe company was also facing the competitive challenges from the low prices import products which were affecting their price strategy resulting in low profits, heavy slump in the automotive market in America also pulled back Atlas to regain its flagship. Due to all these the stock prices dropped down to 20 years low resulting in cutting down of credit rating to junk – bond status. So during this declining phase, the company needs some major transformational changes so that Atlas can regain its position and pride back. 2) Wh y did Harrison focus on an integrated supply chain as a significant element of Atlas’ new strategic plan?As Harrison and his team found that Atlas lacked integrated supply chain capabilities due to which disjointed distribution network was there and various third – partly logistic companies were used which lead to many problems and challenges. And logistics was only considered as a storage and distribution function rather than a strategic supply chain process. As Harrison came out with a strategic plan â€Å" Seven Principle of Growth† for turning around the Atlas’s fate. Harrison special focused on a superior and integrated supply chain.He focused on Integrated Supply Chain because he strongly feels that this would streamline the order and its fulfillment process so that they can be more accurate and easy to use because of their simplicity. The main focus was to deliver â€Å"the right tire to the right place at the right time while keeping costs and in ventories low. † Harrison wants to create an extra edge over the other tire manufacturers by creating the much better service capabilities. 3) What did the UST President, Michael Brogran, do to better understand customer needs? UST business defines Atlas’ s U. S.Tire business, which has the worth of producing, and selling 100 million tires per year and was generating approximately 50 percent of Atlas Revenue. Michael Brogran strongly feels that the superior supply chain was a vision that existed only on paper so he just made a team to understand customer needs in a better way. The main step the team had taken was to work across customer verticals, which were Consumer tires, Commercial Tires and Off Highway Tires. This structure help Atlas to better understand the customer needs in different segment and to meet them in different ways as needs were also different.As the part of this new alignment to understand consumers needs better Brogran facilitates cross company colla boration to identify interactions and collaboration and to share the best practices so that every department can better understand their customer needs for better visibility and control. 4) How did USTSC address the talent gap that they had in the organization? U. S. Tire Supply chain organization’s main objective was to integrate the planning and functional operations which included the responsibilities of contact centers, order management, pricing administration, distribution and delivery, billing and accounts receivable.USTSC was structured so that Atlas can understand & meet the needs of its customers that are classified under various segments likes consumer, commercial and off highway tires. The key goals of USTSC were – Lower the supply chain costs, – Boosting the customer services, – Improvement in the inventory management and associated working capital, – Simplifying the supply chain processes & management of the business. Initially USTSC c ame across major challenge with regards to talent gap.Reduction in headcount from several years impacted the internal supply chain analytical and execution capabilities. Moreover with five different 3PL (third party logistics) the focus was emphasized in managing the suppliers rather than the operations. After a thorough evaluation USTSC concluded that SCM (Supply Chain Management) was never a core competency of Atlas. In order to bridge the gap USTSC obtained assistance from organization that had expertise in planning and managing the complex supply chains.The synergy of these organizations was mutually beneficial to all the parties. Collaboration in supply chain planning, process development and service execution fetched superior results. Due to this synergy Atlas was able to leverage the expertise of Global’s supply chain & Global was in a position to gain a broader insight of Atlas’s requirements and supply chain capabilities. USTSC experienced a synergistic effect because of the Atlas-Global team and experienced more effective customer-supplier 3PL working model. Thus the talent gap was bridged by USTSC . Comment on the effectiveness of Atlas’ efforts to improve team-based strategic collaboration ? With the synergy of Global and Atlas, Atlas was in a position to leverage the expertise of Global’s supply chain & Global was able to derive a broader insight of Atlas’s requirements and supply chain capabilities. Alas-Global USTSC was able to develop team-focused, strategy-driven relationships that were unique in the 3PL industry. The organizations consistently maintained an active & effective coordinated participation across the supply chain sphere.Interactive teams from the three organizations facilitated the planning, developed and executed the key supply chain activities in order to achieve superior supply chain. The collaboration in team was very effective and was result oriented. The SCM team and the steering committee used to do brainstorming sessions to figure out new supply chain initiatives & at the other end the operations team and the SCM team evaluated and ensured the potential projects that could be implemented effectively and are favorable.The relationship building between the organizations was effective and there was a strong executive support from the top management. The organizations attained mutual benefits and they experienced more effective customer-supplier 3PL working model. There was effective synchronization in the strategies with regards to process design and execution. Allocation of resources was well planned and the responsibilities were well defined. More focused on the opportunities available. The integration of the team performance was able to create superior supply chain capabilities within the industry. . As O’Brien, what strategies would you recommend to Atlas to: A) Gain greater control over inventory; B) Optimize the flow of products in the supply chain. In ord er to gain control over inventory & to optimize the flow of products in supply chain: Implementing a centralized system within the 33 company owned regional distribution centers so that the information regarding supply chain, any innovations and critical information are accessible to everyone at one single point of time.Effective integration of the 4 third party logistics providers’ services would generate superior results. Forecasting should be done collaboratively by the Atlas sales group and manufacturing group in order to arrive at accurate estimates. Implementation of the ERP system like SAP would make it easy to figure out inventory analysis.A centralized system like SAP would also create transparency, easy availability of data, accessible to concerned department in a timely manner, constant flow of information, demand and supply of inventory can be balanced and can avoid instances like stock outs or over inventory carrying costs, more reliable and realistic, helps in d ecision making process, implementing a centralized approach in the transportation system, a well defined standard service parameter should be set and should get authorized by the compliance, regular audits and inspections should be done in order to check if there are any violations.

