Outcomes of septal alcoholization in hypertrophic obstructive cardiomyopathy are not enough studied in all centers. The purpose of this study was to determine the outcomes of septal alcoholization in hypertrophic obstructive cardiomyopathy in our hospital. A retrospective and prospective descriptive study focused on all patients aged at least 18 years treated by alcohol septal ablation between July 2005 and June 2010 in the cardiology unit of Clermont-Ferrand teaching Hospital. The inclusion criteria were, hypertrophic obstructive cardiomyopathy with left ventricular outflow tract obstruction ≥ 50 mmHg, symptomatic despite optimal medical therapy. The clinical, paraclinical data and the results of alcohol ablation were collected from medical records of patients and a telephone conversation with the patients or their physicians. These data were analyzed by EPI info 6.04. Eleven patients with average age of 56.27 ± 15, 83 were included of which 81.8% of men. The main indications of alcohol septal were dyspnea stage NYHA II-IV (45.5%), lipothymia (18.2%) and invalidating angina (18.2%). Main electrocardiographic abnormalities were left ventricular hypertrophy and disorders of repolarization with 72.7% each. Minor conductive disorders were found in 45.5% of the cases. The left ventricular outflow tract obstruction was 98.18 ± 25.93 mmHg before alcohol septal ablation and 18.91 ± 31.97 mmHg after a follow-up of 25.64 ± 21.97 months. The success rate was 81.8%. Conductive disorders (45.5%) required the establishment of a definitive pacemaker in 36.4% of the patients. A cardiac defibrillator was implanted at 27.3%. Septal alcoholization was succesful. The prevalence of Hypertrophic cardiomyopathy (HCM) is between 60 to 170/100.000 habitants . Accordist 2005 to June 30th 2010. It was conducted in the department of cardiology and vascular diseases of Clermont-Ferrand teaching Hospital.It is a retrospective descriptive study covering the period of July 1Inclusion criteria: Were consecutively included in the study all patients aged of at least 18 years presenting a hypertrophic obstructive cardiomyopathy (HOCM) with left intraventricular gradient greater than 50 mmHg at rest, remaining symptomatic after failure of optimal medical treatment and/or ventricular pacing, who have received an alcohol septal ablation during the study period.Non-inclusion criteria: Patients with a contraindication to alcohol septal ablation and patients with non-obstructive or asymptomatic hypertrophic cardiomyopathy have been excluded.The alcohol septal ablation procedure consists of several steps:A temporary right ventricular stimulation catheter is set by the femoral vein route. Left heart catheterization confirms the dynamic left intraventricular obstruction and the extent of left intraventricular pressure gradient. Coronarography enables the selection of the artery to be alcoholized. The transthoracic intracoronary injection of contrast under echocardiographic monitoring guides the selection of this artery by showing the target area corresponding to myocardial obstructive bulge. The alcohol septal ablation is performed by a septal intracoronary injection of one milliliter (1ml) of ethanol 95° followed by a second injection of the same volume ten minutes later. The procedure is done under transthoracic echocardiographic , hemodynamic and rhythmic monitoring. Monitoring in a cardiology intensive care unit with telemetry followed by a conventional hospitalization is carried out for at least four days. It focuses on the hemodynamic but importantly on the rhythmic looking rhythmic complications or conduction disorder. Post-septal alcoholization medical treatment is carried out with beta-blockers or calcium channel blockers. In case of supraventricular rhythm disorder, amiodarone is associated. The definitive pacemaker or cardiac defibrillator implantation is performed on a case by case basis according to the complications or associated risks. Post-septal alcoholization follow-up is done at one month then at six months for all patients. The follow-up is clinical, echocardiographic and rhythmic. The frequency of subsequent follow-up  depends on clinical and paraclinical data, on risks and the presence or not of a pacemaker or cardiac defibrillator.Data collection: It was conducted by searching in patient's medical and cardiac catheterization records and a telephone interview with the patient or a member of their families, their physicians and/or their cardiologists. The following variables were collected: age, sex, cardiovascular risk factors, cardiovascular history, comorbidities, clinical and paraclinical data . Holter monitoring, stress test and screening for genetic mutation have been performed for some patients. Indications, results of septal alcoholization as well as complications of the procedure and their treatment were also recorded.Definition: The success of the procedure was defined by obtaining a left intraventricular gradient lower than 30 mmHg in immediate post-procedure or at medium term. Partial success was defined by a significant decrease in left intraventricular gradient without reaching the threshold of 30 mmHg.Data analysis: Data were analyzed using EPI info 6.04 software. The results were expressed as mean ± standard deviation and relative frequency.Epidemiological data: Eleven patients were involved in the present study. The mean age was 56.27 ± 15.83 years (extremes of 20 and 80 years). They were predominantly male (81.8%). The average time between the diagnosis of obstructive cardiomyopathy and septal alcoholization procedure was 51.72 ± 31.60 months (extremes of 1 and 97). Nine patients (81.8%) had at least one cardiovascular risk factor which were mainly age (54.5%), smoking (63.7%) and high blood pressure (27.3%). No family history of HOCM was found.Clinical data: The main circumstances of HOCM discovery were the outcome of heart murmur (36.4%) and chest pain (27.3%) of which 18.2% were associated with a heart murmur and 9.1% with palpitations. Other circumstances of discovery were atrial fibrillation, heart failure and pre-therapeutic assessment for sismotherapy with 9.1% of cases each. Four complications (36.4%) were recorded before septal alcoholization. There were two cases of heart failure (18.2%), a recovered case of cardio-respiratory arrest (9.1%) and one case of atrial fibrillation (9.1%). Cardiac auscultation shown a systolic ejection murmur in all eleven patients at the time of septal alcoholization. The main clinical indications of septal alcoholization besides dynamic obstruction were exertional dyspnea class II-IV of New York Heart Association (NYHA) despite medical treatment (45.5%), malaise/pre-syncope (18.2%) and disabling angina with dyspnea (18.2%). Other indications were recovered cardio-respiratory arrest with persistence of intraventricular gradient under ventricular pacing (9.1%) and blood pressure intolerance to stress test (9.1%). Nine of the eleven patients were medicated with beta-blocker (81.8%) and two others with calcium antagonists (18.2%). Two of them had received pacemaker implantation for HOCM therapy before septal alcoholization.Paraclinical data: The electrocardiogram was in sinus rhythm at the time of the procedure for all patients. It was abnormal for all of them. The main abnormalities were left ventricular hypertrophy and repolarisation disorders with 72.7% for each. Conduction disorders were found in 45.5% of cases. It was bi-fascicular block in 18.2% of cases, a left anterior hemiblock (9.1%), a complete atrioventricular block (AVB3) after his bundle ablation with ventricular stimulation in the context of HOCM and a paroxysmal AVB 3 appareled in a context of recovered sudden death. The transthoracic echocardiography has diagnosed HOCM with an intraventricular gradient exceeding 50 mmHg at rest for all eleven patients. This was an asymmetric HOCM in most cases with a mean septal thickness of 23.09 ± 4.76 mm (extremes of 16 to 33) against an average thickness of 15.91 ± 2.95 mm (12-22 mm) to posterior wall. It was a type 3 of Maron classification in 72.7% of cases. Types 1 and 2 accounted for 18.2% and 9.1% respectively. The average maximum left intraventricular gradient was 128.46 ± 32.50 mmHg (extremes of 87 and 193). Systolic function was preserved and even supernormal for all patients with mean ejection fraction of 74.82% ± 5.93 (extremes of 67 to 84%). The mitral flow was a disorder type relaxation (63.7%) and pseudo-normal type (27.3%). It was normal in 9.1% of cases. A systolic anterior motion of the mitral valve was found in 100% of patients with mitral regurgitation grade 1 to 3 in all eleven patients (100%). A mesosystolic closure of aortic valve was found in 54.5% of cases. The left atrium was dilated in 81.8% of patients while a slight to moderate pulmonary arterial hypertension was observed in 27.3% of them. A fluttering of anterior mitral valve's pillar was found in one case with a left intraventricular gradient of 193 mmHg.A rhythm holter monitoring was performed for seven patients before septal alcoholization (63.6%). A burst of non-sustained ventricular tachycardia (VT) was recorded in one case and idioventricular rhythm in another case. Four patients had received at least one stress testing before septal ablation. It was pathological with blood pressure inadequacy to exercise in one case and not well tolerated with a stop at the first level in another case. Cardiac catheterization with intention to treat was performed in eleven patients. A septal artery suitable for target area was found in ten patients which is 90.9% of the cases. Significant coronary lesions were discovered in two patients (18.2%). Septal alcoholization has actually been performed in ten patients (90.9%). The results of the catheterization and septal alcoholization are summarized in Evolution: The mean follow-up time of patients from the diagnosis of HOCM was 84.46 ± 44.15 months (range 6 to 143). The average follow-up duration after septal alcoholization was 25.64 ± 21.97 months (range 4 to 59). The average hospital stay length was 7.36 ± 4.39 days (range 4 to 18). Septal alcoholization was effective immediately or in the short term in 9 of 10 patients who actually received this therapy. Favorable clinical and hemodynamic course rate was 81.8% in all of eleven patients. One patient (9.1%) had clinical improvement. The last patient who didn't receive septal alcoholization because of lack of a suitable septal artery remained symptomatic (9.1%). No death was recorded during follow-up. th month monitoring of the previously implanted pacemaker. A transitional systolic dysfunction was observed in post procedure (ejection fraction of the left ventricle to 40%) in an elderly patient with multiple complications . Two clinical events justifying rehospitalization were recorded later after septal alcoholization: heart failure on bronchial infection in a patient with chronic obstructive pulmonary disease and recurrence of atrial fibrillation treated with amiodarone for the patient with septal alcoholization failure.A pacemaker was implanted in four patients after septal alcoholization. The average time for permanent pacemaker implantation after alcoholization was 4.2 ± 2.56 days (range 2 to 8). Indications were AVB3 (two cases) and trifascicular block (two cases). The conduction disorders regressed in two patients within the same day of the procedure. The second case was after-hospitalization during the 5The treatment of hypertrophic cardiomyopathy is well codified today –8. SeptaA significant reduction of systolic anterior motion and mitral regurgitation was also obtained in 81.8% and 72.8% respectively. This evolution is typical. The rate of complications requiring permanent pacing has significantly improved (5-10%) along with technology and of the staff experience –11. TheyWe report bursts of ventricular tachycardia in three patients 27.3%) with recurrent ventricular tachycardia treated with ATP (9.1%) and two bursts of non-sustained new-onset ventricular tachycardia 18.2%). The VT even non-sustained is an independent factor of poor prognosis of HCM because it is responsible for sudden death . Cases o.3% with .2%. The The septal alcoholization was clinically and hemodynamically effective. One third of patients presented after a burst of non-sustained ventricular tachycardia and 27.3% received a new implantable cardioverter defibrillator. One third of patients received a new pacemaker for conduction disorder.Septal alcoholization is indicated in symptomatic obstructive hypertrophic cardiomyopathy despite optimal medical treatment;The complication rate or permanent pacing varies according to the teams and their experience.Our study takes stock of the first years of this technique in our center and confirms that prior conduction abnormalities increase the risk of permanent pacing.The authors declare no competing interest.


