Michel Roussey

Neonatal screening for cystic fibrosis in Brittany, France: assessment of 10 years' experience and impact on prenatal diagnosis

BACKGROUND Neonatal screening for cystic fibrosis has been a subject of debate over the past few years. This study assesses 10 years of neonatal screening in Brittany, France, and examines its impact on prenatal screening of subsequent pregnancies in couples with an affected child. METHODS The study included all the neonates screened for cystic fibrosis in Brittany from Jan 1, 1989, to Dec 31, 1998. The screening consisted of an immunoreactive trypsinogen assay from dried blood spots, plus, from 1993, mutation analysis. Data were collected on incidence of cystic fibrosis, and genotypic and biochemical characteristics. The use of prenatal screening of subsequent pregnancies in affected families was also investigated. FINDINGS Of the 343,756 neonates screened, 118 children with cystic fibrosis were identified, giving an incidence of one in 2913. All mutated alleles were characterised: 34 different mutations resulting in 36 genotypes were detected. The introduction of DNA analysis into the protocol greatly reduced the recall rate and increased the sensitivity of the test. The mean cost of the screening programme was US

The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening

2.32 per screened child. 39 (34%) of the families identified by neonatal screening opted for subsequent prenatal diagnosis at least once. 12 couples would have benefited from this procedure while their first child was still symptom-free. 42 healthy children were born, and 18 pregnancies were terminated (therapeutic abortion rate of 100%). INTERPRETATION We have shown the feasibility of neonatal screening for cystic fibrosis in Brittany. Through the detection of a large range of mutations, neonatal screening provides the opportunity for more reliable prenatal diagnosis and cascade screening. The neonatal screening programme described here could provide a good model for other countries intending to initiate such a scheme.

Dose-dependent in vitro effect of recombinant human DNase on rheological and transport properties of cystic fibrosis respiratory mucus.

Background: Cystic fibrosis (CF) is caused by compound heterozygosity or homozygosity of CF transmembrane conductance regulator gene (CFTR) mutations. Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice. Methods: The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (pR117H), on either intron 8 T5 or T7 background, and F508del (pF508del) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. Results: Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns. The disease phenotype was predominantly mild; one child had classical CF, and three adults’ severe pulmonary symptoms. In 5245 healthy adults, pF508del was 1.06%, pR117H;T7 0.27% and pR117H;T5<0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%. Conclusions: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. The real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes.

Implementation of the French nationwide cystic fibrosis newborn screening program.

Recombinant human deoxyribonuclease (rhDNase) has been demonstrated to reduce the viscosity of purulent cystic fibrosis (CF) respiratory mucus, to improve pulmonary function and to reduce the risk of respiratory tract infectious exacerbations, but its effect on mucus transportability has not so far been investigated. The dose-dependent effect of rhDNase was analysed in vitro on mucus transport rate (tr) by ciliary activity and by simulated cough (cough transport (ct)), as well as on mucus viscosity and surface properties. Purulent CF sputa (n = 15) were incubated for 30 min at 37 degrees C with either rhDNase at three different concentrations (final concentrations 0.2, 2 or 20 micrograms.ml-1 of mucus) or placebo. No significant dose-dependent effect of rhDNase on the mucociliary transport rate was observed when the samples wer statistically analysed together. However, in the larger group of mucus samples (n = 11) with a low initial mucociliary transport rate, the latter was improved at each rhDNase concentration (tr0.2 = 0.69, tr2 = 0.88 and tr20 = 0.87) as compared to placebo (trp = 0.58). In the smaller group of mucus samples (n = 4) with high initial transport rate, a decrease in mucociliary transport rate was observed, particularly at the highest concentration rhDNase assayed, i.e. 20 micrograms.ml-1 of mucus (tr20 = 0.58) as compared to placebo (trp = 0.86). The mucus cough transport was increased by rhDNase (ct0.2 = 25 mm, ct2 = 27.5 mm and ct20 = 31 mm) as compared to placebo (ctp = 23.5 mm).(ABSTRACT TRUNCATED AT 250 WORDS)

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

OBJECTIVES To describe optimization of a nationwide newborn screening program for cystic fibrosis (CF) that combines an immunoreactive trypsinogen (IRT) assay and DNA mutation analysis in dried blood samples at day 3. STUDY DESIGN Data from regional screening laboratories and CF care centers were centralized and periodically analyzed to allow adaptation, thus limiting the number of false-positive cases. RESULTS A total of 2717905 infants were screened between 2002 and 2005. Flow chart protocol was modified twice. First, the IRT d3 cutoff value increased from 60 to 65 microg/L, thus decreasing the percentage of samples requiring mutation analysis from 0.82% to 0.64%. Second, for infants with no mutations using the screening panel, a recall for IRT was performed only if IRT d3 was > 100 microg/L; the percentage of recalls decreased from 0.51% to 0.12%, and the percentage of infants requiring a sweat test decreased from 0.14% to 0.01%. No significant change in the CF detection rate was observed after these 2 modifications. A total of 625 CF cases were detected, and 22 false-negative findings (3.4%) were observed, most of them inevitable, with a low initial IRT. CONCLUSIONS The centralized data analysis led to changes in the screening strategy to optimise the newborn screening program.