Emotion is not very strong Essay

In this research, the correlation coefficient is +0.0563 and this number is closer to +1. It means these two variables do have positive correlation but just a slight positive correlation. In other words, if one variable is large the other one will tend to be large. However, +0.0563 is not very close to +1 and it does not reach half of 1, so it does not mean the more chocolate people have, the happier they will be. The +correlation can prove chocolate can have a slight positive effect on emotion and when people are happy they would like to eat chocolate to share the happiness. The data in the first three questions proved that most people like chocolate because of its taste, and eat it very often. However there is still 20% of people do not like it and do not eat it very often. By considering the fourth question, half of people think that chocolate could make them feel better when they are sad. It means chocolate does affect emotions, and help people escape bad mood. For the last question, 8 out of 10 agreed that eating chocolate is useful to reduce the stress and make people feel free. There is a research which was done by Gordon Parkey, (School of psychiatry, University of New South Wales), and they wrote the following result:  Ã¢â‚¬Ëœ Chocolate can provide its own hedonistic reward by satisfying cravings, but when consumed as a comfort eating or emotional eating strategy, is more likely to be associated with prolongation rather than cessation of a dysphonic mood. Any mood benefits of chocolate consumption are ephemeral.’  So it could be a kind of evidence that chocolate could affect emotion. And it indicates that chocolate has a short-time effect on emotion, but it could not last too long. Summary of analysis:  To sum up, the correlation between chocolate and emotion is positive, but it is not very high. By analyzing the data, chocolate could affect emotion and it may useful to help people to reduce stress and feel free. So the two hypothesizes are correct. However, it has to be admitted that the correlation coefficient is low. So the function of chocolate may be not very efficient for emotion. Conclusion In conclusion, this report uses the survey studies to investigate the correlation between chocolate and emotion.10 questions were made for 10 participants. The correlation coefficient is not very high, and it means although chocolate can affect emotion, it just would be a slight effect. The data indicates that chocolate can help people to be relax and happy.  However, in this research, the correlation between chocolate and emotion is not very strong. The reasons for the low correlation coefficient are various. Firstly, the number of participants is not enough. It is too small to get the general conclusion. Second reason is most of participants are Chinese. Compared with people in western countries, Chinese are not very fond of eating sweets. It is the cultural difference. The third reason is the design of this questionnaire is a little simple, and questions are not enough to investigate such a huge item.

This Is Architecture Trying to Understand Frank Gehry