Therefore, we compared the cytotoxicity of TiF4 with sodium fluoride (NaF)  considering different fluoride concentrations, pH values and experimental models.Titanium tetrafluoride (TiF4 (from 0.15 to 2.45% F), both at native and adjusted pH, for 6 h. Step 2) NIH/3T3 were exposed to NaF or TiF4 varnishes with 0.95, 1.95 or 2.45% F (native pH), for 6, 12 or 24 h. We applied MTT (1st and 2nd steps) and Hoescht/PI stain (2nd step) assays. Step 3) NIH/3T3 were exposed to NaF or TiF4 varnish (2.45% F), at native pH, for 6 or 12 h. The cell stiffness was measured by atomic force microscopy (AFM).Step 1) NIH/3T3 fibroblasts were exposed to mediums containing NaF or TiF4 mediums died, regardless of the F concentration and pH. Step 2) Both varnishes, at 1.90 and 2.45% F, reduced cell viability by similar extents  compared with control, regardless of the type of fluoride. Varnishes with 0.95% F did not differ from control. Step 3) TiF4 and NaF reduced cell stiffness to a similar extent, but only TiF4 differed from control at 6 h.Step 1) All cells exposed to NaF or TiF4 and NaF have similar cytotoxicity. The cytotoxicity was dependent on F concentration and exposure time. This result gives support for testing the effect of TiF4 varnish in vivo.Based on the results of the 3 experimental steps, we conclude that TiF The m4 to prevent tooth demineralization has been investigated since 1997 [4 is highly dependent on the low pH of the agent and the type of vehicle. Previous studies have shown that TiF4 could significantly reduce enamel erosion at its native pH (pH 1.2), but not at a pH buffered to 2.1 [4 varnish  seems to be more efficient than a TiF4 solution against tooth demineralization [The potential of TiFnce 1997 . Its pronce 1997 –11. The d to 2.1  or 3.5 [d to 2.1 . On the lization ,15.4 in the management of dental erosion [4 might impair its clinical use, because of possible adverse side effects such as an astringent effect on the mucosa. A scanning electron microscopy study has demonstrated that 1% TiF4 solution has cytotoxic effects on L929 fibroblasts [4 on the viability and morphology of fibroblasts.Although there is laboratory evidence about the effectiveness of TiF erosion  and cari erosion  comparedroblasts . However4, in comparison with NaF, on fibroblasts using three different experimental approaches. First, fluoride salts were added directly into the medium, simulating a mouthrinse, and cytotoxicity was assessed by MTT assay. Second, fluoride was applied as a varnish in contact with the medium and cytotoxicity was assessed by MTT and Hoechst 33342/propidium iodide (HO/PI) stain assays. Third, fluoride was applied as a varnish in contact with the medium and cytotoxicity was assessed via measurement of cell stiffness using atomic force microscopy (AFM). In addition, the dependence of the effects of fluoride on pH, fluoride concentration and exposure time was also taken into account.Here we aimed to evaluate the toxicity of TiF® CRL1658™) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with antibiotics (100 IU ml-1 penicillin and 0.1 mg ml-1 streptomycin) and 10% (v/v) fetal bovine serum , at 37°C in a humidified atmosphere of 5% CO2. Enzymatic digestion with 0.25% trypsin  was used to harvest cells for experimental analysis.NIH/3T3 fibroblasts  and treated with DMEM (containing antibiotics and serum) containing different TiF4 or NaF concentrations . 4 concentrations, respectively. The pH of NaF-DMEM and TiF4-DMEM solutions was adjusted to 4.5 by addition of 1.28 M phosphoric acid and 0.78 M sodium citrate, respectively [Cells were plated in 96-well microplates at a density of 5 x 10ectively . A posit4 (for the MTT assay) and 5 x 104 (for the other assays) per well (1.5 ml culture medium per well) and treated with varnishes containing TiF4 or NaF  for 6, 12 and 24 h. The varnish was applied to a protrusion (30 mg varnish per well) that was immersed in the medium, allowing fluoride release at a distance of 5 mm from the cells. A positive control consisted of medium with no fluoride. The control medium contained either 1% fetal bovine serum (FBS for the MTT assay) or 10% FBS . A negative control consisted of medium containing sodium dodecyl sulfate (only for the MTT assay). The fluoride content of the media was measured by using an ion-specific electrode  and a miniature calomel electrode , both coupled to a potentiometer , following hexamethyldisiloxane-facilitated diffusion [Cells were plated in 24-well microplates at a density of 10iffusion ,18. Cell-1; Sigma-Aldrich Co. LLC, St. Louis, USA) was added and the cells were incubated with it for 4 h at 37°C in an atmosphere of 5% CO2. The formazan crystals produced were dissolved in dimelthyl sulfoxide . After 30 min, absorbance at 540 nm was measured using a scanning spectrophotometer  [The medium above the cells was removed and the wells were washed with phosphate-buffered saline (PBS). Culture medium containing MTT (0.5 mg mlGermany) . Percent-1 in water), 50% fluorescein diacetate (1.5 mg ml-1 in DMSO), 10% HO (1 mg ml-1 in water)  and 15% PBS. Ten microliters of the samples were placed in glass slabs and covered with coverslip. The area of analysis was 0.030 mm2. Cells were classified as either viable , non-viable cells  or necrotic  using confocal microscopy (63x Leica TCS_SPE). The non-viable and necrotic cells were considered to be dead. The percentages of living and dead cells were quantified [Differential staining with specific fluorochromes was used to distinguish living and dead cells. Cells were trypsinized and suspended in 100 μl PBS and 100 μl of a solution containing 25% PI  were fixed with 4% formaldehyde for 10 min and stained with H+E. Cells were examined using an inverted optical microscope . Cell morphology was classified as follows:-) No reaction: no evidence of morphological changes.((+) Mild reaction: some cells with small morphological changes.(++) Moderate: detachment of some cells, presence of collapsed or rounded cells, few changes in cell number.(+++) Severe: cellular fragments, severe reduction in cell number, collapsed and rounded cells with surface blebs.(++++) Very severe: cell lysis, cell contour loss, severe reduction in cell number, few cells preserved, almost all cells collapsed or totally destroyed, most cells shriveled and wrinkled.5 (7 ml medium per dish) and treated with varnishes containing TiF4 or NaF , or placebo varnish, for 6 or 12 h. The varnish was applied to four protrusions attached to the cover of the Petri dish (30 mg per protrusion). The protrusions became immersed in the medium, allowing fluoride release at a distance of 5 mm from the cells. For positive controls, varnish was omitted. Experiments were carried out in biological triplicate (n = 3 dishes for each condition).Cells were plated in Petri dishes (60 x 15 mm), at a density of 3 x 102, 1 mM CaCl2, 5 mM glucose, 10 mM HEPES, pH 7.4). The stiffness of individual cells (10 curves for each cell) was measured using a Bioscope atomic force microscope  mounted on Axiovert 135M microscope  and controlled by a NanoScope IIIa controller. Silicon nitride AFM cantilevers (spring constant 0.01 N/m) with spherical tips  were used as mechanosensors. A force-distance curve was plotted, relating the bending of the cantilever (or the applied force) to its position relative to the cell. The stiffness of the cell was deduced from the linear slope of this curve (~400 nm). Analyses were carried out using Atomic J Software version 1.6.0 [After washing with PBS, cells were exposed to HEPES-buffered saline solution . Values for percentage cell viability (MTT and HO/PI) and fluoride release were compared using Kruskal-Wallis test followed by Dunn’s test. Cell stiffness values were compared using ANOVA followed by the post-hoc Bonferroni test. Percentage of viability (MTT) and fluoride release were correlated using Pearson’s test. The level of significance was set at 5%.The software GraphPad InStat version 2.0 for Windows  was used. The MTT, HO/PI and F release data passed the normality test (Kolmogorov-Smirnov test), but not the equality of variances test , with no significant differences between the fluoride salts, concentrations, or pH values  and NaF (1.90% and 2.45% F) varnishes significantly released more fluoride than the control and placebo. On the other hand, the varnishes with the lowest fluoride concentrations did not differ from the control and placebo (P < 0.0001). After 12 h, the results among the various groups were similar to those over other periods; however, in this case TiF4 (1.90% F) released more fluoride than placebo/control, while NaF (1.90% F) did not. Differences between the experimental times were found for all groups, except for the control, NaF (0.95% F) and placebo.The fluoride concentrations in the medium as a result of release from the varnishes ranged between 3 and 140 ppm. After 6 and 24 h, the various groups behaved similarly; specifically, TiF4 (2.45% F) and NaF (1.90% and 2.45% F) varnishes significantly reduced the percentage cell viability compared to positive control (10% FBS), similarly to the negative control. No significant difference was found between positive control and 1% FBS control. TiF4 (0.95% and 1.90% F), NaF (0.95% F) and placebo varnishes were not cytotoxic at these times (P < 0.0001). After 24 h, the results among the groups were similar to previous periods, but in this case TiF4 (1.90% F) varnish did have a cytotoxic effect (P < 0.0001).As judged by the results of the MTT assay, after 6 and 12 h, TiF4 was more cytotoxic than NaF. With respect to the experimental periods, the fluoride toxicity was significantly increased after 24 h compared to 6 h and 12 h ; however, toxicities at 6 h and 12 h did not differ from each other.No significant differences were found between the fluoride salts, except at 24 h for 1.90% F, in which TiFP = 0.0004). Hence, cellular viability showed a tendency to decrease as the fluoride concentration of the varnish rose.The percentage cell viability (by MTT assay) and fluoride release from the varnishes showed a moderate but significant inverse correlation  and NaF (1.90% and 2.45% F) compared with the positive control (4 (1.90% F) varnish also showed cytotoxicity  and placebo varnishes were not cytotoxic. Representative images for the 24 h treatments are shown in 4 (1.90% and 2.45% F). For the other treatments, there was no significant difference between the periods.According to the HO/PI assay, after 6 h, there were no significant differences in the percentage of living cells among the various treatments . After 1 control . After 2toxicity . The var4 and NaF varnishes (1.90% and 2.45% F) drastically reduced the number of cells and caused morphological alterations in the cells that remained, while the lowest concentrated fluoride varnishes  caused moderate morphological alterations with only slight changes in cell number. No differences were seen between the fluoride salts  significantly reduced cell stiffness compared with the control and placebo, while the stiffness of cells exposed to NaF varnish (2.45% F) did not differ from the control and placebo. There was no significant difference