Personalized drug therapy in cystic fibrosis: from fiction to reality

SISTEMA DE BIBLIOTECAS DA UNICAMP

REPOSITÓRIO DA PRODUÇÃO CIENTIFICA E INTELECTUAL DA UNICAMP

Versão do arquivo anexado / Version of attached file:

Versão do Editor / Published Version

Mais informações no site da editora / Further information on publisher's website:

http://www.eurekaselect.com/126456/article

DOI: 10.2174/1389450115666141128121118

Direitos autorais / Publisher's copyright statement:

©2015

by Bentham Science. All rights reserved.

DIRETORIA DE TRATAMENTO DA INFORMAÇÃO Cidade Universitária Zeferino Vaz Barão Geraldo

CEP 13083-970 – Campinas SP Fone: (19) 3521-6493 http://www.repositorio.unicamp.br

Current Drug Targets, 2015, 16, 1007-1017 1007

Personalized Drug Therapy in Cystic Fibrosis: From Fiction to Reality

Fernando Augusto de Lima Marson

, Carmen Silvia Bertuzzo

and Jose Dirceu Ribeiro

1_{Department of Medical Genetics, Faculty of Medical Sciences, State University of Campinas, Brazil;} 2_{Department of Pediatrics, Faculty of Medical Sciences, State University of Campinas, Brazil}

Abstract: Personalized drug therapy for cystic fibrosis (CF) is a long-term dream for CF patients,

caregivers, physicians and researchers. After years of study, the fiction of personalized treatment has turned to hope. Basic information about CFTR mutations classes and new treatments is needed if we are to deal properly with the new CF era. The problems involved in this issue, however, should be evaluated with greater care and attention. VX-770 is a new drug available to treat CF patients with some class III CFTR mutations and other drugs are being studied regarding other classes. The scien-tific literature has constantly given information about each therapy, both in vitro and in vivo. The hope

is increasing. Nevertheless the “scientific world” still lacks information about patients´ reality and daily health related practical needs. Clinical trials have showed good evaluation of some drugs so far, but clinical response is a wide spectrum yet to be analyzed: CFTR mutations spectrum, costs related to the treatment with new drugs (for VX-770 therapy), vari-ability of CF clinical expression, limitations to test in vitro drugs, absence of good clinical markers to evaluate drug re-sponse, absence of long-term studies and with patients below six years old, multidrug treatment used to improve the ex-pression response, and finally, the most important problem, who will benefit from the new drugs therapy, are issues that constitute a barrier that should be overcome. Personalized drug therapy may not be a fiction anymore, but it is not yet a reality for all CF patients.

Keywords: CFTR, correctors, cystic fibrosis, personalized drug therapy, potentiators, PTC124, VX-770, VX-809. 1. INTRODUCTION

Cystic fibrosis (CF) (OMIM 219700) is a monogenic dis-ease and can, thus, be seen as a key opportunity to study new genetic therapies to correct protein function [1-5]. Nearly 2,000 mutations were screened in the CF disease [6-8].

CFTR (Cystic Fibrosis Transmembrane Regulator) mutations

are included in six classes. Each class has specific character-istics, considering the presence or absence of active CFTR at the cell surface. If the presence of CFTR expression is con-firmed, a quantitative or qualitative defect will be present in the CFTR protein [9, 10].

Since the discovery of the CFTR gene, the mutation data-base has greatly increased. If we turn ourselves to look at the history of the handling of CF disease, it could be noted that its treatment was constantly associated with drugs aiming to re-solve its clinical presentation and therefore not based on the CFTR alteration. Studies on gene therapy were performed in several centers for CF [11, 12]. The lungs are the main af-fected organ in CF patients. Although the access to the epithe-lium by the airways provides great easiness to gene therapy, no positive outcome was observed to include gene therapy in the treatment routine. Apparently, the reintroduction of the *Address correspondence to this author at the Department of Medical Ge-netics and Department of Pediatrics; Faculty of Medical Sciences; State University of Campinas. Tessalia Vieira de Camargo, 126. Barao Geraldo, Cidade Universitaria “Zeferino Vaz”, CEP: 13083-887, Brazil;

Tels: +55 19 35218902, +55 19 35218994, +55 19 35218983; Fax: +55 19 35218907; E-mail: fernandolimamarson@hotmail.com

CFTR gene into the cells of the lung is more difficult than

initially thought; however, plasmid DNA and hybrid lentivi-rus showed satisfactory results as possible tools to introduce the normal CFTR gene in CF patients [13]. More studies in this field are necessary. The gene therapy is still a long-term dream.

Notwithstanding, a new area of studies on CFTR muta-tions status has already emerged. Nowadays, several studies set their objectives to target therapy for each CFTR class mutation. In this new CF disease era, the personalized drug therapy shows a possibility to treat the disease itself and not only its symptoms [1, 5, 14-16].

2. CFTR MUTATION: A COMPLEX MUTATION DA-TABASE

The year 1989 is a milestone in CF history: The CFTR gene is known as the causal gene of CF disease [17-19], and nearly 2,000 mutations have been screened since then [6-8]. The CFTR gene, one of the biggest genes in the hu-man genome, is localized in the 7q32.1 region [17-19] and codifies the CFTR protein; fostering a rich palette of muta-tions; with specific classes and characteristics. To deter-mine CFTR gene mutations for all CF patients is a hard work. High frequency is observed for F508del (rs113993960, c.1521_1523delCTT) [6-8] mutation in typical CF disease [20, 21]. Still, in admixed populations [20], older CF patients [22] and populations of oriental and African ethnicity [23], the prevalence of F508del mutation is lower and higher heterozygosity is found.

Please provide corresponding author(s)

photograph

CFTR mutations are widely distributed in the CFTR gene

(Fig. 1A), thus, in some CF cases, the screening of the com-plete CFTR sequence should be performed if one is to under-stand the complex mechanisms of CF´s molecular aspects [24].

Each mutation class has a specific response to the CFTR expression and action. Therefore, the understanding of the mechanisms involved in the functions of the new drugs de-pends on the understanding of each class of mutation [10, 25-29] (Fig. 2).

Class I: No protein. This class is associated with no

mRNA transcription. The premature stop codon (nonsense mutation) or deletion and insertion do not permit the CFTR expression. No CFTR is found at the cell surface and no pro-tein synthesis occurs. Example: G542X (rs113993959, c.1624G>T), R1162X (rs74767530, c.3484C>T), W1282X (rs77010898, c.3846G>A), 3120+1G>A (rs75096551, c.2988+1G>A), 1717-1G>A (rs76713772, c.1585-1G>A), 1812-1G>A (rs121908794, c.1680-1G>A), R553X (rs74597325, c.1657C>T) [6-8].

