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 CONCLUSIONS: These results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes.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

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.

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.

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.

Relationships between cholesterol, apolipoprotein E polymorphism and dementia: a cross-sectional analysis from the PAQUID study.

This study assesses the cross-sectional relationship between serum cholesterol level and dementia, controlling for apolipoprotein E (apoE) genotype, in a nested case-control study of 334 elderly French subjects aged 73 and over who participated in the PAQUID study (37 demented subjects and 297 nondemented controls). A diagnosis of dementia was established by two-step screening: (1) psychometric testing and DSM-III-R criteria and (2) neurologist's confirmation. Cholesterol, its fractions and apoE genotype were determined from a blood sample. Elevated high-density lipoprotein cholesterol was associated with a significantly decreased risk of dementia, independent of apoE status and other potential confounding variables, suggesting that cholesterol fractions could be involved in both Alzheimer's disease and vascular dementia.

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.

Compound heterozygote for both rare apolipoprotein E1 (Gly127-->Asp, Arg158-->Cys) and E3(Cys112-->Arg, Arg251-->Gly) alleles in a multigeneration pedigree with hyperlipoproteinaemia.

1. A French multigeneration pedigree with hyperlipoproteinaemia was investigated for the transmission of the rare apolipoprotein E1(Gly127-->Asp, Arg158-->Cys) variant. The proband, a 46-year-old male carrying the rare apoE1 variant, presented a severe type III hyperlipoproteinaemia like his three brothers and his sister. 2. ApoE phenotyping and genotyping showed a discrepancy in the second allele carried by the proband's wife and two of her children, thus suggesting another apoE gene mutation. Cloning and sequencing of the entire exon 4 demonstrated a point mutation at codon 251, leading to an apoE3(Cys112-->Arg, Arg251-->Gly) allele. The proband's wife was normolipaemic and heterozygous for this rare isoform and the common apoE3 protein. The rare apoE3(Cys112-->Arg, Arg251-->Gly) allele has been transmitted to her two daughters. The first, aged 19, was normolipaemic and heterozygous for this allele and the common apoE2 allele. The second, carrying both the rare isoforms apoE1(Gly127-->Asp, Arg158-->Cys) and apoE3(Cys112-->Arg, Arg251-->Gly), presented a hypertriglyceridaemia at the age of 10. 3. The exploration of apoE status associated with plasma lipid levels and lipoprotein profiles in this three-generation pedigree made it possible to describe a compound heterozygote for two mutated alleles, one mutation being located in the N-terminal domain of the apoE protein and the other arising in the C-terminal domain.

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.

Lipid-carbohydrate interactions in post-absorptive non-insulin-dependent diabetic patients.

Substrate competition is an important mechanism of insulin resistance, although its role in the post-absorptive hyperglycemia of NIDDM is not clear: lipid infusion does not raise plasma glucose levels in normal subjects, and total lipid oxidation, the elevation of which is a hallmark of disrupted carbohydrate metabolism, is normal in non-insulin-dependent diabetes mellitus (NIDDM). To examine further these two arguments against the involvement of lipid-carbohydrate interactions in the hyperglycemia of NIDDM, we compared the effect of a 3-hour lipid infusion ('Ivélip') on post-absorptive blood glucose levels, plasma lipids and respiratory exchanges in 15 patients with NIDDM, with that of an infusion of saline in 15 other patients with similar metabolic profiles. The lipid infusion significantly slowed the natural post-absorptive decline in blood glucose levels (saline -0.47 +/- 0.14 and Ivélip -0.10 +/- 0.12 mmol.l-1.h-1, p < 0.05), with marked interindividual differences. Substrate oxidation rates were unchanged during saline infusion, and were immediately (within 30 min) and reciprocally modified by the lipid infusion (lipid oxidation enhanced: 0.90 +/- 0.14 to 1.06 +/- 0.13 mg.kg-1.min-1 at time 30 min, p < 0.05; glucose oxidation inhibited: 1.27 +/- 0.19 to 0.87 +/- 0.18, p < 0.05), but this was not correlated with the alteration in blood glucose levels. In contrast, the increase in plasma lipids was continuous, and positively correlated with the change in blood glucose levels (r = 0.58, p < 0.05 for change of plasma free fatty acids; r = 0.55, p < 0.05 for change of plasma triglycerides, TGs). In line with the Randle mechanism, the lipid infusion affected oxidation rates, but another mechanism, depending on intravascular lipolysis of the infused TGs, was thought to occur in certain individuals whose blood glucose levels rose during the infusion.

Improvement in lipid profiles and triglyceride removal in patients on polyamide membrane hemodialysis.

Cardiovascular morbidity and mortality in hemodialyzed patients are increased due to the frequently abnormal lipid metabolism. It has been reported that this abnormal lipid metabolism could be partially corrected by some highly permeable membranes, such as polysulfone or cellulose triacetate. We investigated the influence of 4 months of dialysis with a polyamide membrane upon the course of lipid parameters in 6 patients presenting a hypertriglyceridemia > 2 mmol/l while on bicarbonate dialysis with a cellulose membrane. Lipid parameters improved after 4 months of hemodialysis with a polyamide membrane. Serum triglyceride and cholesterol levels decreased, while HDL cholesterol and HDL levels rose significantly (p < 0.05). Apolipoprotein B decreased significantly (p < 0.05). Following heparin administration, lipoprotein lipase activity improved (p < 0.02), associated with a decrease apolipoprotein C3 (p < 0.05). The fractional clearance rate of triglycerides rose significantly (p < 0.01). The use of highly permeable polyamide membranes results in a significant improvement in lipid disturbances of dialysis patients due to an increased lipoprotein lipase activity, induced perhaps by the removal of circulating inhibitors such as apolipoprotein C3.

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.

The frequency of the mitochondrial aldehyde dehydrogenase I2 (atypical) allele in Caucasian, Oriental and African black populations determined by the restriction profile of PCR-amplified DNA.

The aldehyde dehydrogenase I (ALDH I) gene codes for a mitochondrial enzyme which plays a major role in hepatic alcohol detoxication. It has been related to alcohol flushing in Orientals bearing the atypical ALDH I2 gene. The variant protein results from a lysine for glutamate substitution at position 487 (G-->A change in exon 12). A procedure for ALDH I2 detection consisting in a differentiation between the 'atypical' allele and the 'wild' allele has been improved through PCR and subsequent MboII digestion. Blood samples collected on anticoagulant or directly absorbed on blotting paper were used for DNA amplification in the presence of two specific oligonucleotidic primers, each one able to incorporate a restriction site in the amplimer. After MboII digestion, PCR products were separated by polyacrylamide gel electrophoresis and then visualized with ethidium bromide. This technique permits a rapid and non-radioactive detection of atypical ALDH I2 on a PCR product without the use of allele specific oligonucleotides. It was applied to the study of ALDH I2 allele frequency in random population samples of three ethnic groups: Caucasians, Orientals and African blacks.

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.

Genotyping of a patient homozygous for a rare apolipoprotein E1 [Gly127-->Asp; Arg158-->Cys] (Weisgraber allele).

We examined the apolipoprotein E polymorphism of an obese patient presenting non-insulin-dependent diabetes, hypertension and moderate lipid disturbances. The apolipoprotein E genotyping carried out from leukocyte DNA using PCR amplification and restriction enzyme digestion demonstrated homozygosity for the rare apoE1[Gly127-->Asp; Arg158--> Cys] (Weisgraber allele). The nucleotide change results in a glycine to aspartic acid substitution at amino acid 127 in the apolipoprotein E2.