AUTOMATION OF CFTR RE-SEQUENCING: TIME AND COSTS ANALYSIS

Posters correlation with survival (8) It is necessary to define criteria for the best selection and timing for candidates to lung transplantation. Registries are an important source of data to investigate clinical and research questions. This review points out that only in some instances (incidence/survival analysis) CF Registries are fully able to answer to questions relevant for research. The main limits concern the analysis of historical cohorts in spite of the large number of patients registered. It should be demanded to standardize definitions of variables, outcome measures and timing of data collection to make results comparable among Registries. P5 USE OF NIV IN THE DRAINING THERAPY: CLINICAL CASE M.A. Ciciretti, L. Ratclif. UOS Cystic Fibrosis Cerignola, Italy Studies show that the use of NIV, in Cystic Fibrosis, has a positive effect on the chronic respiratory insufficiency in patients affected by advanced pulmonary disease and/or in waiting lists for a pulmonary transplant, by improving gas transfers and reducing muscular work. For this reason we decided to use NIV as support to the draining therapy, focusing on the following aims: • improving the compliance of the patient; • improving the efficacy of the physiotherapeutic treatment; • estimating the course of the illness. Clinical case: a male patient, 10 years old, affected by Cystic fibrosis with pancreatic insufficiency and chronically colonized by Pseudomonas aeruginosa. The data in the table referto the 12 months before NIV and to the NIV* months. before NIV

NIV* months

Physiotherapeutic activity

2−3 sessions per day with PEP-Mask

Height Weight Spirometry

130 cm (P) → 132 cm (P) + 28 (P) → 29 Kg (P) + FEV1 57.7% 40 cc with stamps 500 mt. with breaks for cough 19 52 days 56 days (ev) 94 days (os) VAS 2 wheezing cough, tiring expectoration, chest tightness Reduced during physical activity

1 session of NIV and 1 session of PEP-Mask 132 cm (P) → 137.5 cm (P) + 29.2 Kg (P) → 32 Kg (P) + FEV1 68.4%

Sputum 6 WT RX Period in bed Therapy period Compliance Symptomatology Tolerance of effort

40cc + fluid without stamps 540 mt. without cough 9 No day 16 days (ev) 25 days (os) VAS 6 easy expectoration, no chest tightness. Also improved PEP session good (gym)

*Resmed pressometric ventilator VPAP III ST-A with nasal mask.

Draining session with 4−5 cycles if C-PAP per 3 days with 7 cm H2 O → use of Coach 2 (respiratory incentive), 3 fast inspirations and 3 slow ones. → Huffing Conclusions: the use of the ventilator during a draining therapy produced the following results: – Improving the compliance: this was possible because the draining therapy has been less tiring, faster, with no chest tightness, with a better approach of the patient to practise the draining session and to require it in case of obstruction. – Obtaining a better efficacy of the physiotherapeutic treatment: a more flowing sputum, a relevant reduction of the air instability, a better tolerance of effort. – Recording positive effects on the course of the illness: spirometric increase, therapy period reduction, no admission, better saturated-ponderal increase, radiological improvement. P6 COMPARISON OF HIGH FREQUENCY CHEST WALL OSCILLATION (HFCWO) AND PEP-MASK IN HOSPITALIZED PATIENTS WITH CYSTIC FIBROSIS (CF) F. Alatri, B. Giacomodonato, T. Perelli, L. Graziano, M. Varchetta, G. Cimino, L. Locorriere, E. Bonci, S. Quattrucci. Centro Fibrosi Cistica Regione Lazio, Rome, Italy Background: Reducing bronchial obstruction plays a primary role in the rehabilitation of patients with CF. In the last decades treatment has shifted gradually from passive and tiring techniques to methods that foster self-management of pulmonary disease, including the use of mechanical tools to loosen secretions. However, the greater efficacy of such mechanical methods compared to other procedures has not been demonstrated clearly. Aim: To compare the efficacy of HFCWO with that of PEP-Mask in the clearance of bronchial tubes from secretion. Methods: 10 patients with CF (4 males, average age 21.4±7.5 years) hospitalized for a relapse of broncho-pulmonary disease. During hospitalization (average length, 6.1±2.7 days) each patient had daily applications of bronchial disobstruction, one with PEP-Mask and one with HFCWO, each lasting 30 minutes, following standard procedures. The sequence of the two interventions as changed daily. All patients were treated with intravenous antibiotics and aerosol bronchodilators. Oxygen was provided to maintain adequate saturations (SpO2>90%). The following parameters were evaluated: quantity of ejected humid mucus, SpO2, and heart rate (HR) during

