Refining the continuum of CFTR-associated disorders in the era of newborn screening.

Clinical heterogeneity in cystic fibrosis (CF) often causes diagnostic uncertainty in infants without symptoms and in older patients with milder phenotypes. We performed a cross-sectional evaluation of a comprehensive set of clinical and laboratory descriptors in a physician-defined cohort (N = 376; Children's Hospital of Wisconsin and the American Family Children's Hospital CF centers in Milwaukee and Madison, WI, USA) to determine the robustness of categorizing CF (N = 300), cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (N = 19), and CFTR-related (CRMS) metabolic syndrome (N = 57) according to current consensus guidelines. Outcome measures included patient demographics, clinical measures, sweat chloride levels, CFTR genotype, age at diagnosis, airway microbiology, pancreatic function, infection, and nutritional status. The CF cohort had a significantly higher median sweat chloride level (105 mmol/l) than CFTR-related disorder patients (43 mmol/l) and CFTR-related metabolic syndrome patients (35 mmol/l; p ≤ 0.001). Patient groups significantly differed in pancreatic sufficiency, immunoreactive trypsinogen levels, sweat chloride values, genotype, and positive Pseudomonas aeruginosa cultures (p ≤ 0.001). An automated classification algorithm using recursive partitioning demonstrated concordance between physician diagnoses and consensus guidelines. Our analysis suggests that integrating clinical information with sweat chloride levels, CFTR genotype, and pancreatic sufficiency provides a context for continued longitudinal monitoring of patients for personalized and effective treatment.

ECFS standards of care on CFTR-related disorders: Towards a comprehensive program for affected individuals.

After three publications defining an updated guidance on the diagnostic criteria for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (pwCFTR-RDs), establishing its relationship to CFTR-dysfunction and describing the individual disorders, this fourth and last paper in the series addresses some critical challenges facing health care providers and pwCFTR-RD. Topics included are: 1) benefits and obstacles to collect data from pwCFTR-RD are discussed, together with the opportunity to integrate them into established CF-registries; 2) the potential of infants designated CRMS/CFSPID to develop a CFTR-RD and how to communicate this information; 3) a description of the challenges in genetic counseling, with particular regard to phenotypic variability, unknown long-term evolution, CFTR testing and pregnancy termination 4) a proposal for the assessment of potential barriers to the implementation and dissemination of the produced documents to health care professionals involved in the care of pwCFTR-RD and a process to monitor the implementation of the CFTR-RD recommendations; 5) clinical trials investigating the efficacy of CFTR modulators in CFTR-RD and how endpoints and outcomes might be adapted to the heterogeneity of these disorders.

CFTR mutation analysis and haplotype associations in CF patients.

Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies.

Induction of choline phosphotransferase and lecithin synthesis in the fetal lung by corticosteroids.

Rabbit fetuses 23 to 24 days of gestation were injected with either 9-fluoroprednisolone acetate or saline. Three days later the lungs of steroid-treated animals showed a significant increase in lecithin concentration and cholinephosphotransferase activity. In addition, lung slices from these animals incorporated more [(14)C]choline into lecithin. The rise in enzyme activity and [(14)C]choline incorporation was blocked by prior treatment of fetuses with cycloheximide but not by treatment with actinomycin D. It is proposed that the corticosteroids induce de novo synthesis of the lung enzyme, which in turn leads to increased synthesis of lecithin through the choline incorporation pathway. Furthermore, it appears that the site of regulation involves translation of messenger RNA.

The occurrence and effects of human vitamin E deficiency. A study in patients with cystic fibrosis.