Neonatal screening of cystic fibrosis: diagnostic problems with CFTR mild mutations

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.

Psychological Effects of False-Positive Results in Cystic Fibrosis Newborn Screening: A Two-Year Follow-Up

SummaryNewborn screening (NBS) of cystic fibrosis (CF) was implemented throughout the whole of France in 2002, but it had been established earlier in three western French regions. It can reveal atypical CF with one or two known CFTR mild mutations, with an uncertain evolution. The sweat test can be normal or borderline. In Brittany, from 1989 to 2004, 196 CF cases were diagnosed (1/2885 births). The incidence of atypical CF diagnosed by NBS is 9.7% (19 from 196). The outcome of 17 (2 lost of view) has been studied, with 9 other atypical CF cases diagnosed by NBS in two other regions. The follow-up period extends from 0.25 to 19.8 years (NBS implemented in Normandy in 1980) with mean age 4.6 years. The most frequent mild mutation is R117H ISV8−7T (50%). At the time of the last visit, nutritional status is normal. All these CF patients are pancreatic sufficient. Only one patient exhibits respiratory infections, whereas 7 others have them intermittently. Two of them had intermittent Pseudomonas aeruginosa colonization at 2.8 and 6.5 years. Mean Shwachman score is 96.7, mean Brasfield score is 22.8. Eight children have had lung function tests (mean follow-up of 10 years): mean FVC was 99% of predicted, mean FEV1 101%, but one of them has FEV1 of 48%. Predicting the phenotype of these atypical CF patients remains difficult, thus complicating any genetic counselling. A regular clinical evaluation is necessary, if possible by a CF unit, because CF symptoms may appear later.

Blood concentrations of pancreatitis associated protein in neonates: relevance to neonatal screening for cystic fibrosis

OBJECTIVE To evaluate parental stress after a false-positive result at the time of the cystic fibrosis (CF) newborn screening (NBS), attributable to heterozygotism or persistent hypertrypsinemia. STUDY DESIGN A prospective study was conducted in 86 French families at 3, 12, and 24 months after NBS. A psychologist conducted interviews with a questionnaire, the Perceived Stress Scale, and the Vulnerable Child Scale. RESULTS Overall, 96.5% of parents said they had been anxious at the time of the sweat test. However, 86% felt entirely reassured 3 months after the test. The mean Perceived Stress Scale score did not differ from that observed in the French population. Mean Vulnerable Child Scale scores were high, associated with a low Parental Perception of Child Vulnerability. These results did not differ significantly at 1 and 2 years. In total, 86% to 100% of families no longer worried about CF. All parents stated that they would have the test performed again for another child. CONCLUSIONS CF NBS can lead to false-positive results, causing parental anxiety, which quickly decreases after a sweat test performed soon after the phone call.

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

AIM To determine whether pancreatitis associated protein (PAP) is a marker for cystic fibrosis which could be used in neonatal screening for the disease. METHODS PAP was assayed on screening cards from 202 807 neonates. Babies with PAP ⩾ 15 ng/ml, or ⩾ 11.5 ng/ml and immunoreactive trypsinogen (IRT) ⩾ 700 ng/ml were recalled for clinical examination, sweat testing, and cystic fibrosis transmembrane regulator (CFTR) gene analysis. RESULTS Median PAP value was 2.8 ng/ml. Forty four cases of cystic fibrosis were recorded. Recalled neonates (n=398) included only 11 carriers. A receiver operating characteristic curve analysis showed that PAP above 8.0 ng/ml would select 0.76% of babies, including all those with cystic fibrosis, except for one with meconium ileus and two with mild CFTR mutations. Screening 27 146 babies with both PAP and IRT showed that only 0.12% had PAP > 8.0 ng/ml and IRT > 700 ng/ml, including all cases of cystic fibrosis. CONCLUSION PAP is increased in most neonates with cystic fibrosis and could be used for CF screening. Its combination with IRT looks promising. Key messages CF neonatal screening with PAP is technically feasible in the same environment as other neonatal screenings (PKU, hypothyroidism) CF neonatal screening with PAP alone performs similarly to screening with IRT alone, with less carrier detection Combining PAP with IRT for CF neonatal screening could be as efficient as the IRT/DNA strategy, but would be cheaper and incur limited carrier detection If a legal requirement for informed consent before DNA testing has expired, the PAP/IRT strategy would be an alternative to the IRT/DNA strategy

Spatial and temporal distribution of cystic fibrosis and of its mutations in Brittany, France: a retrospective study from 1960

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 ≥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.