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each application of draining physiotherapy, dyspnea and mood assessment using a visual analogue scale (VAS) at beginning and end of each treatment, FEVI and WT6min at the start and end of hospitalization, and end-study evaluation questionnaire. Statistical significance was evaluated with the Paired T-Test and Wilcoxon Signed Rank Test. Results: Minimum SpO2 during PEP-Mask was 89.3±3.1 vs. 87.2±3.5 during HFCWO (p < 0.01); average quantity of sputum per treatment was 16±9.6 with PEP-Mask vs. 10.3±6.5 ml with HFCWO (p < 0.02); HR peak during PEPMask was 116.5±8.8 b/m vs. 120±6.7 b/m for HFCWO (p = 0.08); evaluation questionnaire showed a perceived greater draining efficacy with PEP-Mask vs. HFCWO (p < 0.02). No significant differences were identified for the other parameters. Conclusions: Compared to HFCWO, treatment with PEP-Mask is associated with greater draining efficacy, less desaturation, and milder tachycardia in patients with CF. Studies with larger sample size and longer follow up are warranted. P7 EFFICACY OF HIGH FREQUENCY CHEST COMPRESSION (HFCC) IN THE CLEARANCE OF MUCUS IN PATIENTS WITH CYSTIC FIBROSIS (CF) M. Ambroni, V. Mazzotti, E. Balestri, S. Dall’Ara, F. Gobbi, F. Lupi, F. Battistini. Centro Regionale Fibrosi Cistic, Cesena, Italy Aims: To determinate the efficacy of HFCC (using the VEST) in the clearance of mucus, by comparing it to the amount of expectorated mucus obtained with PEP-mask. HFCC is made through a system with an inflatable vest, that rapidly inflates and deflates to compress and release the chest wall, creating an airflow inside the lungs. This process moves mucus towards the larger airways, where it can be coughed up and expectorated. PEP-mask is a positive expiratory pressure generated through a face-mask. Methods: 10 CF patients (M:W=8:2; mean age=24.3 yrs, range=9−43 yrs), clinically stable and undergoing their usual therapies (mean FEV1=54.79%, range=26−93), were randomly recruited. They came twice a week in a 2 weeks period to perform both VEST and PEP-mask therapy each week (1st day VEST, 2nd day PEP, 3rd day PEP, 4th day VEST). %SaO2 and cardiac frequency were checked with pulseoximeter every 2 min during each session. We evaluated the emotional state and the fatiguing using VAS scales, before and after each treatment. The dry expectoration was weighted in laboratory. HFCC consisted on a VEST treatment of 30 min with 3 phases: the first consisted of 7 min in a low frequency (8 Hz) and pressure (P = 4), the second consisted of 7 min at 10 Hz and P = 5, and the third of 7 min at 15 Hz and P = 6. Every phase was followed by a pause of 3 min where they did 3 cough bouts and expectorated. They did a total of 9 cough bouts followed by expectorations. PEP-mask consisted of a 30 min treatment. Patients did 7 min of PEP (with a pause of 20 sec every 2 min) followed by a pause of 3 min where they did 3 cough bouts and expectorated for 3 times. They did a total of 9 cough bouts followed by expectorations. Results: See the table.

Sputum gr % SaO2 mean Peak %SaO2 desat % pz VAS amusing % pz VAS fatiguing

VEST

PEP-mask

P value

34.09±10.28 95.18±1.79 91.90±3.29 30 45

34.91±9.84 95.49±1.38 91.30±3.05 10 45

0.483 0.293 0.441

Conclusions: VEST is as much effective as PEP-mask technique in clearing sputum from airways of CF patients. No differences neither in the amount of sputum expectorated nor in peak SaO2% desaturation were showed. VEST was perceived as less distressing than PEP-mask, in the same fatiguing condition. So the VEST may be used as substitute or integrative to PEP, to create an alternative to the classic chest physical therapy. Reference(s) JC Darbee, JF Kanga, Physiologic evidence for HFCWO and positive expiratory pressure breathing in hospitalized subjects with cystic fibrosis. Physical Therapy, Vol. 85(12), 2005, 1278–1289 P8 AUTOMATION OF CFTR RE-SEQUENCING: TIME AND COSTS ANALYSIS G. Ferraguti1 , S. Pierandrei1 , S.M. Bruno1 , F. Ceci1 , A. Di Gioia1 , S. Quattrucci2 , R. Strom1 , M. Lucarelli1 . 1 Dept. of Cellular Biotechnologies and Hematology, 2 Dept. of Pediatrics, Regional CF Centre, “La Sapienza” University of Rome, Italy Among methods for mutational scan of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, direct sequencing ensures the highest degree of de-

