Diagnosis Of CF Research Articles

39 Evaluation of the Nanoduct sweat test method after newborn screening for cystic fibrosis

Objectives: The approved methods for sweat testing, Gibson-Cooke Pilocarpine Iontophoresis (GCQPIT) or the Wescor Macroduct® Sweat Test system, have many steps that can and do lead to errors. The GCQPIT method is 54 years old, was never intended for newborns, has scared parents and burned infants. Thus, there is a critical need to improve sweat testing technology. Methods: The CF Quantum® Sweat Test (CFQT) System consists of: a. a controller and electrode set that is worn on the arm for pilocarpine iontophoresis; b. a chloride test patch that collects the sample. Chloride ions in the sweat sample come into contact with silver chromate in the test patch, and an ion exchange reaction occurs which creates silver chloride, an insoluble white precipitate formed in the center of the patch; c. a CF Analyzer utilizes a camera and proprietary software that scans the reacted chloride test patch and calculates the concentration of chloride and volume of sweat collected. Herein are preliminary results of a multicenter study. Patients with a previous diagnosis of CF or referred to the sweat testing lab had bilateral GCQPIT or Macroduct® and bilateral CFQT tests performed. Thus far, 57 subjects with a known diagnosis of CF and 56 subjects referred to the sweat test lab have had both tests performed. There is excellent agreement of sweat chloride values between GCQPIT/Macroduct® and CFQT with a Pearson correlation coefficient of r = 0.97. In a post-test questionnaire, 85% of parents/patients preferred the CFQT. Conclusion: The CFQT yields sweat chloride values that are equivalent to that of GCQPIT/Macroduct sweat testing. Supported by: Legacy of Angels Foundation, PolyChrome Medical, Inc.

34 Children with a late diagnosis of CF despite a long-established newborn screening programme

Objectives: Neonatal screening for CF has been continuous in New Zealand (NZ) for 30 years. Initially this was by two blood immunoreactive trypsin (IRT) levels until 1996 when a three gene detection replaced the second IRT measurement. Aim: To determine the number of children with a late diagnosis and why they were not detected despite our long term newborn screening programme. Methods: Retrospective review of all children with CF under full care at Starship Children’s Hospital, Auckland, NZ from 2003–2012 which includes patients up to 18 years of age to therefore determine missed on screening from 1985–2012 in the greater Auckland region with a general population of 800,000 to 1.2 million over this time. Results: In the last decade 100 children with CF were in this clinic of whom 18 had a late diagnosis, mean age 14 months (range 6 weeks to 11 years). Eight of them were born overseas in regions without a newborn screening programme at that time. Ten were born in NZ; 4 were pancreatic sufficient and 6 were pancreatic insufficient. Nine were missed as the IRT level was not in the highest 1% and therefore the gene determination was not undertaken, or they had “uncommon” gene, undetectable on screening. One child’s parents refused newborn screening. All ten had recurrent pneumonias, 2 had established bronchiectasis, 4 FTT and 1 had severe liver disease leading to the diagnosis. Conclusion: Even with an established newborn screening programme in place, cystic fibrosis should be considered in the appropriate clinical setting.

197 Influence of inhalation mode on aerosol lung deposition in patients with cystic fibrosis PART 1: Pharmacokinetic data as representative of lung deposition

Objectives: To investigate the impact of two different inhalation flow maneuvers (TIM = slow and deep inhalation and TBM = normal tidal breathing) on aerosol lung deposition in patients with CF using pharmacokinetic parameters as a representation of lung deposition. Methods: Randomized, open-label, cross-over study. The study group consisted of 18 adult patients with a confirmed diagnosis of CF (genetic analysis). Each patient inhaled a tobramycin solution twice during separate study visits: once in TIM and the other time in TBM mode. Blood samples were collected in order to model tobramycin pharmacokinetics. Outcome measurements: Relative bioavailability (Frel) of tobramycin is defined as the ratio of AUCTIM to AUCTBM, in which the AUC represents the plasma concentration area under the curves. A ratio greater than 1 indicates higher lung deposition for TIM compared to TBM. Individual pharmacokinetic parameters were calculated and assimilated with patient tobramycin serum values using a computerized CF-based Bayesian population model (MW-Pharm, Mediware). Results: Frel was 1 or greater for all patients (mean = 1.55, sd = 0.39, 95%CI = 1.37-1.73). In addition, mean Frel was significant higher than the value of 1 (mean difference = 0.55, p