The role of vitamin E in human nutrition was studied by investigation of patients with cystic fibrosis (CF) and associated pancreatic insufficiency. Vitamin E status was assessed by measurement of the plasma concentration of the principal circulating isomer, alpha-tocopherol. Results of such determinations in 52 CF patients with pancreatogenic steatorrhea revealed that all were deficient in the vitamin. The extent of decreased plasma tocopherol varied markedly but correlated with indices of intestinal malabsorption, such as the serum carotene concentration and percentage of dietary fat absorbed. Supplementation with 5-10 times the recommended daily allowance of vitamin E in a water-miscible form increased the plasma alpha-tocopherol concentrations to normal in all 19 CF patients so evaluated. Studies on the effects of vitamin E deficiency focused on possible hematologic alterations. An improved technique was developed to measure erythrocyte hemolysis in vitro in the presence of hydrogen peroxide. While erythrocyte suspensions from control subjects demonstrated resistance to hemolysis during a 3-h incubation, all samples from tocopherol-deficient CF patients showed abnormal oxidant susceptibility, evidenced by greater than 5% hemoglobin release. The degree of peroxide-induced hemolysis was related to the plasma alpha-tocopherol concentration in an inverse, sigmoidal manner. The possibility of in vivo hemolysis was assessed by measuring the survival of (51)Cr-labeled erythrocytes in 19 vitamin-E deficient patients. A moderate but statistically significant decrease in the mean (51)Cr erythrocyte half-life value was found in this group. Measurement of erythrocyte survival before and after supplementation of 6 patients with vitamin E demonstrated that the shortened erythrocyte lifespan could be corrected to normal with this treatment. Other hematologic indices in deficient subjects, however, were normal and did not change upon supplementation with vitamin E. It is concluded that CF is invariably associated with vitamin E deficiency, provided that the patient in question has pancreatic achylia and is not taking supplementary doses of tocopherol. Concomitant hematologic effects consistent with mild hemolysis, but not anemia, occur and may be reversed with vitamin E therapy. Patients with CF should be given daily doses of a water-miscible form of vitamin E to correct the deficiency.

Analysis of changes in optical fibers during arc-fusion splicing by use of quantitative phase imaging.

A non-interferometric imaging technique in conjunction with Abel inversion is used to directly and quantitatively examine the changes in optical fibers due to the heating produced during arc-fusion splicing as a function of fusion arc parameters. Phase images in the vicinity of a fusion splice are obtained using Quantitative Phase Microscopy, allowing the refractive-index change to be reconstructed with high spatial resolution. This simple, nondestructive method confirms that, for a fixed arc current, while the fusion time increases, the refractive-index of both fiber cores within the fusion region decreases in magnitude, the core region broadens, and the axial gradient decreases.

Improving the time efficiency of the Fourier synthesis method for slice selection in magnetic resonance imaging.

The design of slice selective pulses for magnetic resonance imaging can be cast as an optimal control problem. The Fourier synthesis method is an existing approach to solve these optimal control problems. In this method the gradient field as well as the excitation field are switched rapidly and their amplitudes are calculated based on a Fourier series expansion. Here, we provide a novel insight into the Fourier synthesis method via representing the Bloch equation in spherical coordinates. Based on the spherical Bloch equation, we propose an alternative sequence of pulses that can be used for slice selection which is more time efficient compared to the original method. Simulation results demonstrate that while the performance of both methods is approximately the same, the required time for the proposed sequence of pulses is half of the original sequence of pulses. Furthermore, the slice selectivity of both sequences of pulses changes with radio frequency field inhomogeneities in a similar way. We also introduce a measure, referred to as gradient complexity, to compare the performance of both sequences of pulses. This measure indicates that for a desired level of uniformity in the excited slice, the gradient complexity for the proposed sequence of pulses is less than the original sequence.

Fetal lung surfactant lipid synthesis from glycogen during organ culture.

The role of fetal lung glycogen as a precursor for lipids during late gestational development was explored by a combination of in vivo labeling with [U-14C]glucose, administered directly to rat fetuses at 18.5 days, and in vitro assessment using an organ explant culture system. Our major objectives were to demonstrate that radioactivity was transferred specifically and preferentially to surfactant lipids, as glycogenolysis occurred, and to determine the molecular distribution of 14C labeling in newly synthesized phosphatidylcholine (PC). Surfactant and residual (non-surfactant) lipids were separated by sucrose density gradient centrifugation, and other subcellular fractions such as microsomes were isolated by subsequent centrifugations. After 72 h of culture, there was a 5.7-fold increase in the concentration of PC in the surfactant fraction, which contributed 8.8% of total PC at the beginning and 29.6% (P less than 0.001) at the end of the 72 h period. The labeling of PC in the surfactant fraction increased markedly during culture, but there was no significant change in the residual fraction or microsomal PC. Hydrolysis of surfactant PC indicated that the radioactivity was predominantly located in the fatty acyl portion of the molecule, both before and after culture; however, PC glycerol labeling also increased significantly during culture. The distribution of PC radioactivity was similar in the residual fraction and microsomes, with the majority of 14C in the fatty acids. Neutral lipid radioactivity also increased significantly in both the surfactant (240%) and residual (136%) fractions. Quantitation of the changes in radioactivity among subcellular components during lung explant culture indicated that the greatest decrease occurred in glycogen, whereas only lipids, particularly those of the surfactant fraction, were found to show significant increases. These results support the hypothesis that glycogen, which accumulates in fetal lung prior to augmented surfactant production, can supply precursors for synthesis of functionally essential pulmonary phospholipids.

