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.

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.

[Study of an optimal biological profile for demonstrating pancreatic involvement in acute abdominal syndrome]. / Recherche d'un profil biologique optimal pour la mise en évidence d'une atteinte pancréatique devant un syndrome abdominal aigu.

The authors have studied several seric, plasmatic and urinary constituents in patients hospitalized for an acute abdominal syndrome to be able to characterize an eventual pancreatic lesion; mainly seric and urinary amylase as well as its isoenzymes, lipase, liver profile and trypsin. In acute pancreatitis, the means of the maximal increases of seric amylase, lipase and trypsin are respectively: 10.7; 21.6 and 19.2 X N (upper normal limit) whereas in chronic pancreatitis, these elevations are 6.5 X N for amylase and 9.5 XN for lipase. The authors observed at J1 (first day of hospitalisation) and at J2 an increase in seric amylase, lipase and/or liver profile respectively in 95, 90 and 25 p. cent of acute pancreatitis; in 86, 86 and 14 p. cent of chronic pancreatitis and 43, 39 and 86 p. cent of bili duct diseases. In conclusion, it appears compulsory to run a liver profile with the pancreatic enzymes (amylase and lipase) to diagnose a pancreatitis in presence of an acute abdominal syndrome.

[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.

[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).

[Maternal alpha-fetoprotein. Its levels in the eluate of dried blood for screening antenatally defects in the closure of the neural tube (author's transl)]. / L'alpha-foetoprotéine maternelle. Dosage dans l'éluat de sang séché pour le dépistage anténatal des défauts de dermeture du tube neural.

Taking levels of alpha-feto-protein serially from the mother's blood between the 16th and 18th week of pregnancy gives rise to the possibility of screening more than 80 per cent of cases of anancephaly and spina bifida. Because of the seriousness and the frequency of these fetal malformations (12 in 10,000 deliveries) in our district, routine antenatal screening would be worth while. Because of this we suggest that a radioimmune technique for measuring the levels of AFP in dried blood eluate should be carried out. It is as reliable as measuring the levels in blood serum. The way of obtaining the sample that is used makes it suitable for mass screening. We have established the normal values of AFP measured by our method between the 15th and 22nd weeks of pregnancy. These make it possible to define the upper limits of warning that are suitable for screening on a regional basis. During this study we found a case of anencephaly in which the level of maternal AFP was significantly higher than the limits we had worked out.

[Systematic neonatal screening for mucoviscidosis using an immunoreactive trypsin blood assay. Evaluation of 80,000 tests]. / Dépistage néonatal systématique de la mucoviscidose par dosage de la trypsine immunoréactive sanguine. Bilan de 80 000 tests.

The development of a simple and reliable technique of trypsin radioimmunoassay in blood eluates allowed the neonatal screening for cystic fibrosis in a province of France. The study of the distribution of blood immunoreactive trypsin levels in 79,880 five day-old neonates led to choose a cut-off value of 900 micrograms/l; this choice gives a false positive rate of 3 in 1,000. The evolution of trypsin levels according to age during the first weeks of life leads to select a "two-phase" screening strategy: only neonates whose high neonatal trypsin levels persist after the second week of life (4 in 10,000) will undergo sweat test. Twenty-three cases of cystic fibrosis were diagnosed in this study, which gives an incidence of 1 in 3,470.

[Radioimmunoassay of trypsin in dried blood. Importance for the neonatal detection of cystic fibrosis (author's transl)]. / Dosage radio-immunologique de la trypsine dans le sang séché. Intérêt pour le dépistage néonatal de la mucoviscidose.

The demonstration of very high levels of immunoreactive trypsin in the blood of newborn infants with cystic fibrosis has provided a new way of detecting the disease soon after birth. A radioimmunoassay of trypsin in the eluate of blood dried on filter paper has now been developed. The sensitivity and accuracy of the method, as well as the good correlation observed between the values obtained and those of the conventional plasma assay, indicate that it is reliable and well adapted to the newborn. The new assay can easily be inserted into the present system of neonatal disease detection. A preliminary assessment of more than 5,000 tests enables the authors to report an early diagnosis of proven cystic fibrosis and to discuss an essential aspect of mass-detection methods: the indicence of false-positive results.

Trypsin-binding immunoglobulin G in cystic fibrosis, allergic, and normal children.

Using a simple and sensitive radioassay, we have determined the frequency of trypsin-binding (TB) immunoglobulin G (IgG) as a function of age in cystic fibrosis (CF) patients, in allergic patients, and in control subjects. These IgGs appear during the 1st years of life. Their frequency is maximum between 3 and 20 years of age and decreases during adulthood. In allergic children TBIgG appears later than in CF children and the maximum frequency (32%) is intermediate between those observed in CF and in control children (66 and 19.6%, respectively). We suggest that an endogenous form of trypsin is involved in the phenomenon.

Free thyroxin measured in dried blood spots: results for 10 000 euthyroid and 29 hypothyroid newborns.

We measured free thyroxin (FT4) in dried blood samples from 10 000 euthyroid and 29 hypothyroid newborns (three with transient hypothyroidism and 26 permanent). In euthyroids, the mean FT4 concentration was 16.9 (SD 4.5) pmol/L. Preterm babies had low concentrations of FT4, and values were closely related to birth weight when less than 2500 g. Assay of blood-spot FT4 appears to be specific as a test for hypothyroidism screening, yielding 0.42% false positives when a mean minus 2 SD cutoff value (8 pmol/L) was used. Correcting FT4 for birth weight further decreased the false-positive rate, to 0.07%. In all three newborns with transient hypothyroidism, FT4 was less than 8 pmol/L. In 25 of 26 permanent hypothyroids, FT4 ranged from undetectable to 5 pmol/L; in the 26th infant, who had a large ectopic gland, it was 8.2 pmol/L. We believe that FT4 assay offers an attractive improvement over total T4, whether performed as the first screening test or as a confirmatory test in thyrotropin screening programs.

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.