WS10.6 Organ-specific CFTR dysfunction: Comparison of biomarkers for CF diagnosis and CFTR modulator effects

Alterations on mRNA processing are known to cause variable CF disease manifestations which are difficult to predict. Thus, characterization of the impact of these CFTR mutations in native tissues becomes crucial for disease prognosis and choice of adequate therapies. Here, we aimed to characterize CFTR mutations affecting mRNA processing. CFTR transcripts were analysed by semi-quantitative RT-PCR upon extraction from native tissues of patients with F508del in one allele and one the following in the other: 711+1G>T, 1584+18672A>G, 1717−1G>A, 1812−1G>A, 3120+1G>A. Comparison of transcripts relative levels from both alleles (reference-F508del mRNA levels) show that 1717−1G>A, 1812−1G>A, 3120+1G>A and 1716+18672A>G induce transcripts degradation (63−80%). Notwithstanding, for 711+1G>T mutation no decay of the transcripts was observed. Minigenes with IVS4art+IVS5 into fulllength CFTR cDNA were constructed to further characterize this abnormal mRNA processing. HEK cell expressing the wt and 711+1G>T, 711+3A>G, 711+5G>A mutant minigenes are analysed by RT-PCR and Western blot. Correct processing of CFTR transcripts and protein was observed in the wt minigenes. In contrast, all mutant minigenes originated alternative transcripts resulting in complete absence or misprocessed protein. Compounds like Kinetin and EGCG shown to correct splicing of transcripts from other genes are being tested in these CFTR minigenes. These data may be of high relevance for CF personalized medicine for patients with these splicing mutations. Work supported by PEst-OE/BIA/UI4046/2011 grant and Ciencia2008 fellowship (FCT, Portugal) to ASR and Gilead MED-2012–131 grant. WS10.6 Organ-specific CFTR dysfunction: Comparison of biomarkers for CF diagnosis and CFTR modulator effects L. Pinders-Kessel1, S. Scheinert1, M. Klosinski1, D. Staab2, N. Derichs1,2. 1Charite Universitatsmedizin Berlin, Pediatric Pulmonology and Immunology CFTR Biomarker Centre & Translational CF Research Group, Berlin, Germany; 2Charite Universitatsmedizin Berlin, Christiane Herzog Cystic Fibrosis Centre Pediatric Pulmonology and Immunology, Berlin, Germany

PRM76 - Stated time-allocation, adherence, and health-outcome tradeoff preferences in cystic fibrosis households

Objective: To quantify preferences for allocating hypothetical time savings in cystic fi brosis (CF) inhaled-antibiotic treatment among health care and non–health care activities. Methods: Adult patients with a selfreported physician diagnosis of CF and parents of juvenile patients with CF in the United States (US) who have Pseudomonas aeruginosa in their sputum culture at least twice a year completed a Web-enabled, discretechoice experiment survey. Respondents evaluated treatmentchoice and routine-choice alternatives followed by stated adherence for themselves and other people. Questions required evaluating pairs of hypothetical inhalation devices and routine profi les defi ned by inhalation device features, side effects, time spent on inhaled-antibiotic treatment, time spent on hypertonic saline, time spent on other CF-care activities, and lung function measured as forced expiratory volume in 1 second (FEV 1 ). Respondents also answered a question indicating how they would allocate 80 hypothetical minutes of time savings per day among CF-care and non–CF-care activities (work, sleep, family activities, and social activities). Using a conceptual framework describing optimal time allocations within a health production function, random-parameters logit models were used to quantify tradeoff preferences among effi cacy, treatment-time adherence, and competing time demands. Results: The fi nal sample included 174 adult patients and 130 parents. On average, adult patients would spend 9% of the time saved on additional CF-care activities, and the remaining time would be almost evenly split between work, social activities, and sleep. Juvenile patients would spend 11% of the time saved on additional CF-care activities and about 2.5 times more minutes in social activities than in work and 2 times more minutes in social activities than in sleep.