Class II: No traffic. The most frequent CFTR mutation,

which is responsible for the major part of alleles in the CF patients, is found in this class. F508del mutation shows wide frequency among CF patient worldwide, mainly in Cauca-sian patients [30]. For class II mutations, the CFTR synthesis is partial. After the transcription and translation, the CFTR shows a truncated structure. The cellular mechanisms recog-nize the error and the protein is degraded in the endoplasmic reticulum. No or little protein is present at the cell surface. Other mutations in this class are: A561E (rs121909047, c.1682C>A), N1303K (rs80034486, c.3909C>G), R1066C (rs78194216, c.3196C>T), G85E (rs75961395, c.254G>A) [6-8].

Class III: No function. CFTR III mutations affect the

regulation of CFTR channels. Most of these mutations are found in the NBDs and interfere with ATP binding to these domains or with the stimulation of the channels by ATP, resulting in a decrease of net Cl- _{transport activity (Fig.} 1B-D, complete CFTR structure is showed). CF patients with

mutation in this class show CFTR protein at the cell surface, but no activity is found. Example: G551D (rs75527207, c.1652G>A), S549N (rs121908755, c.1646G>A) [6-8].

The same clinical prognosis is reported for mutations of classes I, II and III. CF patients with these three classes are frequently considered as typical CF disease. Typical CF in-cludes patients with all symptomatology, such as lung and digestive disease, lower body mass index, sweat test with values above 60mEq/L, males with azoospermia, high fre-quency for meconium ileus, diabetes mellitus and os-teopenia/osteoporosis, early colonization of lungs by

Pseu-domonas aeruginosa, early diagnosis and bad clinical

evolu-tion [31].

Class IV: Less function. As a channel, CFTR protein is a

pore. The main function of the CFTR is to translocate the chlo-ride between the intra and extracellular domains (Fig. 1E). The structure responsible to create the linkage between those do-mains is the MSD (membrane-spanning dodo-mains). Mutations of class IV alter the characteristics of the pore. In this mutation class, CFTR is present at the cell surface, but less function is

observed. There is residual function, with normal amount of CFTR protein. A qualitative defect is present. Example: I618T (rs139468767, c.1853T>C), R334W (rs121909011, c.1000C>T), P205S (rs121908803, c.613C>T) [6-8].

Class V: Less protein. CF is a recessive disease; two

cop-ies of mutations should be present, one maternal and one paternal. In this case, CF patients have their parents with 50% of CFTR active, if the best condition is considered. Thus, only partial CFTR expression causes absence of CF clinical manifestation. No specific or “magic” number to provide this information is found in the literature. Nonethe-less, studies show that values equal or above 25% of CFTR expression are associated with no clinical symptoms of CF disease [32]. In this case, CF patients with class V mutation have a residual expression of normal CFTR. However, the amount of expression is associated with clinical symptoma-tology. The mutations included in this class generally are splicing mutations, generating both aberrant and normal pro-tein. Example: 3272-26A>G (rs76151804, c.3140-26A>G), G576A (rs397517979, c.1227G>C) [6-8].

Class VI: Less stable. Class VI is the newest class. It is

associated with no stabilization of CFTR protein at the cell surface by decreasing in anchoring/retention of the protein or in deletion mutant that takes out the CFTR protein initiation codon. Example: 4428insGA (rs397508709, c.4296_4297insGA), Q1412X (rs397508702, c.4234C>T) [6-8].

CF patients with classes IV, V and VI are associated with less severe CF; so the atypical CF denomination term is used. A residual function is present in all these CF patients, with a qualitative or quantitative CFTR protein alteration, however the complete absence of disease is, thus, not possible.

3. SPECIFIC DRUGS THERAPY: ONE MUTATION CLASS, ONE DIFFERENT DRUG?

Wide genotypes combinations for CFTR mutations are possible, frequently producing complex alleles with different clinical and functional outcomes [6, 10, 33, 34]. Understand-ing these complex combinations seems a “magical way” to the geneticists, once such complexity consists in a boundary between the laboratory expertise and the clinical practice. Genetic counseling by CF expertise professionals is made necessary to cope with the increase of novel mutations in the

CFTR gene [29] and with the clinical variability among CF

patients with same genotypes.

Nowadays, the CF disease can be considered as a key-opportunity to foster personalized drug treatment. Each pa-tient receives a specific amount of enzymes [35]. Antibiotic therapy is dependent on patient´s colonization, multi-drug resistance microorganisms, weight, age and clinical condi-tions. Therefore, new studies are increasingly necessary to optimize antibiotic management in CF patients [36-38]. The same idea is important to bronchodilators and corticoids treatment [36, 37] and dietary balancing according to each patient´s condition to provide better clinical evolution [39]. In cases with the following comorbidities, specific treatment should be performed: diabetes melittus [40], osteoporo-sis/osteopenia [41, 42], nasal polyposis [43] and meconium ileus [44]. Physiotherapeutic techniques play an important

Fig. (1). A. CFTR mutation distribution. B. CFTR gene containing introns (yellow) and exons (brown). C. mRNA before maturation showing

the sequence that causes the different constituents of the mature CFTR protein, the introns are shown in (gray), and protein subunits in the second color Scheme D. D. CFTR in the membrane surface with its constituents. E. The blue arrow from left to right in the figure shows, respectively, (i) CFTR protein transportation at the cell flux to cell surface (blue arrow inside the cell), after synthesized (ii) the activity at cell surface as chloride channel, and (iii) details about the CFTR protein structure (green). CFTR, cystic fibrosis transmembrane regulator; MSD, membrane-spanning domains; R, regulatory domain; NBD, nucleotide-binding domain. (The color version of the figure is available in the electronic copy of the article).

A561E Æ class II 2183AA>GÆ class I I618T Æ class IV G85E Æ class II F508del Æ class II

G542X Æ class I R553X Æ class I 1584-18672pbA>G Æ class V 3120+1G>A Æ class I 3272-26A>G Æ class V D1152H Æ class IV W1282X Æ class I N1303K Æ class II A CFTR gene 5´ 5´ 3´3´ 1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14a14b 15 16 17a 18 19 20 21 22 23 24 P205SÆ class IV R334W Æ class IV 4428insGAÆ class IV or VI B 17b 5 5 33 Exon Intron 25 kilobases CFTR mRNA (primary strcture) C N C

CFTR protein in the cell

surface _{Cell surface}

D

R domain

MSD1 and MSD 2 NBD1 and NBD2

E

CHLORIDE

Fig. (2). Personalized genetic drug therapy by CFTR mutation class. First is shown the CFTR protein mechanisms since transcription to cell

surface anchoring and function. Defective CFTR protein is observed follow by class I, II, III and IV. Reduced CFTR protein function is ob-served follow by class V and VI. Class I: No production by no transcription. Class II: CFTR processing error. CFTR protein is degraded in endoplasmic reticulum. Class III: CFTR protein regulation with defect. CFTR mutations related with the R domain expression. Class IV: Conduction is altered by mutations related by membrane-spanning domains. Class V: Reduced amount of CFTR protein at cell surface. How-ever, normal activity is present. Class VI: Low CFTR function stability. CFTR, cystic fibrosis transmembrane regulator; red (X) indicates absence of CFTR protein by premature stop codon (class I), absence of CFTR protein at cell surface (class II) or absence of CFTR function (class III); black continuous arrows indicates normal chloride transport; black no continuous arrows indicates residual chloride transport. The number of CFTR protein at cell surface is related with CFTR gene expression.

role on the management of CF lung disease [45] and each CF patient should have their psychical limit analyzed. CF is a medicine compendium and personalized medicine is not a novelty for the clinical practice. Directive treatment should be performed to each CF patient.

