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

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 (p(R117H)), on either intron 8 T5 or T7 background, and F508del (p(F508del)) 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, p(F508del) was 1.06%, p(R117H;T7) 0.27% and p(R117H;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 CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes.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.

Analysis of the effect of aluminum in drinking water and transferrin C2 allele on Alzheimer's disease.

Although highly controversial, the hypothesis of a link between aluminum (Al) in drinking water and Alzheimer's disease (AD) has been supported by several epidemiological studies. Transferrin (Tf) is a major transport protein for both iron and Al. Moreover, it has been demonstrated that defective binding of iron and Al to the Tf variant C2 could be present in AD. Individuals carrying the Tf C2 allele might therefore be at greater risk of developing AD. We investigated whether the Tf C2 allele might be responsible for susceptibility to AD in a sample of 292 subjects (with 55 AD) aged > or = 75 years from south-west France, some exposed to high levels of Al in tap water (n = 181 subjects) and others to low levels of Al (n = 111 subjects). We also examined the combined genetic effects of Tf C2 and epsilon4 allele of apolipoprotein E gene (ApoE). Logistic regression analysis showed that neither Tf C2 nor its interaction with Al or with the epsilon4 allele of the ApoE were significantly associated with the risk of AD.

Primary sequence of the beta-chain of Badger haemoglobin.

Badger (Meles meles) haemoglobin was purified by paper electrophoresis and converted into globin. Chain separation was carried out on a CM-cellulose column in the presence of 8 M urea. The beta-chain was aminoethylated, purified by gel filtration and submitted to tryptic digestion. A fingerprint obtained with the enzymic digests showed 17 distinct ninhydrin-positive spots from which 20 pure peptides were isolated by further electrochromatographic separations. These peptides were sequenced using Dansyl-Edman and Ptc-Edman degradation techniques. The presence of amide residues was confirmed after aminopeptidase M hydrolysis. Taking human haemoglobin beta-chain as a model, the covalent structure could be completely resolved without the help of any further overlapping technique. The following substitutions were noted (badger/human, position): Ala/Pro5, Ser/Ala13, Tyr/Phe41, Asp/Glu43, Ser/Ala70, Glu/Asp73, Lys/Ala76, Asn/His77, Lys/Thr87, Lys/Arg104 and Gln/Pro125. A comparison with other haemoglobin beta-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of beta-chain of known structure in the order of Carnivores.

Identification of a new apolipoprotein E variant (E2 Arg142-->Leu) in type III hyperlipidemia.

A new rare apolipoprotein E mutant was identified as we were investigating the apolipoprotein E genotype of patients with type III hyperlipidemia (HLP III). The unusual DNA restriction fragment length polymorphism profile and then the sequence analysis of a PCR amplified fragment of the proband's apo E gene revealed a simple base substitution (G-->T) at nucleotide 3836. This mutation leads to the replacement of arginine by leucine at position 142 of the mature protein. The proband carried the mutant allele at the heterozygous status with an epsilon 3 allele. Subsequently, analysis of the proband's father's apo E gene showed that same mutated allele associated with an epsilon 2 allele. The two subjects presented a dysbetalipoproteinemia in which this new apo E variant could be implicated.

Effect of lipid infusion on postabsorptive glucose metabolism in non-insulin-dependent diabetic patients.

The effect of a lipid infusion on postabsorptive glucose metabolism was investigated in non-insulin-dependent diabetes mellitus (NIDDM) patients. During a basal period, plasma free fatty acids (FFA) and triglycerides, postabsorptive glycemia and insulinemia, and glucose turnover and metabolic clearance rates (MCRs) were measured in 10 NIDDM patients. After the basal measurement, five patients received a lipid infusion, and the others received saline. Lipid infusion prevented the decrease in postabsorptive glycemia observed in the saline group (P < .01). The principal mechanism for this effect was a reduction in glucose MCR (30.8 +/- 5.7 v 47.2 +/- 3.4 mL/m2/min, P < .05), which was absent in control tests (50.4 +/- 8.1 v 47.9 +/- 4.7 mL/m2/min, NS). The absence of the compensatory insulin release observed in normal subjects may play a role in this response. The prolonged postabsorptive hyperglycemia induced by lipid infusion in NIDDM patients is further evidence for the detrimental effect of the lipid-carbohydrate interactions described by Randle.

