14 Genetic analysis results of our cystic fibrosis patients

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11 Neonatal screening helps to prevent development of anemia in cystic fibrosis patients M. Gharibzadeh Hizal1, C. Kucukkayikci2, S. Esref1, G.D. Tugcu1, N. Emiralioglu1, E. Yalcin1, D. Dogru Ersoz1, U. Ozcelik1, N. Kiper1. 1Hacettepe University Ihsan Dogramaci Children Hospital, Pediatric Pulmonology, Ankara, Turkey; 2Hacettepe University Medical Faculty, Ankara, Turkey Objectives: In Turkey, cystic fibrosis (CF) newborn screening was added in the newborn screening program on 1 January 2015. We aimed to compare hemoglobin levels and nutritional status of patients diagnosed by newborn screening with patients diagnosed after presenting with symptoms in our tertiary care center. Methods: In this study, clinical and laboratory findings of 34 CF patients at first presentation were evaluated retrospectively in our center. Data of 19 CF patients (Group 1) who were diagnosed after positive newborn screening results were compared with data of 15 CF patients (Group 2) who were diagnosed after presenting to our center with symptoms. Hemoglobin, mean corpuscular volume, red cell distribution width, vitamin, albumin, values, sputum culture results, presence of anemia and pancreatic insufficiency were compared between 2 groups. Anemia was defined as “reduction in hemoglobin value by 2 standard deviations under the mean value of that age group.” Results: The median age of patients at diagnosis was 1.47 ± 0.63 months in Group 1 and 3.6 ± 2.72 months in Group 2. Female to male ratio was 10:9 (52.6/47.4%) in Group 1 and 12:3 (80/20%) in Group 2. Anemia was present in 2 (10.5%) patients in group 1 and 7 (46.6%) patients in group 2. There were no differences in pancreatic insufficiency between two groups. Most common symptoms at presentation were failure to thrive and PseudoBartter syndrome in Group 2. Sputum samples at time of diagnosis were positive in 6 patients in Group 1 and 5 patient in Group 2. Conclusion: Anemia is associated with increased morbidity and mortality in many chronic diseases. Detection of cystic fibrosis patients by routine neonatal mass screening prevents development of anemia, which develops early in disease course. Starting early treatment may have a role in this positive effect.

Chloride (VN < 30 mmol/L): MM = 63,75/SM = 83,62 FE (VN > 200 mg/g): MM = 300,36/SM = 23,42 Conclusion: Our results showed that severity of mutations are only related to the pancreatic sufficiency/insufficiency but not to the chloridium levels. In patients with severe mutations we observed higher levels of IRT and lower levels of FE due to their pancreatic insufficiency. Nevertheless, there was no difference between chloride in sweat in both groups. 13 Saliva for newborn screening for CF A.C. Goncalves1, A.M. Morcillo1, E.A. Lomazi1, I.A. Paschoal2, J.D. Ribeiro1, R.H. Mendonça3, C.E. Levy4, A.F. Ribeiro1. 1Universidade Estadual de Campinas, Pediatrics, Campinas, Brazil; 2Universidade Estadual de Campinas, Medical Clinic, Campinas, Brazil; 3Universidade Estadual de Campinas, Campinas, Brazil; 4Universidade Estadual de Campinas, Clinical Pathology, Campinas, Brazil

12 A comparative study of fecal elastase, immunoreactive trypsinogen, chloride in sweat and severity of the mutation in patients with cystic fibrosis