Impaired CFTR function in mild cystic fibrosis associated with the S977F/T5TG12complex allele in trans with F508del mutation

The S977F mutation (c.2930C>T) in the CFTR gene (CFTR/ABCC7) is extremely rare. We describe the case of an adult patient carrying the complex allele S977F/T5TG12 in trans with the F508del mutation. Mild respiratory manifestations arose in adulthood associated with azoospermia, acute pancreatitis, minor hemoptysis and Cl(-) levels ranging from 40 to 42 mEq/L. Diagnosis was confirmed by repeated NPD measurements, genetic DHPLC analysis and a recently described functional assay measuring cAMP-dependent cell depolarization in peripheral blood monocytes. NPD measurements, DHPLC and monocyte functional assay (CF index=-18). Results were consistent with a CF phenotype. The combined application of DHPLC and NPD analysis in the algorithm for CF diagnosis appears useful for the management of similar cases. In addition, the novel monocyte functional assay might contribute to improve our diagnostic capability, counseling and better treatment of these challenging clinical cases.

Consequences of partial duplications of the human CFTR gene on cf diagnosis: Mutations or ectopic variations

CFTR exon 10 and its flanking regions are duplicated in the human genome. These duplications present mutations compared to the normal exon 10 sequence. Due to the polymorphic sequence of the 3' intron 9 sequence, it may appear difficult to sequence exon 10 and some mutations described in this exon could, in fact, be variations observed in an ectopic duplicated sequence. In our previous work we described a methodology to carry out PCR only of exon 10 and not of ectopic regions. In this work, we analyzed mutations described in the CF data base as being CFTR mutations but also found in ectopic regions: c.1392G>T, c.1338_1339delAT, c.1235delC, and c.1247A>G. We have shown that these mutations appear to be authentic mutations in CFTR exon 10 and not ectopic variations in analyzed patients. These mutations validate the usefulness of our new strategy in the mutation analysis of this region of CFTR.

S123 The Role of Nasal Potential Difference Testing in Difficult Cases of Possible Cystic Fibrosis

The European CF Diagnostic Working Group guidelines include nasal potential difference (nPD) testing when conventional tests (sweat chloride/CFTR mutation analysis) are inconclusive. This specialist measure of epithelial ion transport is...

CFTR haplotypes in northern Iranian population

BackgroundCystic fibrosis (CF) is a multiorganic autosomal recessive disorder, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). CF is highly heterogeneous in Iranian population and molecular diagnosis based on direct identification of mutations is not completely efficient. The use of polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations. Methods60 CF patients and 53 fertile normal subjects originating from North of Iran were analyzed for F508del mutation and c.1210-12T(5_9), c.1408A>G and c.744-33GATT(6_8) polymorphisms. Resultsc.1210-12T[7] is the most prevalent allele in normal individuals and CF non-F508del patients with 87.7%and 86.7% frequencies respectively. c.1408A>G survey showed that frequency of allele G and A is nearly equal in both non-F508del CF patients and normal individuals. c.744-33GATT(6_8) study showed that 7 repeat is the most prevalent allele in normal individuals and non-F508del CF patients with 80.2% and 82.1% frequencies respectively. The [c.1408A; c.1210-12T[9]; c.744-33GATT[6]] haplotype was only associated with mutant alleles including F508del. ConclusionsThe allelic distribution and heterozygosity results suggest that c.1408A>G, c.1210-12T(5_9) and c.744-33GATT(6_8) can contribute to carrier detection and prenatal diagnosis of CF in Iranian families with previous history of the disease.