Unlike clinical manifestation, in laboratory work and re-search we can form classification groups. CFTR mutations were put in six classes and each class is analyzed for its characteristics [10, 25-29, 46]. This is a short-term way to study CF pathology and it is not the true reality for CF carri-ers. CFTR mutation showed wide variability between two CF patients for the same genotype [33]. If we consider all geno-types possibilities, we are only a step away from personal-ized drug therapy for CF disease.

To facilitate, the studies analyze the correctors and the potentiators drugs for CFTR protein and for the CFTR classes considering one specific mutation. In cases with good results, the reproducibility is necessary to other mutations from the same class. Four main “types” of CF drugs are be-ing studied. The drugs have the objective of correctbe-ing, po-tentiating or stabilizing the CFTR protein or to enable the transcription of the CFTR gene in cases of premature stop codon. The CF disease has the special conditions to promote the mutation-targeted therapy, once the defects in a single gene lead to the defective proteins that cause CF disease [4].

The main studies of CF drugs could be analyzed as fol-low, according to CFTR mutation class:

Class I: Premature stop codon are included in this class

being no protein present. Read-through compounds, in cases of nonsense mutations, are tested in treatment of CF disease and other genetic disorders [47]. The main compounds in-cluded are the aminoglycoside antibiotics and PTC124 (Ata-luren; C15H9FN2O3) that over-read the premature stop codon,

permitting the translation until the normal stop codon.

Aminoglycoside antibiotics do not repair the RNA di-rectly but bind themselves to the ribosomes and cause the insertion of a near cognate amino-acyl tRNA into the ribo-somal A site. This process suppresses translational fidelity and allows the ribosome to read through the PTC and to pro-duce full-length protein [16].

As aminoglycoside antibiotics, tobramycin, gentamycin and amikacin exhibit read-through ability on PTCs and pref-erentially in absence of nonsense-mediated mRNA decay [16, 48].

PTC124 is a non-aminoglycoside with read-through function. Clinical trials have showed controversial informa-tion about PTC124 and CF clinical outcome. However, a recent phase III trial showed that PTC124 did not improve lung function in the overall population of nonsense-mutation CF patients who received this treatment; but it might be beneficial for patients not taking chronic inhaled tobramycin [49]. Furthermore, the literature shows that children with nonsense mutation CF, PTC124 can induce functional CFTR production by the high proportion of nasal epithelial cells expressing apical full-length CFTR protein [50].

Class II: No traffic problem can be solved with rescue

molecules, which are known as correctors. Correctors act by two mechanisms: (i) through stabilizing the protein native state with pharmacological chaperones that bind directly to the channel protein; and (ii) through altering the activity of the transcriptional, folding, or membrane trafficking machin-ery or blocking the degradation of partially folded, but func-tional CFTR at the endoplasmic reticulum or the plasma membrane by the so-called proteostasis regulators [1, 16]. The studied drugs are VX-809 (Lumacaftor; C24H18F2N2O5)

and VX-661 (C26H27F3N2O6).

Once the prevalent CFTR gene mutation is F508del, re-searches and clinical trials targeted to class II are important

Wt-CFTR

I

II

III

IV

V

VI

X

X

X

X

DEFECTIVE PROTEIN

REDUCED

PRODUCTION PROCESSING REGULATION CONDUCTANCE AMONT OF

FUNCTIONING PROTEIN

CELL SURFACE STABILITY

to CF disease. VX-809 function is not able to normalize the CFTR expression. Phase II trial showed improved sweat test, but no response of FEV1% (Forced expiratory volume in first

second of forced vital capacity) after 28 days of therapy in F508del homozygous patients [51]. However, a long-term therapy could improve the data and better understanding of CFTR expression.

Nowadays, studies showed that F508del-CFTR contains two folding defects. Studies on second site suppressor muta-tions and evolved sequences coupled to the F508del residue and instability of the NBD1-MSD2 (nucleotide binding do-mains 1 - membrane-spanning dodo-mains 2) interface [52, 53]. In this context, multidrug therapy with a combination of a NBD1 domain stabilizer and a NBD1-MSD2 interface stabi-lizer may be required to be effective as a therapy [54]. Con-ceivably, the parallel targeting of multiple conformational defects by separate small molecule correctors will allow wild type folding of the mutant protein [55] and obviate the need for a potentiator, such as the VX-770 (Ivacaftor; C24H28N2O3) [56-58]. A clinical trial phase II showed the

CFTR protein expression of 50% in CF patients undergoing VX-809/VX770 therapy [59]. This new highlight increased the possibility of clinical trial phase III, considering both drugs at the same time, by a long-term therapy [60].

Class III: No function CFTR protein is present to

pro-mote the pore opening. Drugs target to open the pore are known as potentiators. The first therapeutic agent considered to this therapy was the VX-770 target to G551D mutation [56]. Unfortunately, the class III mutation is very rare in many countries. The VX-770 promotes the channel opening and the passage of chloride. The FDA (US Food and Drug Administration) recently considered the VX-770 for eight other CFTR mutations: G178R (rs80282562, c.532G>A), S549N, S549R (rs121909005, rs121908757, c.1647T>G), G551S (rs121909013, c.1651G>A), G1244E (rs267606723, c.3731G>A), S1251N (rs74503330, c.3752G>A), S1255P (rs121909041, c.3763T>C) and G1349D (rs193922525, c. 4046G>A) [6-8].

Phase III trials oral VX-770 treatment for G551D CF pa-tients led to rapid and sustained improvements in FEV1%

and body weight, and to a reduction of sweat chloride and pulmonary exacerbations [61]. The same was not observed for F508del homozygous patients treated with VX-770, without previous corrector induction. For class II mutation, a potentiator alone is not able to correct the CFTR expression [62, 63].

Recently, CF patients with an FEV1 < 40% predicted

were enrolled to study the VX-770 therapy. Patients under-going VX-770 therapy had median FEV1 improved, median

weight improved, median inpatient IV antibiotic days de-clined from 23 to 0 d/y and median total IV treatment days decreased from 74 to 38 d/y. Changes in pulmonary function and IV antibiotic requirements were significant compared with control subjects. In this context, VX-770 was effective in G551D CF patients with severe pulmonary disease [64-66].

To improve the data, VX-770 was studied for multiple missense CFTR mutations related with mild phenotype, and a wide response was observed [67]. These in vitro studies

along with in vivo measures of residual CFTR function could help stratify CF patients who have different CFTR genotypes to determine the potential clinical benefit of VX-770.