Objective: To evaluate the use of saliva for Cystic Fibrosis screening. Methods: Patients (n = 49) with 2 dosages of altered IRT (Immunoreactive Trypsinogen) were referred for sweat testing, according to the Newbon Screening Program of the State of São Paulo Brazil. Saliva samples were collected for three minutes. The chloride ion was measured by the direct selective ion electrode technique (ABL Radiometer®, model 835, Denmark). In sweat, chloride was analyzed by titration. Results: Mean age: Control group (n = 8) = 0.44, CF group [n = 31(F508del/ F508del (n = 5), F508del/other (n = 18) and other mutations (n = 8)] = 1.95 years. The concentration of chloride in sweat as well as in saliva is higher in patients with CF (Mean = 120.36 mmol/L; SD = 29.12 mmol/L) when compared to individuals without the disease (Mean = 31.93 mmol/L, SD = 18.87 mmol/L) ( p < 0.001). Statistical comparison between Heterozygous and Homozygous: Sweat Chloride: p = 0.218, Saliva Chloride: p = 0.436; Sweat Sodium p = 0.710 Saliva sodium: p = 0.445. The Spearman’s correlation coefficient for saliva chloride in relation to: 1°IRT = 0.36 ( p = 0.027); ( p < 0.001) and chloride in saliva versus sweat chloride = 0.35 ( p = 0.022), showing that there is a positive correlation between the variables. Conclusion: The concentrations of IRT, chloride in sweat and saliva were higher in CF subjects, suggesting the possibility of using the chloride dosage in the saliva by gasometry equipment, for screening and/or diagnosis of CF.

J. Mora Vallellano1, I. Delgado-Pecellín1, M.E. Quintana-Gallego1, L. González García1, M. Ferro Conde1, A.I. Álvarez Rios1, C. Delgado-Pecellín1. 1 Virgen del Rocío University Hospital, Seville, Spain

14 Genetic analysis results of our cystic fibrosis patients

Objectives: Cystic Fibrosis (CF) is caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance) gene. Severe CFTR mutations are related to pancreatic insufficiency whereas mild mutations are not. This condition could be evaluated by measuring the fecal elastase (FE). CF diagnosis is performed in three steps: screening by “Double IRT” (immunoreactive trypsinogen), sweat test (“Gold Standard”) and genetic study. The aim of this study is to compare the severity of the mutation to several parameters utilized for the diagnosis of CF (2ndIRT, chloride in sweat and FE) during 2011–2016. Methods: 35 patients were studied (14 men, 21 women). Levels of 2ndIRT were measured by immunofluorescence, chloride in sweat by ion selective electrode, FE by enzyme immunoassay. A genetic study was performed for each patient. Two groups were made according to the severity of mutations (Severe-mutation-SM): 21 patients, Mild-mutationMM: 14). SPSS v.20 (IBM) was used for the statistical study. The Shapiro-Wilks test showed a non-normal distribution of our variables. The difference between both groups was studied by the Kruskal-Wallis test and the Mann-Whitney U test was used for comparison. Results: The Kruskal-Wallis test showed significant differences for FE ( p < 0.001) and 2ndIRT ( p < 0.001), but not for chloride in sweat ( p = 0.098). These results were confirmed with the means observed by the Mann-Whitney U test: IRT (VN < 40 ng/mL): MM = 60,65/SM = 224,71

E. Atag1, N. Bas Ikizoglu1, P. Ergenekon1, Y. Gokdemir1, E. Erdem Eralp1, F. Karakoc1, R. Ersu1, B. Karadag1. 1Marmara University, Pediatric Pulmonology, Istanbul, Turkey Objectives: More than 2,000 CFTR mutations were identified and the recent treatment strategies are based on the genetic studies. CFTR mutations vary in their frequency and distribution in different populations. The aim of this study was to determine the CF mutation distribution via whole gene sequencing analysis among Turkish CF patients who were followed up at Marmara University Pediatric Pulmonology Department. Methods: Previously diagnosed CF patients with genetic analysis were included in the study. CFTR gene exons 1–27 were amplified by polymerase chain reaction (PCR) and then whole DNA sequencing was performed. Results: 237 patients were enrolled to the study. 172 (73%) patients had at least 2 mutations whereas 36 (15.2%) patients had only one mutation detected. In 28 (11.8%) of the patients no mutation was identified. A total of 382 mutations and 50 variations were detected. Out of these mutations; 125 (32.7%) were F508del mutations; 27 (7%) were 1,677 delTA, 26 (6.8%) 2,789 + 5G > A, 20 (5.2%) were N1303K, 20 (5.2%) were 2183AA- > G. 42 (18.1%) of our patients’ CFTR mutations were not described in CFTR2 (The Clinical and Functional TRanslation of CFTR) database. Novel CFTR mutations and deletions that have never been published were detected in 5 and 3 of our patients, respectively. Conclusion: Many mutations that have not yet been defined in CFTR2 database have been detected in Turkish CF patients. Follow up of CF

