[National neonatal screening program for cystic fibrosis: management and organization]. / Le programme national de dépistage néonatal de la mucoviscidose: mise en place et organisation.

France has decided to add to the national neonatal screening program (Phenylketonuria, Hypothyroidism, Congenital Adrenal Hyperplasia, Sickle cell disease) the screening of cystic fibrosis (CF). The screening of CF will be implemented in all regions of France by the end of 2002 and will cover all newborn (near 800,000/year). Based on the recommendation of the French Screening Foundation, the project has been approved by the Health Ministry and will be financed by the social security. CF neonatal screening is now technically feasible and reliable. The proposed methodology includes: immunoreactive trypsin (IRT) dosage on all newborns at day 3 (by radioimmunology "Cis Bio" or immunofluorescence "Delfia") followed by genotype CFTR analysis if IRT level is above 60 micrograms/L. Screening for 29 mutations is planned. If genotype is negative, control of IRT at day 21 will be obtained. Several requirements are included in the program: a protocol of care for the newly diagnosed CF in a specialised CF center; information to all parents of newborns; results of CFTR genotype has to be given during a clinical visit, even if negative. This screening program should allow to screen 98% of the cystic fibrosis patients before the age of 1 month. In order to ensure perfect efficacy, the CF screening program will be evaluated and modified if necessary.

[Evaluation of diagnosis and follow-up in screened children with cystic fibrosis in Normandy]. / Evaluation du diagnostic et du suivi de la cohorte normande d'enfants dépistés atteints de mucoviscidose.

The neonatal screening programme in Normandy (France) allowed the formation of a homogenous cystic fibrosis (CF) cohort of 150 children diagnosed between 1980 and 1997. At the time of this retrospective study, 11 were deceased, out of which nine had meconium ileus (eight deaths after surgery, one at 5 years of age). Sixty children born between 1980 and 1993 in the Basse-Normandie region were followed up during a mean 80 months following similar protocols. The mean age at diagnosis was 41 days (SD = 27 d) for infants without meconium ileus. The occurrence of Pseudomonas aeruginosa (P. aeruginosa) infection and chronic colonization was studied using a monovariate followed by a multivariate analysis including the following variables: sex; meconium ileus; anthropometric data at birth and at diagnosis; pancreatic insufficiency; radiological data (Brasfield score); microbiology data at diagnosis; and genetic data. P. aeruginosa infection appeared earlier in children with pancreatic insufficiency (OR = 2.2; p < 0.05) or with radiological abnormalities (Brasfield score < 21) at diagnosis (OR = 3.9; p < 0.05). Meconium ileus (OR = 5.3; p < 0.01), pancreatic insufficiency (OR = 3.8; p < 0.01) and Brasfield score < 21 at diagnosis (OR = 5.6; p < 0.001) were prognosis factors for early chronic P. aeruginosa colonization. In CF children without meconium ileus, the major risk factor found through multivariate analysis for earlier infection and for earlier chronic colonization by P. aeruginosa was a diagnosis delay > 40 days (respectively OR = 4.6; p < 0.001 and OR = 10.4; p < 0.005). These results must be compared with the lower Brasfield score at diagnosis in infants diagnosed after 40 days of life (p < 0.01).

Blood immunoreactive trypsinogen concentrations are genetically determined in healthy and cystic fibrosis newborns.

Newborns with cystic fibrosis (CF) have increased blood immunoreactive trypsinogen concentrations. When screening for CF in the newborn by immunoreactive trypsinogen measurement, an abnormally high proportion of healthy deltaF508 carriers is found among false-positive neonates, suggesting that a relationship could exist between immunoreactive trypsinogen concentration at birth and the genetic status. Therefore, this study analysed the possible relationships between neonatal blood immunoreactive trypsinogen concentrations and genotype in 1842 healthy newborns and 111 CF patients detected by a neonatal screening programme. A close correlation was found between immunoreactive trypsinogen and deltaF508: the probability of a healthy newborn being a carrier of this mutation increased regularly with the neonatal immunoreactive trypsinogen concentration. In CF patients, there was a significant difference between deltaF508 homozygotes and deltaF508/X (X = other mutation) compound heterozygotes with respect to the mean neonatal blood immunoreactive trypsinogen concentration. CF neonates with two mutations affecting the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator protein had significantly higher mean immunoreactive trypsinogen concentrations than patients with one mutation affecting a membrane-spanning domain. The data strongly suggest that the neonatal immunoreactive trypsinogen concentration is, in part, genetically determined, with a wide range of variations, similar to the features which have been shown for the relations between the genotype and clinical phenotypes of CF patients.

Neonatal screening for cystic fibrosis: present and future.