Potentially, the VX-770 response reducing of underlying sinus disease measured by computed tomography scan was noted in G5551D CF patients, suggesting a disease modify-ing effect [61, 68]. The time to start the treatment can im-prove the study’s results.

Class IV: Less function occur. Two ways could be used

to correct this problem. First, we can increase the amount of CFTR at the cell surface by correctors, or increase the chan-nel opening by potentiators. If the protein amount or open channels increases, the chloride translocation will be better [1, 16]. Studies considering this class should be done to im-prove the literature about it.

Class V: Less CFTR protein production. The same

mechanisms of class V can be used. However, we can empa-thize the opening of the normal channel by potentiators or increase the CFTR amount by correctors. Another special factor that can be considered is the antisense oligonucleo-tides to silence the aberrant mRNA and improve the expres-sion of the normal allele [1, 16]. Approaches to correct class V CFTR mutation can be viewed in the literature and results are clear. For A455E mutation, co-transfection with the trun-cation mutant Δ264 CFTR rescued the mature C band by transcomplementation. Other potential compound therapy are correctors C3 and C4 that rescued A455E [69].

Class VI: Less CFTR protein stable at the cell surface.

The protein is present at the cell surface, although there is an accelerated turnover to protein degradation. To promote the anchoring/retention, stabilizers can be used as Rac1 signal, which promotes anchoring to actin cytoskeleton via NHERF1 (Na+/H+ Exchanger Regulatory Factor), for exam-ple, the HGF (Hepatic Growth Factor) can be considered. For this class, corrector and potentiator could be used pro-moting bigger amount of CFTR or better opening for the present channels [1, 16].

To speak about each class is a didactic way to solve a complex problem. To promote the treatment, it takes one drug to correct one class of CFTR mutation. However, to correct the CFTR mutation and normalize the CFTR protein expression is not simple. Multidrug should be used (Fig. 3). Modulation of CF disease by the CFTR mutation is intrinsic. Each new drug has in vitro provided conditions to trials, but the response is not good yet, clinical response are ambigu-ous, but optimist vision should be considered as the literature showed [1, 5, 16]. Again, each class mutation will have the specific personalized “new” drug therapy, considering the

CFTR mutation, drug response, and other facts not studied

yet, as the population ethnicity and modifier genes related with CF disease and drug function [5].

The complex mechanisms to treat the CF disease consid-ering the CFTR mutations can be viewed by the F508del mutation. To find the treatment to CF disease for F508del mutation is a fight that researchers all over the world are willing to face [60]. VX-809 showed promising results, but the amount and function of rescued F508del protein was not enough to provide a treatment. Therefore, the use of other correctors will be necessary. The correctors enable the way

Fig. (3). Personalized genetic therapy for cystic fibrosis disease. ER, endoplasmic reticulum; CFTR, cystic fibrosis transmembrane regulator.

Red (X) indicates absence of CFTR protein by premature stop codon (class I), absence of CFTR protein at cell surface (class II) or absence of CFTR function (class III); black continuous arrows indicates normal chloride transport; black no continuous arrows indicates accelerated turnover of CFTR protein. The number of CFTR protein at cell surface is related with CFTR gene expression; blue arrow show the way to have the complete CFTR expression considering specific drugs therapy. First for class I mutation, read through compounds should be used to perform the translation for the stop codon mutation. Second, for the first cell (class II) the corrector drug protects the CFTR protein to ER degradation, increasing activate CFTR at cell surface, and for second (class V) and third cells (class VI), the corrector promote increase amount of active CFTR. Third, for class VI mutation related with accelerated CFTR turn over, stabilizers compounds enables better CFTR anchoring and decrease turn over from cell surface. Fourth, for class III, potentiator drug increase the CFTR function acting in the domains related with the channel open. Fifth, for first cell class II mutation is show, and the potentiator should be used after the corrector drug admini-stration, for the second cell, class IV and V, the potentiator can improve the CFTR activity and to enable better chlorite passage, and for third cell, class VI mutation can be treated with potentiator that enable bigger amount of chlorite passage through the protein. Sixth, the active CFTR protein is shown in normal cell surface. [Adapted from Bell et al., 2014] [1].

to surface, but do not correct the protein. To correct the func-tion, a potentiator is necessary. A mutant protein showed another characteristic: once the retention/anchoring at cell surface is short, stabilizers will promote a better prognostic when used alongside other drugs [70]. One class mutation is not “corrected” by one drug, but by several. The cellular mechanisms in vitro are easier to understand, but the evalua-tion in vivo is not easy.

If the class I CFTR mutation is taken into account, the read-through compounds to over-read the premature stop

codon will be added. Multidrug maybe are the future for CF treatment. Each mutation class will receive a specific ther-apy.

4. DIAGNOSIS AND DRUGS USE: WHEN IS DRUG TREATMENT TO BE STABLISHED?

CF diagnosis has improved in the last years [71]. Sweat test has been widely applied. However, the sweat test is per-formed when clinical symptoms or positive neonatal screen-ing are present [71, 72]. The presence of a clinical symptom

II

No active CFTR protein by premature termination codon

Æ

CFTR MUTATION AND CELL EFFECT _{AND DRUG THERAPY}CELL EFFECT

II

II

II

V

V

Æ READ THROUGHT COMPOUNDS permits

the translation

To promote the folding of mutant CFTR protein Æ CORRECTORS compounds (no ER

x x _{x x} DR

V

V

VI

VI

protein Æ CORRECTORS compounds (no ER degradation)

CFTR retention/anchoring in cell surface Æ DR U G SQ UE M E TO

VI

VI

III

III

CFTR channel with no functional

x x x x x x x x x x x x ST AB I LZ ER TH E

II

II

IV

IV

III

III

In cases that CFTR protein is present x PR OT EI N

V

V

VI

VI

no perfect function Æ POTENTIATORS

is indicative that no normal/minor function of CFTR protein and organ disease are present. However, several countries around the world have promoted neonatal screening by the dosage of immunoreactive trypsinogen, in which patients are included in health programs to perform the CF diagnosis by the sweat test [73]. In this way, the diagnosis improved and the time for this is made shorter every day, with controver-sial results about the clinical evolution [74-76].

One important aspect related to this is the CFTR mutation screening. Several countries have improved the database on

CFTR mutations and a new era for the CF diagnosis by CFTR mutation screening has arisen [77-79]. The knowledge

of geographical distribution and number of patients that could benefit from the new drugs is well known, mainly in developed countries [80].

Thus, genetic therapy will be a choice of physicians, pa-tients, caregivers, health systems, and pharmacology indus-tries. The time to start the treatment is not a well-known fac-tor and studies should be performed to understand this facfac-tor for CF patients around the world.