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Posters

tection rate. In a previous work we have described a procedure allowing an extensive re-sequencing (by cycle sequencing, Applied Biosystems) of all exons and of the adjacent intronic zones of CFTR (30 amplicons, each subjected to forward and reverse sequencing), that can fit in a 8 × 12 (= 96-well) format, with simultaneous examination of 11 subjects (plus one blank). We are presenting here an almost fully automated version of this method, based on the combined use of a Hamilton 4-channels StarLet robotics system and an Applied Biosystems ABI PRISM 3100 Avant genetic analyzer (4-capillaries), with an analysis of time and costs of this automated procedure and an assessment of its advantages. The CFTR complete scan of 11 subjects actually starts the first day with the assembly of the first plate, followed by 13 days of ABI PRISM time (in the meantime, all plates could be prepared and run one after the other). By using our personalized CFTR-template, the large set of sequencing data produced is easily processed by SeqScape software for sequence analysis (Applied BioSystems). One additional day is then needed for software analysis of the last plate. The limiting step in this process is the development of the electropherograms by the 4-capillaries genetic analyzer, since approximately 80 hours are required for a full analysis of 96 samples (forward and reverse, run in duplicate). A different hardware configuration (16-channels, instead of 4, for both instruments) markedly reduces the overall time to 5 days, 3 of which of ABI PRISM time. The overall cost for a complete CFTR scan by this automated method, including consumables (tips, plates, capillary arrays) and reagents (Taq polymerase, POP-6 polymer and BigDye terminators), is about 240 euros/subject. A very limited human involvement is needed after robotics programming. The overall costs of this procedure, especially if its high detection rate is taken into account, are comparable to those of other scanning techniques and also of methods focused on limited number of mutations. For a high productivity, the use of a 16-analytical channel instrument is advised. S. P. was partially supported by a grant from “Associazione Laziale Fibrosi Cistica”. P9 CYSTIC FIBROSIS MUTATIONS: GENOTYPE-PHENOTYPE RELATIONSHIP IN PATIENTS DIAGNOSED BY NEONATAL SCREENING I. Tardivo1 , A. Veljkovic1 , C. Risso1 , G. Restagno2 , C. Mari2 , A. Lezo1 , E. Bignamini1 . 1 Pediatric CF Center Piedmont and Valle d’Aosta – OIRM – Turin, Italy; 2 Molecular Genetics Service – OIRM – Turin, Italy Although there are more than 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, most of them are uncommon and only limited informations exist regarding genotype–phenotype correlation. In this survey we retrospectively examined our screened population of CF patients, diagnosed from July 2000 to May 2007, in order to investigate the relationship between their clinical status and genotype. Patients were classified depending on the CFTR mutation class on each chromosome. They were subsequently categorized into two groups according to whether the CFTR protein reached the epithelial surface (Group 2: at least one mutation class type III-IV-V) or not (Group 1: type I or II mutation class on both chromosome). We considered age, sex, neonatal screening results, sweat test values, pancreatic sufficiency and Pseudomonas aeruginosa first infection. From July 2000 to May 2007 we diagnosed 73 patients (40 females and 23 males) for whom it was possible to identify both CFTR mutations; they actually have a mean age of 3.92 years. We identified in Group 1 52 patients (27 M and 25 F) with a mean age of 3.75 years and in Group 2 21 patients (6M and 15 F) with a mean age of 4.3 years. Six patients of Group 1 (8.6%) vs 3 patients (7%) of Group 2 resulted as false negative at the neonatal screening. The mean sweat test value was 100.17 mEq/l (IC 95% range 86.28–114.06) for the first group of patients and 84.41 mEq/L (IC 95% range 67.93–100.89) for the second group with p: 0.18. With regard to the pancreatic status 35 patients out of 52 (67.3%) in Group 1 have pancreatic insufficiency vs 2 patients out of 21 (9.5%) in Group 2 (p < 0.05). Thirtythree patients of our population have had first Pseudomonas aeruginosa infection: 27/52(51.9%) in Group 1 of patients and 6/21 (28.5%) in Group 2. The mean age of acquisition was for Group 1 1.12 years vs 1.36 years for Group 2 (p: 0.6), but the patients who are actually negative for Pseudomonas aeruginosa infection are 25/52 in the first group with a mean age of 2.8 years and 15/21 in the second group with a mean age of 4.01 years (p: 0.08). Conclusions: screening diagnosed patients belonging to first and second class mutation genotype present an earlier Pseudomonas aeruginosa infection in respect of patients group having other mutations (class type III-IV-V). P10 CFTR MUTATION FREQUENCY IN CHILDREN DIAGNOSED BY NEONATAL SCREENING I. Tardivo1 , A. Veljkovic1 , G. Restagno2 , I. Esposito1 , G. Cordola1 , E. Bignamini1 . 1 Pediatric CF Center Piedmont and Valle d’Aosta – OIRM – Turin, Italy; 2 Molecular Genetics Service – OIRM – Turin, Italy Neonatal screening for CF has been introduced in July 2000 as investigational procedure in Piedmont Region (Italy). Until May 2007 96 children have been

