The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era

Abstract

Cystic fibrosis (CF) is the most common inherited disorder of childhood. The diagnosis of CF has traditionally been based on clinical features with confirmatory evidence by sweat electrolyte analysis. Since 1989 it has been possible to also use gene mutation analysis to aid the diagnosis. Cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has advanced our understanding of CF, in particular the molecular basis of an expanded CF phenotype. However, because there are over 1000 mutations and 200 polymorphisms, many without recognised effects on CFTR, the molecular diagnosis can be troublesome. This has necessitated measurement of CFTR function with renewed interest in the sweat test. This review provides an overview of the clinical features of CF, the diagnosis and complex genetics. We provide a detailed discussion of the structure and function of CFTR and the classification of CFTR mutations. Sweat electrolyte analysis is discussed, from the physiology of sweating to the rigours of a properly performed sweat test and its interpretation. With this information it is possible to understand the relevance of the sweat test in the genomic era.

Figure 1

Schematic diagram of the proposed structure of CFTR. A member of the ABC family, CFTR consists of a tandem repeat of the ABC motif. This motif comprises a membrane-spanning domain (composed of six transmembrane stretches of amino acids) followed by an nucleotide-binding domain (NBD). In CFTR, the two occurrences of this motif are separated by a regulatory (R) domain. Each NBD is able to bind and hydrolyse ATP to operate chloride (Cl-) channel function: hydrolysis of ATP by NBD-1 opens the chloride channel, while ATP hydrolysis by NBD-2 closes the channel. Channel function is further regulated by phosphorylation of serine residues in the R domain. Lyczak JB, Cannon CL. and Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev. 2002 Apr;15:194-222. With permission from American Society for Microbiology Journals Department.

Figure 2

Five classes of CF-related gene mutations are displayed with differing ways of impairing CFTR function. Normally (top panel), CFTR is transcribed into messenger RNA followed by post-translational modifications, including folding, glycosylation and trafficking via the Golgi apparatus to the cell membrane. Among disease-related mutations (bottom panels): Class 1 mutations prevent translation. Class 2 mutations are misfolded and unable to escape the endoplasmic reticulum where they are degraded. Class 3 reach the cell membrane but do not respond to cAMP stimulation. Class 4 are trafficked to the cell membrane and respond to stimuli but have decreased chloride conductance. Class 5 mutations result in decreased abundance of CFTR. Some mutations from this class produce a small percentage of correctly spliced mRNA, resulting in a milder phenotype. Reprinted with permission from Tsui L-C, Durie P: Genotype and cystic fibrosis. Hospital Practice 1997; 32: 115–142. With the permission of The McGraw-Hill Companies.

Figure 3

Relation between CFTR activity and the clinical manifestations of CF. Left panel shows estimate of CFTR activity from published data in the literature. Right panel shows descending order of tissue sensitivity to CFTR deficit. Decreased levels of normal CFTR activity may be involved in various clinical phenotypes, ranging from the normal phenotype to the phenotypes of CBAVD, cystic fibrosis with pancreatic sufficiency, and cystic fibrosis with pancreatic insufficiency. CBAVD = congenital bilateral absence of vas deferens.

Figure 4

Schematic representation of sweat production and electrolyte transport by the sweat gland. This figure shows the reabsorptive and secretory duct. Note that cAMP-dependent and calcium-dependent chloride secretion appear to occur in two different types of cells in the secretory coil. The CFTR chloride channel is indicated by shading; all other channels, transporters and pumps are indicated by open symbols. Sodium (Na⁺) absorption in the sweat duct occurs by sodium entry into the cell across the apical membrane driven by a favourable electrochemical gradient. Sodium then exits across the basolateral membrane in exchange for potassium (K⁺) on the Na⁺/K⁺- ATPase transporters, which maintains a low in-tracellular Na⁺ concentration. These processes are the same in normal and CF sweat gland duct. Na⁺ transport establishes the ion concentration and voltage gradients that drive passive chloride absorption. When CFTR is defective, chloride fails to follow Na⁺ absorption, preventing the sodium and chloride absorption, increasing their concentrations in the sweat. This figure is reproduced from Welsh MJ, Ramsay BW, Accurso F, Cutting GR. Cystic Fibrosis. In: Scriver ABC, Sly WS, Valle D, editors. The Molecular and Metabolic Basis of Inherited Disease. New York: McGraw-Hill; 2001. p. 5150; with permission from The McGraw-Hill Companies.

Figure 5

The Wescor Macroduct Sweat Collector. The secreted sweat is forced through the central hole by hydrostatic pressure and is collected in the microbore tubing. A blue water-soluble dye at the base of the Wescor collector allows easy assessment of the volume produced at any time during collection.

Figure 6

Calculation of average sweat rate and the minimum acceptable weight/volume.