Background: Previous research indicates that total cholesterol levels increase with age during young adulthood and middle age and decline with age later in life. This is attributed to changes in diet, body composition, medication use, physical activity, and hormone levels. In the current study we utilized data from the Framingham Heart Study Original Cohort to determine if variations in apolipoprotein E (APOE), a gene involved in regulating cholesterol homeostasis, influence trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio from midlife through late life. Methods: Cholesterol trajectories from midlife through late life were modeled using generalized additive mixed models and mixed-effects regression models. Results: APOE e2+ subjects had lower total cholesterol levels, higher HDL cholesterol levels, and lower total: HDL cholesterol ratios from midlife to late life compared to APOE e3 and APOE e4+ subjects. Statistically significant differences in life span cholesterol trajectories according to gender and use of cholesterol-lowering medications were also detected. Conclusion: The findings from this research provide evidence that variations in APOE modify trajectories of serum cholesterol from midlife to late life. In order to efficiently modify cholesterol through the life span, it is important to take into account APOE allele status. There is a negative perception associated with cholesterol among the general public due to the frequently observed relationship between high cholesterol and adverse health events, such as heart attack and stroke . This pePrevious research indicates that total cholesterol levels and LDL cholesterol levels increase with age among young and middle age adults and decline with age later in life ,22,23,24APOE), a gene involved in regulating cholesterol homeostasis.Previous studies have reported that the effect of age on cholesterol remains significant even after adjusting for changes in diet, physical activity and the use of statins and other lipid lowering medications  suggestiAPOE. APOE is located on the long arm of chromosome 19 (19q13.2) and includes three common alleles  resulting in six distinct genotypes. The allelic frequency of the APOE e2, e3, and e4 alleles among Caucasians is approximately 7%, 78%, and 15%, respectively [APOE genotypes effect the rate of cholesterol absorption [APOE e3/e3 genotype tend to have lower total cholesterol than adults with either the APOE e3/e4 or APOE e4/e4 genotypes, but higher total cholesterol compared to adults with either the APOE e2/e4, e2/e3 or e2/e2 genotypes [APOE e2/e2 genotype are at an increased risk for type III hyperlipidemia [Several genes encode proteins that are critical to the absorption, transport, and excretion of cholesterol ,42. One ectively . The apoectively . While tsorption ,46 adultenotypes ,48,49. Wipidemia , a condiAPOE genotypes and serum cholesterol has led to considerable research on the relationship between APOE polymorphisms and diseases associated with high cholesterol. The APOE e4 allele is associated with an increased risk for cardiovascular diseases [APOE e4 allele is also an established risk factor for dementia, in particular Alzheimer’s disease (AD) [APOE e2 allele is associated with a decreased risk for AD relative to the APOE e3 allele [APOE has contributed to research on the relationship between serum cholesterol levels and AD risk. Interestingly, the association between serum cholesterol and AD appears to depend upon the age in which cholesterol is measured. High total cholesterol levels during middle age are associated with an increased risk for AD during old age [et al. [APOE allele status. This justifies research to examine if trajectories of serum cholesterol from midlife through late life differ according to APOE allele status. The observed relationship between diseases , includidiseases , and cordiseases . The APOase (AD) , whereas3 allele . The con old age , but old old age  and high old age . Further [et al.  observedAPOE genotypes. However, it is not clear how APOE polymorphisms influence the longitudinal trajectories of cholesterol from midlife through late life. Accordingly, the purpose of this study is to characterize the longitudinal trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio according to APOE e2, e3, and e4 allele status utilizing data from the Framingham Heart Study (FHS) Original Cohort.The apoE alleles clearly modulate cholesterol homeostasis as indicated by the differences in cholesterol concentrations among adults according to APOE genotype data is available for a total of 663 subjects. For the purposes of the current study, subjects were classified as APOE e2+ (e2/e2 or e2/e3 genotype), APOE e3 (e3/e3 genotype), or APOE e4+ (e3/e4 or e4/e4). Subjects with the APOE e2/e4 genotype (n = 11) were excluded from the final sample due to the low sample size and conflicting influence that the APOE e2 and APOE e4 alleles have on cholesterol levels. Because the purpose of this study was to examine cholesterol trajectories beginning in midlife, nine subjects that were not between 30 and 55 years of age during the first clinical examination were excluded from the final sample. Finally, all subjects had two or more repeated measures for total cholesterol; 21 subjects who had fewer than two consecutive repeated measures for HDL cholesterol were excluded from the final sample. The final sample included 596 subjects. The FHS is an ongoing prospective cohort study of residents from Framingham Massachusetts, United States, created with the goal of generating knowledge on the onset and progression of cardiovascular diseases, as well as genetic and environmental risk factors for these diseases. The FHS Original Cohort was initiated in 1948 and includes adults who did not have a cardiovascular disease upon entry into the study . A totalMeasures for total cholesterol and HDL cholesterol were collected from subjects of the FHS Original Cohort using standard laboratory procedures . FastingAPOE allele status were modeled using generalized additive mixed models [ith subject during the jth observation given the covariates included in the model (see Covariates), and bi is a subject-specific random effect that allows for the baseline value of cholesterol to vary for each subject. Xi is a k-length vector comprising the k time-invariant covariates of subject i and β is a k-length vector of the fixed effects for the corresponding k covariates; xij is the age of the ith subject during the jth observation; se2, se3, and se4 are the allele-specific smoothing functions for subjects who were APOE e2+, e3, and e4+, respectively, APOEi is the allele status for the ith subject; and I allows for the trajectories of cholesterol to vary according to APOE allele status. The smoothing function (thin plate regression splines) models the potentially non-linear trajectory of cholesterol with advancing age. An advantage of the thin plate regression spline function over other smoothing functions is that the number and placement of knots that control the flexibility of the model do not need to be specified [is by identifying the function f; and f.The trajectories of total cholesterol levels, HDL cholesterol levels, and total: HDL cholesterol ratios from midlife through late life according to d models  (GAMM) id models  in R verpecified . The morpecified  providinpecified . A detaipecified . BrieflyAPOE allele status to be visually examined. The plots of the estimated trajectories for the adjusted models were obtained by predicting the average values of serum cholesterol based on age and the values of the covariates included in the model. These trajectories represent the average trajectory of serum cholesterol according to APOE allele status controlling for the effects of the covariates. The degree to which the trajectories of cholesterol departed from linearity is reflected by the effective degrees of freedom (EDF) of the smoothing parameter  . The EDFAPOE allele status or other variable of interest. Since the EDF of the smooth term can be interpreted as an x degree polynomial, we used the EDF values obtained from the GAMM as estimates for polynomial terms in mixed-effects regression models [GAMM allow for between group comparisons to be made based on the 95% confidence intervals of each measure of serum cholesterol, but a challenge when using this method is assessing statistically significant differences in the estimated trajectories of serum cholesterol according to n models . This alAPOE allele status. Age during the baseline clinical examination was included as a covariate to differentiate between-subject from within-subject variability for the change in serum cholesterol with age. Educational attainment was initially recorded according to the following categories: (1) none, (2) fourth grade or less, (3) fifth, sixth or seventh grade, (4) completed grade school, (5) some high school, (6) graduated high school, (7) some college, (8) college graduate or(9) post graduate. For the purposes of this study, educational attainment was dichotomized according to receiving a high school (HS) degree (≤HS or >HS). From clinical examination 7 through 21, subjects were asked if they were using cholesterol-lowering drugs, and it was not until clinical examination 22 that subjects were asked which type of medication they used . Since specific cholesterol-lowering medications were not assessed until the 22nd clinical examination, subjects were dichotomized as having reported ever using a cholesterol-lowering medication (yes/no). The age in which subjects first started using cholesterol-lowering medications was included as a covariate in a subsequent analysis to account for differences in the timing in which subjects started taking cholesterol-lowering medications. Also, an analysis limited to only women that controlled for the use of supplemental estrogen and age of menopause was also conducted. During the 1st through 14th clinical examinations, women were asked the age in which menses had stopped for one year or more and the cause of cessation . The average age of menopause was 47.5 years , but significant differences in the average age of menopause according to cause of cessation were detected  49.9 years, SD = 3.4; surgery (n = 108) 42.3 years, SD = 7.1; other (n = 3) 41.7 years, SD = 7.6; F-value = 92.9, p < 0.01). Therefore, the cause of cessation of menses was included as a covariate in this analysis. The use of supplemental estrogen  was assessed during clinical exams 17–27. Subjects were dichotomized as having reported ever using supplemental estrogen (yes/no). Subjects were asked during clinical exams 2, 4, 7–15, and 17 if they were consuming a diet meant to control high cholesterol, diabetes, hypertension, weight, or other low calorie diet. Subjects were dichotomized as reported ever consuming a restricted diet (yes/no). Systolic blood pressure, diastolic blood pressure, height (inches) and weight (pounds) were collected during each clinical examination. Smoking status was assessed during clinical exams 4–27. Blood sugar levels were measured during clinical exams 1, 2, 4, 6, 8–23, 26 and 27. Body mass index (BMI) was calculated as (weight/height2) × 703. Smoking status [Data for several potential confounding variables were collected during each clinical examination using standard laboratory procedures and a medical history interview. An overview of the covariates included in the present analysis and the years in which they were collected is provided in g