The importance of functional tests to assess the effect of a new CFTR variant when genotype-phenotype correlation is not possible.

In vitro functional tests aimed to investigate CFTR dysfunction appear critical to help elucidate the functional impact of new variants of uncertain clinical significance and solve inconclusive cases, especially in early deceased newborns.

CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders.

BACKGROUND: The high frequency of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutation p.Arg117His in patients with congenital bilateral absence of the vas deferens (CBAVD) and in newborns screened for CF has created a dilemma. METHODS: Phenotypic and genotypic data were retrospectively collected in 179 non-newborn French individuals carrying p.Arg117His and a second CFTR mutation referred for symptoms or family history, by all French molecular genetics laboratories, referring physicians, CF care centres and infertility clinics. RESULTS: 97% of the patients had the intronic T7 normal variant in cis with p.Arg117His. 89% patients were male, with CBAVD being the reason for referral in 76%. In 166/179 patients with available detailed clinical features, final diagnoses were: four late-onset marked pulmonary disease, 83 isolated CBAVD, 67 other CFTR-related phenotypes, including 44 CBAVD with pulmonary and/or pancreatic symptoms and 12 asymptomatic cases. Respiratory symptoms were observed in 30% of the patients, but the overall phenotype was mild. No correlation was observed between sweat chloride concentrations and disease severity. Five couples at risk of CF offspring were identified and four benefited from prenatal or preimplantation genetic diagnoses (PND or PGD). Eight children were born, including four who were compound heterozygous for p.Arg117His and one with a severe CF mutation. CONCLUSIONS: Patients with CBAVD carrying p.Arg117His and a severe CF mutation should benefit from a clinical evaluation and follow-up. Depending on the CBAVD patients' genotype, a CFTR analysis should be considered in their partners in order to identify CF carrier couples and offer PND or PGD.

p.Ser1235Arg should no longer be considered as a cystic fibrosis mutation: results from a large collaborative study.

Among the 1700 mutations reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a missense mutation, p.Ser1235Arg, is a relatively frequent finding. To clarify its clinical significance, we collected data from 104 subjects heterozygous for the mutation p.Ser1235Arg from the French CF network, addressed for various indications including classical CF, atypical phenotypes or carrier screening in subjects with or without a family history. Among them, 26 patients (5 having CF, 10 CBAVD (congenital bilateral absence of the vas deferens) and 11 with CF-like symptoms) and 14 healthy subjects were compound heterozygous for a second CFTR mutation. An exhaustive CFTR gene analysis identified a second mutation in cis of p.Ser1235Arg in all CF patients and in 81.8% CBAVD patients. Moreover, epidemiological data from >2100 individuals found a higher frequency of p.Ser1235Arg in the general population than in CF or CBAVD patients. These data, added to the fact that in silico analysis and functional assays suggest a benign nature of this substitution, give several lines of evidence against an association of p.Ser1235Arg with CF or CBAVD.

Clinical phenotype and genotype of children with borderline sweat test and abnormal nasal epithelial chloride transport.

RATIONALE: The diagnosis of cystic fibrosis (CF) is based on a characteristic clinical picture in association with a sweat chloride (Cl(-)) concentration greater than 60 mmol/L or the identification of two CF-causing mutations. A challenging problem is the significant number of children for whom no definitive diagnosis is possible because they present with symptoms suggestive of CF, a sweat chloride level in the intermediate range between 30 and 60 mmol/L, and only one or no identified CF-causing mutation. OBJECTIVES: To investigate the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the airways of children with intermediate sweat tests and inconclusive genetic findings in correlation with clinical phenotype and genotype. METHODS: We developed a composite nasal potential difference (NPD) diagnostic score to discriminate patients with CF from non-CF patients. We tested NPD in 50 children (age, 6 mo to 18 yr) with equivocal diagnoses and correlated the NPD diagnostic score with clinical phenotypes and genotypes. MEASUREMENTS AND MAIN RESULTS: Fifteen of the 50 children had NPD scores in the CF range. Eight of the 15 carried two CFTR mutations compared with only 5 of the 35 children with normal NPD scores (P = 0.01). They were significantly younger at evaluation and had recurrent lower respiratory tract infections, chronic productive coughs, and chronic Staphylococcus aureus colonization significantly more often than the 35 children with normal NPD results. CONCLUSIONS: Evaluation of CFTR function in the nasal epithelium of children with inconclusive CF diagnoses can be a useful diagnostic tool and help clinicians to individualize therapeutic strategy.

Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis.

BACKGROUND: A challenging problem arising from cystic fibrosis (CF) newborn screening is the significant number of infants with hypertrypsinaemia (HIRT) with sweat chloride levels in the intermediate range and only one or no identified CF-causing mutations. OBJECTIVES: To investigate the diagnostic value for CF of assessing CF transmembrane conductance regulator (CFTR) protein function by measuring nasal potential difference in children with HIRT. METHODS: A specially designed protocol was used to assess nasal potential difference (NPD) in 23 young children with HIRT (3 months-4 years) with inconclusive neonatal screening. Results were analysed with a composite score including CFTR-dependent sodium and chloride secretion. Results were correlated with genotype after extensive genetic screening and with clinical phenotype at follow-up 3 years later. RESULTS: NPD was interpretable for 21 children with HIRT: 13 had NPD composite scores in the CF range. All 13 were finally found to carry two CFTR mutations. At follow-up, nine had developed a chronic pulmonary disease consistent with a CF diagnosis. The sweat test could be repeated in nine children, and six had sweat chloride values >or=60 mmol/l. Of the eight children with normal NPD scores, only two had two CFTR mutations, both wide-spectrum mutations. None had developed a CF-like lung disease at follow-up. The sweat test could be reassessed in five of these eight children and all had sweat chloride values <60 mmol/l. CF diagnosis was ruled out in six of these eight children. CONCLUSION: Evaluation of CFTR function in the nasal epithelium of young children with inconclusive results at CF newborn screening is a useful diagnostic tool for CF.