1 The role of CFTR genetic testing in diagnosis of cystic fibrosis

The large number of different mutations in CFTR gene combined with marked variation in their distribution and frequency make the genetic diagnosis of CF rather difficult. The aim of the study was to estimate the detection rate of CFTR mutations in our patients according to the results from the genetic testing performed at Research Center for Genetic Engineering and Biotechnology (Skopje). Methods: CFTR genotype results from 140 patients who had confirmed clinical diagnosis of CF were analyzed. The mainly used molecular analysis methods included: PAGE of PCR-amplified fragments of DNA for the detection of F508del; Inno-LiPA CFTR17 and Inno-LiPA CFTR19 panels for identification of 36 CFTR mutations; and DGGE mutations screening, followed by sequencing of samples showing an abnormal pattern. Results: Mutation detection rate in our group of patients was 83%. The frequency of the most common mutations was: F508del (69.2%), G542X (6.1%), N1303K (2.9%), 621+1G>T (1.1%) and CFTRdel2,1(21kb) (1.1%). Six DNA samples from CF patients in which one or no CFTR mutations have been identified were sent for analysis in Laboratoire de Genetique Moleculaire, Brest (France). With more extensive screening/sequencing protocols and methods for identification of large rearrangements, all unknown mutations were identified. Conclusion: The diagnosis of CF remains clinical. Indetermination of CFTR mutations arouse doubts in families about the accuracy of diagnosis. Because of genetic heterogeneity of CF in our region, there is a need for additional methods for CFTR gene analysis to maximize mutant allele detection rate, which will have positive implications for the genetic counseling of families.

28 Earliest features of cystic fibrosis

Introduction: Nasal potential difference (NPD) test has long been used to assist in the diagnosis of CF and more recently as an outcome measure in clinical trials of new CF therapies. This test has also been adapted to the mouse nose. Objectives: We aimed at evaluating variability of the NPD measurements in CF patients displaying two severe CFTR mutations and in sex-matched healthy controls. NPD recorded from F508del-CF and normal wild-type mice were also compared. Methods: In each setting, tests were performed by a single qualified operator. In the clinical setting, the latest CFTT-TDN SOP was followed. A total of 80 tracings were obtained from 10 patients (23.2 y; range 14 to 32) and 10 healthy subjects (34 y; range 24 to 53), each tested twice, in both nostrils. Two CF and two controls were excluded from the statistical data analysis due to the presence of a single non interpretable NPD tracing (4/80, 5%). To achieve equal sample size, tests were obtained from 8 CF mice and normal wild-type. Comprehensive multivariate analysis of paired data showed a good reproducibility of NPD parameters in the clinical and the pre-clinical setting; lower variability was observed in mice. However, 95% repeatability limits of NPD parameters were large indicating a large measurement error, poor precision and low within-subject repeatability. In both settings, chloride secretion was shown to be the most reproducible and repeatable parameter. Conclusion: In human as in mice, NPD showed good reproducibility but limited between-subject repeatability.

WS8.4 Consequences of partial duplications of the human CFTR gene on CF diagnosis: mutations or ectopic variations

WS8.4 Consequences of partial duplications of the human CFTR gene on CF diagnosis: mutations or ectopic variations

PWE-276 Validation of fibroscan as a screening tool for cystic fibrosis associated liver disease in an adult population

IntroductionCystic fibrosis (CF) associated liver disease affects up to 41% of CF sufferers and may progress to cirrhosis with associated complications.1 Diagnosis is frequently made without biopsy. A recent study...

S44 Lung clearance index (LCI) and FEV1 correlate equally with treatment burden as measured by cystic fibrosis questionnaire-revised (CFQ-R)

IntroductionLCI derived from multiple breath washout (MBW), measures the elimination of an inert marker gas during tidal breathing and is a sensitive measure of ventilation inhomogeneity in CF. LCI is...

