ECFS standards of care on CFTR-related disorders: Towards a comprehensive program for affected individuals.

After three publications defining an updated guidance on the diagnostic criteria for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (pwCFTR-RDs), establishing its relationship to CFTR-dysfunction and describing the individual disorders, this fourth and last paper in the series addresses some critical challenges facing health care providers and pwCFTR-RD. Topics included are: 1) benefits and obstacles to collect data from pwCFTR-RD are discussed, together with the opportunity to integrate them into established CF-registries; 2) the potential of infants designated CRMS/CFSPID to develop a CFTR-RD and how to communicate this information; 3) a description of the challenges in genetic counseling, with particular regard to phenotypic variability, unknown long-term evolution, CFTR testing and pregnancy termination 4) a proposal for the assessment of potential barriers to the implementation and dissemination of the produced documents to health care professionals involved in the care of pwCFTR-RD and a process to monitor the implementation of the CFTR-RD recommendations; 5) clinical trials investigating the efficacy of CFTR modulators in CFTR-RD and how endpoints and outcomes might be adapted to the heterogeneity of these disorders.

ECFS standards of care on CFTR-related disorders: Diagnostic criteria of CFTR dysfunction.

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD : (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.

Do common in silico tools predict the clinical consequences of amino-acid substitutions in the CFTR gene?

Computational methods are used to predict the molecular consequences of amino-acid substitutions on the basis of evolutionary conservation or protein structure, but their utility in clinical diagnosis or prediction of disease outcome has not been well validated. We evaluated three popular computer programs, namely, PANTHER, SIFT and PolyPhen, by comparing the predicted clinical outcomes for a group of known CFTR missense mutations against the diagnosis of cystic fibrosis (CF) and clinical manifestations in cohorts of subjects with CF-disease and CFTR-related disorders carrying these mutations. Owing to poor specificity, none of tools reliably distinguished between individual mutations that confer CF disease from mutations found in subjects with a CFTR-related disorder or no disease. Prediction scores for CFTR mutations derived from PANTHER showed a significant overall statistical correlation with the spectrum of disease severity associated with mutations in the CFTR gene. In contrast, PolyPhen- and SIFT-derived scores only showed significant differences between CF-causing and non-CF variants. Current computational methods are not recommended for establishing or excluding a CF diagnosis, notably as a newborn screening strategy or in patients with equivocal test results.

Sweat gland bioelectrics differ in cystic fibrosis: a new concept for potential diagnosis and assessment of CFTR function in cystic fibrosis.

BACKGROUND: For nearly 50 years the diagnosis of cystic fibrosis (CF) has depended on measurements of sweat chloride concentration. While the validity of this test is universally accepted, increasing diagnostic challenges and the search for adequate biomarker assays to support curative-orientated clinical drug trials have created a new demand for accurate, reliable and more practical CF tests. A novel concept is proposed that may provide a more efficient real-time method for assessing CFTR function in vivo. METHODS: Cholinergic and beta-adrenergic agonists were iontophoresed to stimulate sweating. The bioelectric potential from stimulated sweat glands (SPD) was measured in vivo using a standard ECG electrode applied to the skin surface. SPD and sweat chloride concentrations were compared in cohorts predicted to express a range of CFTR function as presented by healthy controls (HC), heterozygotes (Hz), pancreatic sufficient (CFPS) and pancreatic insufficient patients with CF (CFPI). RESULTS: The median SPD was hyperpolarized in patients with CF compared with control subjects (-47.4 mV vs -14.5 mV, p<0.001). In distinguishing between control and CF subjects, SPD (area under receiver operator curve (AUC) = 0.997) was similar to sweat chloride concentration (AUC = 0.986). Sequential cholinergic/beta-adrenergic sweat stimulation dramatically depolarised the SPD in patients with CF (p<0.001) but had no effect in control subjects (p = 0.6) or on the sweat chloride concentration in either group (p>0.5). Furthermore, the positive SPD response was larger in CFPI than in CFPS subjects (p = 0.04). CONCLUSION: These results support the concept that skin surface voltages arising from stimulated sweat glands can be exploited to assess expressed CFTR function in vivo and may prove to be a useful diagnostic tool.

Does the Macroduct collection system reliably define sweat chloride concentration in subjects with intermediate results?

OBJECTIVES: The sweat test remains the current diagnostic gold standard for CF disease. Many CF testing centres have switched from the Gibson and Cooke to the Macroduct. Since the validity and sensitivity of Macroduct has not been tested in patients with intermediate sweat chloride concentrations, we compared both methods simultaneously including subjects expected to have intermediate results. DESIGN AND METHODS: We prospectively evaluated controls, obligate heterozygotes, patients with CF and with an uncertain diagnosis of CF (congenital absence of the vas deferens, pancreatitis and sinopulmonary disease). RESULTS: We assessed 82 subjects (3.7-60.1 years); 14 healthy controls, 7 obligate heterozygotes, 20 CF (15 pancreatic insufficient, 5 pancreatic sufficient), and 41 with unproven diagnosis. Mean test difference was close to 0 (95% CI+/-20 mmol/L) and test values were highly correlated (r=0.93, p < or =0.0001). Discrepancies between the two testing methods occurred in 22% of subjects. CONCLUSION: Sweat chloride measured by Macroduct highly correlates with Gibson and Cooke for concentrations in all ranges, including the intermediate range. This study reveals the limitations of sweat testing for excluding a diagnosis of CF since 38% of subjects had intermediate range results.

Amino acid transport in the renal proximal tubule.

In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na+, Cl- and K+) or use an H+-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects.