between the effects of TiF4 and NaF varnishes  varnishes partially release fluoride along to 12h in contact with water and artificial saliva (in a range of 15–25 ppm F) [Despite some studies have shown cytotoxicity of NaF, it has been considered safe especially when included in varnish. Varnish has some advantages compared to mouthrinse or gel, due to its adherence to the tooth surface, allowing a longer contact time between fluoride and enamel (from 6h to 12h). A previous study has shown that NaF and TiF5 ppm F) , amount 5 ppm F) –38.4, both at high and similar fluoride concentrations as those found in products indicated for professional application. Experiments using solutions containing different fluoride concentrations were aimed at simulating the application of TiF4 as a mouthrinse. The concentrations were chosen according to previous studies [Our study aimed to compare the cytotoxicity potential of NaF and TiF studies ,15, whic studies , is impo studies . A pH va studies .4 might be related to its low pH [4 and NaF had comparable cytotoxic effects. Our data disagree with a previous report that 1% TiF4 (pH 1.35) at native pH is more toxic than NaF (pH 8.45), which was mainly attributed to the lower pH of the first [4 solution, could have a cytotoxic effect similar to those caused by the low pH itself. Thus, it seems that the increase in the pH of TiF4 solutions might not benefit the patient, at least not through the use of sodium citrate.It has been reported that the toxicity of TiFs low pH . When aphe first . In our 4 data and previous results [The differences between our TiF results  could be results  applied  results  used sca results  and viab results , and so 4 into a varnish for professional application because varnishes have been shown to be better than an equivalent solution for the prevention of dental caries and erosion [4 and NaF varnishes partially released their fluoride content in DMEM, and we found a negative correlation between fluoride release and viability. In respect to the analyzed variables, the two highest fluoride concentrations tested here were cytotoxic, reducing viability and causing morphological changes, while the lowest fluoride concentration was not. Additionally, a 24 h treatment led to lower cell viability and survival than 6 h and 12 h treatments. On the other hand, TiF4 and NaF varnishes were similarly cytotoxic.We propose the incorporation of TiF erosion ,43. In oInkielewicz-Stepniak et al.  showed a4 against enamel erosion is greater when a highly concentrated solution is applied. Comar et al. [4 varnishes rises. Generally, dental varnishes contain a range of 2.26% to 6% F [4 varnish in further clinical trials, since it was not shown to be more toxic than NaF varnish, which is already widely used clinically.Vieira et al.  have shor et al.  also sho to 6% F , and the to 6% F ,48. Henc4 toxicities. We analyzed stiffness over a depth of 400 nm, which reveals mechanical alterations related to the cytoskeletal proteins beneath the plasma membrane [Lee et al. and Subbmembrane . Considemembrane ,53, we s4 is so far unexplored as yet, although it is likely that similar processes are involved.The mechanism underlying the cytotoxic effects of fluoride has been extensively investigated. Jeng et al.  showed t4 and NaF have similar cytotoxic effects on fibroblast viability, stiffness and morphology. The cytotoxic effects mainly depend on the fluoride concentration and exposure time. This result gives support for testing the effect of TiF4 varnish in vivo.Based on the results of the 3 experimental steps, we have shown that TiF


True CKD incidence and mortality rates were computed for the whole study period, and prevalence for the intervals 1982–1994 and 1995–2007. Estimated age-sex stratified CKD incidence rates were computed using illness-death models of the observed prevalences, and of the whole-period mortality rate ratio of CKD to non-CKD persons. Among 1201 participants, 616 incident events of CKD occurred during a median follow-up of 5.6 years. Observed CKD prevalence was 56.9% (95%CI 53.7–60.0) and 48.0% (95%CI 45.2–50.8) in women; 54.0% (95%CI 49.9–58.1) and 49.6% (95%CI 46.0–53.3) in men, across the two periods. Mortality rate was 2.5 (95%CI 1.9–3.3) times as high in women with CKD and 1.6 (95%CI 1.3–2.1) times as high in men with CKD, compared to women or men without CKD. In women, estimated CKD incidence increased linearly from 25.6 (95%CI 4.2–53.0) to 128.6 (95%CI 77.1–196.6) with each 5-year age group up to 69 years, and to 99.8 (95%CI 38.7–204.7) at age ≥70. In men, estimated CKD incidence increased form 28.5 (95%CI 3.8–71.2) at age 20–24 years to 118.7 (95%CI 23.6–336.7) at age ≥70. Age-sex-stratified estimated incidence reflected the magnitude and directional trend of the true incidence and were similar to the true incidence rates (p>0.05 for difference) except for age 20–24 in women (p = 0.008) and age 25–29 in men (p = 0.002). In conclusion, the estimated and observed incidence rates of CKD agree well over 25 years of observation in this well characterized population with type 2 diabetes.The objective was to estimate chronic kidney disease (CKD) incidence rates from prevalence and mortality data, and compare the estimates with observed (true) incidence rates in a well-characterized population with diabetes. Pima Indians aged 20 years and older with type 2 diabetes were followed from 1982 through 2007. CKD was defined by estimated GFR (eGFR) <60 ml/min/1.72 m Chronic kidney disease (CKD) affects 39.2% of adults with diabetes in the United States . When adIn this study, we use observations from a well-characterized population with type 2 diabetes to estimate CKD incidence rates from observed prevalence and mortality data, and then compare these estimates with true CKD incidence rates in this population. We demonstrate that estimating CKD incidence using the illness-death model represents a reliable method for estimating incidence rates from cross-sectional studies.A longitudinal study of diabetes and related conditions was conducted in the Gila River Indian Community in southern Arizona from 1965 through 2007 , and abs2 or ACR ≥30 mg/g.Diabetes was diagnosed by a 2-hour post-load plasma glucose level ≥11.1 mmol/L (200 mg/dL). The date of diabetes diagnosis was determined from these research examinations or from review of clinical records if diabetes was diagnosed during routine medical care. Serum creatinine was measured by a modified Jaffé reaction with calibration traceable to an isotope-dilution mass spectrometry measurement procedure . AlbuminThe study was approved by the Review Board of the National Institute of Diabetes and Digestive and Kidney Diseases. Each participant gave written informed consent.r were computed among those with pre-dialysis CKD as the count of transitions to the "non-CKD" state (numerator) divided by the person-time at risk for remission (denominator); only the first remission event was counted. Observed prevalence of CKD was computed for the calendar intervals 1982–1994 and 1995–2007 using data from the research examination closest to the midpoint of each period. Observed death rates were computed for the whole study period as the number of subjects without or with CKD who died of any cause per 1,000 person-years of follow-up. The period at risk for the without CKD death rate began at the first diabetic examination and ended at the date of death, onset of CKD, or December 31, 2007, whichever came first. The period at risk for those with CKD death rate began at the first diabetic examination with CKD and ended at the date of death or December 31, 2007, whichever came first.Observed incidence of CKD was calculated for the whole study period as the number of new CKD events per 1,000 person-years at risk. Follow-up extended from the date of the first diabetic research examination after the age of 20 years without CKD to first date with CKD or, in those who did not develop CKD, to the date of the last research examination within the study period. The age- and sex-specific remission rates a at calendar time t are either without CKD or with CKD. Persons may change status according to the transition rates i, r, m0, m1, where i represents CKD incidence, r is the CKD remission rate, m0 the death rate among persons with diabetes without CKD, and m1 the death rate among persons with both diabetes and CKD.To estimate incidence of CKD we used the illness-death model shown in i leads to the following equation:Equation (3) in Brinks and Landwehr  solved fp represents change of prevalence in time , rp is remission rate, m0 death rate without CKD, m1 the death rate with CKD, and p is the prevalence in the middle (t*), between the first point in time (t1) and the second (t2), i.e.p is estimated from the two surveyed prevalences:a = age [Where ∂a = age .We then compared the incidence rate estimated via Eq  with the2). Mean arterial pressure (MAP) was calculated as 2/3 diastolic arterial pressure + 1/3 systolic arterial pressure. A χ2 tests for general association were used to examine relationships between time period, hypoglycemic and antihypertensive treatment; median values were compared by the Kruskal–Wallis test.Baseline clinical and demographic features of the study population are presented as median and interquartile range (IQR). Body mass index (BMI) was defined as weight divided by the square of height  with type 2 diabetes and without CKD at baseline, 616  incident events of CKD occurred during a median of 5.6 years of follow-up (IQR 2.9 to 9.9 years); 19 of these represented incident dialysis cases. The observed age- and sex-stratified incidence rates of CKD are shown in 2, the overall incidence of CKD was 25.4 events/1,000 person-years (95% CI 21.3–30.1) in men and 21.9 events/1,000 person-years (95% CI 19.3–24.8) in women.Among those with pre-dialysis CKD, 341  experienced remission of CKD during a median follow-up of 4.8 years (IQR 2.3 to 9.6 years); all except three of these remissions were due to reduction of ACR from ≥30 mg/g to <30 mg/g. The overall CKD remission rate was 50.2 events/1,000 person-years (95% CI 45.0–55.8); CKD remission rate was 52.9 events/1,000 person-years in women (95% CI 46.6–60.0), and 44.0 events/1,000 person-years in men (95% CI 35.5–54.0) . In the Observed CKD prevalence estimates for the first and second periods are shown in The observed age- and sex-stratified death rates, overall and by presence of CKD, are shown in Age-sex stratified observed  and estimated incidence rates are shown in The estimated incidence rate of CKD based on the illness-death model agreed well with the observed incidence of CKD in this well characterized population with type 2 diabetes. Estimated incidence rates, computed from observed prevalence and mortality data, reproduced the true incidence rates particularly well in women, largely due to a greater number of women than men in each 5-year age group.Why is it important to know incidence rate of CKD? Although the term ‘epidemic’ has been used in recent years to describe the increasing prevalence of CKD, a true epidemic would require an increasing incidence of the disease. Unlike