Sweat Chlorides in Salt-Deprived Cystic Fibrosis Heterozygotes

Sweat chlorides of 10 sets of parents of children with cystic fibrosis and 11 controls were studied in an attempt to develop a test for the diagnosis of cystic fibrosis heterozygotes by subjecting both the parents and controls to a low sodium diet and comparing sweat chloride values as the diet progressed. It was hoped that the sweat chloride levels of the parents, the heterozygotes, would remain stationary throughout the diet, since their children, the homozygotes, reveal this finding under similar conditions of salt deprivation. The sweat chloride levels of the controls, because of effects of aldosterone, were expected to decrease steadily from the commencement of the diet to its termination.A decrease in sweat chloride values of similar magnitude was found in both parents and controls as the diet continued. It is concluded that the study of sweat electrolyte levels in salt-deprived subjects is of no value in the diagnosis of cystic fibrosis heterozygotes.     

Insulin iontophoresis in cystic fibrosis.

Insulin and its vehicle without insulin were administered separately by iontophoresis to patients with cystic fibrosis (CF), obligate heterozygotes, and healthy controls. The resultant sweat chloride concentration after treatment with both preparations was compared in each individual. No difference after the two treatments was found in the control sample. A decrease in sweat chloride concentration after insulin iontophoresis in comparison with the vehicle was observed in both the CF (P smaller than 0.005) and heterozygote (P smaller than 0.01) samples. These observations suggest an involvement of insulin in CF and a possible role of insulin in sweat gland function.     

Sweat tests to diagnose cystic fibrosis in adults.

Twenty five patients with cystic fibrosis and 25 controls were studied to define a sweat sodium concentration in adults that could be taken as diagnostic of cystic fibrosis. Some of the controls had a sweat sodium concentration of over 50 mmol(mEq)/l, and thus cystic fibrosis should be diagnosed in an adult only when two measurements of sweat sodium concentration are above 70 mmol/l. In cases in which the sweat sodium concentration was borderline a suppression test using fludrocortisone improved the accuracy of diagnosis; this test entails recording the lowest concentration reached after administration of the drug. A scatter diagram of the baseline sweat sodium concentrations plotted against the lowest concentration attained after suppression with fludrocortisone may aid the diagnosis further.     

Cystic Fibrosis of the Pancreas—Diagnosis by Sodium Electrode Sweat Tests

One of the most difficult and unreproducible procedures in clinical laboratories has been the measurement of electrolytes in sweat. The iontophoresis techniques for the diagnosis of cystic fibrosis of the pancreas, which are widely used, are fraught with difficulties.Measurement of sweat electrolytes with sodium or chloride electrodes is gradually replacing the iontophoresis methods. A simple modification of the sodium electrode technique was used for diagnosis in 11 cystic fibrosis patients and in 260 normal children. The values obtained with the sodium electrode clearly separated the normal children from those with cystic fibrosis.     

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described. Received: 25 August 1997 / Accepted: 20 January 1998     

Abnormal sweat electrolytes in symptomatic human immunodeficiency virus infection in a child.

A 3 1/2 year old girl presented with failure to thrive and a five month history of diarrhoea and recurrent cough. The results of sweat sodium tests suggested a diagnosis of cystic fibrosis; but atypical organisms were found (Haemophilus influenzae, Candida albicans, but no Staphylococcus aureus), she failed to respond to treatment, and her sweat sodium concentrations fell in response to fludrocortisone. She also had hyperglobulinaemia, neutropenia, and reduced numbers of T4 lymphocytes, which prompted the performance of a test for antibody to human immunodeficiency virus (HIV). This proved positive, and she was treated with co-trimoxazole, zidovudine, and human immunoglobulin. Both parents and two siblings were also positive for HIV, though all had normal sweat sodium concentrations. Children with symptoms suggestive of cystic fibrosis but who also show atypical features, as in this case, should have their HIV state checked.     

Kinetic analysis of chloride efflux from normal and cystic fibrosis fibroblasts

Chloride permeability in 9 cystic fibrosis- and 11 normal-skin fibroblast lines has been investigated. Chloride efflux, under steady-state conditions, involves two intracellular compartments characterized by slow- and fast-rate constants of efflux. We show here that the fast rate constant in cystic fibrosis cells is reduced by 25% in comparison with controls. The data presented support recent studies indicating that isolated sweat glands and respiratory epithelia of patients suffering from cystic fibrosis have an unusual low permeability to chloride ions compared to control epithelia. It is concluded that variation in chloride transport can successfully be studied in cultured fibroblasts, which are not directly involved in the pathology of the disease.     