Evidence for the existence of a single enzyme catalyzing the phosphorylation of choline and ethanolamine in primate lung.

Choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) has been isolated and purified 1000-fold from adult African Green monkey lung with a yield of 10%. The purified enzyme also phosphorylated ethanolamine (ratio of ethanolamine kinase to choline kinase = 0.30). This ratio remained constant throughout the purification procedure. The Km for choline (3.0 - 10(-5) M) was lower than that of ethanolamine (1.2 - 10(-3) M.) Choline was also found to inhibit ethanolamine kinase activity by 50% at a concentration of 0.005 mM, while ethanolamine inhibited choline only at very high concentrations (100--150 mM). When the enzyme was subjected to inactivation by heat, hemicholinium-3, trypsin digestion, and p-hydroxymercuribenzoate, both ethanolamine kinase and choline kinase activities were destroyed at the same rate. Freezing and thawing in the absence of glycerol also destroyed both activities at the same rate. Based on these findings, we conclude that in adult African Green monkey lung tissue, there is only one enzyme for the phosphorylation of ethanolamine and choline, and that choline phosphorylation predominates.

Relationships among surfactant fraction lipids, proteins and biophysical properties in the developing rat lung.

Lung development is associated with increases in specific phospholipids and proteins that function as critical pulmonary surfactant components. Attempts to characterize the pattern of surfactant development in fetal rat lungs have been hampered by the lack of a micromethod which will permit quantitative isolation of surface active components from small tissue specimens. As part of studies designed to elucidate the metabolic regulation of lung development in the rat, we developed sucrose density gradient centrifugation procedures to separate pulmonary phospholipids and proteins into a presumed surfactant (S) fraction and a residual (R) fraction. Electron microscopy of S pellets from mature fetuses identified predominant lamellar bodies and minimal contamination; incubation with 5 mM CaCl2 induced the appearance of tubular myelin figures, implying functional potential. This was confirmed by demonstrating low surface tension (less than 1 dyn/cm) in S, but not R, fractions at term gestation (21.5 days) and in 1-day-old neonatal lung isolates, based on dynamic measurements using the oscillating bubble technique. Surface activity was also high in the S pellets from fetuses at 20.5 days of gestation; however, at 19.5 days, minimum surface tension values of at least 19 dyne/cm were seen. These results correlated directly with biochemical analyses which indicated striking increases in three surfactant-associated proteins (SP-A, SP-B, and SP-C) after 19.5 days of gestation; a finding in agreement with previously reported data on the developmental increase of disaturated phosphatidylcholine in fetal rat lung. We conclude that isolation of S fraction components is valuable for demonstrating maturation of the fetal rat lung and may provide a useful tool for the study of regulatory mechanisms influencing surfactant production and function.

Complications of pregnancy and fetal development.

Although the outcome of pregnancy for women with diabetes mellitus has improved in recent years, the infant of the diabetic mother has an increased risk of major clinical problems, particularly in the early neonatal period. These include birth injury due to macrosomia, neonatal hypoglycemia, respiratory distress syndrome, and serious congenital anomalies. Because of the great difficulties encountered during attempts to investigate these problems in clinical research protocols, there is a continuing need to establish appropriate animal models of the diabetic pregnancy. Studies carried out over the past decade, primarily with chemically-induced diabetes have suggested techniques which might be useful. In general, the choice of the animal to be studied will depend on the hypotheses being addressed. For instance, small animals such as rabbits and rats made diabetic with streptozotocin have been successfully used for investigation of fetal lung development. Furthermore, the rat model has been helpful for evaluation of fetal anomalies associated with maldevelopment of the spine and central nervous system. Larger animals, such as the nonhuman primate, are more appropriate for studying placental function and amniotic fluid composition in diabetic pregnancies. The task group on pregnancy and fetal development recommends that animal models of diabetes mellitus be used for a more extensive hormonal and metabolic characterization of diabetic mothers during pregnancy, for investigation of placental physiology with respect to the transfer of substrates from mother to fetus, for systematic and comprehensive study of mechanisms controlling fetal lung development, and for delineation of the pathophysiology of neonatal hypoglycemia. It is further recommended that animal models of spontaneous diabetes such as the BB/W rat be used in future studies dealing with pregnancy and fetal development. Because females with spontaneous diabetes show reduced conception rates, there is a pressing need to enhance the fertility of these animals in order to intensify studies on fetal development.