prevalence, CKD incidence rate is specifically reflecting exposure to particular risk factors and therefore its measure helps characterize factors contributing to disease onset and progression, estimate how changes in these exposures impact development of disease over time, and evaluate the effectiveness of prevention strategies and health interventions. Knowing how much of the prevalence is due to incidence of a disease over time has therefore important implications for the allocation of limited public and healthcare resources for the prevention and treatment of kidney diseases.Except for reports on the incidence of end-stage renal disease , 17, tru2, was 24.8 events per 1000 person-years (95% CI 21.9–27.9) between 2007–2012 [2 was 21.5 (95% CI 19.9–23.1) per 1,000 person-years during a mean follow-up of 9.9 years; incidence rates among women and men were 28.5 (95% CI 26.2–31.1) and 13.3 (95% CI 11.6–15.2), respectively [A study among 14,873 participants in the community-based, multicenter, biracial Atherosclerosis Risk in Communities Study (ARIC), used different case definitions to estimate CKD incidence during nine years of follow-up . Because007–2012 . The inc2. Thus, accounting for the remission rate in the differential equations will substantially improve the incidence model when all stages of CKD are considered, but may not have any impact on the modelling when CKD is defined by GFR <60 ml/min/1.73 m2 only.Accounting for the remission rate of CKD is important when estimating CKD incidence by the illness-death model. A substantial proportion of persons with moderate albuminuria regress to normoalbuminuria, either spontaneously or as a result of treatment, indicating a measure of reversibility of initial kidney injury rather than inevitable progression to ESRD. Generally, the proportion of persons with type 2 diabetes who regress from moderate to normoalbuminuria is 30%-54%, while the frequency of progression to overt proteinuria is 12%-36% –23. In aThe applicability of the illness-death model for extracting incidence density estimates from prevalence and mortality data has been demonstrated previously for chronic diseases such as renal replacement therapy , dementiThe strength of the study derives from including a population in whom type 2 diabetes and CKD are known with greater accuracy than in other studies because of systematic periodic testing as part of the longitudinal follow-up. The study limitations relate to 1) the inclusion of a homogenous cohort of Pima Indians, however, these results are intended to add to previous ones conducted in predominantly Caucasian populations; 2) it is the first time that the method is applied to such a long period, covering 25 years of data. Many changes might have happened during that time, including changes in risk factors, in treatment and mortality, all of which might not be captured in two cross-sections. Even so, the magnitudes of the estimated and observed incidences are similar. Another important limitation is our method of determining if the true and estimated incidences are the same. We tested the null hypothesis that the true and estimated incidences are the same. In effect, this says that they will be declared the same unless the data provide strong evidence that they are different. This could be addressed in some future study by the use of equivalence testing, instead of difference testing.In conclusion, over 25 years of observation, the estimated incidence rate of CKD based on the illness-death model agreed well with the observed (true) incidence of CKD in Pima Indians with type 2 diabetes. Although we used longitudinal data to compute CKD remission rates, persistence and/or regression of albuminuria can be easily obtained in subsets of cross sectional populations with repeated ACR and eGFR measurements, as has been done for the NHANES population . The preS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


T1 mapping is a rapidly evolving field and is emerging as a novel quantitative tissue characterisation technique with potential applications in fibrosis, oedema, fat, iron and other patho-physiology. Its significance in heart failure is not yet fully explored but has the potential to add insights into disease processes and aid in refining diagnosis.242 consecutive consenting patients referred for CMR between December 2014 and June 2015 were scanned on a 1.5T Magnetom Avanto  using a MOLLI . In all cases on the basal short axis slice, a single, slender ROI was placed in the mid wall of the septum with meticulous attention to avoid partial volume of blood/and or scar areas to derive the native T1 in msec. The patients were grouped by ejection fraction: <45%, 45-55% and >55%. 15 healthy volunteers were also used as a control group. Ejection fraction (EF), LV volumes, LV mass and BSA indexed left atrial area (LAAi) were also acquired. Comparison was performed using ANOVA and linear regression with adjustment for demographic variables that were found to vary between groups (age and BSAi LV mass).Baseline characteristics are given in the table. Unadjusted LAAi values were similar in the EF 44-55% and EF >55% groups 13.3 ± 3.5) but significantly higher in the EF <45% group , see figure 3.3 ± 3.5On pair wise comparisons between groups, there was no difference in T1 values between the <45% and 45-55% group in all analyses. The T1 difference between the 45-55% and >55% groups was significant only after adjusting for age, sex and LV mass p = 0.04. The control group septal T1 significantly varied from both the <45% and 45-55% groups (p => 0.001 and p = 0.03 respectively after adjusting for age and sex). There was no difference between the Septal T1 between the control group and the >55% group (p = 0.9 after adjusting for age and gender).In a large cohort of consecutive patients, we have demonstrated that native T1 is prolonged in patients with low EF. Based on prior study, this is likely to reflect diffuse fibrosis. In addition we have shown that Native T1 is not different between patients referred for CMR with normal EF and healthy volunteers.


Well differentiated keratinized squamous component as a part of urothelial carcinoma can be easily appreciated; however non-keratinizing squamous differentiation closely resembles urothelial differentiation. In addition prognostic significance of CK 5/6 expression in the absence of apparent squamous differentiation is still unclear. Therefore, in the present study we aimed to evaluate the frequency of CK 5/6 expression in 127 cases of urothelial carcinoma and its prognostic significance in loco-regional population.Positive CK5/6 expression was noted in 6.3% (8 cases) and 13.4% (17 cases) revealed focal positive CK 5/6 expression. On the other hand, 80.3% (102 cases) showed negative CK5/6 staining. Significant association of CK5/6 expression was noted with tumor grade and muscularis propria invasion, however no significant association was noted with overall and disease free survival. On the basis of the results of our study we can conclude that CK5/6 is an independent prognostic biomarker in urothelial carcinoma and therefore can be used in the prognostic stratification of the patients with bladder cancer. Bladder cancer is among one of the most common malignancy in males worldwide and its incidence is even higher in developing countries owing to certain endemic infections , 2. UrotTotal 240 diagnosed cases of urothelial carcinoma specimens were selected from records of pathology department. All patients underwent surgeries at Liaquat National hospital, Karachi from January 2010 till December 2014 over a period of 5 years. The study was approved by research and ethical review committee of Liaquat National Hospital and informed written consent was taken from all patients at the time of surgery. Hematoxylin and eosin stained slides and paraffin blocks of all cases were retrieved and new sections were cut when necessary. Slides of all cases were reviewed by two senior histopathologists and pathologic characteristics like histologic type, tumor grade, lamina propria invasion, muscularis propria invasion were evaluated. Clinical records of 61 patients were available and are thus reviewed from institutional records to evaluate history of radiation and chemotherapy and recurrence status. Moreover, representative tissue blocks of 127 cases were available for CK5/6 immunohistochemistry.CK5/6 IHC was performed by using FLEX Monoclonal Mouse Anti-human Cytokeratin 5/6, clone D5/16 B4 (Lot No. 20042129) by DAKO envision method according to manufacturers protocol on 127 cases of urothelial carcinoma (on representative tissue blocks). Results of IHC staining were interpreted by two senior histopathologists with more than 5 years experience of reporting histopathology and immunohistochemistry and they were blinded by other histopathological features of the tumors. For quantification, at least 1000 cells were counted in 10 HPFs (40×). Intermediate to strong cytoplasmic and membranous staining in more than 10% of tumor cells was considered positive. Weak to intermediate staining in < 10% was taken as focal positive, while no staining was considered as negative  were excluded from the study.P value ≤ 0.05 was taken as significant.Statistical package for social sciences (SPSS 21) was used for data compilation and analysis. Mean and standard deviation were calculated for quantitative variables. Frequency and percentage were calculated for qualitative variables. Chi square was applied to determine association. Student t test or Mann Witney test were applied to compare difference in means among groups. Survival curves were plotted using Kaplan–Meier method and the significance of difference between survival curves were determined using log-rank ratio. Mean age of patients was 63.23 + 13.9 years with male to female ratio of 3:1. 95.8% specimens were of transurethral resections. 50.8% (122 cases) were of high grade morphology, whereas 49.2% (118 cases) showed low grade histology. Lamina propria invasion was seen in 30.4% (73 cases), while muscularis propria invasion was noted in 22.9% (55 cases). Mean follow up of patients involved in the study was 22.0 + 13.74 months and recurrence was seen in 45.9% (28 cases) as presented in Table Positive CK5/6 expression was noted in 6.3% (8 cases) and 13.4% (17 cases) revealed focal positive CK 5/6 expression. On the other hand, 80.3% 102 cases) showed negative CK5/6 staining. Significant association of CK5/6 expression was noted with tumor grade and muscularis propria invasion, however no significant association was noted with lamina propria invasion and disease free survival  in up to 50% of cases and is associated with poor disease progression  performeOne of the major limitations of our study was that we performed only single biomarker of squamous differentiation in our study; use of multiple markers like CK5/14 and CK4/14 could increase the sensitivity of the study. However, on the basis of the results of our study we can conclude that CK5/6 is an independent prognostic biomarker in urothelial carcinoma and therefore can be used in the prognostic stratification of the patients with bladder cancer.