Low abundance of sweat duct Cl- channel CFTR in both healthy and cystic fibrosis athletes with exceptionally salty sweat during exercise

To understand potential mechanisms explaining interindividual variability observed in human sweat sodium concentration ([Na(+)]), we investigated the relationship among [Na(+)] of thermoregulatory sweat, plasma membrane expression of Na(+) and Cl(-) transport proteins in biopsied human eccrine sweat ducts, and basal levels of vasopressin (AVP) and aldosterone. Lower ductal luminal membrane expression of the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) was observed in immunofluorescent staining of sweat glands from healthy young adults identified as exceptionally "salty sweaters" (SS) (n = 6, P < 0.05) and from patients with cystic fibrosis (CF) (n = 6, P < 0.005) compared with ducts from healthy young adults with "typical" sweat [Na(+)] (control, n = 6). Genetic testing of healthy subjects did not reveal any heterozygotes ("carriers") for any of the 39 most common disease-causing CFTR mutations in the United States. SS had higher baseline plasma [AVP] compared with control (P = 0.029). Immunostaining to investigate a potential relationship between higher plasma [AVP] (and sweat [Na(+)]) and ductal membrane aquaporin-5 revealed for all groups a relatively sparse and location-dependent ductal expression of the water channel with localization primarily to the secretory coil. Availability of CFTR for NaCl transport across the ductal membrane appears related to the significant physiological variability observed in sweat salt concentration in apparently healthy humans. At present, a heritable link between healthy salty sweaters and the most prevalent disease-causing CFTR mutations cannot be established.     

Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience.

OBJECTIVE--To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis. DESIGN--Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (delta F508, delta I506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus or family history of cystic fibrosis. SETTING--South Australian Neonatal Screening Programme, Adelaide. SUBJECTS--All 88,752 neonates born in South Australia between December 1989 and December 1993. INTERVENTIONS--Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. MAIN OUTCOME MEASURES--Identification of all children with cystic fibrosis in the screened population. RESULTS--Of 1004 (1.13%) neonates with immunoreactive trypsinogen > or = 99th centile, 912 (90.8%) had no identifiable mutation. 23 neonates were homozygotes or compound heterozygotes; 69 carried one identifiable mutation, of whom six had positive sweat tests. Median age at clinical assessment for the 29 neonates with cystic fibrosis was 3 weeks; six had meconium ileus and two had affected siblings. 63 neonates were identified as carriers of a cystic fibrosis mutation. Extra laboratory costs for measuring immunoreactive trypsinogen and direct gene analysis were $A1.50 per neonate screened. CONCLUSION--This strategy results in early and accurate diagnosis of cystic fibrosis and performs better than screening strategies based on immunoreactive trypsinogen measurement alone.     

Cystic fibrosis: present status and future prospects in detection of patients and carriers.

Development of a sensitive, easily performed, reliable test would be an important advance in detecting cystic fibrosis, improving genetic counselling and providing early effective treatment. The sweat chloride test, which is reliable in diagnosis, is technically too difficult for a screening program, and only reliably detects homozygotes. In contrast, the meconium test for detecting homozygote newborns is simple, inexpensive, reasonably specific but its general application has yet to be evaluated. Detection of serum components is the basis of two new tests to distinguish patients with cystic fibrosis and carriers. The effect of these serum components on ciliary activity is the principle of one test, an extremely difficult procedure that is subjective and lacks sufficient specificity for routine use. The second test, in which serum components are separated by isoelectric focusing, may provide an objective biochemical means of detecting both homozygotes and heterozygotes.     

Cystic fibrosis: the 'bicarbonate before chloride' hypothesis.

The specific effects of some mutations that cause cystic fibrosis suggest that reduced HCO(3)(-) transport is the key to understanding cystic fibrosis pathology. But there is a puzzling discrepancy between measures of CFTR-mediated chloride conductance in expression systems and the sweat chloride values of patients.     