diagnosed at our CF Center: 19 patients out of 96 resulted false negative at the neonatal screening and therefore were diagnosed by symptoms. The genetic diagnosis is performed at first instance by the analysis of the 31 most common mutations using the OLA-PCR-SCS method and then, if necessary, by the whole gene scan with DHPLC method. In 45 patients out of 96 it has been necessary to apply DHPLC: in 15 patients out 45 the neonatal screening has resulted false negative. Using DHPLC both mutations were detected in 30 out of 45 patients; in 15 cases only one mutation (13 cases) or no mutation (2 cases) were found: 7 patients of this group had been false negative at neonatal screening. Mutations detected by DHPLC belong to mutation class I in 45.83% of cases, to class IV in 16.67% and to class III in 4.17% of cases. The table shows the class of mutation detected in our population with OLA and DHPLC. Mutation class

Frequency

Percentage

Cumulative percentage

II I N IV V III Total

111 30 24 17 5 2 189

58.73 15.87 12.70 8.99 2.65 1.06 100.0

58.73 74.60 87.30 96.30 98.94 100.00

Conclusions: neonatal screening has permitted to modify patient’s population, detecting also uncommon mutations; furthermore neonatal screening seems to be more sensitive for patients with mutation class I or II. P11 PHENOTYPIC VARIABILITY IN A FAMILY WITH PANCREATITIS AND CYSTIC FIBROSIS SHARING COMMON MILD CFTR MUTATION V. Lucidi1 , F. Alghisi1 , A. Angioni2 , A.C. Tomaiuolo2 , M.R. D’Apice3 , S. Gambardella3 , B. Russo1 , S. Bella1 , E. Fiscarelli4 , G. Novelli3 . 1 U.O.S. of Cystic Fibrosis, Bambino Ges`u Pediatric Hospital, Rome, Italy; 2 Laboratory of Cytogenetics and Molecular Genetics, Bambino Ges`u Pediatric Hospital, Rome, Italy; 3 Dept. of Biopathology and Diagnostic Imaging, Tor Vergata University, Rome, Italy; 4 Bacteriology Laboratory, Bambino Ges`u Pediatric Hospital, Rome, Italy Background: Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene cause Cystic Fibrosis (CF). They are also involved in other diseases, such as idiopathic pancreatitis. Case-report: We present a case-report of a family composed by four persons in which genetic analysis showed an identical homozygous genotype (D1152H/D1152H) in the mother and her two sons, associated with different clinical expressions. In fact, only the second born male had a pathological sweat test and a diagnosis of a mild form of classic CF, with a minimal lung disease and pancreatic sufficiency. On the other hand, the mother and the first born female had an exclusive pancreatic involvement and signs of chronic pancreatitis: the mother had only one clinical expression in her life referable to an acute pancreatitis, whereas her daughter was affected by recurrent episodes of pancreatitis confined to her first years of life. Both patients showed normal chloride values at the sweat test. Conclusions: This report confirms the difficulties for diagnosing atypical CF and emphasizes the role of CFTR mutations in the pathogenesis of idiopathic pancreatitis. In particular, D1152H mutation is strictly associated with atypical expression of CF and pancreatic sufficiency. The phenotypic variability seen in this family could be determined by other non-CFTR genes acting as possible modifiers of disease expression; alternatively, gender differences may be involved in the determination of CF phenotype, as suggested by several observations in literature. P12 DIAGNOSIS IN ATYPICAL CF: A CASE-REPORT TO LEARN V. Lucidi1 , F. Alghisi1 , A. Angioni2 , A.C. Tomaiuolo2 , M.R. D’Apice3 , B. Russo1 , S. Bella1 , E. Fiscarelli4 , G. Novelli3 . 1 U.O.S. of Cystic Fibrosis, Bambino Ges`u Pediatric Hospital, Rome, Italy; 2 Laboratory of Cytogenetics and Molecular Genetics, Bambino Ges`u Pediatric Hospital, Rome, Italy; 3 Dept. of Biopathology and Diagnostic Imaging, Tor Vergata University, Rome, Italy; 4 Bacteriology Laboratory, Bambino Ges`u Pediatric Hospital, Rome, Italy Background: Non-classic Cystic Fibrosis (CF) currently represents a difficult entity to define for clinicians, especially at the early stage of the disease, because of unusual presentation and/or late onset of symptoms. Moreover, the sweat test may show border-line or normal values and the diagnosis is mainly based on clinical features and follow-up. Genetic analysis of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may identify one severe or mild mutation in