Assessment of vitamin and trace element supplementation in severely burned patients undergoing long-term parenteral and enteral nutrition.

The efficacy of an oral supplement of vitamins and trace elements during a longterm artificial parenteral and enteral nutrition was investigated for 3 months in patients with extensive burns. Thirty severely burned patients (22 male, 8 female, age 41 +/- 18 years, range 23-59 years, 33 +/- 12% total body surface area burn, 22% +/- 8 full thickness burn surface area) were included. Every 10 days, from day 10 until day 90, we determined serum levels of: *vitamins B1, B12, A, E, *folic acid, *copper, zinc, iron, *transferrin, albumin, prealbumin, total proteins, *fibronectin, retinol binding protein (RBP), *calcium, *phosphorus, *triglycerides, *total cholesterol, *C reactive protein (CRP), *erythrocyte folic acid. The mean daily nutritional support was 60 Kcals and 0.4 g N per kg of body weight, 70% enterally and 30% parenterally administered, with enteral vitamin and trace element supplementation. On day 10, there was a decrease of the serum level of 19/20 parameters. For 8 parameters (vitamin A, total cholesterol, iron, transferrin, fibronectin, phosphorus, RBP, total proteins), the level was lower than usual. Between day 10 and day 20, a significant normalization of 6 of them was noted, the average levels of transferrin and iron remaining below normal values until day 50. There was a significant decrease in C-reactive protein levels, however above normal limits. No deficiency in vitamins or trace elements was found. Cyclic variations of serum levels occurred which may be more related to volemic, hydroelectrolytic, endocrine and inflammatory disorders than to nutritional problems.

Morphologic structure of bile canaliculi after bile duct ligation in the rat. A time-course study.

Changes in bile canaliculi (BCs) after bile duct ligation (BDL) are heterogeneous throughout the liver lobule. Male Wistar rats were killed five hours, 20 hours, four days, or eight days after BDL, and BCs were counted according to their shape (1, normal; 2, elongated with microvilli; 3, dilated, devoid of microvilli; or 4, filled with blebs). Compared with controls, the number of BCs doubled after BDL, less than 10% of the BCs were of type 3. As time elapsed, the ratios of types 3 and 4 increased; however, their combined ratio always was below 50%, and remained stable between days 4 and 8. Bilirubin levels peaked between days 2 and 4, then remained stable. The heterogeneous changes in BCs could be explained by the heterogeneous tightness of tight junctions. Decrease in cell size and increase in the size of BC branches could favor contact between bile and blood.

[Optimization of a spectrophotometry assay of total and oxidized blood glutathione: comparison with a fluorimetric method]. / Optimisation du dosage spectrophotométrique du glutathion sanguin total et oxydé: comparaison avec une méthode fluorimétrique.

We developed a method for the enzymatic assay of glutathione which is easy to practice, rapid, specific, based on the reaction of the thiol group of glutathione with dithiobis-nitrobenzoic acid after the action of glutathione reductase in the presence of NADPH. This spectrophotometric technique allowed, on the one hand, the determination of total glutathione and on the other hand, that of oxidized glutathione (disulfide), after the blockage of reduced glutathione by 2-vinyl-pyridine. The improvements of the assay of blood glutathione concerned the sample preparation, the reaction sensitivity, thanks to a better definition of the optimal pH and a reduction ot the blockage time by 2-vinyl-pyridine in well defined operating conditions. We compared the performances of our technique with a fluorimetric method. We used our method for the determination of total and oxidized blood glutathione in a control population.

[CFTR gene analyis in 207 patients with cystic fibrosis in southwest France: high frequency of N1303K and 1811+1.6bA>G mutations]. / Etude du gène CFTR chez 207 patients du Sud-Ouest de la France atteints de mucoviscidose: fréquence élevée des mutations N1303K et 1811 + 1,6 kbA > G.