CO16 COW'S MILK CHALLENGE INCREASES WEAKLY ACIDIC REFLUX IN CHILDREN WITH COW'S MILK ALLERGY AND GERD

Results: All patients under 2 years of age were normal for weight. Between 2 and 20 years, 18/143 (12.6%) were underweight and 48/143 (33.6%) were “at risk”. Over 20 years 6/12 (50.0%) “at risk” and no one was underweight. We confirm the positive relationship between FEV1% and nutritional status (FEV1% M±DS 98.03±24.11 in normal weight vs 67.05±25.67 in underweight; p 20 yrs BMI “at risk” M±DS T0 18.50±9.57 vs T1 20.11±10.52). In patients between 2 and 20 years the percentage of patients less than the 10th BMI percentile and between 10th and 50th BMI percentile decreased from 18/143 to 4/143 (12.6% vs 2.7% p<0.001) and from 48/143 to 34/143 (33.6% vs 23.7%, p<0.001) respectively. Conclusion: Our study confirms the crucial role of an early diagnosis of CF in order to prevent nutritional failure. Individual management with an aggressive nutritional treatment in CF patients in or at highest risk for malnutrition can increase rates of adherence to nutrition recommendation for improving clinical outcomes.

CO15 OESOPHAGEAL MUCOSAL DILATED INTERCELLULAR SPACES (DIS): IS IT A REAL ULTRASTRUCTURAL MARKER OF GERD PHENOTYPE? A CHILDREN POPULATION STUDY

Results: All patients under 2 years of age were normal for weight. Between 2 and 20 years, 18/143 (12.6%) were underweight and 48/143 (33.6%) were “at risk”. Over 20 years 6/12 (50.0%) “at risk” and no one was underweight. We confirm the positive relationship between FEV1% and nutritional status (FEV1% M±DS 98.03±24.11 in normal weight vs 67.05±25.67 in underweight; p 20 yrs BMI “at risk” M±DS T0 18.50±9.57 vs T1 20.11±10.52). In patients between 2 and 20 years the percentage of patients less than the 10th BMI percentile and between 10th and 50th BMI percentile decreased from 18/143 to 4/143 (12.6% vs 2.7% p<0.001) and from 48/143 to 34/143 (33.6% vs 23.7%, p<0.001) respectively. Conclusion: Our study confirms the crucial role of an early diagnosis of CF in order to prevent nutritional failure. Individual management with an aggressive nutritional treatment in CF patients in or at highest risk for malnutrition can increase rates of adherence to nutrition recommendation for improving clinical outcomes.

Factors Associated With Parental Perception of Child Vulnerability 12 Months After Abnormal Newborn Screening Results

We identified factors associated with elevated parental perceptions of child vulnerability (PPCV) 12 months after newborn screening (NBS) of 136 children: healthy, normal results (H, n = 37), cystic fibrosis carriers (CF-C, n = 40), congenital hypothyroidism (CH, n = 36), and cystic fibrosis (CF, n = 23). Controlling for infant and parent characteristics, mixed logit structural equation modeling showed direct paths to elevated PPCV included parent female sex, CF diagnosis, and high documented illness frequency. PPCV was positively associated with maternal parenting stress. Infants with CF and CF carriers had significantly more documented illness frequency than H group infants. The CH group did not differ significantly from the H group and had no paths to PPCV. Unexpectedly high documented illness frequency among infants who are CF carriers warrants further investigation.

370* Increasing incidence of cystic fibrosis being diagnosed in adulthood over time: an analysis of two decades of care

Objectives: 5−10% of CF patients are diagnosed as adults. They often present with non-classical CF and suffer a milder clinical course. We postulate an increased incidence of adult CF diagnoses. We sought to determine if the adult CF, diagnostic characteristics and clinical course differed between 2000–2009 and 1990–1999. Methods: Patients attending CF centres in Northern Ireland from 1990 with a diagnosis of CF after the age of 16 were identified. Comparison of their demographics and biostatistics was made and a F508del homozygous cohort used as a control. Results: Fifty-two patients were identified. There was an increased incidence of individuals diagnosed with CF in adulthood in the latter decade (12 vs 40). Mean age at diagnosis was 30.4(±4.0) yrs in the 90’s and 43.2(±2.7) yrs in the 00’s (p = 0.02). The majority of patients presented with respiratory manifestations in both cohorts. Mean FEV1% predicted at presentation in the 90’s group 71.3% (±7.6) and the 00’s group 76.9% (±4.1) (p = 0.52). Class 1−3 mutations represented 75% of the 90’s group and 50% of the 00’s (p = 0.18), Class 4 mutations 25% of 90’s and 2.5% of 00’s (p = 0.03) and unknown mutations accounted for 0% of 90’s and 50% of 00’s (p = 0.002). A comparison of FEV1 % decline between the late diagnosis group (0.46ml/yr) and F508del homozygous patients (1.64ml/yr) (p = 0.02). Conclusions: Owing to the increased recognition of the heterogeneity of CF, this diagnosis is increasingly being made in adulthood. The majority of patients in both cohorts presented with Bronchiectasis. While having a milder clinical course these patients still suffer a considerable burden of disease and have a declining respiratory status.