status , BMI [72g status  have beeg status ,75. A toAPOE e2+ , 406 were APOE e3, and 129 were APOE e4+ . The final sample was predominantly female and the majority of subjects had a high school degree or less. There were no statistically significant differences in APOE allele status according to any of the characteristics listed in 2χ = 44.4, p < 0.01) and late life (men = 79 (33.3%), women = 74 (21.3%), 2χ = 56.6, p <0.01), had high average BMI during midlife  and late life , higher average blood sugar during late life, but not midlife, , higher average systolic blood pressure during midlife, but not late life, , and higher average diastolic blood pressure during midlife  and late life .The descriptive characteristics of subjects of included in the final sample are provided in APOE allele status, are presented in APOE status and the shaded regions represent the point-wise 95% confidence intervals for each estimated measure. The degree of non-linearity for the trajectories as reflected by the estimated EDF of the smoothing term, and the model fit by adjusted R2, which is interpreted as the proportion of total variance explained by the model with an adjustment for model complexity, are presented in The unadjusted and adjusted trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio, according to APOE e2+, e3, and e4+ subjects all followed similar non-linear trends based on the EDF values  and quartic terms, while the cubic term approached statistical significance.Total cholesterol trajectories for APOE e2+ subjects had higher HDL cholesterol levels compared to APOE e3 and APOE e4+ subjects  and cubic  main effect polynomial terms were statistically significant.In general, subjects . APOE e2APOE e2+ subjects maintained significantly lower total: HDL ratios from midlife through late life compared to APOE e3 and APOE e4+ subjects and these differences increased with advancing age. There were little to no differences in the trajectories of total: HDL cholesterol ratio according to APOE allele status. A mixed-effects model that included linear through quartic polynomial terms for age and interactions with APOE allele status detected a statistically significant interaction between age and APOE allele status . The respective interactions between APOE allele status and the other three polynomial terms were not statistically significant. All subjects maintained consistent total: HDL cholesterol ratio until 70 years of age when the trajectories began to decline . APOE e2APOE allele status by linear , quadratic , and cubic  interaction terms. The trajectories of serum cholesterol levels stratified according to use of cholesterol-lowering medications are presented in the first row of p = 0.01) and cubic trends  in HDL cholesterol according to age for all subjects, but only the APOE allele status by age interaction term was statistically significant . Medication users had higher HDL cholesterol levels during midlife compared to non-medication users. Medication users, however, did have a slight decline in HDL cholesterol until 80 years of age, whereas non-medication users displayed a gradual increase until 60 years of age, which was followed by a slight decline. Both medication users and non-users exhibited an increase in HDL cholesterol levels following 80 years of age. These findings are supported by the results of a mixed-effects regression model that included linear, quadratic, and cubic polynomial terms for age. There were significant linear .Finally, subjects with a history of using cholesterol-lowering medications maintained significantly higher total: HDL cholesterol ratio compared to non-medication users from midlife through late life row 1, . HoweverAPOE allele status adjusting for the effects of age of first using cholesterol-lowering medications. APOE e2+, e3, and e4+ subjects had similar trajectories of serum total cholesterol through 50 years of age. Total cholesterol continued to increase among APOE e4+ subjects until 60 years of age, whereas total cholesterol began to decline at 50 years of age for APOE e3 and APOE e2+ subjects. The EDF values for the smooth terms were similar for APOE e4+, e3, and e2+ subjects (p = 0.04) and quartic  polynomial terms according to APOE allele status while the cubic interaction term approached statistical significance .The second row of subjects . HoweverAPOE e4+ had significantly higher HDL cholesterol levels during midlife compared to APOE e3 and APOE e2+ subjects , but there was not a statistically significant interaction between age and APOE allele status. row two, . APOE e4ic trend . There wAPOE e4 allele status .There was substantial overlap in the 95% confidence intervals for estimated values of total: HDL cholesterol ratio according to row two, . Based orow two, , APOE e4ajectory . The ratAPOE allele status. This model detected statistically significant differences in the linear , quadratic , quartic , and quintic  polynomial terms according to APOE allele status.Cholesterol trajectories stratified according to gender are presented in the first row of APOE allele status supports this finding. There were no significant differences in any of the polynomial terms included in this model according to APOE allele status. Both men and women maintained consistent levels of HDL cholesterol  and the quadratic interaction term approached statistical significance .Finally, men maintained significantly higher total: HDL cholesterol ratio compared to women from midlife through late life , and less likely to have been a smoker during midlife (n = 95 (72.5%), n = 189 (42.4%), 2χ = 37.8, p < 0.01) and late life (n = 57 (43.8%), n = 96 (21.1%), 2χ = 30.7, p < 0.01). An explanation for the observed decline in total cholesterol levels, increase in HDL cholesterol levels and decline in total: HDL cholesterol ratio is that subjects with healthy cholesterol levels are more likely to reach very old age. The majority of subjects included in the final sample lived beyond 80 years of age , which included 133 subjects who lived past 90 years of age. Compared to subjects who did not live beyond 80 years of age, subjects who were 80 years of age or older were more likely to be female (n = 290 (63.0%), n = 64 (47.1%), Since there were statistically significant differences in the characteristics of the final sample according to longevity, a subsequent analysis was conducted in which the trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol were stratified according to longevity . These models adjusted for the effects of age, educational attainment, smoking, systolic and diastolic blood pressure, blood sugar, BMI, restricted diet, and use of cholesterol-lowering medications. p < 0.01), quadratic , quartic , and quintic  polynomial terms. The trajectories for total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio are presented in the first row of p < 0.01), but there was not a statistically significant interaction between age3 and longevity. There were little to no differences in the estimated trajectories of HDL cholesterol according to longevity. This is supported by the findings from a mixed-effects model that included linear, quadratic, and cubic polynomial terms for age and interactions with longevity status. In this model, the cubic polynomial term was statistically significant . Finally, subjects who did not live past 80 years had a linear decline in total: HDL cholesterol ratio, whereas total: HDL cholesterol ratio for subjects who lived past 80 and 90 years of age did not begin to decline until approximately 65 years of age. Interestingly, subjects who lived past 90 years of age had low total: HDL cholesterol ratio that increased until approximately 60 years of age. This visibly apparent difference in the trajectory of total: HDL cholesterol ratio is detected in a mixed-effects model that included linear, quadratic, and cubic terms for age in which the interaction term between ageAPOE allele status differed once subjects who did not live beyond 80 years were removed. These models adjusted for the effects of age, educational attainment, smoking history, average systolic and diastolic blood pressure, average blood sugar, average BMI, restricted diet, and use of cholesterol-lowering medications. It should be noted that APOE e3 subjects remained in the FHS Original Cohort longer compared to APOE e2 and APOE e4 subjects as indicated by the five more years of observation for APOE e3 subjects  and quartic  polynomial terms for age. As was observed in the initial analysis, there were no statistically significant interactions between any of the polynomial terms for age and APOE allele status in the mixed-effects model for HDL cholesterol. Finally, the mixed-effects model of total: HDL cholesterol for subjects who lived beyond 80 years of age detected a statistically significant interaction between age and APOE allele status , whereas the interactions between APOE allele status and other polynomial terms  were not statistically significant. This is consistent with the results from the mixed-effects model that included the entire final sample. To summarize, these findings provide evidence that the results from the initial analysis are not due to a healthy survivor bias. A final analysis limited to the 460 subjects who lived beyond 80 years of age  was conducted to assess if trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio according to subjects . There wsubjects ; Table 6subjects ; Table 3APOE allele status. Results from the GAMMs indicated that APOE e2+ subjects maintained lower serum total cholesterol levels, higher HDL cholesterol levels, and lower total: HDL cholesterol ratios from midlife through late life compared to APOE e3 and APOE e4+ subjects after controlling for the effects of age gender, educational attainment, smoking, use of cholesterol-lowering medications, systolic and diastolic blood pressure, blood sugar, diet restriction, and BMI. Similar findings were observed when subjects who did not live beyond 80 years of age were excluded. Further analysis using mixed-effects regression models detected statistically significant interactions between polynomial terms for age and APOE allele status for trajectories of serum total cholesterol. This provides evidence that trajectories of serum total cholesterol from midlife through late life differ according to APOE allele status. However, a mixed-effects regression model for HDL cholesterol did not detect any statistically significant interactions between APOE allele status and polynomial terms for age. This suggests that there is a between group effect for APOE on HDL cholesterol levels, but there are not substantial differences in HDL cholesterol trajectories according to APOE allele status. Finally, there was a statistically significant interaction between age and APOE allele status in the mixed-effects model for total: HDL cholesterol ratio, but the interactions between APOE allele status and quadratic, cubic, and quartic polynomial terms were not statistically significant. These findings, when interpreted with the results from the GAMM, indicate there is a significant between group effect for APOE allele status on total: HDL cholesterol trajectories and that APOE e2+ subjects have a greater overall decline in total: HDL cholesterol ratio than APOE e3 or APOE e4+ subjects. The findings from this study present new evidence for age related changes in total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio according to APOE allele status. In the current study, serum