The cystic fibrosis transmembrane conductance regulator gene and ion channel function in patients with idiopathic pancreatitis

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations are associated with cystic fibrosis (CF)-related monosymptomatic conditions, including idiopathic pancreatitis. We evaluated prospectively enrolled patients who had idiopathic recurrent acute pancreatitis or idiopathic chronic pancreatitis, healthy controls, CF heterozygotes, and CF patients (pancreatic insufficient or sufficient) for evidence of CFTR gene mutations and abnormalities of ion transport by sweat chloride and nasal potential difference testing. DNA samples from anonymous blood donors were controls for genotyping. At least one CFTR mutation or variant was carried in 18 of 40 patients (45%) with idiopathic chronic pancreatitis and in 6 of 16 patients (38%) with idiopathic recurrent acute pancreatitis but in only 11 of the 50 controls (22%, P=0.005). Most identified mutations were rare and would not be identified in routine genetic screening. CFTR mutations were identified on both alleles in six patient (11%). Ion transport measurements in patients with pancreatitis showed a wide range of results, from the values in patients with classically diagnosed CF to those in the obligate heterozygotes and healthy controls. In general, ion channel measurements correlated with the number and severity of CFTR mutations. Twelve of 56 patients with pancreatitis (21%) fulfilled current clinical criteria for the diagnosis of CF, but CFTR genotyping alone confirmed the diagnosis in only two of these patients. We concluded that extensive genotyping and ion channel testing are useful to confirm or exclude the diagnosis of CF in the majority of patients with idiopathic pancreatitis.     

K dependence of the Na-K pump is abnormal in erythrocytes from patients with cystic fibrosis and obligate heterozygotes

The affinity of the Na-K pump for K was significantly (P less than .001) lower in erythrocytes from patients with cystic fibrosis (Km 4.6 +/- 0.35 mM; n = 26) or from heterozygotes (Km 3.9 +/- 0.57 mM; n = 12) than in controls (Km 2.2 +/- 0.10 mM; n = 20). The affinity of the Na-K pump for K was lower in normal erythrocytes than in normal fibroblasts which may explain the variability in the severity of involvement of different organs in cystic fibrosis. We have now shown in human skin fibroblasts and erythrocytes, that the K affinity of the Na-K pump is lower in patients with cystic fibrosis than in controls. Since the abnormality is also present in erythrocytes from heterozygotes who are clinically normal, it is likely that this abnormality is closely related to the genetic defect in cystic fibrosis.     

Controlled trial of serum isoelectric focusing in the detection of the cystic fibrosis gene

Summary Three independent observers assessed the discriminating power of serum isoelectric focusing in detecting the presence of the cystic fibrosis gene. On the basis of average scores, four out of 23 cystic fibrosis patients, six out of 22 heterozygotes, and three out of 16 controls were misclassified. However, the mean scores for the cystic fibrosis and heterozygote groups were significantly different to that for the control group. It is concluded that isoelectric focusing is insufficiently reliable to be used for diagnosis or heterozygote detection in cystic fibrosis, but that it does provide evidence for the presence of a protein associated with the mutant gene.     

Decreased formation of alpha 2-macroglobulin-protease complexes in plasma of patients with cystic fibrosis.

Alpha₂-macroglobulin from patients with cystic fibrosis is shown to have reduced binding with papain, trypsin, and thrombin. The obligate heterozygotes for cystic fibrosis revealed intermediate values between the controls and the patients. Since papain and trypsin are not plasma endopeptidases, it becomes evident that the absence of α₂-macroglobulin-protease complex in cystic fibrosis is due to a molecular defect within the macroglobulin.     

First report of c. 1499G>C mutation in a 6-month-child with cystic fibrosis

So far, more than 1800 mutations identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this case report, we presented first report of c. 1499G>C mutation in a 6-month-old girl with cystic fibrosis (CF) diagnosis. A 6-month-old girl with weakness and meconium Ileus referred to the pediatric clinic in Ilam, in the west of Iran. Patient''s skin was dark and suffered from bronchiectasis. The sweat test was performed, and the concentration of chloride and sodium in patient''s sweat was 130-135 mmol/L and 125-128 mmol/L, respectively. The exon 10 mutation analysis of a CF patient was performed. CFTR mutation analysis revealed the identification of 2 mutations in patient, the mutations were p.F508del (ΔF508) and c. 1499G>C (cd500), respectively. The mutation c. 1499G>C (cd500) were found for the first time in the world. Assessing this mutation in future study and genetic investigation is recommended.     