UNLABELLED: The large molecular heterogeneity in cystic fibrosis (CF) represents the main difficulty for the genotype characterization. Moreover, numerous studies have reported considerable variations in frequencies of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in different populations. MATERIAL AND METHODS: We analyzed the genotype of 207 CF children living in southwest France. RESULTS: Among 50 identified mutations, we report for some of them a widely modified incidence compared with those observed in other regions of France. These differences were more significant in the subset of the CF chromosomes originating in southwest France. Thus, the 1811 + 1.6 kbA > G mutation, rarely observed in the other French regions (< 0.5%), proved to be, with a frequency of 8.8%, the most frequent mutation after the F508 deletion (57%). The frequencies of N1303K, 1811 + 1.6 kbA > G and R334W mutations were also clearly increased: 7.9 and 2.6%, respectively. CONCLUSION: We show that the southwest of France is characterized by a specific mutational spectrum. We consider that these regional data on the spectrum of CF mutations are crucial to develop more accurate and less expensive molecular screening strategies for cystic fibrosis in France.

[Pathology of the human apolipoprotein E gene]. / Pathologie du gène de l'apolipoprotéine E humaine.

Apolipoprotein E (apo E) is a polymorphic glycoprotein that plays an essential part in the binding to receptors for the uptake of chylomicrons and VLDL remnants and of LDL. The three major isoforms are E3 (Cys112/Arg158), E4 (Arg112/Arg158) and E2 (Cys112/Cys158). The apo E genetic variation has a great impact. In most of type III familial hyperlipoproteinemias (HLP), E2 is implicated at the homozygote status. In other cases, rare alleles are directly responsible for dominant type III HLP. Apo E polymorphism is an essential determinant in the interindividual variations of lipids in healthy subjects in various populations. Its influence can be significant on the efficacy of nutritional or therapeutic interventions. The allele epsilon 4 appears to be associated with an increased risk of premature atherosclerosis. Recently, epsilon 4 was demonstrated to be associated with an early Alzheimer's disease onset. Apo E polymorphism contributes to the lipid disorders in diabetes and obesity. The analysis of apo E polymorphism can be carried out with two conceptually different approaches. The first one is based on the separation of plasma isoforms of the protein by isoelectric focusing or bidimensional electrophoresis. The other one consists in the application of molecular biology techniques (PCR and endonuclease restriction profiles) for a detection of the common alleles and of several rare alleles, avoiding the possible errors of the phenotyping technique of the apo E protein. The application of genetic engineering allows a better understanding of the role played by apo E towards its receptors and in other molecular interactions which are not well known up to now.

Vision screening in children by Plusoptix Vision Screener compared with gold-standard orthoptic assessment.

BACKGROUND/AIMS: To evaluate a new autorefractor, the Plusoptix Vision Screener (PVS), as a screening tool to detect risk factors for amblyopia by comparing it with gold-standard orthoptic vision screening in children. METHODS: Community-based screening study including 288 children age 4-7 years who were screened with the PVS and by orthoptic assessment (distance acuity, cover test, extraocular movements, 20 PD prism test, Lang stereotest). Follow-up comprehensive eye examination of screening-positive children included manual cycloplegic retinoscopy. RESULTS: Testability was high for both methods. Orthoptic screening identified 36 children with reduced vision and/or factors associated with amblyopia (referral rate 12.5%). The PVS identified 16 children with potential vision problems (referral rate 5.6%), indicating only moderate sensitivity (44%; 95% CI 27.9 to 61.9%), but high specificity (100%; 95% CI 98.5 to 100%) to detect factors associated with amblyopia. The PVS underestimated visually significant refractive errors. CONCLUSIONS: Use of the PVS as single screening test in young children may miss a significant number of children with amblyopia or amblyogenic risk factors.

Plusoptix Vision Screener: the accuracy and repeatability of refractive measurements using a new autorefractor.