46 Delay in meconium emission: should it be considered a risk for cystic fibrosis?

Background: Ten to 15% of CF infants present with meconium ileus (MI), that sometimes can lead to intestinal malformation. Serum immunoreactive trypsin (IRT) levels may be normal and newborn screening (NBS) could be erroneously considered negative. In our CFNBS protocol newborn babies presenting problems with meconium passage (delay in meconium emission − MD − included) undergo sweat test also if IRT is negative Aim: The aim of this study is to investigate the incidence of CF in neonates with MI and MD to determine if infants with this last condition have to be submitted to CF diagnostic test. Patients and Method: A retrospective 5-year study examines neonates screened in our Center that presented problems in meconium passage, with regard to IRT values and CF diagnosis. Results: In a 5-year period (2006–2010) we screened 172,185 newborns and diagnosed 50 CF. Eigthy-two newborns were reported having some problems related to meconium passage: 13 were referred as MI (group 1), 7 as intestinal atresia (IA) (group 2), 62 as MD (group 3). In group 1 8/13 had IRT positive, 6/13 were diagnosed as CF and one of them had IRT normal value; 2 died without diagnosis. In group 2 6/7 had IRT negative, 2/7 had CF and one of them was IRT negative. In group 3 nobody out 62 had IRT positive, nor positive sweat test. Conclusion: In this series 12% of CF neonates presented with MI and/or IA, and 2 of them were IRT negative confirming that CF with MI and IA can have negative NBS. Delay in meconium pass is therefore, asscociated neither with positive screening nor with CF, suggesting that newborns with MD and IRT negative don’t require further analysis for CF.

47 CFTR gene mutations and the risk for developing a pancreatic cancer: is it only a consequence of chronic inflammation?

Background: Ten to 15% of CF infants present with meconium ileus (MI), that sometimes can lead to intestinal malformation. Serum immunoreactive trypsin (IRT) levels may be normal and newborn screening (NBS) could be erroneously considered negative. In our CFNBS protocol newborn babies presenting problems with meconium passage (delay in meconium emission − MD − included) undergo sweat test also if IRT is negative Aim: The aim of this study is to investigate the incidence of CF in neonates with MI and MD to determine if infants with this last condition have to be submitted to CF diagnostic test. Patients and Method: A retrospective 5-year study examines neonates screened in our Center that presented problems in meconium passage, with regard to IRT values and CF diagnosis. Results: In a 5-year period (2006–2010) we screened 172,185 newborns and diagnosed 50 CF. Eigthy-two newborns were reported having some problems related to meconium passage: 13 were referred as MI (group 1), 7 as intestinal atresia (IA) (group 2), 62 as MD (group 3). In group 1 8/13 had IRT positive, 6/13 were diagnosed as CF and one of them had IRT normal value; 2 died without diagnosis. In group 2 6/7 had IRT negative, 2/7 had CF and one of them was IRT negative. In group 3 nobody out 62 had IRT positive, nor positive sweat test. Conclusion: In this series 12% of CF neonates presented with MI and/or IA, and 2 of them were IRT negative confirming that CF with MI and IA can have negative NBS. Delay in meconium pass is therefore, asscociated neither with positive screening nor with CF, suggesting that newborns with MD and IRT negative don’t require further analysis for CF.