Patients with mutations in Gsalpha have reduced activation of a downstream target in epithelial tissues due to haploinsufficiency.

CONTEXT: Patients with Albright hereditary osteodystrophy (AHO) have defects in stimulatory G protein signaling due to loss of function mutations in GNAS. The mechanism by which these mutations lead to the AHO phenotype has been difficult to establish due to the inaccessibility of the affected tissues. OBJECTIVE: The objective of the study was to gain insight into the downstream consequences of abnormal stimulatory G protein signaling in human epithelial tissues. PATIENTS AND DESIGN: We assessed transcription of GNAS and Gsalpha-stimulated activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in AHO patients, compared with normal controls and patients with cystic fibrosis. MAIN OUTCOME MEASURES: Relative expression of Gsalpha transcripts from each parental GNAS allele and cAMP measurements from nasal epithelial cells were compared among normal controls and AHO patients. In vivo measurements of CFTR function, pulmonary function, and pancreatic function were assessed in AHO patients. RESULTS: GNAS was expressed equally from each allele in normals and two of five AHO patients. cAMP generation was significantly reduced in nasal respiratory epithelial cells from AHO patients, compared with normal controls (0.4 vs. 0.6, P = 0.0008). Activation of CFTR in vivo in nasal (P = 0.0065) and sweat gland epithelia (P = 0.01) of AHO patients was significantly reduced from normal. In three patients, the reduction in activity was comparable with patients with cystic fibrosis due to mutations in CFTR. Yet no AHO patients had pulmonary or pancreatic disease consistent with cystic fibrosis. CONCLUSIONS: In humans, haploinsufficiency of GNAS causes a significant reduction in the activation of the downstream target, CFTR, in vivo.

Variant cystic fibrosis phenotypes in the absence of CFTR mutations.

BACKGROUND: Cystic fibrosis is a life-limiting autosomal recessive disorder with a highly variable clinical presentation. The classic form involves characteristic findings in the respiratory tract, gastrointestinal tract, male reproductive tract, and sweat glands and is caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR ) gene. Nonclassic forms of cystic fibrosis have been associated with mutations that reduce but do not eliminate the function of the CFTR protein. We assessed whether alteration in CFTR function is responsible for the entire spectrum of variant cystic fibrosis phenotypes. METHODS: Extensive genetic analysis of the CFTR gene was performed in 74 patients with nonclassic cystic fibrosis who had been referred by 34 medical centers. We evaluated two families that each included a proband without identified mutations and a sibling with nonclassic cystic fibrosis to determine whether there was linkage to the CFTR locus and to measure the extent of CFTR function in the sweat gland and nasal epithelium. RESULTS: Of the 74 patients studied, 29 had two mutations in the CFTR gene, 15 had one mutation, and 30 had no mutations. A final genotype of two mutations was more common among patients who had been referred after screening for common cystic fibrosis-causing mutations identified one mutation than among those who had been referred after screening had identified no such mutations (26 of 34 patients vs. 3 of 40 patients, P<0.001). Comparison of clinical features and sweat chloride concentrations revealed no significant differences among patients with two, one, or no CFTR mutations. Haplotype analysis in the two families revealed no linkage to CFTR. Although each of the affected siblings had elevated sweat chloride concentrations, measurements of cyclic AMP-mediated ion and fluid transport in the sweat gland and nasal epithelium demonstrated the presence of functional CFTR. CONCLUSIONS: Factors other than mutations in the CFTR gene can produce phenotypes clinically indistinguishable from nonclassic cystic fibrosis caused by CFTR dysfunction.

Phenotypic and genetic characterization of patients with features of "nonclassic" forms of cystic fibrosis.

OBJECTIVE: To determine which features of incomplete or "nonclassic" forms of cystic fibrosis (CF) are associated with deleterious CF transmembrane conductance regulator gene ( CFTR ) mutations, and to explore other etiologies for features not associated with deleterious CFTR mutations. STUDY DESIGN: Clinical features were compared between 57 patients with deleterious mutations in each CFTR and 63 with no deleterious mutations. The Shwachman Bodian Diamond syndrome gene ( SBDS ) was sequenced to search for mutations in patients with no deleterious CFTR mutations and steatorrhea to determine if any had unrecognized Shwachman-Diamond syndrome (SDS). RESULTS: The presence of a common CF-causing mutation, absence of the vas deferens, and Pseudomona aeruginosa in the sputum correlated with the presence of two deleterious CFTR mutations, whereas sweat chloride concentration, diagnostic criteria for CF, and steatorrhea did not. However, sweat chloride concentration correlated with CFTR mutation status in patients infected with P aeruginosa. One patient had disease-causing mutations in each SBDS . CONCLUSIONS: Presence of a common CF-causing mutation, absence of the vas deferens and/or P aeruginosa infection in a patient with features of nonclassic CF are predictive of deleterious mutations in each CFTR , whereas steatorrhea in the same context is likely to have etiologies other than CF transmembrane conductance regulator (CFTR) dysfunction.

Mutations in the beta-subunit of the epithelial Na+ channel in patients with a cystic fibrosis-like syndrome.

Cystic fibrosis (CF) is an autosomal recessive disorder of Cl(-) and Na(+) transport. The vast majority of CF patients have deleterious mutations in an epithelial Cl(-) channel called the CF transmembrane conductance regulator (CFTR). In contrast, defects in the epithelial Na(+) channel (SCNN1) have been associated with phenotypes dominated by renal disease (systemic pseudohypoaldosteronism type I and Liddle syndrome). We report two non-classic CF patients without CFTR mutations who have novel deleterious mutations in the beta-subunits of SCNN1 in the absence of overt renal disease.

A variable dinucleotide repeat in the CFTR gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing.

Dinucleotide repeats are ubiquitous features of eukaryotic genomes that are not generally considered to have functional roles in gene expression. However, the highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Using a CFTR minigene system, we studied TG tract variation and observed the same correlation between dinucleotide repeat number and exon 9 splicing efficiency seen in vivo. Replacement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacements of the TG tract with sequences that can self-base-pair suggested that the formation of an RNA secondary structure was associated with efficient splicing. However, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal. Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Taken together, these data indicate that dinucleotide repeats can form secondary structures that have variable effects on RNA splicing efficiency and clinical phenotype.

Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.

An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.