total cholesterol levels increased with age during midlife but declined during old age. These results are consistent with other studies that have examined age related changes in serum total cholesterol [APOE allele status. This increasing trend in HDL cholesterol with age as been reported previously [Prior research has focused on age-related changes to total and HDL cholesterol levels through the lifespan and the present analysis is the first study to our knowledge that has examined trajectories of serum cholesterol from midlife through late life stratified according to lesterol ,22,24,25eviously  in HDL cAPOE allele status were not the objective of the present analysis. Future research should examine how sex may modify the relationship between APOE allele status and trajectories of serum cholesterol levels from midlife through late life.Previous studies have also assessed sex differences in serum cholesterol levels. The findings from the GAMM and mixed-effects models provide evidence for significant differences in the trajectories of serum cholesterol between men and women. Women had lower serum total cholesterol during midlife, but higher total cholesterol during late life compared to men. Women also had significantly higher HDL cholesterol and total HDL cholesterol and lower total: HDL cholesterol ratio from midlife through late life compared to men. These results are consistent with previous research ,76. It sThe decline in total cholesterol with increasing age among older adults observed in the current study may be the result of a survivor effect in which subjects at an increased risk for mortality due to diseases caused by elevated cholesterol die and subjects with healthy cholesterol levels remain in a study into old age . In the Certain limitations in this study need to be acknowledged. First, we limited the number of covariates included in the adjusted models by using midlife and late life values for potentially important covariates such as smoking status, measures for systolic and diastolic blood pressure and blood sugar, use of cholesterol-lowering medications, and BMI. This approach allowed us to account for changes in these covariates from midlife through late life, but may have oversimplified the relationship between these variables and trajectories of serum cholesterol levels. Furthermore, we controlled for the use of cholesterol-lowering medications by dichotomizing subjects as ever using medications . It has been suggested that adding a constant value to observed values for subjects receiving treatment is more appropriate when controlling for treatment effects than including treatment status as a dichotomous covariate . These lAPOE allele status. Between-group differences in HDL cholesterol according to APOE allele status were also detected. In summary, this study presents evidence that serum total cholesterol and total: HDL cholesterol ratio trajectories from midlife through late life differ according to APOE e4 allele is associated with an increased risk for several aging related diseases, including AD [APOE e2 allele has been associated with a decreased risk for these diseases [APOE allele status and how APOE allele status influences serum cholesterol levels from midlife through late life. A plausible hypothesis is that the increased risk for cognitive and vascular diseases among older adults who carry one or more APOE e4 alleles may be due, in part, to higher total cholesterol and lower HDL cholesterol from midlife through late life, whereas the decreased risk for these diseases associated with the APOE e2 allele may be due to the lower total cholesterol and higher HDL cholesterol across the life span. This hypothesis is based on interpreting the findings of the present analysis within the context of findings from previous research. Additional research is necessary to determine if reducing total cholesterol and increasing HDL cholesterol decreases the risk for cognitive and vascular diseases among adults who carry one or more APOE e4 alleles.The findings from this study have important implications to public health. First, we observed that adults who lived past 90 years of age had higher total cholesterol during late life compared to adults who did not live past 80 or 90 years of age. This has potentially important implications for the treatment of high cholesterol with statins and other cholesterol-lowering medications because these findings suggest that higher cholesterol may be associated with longevity. An argument can be made that it may be harmful to prescribe older adults statins or other medications to lower cholesterol based on evidence that low serum cholesterol and a decline in serum cholesterol are associated with increased mortality ,90. Howeuding AD  and carduding AD , whereasdiseases . To effi


Thalassemia and thalassemic hemoglobinopathies pose serious health problem leading to severe morbidity and mortality in Indian population. Plethora of hemoglobin variants is prevalent in multiethnic Indian population. The aim of the present study was to analyze laboratory aspects, namely, hematological profile and HPLC findings of the hemoglobin variants detected, and to discuss problems that we faced in diagnosis in a routine clinical laboratory. We screened a total of 4800 cases in a hospital based population of North India in a 2-years period of by automated HPLC method using the Variant Hemoglobin Testing System  under the experimental conditions specified by the manufacturer. Whole blood in EDTA was used and red cell indices were determined using automated hematology analyzer. We detected 290 cases with abnormal variants in which beta thalassemia was the most common followed by hemoglobin E. Here, we discuss the laboratory aspects of various hemoglobin disorders and diagnostic difficulties in cases like borderline HbA2 values, presence of silent mutation, alpha thalassemia gene, and few rare variants which at times require correlation with genetic study. Special attention was given to HbA2 level even in presence of a structural variant to rule out coinheritance of beta thalassemia gene. Thalassemia is an autosomal recessive inherited group of disorders of hemoglobin synthesis characterized by the absence or reduction of one or more of the globin chains of hemoglobin. The structural variants result from substitution of one or more amino acids in the globin chains of the hemoglobin molecule . PlethorDetection of asymptomatic carriers by reliable laboratory methods is the cornerstone of prevention of this serious health problem. Cation exchange high performance liquid chromatography (CE-HPLC) has become the preferred technique suitable in Indian scenario, as it can detect most of the clinically significant variants. The simplicity of the automated system with internal sample preparation, superior resolution, rapid assay time, and accurate quantification of hemoglobin fractions makes this an ideal methodology for the routine clinical laboratory , 3.With increasing global awareness and mass screening programs undertaken at various levels by health care system, the responsibility for laboratory personnel has greatly enhanced in detection and prevention of this problem. Awareness about the diagnostic problems as well as their solutions is very important so that one does not miss a single case.Many studies have been published from India on thalassemia and thalassemic hemoglobinopathies mostly putting emphasis on epidemiology and screening –6.Very few studies are available on the approach of diagnosis and problems in routine diagnosis, especially in laboratories with limited resources.The aim of the present study was to analyze laboratory aspects, namely, hematological profile and HPLC findings of the hemoglobin variants detected, and to discuss problems that we faced in diagnosis in a routine clinical laboratory.μl) not responding to conventional treatment, clinically suspected cases of hemoglobinopathy, antenatal, and other cases coming to the department for thalassemia screening. A 5 mL intravenous blood sample was collected in EDTA anticoagulant. Red cell indices were measured on an automated haematology analyzer (Sysmex KX 21).This was a prospective study carried out in the Department of Pathology and Thalassemia Control Cell, Hindu Rao Hospital, Delhi, for 2 years' period. A total of 4800 cases were screened for presence of thalassemia or any structural variant. These included all cases of microcytic hypochromic anaemia  under the experimental conditions specified by the manufacturer .The Variant II Beta Thalassemia Short Program utilizes principles of ion-exchange high-performance liquid chromatography (HPLC). The samples are automatically mixed and diluted on the Variant II Sampling Station (VSS) and injected to the analytical cartridge. The Variant II Chromatographic Station (VCS) dual pumps deliver a programmed buffer gradient of increasing ionic strength to the cartridge, where HbA2/F are separated based on their ionic interaction with the cartridge material. The separated HbA2/F then pass through the flow cell of the filter photometer where the changes in absorbance at 415 nm are measured. An additional filter at 690 nm corrects the background absorbance. The Variant II CDM (CDM) Software performs reduction of raw data collected from each analysis. To aid in the interpretation of results, windows have been established for the most frequently occurring hemoglobins based on the characteristic retention time. For each sample a sample report and a chromatogram are generated by CDM showing all hemoglobin fractions eluted, their retention times, the area of the peaks, and values of the fractions.Elution buffers : sodium phosphate buffer.Whole blood primer: lyophilized human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as preservative.HbA2/F calibrator/diluent set: lyophilized human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as preservative analytical cartridge. Diluent contains deionized water.Wash/diluent solution: deionized water.Control: normal  and abnormal  controls.