Reclassifying inconclusive diagnosis for cystic fibrosis with new generation sweat test

Abstract

The biological diagnosis of cystic fibrosis (CF) is based on a sweat chloride (Cl − ) concentration (SCC) ⩾60 mmol•L −1 and/or the identification of two allelic CF-causing variants [1]. Diagnosis of CF can be challenging in subjects with intermediate SCC between 30 and 59 mmol•L −1 and only one CF-causing variant detected. A proportion of these individuals with a query diagnosis for CF (Q) are simple carriers but a subset carries two variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, some of which are of unknown significance. This includes patients referred for symptoms evocative of CF and infants with an inconclusive diagnosis after newborn screening. In such cases, diagnostic algorithms recommend performing CFTR functional investigations, such as trans-epithelial nasal potential difference (NPD) measurement and intestinal current measurement (ICM) in rectal biopsies [1]. However, these tests, which require expensive equipment, are only performed in a few expert laboratories [2,3]. The beta-adrenergic (β-adr) sweat secretion test (SST), which evaluates the specific CFTR activity in the sweat gland acinus, might be an alternative test for CFTR functional exploration [4]. We investigated β-adr SST measured by evaporimetry in a cohort of Q-patients in comparison to NPD and ICM. The diagnosis performance of the test was related to final patient classification after extensive genetic screening of the CFTR gene.We enrolled 24 symptomatic Q-patients with borderline SCC including 14 children (mean±SEM 12.5±1.1 years old) and 10 adults (41.9±5.7 years old), age range of 4.0 to 66.4 years old (figure 1a). Written informed consent was collected for all patients (IRB, Clinical trial number RCB2016-A01923-48, 2018).The β-adr SST was performed using evaporimeter probes (CyberDerm, PA, USA) as we previously published [5]. Maximal β-adr sweat rate was normalised to the maximal cholinergically stimulated sweat rate by calculating the ratio of β-adr peak to carbachol peak (β-adr ratio). Tracings were considered if: 1) sweat rate was stable at the reference probe during the whole test; 2) carbachol peak was >30 g water loss•m −2 •h −1 ; and 3) atropine injection completely inhibited the cholinergic response. The discrimination threshold for CF was calculated by receiver operating characteristic curve ⩽4.5 g water loss•m −2 •h −1 for β-adr peak and 0.08 for β-adr ratio, based on our reference population composed of adolescents or young adults (31 healthy controls, 20 with CF and four heterozygotes).CFTR activity in the nasal epithelium was assessed by the sum of the changes in NPD after perfusion with the low Cl − solution and isoproterenol (Δtotal Cl − response, ΔTCR) and in the intestinal mucosa by the change in short circuit current (IsC) after addition of forskolin (Δforskolin) [2,3].Search for frequent CF-causing variants was followed, when necessary, by extensive analysis of all coding regions using next generation sequencing [6]. The CFTR variants were classified as follows: CF-causing; variants...

Proteomic profiling of sweat in patients with cystic fibrosis provides new insights into epidermal homoeostasis

“…Patient 13 carried the rare missense variant p.Phe191Val (F191 V) in trans of F508del and had a normal chloride sweat concentration at 22 mmol/L. However, β ‐adrenergic sweat secretion rate was in the CF range, providing insight for a genotype with minimal CFTR activity 8 . Patient 20 carried the 2789 + 5G > A splice site mutation, known to be associated with a small amount of normally spliced transcripts 9 .…”

β‐adrenergic sweat test in children with inconclusive cystic fibrosis diagnosis: Do we need new reference ranges?

“…However, the β‐adrenergic:cholinergic ratio, which is less influenced by sex, may be more useful than peak β‐adrenergic secretion in young children as supported by our data of children with Δβ‐adrenergic < 4.5 TEWL, where β‐adrenergic:cholinergic ratio > 0.05 excluded CF/CFTR‐RD in all but one child. However, further studies are needed to establish optimal age and sex‐dependent reference ranges of the BAST in children as conflicting data in older children has been published suggesting established reference ranges are valid 11 …”

“…Minor adverse events occurred in five participants: one healthy adult had anxiety, one child had inconsolable crying, and three participants (one adult and two children) had redness at the injection site, which resolved without intervention. However, the β-adrenergic:cholinergic ratio in children was >0.05 in all but one child who was heterozygous with c.1210-34TG [11]T [5] variant (participant no. 4).…”

“…The novel CFTR‐dependent β‐adrenergic sweat test (BAST) measures rate of sweat production by evaporimetry or sweat droplet imaging 6,8–10 . BAST is easier to perform and less invasive than NPD and ICM and demonstrates a greater ability than SCT to discriminate between CF, CFTR‐RD, CFTR heterozygotes and subjects without CFTR variants 6,11 . The BAST is safe, validated, and well tolerated in adults but the diagnostic utility, feasibility and safety in children and populations with diverse ethnicity is not well established 12 .…”

“…11 There are several limitations to this study that we considered when interpreting our results. First, we did not do extensive genetic testing in healthy adult controls due to budgetary constraints, thus it is possible that some were asymptomatic CFTR heterozygotes, which could have lowered their β-adrenergic secretion.…”