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patients with mutations and deletions that was not defined earlier may contribute to determining the phenotypes of CF patients with these novel mutations. 15 Analysis of cystic fibrosis gene mutations at national level in R. Macedonia T. Jakovska-Maretti1, S. Fustik1, L. Spirevska1. 1University Pediatric Clinic, Skopje, Macedonia (the Former Yugoslav Republic of ) Genotype characterization is essential to improve our knowledge about cystic fibrosis (CF) in R. Macedonia. Aim of the study was genotype characterization of CF population in 8 different regions in R. Macedonia in the period from 1996 to 2016 year. Demographic registry was analyzed from data collected at the CF center at the University Pediatric Clinic in Skopje, R. Macedonia. Methods: Clinical data of CF patients were reviewed and analyzed according to demographic indicators such as geographic family origin, CFTR genotype, pancreatic insufficiency and chronic pathogenic bacteria. Results: 153 CF patients were obtained, most of them (78,4%) had F508del., from which 10,13% were found in first region,3,6% in second,13,07% in third, 2,6% in forth, 5,9% in fifth, 8,8% in sixth region, 5,9% in seventh and 28,4% in the eight region. G542X was found total 5, 23%, and was not found in the third region. In the sixth and eight region was found 1, 63%, in the forth 0, 65% and in first, second, fifth and seventh 0, 33%. N1303 was found total 2, 29%, and was found in eight region with 1, 3% and with 0, 33% in third, fifth and sixth region. E822X was found only in third region with 0, 65%. R1662X was found only in forth region with 0, 65%. 457TAT → G was found only in fifth region with 0, 65%. 621 + G → T was found in the eight region with 0, 65%. Y1092 was found in eight region with 0, 65%. CFTRdel 21 kb was found in seventh region with 0, 65%. Conclusions: Molecular basis of CF in R. Macedonia is highly heterogenous. The most common mutation in CFTR is F508del, followed by G542X and N1303K. The R1662X, 457TAT → G and CFTRdel 21kb were found with some frequency in southern part of the country compared with other mutations. It would be beneficial if the detection of these mutations would be incorporated in routine diagnostic analysis. 16 Frequency of 7 CFTR gene mutations in cystic fibrosis patients in a mixed population in the state of Bahia, Brazil L. Ribeiro Mota1, R.L. Leite Ferreira de Lima1, P. Horejs Bittencourt1, N. Araújo Sena1, M.B. Perreira Toralles1, E.L. Souza1. 1Federal University of Bahia, Salvador, Brazil Objectives: To determine the frequency of 7 mutations (F508del, G542X, G551D, R553X, 3120 + 1G → A, R334W and R1162X) in Cystic Fibrosis patients assisted at a Teaching Hospital in Salvador, Brazil. Methods: It was a cross-sectional study involving 60 Cystic Fibrosis patients with ages from 0 to 20 years. The molecular analysis for mutations was performed using standard methods. Results: Five mutations were identified and the allele frequencies observed were: F508del (30.8%), 3120 + 1G → A (5%), G542X (3.3%), R1162X and R334W (2.5%). The mutations G551D and R553X were not found. Around 40% of the CF patients had their genotype determined. Conclusion: The F508del mutation was the most commonly identified; however, it had a lower frequency than the one found in Brazilian Cystic Fibrosis Registry (BCFR)-47.5%- and in Caucasian populations (∼70%). The frequency of 3120 + 1G → A mutation was higher than theone observed in the BCFR (1.2%). The occurrence of G542X mutation was higher than that found in Caucasians (2.4%) but lower than the BCFR data (4.3%). The frequency of R334W and R1162X was superior to that observed in BCFR, 1.25 and 1%, respectively. The results highlight the substantial ethnic/racial diversity of the analyzed population and the importance to have regionspecific mutation panels.

