[Italian Cystic Fibrosis Registry (ICFR). Report 2021-2022]. / Registro Italiano Fibrosi Cistica (RIFC). Rapporto 2021-2022.

INTRODUCTION: Italian Cystic Fibrosis Registry (ICFR) collects data of patients with cystic fibrosis (CF) through the collaboration with Italian CF referral and support Centres (Italian law 548/93). It aims at analysing medium and long-term clinical and epidemiological trends, identifying healthcare needs at regional and national levels, contributing to healthcare programmes, and resource allocation. Italian data are also compared at international level through the collaboration with the European CF Registry for sharing epidemiological data on general aspects like CF epidemiology and specific topics such as the use of CFTR modulators. OBJECTIVES: The purpose of this Report is to provide updated demographic and clinical data of the Italian FC population for the years 2021 and 2022, to contribute essential information for the implementation of projects aimed at improving the management of patients affected by this disease. DESIGN: Analyses and results presented in this Report pertain to patients currently under care at Italian National Referral and Support Centres for Cystic Fibrosis and Paediatric Hospital 'Bambino Gesù' in the 2021-2022 period. Data were submitted by clinical Centres through a dedicated web-based software and underwent dual quality control (QC) measures: automated quantitative QC within the software and secondary QC at the European level before the integration into the European Cystic Fibrosis Registry. These measures ensure data completeness, accuracy, and longitudinal consistency with European core data. SETTING AND PARTICIPANTS: A total of 27 CF Centres, including referral and support centres, as well as 'Bambino Gesù' Children's Hospital CF centre, submitted their data to ICFR for the years 2021-2022. Althourgh CF Centres in Verona and Messina do not use the ICFR software, their data are centrally collected and subsequently forwarded to the European Registry. Data from service centres in Treviso and Rovereto are transmitted via the Verona CF Centre. Data from Sardinia Centre are currently unavailable. RESULTS: The results section provides a comprehensive overview of various aspects of CF epidemiology and patient characteristics. 1.Demography: in 2021 and 2022, 5,977 and 6,077 CF patients were respectively included in the ICFR, with median ages of 23.3 and 23.7 years. The prevalence rates were 10.1 and 10.3 per 100,000 residents in Italy for the respective years, with males comprising 51.6% on average. The distribution by age showed a higher frequency among patients aged 7 to 35 years; adult patients constituted 63.5% on average in both years. 2. Diagnosis: most CF patients were diagnosed before the age of two (mean value 57.9%), with a significant percentage diagnosed in adult age (35.4% in 2021 and 25.6% in 2022). 3.New diagnoses: there were 113 new diagnoses in 2021 and 121 in 2022, with estimated incidences of 1 in 9,097 living births in 2021 and 1 in 6,232 in 2022. 4. Genetics: genetic analyses were conducted on 99.9% of patients, revealing CFTR gene mutations in over 98% of cases. The F508del mutation was the most common (44% of alleles in 2021), with 18% of patients having at least one "residual function" mutation. Gating mutations were present in 3.4% of Italian patients, while 20% had at least one-stop codon mutation. 5.Lung function: lung function, measured by percent predicted (pp)FEV1 (Forced Expiratory Volume in the first second) progressively declined before adulthood, with the majority of paediatric patients (92.8% in 2021 and 93.8% in 2022) maintaining a ppFEV1≥70%. 6.Nutrition: critical periods for nutrition were identified as the first 6 months of life and adolescence, with higher prevalence of malnourished male adolescents compared to females. Suboptimal BMI values were more common in adult females (28.7% in 2021 and 26.9% in 2022) compared to males (14.2% in 2021 and 12.6% in 2022). 7. Complications: CF-related liver disease without cirrhosis was prevalent in patients under 18 years (21.9% in 2021 and 21.2 in 2022), while CF-related diabetes was most frequent in adults (24.2%). 8.Transplantation: over the two-year period, 28 patients underwent double-lung transplantation, with median ages of 29.1 in 2021 and 35.3 in 2022, respectively. Median waiting times ranged from 9.4 to 11.6 months. 9.Microbiology: chronic Pseudomonas aeruginosa infection affected 37.2% of adult patients in 2021 and 36.0% in 2022, compared to 7.4% and 6.5% in paediatric patients. Staphylococcus aureus infection rates were 34.6% and 42.2% in 2021 among adults and 34.4% and 36.7% in 2022 among paediatric patients. 10. Mortality: a total of 34 patients died during the 2021-22 period (19 females, 15 males), with median ages at death of 43.7 years in 2021 and 46 years in 2022 (excluding transplanted patients). CONCLUSIONS: The present Report is an update of the data published in the past years and summarizes the main epidemiological and clinical data regarding Italian CF subjects in the years 2021 and 2022. The number of patients registered in 2021 was 5,977, while in 2022 was 6,077. The population coverage estimates for 2022 to be around 97%. In 2020, 60.5% of patients were older than 18 years, in 2022 adult patients account for 63.5% of the Italian CF population. Over the years, therefore, an increase in the median age of Italian CF patients has been observed, reaching 23.7 years in 2022. The absolute number of new diagnoses per year remains substantially unchanged over the years (a total of 234 in the period under review). The median age at diagnosis in 2022 was 2.5 months, 62.6% of subjects are really diagnosed within the first year of life and almost 90% of them are diagnosed through neonatal screening. In 2022, almost all patients underwent genetic analysis (99.9%). Data collected confirm the great variability among Italian CF patients. As regards respiratory function, what is reported in previous reports is here confirmed, with an ever-increasing percentage of subjects under the age of 18 having normal respiratory function, moreover, less than 1% of paediatric patients has a severe lung function (ppFEV1<40). The marked improvement in this indicator in the adult population seems to be mainly due to the introduction from 2021 in Italy of therapy with highly effective CFTR modulators. At the same time, the close positive correlation between nutritional status and respiratory function is confirmed for the adult population. As regards chronic infection by Pseudomonas aeruginosa, in 2022, a reduction in the percentage of chronic infection is observed both among adults (36% vs 38.8% in 2020) and in paediatric patients (6.5% vs 7.6% in 2020). The most frequent complication in both paediatric and adult populations is liver disease (respectively, in 24.2% and 41.3% of subjects). In the two-year period, 34 patients died; their median age at death was between 43 and 46 years (transplant patients excluded); only two patients under the age of 18 died in the period 2021 and 2022, confirming once again that mortality in paediatric age is a rare event. The data presented in this Report shows how the register can be a national and international point of reference for CF patients and the scientific community, a tool for describing the Italian CF population over the years, and a starting point for planning epidemiological studies and clinical studies.