μL of sample in the tube. In such case, sample was manually prediluted. Predilution was carried out by mixing 1.0 mL wash/diluents with 5 μL of whole blood sample.Five milliliters (5 mL) of whole blood was collected in a vacuum collection tube containing EDTA which can be stored at 2–8 degrees C for maximum 7 days if processing is delayed. No preparation was required unless the sample was in a tube other than the recommended tube or there was less than 500 HbA2/F calibrators and normal and abnormal controls were analyzed at the beginning of each run.Reports and chromatograms generated were studied and interpreted by observing HbA2 and F concentration for beta thalassemia and retention time and area percentage of other peaks and windows for structural variants. Each chromatogram shows peaks of Hb A0, A2, and Hb F along with C window, D window, S window, and two minor peaks, P2 and P3. Several hemoglobin variants elute same window; they were provisionally diagnosed by retention time and area percentage keeping in mind the ethnicity of the patients.Other relevant tests were done, for example, sickling test as supporting evidence of Hb S. Family study was carried out whenever possible and correlation with findings of Hb electrophoresis result was done in few cases.Among 4800 cases screened, 290 6.04%) cases were detected with abnormal hemoglobin in this study. Presumptive identification of hemoglobin variants was made primarily by retention time (RT) windows and area percent; however geographical factor, ethnicity, and clinical presentation were also taken into consideration. Distribution of hemoglobin variants identified is shown in .04% caseGenotypes of some structural variants are shown in As expected, beta thalassemia trait (BTT) was the most common hemoglobin variant (74.48%) detected in our study with elevated HbA2 level (>3.5%) and RT 3.63–3.69 min. Majority were asymptomatic and detected during carrier screening and family studies.Nine cases of beta thalassemia were detected which were classified as either major or intermedia depending upon clinical severity. HbF was raised (20.7–97.1%), with variable HbA2 (2.4–5.7%). Cases of thalassemia major presented within 1st year of life. Parental study was done to find out carrier status and to confirm the diagnosis.Three cases showed raised HbF (13.7–19.7%), normal HbA2 values (2.2-2.3%), and normal Hb level, raised RBC count, and reduced MCV and MCH levels. Parental study was done with a provisional diagnosis of delta beta thalassemia trait and DNA study was advised.Hemoglobin E (HbE) was found to be the most common structural variant with raised peak in A2 window (RT 3.76–3.78 min). A total of 28 cases of Hb E trait were detected with peak area ranging from 18.5 to 39% and mild elevation of HbF level. Two cases of homozygous Hb E were detected showing 77–83% HbE and 2–5% HbF. Hb level was mildly reduced with raised RBC counts and reduced MCV and MCH values. Parental study was carried out for confirmation.Two cases of double heterozygous of HbE and beta thalassemia trait were found showing peak in HbA2 window (53.5–69.7%) and raised HbF level (5–14.5%). Again, parental and family study was carried out for confirmation.Nineteen cases showed peak in the D window (RT 4.13–4.15 min), indicating presence of structural variant hemoglobin D (Hb D) Punjab. Among these, 15 were HbD Punjab heterozygote showing Hb D level 31–40% and near normal RBC parameters. Four cases had HbD level 77–81.3%, with raised HbA2 levels (3.8–6%). These cases showed reduced hemoglobin, MCV, and MCH levels. They were provisionally diagnosed as double heterozygous of Hb D and beta thalassemia trait. Parental study and genetic study were done for confirmation.Nine cases had peak in S window (RT 4.27-4.28 min), indicating presence of hemoglobin S (HbS). Eight of them had HbS level 25–40%, HbF-0.2–7.4%. Mildly raised HbA2 (3.3–3.7%) level was seen in 5 of them. There was one case with marked reduction in Hb, MCV, and MCH levels and HbA2 10% and HbS 65.4%. Parental study was advised to confirm the double heterozygous status. Sickling test was found to be positive in all these cases.One case showed elevated P3 peak 20%, with RT 1.81 min, suggesting Hb J Meerut with reduced MCV and MCH values. Iron studies and family study were correlated.One case showed elevated peak (RT 1.37 min), 48.8% with normal hematological parameters. It was provisionally diagnosed as Hb Hope after further confirmation by hemoglobin electrophoresis. Family study and DNA analysis were advised.India is an ethnically diverse country with marked regional variation. This diversity is reflected in the presence of different hemoglobin variants in different ethnic groups. Due to migration, there is constant mixing of peoples from different regions. Many of these abnormal variants are of little clinical significance in heterozygous state, but when combined with other variants they may give rise to severe disease. Therefore there is always a need for a screening method which can detect maximum variants. HPLC has the advantage of quantifying Hb F and Hb A2 along with detecting other variants in a single screening test.Besides HPLC, there are other analytical procedures used for detection of thalassemia and hemoglobinopathies such as alkaline and acid electrophoresis, Hb A2 quantification by ion-exchange column chromatography, and Hb F quantification by alkali denaturation and radial immunodiffusion (9). Electrophoretic method does not separate all variants from each other, and it is recommended by screening programs such that further tests should always be carried out to confirm the presumed identity of an abnormal variant. None of the above-mentioned methods can detect multiple hemoglobin fractions in a single step procedure. HPLC has many advantages over these methods and over the past decades it has evolved as an excellent and powerful diagnostic tool for identification of most of the clinically significant Hb variants especially to beta thalassemia trait owing to its quantitative power and automation. HPLC is sensitive, specific, reproducible, and less time consuming and requires less man power. Hence, it is ideal for a routine clinical laboratory with high work load.β-thalassaemia, common variants were encountered with various incidences: HbE, HbD Punjab, and HbS—being the most common along with a rare variant Hb Hope.Our study was carried out in a tertiary care hospital in Delhi, which represented not only north Indian population but a large migrant population of eastern and north eastern parts of the country also. Apart from Thalassemia being the major concern in this study, quantitation of HbA2 and Hb F levels by HPLC was of prime importance in our laboratory where facilities for genetic studies are not available. However, parental study is also of great help in arriving at a conclusive diagnosis before referring patients for costly genetic studies.HbA2 is constantly elevated in heterozygous beta thalassemia carriers with values ranging from 3.5 to 7% with a mean of 5% . During Iron deficiency anaemia is a very common occurrence in most of the screening populations, namely, school children and pregnant women in India. There are several studies regarding the impact of iron deficiency on HbA2 level and controversy over its significance in screening of beta thalassemia trait , 15, 16.The variation of cut off value as well as borderline value by the same method leads to confusion resulting in underdiagnosis or overdiagnosis. In such situations, genetic counseling with DNA analysis should be recommended. But in a resource-limited country where population is huge but facilities of genetic or molecular tests are available only in a few research laboratories, we may refer only antenatal cases for genetic studies if one partner is a trait. For other cases, we can opt for parental study if possible or iron studies before referring them for costly genetic test.On the other hand, there were few cases with symptomatic refractory microcytic hypochromic anaemia with borderline, normal, or reduced HbA2 levels. These cases should be investigated for presence of alpha thalassemia or its coinheritance with beta thalassemia gene. Alpha thalassemia is by far the commonest hemoglobinopathy in India, with highest prevalence in Punjabi population in the northern region . MoleculHb E, the most common structural variant in our study, is one of the world's important mutations. Hb E trait, homozygous Hb E disease (Hb EE), and Hb E beta thalassemia are common in north eastern part of the country . Hb E trWe observed that distinction between homozygous HbE disease and HbE beta thalassemia sometimes become difficult as both of these conditions show variably elevated HbE and Hb F with marked microcytic hypochromic picture. Hb D Iran also comes as a differential diagnosis. Compared to Hb E trait, Hb D Iran tends to have more area percentage at the window of Hb A2 . Parental study, if possible, may easily resolve the problem. Otherwise, genetic study should be done.Hb D Punjab was the second most common structural variant in our study, mostly presented as asymptomatic heterozygous condition with normal hematological parameters. Hb D Punjab occurs with greatest prevalence, that is, 2% among Sikhs in Punjab, and in Gujarat, reported prevalence is 1% . Hb D PuIt has been reported that coinheritance of beta thalassemia and HbD Punjab may result in symptomatic disease and its detection is important in prenatal diagnosis. In cases, where HPLC or Hb electrophoresis shows very high HbD level and negligible HbA, one should look for raised HbA2 level and do parental study or DNA analysis for confirmation of coinheritance.A total of nine cases of Hb S were detected of which 5 cases showed elevated Hb A2 level (3.3−3.7%) with 25–40% HbS and moderate to severe anaemia. The mean values of HbA2 have been reported to be elevated in sickle cell syndrome in previous reports , 24. Theδβ-thalassemia with β-thalassemia can result in a severe disorder.We reported 3 cases of delta-beta thalassemia trait. Delta-beta thalassemia and hereditary persistence of fetal hemoglobin (HPFH) constitute a heterogeneous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). The distinction between HPFH and delta-beta thalassemia is subtle and should be confirmed by alpha-beta-globin chain synthesis ratio and DNA analysis since the distinction between these two conditions is not always possible from routine hematologic analyses. It is important to differentiate between these two conditions especially in antenatal screening because HPFH is clinically asymptomatic, but interaction of Hb J Meerut was another rare variant detected in our study. This clinically asymptomatic variant has been reported to interfere with HbA1c estimation  and to mAmong the rarely encountered variants we detected one case of Hb Hope. In cation exchange HPLC method, it may be mistaken as high HbA1c, as it comes in the same position as HbA1c. Hb Hope (beta 136(H14) Gly → Asp (GGT → GAT)) is one of the unstable haemoglobin variants of the beta-globin chain, which has been demonstrated in people of various ethnic backgrounds. Few case reports of Hb hope and its association with thalassemia and Hb E have been published from southeast Asia , 28. ThiIt has been reported that one can detect HbH form of alpha thalassemia on HPLC by visual analysis of the chromatogram plot as it produces a sharp peak before the start of integration . We, howThis automated HPLC system is intended for separation and determination of area percentage for HbA2 and HbF and for qualitative detection of abnormal hemoglobins. With the integration of proper algorithms involving retention time, % Hb, and peak characteristics, a clinical laboratory is capable of identifying 75% of the common variants encountered without the need for confirmatory studies such as alkaline and acid electrophoresis . In the HPLC has some limitations, including falsely decreased HbA2 levels in patients with the HbD Punjab trait, falsely increased HbA2 levels in patients with HbS, and coelution of various Hbs, including HbE, Hb Osu Christianborg, HbG Coushatta, and Hb Lepore with HbA2 . InterfeTo conclude, RBC indices, HPLC finding, and family study are sufficient to detect and manage most of the hemoglobin variants prevalent in this country. However, one has to be aware of the limitations and problems associated with the diagnostic methods to avoid false negative diagnosis in day to day practice. Genetic studies are indicated to confirm borderline cases and to detect silent carriers of beta thalassemia, alpha thalassemia, and rare and novel variants in routine practice. The present study conducted using HPLC reflects the magnitude of thalassemia and hemoglobinopathies in a small hospital based population which may be in fact the tip of an iceberg, but this type of study can definitely help to increase awareness among both health care givers and general population.