Cortisol-dihydrotachysterol antagonism in a patient with hypoparathyroidism and adrenal insufficiency: apparent inhibition of bone resorption.

This report describes a case of chronic mucocutaneous candidiasis with associated hypoparathyroidism and acutely developed adrenocortical insufficiency. The latter was heralded by hypercalcemia. Upon the institution of cortisol therapy, while still under the effects of a vitamin D analog dihydrotachysterol (DHT), the patient exhibited severe hypocalcemia and tetany. Since calcium intake was minimal during this period of presumed corticosteroid-DHT antagonism, it is suggested that the cortisol disturbed calcium homeostasis by in inhibiting bone calcium resorption.

Intravenous infusion of lipid for the prevention of essential fatty acid deficiency in premature infants.

This study was designed to determine the amount of linoleic acid required to prevent essential fatty acid deficiency in premature infants. This was achieved by infusing 1 g intravenous lipid.kg-1.d-1 over 18-20 h beginning on day 2 and increasing by 0.5 g.kg-1.d-1 to a maximum of 3 g.kg-1.d-1. The actual mean amounts of linoleic acid administered (mg/kg) were 613 on day 2, 767 on days 3 and 4, 862 on day 5, and 1062 on day 6. None of the neonates managed in this fashion showed a high triene-tetraene ratio on days 3 or 7 (in contrast to control subjects, 80% whom were abnormal). Plasma triglycerides and nonesterified fatty acids (NEFAs) increased during the infusions but not to concentrations indicative of fat intolerance. The rise in NEFAs was associated with evidence of bilirubin displacement from circulating albumin but this occurred to only a modest degree.

Megavitamin E supplementation in man.

To assess possible toxic and/or beneficial effects of vitamin E supplementation, a group of 28 adults voluntarily ingesting 100 to 800 IU/day of tocopherol for an average of 3 years were evaluated in this study. Half of the subjects claimed a feeling of improved health or well being, but no specific beneficial effects were noted consistently; the other half indicated no change in health status after beginning vitamin E supplements. No gross evidence of toxicity was apparent on reviewing past medical histories with the subjects. Plasma alpha-tocopherol was found to be elevated significantly in the group from 650 micrograms/100 ml (control mean) to 1,340 micrograms/100 ml; however, 25% of the values were within 2 SD of the control mean. Plasma alpha-tocopherol levels did not correlate with total daily dose but did relate to plasma triglyceride and cholesterol concentrations. Total plasma carotenoids were also significantly increased along with vitamin A levels; the former did not correlate with plasma vitamin E, whereas the latter showed a significant correlation. Laboratory screening for toxic side effects of vitamin E supplementation by performance of 20 standard clinical blood tests failed to reveal any disturbance in liver, kidney, muscle, thyroid gland, erythrocytes, leukocytes, coagulation parameters, or blood glucose. It is concluded that megavitamin E supplements in this group produced no apparent toxic side effects and that subjective claims for beneficial effects were highly variable.

Vitamin E, vitamin A and essential fatty acid status of patients hospitalized for anorexia nervosa.

A group of 17 patients hospitalized for anorexia nervosa was assessed for nutritional status with respect to the essential fatty acids and vitamins E and A. Plasma aliquots available from blood drawn upon hospital admission were frozen and later extracted for total lipids. The total lipids were then fractionated by thin layer chromatography to isolate the phospholipids (PL). The fatty acid profile of both total plasma and PLs was determined by gas chromatography. High pressure liquid chromatography was used to quantitate the tocopherol isomers and retinol (vitamins E and A, respectively). The anorexic patients showed below normal weight-for-height, decreased percent body fat, and a high incidence of secondary amenorrhea. Of the four tocopherol isomers measured, only the beta + gamma fraction was significantly lower than the normal value (0.16 +/- .06 mg/dl vs 0.25 +/- 0.51 mg/dl, p less than .001). The essential fatty acids, linoleate and linolenate, were lower in the PL fraction of the anorexic group (linoleate, 19.35 +/- 5.65 vs 24.96 +/- 2.24, p less than .01); linolenate, none detected vs 0.16 +/- 0.13, p less than .05). Other indicators of essential fatty acid status differed from the normal group in the direction of deficiency (total omega 6, 31.44 +/- 7.41 vs 37.42 +/- 3.40, p less than .05; 20:4 omega 6/18:2 omega 6, 0.49 +/- 0.14 vs 40 +/- 0.02, mean +/- standard deviation, p less than .01). Eicosatrienoic acid, however, was not detected in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutritional assessment and management in cystic fibrosis: a consensus report. The Consensus Committee.