5. IF THESE PATIENTS ARE NEWBORNS, COULD THE NEW DRUGS THERAPY BE USED?

This is an important question. One that remains without answer. The trial for Ivacaftor and G551D mutation showed mild alteration of the disease. No important lung function evaluation was achieved [81]. However, the patient´s age were high (> six years old) and previous lung diseases were observed. The new drugs are not responsible to modulate lung structure. After initial lung disease, the damage could not be recovered to healthy lung. We do not dispose, indeed, of excellent results from the trials, but considering newborns and old patients could be a way to determine the correct time to promote personalized therapy. Time is necessary to pro-mote better understanding of the new drugs functions in old-est patients and to enable their use in newborns. We are in the onset of a field and the options are wide. Studies will gradually show the correct way to treat newborns by the new drugs. However, the progression of lung disease [82] could be an “accelerator” to promote early intervention by the new drugs.

6. ELDERLY CYSTIC FIBROSIS PATIENTS: WHAT WE SHOULD DO?

Can elderly CF patients with severe/mild mutations have satisfactory response by new drugs therapy? It is another question without answer. Severe CFTR mutations are associ-ated with typical CF disease [10, 83]. The lung is the princi-pal affected organ causing highest comorbid and mortality. The reduction of lung function is a natural process; although in CF patients, the drop is higher than the normal [82]. It is not possible, to recover the lung function affected by the CF disease considering the structure damage. If a better quality of life and life expectancy is observed by new drugs therapy, it should be used [66, 81, 84]. However, we are dealing with two different CF patients´ population: (i) patients with early diagnosis by clinical symptomatology followed by positive sweat test: (ii) elderly CF patients with diagnosis by clinical aspect, as obstruction of the vas deferens, lung disease with-out important clinical manifestation, and sometimes

diagno-sis without a special cause in advanced age [22, 66]. The first group has generally CFTR mutation from class I, II and III; the second group is a part of a not well studied CF popula-tion, and CFTR mutation from class IV, V and VI are fre-quently present [22]. It will be necessary for the first group to promote CF personalized treatment, but without clinical importance, should the lung disease for the second group be treated by the new drugs? Studies considering this fact should be performed, although, studies with CF patients with class I, II and III should be performed first, considering, the fastest lung damage, associated with severe CFTR mutation and early age [83, 85].

7. A TYPICAL CYSTIC FIBROSIS DISEASE: IS THERE A SPECIFIC DRUG FOR EVERY SPECIFIC MUTATION?

In general population, no screened CF patients is present. Difficulty of molecular diagnosis of CF patients are frequent in atypical cases [86]. Atypical cases are associated generally with absence or partial CF clinical manifestation and CFTR expression. As mentioned before, the importance of the new drugs is not clear. Furthermore, the CFTR mutation screen-ing, in atypical CF disease is complex [86]. The complete sequencing should be performed to determine the CFTR mu-tation. In atypical CF disease, mutations of classes IV, V and VI are frequent [22, 86]. The CFTR mutation is present, but the necessity of the “common” medication is not well known, as the clinical outcomes [87]. In these cases, the fol-low-up of CF patients is performed and only the complica-tions are treated. If the personalized drug therapy enables better prognosis, it should be used - but not in all atypical CF patients. The idea of one mutation class, and one drug, oc-curs again, but the symptomatology should be clear. It is necessary to evaluate the pros and cons.

8. IMPROVING THE TREATMENT. IMPROVING THE COSTS.

Each study, each new drug, each trial has a price includ-ing CF disease [88, 89]. CF disease is the most frequent autosomal recessive monogenic disorder: if we consider the diagnosed CF population worldwide, 70,000 cases of CF can be found [90]. Mutation class can classify CF population in groups and the frequency of each mutation is different among ethnicities [91]. To study a non-common disorder, to promote better prognostic is important, but the costs are ele-vated [89]. Each step to put a new drug viable is high, and with the years passing by, the pharmaceutical industry should receive the money used as fast as possible.

The government should be prepared to pay for this treat-ment. The CF population need to have this treattreat-ment. The studies should continue. This fact in CF disease is a long history. As CF disease is a rare disorder, the public health system need to fight to improve the treatment to it. The gov-ernment, sometimes, has no idea about it. However, nowa-days, with the greater access of information, the CF is a known disease, and a new era is in front of us.

Clinical knowledge, laboratorial studies, CF societies and parents associations perform a promising way to improve the life expectancy and quality of life of the CF patients [92]. New drugs can be viable, as the VX-770 [81] and VX-809

[84], but they are very expensive for the CF patients [88, 93]. We need to think about how to promote progress, how to deal with the pharmaceutical industry and life hope, at the same time, respecting with equal values: two different rights? These questions are important, but are lost/forgotten when we see the information about a new treatment. Every-thing has a cost, but we need to possibility the treatment cor-rectly considering the patients financial condition. In addi-tion, the amount received by the treatment should be propor-tional to the gains and losses.

Double response in CF disease is clear in the literature. The drugs can be affordable [94] or unaffordable [95]. The drugs are in the laboratory gates. We need to think about this issue. We need “to run” to promote the “conscious hope” - to speak to CF patients the truth. The genetic counseling is pri-mordial to give the correct information to CF patients. The drugs have a price, but considering the patient´s hope is nec-essary [88].

9. NO MORE FICTION. HOWEVER, IS THE PER-SONALIZED DRUG THERAPY A REALITY FOR ALL CYSTIC FIBROSIS PATIENTS?

For the personalized drugs treatment we need to consider important aspects related with the CF disease. Gradually, the boundaries around the CF should be overtaken to improve and enable the use of the new drugs.

First, the correct diagnosis of all CF patients should be performed, in this context, typical and atypical CF patients should be enrolled. Another aspect to be taken into consid-eration are the patients without diagnosis. CF subjects with residual CFTR function are missing for the CF diagnosis worldwide. To find and to perform the diagnosis of these patients is an issue that should be considered to enable the correct treatment. In cases with borderline values in the sweat test, complementary tests could be used, as the evaporimeter [96].

Second, the wide spectrum of CFTR mutations are well known. However, to screen CFTR mutations, for all patients is not a reality. New molecular technologies can be used. The price is lower every day, but the costs are high yet for the major part of the CF patients, hospitals, CF centers and countries. Each mutation has an activity on CFTR function. In addition, to know the importance, expression and function of each mutation can be a boundary. If the complete CFTR mutation screening was performed, variants without estab-lished condition were screened; then clinical studies should be performed.

Considering the CFTR mutation of each population [30] can enable the direct screening for specific mutation with high frequency for this population. However, the F508del should be the first mutation screened, once it is the most common CFTR mutation worldwide [97].

If the screening is to be done, unknown CFTR mutations should be analyzed in vitro and in vivo to enable to know the correct function of this variant and the association with the CFTR expression. To do so is to thread a troubled track. In

vitro response is not the same than in vivo. The low number

of CF patients in rare CFTR mutation variants is a problem to association studies. Therefore functional tests should be

performed. A further problem is to determine the correct and potential alteration in the function of CFTR residual muta-tion, if the complete CFTR screening is done. Variants com-binations can be the responsible factor for CF disease. How-ever, isolated variants can show no association with the dis-ease.