[Italian Cystic Fibrosis Registry (ICFR). Report 2019-2020]. / Registro italiano Fibrosi Cistica (RIFC). Rapporto 2019-2020.

INTRODUCTION: Italian cystic fibrosis registry (ICFR) collects data from cystic fibrosis (CF) patients through the collaboration with Italian CF referral and support Centres (Italian law 548/93). ICFR contributes: • to the analysis of medium and long term clinical and epidemiological trends of the disease; • to the identification of the main health care needs at regional and national level to contribute to the Health Care programmes and to the distribution of resources; • to the comparison of the Italian data with international ones. This latter is based on the collaboration with the European CF registry and, due the COVID-19 pandemic emergency, with important global projects. OBJECTIVES: The purpose of this Report is to update the demographic and clinical data of the Italian FC population in the years 2019 and 2020, contributing to the information necessary to implement projects to improve the management of patients affected by this disease. DESIGN: Analyses and results described in the present Report are referred to patients currently followed at the Italian National Referral and Support Centres for Cystic Fibrosis in the 2019-2020 period. Data were sent by clinical Centres through a dedicated web-based software. Data undergo a double quality control (QC): the first is automatically performed by the software (quantitative QC), the second is performed at a European level (before the inclusion of the Italian data within the European Cystic Fibrosis Registry). These QCs assure the completeness and the accuracy of data as well as their longitudinal consistency with the European core data. SETTING AND PARTICIPANTS: A total of 29 CF Centres (referral and support centres and 'Bambino Gesù' Children's Hospital CF centre) sent to ICFR their data referred referred to years 2019-2020. CF Centres of Verona, Messina, and Palermo (this latter only for 2019) do not use the ICFR software; however, their data are firstly collected in a centralized manner, then sent to the European Registry. Data from support centres of Treviso and Rovereto are sent through the Verona CF Center. Finally, data from Sardinia Centre are still missing. RESULTS: The present Report has been organized into 10 sections. 1. Demography: in 2019, 5,585 CF patients were registered in the ICFR and 5,801 in 2020; median age was 21.6 years in 2019 and 22.4 years in 2020. Prevalence was 9.36/100,000 and 9.79/100,000 residents in Italy in 2019 and in 2020, respectively. Male percentage was 51.5% in 2019 and 2020 and CF distribution by age range showed higher frequency in patients aged 7 to 35 years. Adult patients (aged more than 18 years) were 59.5% on average in both years. 2. Diagnoses: most of the CF patients were diagnosed before two years of age (median value 68.5%); a significant percentage of patients (12.9% in 2019 and 13.4% in 2020) was diagnosed in adult age. 3. New diagnoses: new diagnoses were 136 in 2019 and 96 in 2020. Estimated incidence was 1/5.568 living births in 2019 and 1/7.369 in 2020. 4. Genetics: 99.9% of patients underwent genetic analyses and in 98.2% of these patients a mutation in Cystic Fibrosis Transmembrane Regulator (CFTR) gene was identified. The F508del mutation was the most frequent (identified in 44.7% allele; 2019 data). Furthermore, on average 17.3% of patients had at least one 'residual function' mutation. At least one gating mutation is present in 3.3% of Italian patients. Finally, 20.5% of patients had at least one stop codon mutation (class 1). 5. Lung function: percent predicted FEV1 (Forced Expiratory Volume in the first second) progressively declined before adult age, in accordance with the natural history of the disease. The majority of paediatric patients (6-17 years of age), i.e., 86.7% in 2019 and 90.5% in 2020, had percent predicted FEV1 >=70%; whereas paediatric patients with a FEV1% >=40% are less than 2% in the study period. 6. Nutrition: the two most critical periods are the first 6 months of life and adolescence. Prevalence of malnourished adolescent males (12-17 years of age) is higher than the prevalence observed in females. Increasing percentages of female patients with a suboptimal BMI value (33.5% and 31.4%, respectively, in 2019 and 2020) are observed in adult age. 7. Complications: in 2019, CF-related liver disease without cirrhosis was the main complication both in patients aged less than 18 years (20.3% on average) and in adults (37.5%). CF-related diabetes was also frequent in CF adults (23.4%). 8. Transplantation: in 2019-2020, 64 patients received a double-lung transplantation. Median and range of age were 33 years (12.29-57.46) in 2017 and 32.9 (16.5-53.6) years in 2020. Median waiting times for lung transplantation in the two-year period ranged from 6 to 8 months. 9. Microbiology: percentage of adult patients with chronic Pseudomonas aeruginosa infection was 41.6% in 2019 and 38.8% in 2020 vs 14.3% in 2019 and 7.6% in 2020 in paediatric age. Staphylococcus aureus infection is present in 31.1% and 35.9% of adult patients in 2019 and in 33.5% and 34.7% of paediatric patients in 2020. 10. Mortality: a total of 51 patients died in the 2019-2020 period (28 females and 23 males); median age at death was 35.7 years in 2019 and 39 years in 2020 (transplanted patients are not included). CONCLUSIONS: The present report shows that the Italian CF population is growing (4,159 in 2010 vs 5,801 in 2020). Median age of patients increased in the 2010-2020 period (17 years in 2010 vs 22.4 years in 2020). Prevalence of adult patients is increasing (in 2020, 60.5% of patients is more than 18 years old). About 68.5% of new patients is diagnosed within the second year of life and median age at death (transplanted patients not included) increased in 2020 up to 39 years (in 2018 this value was 35.8). Some statistical differences between 2019 and 2020 are mainly due to the absence of about 200 patients not included in 2019 data by a participating centre for a technical problem.

Mannose-Binding Lectin (MBL) and Gap Junction Protein Alpha 4 (GJA4) Gene Heterogeneity in Relation to Severity of Clinical Disease in Cystic Fibrosis.