The use of doubled haploids in onion breeding is limited due to the low gynogenesis efficiency of this species. Gynogenesis capacity from Spanish germplasm, including the sweet cultivar Fuentes de Ebro, the highly pungent landrace BGHZ1354 and the two Valenciana type commercial varieties Recas and Rita, was evaluated and optimized in this study. The OH-1 population, characterized by a high gynogenesis induction, was used as control. Growing conditions of the donor plants were tested with a one-step protocol and field plants produced a slightly higher percentage of embryogenesis induction than growth chamber plants. A one-step protocol was compared with a two-step protocol for embryogenesis induction. Spanish germplasm produced a 2–3 times higher percentage of embryogenesis with the two-step protocol, Recas showing the highest percentage (2.09%) and Fuentes de Ebro the lowest (0.53%). These percentages were significantly lower than those from the OH-1 population, with an average of 15% independently of the protocol used. The effect of different containers on plant regeneration was tested using both protocols. The highest percentage of acclimated plants was obtained with the two-step protocol in combination with Eco2box (70%), whereas the lowest percentage was observed with glass tubes in the two protocols (20–23%). Different amiprofos-methyl (APM) treatments were applied to embryos for chromosome doubling. A similar number of doubled haploid plants were recovered with 25 or 50 μM APM in liquid medium. However, the application of 25 μM in solid medium for 24 h produced the highest number of doubled haploid plants. Somatic regeneration from flower buds of haploid and mixoploid plants proved to be a successful approach for chromosome doubling, since diploid plants were obtained from the four regenerated lines. In this study, doubled haploid plants were produced from the four Spanish cultivars, however further improvements are needed to increase their gynogenesis efficiency. Allium cepa L.) is a valuable crop for food and medicinal purposes, ranking second after tomato in the list of vegetables cultivated worldwide, with production of over 90 million tons on 4.7 million ha  plants. Two gynogenesis induction protocols, previously described in the literature, different plant containers for plant regeneration, and APM treatments for chromosome doubling, were assayed. Somatic regeneration was also tested as an alternative approach for chromosome doubling of haploid (H) gynogenetic plants.Research was carried out from 2012 to 2014 in experimental plots in a shade house located at 41°39′N latitude. In 2012, two Valenciana type commercial varieties Recas (Veronsa) and Rita (kindly provided by Ramiro Arnedo S.A.) were used, as well as a half-sib family from the breeding program carried out with the cultivar Fuentes de Ebro, a landrace known for its mild and sweet flavor , coconut (30%), sand (20%), and N:P:K (14:16:18) with micronutrient fertilizer . In April, the plantlets were transplanted directly to soil in the field under natural conditions and bulbs were collected for repose in September. In order to obtain flower heads, the bulbs were planted in November in plastic pots (4 l) and grown in the field under natural conditions. One month before bolting, some bulbs were transferred to a growth chamber exposed to 16 h photoperiod at a continuous temperature of 15–18°C and an illumination of 300 μmol mIn February 2012, seeds of donor plants from Fuentes de Ebro, BGHZ1354, Recas, and OH-1 were sown, grown and harvested as described previously. In November 2012, the bulbs were transplanted directly to soil in the field under natural conditions for inflorescence development in 2013. Whole umbels were harvested from mid-May to the end of June.−1 dichloroisocyanuric acid disodium salt with two drops of Tween 80 for 10–12 min and followed by 4–5 rinsed with sterile distilled water.The whole umbel was harvested when 30% of the flowers were at three to 4 days before anthesis Figure . Flowers1 medium  was applied in liquid or solid elongation medium and consisted of BDS (x1/2) with 15 g lIn 2013, two experiments were performed. In the first, glass tubes were sealed with plastic or cellulose plugs . In the second, glass tubes with plastic plugs were compared with Magenta boxes and polypropylene boxes with an aeration filter . Two to three plants were plated in each Magenta box and 4–6 plants in the Eco2box. Good quality embryos from the OH-1 population were randomly distributed in different containers.In 2012, APM treated embryos were cultured in solid elongation medium in individual glass tubes with plastic plugs (150 × 25 mm). In 2013, glass tubes with plastic glass and Eco2boxes were used for plant regeneration from Spanish germplasm.−2 s−1 provided by Phillips Master TLD Super 80 58 W/840 and OSRAM 30 W/2700 K.Flower buds and plantlets were incubated in a growth chamber at 24°C with 16 h photoperiod and 100 μmol mAfter 6–10 weeks in elongation medium, rooted, healthy plants were transferred to 110 mm Ø pots containing soil (3:2 peat:vermiculite), covered with a plastic glass for 10 days and planted in a greenhouse. Plants were watered with Hoagland nutrient solution   and filtered through a 30 μm nylon filter. Samples were analyzed in a PAS cytometer (Partec). Leaves from young seedlings were used as control.The following variables were calculated: percentage of gynogenesis induction , percentage of embryogenesis induction (number of embryos/100 flowers), percentage of acclimated plants (number of acclimated plants/100 embryos), percentage of hyperhydricity (number of hyperhydrated plants/100 embryos), percentage of H, mixoploid (n/2n), DH and tetraploid as the number of plants/100 acclimated plants. All experiments were established in a completely randomized design. Percentages of gynogenesis induction, embryogenesis induction, acclimated plants, hyperhydricity, ploidy levels and globular structures were analyzed using the Chi Square test by the FREQ procedure.Bulbs from Fuentes de Ebro, Recas, and Rita were grown in the field and in a growth chamber. A total number of 9934 flower buds from the three cultivars were grown in 2012 following the protocol described by Jakše and Bohanec  Protoco, stayed Well-developed gynogenetic embryos Figure  were treSince low percentages of embryogenesis induction and acclimated plants were obtained in 2012, the two-step protocol described by Michalik et al.  .In order to reduce plant hyperhydricity, two experiments were performed to assay containers with different levels of ventilation, using OH-1 embryos obtained with both induction protocols Table . In the In the second experiment, different types of plant containers were compared; glass tube with plastic plug, Eco2box and Magenta box Table . An inteTwo concentrations of APM were applied in liquid medium to OH-1 embryos to optimize the percentage of chromosome doubling Table . No signIn a second experiment, APM application was compared in solid and liquid media. No significant differences in the percentage of acclimated plants were observed between the application of APM for 24 h in solid and liquid media, but there was a slight increase in the solid medium (from 27 to 40%). APM application in solid medium for 72 h slightly decreased plant survival and produced the lowest percentage of haploid and diploid plants, but up to 57% of mixoploid plants. The highest percentage (35%) and number of DH plants was reached when 25 μM APM was applied in solid medium for 24 h.Plant production for the onion breeding program was carried out in parallel with experiments described above for the optimization of DH plant production. Protocol A was followed and embryos were treated with 25 μM APM in liquid medium for 24 h as performed in 2012, but embryos were transferred to Eco2boxes or glass tubes for plant elongation. A total number of 15,064 flower buds from plants grown in the field from Fuentes de Ebro, Recas, BGHZ1354 and OH-1 were inoculated , with close rates to those obtained from OH-1. Fuentes de Ebro and Recas reached almost 17%. These percentages were significantly higher than those obtained in 2012  and tetraploid plants were also produced from H and mixoploid plants.The availability of protocols for onion DH production represents a unique opportunity to have completely homozygous and stable inbred lines. Gynogenesis capacity from Spanish germplasm has been evaluated and different protocols have been assayed to increase the number of acclimated plants, in order to introduce pure inbred lines in an onion breeding program, to improve bulb size, uniformity and storability  has been described in an onion inbred line  could account for the higher gynogenesis efficiency. Higher percentages of embryogenesis were also reported with Polish onion cultivars, with media containing 2iP and NAA (Michalik et al., The presence of an unbalanced gaseous environment in the culture container could lead to plant hyperhydricity (Kozai and Smith, Several duplication agents and different strategies have been used for chromosome doubling of onion haploid embryos/plants (Campion et al., in vitro adventitious somatic regeneration (Alan et al., An alternative approach for chromosome doubling in onion is In the first study using this strategy for chromosome doubling 60% of the somatic plants regenerated from haploid flowers were spontaneously doubled (Alan et al., The gynogenesis capacity of Spanish germplasm has been evaluated. This germplam showed a low capacity for embryogenesis induction, the sweet cultivar Fuentes de Ebro being the most recalcitrant. For the first time comparison of two protocols has been addressed, the protocol described by Michalik et al.  being moThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Current debate in type 2 diabetes (T2DM) has focused on shortened leukocyte telomere length (LTL) as the result of a number of possible causes, including polymorphisms in mitochondrial uncoupling proteins (UCPs) leading to oxidative stress, telomere regulatory pathway gene polymorphisms, or as a direct result of associated cardiovascular complications inducing tissue organ inflammation and oxidative stress. There is evidence that a heritable shorter telomere trait is a risk factor for development of T2DM. This review discusses the contribution and balance of genetic regulation of UCPs and telomere pathways in the context of T2DM. We discuss genotypes that are well known to influence the shortening of LTL, in particular OBFC1 and telomerase genotypes such as TERC. Interestingly, the interaction between short telomeres and T2DM risk appears to involve mitochondrial dysfunction as an intermediate process. A hypothesis is presented that genetic heterogeneity within UCPs may directly affect oxidative stress that feeds back to influence the fine balance of telomere regulation, cell cycle regulation and diabetes risk and/or metabolic disease progression. Telomere length varies from 4–20 kb in humans and telomere shortening is thought to be key mechanistic event in cellular aging –3. ShortThere is certainly a growing trend to report telomere length in population studies that encompass traditional metabolic and cardiovascular risk factors. The large community population Framingham heart study has demonstrated that cardiovascular risk factors such as hypertension and/or insulin resistance are associated with shorter human LTLs . ShortenDiabetes complicated by cardiovascular disease is associated with increased mortality and higher levels of oxidative stress and inflammation, which may accelerate telomere shortening and associated premature cell senescence –22. ImpoIt is generally accepted that shortened telomeres may independently contribute to clinical metabolic syndrome progression via directly affecting cellular metabolic rate and influencing neighboring cell function –30. For Several studies have reported positive associations between LTL and T2DM. LTL has been shown to be shorter in patients with T2DM and also correlates with time of onset and duration of disease , 33, 34.The three common isotypes of UCP are UCP1, mainly expressed in brown adipose tissue, UCP2, expressed widely in most cells and UCP3, expressed mainly in striated muscle . UCP2 haUCP1, UCP2, UCP3) suggests they may have evolved from a common ancestor or are the result of gene duplication events [UCPs are located in the inner mitochondrial membrane and mediate proton leakage, to regulate the production of ATP and reduce oxidative stress , 39. Then events . Peroxisn events  and are n events , 43 Inten events , ameliorUCP2 gene variant −866G < A (rs659366), T2DM and telomere length [Two studies have explored the association between the e length , 38. Onee length   gene, was found in South Asians [General population studies have identified several genes through genome-wide association studies as a locus for cross-sectional individual variation in LTL , 65. A nh Asians . Oligonuh Asians . Severalh Asians , 67. In h Asians . The OBFh Asians . OBFC1 ih Asians , 69, sugh Asians .The relevance of OBFC1 is supported by the finding of Maubaret et al. , where aOBFC1 gene, which is associated with LTL, and its association with T2DM development. In addition, another nine genes, including TERT, TERC, TEP1, TRF1, TRF2, POT1, and TPP1 were exmined [Another independent study also provides positive evidence for the association between OBFC1 and LTL. You et al. examined rs4387287 on the  exmined . The stuIn summary, this review suggests that short LTL has been identified in a limited number of population studies as a risk factor for the development of T2DM. The shortening of LTL relates to both OBFC1 genotypes and telomerase genotypes such as TERC. Interestingly, the interaction between short telomeres and T2DM risk appears to involve mitochondrial dysfunction as an intermediate process, suggesting a mitochondrial axis where underlying gene regulation of telomerase-related pathways is important. We suggest that genetic heterogeneity within UCPs may directly affect oxidative stress that feeds back to influence the fine balance of telomere regulation and cell cycle regulation. This in turn may, in part, be related to both the development of T2DM and enhancing the rate of complications once the disease is present. The interaction of cardiovascular disease in association with the development of T2DM is important and is likely to associate with some genetic components of telomere regulation, but not with all. Overall, the accumulated data in this review illustrates the emerging influence of genetic factors on LTL and T2DM and interplay with mitochondrial function. We suggest that there is a likely need to explore the biological and pathological interactions between UCP, telomerase and shelterin regulatory pathways. These studies are required to adequately describe the precise role of mitochondria on LTL shortening with respect to both acute and chronic pathological events. A greater understanding of the genetic influences on telomere length and T2DM is likely to facilitate risk stratification amongst patients, as genomics is progressively embraced as a clinical tool in the management of this chronic illness.