Drugs will be mutation specific or class mutation spe-cific. However, it is necessary to study the drugs activity to each mutation before its implementation for CF patients. Transversal clinical data studies of drug response are not the best way to approach this issue. Spirometry, clinical scores, sweat test values, body mass index and another clinical data are associated with clinical severity, but show wide variation and are not associated directly with the CFTR activity after drug use.

Patients currently tested to new drugs therapy are CF pa-tients older than six years old and with lung deterioration. Therefore, showing the drug response can be a problem, con-sidering the already established disease. To overcome these barriers, in vitro studies can be used. Groups around the world are studying the drug response by rectal biopsies [28] and cell culture from lungs by Ussing chamber [1], but other tools should be better understood before being implemented in CFTR expression studies, as it is the case with the nasal potential difference.

We need to know the total CFTR channel activity by the amount of CFTR as well as CFTR function and activity. This can be evaluated by the formula - total CFTR channel activ-ity = [N CFTR channel in the cells x (N CFTR channels at the cell surface - N CFTR channels internalized from sur-face) x Po - open probability x conductance)] [1]. This is a primordial fact that should be included in the drug function studies.

CONCLUSION

Personalized genetic medicine for CF disease is a long date dream. Laboratorial conditions showed good drug re-sponse for CFTR mutations included in all classes, although clinical trials are conflicting about the results. It is well known that the first idea - one drug, one CFTR class muta-tion - is not correct. The CF disease can be more difficult to treat by personalized genetic medicine than the first reports showed. A long distance should also be covered until the drugs are available for use. The most prevalent mutation, F508del, showed good results for personalized medicine, but to achieve this therapy is not easy yet. It is important to con-sider the CFTR mutation screening, the correct CF diagnosis, the drugs price [98] as well as the studies limitation in vivo and in vitro. Indeed, the drugs are not a fiction, but the same cannot be said for the treatment by them. We could support an optimistic point of view Trying to conciliate the CF pa-tient’s disease severity, family expectation, political limita-tions, CF centers expertise, drugs distribution and price. Maybe this will be the newest limitation for the "dream" of CF patients. The space between fiction and reality in the CF disease treatment is a short place for dreams. However, the dream of each CF patient should be considered before pro-moting the conciliation between scientific studies and clini-cal trials. Before speaking about a possible treatment for a patient, to think in the possibility of turning a dream into

nightmare should be evaluated. We are far away from the goal, but walking forward to meet it.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flict of interest.

ACKNOWLEDGEMENTS

Authors thank: (i) http://www.laboratoriomultiusuario. com.br/ for work contribution; (ii) FAPESP - 2011/12939-4 to provide research funds to FALM; Cystic Fibrosis Group from State University of Campinas - Antonio Fernando Ribeiro, Maria Angela Gonçalves de Oliveira Ribeiro, Carla Cristina de Souza Gomes, Gabriel Hessel, Maria de Fatima Servidoni, Adyleia Aparecida Dalbo Toro Contrera, Carlos Emilio Levy, Luciana Cardoso Bonadia, Tais Daiene Russo Hortencio, Katia Alberto Aguiar, Renan Mauch, Aline Cris-tina Gonçalves, Andreia Bieger, Larissa Furlan, Lucas Moraes Brioschi, Eulalia Sakano and Roberto José Negrão Nogueira.

REFERENCES

[1] Bell SC, De Boeck K, Amaral MD. New pharmacological ap-proaches for cystic fibrosis: Promises, progress, pitfalls. Pharmacol Ther 2014; 145: 19-34.

[2] Fanen P, Wohlhuter-Haddad A, Hinzpeter A. Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies. Int J Biochem Cell Biol 2014; 52: 94-102.

[3] Lane MA, Doe SJ. A new era in the treatment of cystic fibrosis. Clin Med 2014; 14(1): 76-8.

[4] Pettit RS, Fellner C. CFTR modulators for the treatment of cystic fibrosis. P T 2014; 39(7): 500-11.

[5] Amaral MD. Novel personalized therapy for cystic fibrosis: treat-ing the basic defect in all patients. J Intern Med 2014. In print. [6] www.CFTR.2.org

[7] http://www.ncbi.nlm.nih.gov/gene/1080 [8] http://www.genet.sickkids.on.ca/app

[9] Lommatzsch ST, Aris R. Genetics of cystic fibroosis. Semin Respir Crit Care Med 2009; 30(5): 531-8.

[10] Fanen P, Wohlhuter-Haddad A, Hinzpeter A. Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies. Int J Biochem Cell Biol 2014; 52: 94-102.

[11] Davies LA, Nunez-Alonso GA, McLachlan G, et al. Aerosol deliv-ery of DNA/liposomes to the lung for cystic fibrosis gene therapy. Hum Gene Ther Clin Dev 2014; 25(2): 97-107.

[12] Suk JS, Kim AJ, Trehan K, et al. Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier. J Control Rel 2014; 178: 8-17.

[13] Gill DR, Hyde SC. Delivery of genes into the CF airway. Thorax 2014; 69(10): 962-4.

[14] Clancy JP, Jain M. Personalized medicine in cystic fibrosis: dawn-ing of a new era. Am J Respir Crit Care Med 2012; 186: 593-7. [15] Wilschanski M. Novel therapeutic approaches for cystic fibrosis.

Discov Med 2013; 15(81): 127-33.

[16] Ikpa PT, Bijvelds MJ, de Jonge HR. Cystic fibrosis: toward per-sonalized therapies. Int J Biochem Cell Biol 2014; 52: 192-200. [17] Kerem B, Rommens JM, Buchanan JA, et al. Identification of the

cystic fibrosis gene: genetic analysis. Science 1989; 245(4922): 1073-80.

[18] Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245(4922): 1066-73.

[19] Rommens JM, Zengerling-Lentes S, Kerem B, et al. Physical local-ization of two DNA markers closely linked to the cystic fibrosis lo-cus by pulsed-field gel electrophoresis. Am J Hum Genet 1989; 45(6): 932-41.

[20] Bieger AM, Marson FA, Bertuzzo CS. Prevalence of ΔF508 muta-tion in the cystic fibrosis transmembrane conductance regulator gene among cystic fibrosis patients from a Brazilian referral center. J Pediatr (Rio J) 2012; 88(6): 531-4.

[21] De Boeck K, Zolin A, Cuppens H, et al. The relative frequency of CFTR mutation classes in European patients with cystic fibrosis. J Cyst Fibros 2014; 13(4): 403-9.

[22] Bonadia LC, Marson FAL, Ribeiro JD, et al. CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease. Gene 2014; 540(2): 183-90.

[23] Rohlfs EM, Zhou Z, Heim RA, et al. Cystic fibrosis carrier testing in an ethnically diverse US population. Clin Chem 2011; 57(6): 841-8.

[24] Tsui LC, Dorfman R. The cystic fibrosis gene: a molecular genetic perspective. Cold Spring Harb Perspect Med 2013; 3(2): a009472. [25] Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med

2005; 352: 1992-2001.