BACKGROUND: Recently, we provided evidence that a single nucleotide polymorphism (SNP), rs41266431, on the gap junction protein alpha 4 (GJA4) gene, acts as a modifier for clinical disease severity in patients with cystic fibrosis (CF). These features are very similar to those of variants of the mannose-binding lectin (MBL). This study aimed to clarify whether the clinical disease phenotype associated with GJA4 variants is independent of MBL variants. METHODS: One hundred and twelve patients with homozygous F508del (mean age, 27.6 years; m/f, 61/51) were recruited from the CF centers of Bonn, Frankfurt, and Amsterdam. A sequence analysis was performed for GJA4 and MBL. The clinical phenotype was assessed over three years using pulmonary function tests, body mass index, Pseudomonas aeruginosa colonization, diabetes mellitus, survival to end-stage lung disease, and inflammatory markers. RESULTS: A clinically relevant SNP of GJA4 was identified by sequence analysis. Pulmonary function (FVC% pred, mean 78/85; p < 0.055) and survival to end-stage lung disease were lower (p < 0.043) for this variant (rs41266431) in carriers homozygous for the G variant (n = 82/112; 73%) than in other carriers. Serum MBL (820/372 ng/mL, p < 0.001) was significantly higher in "MBL-sufficient" genotypes (n = 79/112; 71%) than in "MBL-insufficient" genotypes, and a trend for a significant difference in BMI percentiles (35.2/23.8; p < 0.059) was observed. For the MBL-sufficient genotype (median age at death, 38/26 years), there was a trend for better survival (p < 0.076). There was no augmentation by gene-gene interaction between MBL and GJA4 variants for any outcome parameter. CONCLUSIONS: The clinical disease phenotype associated with GJA4 variants is independent of MBL variants. MBL-sufficient variants were associated with superior BMI and a trend for better survival than MBL insufficient variants.

[Italian Cystic Fibrosis Registry (ICFR). Report 2017-2018]. / Registro italiano Fibrosi Cistica (RIFC). Rapporto 2017-2018.