Using whole exome sequencing to predict aberrations in tumours is a cost effective alternative to whole genome sequencing, however is predominantly used for variant detection and infrequently utilised for detection of somatic copy number variation.We propose a new method to infer copy number and genotypes using whole exome data from paired tumour/normal samples. Our algorithm uses two Hidden Markov Models to predict copy number and genotypes and computationally resolves polyploidy/aneuploidy, normal cell contamination and signal baseline shift. Our method makes explicit detection on chromosome arm level events, which are commonly found in tumour samples. The methods are combined into a package named ADTEx (Aberration Detection in Tumour Exome). We applied our algorithm to a cohort of 17 in-house generated and 18 TCGA paired ovarian cancer/normal exomes and evaluated the performance by comparing against the copy number variations and genotypes predicted using Affymetrix SNP 6.0 data of the same samples. Further, we carried out a comparison study to show that ADTEx outperformed its competitors in terms of precision and F-measure.http://adtex.sourceforge.net/.Our proposed method, ADTEx, uses both depth of coverage ratios and B allele frequencies calculated from whole exome sequencing data, to predict copy number variations along with their genotypes. ADTEx is implemented as a user friendly software package using Python and R statistical language. Source code and sample data are freely available under GNU license (GPLv3) at The online version of this article (doi:10.1186/1471-2164-15-732) contains supplementary material, which is available to authorized users. Tumourigenesis is associated with the acquisition of genomic aberrations , 2 inclua priori[Computational methods have been published for detecting CNAs in targeted resequencing data including whole exome sequencing , 12–19, a priori which isa priori while Control-FREEC [Simultaneous generation of depth of coverage (DOC) ratios and B allele frequencies (BAFs) would facilitate the identification of aneuploidy and polyploidy present in cancer samples. A diploid genome would only have BAFs of 0, 0.5 and 1, corresponding to AA, AB and BB genotypes, whereas, for example, a triploid genome, assuming no tumour heterogeneity or normal contamination, would have allele ratios of 0, 0.33, 0.67 and 1 regions corresponding to AAA, AAB, ABB and BBB genotypes and a baseline DOC ratio of one which is similar to a diploid tumour genome. To generate ratios and allele frequencies from WES data, we need to align them to the reference genome and identify the SNPs. Another issue impeding the use of WGS and WES for somatic CNA identification is non-tumour cell contamination, which is present in the majority of tumour tissues. Normal DNA contamination attenuates the signal-to-noise ratio in BAF and coverage ratio signals by altering their values towards a normal diploid genome pattern. Consequently, a high normal cell contamination would make it impossible to differentially detect somatic variations in tumour cells. Among the previously published methods, ExomeCNV [ol-FREEC  does notol-FREEC , 21 suggWhen analysing exome sequencing data, it is important to overcome the intrinsic noise present in data itself, which hinders its ability to accurately predict CNAs. Programs such as XHMM  and CoNIOverall, the three above mentioned issues, (i) noise in WES data, (ii) ploidy and (iii) normal cell contamination in tumour samples have not been simultaneously evaluated by any of the computational methods applicable to WES tumour data. Although our previously proposed method  considerADTEx consists of two HMMs to predict copy number alterations and genotypes in WES data of paired tumour/normal samples. Two types of signals were generated from the exome data, DOC ratios and BAFs. Copy number analysis using ratios can be complemented by the computation of BAFs to determine ploidy and zygosity. Here, we propose to apply these two types of signals to predict the zygosity state of segments in the genome targeted by exome capture.One HMM is used to predict CNAs, which in combination with BAF signal can be used to estimate ploidy of the tumour and predict the absolute copy numbers. A second HMM is used to predict zygosity or genotype of each CNA segment. The overall framework of the method is given in Additional file The parameters in our two HMMs were trained using an expectation maximisation (EM) algorithm . Given tWe used 17 paired ovarian tumour/normal samples to evaluate the performance of our proposed method , which were therefore used in the genotype prediction evaluation. A representative comparison between exome results and SNP 6.0 results are shown in Figure We evaluated ADTEx predicted CNAs against those predicted by ASCAT on SNP 6.0 array data, which we assumed as the ground truths. Sensitivity, specificity, precision and accuracy were computed for each sample Figure . As showFor large scale (>0.1 of the chromosome) CNA events, we further assessed the performance of ADTEx based on the number of events detected by the method. According to the results from SNP arrays, there were 150 large scale events in all samples  were reported as median sensitivity of 96.8% and specificity of 98.2% and are summarised in Additional file Next, we evaluated the performance of ADTEx on high-grade serous ovarian adenocarcinoma samples sequenced as part of TCGA project . We downTo evaluate the somatic CNA detection of ADTEx, we also downloaded the raw Affymetrix SNP 6.0 files of the same samples from TCGA data portal. Then, as before, we carried out CNA detection on SNP 6.0 data using ASCAT algorithm . These rIn detecting somatic CNA, ADTEx showed median sensitivity of 93.7%, precision 79.3% and F-measure of 83.0%. F measure was computed using the following equation.The largest detected CNA segment was 242,433,351 bp long in length and smallest detected CNA was 120 bp long. On average 1,035 CNA segments per tumour were detected by ADTEx  F-measure values for ADTEx, ExomeCNV, VarScan2 and Control-FREEC were 91.5% (92.3%), 76.5% (90.6%), 82.0% (84.7%) and 76.0% (82.5%) respectively. Overall, both mean and median performance scores of ADTEx were better compared to other methods. VarScan2 ranked second and ADTEx showed 12% increase in terms of mean F measure over VarScan2. As per the Table Figure SNP genotyping arrays cannot be used to detect smaller-size CNAs <1Mbp) due to limited resolution. To assess the performance at this level, we compared smaller (<1Mbp) CNAs detected by ADTEx with three other exome based methods. Overall, ADTEx, ExomeCNV, VarScan 2 and Control-FREEC detected 448, 7167, 4618 and 1494 CNA events, respectively, of which 79%, 94%, 93% and 82% were identified as smaller than 1Mb. Identification of high percentages of smaller events by all four methods can be attributed to the sparse nature of WES data. As ADTEx performs noise reduction in WES data, many of the smaller CNAs predicted by other three methods are integrated into larger CNAs predicted by ADTEx. The overlap between other methods and 354 smaller CNAs identified by ADTEx is given in Additional file Mbp due tControl-FREEC predicts LOH and ASCNA events similar to ADTEx, hence we compared ADTEx LOH and ASCNA predictions against the results from Control-FREEC  genotype status of SNPs in each of the CNA segment predicted by ADTEx. We evaluated the performance of Amarasinghe et al., 2013 [We described a copy number predicting algorithm in Amarasinghe et al., 2013 . The mail., 2013  are i) nl., 2013  using thl., 2013  on SNP 6l., 2013 . FurtherWe carried out computer simulations to evaluate the effect of normal cell contamination on CNA and LOH predictions by ADTEx. We use OV1 data sample to generate different combination of data sets with 0.1 to 0.7 normal cell contamination and read depths of 150X , 60X and 400X. The original normal cell contamination is predicted as 25%, therefore the expected contamination levels can be calculated as 0.325, 0.4, 0.475, 0.55, 0.625, 0.7 and 0.775 respectively. For original coverage, ADTEx accurately predicted the normal cell contamination up to 0.7 (Pearson correlation 0.99). Additional file Chromosome arm level or full chromosomal CNAs are commonly observed across many tumour types , 29. DifADTEx explicitly predicts chromosome arm level events based on the results generated by exon level copy number predictions. The distribution of DOC ratios of copy neutral regions is calculated from the exon level result. Then, statistical confidence level (assuming no CNA) for each chromosome arm considering the mean DOC ratio is produced based on the calculated distribution. At the 0.05 confidence level we were able to detect 44 chromosome p/q level events (15 gains and 29 losses) and 30 full chromosome CNA events (15 of each gains and losses) in the 17 in-house ovarian tumour samples. According to the carefully curated list of chromosome arm level CNAs  paired ovarian tumour/normal samples captured using three different targeted capture platforms and sequenced using Illumina Genome Analyzer II or HiSeq2000 sequencers. Further, to our knowledge ADTEx is the only method that predicts chromosome arm level CNA in WES data.We have described a new approach to infer somatic CNAs and genotype states in WES data from tumour samples. Our method both models and evaluates tumour related attributes in WES data. Further, the proposed method explicitly predicts chromosome arm level CNA events, which are commonly found in many tumour types. We implemented this approach in a software called “Aberration Detection in Tumour Exome (ADTEx)”, which is freely available under GNU General Public Licence v3 (GPLv3). To our knowledge, ADTEx is the first attempt to computationally derive absolute copy numbers and genotypes using WES data from tumour samples without any We demonstrated the superior performance of ADTEx compared to existing methods. Most importantly, we compared the performance of ADTEx against the results generated by ASCAT on Affymetrix SNP 6.0 data and showed that our method can produce results consistent with SNP array data, the gold standard for detecting CNAs. We believe that the integrated CNA and LOH predictions in ADTEx will greatly improve the type and usefulness of the data generated in large WES studies.However, We have not addressed