Issues of mass screening for prostate cancer rather controversial since 2013 in 11 regions of Kazakhstan introduced a population-based screening for prostate cancer, so we need to evaluate its results.In different regions of Kazakhstan during 2013–2015, a total of 321548 prostate-specific antigens (PSA) were determined in men aged 50–66 yr, under the Prostate Health Index (PHI) and transrectal ultrasonography (TRUS) guided prostate biopsy with histological examination.PSA level up to 4 ng/ml in 310870 (96.7%) men, PSA level between 4 and 10 ng/ml in 8 624 (2.7%) men, PSA level above 10 ng/ml in 2054 (0.6%) men. PHI was identified in 5716 (1.8%) men, of which 2867 cases were with PHI ≥ 25 (35.9%). Totally, 3680 biopsies (1.1%) of the prostate were performed. As part of the screening, 2870 cases (0.88%) of benign prostatic hyperplasia and prostatic intraepithelial neoplasia were found. Of 742 cases of prostate cancer (0.23%) were revealed. The stages of prostate cancer screening were as follows: stage I in 172 men (23.2%), stage II in 444 men (59.8%), stage III in 98 men (13.2%) and stage IV in 28 (3.8%) men. The indicators of prostate cancer early diagnosis in the I–II stages were bigger in the “screening regions” than in the “traditional diagnostics” regions: RR 1.35 95% CI (1.24 – 1.46), OR 1.84 95% CI (1.58–2.15). Prostate cancer was detected at I–II stages in the “screening” regions only by screening vs traditional diagnostics, with RR 1.64 95% CI (1.56 – 1.73), OR 4.77 95% CI (3.87–5.87).Implementation of screening can improve the diagnosis of prostate cancer in the early stages. According to the GLOBOCAN database , 1.1 milFollowing the Health Development State Program of the Republic of Kazakhstan, prostate cancer screening program was launched in 2013, given the trend towards low ratio of mortality to morbidity and low 5-year survival rate due to late diagnosis. According to WHO Regional Office for Europe, introduction of mass screening programs at a national level remains rather controversial  for PSA. Primary health care staff takes the blood samples for the pre-analytical processing and delivery to the central laboratory in each region equipped with ICMA analyzers. The methods for in-depth diagnosis are as follows: the PHI is identified when the value of total PSA is at the level of 4–10 ng/ml by Hybritech calibration. The method of TRUS with 8 cores prostate biopsy is used if the patient’s PHI is ≥ 25, and in the patients with PSA ≥ 10 ng/ml. Histomorphology of biopsy material is examined in the pathology lab of an oncologic center or a central regional pathology office following the generally accepted international standards and classification with indication of the Gleason score . The data of Kazakhstan Cancer Registry from 2001 to 2015 and the MedInform Company reports on the results of preventive medical examinations for prostate cancer according to the forms completed in the primary care organizations were used for the analysis. The dynamics of prostate cancer was analyzed using Kazakhstan Cancer Registry, official reports of the regional oncologic centers on primary patients registered in 2013–2015 and notices about patients with first-ever diagnosis of prostate cancer for the 2013–2015.PSA level was examined in 321548 men during 2013–2015, as part of a screening study. Total PSA level up to 4 ng/ml was identified in 310870 males (96.7%), the level of total PSA between 4 to 10 ng/ml - in 8624 men (2.7%), and total PSA level above 10 ng/ml was found in 2054 men (0.6%). At the same time, hemolysis or chylesis observed in the blood samples of 1604 men (0.5%) were due to non-compliance with pre-analytical phase of the algorithm. The patients had to reproduce blood samples for PSA. PHI is found in only 5716 men (1.8% of those surveyed), which comprised only 66.3% of the 8624 men in whom PHI had to be identified according to the screening algorithm, of these 2867 cases (35.9%) had a level of PHI ≥ 25. Total number of prostate biopsies was 3680 (1.1%), making 74.8% of the indicated, according to the screening algorithm . The screening revealed 2870 cases (0.88%) of benign prostatic hyperplasia and prostatic intraepithelial neoplasia, it revealed also 742 cases of prostate cancer (0.23% of those surveyed). To reveal one patient with prostate cancer 433.3 total PSA tests 7.7 PHI level measurements and 5 biopsies with histological examination were conducted.After the introduction of screening for 3 yr the epidemiological situation in Kazakhstan has changed, with an increased morbidity of 2.5 per 100000 people per year, or 480 newly diagnosed patients, whereas the ratio of mortality to morbidity increased by 15.2% and amounted to 28.2%. The ratio of mortality to morbidity in the regions of “screening” was 25.5%, while in the regions with traditional diagnostics it made 46.7%. An offset age-peak detection of prostate cancer showed a shift from 70–74 yr in 2012 to the screening age 62–66 yr in 2013, 2014 and 2015. In this relatively young age there is a lower incidence of co-morbidities and a high probability of radical treatment of prostate cancer patients. In the regions with traditional diagnostics, there is no change by age in the cohort of patients. In 2014 and 2015 yr , analysiIn the 11 regions where the screening programme was carried out, the prostate cancer was found in 1763 males (58.6%) in the I–II stages, while 1244 (41.4%) screened patients revealed the III–IV stages of the prostate cancer. In the 5 regions where the screening was not carried out, the prostate cancer was detected in 372 men (43.5%) at the I–II stages, with 483 prostate cancer cases at the stages III–IV (56.5%). RR 1.35 95% CI (1.24 – 1.46), OR 1.84, 95% CI (1.58–2.15) Total 6.16 cases should be screened in order to prevent detection of one prostate cancer case in the III–IV stages among all men. The screening allowed to reveal 616 (83%) cases of prostate cancer at stages I–II and 126 (17.0%) at stages III–IV. In these same regions, methods of “traditional diagnosis” revealed 1147 (50.7%) at stages 1118, and I–II (49.3%) at stages III–IV. RR 1.64 95% CI (1.56 – 1.73), OR 4.77 95% CI (3.87–5.87). Total 3.09 cases should be screened in order to prevent detection of one prostate cancer case in the III–IV stages in the men of 50, 54, 58, 62 and 66 yr of age.Determining the level of PSA has revolutionized the diagnosis of prostate cancer . Only twIn recent years, the problem of defining specific forms of PSA is of great interest in screening programs. One of these forms is known as a pro-enzyme form of PSA (pro PSA). Pro PSA, and in particular its truncated form -2pro PSA may be more associated with prostate cancer, compared to the PSA and the use of these forms and relations to the PSA can improve the detection of prostate cancer . CompareThis is the first screening in Kazakhstan, with the target group of only men. Its implementation contributes to men’s responsibility for their health, cancer awareness and consolidating efforts to protect men’s health.The introduction of population screening for prostate cancer in Kazakhstan has improved the diagnosis of prostate cancer in the early stages.Ethical issues  have been completely observed by the authors


Thyroid-associated eye disease (TED), previously known as Graves’ ophthalmopathy is a cosmetically and functionally debilitating disease that is seen worldwide. The aim of this study was to evaluate the prevalence and clinical severity of ocular manifestations of Graves’ disease according to sex, age and duration in northern Iran. Between April 2011 and March 2012, 105 patients with Graves’ disease, underwent ophthalmic examination, including ocular motility, exophthalmometry, intraocular pressure (IOP), slit lamp and fundoscopy. Patients received scores according to modified Werner’s NO SPECS classification.Ocular involvement was found in 70 patients with established Graves’s disease. The mean age was 35.0 years, . The most common ocular findings were exophthalmometric proptosis of more than 20 mm (63.8%), lid lag (55.7%), lid retraction (52.8%) and tearing (38.6%). Almost 70% of patients had bilateral involvement. Elevated IOP was seen in 15 (25.4%) patients, and was significantly related to proptosis (P=0.007). More than half of the patients  had a modified Werner’s NO SPECS score of 3.00. Clinical severity as shown by the increasing number of signs and symptoms per patient was correlated to increasing age  but not to gender (P=0.17).Both functional  and cosmetic  sequels are common ocular presentations in patients with Graves' disease. Proptosis was the most common finding in this study and was associated with elevated IOP. Clinical severity was found to correlate to increasing age. Thyroid-associated eye disease (TED), is a cosmetically disfiguring and functionally incapacitating, chronic, infiltrative ocular disease that is usually associated with Graves' disease . HoweverThere have been only a few studies on clinical features of TED in Iranian patients . In ordeThis was a descriptive cross-sectional study. The study population included all consecutive patients with confirmed diagnosis of Graves' disease who referred to the endocrine clinic of Ayatollah Rouhani Hospital in Babol, Iran, from April 2011 through March 2012. The diagnosis of Graves' disease was based on clinical and laboratory findings of diffuse enlargement of thyroid gland, raised free thyroxin or triiodothyronine levels, and suppressed thyroid-stimulating hormone levels. Information regarding ocular symptoms, family history, smoking history, associated systemic disease and the treatment regimens were obtained. The study had no exclusion criteria and all patients with documented history of Graves' disease with any age, onset or type of intervention were included. A single ophthalmologist performed ocular examinations in all patients.A comprehensive ophthalmic examination including visual acuity, slit-lamp examination, evaluation of eyelids and ocular movements, tonometry with Keeler non-contact tonometer, exophthalmometry with Hertel exophthalmometer, and fundoscopy for evaluation of optic nerve head was performed for all patients. Best-corrected visual acuity (BCVA) was documented using Snellen chart. Intraocular pressure (IOP) was measured in primary position and upward gaze. Retraction of either upper or lower eyelid was defined as any exposed superior or inferior sclera beyond the limbus in the primary gaze. Proptosis was defined as the measurement of antero-posterior protrusion of the globe 20 mm or more from the orbital rim in either eye or any discrepancy in the degree of protrusion of the two eyes by >2 mm . CornealA total of 105 patients with established Graves' disease were studied. 70 patients, 26 (37.1%) males and 44 (62.9 %) females, were diagnosed with Graves’ ophthalmopathy (GO). The age range was from 15-69 years. The mean age of our patients was 35.0 [standard deviation (SD) 13.0]. The mean age for females was 33.0±10.7 and for males 38.1±15.6. 26 (39.4%) patients were under the age 29 years, 27 (40.9%) patients were between 30-49 years and 13 (19.7%) patients were between 50-70 years . Overall, in this study, there were more females involved with TED than males (F/M=1.7/1). The female to male ratio was 1.6 and 2.25 in the younger and the middle age groups respectively, but was 0.6 in the older age group. Even though in the older age group there were more males than females, 8 (61.5%) to 5 (38.5%), this reversal of ratio in older patients was not significant (P=0.17). The most common ocular finding in this study was proptosis in 44 (63.8%) patients that was unilateral in 14 (31.8%) and bilateral in 30 (68.2%). The next common finding was lid lag in 39 (55.7%) patients, of which 10 (14.3%) were unilateral and 29 (41.4%) were bilateral, and lid retraction in 37 (52.8%) patients of which 29 (41.4%) were bilateral. Other signs and symptoms include tearing in 27 (38.6%), eyelid swelling in 23 (32.9%), conjunctival injection 21 (30%), ocular pain in 18 (25.7%) and dry eye in 12 (17.1%). Not all findings were similar in men and women: eyelid swelling (42.3%) and conjunctival injection (53.8%) was more frequent in males whereas ocular pain (32.6%) and dry eye (20.5%) were seen more in females . OphthalThe mean NO SPECS score for males was 2.69±1.01, and for females 2.52±1.04 but the difference was not found to be significant (P=0.5). The average number of ocular signs and symptoms per patient was 4.1 in those younger than 29 years, 4.0 in those between 30-49 years, and 6.7 in those above 50 years . This diThe average number of signs and symptoms was greater in males (5.15) than females (4.27), (P=0.17). Clinical severity, as judged by the increasing number of signs and symptoms, was correlated to increasing age . Elevated IOP was seen in 15 (25.4%) patients, 9 (37.5%) males and 6 (17%) females, however the sex difference was not found to be significant (P=0.07). There were 13 (39.4%) patients with proptosis and increased IOP. There was a positive correlation between increased IOP and exophthalmos (P=0.007) chi squared test. Also, there was a positive correlation between increased IOP and severity of disease. In patients with increased IOP, the mean NO SPECS score was 3.13 and the median was 3. In patients with normal IOP mean NO SPECS was 2.3 and the median was 2. This difference was significant (P=0.029).Patients with elevated IOP had a greater number of signs and symptoms (4.9±2.1) compared to those with normal IOP (4.8±2.7) but the difference was not significant (P= 0.9) . LimitatIn 65% of patients, the presence of TED was concurrent or within 6 months of diagnosis of thyroid disease, 20% between 7-24 months and 15% more than 24 months. There was no correlation between duration of disease and proptosis (P=0.47) or clinical severity (P=0.84). We had only two smokers in this study who were both males. In this study, there was only one patient