INTRODUCTION: On the 15th of November 2020, the National Centre for Rare Diseases of the Italian National Health Institute, clinicians of the Italian National Referral and Support Centres for Cystic Fibrosis, Children's Hospital "Bambino Gesù", Italian Cystic Fibrosis Society, Italian League for Cystic Fibrosis renewed the agreement about CF data flow for a 3-year period. The possibility to access data by third parties is among the most important innovation introduced within the agreement. OBJECTIVES: Aim of the present Report is to improve the know-how of cystic fibrosis (CF) through a better characterization of Italian patients. Furthermore, the present Report aims at improving the care of CF patient. In particular, this Report should contribute to the following objectives: • to analyse the medium- and long-term clinical and epidemiological trends of the disease; • to identify the main healthcare needs at regional and national level, in order to contribute to the healthcare programmes and to the distribution of resources; • to compare Italian data with international ones. DESIGN: Analyses and results described in the present Report are referred to patients currently followed at the Italian National Referral and Support Centres for Cystic Fibrosis in the 2017-2018 period. Data were sent by clinical Centres through a new-committed software. Data underwent a double quality control (QC): the first is automatically performed by the software (quantitative QC), the second is performed at a European level (before the inclusion of the Italian data within the European Cystic Fibrosis Registry). These QCs assure the completeness and the accuracy of data as well as their consistency with the European core data. SETTING AND PARTICIPANTS: The present Report has been organized into 10 sections. 1. Demography: in the ICFR, 5,565 CF patients were registered in 2017 and 5,501 in 2018; median age was 21.4 years in 2017 and 21.2 years in 2018. Prevalence was 9.20/100,000 residents in Italy in 2017 and in 2018. Male percentage was 51.65% in 2017 and 2018, CF distribution by age range showed higher frequency in patients aged 7 to 35 years. Adult patients (aged more than 18 years) were 56.4% on average in 2017 and 2018. 2. Diagnoses: most of the CF patients were diagnosed before two years of age (median value 66.4%); a significant percentage of patients (21.6% in 2017 and 18.3% in 2018) was diagnosed in adult age. 3. New diagnoses: new diagnoses were 162 in 2017 and 142 in 2018. Estimated incidence was 1/5.214 living births in 2017 and 1/5.442 in 2018. 4. Genetics: 99.8% of patients underwent genetic analyses and in 97.1% of these patients a mutation in Cystic Fibrosis Transmembrane Regulator (CFTR) gene was identified. The F508del mutation was the most frequent (44.6% in 2018). Furthermore, 16.3% of patients in 2017 and 16.9% of patients in 2018 had at least one 'residual function' mutation. At least one gating mutation is present in 3.3% of Italian patients. Finally, 20.5% of patients had at least one stop codon mutation (class 1). 5. Lung function: percent predicted FEV1 (Forced Expiratory Volume in the first second) progressively declined before adult age, in accordance with the natural history of the disease. The majority of paediatric patients (6-17 years of age), i.e., 86.70% in 2017 and 90.50% in 2018, had percent predicted FEV1 ≥70%; whereas paediatric patients with a FEV1% ≤40% are less than 2% in the 2017-2018 period. 6. Nutrition: the two most critical periods are the first 6 months of life and adolescence. Prevalence of malnourished adolescent males (12-17 years of age) is higher than the prevalence observed in females. Increasing percentages of adult female patients with a suboptimal BMI value (39.1% and 36.1%, respectively, in 2017 and 2018) are observed. 