the issue of tumour heterogeneity where some components of the tumour biopsy will have different clonal outgrows harbouring different genetic alterations. Clonal heterogeneity will result in an amalgamation of the signals present in both subpopulations which may result in a reduced sensitivity of ADTEx. This could be a future research direction that can be pursued using WES data.i)Increased performance in predicting CNAs by taking all data points that are available (depth of coverage ratios) for the evaluation of first HMM and then calling zygosity on SNP loci in the second HMM.ii)Computational efficiency, achieved through dimensionality reduction in the first stage of HMM.ADTEx processing pipeline is shown in Additional file DOC ratios are calculated as the ratios between average coverage per base of exonic regions in tumour and matched normal samples using the following steps: (i) coverage per base at targeted regions are calculated by BEDTools software , (ii) avBAF is calculated as the ratio between number of B alleles and total number of A and B alleles. Here, ‘A allele’ refers to the reference allele and ‘B allele’ refers to the non-reference allele in DNA sequencing data when the sequence reads are aligned to the reference genome. We calculated SNP loci in both tumour and matched normal samples by applying The Genome Analysis Toolkit (GATK) software . BAF at For example, based on the above formula BAF should be around 0, 0.5 and 1 for genotypes AA, AB and BB, respectively. If a BAF is deviated from these values, it would mean that there is a possibility of copy number alteration. For instance, a BAF value of 0.25 indicates a genotype of AAAB.DOC ratios can be used as an indication of the relevant copy number present in tumour sample compared to the matched normal sample. Copy number of a particular region and the DOC ratio has a direct relationship in non-cancerous samples. For example, ratios of one and two represent copy number two and four respectively. However, due to the presence of extensive abnormalities in tumour samples, mean DOC ratio will differ from the nominal ratio of one. This change in the signal is known as the baseline shift  for different values of contamination  and (v) calculate the minimum summation of distances that would give the best fit for the base ploidy.When the BAFs of tumour samples are present, we fitted HMM for different base ploidy values  and then applied the following steps to determine the base ploidy: (i) Select the SNPs which overlaps with the captured exonic regions, (ii) segment BAFs (bEquation ) of SNPsT represents the copy number of the tumour that is predicted by the HMM. From Equation . The reAs depicted in Table Mirrored BAFs (as defined below) around 0.5 are used as the observations in the proposed HMM.b) of BAF signal are considered as main parameters of the observation distribution. We assumed mirrored BAF signal is normally distributed with state specific mean and a constant b for all states. If BAF of lth SNP is bl, then observation probability distribution function (pdf) for a hidden state Sk with associated copy number CT can be formulated as:Normal cell proportion  is the expected B allele count for state Sk.where, Nd. Further, the transition probability depends on the state specific copy number. For example, if the current observation j, is assigned with a copy number of two, then the model can only transit to ‘hidden state 2’ and ‘hidden state 3’  is the posterior probability of lth SNP to be in state Sk, which is calculated using forward-backward algorithm [In the above Equation , l (Sk) lgorithm . By takiPT = 2α + (1 − α)CT(Sk) and t = EM iteration number. The algorithm is constrained to identify α(t+1) is less than 0 or greater than 0.7, it will be set to 0 or 0.7 respectively.Where Estimated Equation  is used Contains Supplementary Methods, Supplementary Figures S1 - S9 and Supplementary Tables S1 - S12.(PDF 1 MB)Additional file 1:Copy number and genotype profiles of the in-house generated ovarian cancer samples predicted by ADTEx.(PDF 1 MB)Additional file 2:Supplementary Table containing CNA predictions of the TCGA samples made by ADTEx.(ZIP 5 MB)Additional file 3:


Mycobacterium tuberculosis, and associations between susceptibility to tuberculosis and polymorphisms in the genes NRAMP and VDR have been sought in geographically diverse populations. We investigated associations of NRAMP1 and VDR gene polymorphisms with susceptibility to TB in the Venezuelan population. The results suggest the absence of any association between VDR variants FokI, ApaI, and TaqI and susceptibility to tuberculosis. In contrast, the NRAMP1 3′UTR variants were associated with susceptibility to M. tuberculosis infection, as seen in the comparisons between TST+ and TST− controls, and also with progression to TB disease, as shown in the comparisons between TB patients and TST+ controls. This study confirms the previously described association of the NRAMP1 3′UTR polymorphism with M. tuberculosis infection and disease progression.Natural resistance-associated macrophage protein (Nramp1) and the vitamin D receptor (VDR) are central components of the innate and adaptive immunity against  Mycobacterium tuberculosis (Mtb) is much higher, but the great majority of those infected are able to keep the pathogen under control and never develop the disease. Multiple evidence suggests that there is a genetic component involved in determining resistance or susceptibility to TB patients, who are infected but do not develop the disease and who developed the disease, but it is difficult to conduct genetic studies on susceptibility to infectious diseases because of the multifactorial influences of the host, the pathogen, and environmental variables that differ for each disease and even each individual studied. The association of host genetic factors with susceptibility or resistance to TB has been studied extensively using various methods including case-control studies, candidate gene approaches, and family-based and genome-wide linkage analyses that have revealed several candidate genes involved in susceptibility (reviewed in [ NRAMP1 and VDR.Tuberculosis (TB) is the second cause of death worldwide among infectious diseases. In the World Health Organization (WHO) Global Tuberculosis Report for 2014 there were 9.0 million new TB cases in 2013 and 1.5 million TB deaths (1.1 million among HIV negative and 0.4 million among HIV positive), of which 218,875 new cases and relapses occurred in the Americas. In Venezuela, according to the WHO, the incidence of new and relapse TB cases is between 20 and 49 per 100,000 inhabitants [ NRAMP1 and VDR gene polymorphisms with susceptibility to TB in the Venezuelan population.In this study we investigated associations ofThe study included one hundred and ninety-five (195) unrelated and ethnically mixed Venezuelan individuals from the northern region of country, divided into two cohorts, patients and controls.Patients. The ninety-three individuals all had a clinical diagnosis of pulmonary tuberculosis and were seen by the Pneumology Service of the Hospital José Ignacio Baldó, Algodonal, Caracas. There were 52 men (56%) and 41 women (44%), with an age range of 17–70 years. Patients were selected based on the diagnostic criteria of the National Standard Integrated Venezuela Tuberculosis Control Program: two sputum smears positive for acid fast bacilli by microscopy, clinical symptoms characteristic of TB, and a chest X ray consistent with active TB disease.Controls. There were one hundred and two apparently healthy individuals who were known to be exposed to patients with active TB. This group was composed of staff members who had worked at Hospital Dr. José Ignacio Baldó, Algodonal, Caracas, for more than 3 years, and included 25 men (24.5%) and 77 women (75.5%) whose age range was 20–67 years. The controls were classified according to the tuberculin skin test “TST” as follows: positive TST (51/102), negative TST (19/102), or without information (32/102). All controls were without clinical manifestations of TB at the time of blood sampling.Individuals, who were HIV positive or known to have any autoimmune, chronic inflammatory, or other disease, were excluded from the study. Participants in the study signed an informed consent form previously approved by the IVIC Bioethics Committee. NRAMP1 gene were studied by PCR-RFLP technique, using primers and restriction enzymes reported by Taype et al., 2006 [ INT4 (469 + 14G/C), D543 , and 3′UTR . The polymorphic variants of the VDR gene were studied by PCR-RFLP, using primers and restriction enzymes reported by Curran et al., 1999 [Genomic DNA was extracted from blood samples according to the procedure described by Bunce . The poll., 2006 : INT4  were obtained by multiplying the p values by the total number of variables analyzed and were considered significant when p < 0.05 [Allele and genotype frequencies were determined by direct counting. The Hardy-Weinberg equilibrium was calculated with the exact test. The statistical significance of allele frequency differences between patients and controls was estimated by Fisher's exact test using 2 × 2 contingency tables. The Bonferroni corrected p < 0.05 . Relativp < 0.05 . NRAMP genotypes and alleles in controls and TB patients. The allelic and genotypic frequencies of the NRAMP1 polymorphisms showed Hardy-Weinberg equilibrium for INT4 (p = 0.832), D543 (p = 0.296), and 3′UTR (p = 0.594) polymorphisms in the control cohort. There was a significantly increased frequency of homozygous 3′UTR TGTG+/+ genotype in the healthy controls compared to the TB patients . However, the frequency of the heterozygous 3′UTR SNP TGTG+/del  and homozygous 3′UTR SNP del/del genotypes  was higher in the patient group than in the control group, although the corrected p values (pc) were not significant. In addition, there was a significant difference in the distribution of the allele frequencies (TGTG+ and TGTG del) between the controls and the TB patients (pc = 0.018). There was no significant association between INT4 and D543 genotypes with tuberculosis. VDR genotypes and alleles in controls and TB patients. There was Hardy-Weinberg equilibrium for the genotype distributions of FokI (p = 0.074), TaqI (p = 0.066), and ApaI (p = 0.545) polymorphisms in apparently healthy individuals. The data showed that the