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17 Features of mutation spectrum in the Chechen CF patients from Russian Federation N.V. Petrova1, N.J. Kashirskaya1, D.K. Saydaeva2, O.I. Simonova3, E.I. Kondratyeva1, O.G. Novoselova1, T.A. Vasilyeva1, R.A. Bikanov1, E.K. Ginter1, R.A. Zinchenko1,4. 1Federal State Scientific Budgetary Institution «Research Centre for Medical Genetics», Moscow, Russian Federation; 2State budgetary institution “Maternity Hospital” MC CR, Grozniy, Russian Federation; 3Federal State Budgetary Institution “Children’s Health Research Center”, Moscow, Russian Federation; 4Pirogov Russian National Research Medical University, Moscow, Russian Federation Objectives: The spectrum and frequency of CFTR mutations significantly vary in different populations and ethnic groups. Detection of the mutations carried by patients in specific population is critical for the optimization of care and genetic counselling in such a multiethnic country as Russian Federation. Chechens are a Caucasian ethnic group of the Nakh peoples originating in the North Caucasus region. The Chechen people are mainly inhabitants of Chechnya. The aim is to investigate the frequencies of CFTR mutations in Chechen CF patients. Methods: DNA of 26 Chechen CF patients with clinically confirmed diagnosis was tested for 29 routinely screened CFTR mutations (CFTRdele2,3(21 kb), G85E, 394delTT, E92K, R117H, L138ins, 604insA, 621 + 1G > T, R334W, R347P, I507del, F508del, 1677delTA, 1717-1G > A, G542X, G551D, R553X, 2143delT, 2183AA > G, 2184insA, 2789 + 5A > G, 3667insTCAA, S1196X, 3821delT, 3849 + 10kbC > T, 3944delTG, W1282R, W1282X, N1303K). Results: Four of 28 tested CFTR mutations were revealed in studied patients: 1677delTA-55.8% (29/52), E92K-11.5% (6/52), R334W-1.9% (1/52), W1282X-1.9% (1/52). The most common CFTR mutation, F508del, was not detected among Chechen CF patients. The most frequent mutation was 1677delTA. For the first time it had been revealed in Georgian CF patient, and as it turned out the frequency of 1677delTA mutation reached up to 60% of CF alleles in the Mingrelians (sub-ethnic group of Georgians). Also 1677delTA mutation was determined in other ethnic groups of the North Caucasus such as Karachai, Cherkess, Abaza, Ingush, but the frequencies were much lower. E92K mutation was shown to be present at a relatively high frequency in populations that are historically associated with the settlement of Turkic peoples in the Volga-Ural region (Chuvash, Tatars, Bashkirs), but not in North Caucasus populations (Karachai, Nogai). Acknowledgement: The research was partially supported by grants RFFR 17-04-00288; RSF 17-15-01051. 18 Mutation analysis of cystic fibrosis patients in the middle region of Turkey: three centers’ results A.T. Aslan1, T. Sismanlar Eyuboglu1, S. Pekcan2, M. Köse3, F.S. Ezgü4. 1Gazi University Medicine Faculty, Pediatric Pulmonology, Ankara, Turkey; 2 Necmettin Erbakan University Meram Faculty of Medicine, Pediatric Pulmonology, Konya, Turkey; 3Erciyes University Medicine Faculty, Pediatric Pulmonology, Kayseri, Turkey; 4Gazi University Medicine Faculty, Pediatric Metabolism and Genetic, Ankara, Turkey Objectives: Cystic fibrosis (CF) is the most common life shortening disease in caucasian people all over the world. More than 2 thousand mutations were discovered as disease-causing mutations. Mutations are classified in 6 groups according to the CFTR production and functions. Mutation analysis is very important in patients with CF for genetic counselling, learning about disease phenotype and also for new genetic-based treatments. Mutations may change according to the regions, ethnicity and countries. Herein, we aimed to report mutation analysis of CF patients in three CF centers in the middle region of Turkey. Methods: Mutation analysis of CF patients were reviewed between January 2008 and December 2016 in three CF centers. Results: In 8 year-period, 270 CF patients were followed, in 180 patients CF related mutations were detected. In 20 patients only polymorphism, in 160 patients one or two disease-causing mutations were found. In 230 diseasecausing mutations the most common mutations were detected in class 2 (154 mutations) then 1 (38 mutations), 4 (29 mutations), 5 (9 mutations), 3 (3 mutations), respectively. The most common mutation was DeltaF508 in