BACKGROUND: The Plusoptix Vision Screener (PVS) is a new non-cycloplegic videoretinoscopy autorefractor. Refractive accuracy may affect its performance as a screening tool. AIMS: Study 1: To determine the intra- and interobserver variability of PVS measurements. Study 2: To compare PVS measurements with gold-standard manual cycloplegic retinoscopy (MCR). METHODS: Study 1: PVS refraction of 103 children with mean (SD) age 5.5 (0.6) years by two observers. Study 2: PVS and MCR refraction of 126 children with mean (SD) age 5.5 (1.5) years, including 43 children with manifest strabismus >/=5 PD, comparing mean spherical equivalent (MSE) and Jackson cross cylinders J(0) and J(45). RESULTS: Study 1: Repeatability coefficients (observer 1): MSE: 0.63 D, J(0): 0.24 D, J(45): 0.18 D; those of observer 2 were nearly identical. The mean difference (95% limits of agreement) between the two observers for MSE, J(0) and J(45) were, respectively, 0.03 (-0.62 to 0.68 D), -0.008 (-0.25 to 0.23 D) and 0.013 (-0.18 to 0.20) D. Study 2: MSE tended to be lower on PVS than MCR, with differences of up to 8.00 D. Less than 20% of values were within +/-0.50 D of each other. Agreement was better for J(0) and J(45). Strabismus was associated with an odds ratio of 3.7 (95% CI 1.3 to 10.5) of the PVS failing to obtain a reading. CONCLUSIONS: The PVS may underestimate children's refractive error.

Mosaic maternal uniparental isodisomy for chromosome 7q21-qter.

Uniparental disomy (UPD) for several human chromosomes is associated with clinical abnormalities. We report the case of a 2-year-old boy with severe intrauterine and post-natal growth retardation (IUGR/PNGR) and highly variable sweat chloride concentrations. The patient was identified as heterozygous for the F508del mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Unexpectedly, the signal corresponding to the maternally inherited F508del allele appeared much more intense than the paternally derived wild allele. Molecular analysis including polymorphic marker studies, microsatellites and single-nucleotide polymorphisms subsequently showed that the boy was a carrier of a de novo mosaic maternal isodisomy of a chromosome 7 segment while there was a biparental inheritance of the rest of the chromosome. This is the first report of a mosaic partial UPD7. The matUPD7 segment at 7q21-qter extends for 72.7 Mb. The karyotype (550 bands) of our patient was normal, and fluorescence in situ hybridization with probes mapping around the CFTR gene allowed us to rule out a partial duplication. The detection of this chromosomal rearrangement confirms the hypothesis that the 7q31-qter segment is a candidate for the localization of human imprinted genes involved in the control of IUGR and PNGR. It also emphasizes the importance of searching for UPD7 in severe, isolated and unexplained IUGR and PNGR.

Effect of the phosphonic analogue of tyrosine on tyrosine iodination.

Depositing ofDL-1-amino-2-(p-hydroxyphenyl)-ethylphosphonic acid (Tyr-P) on the chicken embryo induced a dose dependent decrease of the iodine uptake by the embryonic thyroid. Tyr-P interfered on iodination of tyrosine when tested with hog thyroid peroxidase (TPO) and with bovine lactoperoxidase (LPO); the analogue was recognized by the two enzymes but its affinity for TPO and LPO was respectively 3 and 7 fold higher compared with that of the natural substrate, suggesting that Tyr-P may act as an iodine trap.

Action of the phosphonic analogue of tyrosine and of other tyrosine derivatives on rat liver tyrosine aminotransferase.

Tyrosine transamination has been investigatedin vitro with a preparation of rat liver tyrosine aminotransferase in the presence of several structural derivatives of the substrate, including the phosphonic analogue. The transamination by tyrosine aminotransferase (TAT) needs the presence in the substrate molecule of free amino and carboxylic groups, a three-carbon aliphatic chain, a para-phenolic hydroxylic function and aL-configuration. Some tyrosine analogues can markedly disturb the Tyr-TAT association: the chief structural modifications are (i) the removal of the free amine function in a compound still possessing a para-hydroxylic and a carboxylic group, (ii) the change of the carboxylic function by another acidic group, especially a phosphonic one, (iii) a disubstitution in positions 3 and 5. In every situation, the presence of a parahydroxylic group is compulsory to observe an inhibitory effect.