with unilateral pallor of the optic disc due to compressive optic neuropathy and one case of bilateral venous congestion of the optic nerve head. Both patients were males and more than 50 years old. We had no cases of sight loss due to corneal opacification.Involvement of the eyes in thyroid disease is a well-known entity. In this study, our aim was to find the relative frequencies of various presenting signs and symptoms of TED in a patient population in northern Iran based on different age groups and gender. Most studies around the world have shown a higher female to male ratio , 8. In oMore than 80% of our patients were under the age 50. Similarly in a study by Besharati (5), 85.3% of patients were below 50 years old. In most studies, the mean age of involvement with TED was lower in females than males. Etezad-Razavi reported a mean of 34.7 years for females and 44.7 years for males, which is slightly different from our mean of 33.0 and 38.1 years for females and males, respectively. Lee  found a The majority of our patients had NO SPECS score or 3 or less which was similar to other studies . There was an increasing number of individual signs and symptoms with increasing age, showing that older patients tend to develop more severe forms of TED (p=0.004). The studies by Besharati , Perros Proptosis was the most common finding in our series (63.8%), followed by lid lag 55.7%), and lid retraction (52.8%). Etezad-Razavi  reported5.7%, andOne notable finding in this study was elevation of IOP. The orbit is a bony structure and any increase in intraorbital volume will cause both displacement of the globe anteriorly and increased intraocular pressure . The assIn a study by Haefliger et al. in 1997 , performIn our study, we had 15 (25.4%) cases of increased IOP in primary position. Increase in IOP can be explained by increased pressure on the globe by enlarging muscle masses and by impediment of episcleral outflow. Indeed in our study, there was a strong correlation between proptosis and increase IOP (P=0.007). Increased IOP was seen more in men (37.5%) than women (17.1%), P=0.078. Persistence of TED may lead to progressive increase of IOP, leading to overt manifestations of glaucoma such as cupping of the disc and visual field loss (18), therefore, follow-up of patients in this regard is warranted.Dry eye has historically been explained by increased tear evaporation from proptotic eyes . NonetheIn our study, we had one case of compressive optic neuropathy that presented with pale discs (2.8%). This patient had severe proptosis, limitation of ocular motility, increased IOP, and periorbital swelling. Kashkouli et al.  reportedSmoking has been shown to correlate with the severity of TED . In our


We evaluated 62 hospitality venues that allowed smoking in Seoul, Korea. A real-time aerosol monitor was used to measure indoor PM2.5 concentrations. Field technicians recorded indoor environmental characteristics. One non-smoking staff member in each hospitality venue was tested for urinary cotinine and total NNAL concentrations. Demographic characteristics were obtained from self-reported staff questionnaires. Natural-log (ln)-transformed PM2.5 concentrations were significantly correlated with the ln-transformed cotinine (r = 0.31) and the total NNAL concentrations (r = 0.32). In multivariable regression analysis, the urinary cotinine concentrations of the staff members were significantly correlated with indoor PM2.5 concentrations; those with the highest concentrations were more likely to be women or staff members that worked in venues with a volume <375 m3. Total NNAL concentrations were significantly correlated only with indoor PM2.5 concentrations. Indoor PM2.5 may be used as an indicator for urinary cotinine and total NNAL concentrations in non-smoking staff members in hospitality venues that allow smoking.The purpose of this study was to determine the relationship between urinary cotinine and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) concentrations in non-smoking staff and the indoor levels of fine particles (PM Secondhand smoke (SHS) contains more than 7000 chemicals, including more than 69 known carcinogens . Exposur2.5), have been used to demonstrate SHS exposure in hospitality venues [2.5 concentrations in hospitality venues [Based on increased evidence of adverse health effects due to SHS exposure, many countries have implemented smoke-free regulations in public places, including hospitality venues. Airborne markers for SHS exposure, such as particulate matter smaller than 2.5 μm .This study used data from a previous study that assessed air quality and biomarker levels in non-smoking staff members at restaurants and bars before and after the implementation of Korean smoke-free regulations . The datmeasured , we sele2.5 concentrations were measured using a real-time photometric aerosol monitor . The monitor was installed with a 2.5-μm impactor and the airflow rate was set at 1.7 L/min. The monitor was calibrated to zero using a high-efficiency particulate absorption filter prior to measurement and was set to a logging interval of 1 min. The measured data from the monitor were adjusted by a factor of 0.295, which was obtained from a previous experiment using a SidePak monitor co-located with a gravimetric measurement [PMsurement .2.5 concentrations were measured for 30 min (n = 30) and outdoor PM2.5 concentrations were measured for 5 min before and after the indoor measurement (n = 10). An average PM2.5 concentration value was determined by averaging 1-min data points from each indoor and outdoor venue. In the venues, the monitor was placed on a table or a seat away from windows, doors, kitchen areas, and direct emission sources . The researchers recorded information on the type of hospitality venue, size, window status, and observation of smoking and counted the numbers of customers and vents in the venue. The number of customers was counted every 5 min and averaged to estimate the average number of customers over the course of the visit. Customer and vent densities were calculated as the number of customers and vents per 100 m2 of indoor area. When outdoor PM2.5 concentrations were measured, the monitor was placed away from direct emission sources, such as smokers and outside vents.The researchers visited restaurants between 18:00 and 20:00 and bars between 20:00 and 24:00 on weekdays. The monitor was hidden in a small bag, with its inlet protruding out of the bag. Indoor PMThe researchers collected spot-urine samples and self-reported staff demographic questionnaires in the venues during staff breaks or setup time. The urine samples were frozen at −70 °C for storage until analysis. The frozen samples were sent to the Center for Clinical Services, National Cancer Center, and the analysis was conducted in a blinded manner to the type of hospitality venue and the smoking history of the participants.The urinary cotinine concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a method modified from a previous study . An AgilThe study used face-to-face questionnaires to obtain the participants’ demographic information . Demogra2.5, urinary cotinine, and total NNAL concentrations were not normally distributed, they were log-transformed for statistical analysis. We present the measured data as geometric means (GMs) and geometric standard deviations (GSDs). Pearson’s correlation test was used to assess the relationship between indoor PM2.5, urinary cotinine, and total NNAL concentrations. We describe indoor PM2.5 according to indoor environmental characteristics, such as the type of hospitality venue (restaurant or bar), volume (<375 m3 or ≥375 m3), customer density (<10 customers/100 m2 or ≥10 customers/100 m2), vent density (<7 vents/100 m2 or ≥7 vents/100 m2), window status (closed or open), and smoking observed (yes or no). The volume (ranging from 90 to 1680 m3), customer density (ranging from 1 to 53 customers/100 m2), and vent density (ranging from 0 to 40 vents/100 m2) were divided into two groups based on their median values. Spearman’s correlation test was used to assess the associations between indoor PM2.5 concentrations and the type of hospitality venue, customer density, and observations of smoking.The normality of the data was assessed using the Shapiro-Wilk test. Since indoor PM2.5, urinary cotinine, and total NNAL concentrations were compared using Student’s t-test for two groups of variables and a general linear model analysis for more than two groups of variables.We described urinary cotinine and total NNAL concentrations according to demographic characteristics, such as sex (women and men), age , employment position (owner or manager and temporary or permanent staff), working hours (<70 h/wk and ≥70 h/wk), whether the participant lived with a smoker at home (yes or no), and the indoor environmental factors. The number of working hours (ranging from 22 to 91 h/wk) was divided into two groups based on median values. Indoor PMp-values < 0.1 identified in the univariable analysis, a multivariable regression analysis was conducted to identify the relationships between urinary cotinine and total NNAL concentrations and indoor PM2.5 concentrations including other variables. A stepwise method was used in the regression models. In all of our analyses, a p-value of 0.05 was deemed significant. SAS 9.3  was used for statistical analysis.Using variables with 2.5 concentrations in the 62 hospitality venues were 65.3 μg/m3 (GSD = 2.2) and 33.8 μg/m3 (GSD = 1.6), respectively. The indoor and outdoor PM2.5 concentrations differed significantly (p < 0.001). In the univariable analysis, several indoor environmental factors were significantly associated with indoor PM2.5 concentrations (p < 0.001). Indoor PM2.5 concentrations were significantly higher in venues with a customer density ≥10 customers/100 m2 than in those with <10 customers/100 m2 (p < 0.001), and were significantly higher in venues where smoking was observed than in venues where smoking was not observed (p < 0.001). However, indoor PM2.5 concentrations were not associated with the volume, vent density, or window status of the venues.The GMs of the indoor and outdoor PMtrations , which w2.5 concentrations were significantly correlated with the type of hospitality venue , customer density , and observation of smoking   and observation of smoking . Customer density was significantly correlated with observations of smoking .Indoor PM< 0.001) . The typ2.5 concentrations were significantly correlated with ln-transformed urinary cotinine concentrations  and with ln-transformed total NNAL concentrations  .Of the 62 urinary samples collected from the staff members, urinary cotinine levels were above the LOQ in 19 samples (31%) and total NNAL values were above the LOQ in 62 samples (100%). The GMs of urinary cotinine and total NNAL concentration of the 62 staff members were 1.8 ng/mg Cr (GSD = 2.8) and 7.3 pg/mg Cr (GSD = 2.5), respectively. Natural log (ln)-transformed indoor PM= 0.011) . Pearsonp < 0.001), and were significantly higher among staff members who worked in bars than in those who worked in restaurants (p = 0.040). They were also significantly higher in staff members who worked in venues with volumes <375 m3 than in those who worked in venues with volumes ≥375 m3 (p = 0.005). The cotinine concentrations of staff members who worked in venues with customer densities of ≥10 customers/100 m2 were significantly higher than those of staff members who worked in venues with customer densities of <10 customers/100 m2 (p = 0.049). Cotinine concentrations were significantly higher in staff members who worked in venues with closed windows than in those who worked in venues with open widows (p = 0.021). Cotinine concentrations were significantly higher in staff members who worked in venues where smoking was observed than in those who worked in venues where smoking was not observed (p < 0.001). However, the cotinine concentrations of staff members were not significantly associated with age, living with a smoker at home, employment position, working hours, or vent density in the venues.Several demographic and indoor environmental factors were significantly associated with urinary cotinine and total NNAL concentrations . Urinaryp = 0.002). The total NNAL concentrations were significantly higher in staff members who worked in venues with volumes <375 m3 versus ≥375 m3 (p = 0.046). The total NNAL concentrations were significantly higher in staff members who worked in venues where smoking was observed than in those who worked in venues where smoking was not observed. However, the NNAL concentrations of staff members were not significantly associated with sex, age, living with a smoker, employment position, working hours, customer or vent densities, or window status.The total NNAL concentrations of staff members who worked in bars were significantly higher than those of staff members who worked in restaurants , and were significantly higher in female staff members than in male staff members (p = 0.001). The urinary cotinine concentrations of staff members who worked in venues with indoor volumes <375 m3 were significantly higher than those of staff members who worked in venues with indoor volumes ≥375 m3 (p = 0.038), and were marginally higher in staff members who worked in venues with closed windows versus open windows (p = 0.065).Urinary cotinine concentrations were significantly associated with indoor PM2.5 concentrations and were marginally associated with volume (R2 = 0.19). The total NNAL concentrations of staff members were positively associated with indoor PM2.5 concentrations (p = 0.012). The total NNAL concentrations were marginally higher in staff members who worked in venues with indoor volumes <375 m3 than in those who worked in venues with indoor volumes ≥375 m3 (p = 0.090). However, window status was not associated with the total NNAL concentrations.Total NNAL concentrations were