Inhibition of amiloride-sensitive epithelial Na(+) absorption by extracellular nucleotides in human normal and cystic fibrosis airways.

Cystic fibrosis (CF) airway epithelia are characterized by enhanced Na(+) absorption probably due to a lack of downregulation of epithelial Na(+) channels by mutant CF transmembrane conductance regulator. Extracellular nucleotides adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) have been shown to activate alternative Ca(2+)-dependent Cl(-) channels in normal and CF respiratory epithelia. Recent studies suggest additional modulation of Na(+) absorption by extracellular nucleotides. In this study we examined the role of mucosal ATP and UTP in regulating Na(+) transport in native human upper airway tissues from patients with 16 patients with CF and 32 non-CF control subjects. To that end, transepithelial voltage and equivalent short-circuit current (I(SC)) were assessed by means of a perfused micro-Ussing chamber. Mucosal ATP and UTP caused an initial increase in lumen-negative I(SC) that was followed by a sustained decrease of I(sc) in both non-CF and CF tissues. The amiloride-sensitive portion of I(SC) was inhibited significantly in normal and CF tissues in the presence of either ATP or UTP. Both basal Na(+) transport and nucleotide-dependent inhibition of amiloride-sensitive I(SC) were significantly enhanced in CF airways compared with non-CF. Nucleotide-mediated inhibition of Na(+) absorption was attenuated by pretreatment with the Ca(2+)-adenosine triphosphatase inhibitor cyclopiazonic acid but not by inhibition of protein kinase C with bisindolylmaleimide. These data demonstrate sustained inhibition of Na(+) transport in non-CF and CF airways by mucosal ATP and UTP and suggest that this effect is mediated by an increase of intracellular Ca(2+). Because ATP and UTP inhibit Na(+) absorption and stimulate Cl(-) secretion simultaneously, extracellular nucleotides could have a dual therapeutic effect, counteracting the ion transport defect in CF lung disease.

Diadenosine polyphosphates activate a Ca(2+)-dependent K(+)-conductance in porcine aortic smooth muscle cells via P2-purinoceptors.

Effects of the diadenosine polyphosphates P(1),P(3)-diadenosine triphosphate (Ap3A), P(1),P(4)-diadenosine tetraphosphate (Ap4A), P(1),P(5)-diadenosine pentaphosphate (Ap5A) and P(1), P(6)-diadenosine hexaphosphate (Ap6A) and of adenosine, ATP, ADP, AMP, UTP on smooth muscle cells from porcine aorta were examined. Membrane voltages and cellular conductances were measured in the slow whole cell configuration of the patch clamp technique. All four diadenosine polyphosphates, adenosine, AMP and ADP predominantly hyperpolarized membrane voltages with only occasional transient initial depolarizations whereas ATP and UTP led to sustained depolarizations. All four diadenosine polyphosphates increased cellular conductances. The effects of Ap5A on membrane voltages were almost completely inhibited by the putative P2-purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 micromol/l) and only partially reduced by the putative A(2)-purinoceptor antagonist 3,7-dimethyl-1-propragyl-xanthine (DMPX, 10 micromol/l) or the Ap4A-receptor antagonist diinosine pentaphosphate (Ip5I, 10 micromol/l). The adenosine-induced hyperpolarization was partially reduced by the putative A(1)-purinoceptor antagonist 8-cyclopentyl-1,3-dipropargylxanthine (DPCPX, 0.1 micromol/l) or by DMPX while PPADS or Ip5I were without effects. Ap5A-induced hyperpolarizations were inhibited by Ba(2+) and clotrimazole but not by glibenclamide. We conclude that diadenosine polyphosphates activate predominantly a Ca(2+)-dependent K(+)-conductance in smooth muscle cells obtained from porcine aorta most likely mediated via P2Y-purinoceptors and possibly partially also by Ap4A receptors.

Na(+)-dependent and -independent amino acid transport systems in immortalized human kidney epithelial cells derived from the proximal tubule.

In the proximal tubule Na(+)-dependent (SDAT) and Na(+)-independent (SIAT) amino acid (AA) transporters are present. The effects of neutral, basic, and acidic AA on membrane voltage (Vm) of immortalized human kidney epithelial (IHKE-1) cells derived from the proximal tubule were examined using the slow whole-cell patch-clamp technique. In the presence of Na+ AA depolarized Vm in a concentration-dependent manner (0.05-5 mM) with Asp = Arg = Glu = 2Cys < Pro = Leu < Phe = AIB = Ala = Pro = Asn < Gly. In the absence of extracellular Na+ a decreased depolarization was seen with most neutral AA (Ala, Pro, Asn, Gly, Phe, and Leu), and the depolarization was increased with Asp, Glu, Arg, and 2Cys (1 mM each). In the absence of Na+ and a reduction in Cl- (5 mM) the depolarization by Arg was reduced. Unlike that predicted for transport by system b0,+ which exchanges neutral against dibasic amino acids, Leu does not hyperpolarize but depolarize Vm of IHKE-1 cells in the absence of extracellular Na+. After removal of Na+ (0 mM) and a reduction in Cl- (5 mM) in the extracellular solution, Leu or Glu hyperpolarized Vm, indicating that IHKE-1 cells possess two different SIAT systems, one Cl(-)-dependent and similar to system b0,+ and one novel Cl(-)-dependent system, which might be a Cl-/AA exchanger and can be blocked by the Cl(-)-channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (10 microM) and 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (50 microM). B system-related AA transporters might be responsible for the C(-)-independent SIAT, since we were able to detect its signal by Northern blot analysis.