7. in 2018, CF-related liver disease without cirrhosis was the main complication both in patients aged less than 18 years (17.0% on average) and in adults (31.5%). CF-related diabetes was also frequent in CF adults (23.4%). 8. Transplantation: in 2017-2018, 83 patients received a double-lung transplantation. Median and range of age were 29.3 years (11.8-60.2) in 2017 and 29.1 (7.8-45.6) years in 2018. Median waiting times for lung transplantation in the two considered years were 8.6 and 7.7, respectively. 9. Microbiology: percentage of adult patients with chronic Pseudomonas aeruginosa infection was 51.3% in 2017 and 46.3% in 2018 vs 15.6% in 2017 and 10.2% in 2018 in paediatric age. Staphylococcus aureus infection is present in 53.4% and 53.5% of adult patients in 2017 and in 41.6% and 37.5% of paediatric patients in 2018. 10. Mortality: a total of 89 patients died in the 2017-2018 period (49 females); median age at death was 33.9 years in 2017 and 35.8 years in 2018 (transplanted patients are not included). CONCLUSIONS: The present report shows that the Italian CF population is growing (4,159 in 2010 vs 5,501 in 2018; +1,342). Quality of data collected has been improved by the drastic reduction of missing data, thanks to the new software for data collection. Median age of patients increased in the 2010-2018 period (17 years in 2010 vs 21.2 years in 2018). Paediatric death is a very rare event. A very low percentage of paediatric population was characterized by severe lung disease (FEV1% <40). Prevalence of adult patients is increasing (56.4% in 2018). Age at diagnosis is decreasing (4.2 months in 2017 vs 3.8 months in 2018). Median age at death (transplanted patients not included) was 33.9 in 2017 and 35.8 in 2018. RIFC is completely compliant with the GDPR (UE 2016/679 regulation) and its role in national and international CF communities is confirmed.

Antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in F508DEL cystic fibrosis.

BACKGROUND: Eluforsen is an antisense oligonucleotide designed to bind to the mRNA region around the F508-encoding deletion and restore the cystic fibrosis transmembrane conductance regulator (CFTR) protein function in the airway epithelium. We assessed the safety and tolerability, pharmacokinetics and exploratory measures of efficacy of inhaled eluforsen in cystic fibrosis (CF) patients homozygous for the F508del-CFTR mutation. METHODS: This randomised, double-blind, placebo-controlled, dose escalation 1b study recruited adult CF subjects with a FEV1 > 70% predicted in four single ascending dose cohorts and four multiple ascending dose cohorts. Primary objectives were safety and tolerability. Secondary endpoints included pharmacokinetics, percent predicted forced expiratory volume in 1 s (ppFEV1), and Cystic Fibrosis Questionnaire-Revised (CFQ-R) Respiratory Symptom Score (RSS). RESULTS: Single and multiple doses of inhaled eluforsen up to 50 mg were safe and well tolerated. A maximum tolerated dose was not established. Systemic exposure was low in all cohorts and lung function remained stable throughout the study. Three of four eluforsen-treated groups in the MAD study demonstrated an improvement in CFQ-R RSS at end of treatment with adjusted mean change from baseline values ranging from 6.4 to 12.7 points. In comparison, there was a mean decrease of 6.5 points in the placebo group from baseline to end of treatment. CONCLUSIONS: Inhaled eluforsen up to 50 mg dosed 3 times per week for 4 weeks was safe and well tolerated, showed low systemic exposure, and demonstrated improvement in CFQ-R RSS, a relevant measure of clinical benefit in CF patients.