significantly associated with indoor PM2.5 concentrations in 62 hospitality venues was 65.3 μg/m3, 1.6-fold higher than the National Ambient Air Quality Standard (NAAQS) for the daily PM2.5 concentration of 35 μg/m3. The levels were above the NAAQS in 72% of the hospitality venues. Of the hospitality venues, indoor PM2.5 concentrations were above the NAAQS in 51% of restaurants and 100% of bars. High levels of indoor PM2.5 concentrations have been reported in hospitality venues not covered by smoke-free regulations. In Scotland, the average indoor PM2.5 concentration in 41 bars before smoke-free regulations were imposed was 246 μg/m3 [2.5 concentration in nine hospitality venues and one bingo hall, before the introduction of smoke-free regulations, was measured at 84 μg/m3 [The GM of indoor PM46 μg/m3 . In Geor84 μg/m3 .2.5 concentrations in hospitality venues. Indoor PM2.5 concentrations were significantly higher in bars, those with customer densities ≥10 customers/100 m2, and those in which smoking was observed. Smoking is a significant emission source, and can contribute 90%–95% of respirable particles in hospitality venues [2.5 concentrations in bars than in restaurants may be explained by the presence of smokers. In this study, smoking was observed in 14% of restaurants and 74% of bars during the measurement of indoor PM2.5 concentrations. Other possible sources in hospitality venues are human activity, cooking, and outdoor air pollution. Human activity, cooking, and smoking may be associated with customer density. When more customers are present in a hospitality venue, these activities are likely to increase and, in turn, indoor PM2.5 concentrations may be associated with customer density. A previous study reported differences between indoor and outdoor PM2.5 concentrations in 109 bars were significantly correlated with the number of patrons  [Several indoor environmental factors were associated with indoor PMy venues . Higher  < 0.05) .2.5 concentrations and the urinary cotinine and total NNAL concentrations of staff members in hospitality venues. Although the correlation coefficients between indoor PM2.5 concentrations and urinary cotinine and total NNAL concentrations were similar, higher correlation coefficients have been found between urinary cotinine and total NNAL concentrations. Previous studies in semi-open-air cafés have found that indoor PM2.5 concentrations were significantly correlated with urinary cotinine concentrations in non-smoking staff  [2.5 concentrations were significantly correlated with urinary total NNAL concentrations  in non-smoking staff [Our study found significant correlations between indoor PM< 0.001)  and thatng staff .2.5 concentrations and the urinary cotinine concentrations of non-smoking staff members were lower in our study than in a previous study conducted in semi-open-air cafés [2.5 concentrations and urinary total NNAL concentrations were slightly lower than in another previous study conducted in semi-open-air cafés [2.5 and urinary cotinine and total NNAL concentrations in our study may be because most urine samples were not collected after work on the day of the measurement of indoor PM2.5 concentrations. Conversely, previous studies collected urine samples from non-smoking staff after their shift at the end of the day, on the same day that the PM2.5 concentrations were measured [2.5 concentrations was −2.4 ± 5.7 days.The correlation coefficients between indoor PMir cafés . The corir cafés . The lowmeasured ,20. In o2.5 concentrations, were significantly associated with urinary cotinine and total NNAL concentrations in hospitality venue staff members. Sex was the only significant factor among the demographic factors for urinary cotinine concentrations. The urinary cotinine concentrations of female staff members were higher than those of male staff members. However, total NNAL concentrations were not associated with any other demographic factor evaluated. Previous studies have reported that demographic factors, such as sex and age, are not associated with urinary cotinine [n = 44) and 59 ± 17 h for male staff members (n = 18). Longer working hours for female staff members may contribute to greater SHS exposure, and this may have resulted in higher urinary cotinine concentrations than those found in the male staff members.In our multivariable analysis, several factors, including indoor PMcotinine  or totalcotinine . PossiblUrinary cotinine and total NNAL concentrations were not consistent by sex. Urinary cotinine and total NNAL concentrations may have yielded different results, because cotinine, a major metabolite of nicotine, is a more specific and sensitive biomarker for SHS exposure than is total NNAL. Cotinine has an average half-life of 16 h  and is d3 were 2.2- and 1.6-fold higher, respectively, than those in venues with indoor volumes ≥375 m3. The findings of this study indicate that staff members who work in hospitality venues with relatively small volumes may be at risk of higher SHS exposure than those who work in hospitality venues with relatively large volumes.Indoor volume was significantly associated with urinary cotinine concentrations in hospitality venue staff members. Indoor volume was marginally associated with urinary total NNAL concentrations. The urinary cotinine and total NNAL concentrations in the hospitality venues with indoor volumes <375 mWindow status was not significantly associated with urinary cotinine or total NNAL concentrations in our multivariable analysis. In addition to window status, vent density was not significantly associated with urinary cotinine or total NNAL concentrations in our univariable analysis. The findings of our study indicate that natural or mechanical ventilation does not protect staff from SHS exposure in hospitality venues.Hospitality venues without indoor smoking showed significantly lower levels of urinary cotinine and total NNAL concentrations. The finding suggested that implementation of smoke-free regulations in hospitality venues could protect staff from SHS exposure. A previous study reported that urinary cotinine concentrations of 40 non-smoking staff member in bars decreased from 35.9 to <5 ng/mL in 6–10 weeks after the implementation of smoke-free regulations in Michigan, USA . These s2.5) [2.5 concentrations in hospitality venues, and another strength is the inclusion of demographic and indoor environmental factors. Total NNAL concentrations, metabolites of carcinogenic NNK, were used to estimate staff exposure to SHS in hospitality venues.Key strengths of our study include the use of one non-specific airborne marker (indoor PM2.5)  and two 2.5)  to chara2.5 concentrations were measured for 30 min at each venue. However, the short measurement time might not cause under or over estimation of indoor air quality. Another limitation is that we did not collect staff urine samples on the same day that PM2.5 measurements were taken. Some urine samples were collected several days before or after measurement of the indoor PM2.5 concentrations. Therefore, the urinary cotinine and total NNAL values of nonsmoking staff in the hospitality venues may not directly reflect SHS exposure on the day of the indoor PM2.5 measurements. The study did not account for non-combustible tobacco products in non-smoking staff member because they were not commonly used.However, our study has several limitations. Restaurants and bars were not selected randomly. The selected hospitality venues and their staff may not be representative of the more general population. We chose different times to visit for the types of venues  because the time might be the most crowded time for each type of venue. Indoor PM2.5 concentrations with respect to demographics and indoor environmental factors. Indoor PM2.5 concentrations were significantly correlated with urinary cotinine and total NNAL concentrations in staff members in the hospitality venues. Including indoor PM2.5, we found the urinary cotinine concentrations in the staff members to be significantly associated with sex and indoor room volume. Total NNAL concentrations in staff members were significantly associated only with indoor PM2.5 concentrations. Our findings suggest that indoor PM2.5 concentrations may be useful indicators for urinary cotinine and total NNAL concentrations in non-smoking staff members in hospitality venues that allow smoking.In total, 62 hospitality venues that allowed smoking and their non-smoking staff members were evaluated to determine the relationships between urinary cotinine and total NNAL concentrations and indoor PM


CF) is usually performed using ethnically targeted panels of selected mutations. This has been recently challenged by the use of expanded, ethnically indifferent, pan‐population panels. Israel is characterized by genetically heterogeneous populations carrying a wide range of CFTR mutations. To assess the potential of expanding the current Israeli preconception screening program, we sought the subset of molecularly unresolved CF patients listed in the Israeli CF data registry comprising ~650 patients.Preconception carrier screening for cystic fibrosis , and next‐generation sequencing of the poly‐T/TG tracts.An Israeli nationwide genotyping of 152 CF causing by the CFTR2 database, and only 4/54 (7.4%) were novel. Molecular diagnosis was reached in 78/152 (51.3%) cases. Prenatal diagnosis of 24/78 (30.8%) cases could have been achieved by including all CFTR2‐causing mutations in the Israeli panel.We identified 54 different mutations, of which only 16 overlapped the 22 mutations included in the Israeli preconception screening program. A total of 29/54 (53.7%) mutations were already listed as CFTR gene mutations suggesting that expanded preconception carrier screening might achieve higher preconception detection rates.Our data reveal an overwhelming hidden abundance of  CFTR, OMIM #602421), the only gene known to be associated with CF (OMIM #219700), is widely accepted and has been repeatedly advocated for by opinion leaders , of which, currently, many have debatable pathogenic effect  using conventional PCR techniques. The intronic CFTR mutation, c.3717+12191C>T, was also analyzed by direct sequencing. PCR products were purified using magnetic particle technology . After purification, all fragments were sequenced in the forward and reverse direction to determine the noted regions. Sequencing was performed on a 3730xl DNA Analyzer , and the resulting sequences were analyzed with the Sequencher software . All identified variants were scored relative to the reference sequences deposited in the National Center for Biotechnology Information (CFTR: [NM_000492.3]). CFTR chromosomal rearrangements of all exons and intron/exon boundaries were analyzed using MLPA . The poly‐T/TG tracts (hg 19_chr7:117188683‐117188689) were studied by means of NGS techniques. For this purpose, amplicons spanning the region of interest were run on the MiSeq system  and aligned using NextGene Software . The minimum required coverage for each sample was 1000 reads. Genotyping was completed at the Genomic Research Center, Gene by Gene, Houston, TX.In most cases, DNA was extracted from buccal swabs and quantitated with a SpectraMax190 . In a minority of cases, genomic DNA was extracted from peripheral leukocytes following standard protocols. Sanger sequencing of the The term mutation is used in this manuscript to describe a variant that is pathogenic or likely pathogenic. As the scope of this project did not allow performing functional studies on each of the identified mutations, they were classified based on available information in the literature and the following bioinformatics tools:The current definition of the mutation in CFTR2: CF‐causing mutation, mutation under evaluation, mutation of varying clinical consequence, and non‐CF‐causing mutation  including relevant supporting data  were identified: c.958T>G;p.Leu320Val, c.2620‐15C>G, c.3607A>G;p.Ile1203Val, and c.4242+13A>G  cases; of these, 7/23 (30.4%) were clinically defined as typical CF and 16/23 (69.6%) as atypical CF. In 51/152 (33.6%) cases included in this study for which no mutations were identified, 8/51 (15.7%) were clinically defined as typical CF and 43/51 (84.3%) as atypical CF.A total of 75/152 49.3%) cases were clinically defined as typical CF  cases. In only 18/77 23.4%) cases, the genotyping and clinical data converged into an established molecular diagnosis. Only 2/18 (11.1%) of the cases carried two CF‐causing mutations per CFTR2. In 16/77 (20.7%), only one mutation was found, and in 43/77 (55.8%) cases, no mutations were detected   who will now benefit from better genetic counseling. Changing demographics expressed by the loss of affiliation to one traditional community or geographic location and the clear occurrence of many different mutations across various communities provides motivation for a uniform pan‐population screening program for CF. The identification of 38 mutations that were not known to be part of the mutations affecting the Israeli population, and the understanding of the potential benefit of expanded preconception screening using a well‐curated list of CF‐causing mutations could lead to a revision of the The laboratory work presented in this study was conducted by Gene by Gene, Ltd., in which D.M.B and C.B. declare stock ownership, and for which G.A. serves as a consultant. O.I. is the chairman of The Cystic Fibrosis Foundation of Israel. The other authors declare no conflict of interest.Table S1. List of mutations comprising the Israeli CF preconception program.Click here for additional data file.Table S2. Suggested pathogenicity of all mutations not listed in the CFTR2 database.Click here for additional data file.Table S3. Detailed genotyping results of all participating patients.Click here for additional data file.Table S4. List of all different mutations identified per ethnicity.Click here for additional data file.