[26] Kreindler JL. Cystic fibrosis: exploiting its genetic basis in the hunt for new therapies. Pharmacol Ther 2010; 125(2): 219-29.

[27] Rogan MP, Stoltz DA, Hornick DB. Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and op-portunities formutation-specific treatment. Chest 2011; 139(6): 1480-90.

[28] Sousa M, Servidoni MF, Vinagre AM, et al. Measurements of CFTR-mediated Cl- secretion in human rectal biopsies constitute a robust biomarker for cystic fibrosis diagnosis and prognosis. PLoS One 2012; 7(10): e47708.

[29] Derichs, N. Targeting a genetic defect: cystic fibrosis transmem-brane conductance regulator modulators in cystic fibrosis. Eur Respir Rev 2013; 22(127): 58-65.

[30] De Boeck K, Zolin A, Cuppens H, et al. The relative frequency of CFTR mutation classes in European patients with cystic fibrosis. J Cyst Fibros 2014; 13(4): 403-9.

[31] Radlović N. Cystic fibrosis. Srp Arh Celok Lek 2012; 140(3-4): 244-9.

[32] Zhang L, Button B, Gabriel SE, et al. CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium. PLoS Biol 2009; 7(7): e1000155.

[33] El-Seedy A, Girodon E, Norez C, et al. CFTR mutation combina-tions producing frequent complex alleles with different clinical and functional outcomes. Hum Mutat 2012; 33(11): 1557-65.

[34] Poulou M, Fylaktou I, Fotoulaki M, et al. Cystic fibrosis genetic counseling difficulties due to the identification of novel mutations in the CFTR gene. J Cyst Fibros 2012; 11(4): 344-8.

[35] Li L, Somerset S. Digestive system dysfunction in cystic fibrosis: Challenges for nutrition therapy. Dig Liver Dis 2014; 46(10): 865-74.

[36] Chmiel JF, Konstan MW, Elborn JS. Antibiotic and anti-inflammatory therapies for cystic fibrosis. Cold Spring Harb Per-spect Med 2013; 3(10): a009779.

[37] Mogayzel PJ Jr, Naureckas ET, Robinson KA, et al. Cystic fibrosis pulmonary guidelines. Chronic medications for maintenance of lung health. Am J Respir Crit Care Med 2013; 187(7): 680-9. [38] Waters VJ, Ratjen FA. Is there a role for antimicrobial stewardship

in cystic fibrosis? Ann Am Thorac Soc 2014; 11(7): 1116-9. [39] Culhane S, George C, Pearo B, Spoede E. Malnutrition in cystic

fibrosis: a review. Nutr Clin Pract 2013; 28(6): 676-83.

[40] Middleton PG, Matson AG, Robinson PD, et al. Cystic Fibrosis Related Diabetes: Potential pitfalls in the transition from paediatric to adult care. Paediatr Respir Rev 2014; 15(3): 281-4.

[41] Bianchi ML, Colombo C, Assael BM, et al. Treatment of low bone density in young people with cystic fibrosis: a multicentre, prospec-tive, open-label observational study of calcium and calcifediol fol-lowed by a randomised placebo-controlled trial of alendronate. Lancet Respir Med 2013; 1(5): 377-85.

[42] Sheikh S, Gemma S, Patel A. Factors associated with low bone mineral density in patients with cystic fibrosis. J Bone Miner Me-tab 2014. (In print).

[43] Mainz JG, Koitschev A. Pathogenesis and management of nasal polyposis in cystic fibrosis. Curr Allergy Asthma Rep 2012; 12(2): 163-74.

[44] Colombo C, Ellemunter H, Houwen R, et al. Guidelines for the diagnosis and management of distal intestinal obstruction syn-drome in cystic fibrosis patients. J Cyst Fibros 2011; 10(2): S24-8. [45] Rand S, Hill L, Prasad SA. Physiotherapy in cystic fibrosis:

opti-mising techniques to improve outcomes. Paediatr Respir Rev 2013; 14(4): 263-9.

[46] Clancy JP. CFTR potentiators: not an open and shut case. Sci Transl Med 2014; 6(246): 246fs27.

[47] Keeling KM, Xue X, Gunn G, Bedwell DM. Therapeutics based on stop codon readthrough. Annu Rev Genomics Hum Genet 2014; 15:371-94.

[48] Altamura N, Castaldo R, Finotti A, et al. Tobramycin is a suppres-sor of premature termination codons. J Cyst Fibros 2013; 12(6): 806-11.

[49] Kerem E, Konstan MW, De Boeck K, et al. Ataluren for the treat-ment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Respir Med 2014; 2(7): 539-47.

[50] Sermet-Gaudelus I, Boeck KD, Casimir GJ, et al. Ataluren (PTC-124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care Med 2010; 182(10): 1262-72. [51] Clancy JP, Rowe SM, Accurso FJ, et al. Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation. Thorax 2012; 67(1): 12-8.

[52] Mendoza JL, Schmidt A, Li Q, et al. Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved se-quences. Cell 2012; 148: 164-74.

[53] Rabej WM, Bossard F, Xu H, et al. Correction of both NBD1 ener-getics and domain interface is required to restore DeltaF508 CFTR folding and function. Cell 2012; 148: 150-63.

[54] Okiyoneda T, Veit G, Dekkers JF, et al. Mechanism-based correc-tor combination rescorrec-tores DeltaF508-CFTR folding and function. Nat Chem Biol 2013; 9: 444-54.

[55] Phuan PW, Veit G, Tan J, et al. Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-CFTR correctors that augment VX-809 maximal efficacy. Mol Pharmacol 2014; 86(1): 42-51.

[56] Wainwright CE. Ivacaftor for patients with cystic fibrosis. Expert Rev Respir Med 2014; 22: 1-6.

[57] Kopeikin Z, Yuksek Z, Yang HY, Bompadre SG. Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels. J Cyst Fibros 2014; 13(5): 508-14. [58] Veit G, Avramescu RG, Perdomo D, et al. Some gating

potentia-tors, including VX-770, diminish ΔF508-CFTR functional expres-sion. Sci Transl Med 2014; 6(246): 246ra97.

[59] www.CFF.org

[60] Cholon DM, Quinney NL, Fulcher ML, et al. Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fi-brosis. Sci Transl Med 2014; 6(246): 246ra96.

[61] Rowe Sm, Verkman AS. Cystic fibrosis transmembrane regulator correctors and potentiators. Cold Spring Harb Perspect Med 2013; 3(7): pii a0061.

[62] Flume PA, Liou TG, Borowitz DS, et al. Ivacaftor in subjects with cystic fibrosis who are homozygous for the F508del-CFTR muta-tion. Chest 2012; 142: 718-24.

[63] Liu X, Dawson DC. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiators Protect G551D but Not ΔF508 CFTR from Thermal Instability. Biochemistry 2014. (In print). [64] Deeks ED. Ivacaftor: a review of its use in patients with cystic

fibrosis. Drugs 2013; 73(14): 1595-604.