frequency of the FF SNP genotype of FokI was higher in the patients than in the control group  although the corrected p value was not significant. No significant difference in the allele frequencies was observed between the TB patients and the controls. NRAMP1-3′UTR variants in the development of tuberculosis, we compared the genotype and allele frequencies between the different groups: TB patients versus TST positive controls, TB patients versus TST negative controls, and TST positive controls versus TST negative controls  and TGTG+/del genotypes . These same differences were conserved when the comparison was made between TST positive controls versus TST negative controls, although the significance was lost with the correction of p values. Additionally, there was a significant increase in the frequency of the TGTGdel allele among TB patients  and TST positive controls  compared to TST negative controls.In order to investigate the possible influence ofcontrols . There w VDR and NRAMP1 genes and susceptibility to infection and disease with Mycobacterium tuberculosis, as indicated by a positive TST, and the development of TB in the Venezuelan population. Vitamin D is an immune-modulator molecule that, via its receptor VDR, can modulate cytokine responses by T cells [ VDR polymorphisms and tuberculosis in different populations [ ApaI, TaqI, or FokI variants and susceptibility to either infection or development of tuberculosis.The aim of the present study was to look for associations between polymorphisms in T cells . Althougulations –26, our ulations –31, did  NRAMP1 gene appear to favor bacterial replication within macrophages and have been associated not only with increased susceptibility to infection by Mycobacterium tuberculosis but also with an increased tendency to develop severe disease [Nramp1  is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication . Some po disease –35. NRAMP1 gene 3′UTR polymorphism and susceptibility to tuberculosis in West Africans [ M. tuberculosis infection, as seen in the comparisons between TST+ and TST− controls, and also with progression to TB disease, as shown in the comparisons between TB patients and TST+ controls.However, different studies have found conflicting results. There was a positive association between theAfricans , KoreansAfricans , ChineseAfricans , and ChiAfricans , but theAfricans , Thai [4Africans , MoroccaAfricans , Danish Africans , BraziliAfricans , and IndAfricans  populatiAfricans . The res 3′UTR polymorphism consists of a 4 bp TGTG deletion located 55 nucleotides downstream the last codon in exon 15 of the NRAMP1 gene, in a region where sequence variation can affect mRNA stability and/or efficiency of protein translation. Therefore, to explain the increased susceptibility promoted by the TGTG+/del genotype and TGTGdel allele, two essential aspects should be considered: (1) the protein encoded by the NRAMP1 gene plays an important role in the phagolysosomal function of pulmonary macrophages and in antigen presentation. The Nramp1 protein becomes activated and fused with lysosomes to digest the engulfed mycobacteria ; (2) Nriewed in . As a coiewed in ). In con


A force sensing touchscreen, which detects touch point and touch force simultaneously by sensing a change in electric capacitance, was designed and fabricated. It was made with single-walled carbon nanotubes (SWCNTs) which have better mechanical and chemical characteristics than the indium-tin-oxide transparent electrodes used in most contemporary touchscreen devices. The SWCNTs, with a transmittance of about 85% and electric conductivity of 400 Ω per square; were coated and patterned on glass and polyethyleneterephthalate (PET) film substrates. The constructed force sensing touchscreen has a total size and thickness of 62 mm × 100 mm × 1.4 mm, and is composed of 11 driving line and 19 receiving line channels. The gap between the channels was designed to be 20 µm, taking visibility into consideration, and patterned by a photolithography and plasma etching processes. The mutual capacitance formed by the upper and lower transparent electrodes was initially about 2.8 pF and, on applying a 500 gf force with a 3 mm diameter tip, it showed a 25% capacitance variation. Furthermore, the touchscreen can detect multiple touches and forces simultaneously and is unaffected by touch material characteristics, such as conductance or non-conductance. As mobile device usage has grown rapidly, so has interest in touchscreen input devices. Earlier research concentrated on push-button input which was used in a variety of industries and applications, such as robots, displays, and information technology fields. However, the transparent touchscreen has recently become widely adopted because of its increased simplicity and ease of use. The touchscreen can recognize touch position by simple contact and perform various functions by double-click motion.i.e., resistive, capacitive, infrared, and acoustic [Their touch-sensing mechanisms can generally be divided into four types, acoustic ,6,7,8,9.2O3:Sn) are typically used in touchscreens and optoelectronic devices because of their high transparency and low resistivity characteristics. However, the force sensing touchscreen proposed in this research, rather than ITO-based material, uses single-walled carbon nanotubes (SWCNTs) as transparent electrodes. This has three main advantages: first, the proposed touchscreen device can simultaneously measure both touch position and touch force, regardless of the type of touch media used. Secondly, it can maintain stability when subjected to stringent environmental conditions and external shocks [Currently transparent electrodes made of indium oxide (ITO) doped with tin  water by an ultrasound vibration process with the aid of surfactant and sodium dodecylbenzenesulfonate. The surfactants and sodium dodecylbenzenesulfonate were used to apply a uniform coat of SWCNTs to the substrate. The dispersed SWCNT coating solution contained 0.05 wt% of SWCNTs and 0.5 wt% of sodium dodecylbenzenesulfonate. After ultrasound treatment, the chopped SWCNTs were separated by weight and refined by a centrifugal system for pure refining. This process removed most of the impurities and the bundles of SWCNTs that had not been broken into small pieces. The transparent electrode coating process created an SWCNT coat of about 400 Ω electric sheet resistance and 85% transmittance at a wavelength of 550 nm. The coating process was performed on a hot plate, which was kept at 90 °C in order to remove the DI water medium in which the SWCNTs were dispersed for spraying .The patterned SWCNT electrodes on the upper PET substrate have a rectangular geometry 4.35 mm in width and 52.85 mm in length and those on the lower glass substrate a rectangular geometry 4.39 mm in width and 87.4 mm in length. In addition, they had electrical resistances of about 6 kΩ and 9 kΩ on the upper and lower substrates, respectively. After completing the patterning process of the upper and lower electrode substrates, the flexible printed circuit (FPC) bonding process was carried out, followed by the silicone gel injection process which formed the force sensing layer of the touchscreen. The FPC for signal processing was bonded using anisotropic conductive film (ACF) made from circular nickel particles 6 μm in diameter. The last fabrication process, the injection of the compressive-elastic dielectric gel material  was achieved using an air pressure injector consisting of a metal needle and an air pressure controller. As silicone gel in solution has some viscosity, a 500 µm thick polycarbonate dam structure was attached to the edges of the electrode substrates before the injection process to prevent the silicone gel from leaking outside. After the injection process, the injected silicone gel was left at room temperature for approximately 12 h to solidify and gain its elastic property. The resulting force sensing touchscreen panel module had a type of sandwich structure.As the fabricated force sensing touchscreen  uses a cThe force sensing touchscreen was composed of an 11 X-channel electrode and a 19 Y-channel electrode so that the total 209 mutual capacitances formed by the channel electrodes could be measured by capacitance measuring apparatus.Another important characteristic of the force sensing touchscreen was the simultaneous detection of both multi-touch and multi-force. As shown in Conventional capacitive touchscreen only reacts to conductive touch such as that of a human finger. However, the force sensing touchscreen also operates with insulating materials, such as Teflon as used in our study. Indeed, the advantage of the force sensing touchscreen is that there are no restrictions on the type of touch material which can be used.A force sensing touchscreen, which is capable of multi-touch and multi-force sensing, was designed and fabricated using SWCNTs as an alternative transparent electrode to conventional ITO-based electrodes. The SWCNT electrodes proved superior to ITO electrodes in their mechanical and chemical characteristics, and have excellent robustness to external environment and shock. The force sensing touchscreen was fabricated with an area of 4 square inches and a thickness of 1.5 mm. It consists of upper and lower transparent electrodes and maintains a uniform mutual capacitance. It shows relatively linear characteristics and has a 25% capacitance variation under a 500 gf force applied with a 3 mm diameter tip. It perceives the touch position and touch force by sensing the simultaneously resulting change in mutual capacitance. The SWCNTs were patterned using plasma etching technology with a gas mixture of oxygen and argon. The micro-patterning process was successfully carried out with a 20 µm gap between the SWCNT electrode channels. The force sensing touchscreen, with its two electrodes and dielectric material, has no restrictions with respect to the conductance or non-conductance of the touch material.The force sensing touchscreen goes beyond the capabilities of existing touchscreens in that it not only recognizes the touch position but can also provide multi-tasking capabilities through simultaneous multi-touch and multi-force sensing. It is therefore applicable to diverse applications, such as mobile devices and flexible display fields that require touch devices and transparent electrodes.