Despite there being effective tests for detecting cystic fibrosis (CF) using newborn screening blood samples, screening in neonates has not had universal approval because of uncertainty about its benefits. After up to 18 y experience, at a recent conference in Caen several aspects attracted universal agreement. There is still major delay in clinical diagnosis after the onset of symptoms. There is short-term benefit in early diagnosis by screening, with reduced morbidity in the first 2 y, evidence of significant nutritional benefits up to the age of 10 y, and probable respiratory benefit over this time frame. There is great potential for research into treatment modalities and no evidence of significant psychological harm to CF babies from early diagnosis. With a screening protocol that includes a DNA test there is some unwanted carrier detection and careful genetic counselling is needed. There is no evidence yet that screening will extend the life of CF patients, so some doubts remain as to its overall effectiveness, and there have been no good studies on comparative costs in screened and unscreened cohorts. Even so, the weight of evidence suggests very worthwhile advantages for screened babies and their families. Because of this, it is unlikely that further trials will take place. It may be that the onus now is on those who do not support screening to justify this stance to parents who may favour it.

The A985 to G mutation of the medium-chain acyl-CoA dehydrogenase gene and sudden infant death syndrome in Normandy.

Medium-chain acyl-CoA dehydrogenase deficiency is the most common genetic defect of hepatic fatty acid oxidation. Clinical signs are somnolence and lethargy potentially leading to coma. Death occurs during the first attack in about 20% of cases, suggesting sudden infant death syndrome. A point mutation (adenine to guanine at position 985) in exon 11 of the medium-chain acyl-CoA dehydrogenase gene accounts for 90% of medium-chain acyl-CoA dehydrogenase deficiency-causing alleles. Such a high prevalence of a single mutation makes it possible to estimate the incidence of medium-chain acyl-CoA dehydrogenase deficiency in the general population and in sudden infant death syndrome. The study was performed by polymerase chain reaction amplification from blood spots on filter paper in 2000 randomly selected newborns (group I) and in 225 infants dead from sudden infant death syndrome (group II). Among 2000 newborns, 17 were found to be heterozygote for the G985 mutation. In group II, one child was found with a single copy of the G985 mutation. So, the estimated frequency of the G985 mutation in the general population was 1/118 and the incidence of medium-chain acyl-CoA dehydrogenase deficiency was calculated as around 1/45,000 in Normandy.

[Screening of A985 to G mutation of medium-chain acyl-CoA dehydrogenase (MCAD) gene in Normandy. Evaluation of the role of MCAD deficiency in sudden infant death]. / Dépistage de la mutation A985-->G du gène de la medium-chain acyl-CoA deshydrogénase (MCAD) en Normandie. Evaluation du rôle du déficit en MCAD dans la mort subite du nourrison.

MCAD deficiency is recognized as the most common hereditary defect of hepatic fatty acid oxidation. Clinical signs are somnolence progressing to lethargy potentially leading to coma. Death is the outcome of the first attack in about 20% of cases, suggesting sudden infant death syndrome (SIDS). A point mutation (adenine to guanine at position 985) in exon 11 of the MCAD gene represents 90% of alleles causing MCAD deficiency. The high prevalence of this mutation allows the estimation of the incidence of MCAD deficiency in the general population and in SIDS. The study was performed after PCR amplification from blood spots on filter paper in 1,432 randomly selected newborns (group I), in 225 SIDS (group II) and in 47 infants of SIDS family (group III). In group I, 10 newborns were found to have the G985 mutation in the heterozygous form. In group II, among 225 SIDS cases, the G985 MCAD mutation was found once in the heterozygote state. In group III, the mutation was not found. The estimated frequency of the mutation was 1/143 in the reference group and the incidence of MCAD deficiency was calculated as 1/67,000 in Normandy.

Retrospective study of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Guthrie cards from a large cohort of neonatal screening for cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride channel, and in individuals with both alleles of the gene mutated, symptoms of CF disease are manifest. With more than 300 mutations so far described in the gene the profile of mutant alleles in a population is specific to its ethnic origin. For an analysis with an unbiased recruitment of the CF alleles in neonates of similar origin (Normandy, France), we have retrospectively analyzed the Guthrie cards of affected newborns, diagnosed by the immunoreactive trypsinogen (IRT) assay. Analysis of the 27 exons of the CFTR gene using a GC clamp denaturing gradient gel electrophoresis (DGGE) assay has enabled us to identify over 96% of the mutated alleles. Two of these were novel mutations. We would like to propose this strategy as an efficient method of retrospective molecular genetic diagnosis that can be performed wherever Guthrie cards can be obtained. Knowledge of rare alleles could be a prerequisite for CF therapy in the future.

[Cystic fibrosis gene mutations in the West of France: clinical application]. / Les mutations du gène de la mucoviscidose dans l'ouest de la France: application clinique.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for the cystic fibrosis phenotype when both alleles are mutated, was cloned and sequenced in 1989. Since then, more than 400 mutations have been reported in the gene, although most of these are rare. We have systematically analysed the entire coding sequence of the CFTR gene in a cohort of patients originating from the West of France (Caen, Brest and Nantes). More than 450 CF children, 914 chromosomes in all, have been exhaustively studied in the three centers. We have been able to characterize more than 90% of the mutations, respectively 93.5%, 99% and 95.8%. Despite the large diversity in the CFTR mutations occurring in CF patients from this area, these results can help to improve genetic counselling, prenatal diagnosis as well as our understanding of the molecular basis of the pathophysiology of cystic fibrosis.