After publication of our article  it was bThis programming error has been corrected in v1.2.0 of the software, available from [We have reanalyzed the data in the paper using v1.2.0. The results are very similar and do not significantly affect any of the manuscript’s conclusions. Notably, none of the numeric values quoted in the main text change.Updated versions of Figs. We would like to thank Jörn Schmiedel for alerting us to this issue, helping us reproduce it and testing the fix.Additional file 2:Supplementary figures. (PDF 423 kb)Additional file 3:Replicate correlation tables. (XLSX 14 kb)Additional file 4:Individually validated variants of the neuraminidase gene. (XLSX 12 kb)Additional file 5:Variants with higher scores than wild-type in the presence of oseltamivir. (XLSX 15 kb)


Fragile X syndrome (FXS) is the most common cause of inherited mental retardation caused by expansion of a (CGG) repeat region up to 1000 repeat in 5' region of the FMR1 gene located in FRAXA locus Xq27.3. To better understand the mechanism involved in expansion of CGG region, the molecular characteristic of the flanking microsatellite markers in the region must be clarify in different populations. We aimed to examine the potential association between specific haplotype and the expanded AC-repeat region in cases and controls chromosomes. Forty unrelated FXS males and 62 unrelated normal males originating from various regions of Iran were haplotyped by analyzing two CA-repeat markers, FRAXAC1 and DXS548. Significant linkage disequilibrium was obtained between DXS548 and FRAXAC1 specific marker alleles and CGG repeat expansion among 40 fragile X cases compared to 62 normal controls. The frequencies of DXS548 and FRAXAC1 longer alleles in patients were significantly higher than that in control group. Two FRAXAC1 long alleles were only observed in cases, possibly due to concatenated mutations. The increase of heterozygosities in fragile X cases  in comparison to the controls  showed a multimodal distribution of fragile X associated alleles. Haplotype analyses with DXS548 and FRAXAC1 markers represented that haplotype distribution in the normal controls and FXS patients were significantly different, representing a weak founder effect. FRAXA, termed FRAXD, FRAXE and FRAXF respectively (Fragile X syndrome (FXS) is the most common cause of inherited mental retardation observed approximately 1 in 4000 males and 1 in 8000 females . Males wectively . FMR1 gene is located in FRAXA locus contained a tandemly repeated trinucleotide sequence (CGG) near its 5' end. The mutation responsible for FXS involves the expansion of this repeat segment. The number of CGG repeats in the FMR1 gene of the normal population varies from 6 to approximately 50. There are two main categories of mutations, premutations of approximately 60 to 250 repeats and full mutations of more than approximately 250 repeats. There is no clear boundary between the upper limit of normal and the lower limit of the premutation range. For this reason alleles with approximately 45-55 copies of the repeat are said to be in the "gray zone". Some alleles in this size range are unstable and expand from generation to generation, while others are stably inherited. A premutation is susceptible to expansion after passage through a female meiosis. The larger size of a mother's premutation the more the risk of expansion to a full mutation in her offspring  using PCR and Southern blot analysis. Controls were 62 age/sex matched unrelated healthy subjects from the same geographic area. Genomic DNA was extracted from peripheral blood leukocytes using a commercial kit (QIAamp DNA Mini Kit). Informed consent was obtained from all participants and the study was approved by the Ethics Committee of the center. CGG Repeat Analysis 5'agccccgcacttccaccaccagctcctcca3' and reverse 5'cgacctgtcaccgcccttcagccttcc3' primers . PCR thermal cycle was performed in 32 cycles. Each cycle consisted of 95 °C denaturizing for 30 seconds, 60 °C annealing for 1 minute and 72 °C extension for 40 seconds. The thermal cycles were started with an initial denaturizing of 95 °C for 5 min and a final of 72 °C extension for 10 min.The DXS548 AC-repeat polymorphism was amplified using forward ns et al . FRAXAC1Evaluation of polymorphism was carried out using denaturing polyacrylamide gel electrophoresis contained 8% polyacrylamide (29:1 acrylamide/bisacrylamide), 7M urea, 1XTBE, 500 μl 10% ammonium persulfate, and 50 μl TEMED. Gels were leaved to polymerize at room temperature for 1 hour. The gel apparatus was DNA sequencing unit (30 X 45 cm) connected to 2000V power supply clamps. PCR products (25 μl) were mixed with 45 μl of loading buffer and denatured at 95 °C for 8 min. Denatured samples were cooled on ice and 8μl of each sample was loaded. Gels were run at 50 μC constant temperature and 100 watts limiting power for about 3 hours (BioRad Power/Pak 3000 power supply). Gels were stained and alleles were visualized using standard silver staining procedure.Statistical AnalysisDXS548 and FRAXAC1 alleles were named by base pair marking . For repExpected marker heterozygosity was calculated using the formula CGG Repeat Analysis General distribution of the CGG repeat within 62 unaffected Iranian population resulted in variable alleles, ranging in size 11-38 repeats, were detected as alleles 26 (13.0%), 29 (24.0%), and 33 (11.0%) being the most common .Allele Polymorphism at DXS548 and FRAXAC1The distribution of DXS548 and FRAXAC1 alleles in subjects of normal controls and FXS is listed in In the 62 normal X chromosomes, we observed 6 alleles in DXS548 locus  that the most frequent DXS548 allele was allele 7 (58%), followed by allele 6 (14.9%) . In FXS P=0.001 and P=0.00, respectively. Heterozygosity of DXS548 was 37.06% in controls and increased to 78.68% in the FXS group. Observed heterozygosities for FRAXAC1 were 53.07% and 35.37% in control and FXS groups, respectively.As shown in Haplotype AnalysisDistribution of haplotypes in 62 normal individuals with different CGG repeats is shown in We detected 21 different normal alleles ranging in size from 11 to 38 CGG repeats as alleles 26 (13.0%), 29 (24.0%) and 33 (11.0%) being the most common. The most common fragile X CGG repeat was 29 copies in our study. These data are in agreement with findings previously reported in Asian population  such as P value <0.001 and chi square 23.66 representing association between longer allele in DXS548 and FXS due to either founder effect or concatenated mutation. The heterozygosity of our normal group (37.06%) is higher than the Chinese (33%) and Thai (16.5%) population which indicate the heterogeneity of the studied Iranian population by us.The increase of heterozygosities has been observed in FXS samples for DXS548 (78.63%) locus relative to the controls (37.07%). The increase of heterozygosity in FXS patients have been observed in nearly every population studied , 19-21 aMoreover the number of longer alleles was higher in FXS samples than that of controls for both tested microsatellite markers. The percentage of longer alleles 2 and 3 for DXS548 in cases group was 12.5% and 25% in comparison to 3.2% and 4.8% in control group respectively. The percentage of longer alleles A and B for FRAXAC1 in FXS group was 10% and 12.5% in comparison to 0.0% in control group. In addition, some short alleles such as 8 and 9 for DXS548 were only observed in control group equal to 6.5%. FRAXAC1 alleles distribution showed five different kinds of allele in our cases  and three  in normal population. The alleles A (21 repeat) and B (20 repeat) were absent in our normal population whereas they were participated in 22.5% of haplotypes in FXS group. Therefore A and B alleles were associated with fragile X chromosomes in our population. The FRAXAC1 locus alleles C (48.3%) and D (45.1%) were modal with a higher frequency in controls, whereas in FXS locus allele C (55%) was modal with a higher frequency in case group. The D and C alleles are the most frequent in Asian and Caucasians populations, respectively . HeterozFMR1 founder chromosomes in Europeans (The DXS548-2 (204bp) allele is known to be common on uropeans , 16, 25,uropeans , 26, whiuropeans -30 whereThe most significant haplotype was 7D represented in 29.0% of the X chromosomes followed by 7C (24.2%) and 6C 8.06%). The modal haplotype 7D, instead of the nearly worldwide haplotype 7C, had been also observed exclusively in Eastern Asian populations  with a significantly lower frequency (1-4%) and the Mexican population 23.8%). Moreover, haplotype 6D detected in 5.3% of the Mexican population is found in most other populations at a comparable frequency . The second modal haplotype 8D was observed in 23.8% of the Mexican population has been reported almost exclusively in East Asian populations (Chinese and Taiwanese) but at a significantly lower frequency (1-4%). Moreover, haplotype 6D detected in 6.45% of the studied population is reported in most other populations at a comparable frequency . The haplotype 6C which is 8.3% among normal populations was found to be the most frequent haplotype with frequency of 17.5% in our fragile X population that followed by 3C, 4C and 7C (10%) each. The 6D haplotype has been found to be the most common haplotype among the fragile X chromosome in white Americans, Chinese, and British population. However, it has not been found among the fragile X chromosomes in ours and the Indian, Thai population . This suggests weak founder effect.In conclusion, the CA repeat analysis at DXS548-FRAXAC1 loci in the normal X chromosomes and Fragile X chromosomes represented 13 and 18 different haplotypes, respectively. A few unique longer alleles A and B for FRAXAC1 was found in FXS chromosomes compared to normal controls which represent a weak founder effect.