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 CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes.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.

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.

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.

[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.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

Analysis of the polymorphism of the tumour necrosis factor (TNF) gene and promoter and of circulating TNF-alpha levels in heart-transplant patients suffering or not suffering from severe rejection.

Plasma TNF-alpha levels are generally higher in heart-graft patients who experience a rejection episode than in those who do not. Because the TNF gene and its promoter are polymorphic, we studied the relationships between genetic variability at the TNF locus, the occurrence of graft rejection and TNF-alpha plasma levels in 62 heart-transplant patients in order to investigate inter-individual differences in plasma TNF-alpha levels after allogeneic stimulation. TNF-alpha was immunoenzymatically measured in blood specimens collected on the same day as endomyocardial biopsy. After PCR amplification of DNA, NcoI and AspHI polymorphisms were characterized by their restriction profiles, TNFa microsatellites by electrophoretic separation on acrylamide and the promoter region by sequencing. Plasma levels and molecular genetic results were compared to the grade of heart graft rejection established according to pathological criteria. In our study, allograft rejection was associated neither with NcoI or AspHI polymorphism nor with nucleotide changes in the TNF-A promote. We observed low TNF-alpha levels in n1/n1 homozygous patients and in subjects with G-->A at position--308 of the promoter sequence. Concerning the polymorphism of the TNFa microsatellite, our results might suggest an association with graft rejection but we have to be very careful in drawing conclusions because of the small size of the sample.

Evaluation of plasma levels of tumour necrosis factor alpha and interleukin-6 as rejection markers in a cohort of 142 heart-grafted patients followed by endomyocardial biopsy.

The rejection reaction after cell or organ transplantation has to be detected as early as possible in order to conduct optimal immunosuppressive treatment. Among the numerous events leading to rejection, cytokine production, especially of tumour necrosis factor alpha, is particularly important. Interleukin-6 and tumour necrosis factor alpha were investigated in 142 heart-grafted patients in order to define an early peripheral non-invasive marker of an acute rejection that could fit well with myocardial biopsy results. Cytokines were immunoenzymatically measured in blood specimens collected on the day of the endomyocardial biopsy. The values were compared to the grade of heart graft rejection established according to pathological criteria. Plasma interleukin-6 and especially tumour necrosis factor alpha determined on the day of the rejection diagnosis were significantly increased in the patient sample with moderate or severe rejection when compared with mean values of interleukin-6 and tumour necrosis factor alpha in the patient sample without rejection or with mild rejection (P = 0.04 and 0.001 respectively). Because high levels of tumour necrosis factor alpha may appear before histological signs, this biological marker could be useful in the follow-up of heart-grafted patients.

Laryngeal and oropharyngeal cancer, and alcohol dehydrogenase 3 and glutathione S-transferase M1 polymorphisms.

In this study the GSTmu phenotype and ADH genotype at the ADH3 locus were investigated in a group of 39 alcoholic men with upper respiratory/digestive tract cancer: 21 with oropharyngeal cancer and 18 with laryngeal cancer. The results are compared with those of a control group of 37 alcoholic men without alcohol-related medical complications. Of the control subjects, 48% were found to be GSTmu deficient [GSTmu(-)] and 19% carried the ADH(3)1/ADH(3)1 genotype. In the laryngeal cancer patients, a significantly elevated frequency of both the GSTmu(-) (78%) and ADH(3)1/ADH(3)1 genotype (56%) was observed, relative to the control group. On the basis of this result, the risk of laryngeal cancer associated with the GSTmu(-) and ADH(3)1/ADH(3)1 genotypic combination within the population of alcoholics was estimated to be 12.9 with a 95% confidence interval of 1.8-92 (P < 0.01) relative to alcoholic individuals who have GSTmu [GSTmu(+)] and are not ADH(3)1/ADH(3)1. Thus, alcoholics who are GSTmu(-) and ADH(3)1/ADH(3)1 have at least an 80% greater risk of developing laryngeal cancer than alcoholics who are GSTmu(+) and who are not ADH(3)1/ADH(3)1. In addition, the oropharyngeal cancer patients had excess frequencies of both GSTmu(-) (62%) and ADH(3)1/ADH(3)1 (43%) relative to the control group, but these excess frequencies were not statistically significant. The GSTmu(-) and ADH(3)1/ADH(3)1 genotypic combination may be a constitutional risk factor for laryngeal cancer among alcoholics.

Plasma cytokines in graft vs host disease and complications following bone marrow transplantation.

A cohort of 201 autologous or allogeneic bone marrow transplanted (BMT) patients were included for studying the evolution of circulating tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) by means of repeated plasma determinations. IL-6 levels were high during major transplant-related complications (TRC) or severe graft vs host disease (GVHD). High levels of TNF-alpha seemed to be associated with chronic GVHD but not with acute GVHD or TRC.

[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.

Improved methods for genotype determination of human alcohol dehydrogenase (ADH) at ADH 2 and ADH 3 loci by using polymerase chain reaction-directed mutagenesis.

The human gene for producing alcohol dehydrogenase (ADH; EC 1.1.1.1) is polymorphic at ADH 2 and ADH 3 loci. Until now, the study of this polymorphism required liver biopsy or allele-specific radioactive probes. We have used directed mutagenesis by the polymerase chain reaction (PCR) to amplify and analyze the genotype of ADH 2 and ADH 3 loci. Thus, we could determine easily and unambiguously the complete genotype at these two loci by using a microsample of blood and restriction fragment length polymorphism after DNA amplification by PCR.

[Nutritional anemias of iron deficiency origin: etiological approach, means of control adapted to Vietnam]. / Anémies nutritionnelles d'origine ferriprive: approche étiologique, moyens de lutte adaptés au Viet-Nam.

Among nutritional anaemias caused by the lack of erythropoietic nutriments, iron deficiency is the most frequent. In developing countries, it mainly comes from supply deficiencies. It can also be due to multiple and successive pregnancies, to pathological iron loss, to absorption defects. The study of the iron content of the main kinds of nutriments available in Vietnam and of their absorbability allows nutritionists to give useful advice to the population. Other means (use of drugs, foodstuffs enrichment) together with nutritional and health education are essential to fight efficiently against this major nutritional disease.