Antisense oligonucleotide eluforsen improves CFTR function in F508del cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. In this study we assessed the effect of antisense oligonucleotide eluforsen on CFTR biological activity measured by Nasal Potential Difference (NPD) in patients with the most common mutation, F508del-CFTR. METHODS: This multi-centre, exploratory, open-label study recruited adults with CF homozygous or compound heterozygous for the F508del-CFTR mutation. Subjects received intranasal eluforsen three times weekly for 4 weeks. The primary endpoint was the within-subject change from baseline in total chloride transport (Cl-free+iso), as assessed by NPD. Secondary endpoints included within-subject change from baseline in sodium transport. RESULTS: In the homozygous cohort (n = 7; per-protocol population), mean change (90% confidence interval) in Cl-free+iso was -3.0 mV (-6.6; 0.6) at day 15, -4.1 mV (-7.8; -0.4, p = .04) at day 26 (end of treatment) and - 3.7 mV (-8.0; 0.6) at day 47. This was supported by improved sodium transport as assessed by an increase in average basal potential difference at day 26 of +9.4 mV (1.1; 17.7, p = .04). The compound heterozygous cohort (n = 7) did not show improved chloride or sodium transport NPD values. Eluforsen was well tolerated with a favourable safety profile. CONCLUSIONS: In F508del-CFTR homozygous subjects, repeated intranasal administration of eluforsen improved CFTR activity as measured by NPD, an encouraging indicator of biological activity.

Clinical and laboratory profile of children with Cystic Fibrosis: Experience of a tertiary care center in Pakistan.

OBJECTIVE: To determine the clinical presentation, diagnostic investigations and laboratory workup done in admitted children with cystic fibrosis at Aga Khan University Hospital Karachi, Pakistan. METHODS: This is athree years retrospective study from January 2013 to December 2015 conducted at The Aga Khan University Hospital Karachi Pakistan, enrolling admitted patient from birth to 15 years of either gender, diagnosed with CF on the basis of clinical features and positive sweat chloride test. Different clinical presentations were noted including initial presentations. Sweat chloride values more than 60mmol/L were labeled as positive and consistent with diagnosis of CF. Available Delta F-508 mutation analyses were noted. Relevant laboratory and radiological investigations including sputum culture and HR-CT chest findings were documented. Results were analyzed using SPSS version 20. RESULTS: Total 43 children were selected according to the inclusion criteria. Chronic cough (69.76%) was the most common initial clinical presentation. Mean age at onset of symptoms was 14.41± 26.18 months and mean age at diagnosis was 47.20 ± 45.80 months Respiratory features were most common in our cohort including chronic productive cough (90.71%), recurrent bronchopneumonia (72.09%) and asthma like presentation (44.19%) with wheezing and cough. 86% patients presented with failure to thrive. Gastroenterological features including steatorrhea were seen in 55.81% patients and 44.19% patients had abdominal distension. Mean sweat chloride value in our population was 82.70± 22.74. Gene analysis for Delta F-508 was identified in 12 (27.90%) patients. Bronchiectatic pulmonary changes on HRCT were seen in 18 patients (41.86%). Pseudomonas grew in 12 patients (27.90%) in sputum cultures at the time of diagnosis. CONCLUSION: Respiratory presentations predominate in CF children followed by gastrointestinal features. Nearly half of our patient had bronchiectatic changes on CT scan chest and more than quarter had pseudomonas colonization in the airways at the time of diagnosis. Delta F-508 mutation was found to be uncommon in our study population. There is significant delay in diagnosing patients with CF.

Cystic Fibrosis Mutation Detection with SPR Biosensor in Real Samples via Multiple Surfaces Binding Method.