[65] Barry PJ, Plant BJ, Nair A, et al. Effects of Ivacaftor in cystic fibrosis patients carrying the G551D mutation with severe lung dis-ease. Chest 2014. (In print).

[66] Whiting P, Al M, Burgers L, et al. Ivacaftor for the treatment of patients with cystic fibrosis and the G551D mutation: a systematic review and cost-effectiveness analysis. Health Technol Assess 2014; 18(18): 1-106.

[67] Van Goor F, Yu H, Burton B, Hoffman BJ. Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function. J Cyst Fibros 2014; 13(1): 29-36.

[68] Sheikh SI, Long FR, McCoy KS, et al. Ivacaftor improves appear-ance of sinus disease on computerized tomography in cystic fibro-sis patients with G551D mutation. Clin Otolaryngol 2014. In print. [69] Cebotaru L, Rapino D, Cebotaru V, Guggino WB. Correcting the

cystic fibrosis disease mutant, A455E CFTR. PLoS One 2014; 9(1): e85183.

[70] Kirby EF, Heard AS, Wang XR. Enhancing the potency of F508del correction: a multi-layer combinational approach to drug discovery for cystic fibrosis. J Pharmacol Clin Toxicol 2013; 1(1): 1007. [71] Padman R, Flathers K, Passi V. Cystic fibrosis infant care

chal-lenges in diagnosis and management in the era of newborn screen-ing. Del Med J 2012; 84(5): 149-55.

[72] Warwick G, Elston C. Improving outcomes in patients with cystic fibrosis. Practitioner 2011; 255(1742): 29-32.

[73] Wagener JS, Zemanick ET, Sontag MK. Newborn screening for cystic fibrosis. Curr Opin Pediatr 2012; 24(3): 329-35.

[74] Maclean JE, Solomon M, Corey M, Selvadurai H. Cystic fibrosis newborn screening does not delay the identification of cystic fibro-sis in children with negative results. J Cyst Fibros 2011; 10(5): 333-7.

[75] Van Devanter DR, Pasta DJ, Konstan MW. Improvements in lung function and height among cohorts of 6-year-olds with cystic fibro-sis from 1994 to 2012. J Pediatr 2014;165(6): 1091-7.

[76] Vernooij-van Langen AM, Gerzon FL, Loeber JG, et al. Differ-ences in clinical condition and genotype at time of diagnosis of cystic fibrosis by newborn screening or by symptoms. Mol Genet Metab 2014; 113(1-2): 100-4. http://www.ncbi.nlm.nih.gov/pub- med?term=Dankert-RoelseJE%5BAuthor%5D&cauthor=true& cauthor_uid=25077434.

[77] Lilley M, Christian S, Hume S, et al. Newborn screening for cystic fibrosis in Alberta: Two years of experience. Paediatr Child Health 2010; 15(9): 590-4.

[78] Earley MC, Laxova A, Farrell PM, et al. Implementation of the first worldwide quality assurance program for cystic fibrosis multi-ple mutation detection in population-based screening. Clin Chim Acta 2011; 412(15-16): 1376-81.

[79] Cordovado SK, Hendrix M, Greene CN, et al. CFTR mutation analysis and haplotype associations in CF patients. Mol Genet Me-tab 2012; 105(2): 249-54.

[80] Boyle MP, De Boeck K. A new era in the treatment of cystic fibro-sis: correction of the underlying CFTR defect. Lancet Respir Med 2013; 1(2): 158-63.

[81] McKone EF, Borowitz D, Drevinek P, et al. Long-term safety and efficacy of ivacaftor in patients with cystic fibrosis who have the Gly551Asp-CFTR mutation: a phase 3, open-label extension study (PERSIST). Lancet Respir Med 2014; 2(11): 902-10.

[82] Li Z, Sanders DB, Rock MJ, et al. Regional differences in the evolution of lung disease in children with cystic fibrosis. Pediatr Pulmonol 2012; 47(7): 635-40.

[83] Ferril GR, Nick JA, Getz AE, et al. Comparison of radiographic and clinical characteristics of low-risk and high-risk cystic fibrosis genotypes. Int Forum Allergy Rhinol 2014. In print.

[84] Boyle MP, Bell SC, Konstan MW, et al. A CFTR corrector (lu-macaftor) and a CFTR potentiator (ivacaftor) for treatment of pa-tients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial. Lancet Respir Med 2014; 2(7): 527-38.

[85] Lerín M, Prados C, Martínez MT, et al. Cystic fibrosis in adult age. Rev Clin Esp 2014; 214(6): 289-95.

[86] Dal'Maso VB, Mallmann L, Siebert M, et al. Diagnostic contribu-tion of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis. J Bras Pneumol 2013; 39(2): 181-9. [87] Schram CA. Atypical cystic fibrosis: identification in the primary

care setting. Can Fam Physician 2012; 58(12): 1341-5, e699-704. [88] Kaiser J. Personalized medicine. New cystic fibrosis drug offers

hope, at a price. Science 2012; 335(6069): 645.

[89] Leonard A, Leal T, Lebecque P. Mucoviscidosis: CFTR mutation-specific therapy: a ray of sunshine in a cloudy sky. Arch Pediatr 2013; 20(1):63-73.

[90] Jennings MT, Riekert KA, Boyle MP. Update on key emerging challenges in cystic fibrosis. Med Princ Pract 2014; 23(5): 393-402.

[91] Zvereff VV, Faruki H, Edwards M, Friedman KJ. Cystic fibrosis carrier screening in a North American population. Genet Med 2014; 16(7): 539-46.

[92] von der Hardt H, Schwarz C, Ullrich G. Adults with cystic fibrosis. It's not just about longevity. Bundesgesundheitsblatt Gesundheits-forschung Gesundheitsschutz 2012; 55(4): 558-67.

[93] The Lancet Respiratory Medicine. Surging forwards with cystic fibrosis care. Lancet Respir Med 2013; 1(2): 91.

[94] Bilton D. Personalised medicine in cystic fibrosis must be made affordable. Paediatr Respir Rev 2014; 15(1): 6-7.

[95] Balfour-Lynn IM. Personalised medicine in cystic fibrosis is unaf-fordable. Paediatr Respir Rev 2014; 15(1): 2-5.

[96] Quinton P, Molyneux L, Ip W, et al. β-adrenergic sweat secretion as a diagnostic test for cystic fibrosis. Am J Respir Crit Care Med 2012; 186(8): 732-9.

[97] Marson FA, Bertuzzo CS, Ribeiro MA, et al. Screening for F508del as a first step in the molecular diagnosis of cystic fibrosis. J Bras Pneumol 2013; 39(3): 306-16.

[98] Cooney D. New cystic fibrosis drug paves the way for orphan diseases. Lancet Respir Med 2013; 1(2): 104.

Received: September 16, 2014 Revised: October 20, 2014 Accepted: November 14, 2014