Food-dependent Cushing's syndrome mediated by aberrant adrenal sensitivity to gastric inhibitory polypeptide.

BACKGROUND: Some patients with Cushing's syndrome have nodular adrenal hyperplasia. In most the disease is corticotropin-dependent, but in others it is corticotropin-independent. The cause of the adrenal hyperplasia in the latter patients is not known. METHODS: We studied a 49-year-old woman with Cushing's syndrome and nodular adrenal hyperplasia in whom food stimulated cortisol secretion. Plasma cortisol concentrations were measured in response to the ingestion of mixed meals, glucose, protein, and fat and after the administration of various gastrointestinal and other types of hormones. We also studied the ability of the long-acting somatostatin analogue octreotide to prevent the food-induced increase in plasma cortisol concentrations and to ameliorate the clinical manifestations of Cushing's syndrome in this patient. RESULTS: The patient's fasting plasma cortisol concentrations were subnormal, ranging from 3.0 to 7.5 micrograms per deciliter (83 to 207 nmol per liter), and they increased to as high as 16.5 micrograms per deciliter (455 nmol per liter) after a mixed meal. Her urinary cortisol excretion ranged from 164 to 250 micrograms per day (453 to 690 nmol per day) and could not be suppressed by a large dose of dexamethasone. Plasma corticotropin concentrations were virtually undetectable at all times. The ingestion of glucose, protein, and fat increased plasma cortisol concentrations to 3.6, 2.2, and 4 times the base-line value, respectively; the meal-induced and glucose-induced increases were inhibited by octreotide. The infusion of gastric inhibitory polypeptide (GIP) increased the patient's plasma cortisol concentration to 3.7 times the base-line value, but had no effect in normal subjects. The patient's fasting plasma GIP concentrations were normal both before and after a meal, and there was a close correlation between her plasma cortisol and GIP concentrations. Treatment with octreotide decreased urinary cortisol excretion and ameliorated the clinical manifestations of Cushing's syndrome. CONCLUSIONS: The development of aberrant adrenal sensitivity to GIP can result in food-dependent adrenal hyperplasia and therefore in Cushing's syndrome.

Free thyroxin measured in dried blood spots from normal, low-birth-weight, and hypothyroid neonates.

We have adapted a new radioimmunoassay for free thyroxin (FT4) measurement in dried blood spots for use in neonatal screening for hypothyroidism. The method is easy, fast, and cheap. Within-assay and between-assay CVs are respectively 9.6% and 13.2%. In 997 neonates three days postpartum with normal thyrotropin concentrations, the mean FT4 concentration was 27.2 pmol/L (SD 7.3 pmol/L). There was no significant difference in mean FT4 concentration between boys and girls. FT4 concentrations increased linearly with birth weight or with gestational age, as expressed by multiple linear regression: FT4 (pmol/L) = 0.0016 birth weight (g) + 0.6931 gestational age (weeks) - 4.8772. Only gestational age significantly affected the FT4 value. For five hypothyroid infants tested on day three postpartum, FT4 values were all below the 1st percentile of values from healthy neonates. Thus, when the neonatal concentration of thyrotropin is above normal, FT4 measured in the same sample can provide a reliable earlier diagnosis of hypothyroidism.

The application of PCR amplification and the polymorphic marker KM.19 to dried blood spots: comparison with deletion 508 for the confirmation of the neonatal screening test for cystic fibrosis.

Cystic fibrosis (CF) screening by measurement of immunoreactive trypsin (IRT) lacks specificity: only 9% of hypertrypsinemic neonates have CF. We have studied retrospectively 114 hypertrypsinemic samples (including 37 CF) for KM.19 polymorphic DNA marker and made risk calculations. If the neonate is homozygous for KM.19 allele 2, the risk of CF rises to 55%; if homozygous for allele 1, the risk is very low (less than 1%) and if heterozygous, the risk is intermediate (4%). In a prospective study including 28,000 IRT tests, 76 neonates with IRT greater than 800 micrograms/L have been identified: 16 were homozygous for allele 2 (8 CF), 30 for allele 1 (1 CF), and 30 were heterozygotes (no CF). Deletion 508 was present in 10 neonates: 4 homozygotes (4 CF) and 6 heterozygotes (3 CF). Two CF did not carry any copy of deletion 508. We have studied 181 (presumably non-CF) neonates with IRT greater than 600 micrograms/L. The KM.19 genotypes distribution is significantly different from the one expected in the French population: homozygotes for allele 2 are more numerous. Furthermore, heterozygotes for deletion 508 are 1 in 15 (expected: 1 in 42). In conclusion, molecular biology in dried blood spots can enhance the specificity of CF neonatal screening, but IRT and genotype may not be independent.