This report is a summary of a meeting convened by the Cystic Fibrosis Foundation to develop a consensus among nutrition specialists and cystic fibrosis care givers regarding optimal nutritional management of patients with cystic fibrosis. The first section of the report provides a rationale for emphasizing nutritional management of this genetic disorder. The multiple factors causing malnutrition and a negative energy balance are outlined. The second section provides guidelines for routine assessment of nutrition in these patients. Five categories of nutritional status are defined based on ideal weight for height, age, and gender. These categories are used to formulate a graded response for nutritional intervention. Recommendations are provided for routine dietary supplements, vitamin supplements, and pancreatic enzyme replacement. The primary aim of this report is to educate clinicians as to the importance of frequent assessments and early intervention.

Changes in erythrocyte and plasma carnitine concentrations in preterm neonates.

Erythrocyte and plasma total, free, and acyl carnitine concentrations in 13 low birthweight, preterm infants were determined between birth and 21 days of age. Although erythrocytes contributed 73.6 +/- 4% (mean +/- SD) of total blood carnitine at birth, the contribution by day 14 declined to 42.2 +/- 14.1. Linear regression analysis showed no significant correlation between plasma and erythrocyte concentrations. At 3 wk erythrocyte total carnitine concentrations were similar to adult values, but erythrocyte acyl carnitine concentrations were markedly lower. Although a significant (p less than 0.05) positive correlation between plasma carnitine concentrations and mean daily intake from birth was found at 7, 14, and 21 days of age (r = 0.66, 0.87, and 0.88, respectively), no significant relationships between erythrocyte carnitine concentrations and carnitine intake could be demonstrated by linear regression analysis. It appears that the carnitine present in plasma and erythrocytes represents two separate pools which are influenced by different factors in preterm infants.

Essential fatty acid deficiency in premature infants.

To better characterize essential fatty acid (EFA) deficiency in neonates, we assessed 63 premature infants by serial determinations of plasma fatty acids for the level of linoleic acid, the presence of an abnormal trienoic acid (5,8,11-eicosatrienoic acid [20:3 omega 9]), and the ratio of this compound to arachidonic acid, ie, the triene-tetraene ratio. The data indicated that at age 7 d, 67% of these infants had low plasma linoleic acid levels, 62% showed readily detectable 20:3 omega 9, and 44% had a high triene-tetraene ratio. Infants fed by age 2 d had a normal mean linoleate level at 7 d and none showed detectable 20:3 omega 9 by 10 d. In contrast, infants who were not fed until 7 d showed a very high incidence of abnormal fatty acid status. By maintaining a daily record of linoleate intake, we calculated from regression models that the average amount required to achieve normal fatty acid nutrition was 1.19 g.kg-1.d-1.

An evaluation of vitamin E status in premature infants.

Prematurely born, low birth weight infants are abnormal by their very existence ex utero. Thus, the well-documented finding in such infants of low plasma vitamin E concentrations when compared to the adult poses philosophical and pragmatic difficulties as to whether or not a true deficiency state exists: do these low levels represent age-adjusted reference values or do they in fact represent a deficiency state, warranting treatment? We examined multiple measures of vitamin E status in 62 prematurely born, low birth weight infants in order to address this issue. Mathematical and statistical modeling of these measures during the first 21 days of life lead us to conclude that the ex utero antioxidant protective role of vitamin E is best achieved at plasma concentrations of tocopherol very close to those observed in the adult; specifically, when total tocopherol is greater than 0.64 mg/dl and alpha-tocopherol is greater than 0.50 mg/dl. Thus, at birth, a true deficiency in vitamin E exists for most preterm, low birth weight infants and early treatment is warranted.

Essential fatty acid deficiency after hepatic portoenterostomy for biliary atresia.

Gas chromatography was used to determine the fatty acid composition of total lipids extracted from plasma and erythrocytes of five patients who had received an hepatic portoenterostomy for treatment of extrahepatic biliary atresia. Three patients, including one with successful surgery, demonstrated evidence of essential fatty acid deficiency, including decreased levels of linoleic and arachidonic acids with concomitant increases in palmitoleic and oleic acids. In two of these patients, the ratio of 5,8 11-eicosatrienoic acid to arachidonic acid ("triene/tetraene") exceeded 0.3, diagnostic of essential fatty acid deficiency. Even patients with successful hepatic portoenterostomy are at risk to develop essential fatty acid deficiency.