AIM AND OBJECTIVE: Surface Plasmon Resonance (SPR) based biosensor system was developed for the detection of Delta F508 (ΔF508del) Cystic Fibrosis (CF) mutation in both synthetic and real samples. MATERIAL AND METHOD: In order to provide an effective hybridization between probe and the Polymerase Chain Reaction (PCR) amplicons (target), streptavidin was bound to the surface and biotin-tag probe was sent to the streptavidin-coated surface. For the target preparation, blood samples were collected from the patients who suffer from CF. Following the DNA isolation; samples were amplified with PCR with biotin-tag. Before sending the biotin-tag PCR amplicons onto the modified surface, amplicons were also interacted with the helper oligonucleotides to prevent re-annealing of the denatured DNA strands. This kind of 'multiple surface binding' method helps increasing the sensitivity of the detection. RESULTS: The limit of detection (S/N= 3) was calculated as 12.24 pico-mole/ml for PCR-like synthetic long target sequence and 13x105 molecules for real samples in less than half an hour. CONCLUSION: Using the both biotin-tag probe and the helper oligonucleotides together, hybridization was achieved much more efficiently than traditional denaturation protocols for real samples and biotinfree hybridization detection. To the best of our knowledge, the procedure described in this study is one of the simplest, rapid and sensitive methods for CF mutation detection with SPR based biosensor system in real samples.

Molecular basis of cystic fibrosis disease: an Indian perspective.

Cystic fibrosis is a common autosomal recessive disorder usually found in population of white Caucasian descent. Now it is well documented the presence of CF disease in India with the advancement of laboratory testing. As once it was thought non existence of this disease in our population. Most of the phenotype of CF disease was in accordance of western population. Genetic analysis of CFTR gene in Indian CF patients revealed that most common mutation was delta F508 mutation. However, it was less than Caucasian population. CFTR mutations are also a causative factor in the pathogenesis of male infertility due to obstructive azoospermia. There are two most common mutation viz. IVS8-T5 and delta F508 which are responsible for congenital absence of vas deferens in male infertility patients. Elevated levels of sweat chloride at two occasions along with the presence of two mutations in CFTR gene was gold standard method for diagnosis of CF disease. It is noteworthy here that due to magnitude of Indian population, the total CF disease load would be more than many European countries. Clinical data demonstrate the prevalence of both classical and genetic form of CF in India.

Frecuencia de la mutación F508del en estudiantes de la Facultad de Medicina de la Universidad del Rosario, Bogotá, Colombia / Carrier frequency of F508del mutation of cystic fibrosis in medical students from Universidad del Rosario, Bogotá, Colombia

Introducción: La fibrosis quística es la enfermedad letal autosómica recesiva más frecuente en caucásicos, donde su incidencia es de 1 en 2000 nacidos vivos. Se debe a mutaciones en el gen CFTR, de las cuales la más frecuente es la F508del presente en 66% de los enfermos y en 1 de cada 25 personas sanas de origen caucásico. Objetivo: Identificar la tasa de portadores de la mutación F508del en una muestra de estudiantes de la Facultad de Medicina de la Universidad del Rosario.Materiales y métodos: Se determinó la presencia de la mutación F508del mediante PCR y análisis de heterodúplex en 110 estudiantes de IV y VII semestre de la Facultad de Medicina. Resultados: Se obtuvieron 4 heterocigotos para la mutación F508del, es decir, una frecuencia de portadores de 1 en 27 estudiantes. Conclusiones: La frecuencia de portadores de esta mutación en la población analizada es considerable, e indica que hay un alto número de personas en riesgo de heredar la enfermedad y que deben recibir asesoramiento genético. Es necesario aumentar el tamaño de la muestra para obtener datos representativos de la población colombiana.

Introduction: Cystic fibrosis (CF) is the most frequent autosomical recessive disorder in Caucasian population with an incidence of 1 in 2000 newborns. The disease is caused by mutations in the cftr gene, but the most common mutation is F508del, which accounts for 66% of CF chromosomes worldwide and a carrier frequency for Caucasian population of 1 in 25. Objective: To determine the carrier frequency of the F508del mutation in 110 unrelated, healthy students from the Facultad de Medicina, Universidad del Rosario. Methods: The presence of F508del mutation using PCR and heteroduplex analysis was determined. Results: Only four heterozygotes for F508del mutation were discovered. This represents a carrier frequency of 1 in 27 students. Conclusions: This estimated frequency of F508del carriers is higher than expected, encouraging further screening in normal control individuals from different regions of Colombia.