The limitations of sweat electrolyte reference intervals for the diagnosis of cystic fibrosis: a systematic review

The sweat test has been used for more than 50 years for the diagnosis of cystic fibrosis (CF) and remains an important diagnostic test in the genomic era. The currently used reference intervals for sweat electrolytes are applied to all patients regardless of age or sex. We performed a systematic review to summarise the studies with published reference values of sweat electrolyte concentrations for the diagnosis of CF. The MEDLINE (from 1950), EMBASE (from 1980) and PubMed (from 1950) databases were searched for English language studies. An abstract was also found by hand-searching. The search generated 1136 articles that matched the search key terms. Of these, 17 studies that contained data on sweat electrolyte concentrations were included in the analysis. Among these, seven studies did not perform the sweat test in accordance with current international and Australian guidelines. Of the ten remaining studies, four reported both the sweat sodium and chloride concentrations and six reported sweat chloride concentration only. A major limitation of these studies was the subject selection. Most recruited patients with various medical conditions including respiratory diseases or undefined recruitment criteria, whilst some did not report the subjects’ age and some had small subject numbers. Only one study performed mutation analysis to determine carrier status. No study used appropriate statistical analysis to develop a sweat chloride reference interval. The literature review yielded no studies that reliably developed reference intervals for sweat electrolyte concentrations. The limitations of the studies highlight the need for reliable age-related reference intervals for sweat electrolyte concentrations in healthy subjects.     

Targeted extended cystic fibrosis mutation testing on known and at-risk patients and relatives

This paper reports mathematically derived residual risks of being a carrier or being affected with cystic fibrosis following various screening scenarios to assist in interpreting test results and advising patients. While parental screening with 23 American College of Medical Genetics (ACMG) cystic fibrosis mutations defines the 64% of affected U.S. Caucasian fetuses with two detectable mutations, newborn screening for elevated immunoreactive trypsinogen (IRT) and sweat chloride identifies an additional 36% of affected newborns with zero or one detected mutation. The relatives of these affected newborns with less than two detectable mutations have higher posterior (after) 23 mutation-negative test risks of carrying undetected mutations. These calculations emphasize how knowledge of the mutations in the related affected patient substantially improves upon the quality of after-test advice to patients. Furthermore, negative tests of the partner without a family history and/or more extensive cystic fibrosis transmembrane conductance regulator (CFTR) gene testing also increases the likelihood that a negative report is truly negative. When a newborn patient with zero or one detected CFTR mutation has an inconclusive sweat test result, the sweat test should be repeated before ordering additional often unnecessary CFTR gene sequencing. Given the same composite mutation panel test accuracy, a higher proportion of reported test results would be correct during parental screening than when testing at-risk fetuses or symptomatic newborns. Prenatal and newborn screening would be enhanced substantially by medical professionals offering copies of all positive parental and newborn test reports to the parents to share with their relatives. These principles are likely to be applicable to other genetic diseases as the most common mutation frequencies are reported.     

An outward-rectifying potassium channel in primary cultures of sweat glands from cystic fibrosis subjects

We have previously described a high conductance calcium-activated 'maxi K' channel in primary cultures of human eccrine sweat gland cells both from normal subjects and those with cystic fibrosis. In further studies we have now identified a potassium-selective channel of much lower conductance which shows outward-rectification and which is present in sweat glands isolated from cystic fibrosis subjects. In experiments with inside-out patches using symmetrical pipette and bath solutions containing 140 mM K+ the channel showed an outward slope conductance (at +50 mV) of approximately 26 pS and an inward conductance (at -50 mV) of approximately 11 pS. When K+ in the bath was replaced by Na+ the reversal potential shifts to reveal a permeability ratio PK/PNa approximately 40 Unlike the maxi-K+ channel, the outward-rectifying channel does not show sensitivity to Ca2+. Channels were found in cells cultured from the glands of four out of five cystic fibrosis subjects. In cells cultured from 30 subjects who did not have cystic fibrosis, an outward-rectifying potassium channel was seen in only one out of approximately 3000 patches.     

Immunolocalization of band 3 protein in normal and cystic fibrosis skin

Current evidence indicates that the defect in cystic fibrosis (CF) involves chloride transport in various epithelial cells. The sweat gland, one site of altered chloride transport in CF, was examined immunocytochemically for localization of a chloride-channel membrane protein, designated band 3 protein. Immunoreactivity was observed in sweat duct cell membranes of both normal and CF samples, whereas secretory coil regions were entirely unreactive. No difference was observed in the pattern or intensity of immunoreactivity between the two groups at the light microscopic (LM) level of resolution.