Renal impairment in children with cystic fibrosis.

BACKGROUND: Due to the improvement in life expectancy in cystic fibrosis (CF), co-morbidities such as renal function impairment may be more frequent. AIM: To determine the prevalence of renal disease in children with CF and to identify associated risk factors. METHODS: A single-center retrospective study analyzing the genetic, clinical and therapeutic characteristics of 112 children. The estimated glomerular filtration rate (GFR), microalbuminuria and lithiasic risk factors were assessed. RESULTS: The median calculated GFR (Schwartz) was 123, 161 and 155ml/min/1.73m(2) in children aged 1, 6 and 15years, respectively. The cumulative dose of aminoglycosides was not correlated to GFR. Microalbuminuria was present in 22/38 patients. Hyperoxaluria was observed in 58/83 patients and was associated with a severe genotype, pancreas insufficiency and liver disease. Hypercalciuria, hyperuricuria and hypocitraturia were identified in 16/87, 15/83 and 57/76 patients, respectively. CONCLUSION: Renal impairment in CF has various presentations. There appears to be low levels of renal impairment in children with CF. However, the risk of oxalocalcic urolithiasis is enhanced, and GFR may be underestimated by the Schwartz formula. Further studies using measured GFR techniques are thus warranted.

Influence of the duplication of CFTR exon 9 and its flanking sequences on diagnosis of cystic fibrosis mutations.

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact, present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions.

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening.

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.

Rapid and accurate detection of the CFTR gene mutation 1811+1.6 kbA>G by real-time fluorescence resonance energy transfer PCR.

The CFTR gene mutation 1811+1.6 kbA>G has been reported as associated with a severe phenotype of cystic fibrosis with pancreatic insufficiency. This mutation has been identified as a rather common one in the South West of France and in the Iberian Peninsula. Because of the precise geographical origin of the subjects and its frequency, the mutation has to be investigated with accuracy. We have developed an original real-time Fluorescence Resonance Energy Transfer (FRET) PCR assay for genotyping the mutation 1811+1.6 kbA>G. It is based on the amplification of a region spanning the mutation with simultaneous detection of the amplicon by hybridization with a bi-probe followed by a melting curve analysis. The results obtained are identical with those resulting from either restriction fragment length polymorphism analysis or sequencing. The distinction between the wild type and the mutation 1811+1.6 kbA>G is easy because the corresponding melting points shows a difference of 6 or 9.5 degrees C depending on the associated SNP A/T located 16 bp downstream. We demonstrated that a FRET assay showed enough sensitivity to discriminate between two nucleotide polymorphisms (SNPs) in the sequence of the sensor. In conclusion, this method is specific, fast, easy to perform, reproducible, inexpensive as it uses only one bi-probe and well adapted to daily practice.

Isolated idiopathic chronic pancreatitis associated with a compound heterozygosity for two mutations of the CFTR gene.

We report the case of a patient suffering from idiopathic chronic pancreatitis (ICP) and compound heterozygous for mutations G542X and S1235R of the cystic fibrosis transmembrane regulator (CFTR) gene. The patient had normal sweat test and no other clinical sign usually linked with a typical or moderate pathology (bronchiectasis, nasal polyposis, congenital absence of the vas deferens) of the CFTR gene. G542X is a severe mutation, which is usually found in classical cystic fibrosis when associated with other severe mutations. S1235R is a quite rare abnormality recently reported as being potentially pathogenic when combined in trans with a second CF mutation. Our case is quite similar to the only other six patients in the literature in whom only the pancreas is affected and who bear a rare mutation with moderate effect. The history and the clinical features of our patient indicate an unambiguous isolated ICP in which the presence of the S1235R mutation--in trans with regard to G542X--is likely responsible for the ICP phenotype. This case could throw light on some of the as yet poorly known abnormalities of the CFTR gene in the ICP phenotype.

CFTR genotypes in patients with normal or borderline sweat chloride levels.

In recent years, some patients bearing "atypical" forms of cystic fibrosis (CF) with normal sweat chloride concentrations have been described. To identify the spectrum of mutant combinations causing such atypical CF, we collected the results of CFTR (ABCC7) mutation analysis from 15 laboratories. Thirty patients with one or more typical symptoms of the disease associated with normal or borderline sweat chloride levels and bearing two CFTR mutations were selected. Phenotypes and genotypes of these 30 patients are described. A total of 18 different CFTR mutations were observed in the 60 chromosomes analysed. F508del was present in 31.6 % of the mutated chromosomes and 3849+10kbC>T in 13.3 %. R117H, D1152H, L206W, 3272-26A>G, S1235R, G149R, R1070W, S945L, and the poly-T tract variation commonly called IVS8-5T were also observed. The relative frequency of CFTR mutations clearly differed from that observed in typical CF patients or in CBAVD patients with the same ethnic origin. A mild genotype with one or two mild or variable mutations was observed in all the patients. These findings improve our understanding of the distribution of CFTR alleles in CF with normal or borderline sweat chloride concentrations and will facilitate the development of more sensitive CFTR mutation screening.