Controlled trial of serum isoelectric focusing in the detection of the cystic fibrosis gene

Summary Three independent observers assessed the discriminating power of serum isoelectric focusing in detecting the presence of the cystic fibrosis gene. On the basis of average scores, four out of 23 cystic fibrosis patients, six out of 22 heterozygotes, and three out of 16 controls were misclassified. However, the mean scores for the cystic fibrosis and heterozygote groups were significantly different to that for the control group. It is concluded that isoelectric focusing is insufficiently reliable to be used for diagnosis or heterozygote detection in cystic fibrosis, but that it does provide evidence for the presence of a protein associated with the mutant gene.     

Demonstration of a factor in the serum of homozygotes and heterozygotes for cystic fibrosis by a non-biological technique.

A factor has been isolated from serum of homozygotes and obligate heterozgotes for cystic fibrosis using isoelectric focusing and disc electrophoresis as analytical methods. The factor is focused within an IgG-fraction with an isoelectric point of pH 8 to 9 but differs from IgG in its lower molecular weight. It is thus similar to, if not identical with, the ciliary dyskinesia factor.     

Decreased inhibition of platelet aggregation by PGE1 in children with cystic fibrosis and their parents

PGE₁ inhibited ADP-induced platelet aggregation in children with cystic fibrosis and their parents to a much lesser extent than in normal controls. We suggest that this may be a reliable test for heterozygote carriers of cystic fibrosis.     

Sweat Chlorides in Salt-Deprived Cystic Fibrosis Heterozygotes

Sweat chlorides of 10 sets of parents of children with cystic fibrosis and 11 controls were studied in an attempt to develop a test for the diagnosis of cystic fibrosis heterozygotes by subjecting both the parents and controls to a low sodium diet and comparing sweat chloride values as the diet progressed. It was hoped that the sweat chloride levels of the parents, the heterozygotes, would remain stationary throughout the diet, since their children, the homozygotes, reveal this finding under similar conditions of salt deprivation. The sweat chloride levels of the controls, because of effects of aldosterone, were expected to decrease steadily from the commencement of the diet to its termination.A decrease in sweat chloride values of similar magnitude was found in both parents and controls as the diet continued. It is concluded that the study of sweat electrolyte levels in salt-deprived subjects is of no value in the diagnosis of cystic fibrosis heterozygotes.     

Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience.

OBJECTIVE--To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis. DESIGN--Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (delta F508, delta I506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus or family history of cystic fibrosis. SETTING--South Australian Neonatal Screening Programme, Adelaide. SUBJECTS--All 88,752 neonates born in South Australia between December 1989 and December 1993. INTERVENTIONS--Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. MAIN OUTCOME MEASURES--Identification of all children with cystic fibrosis in the screened population. RESULTS--Of 1004 (1.13%) neonates with immunoreactive trypsinogen > or = 99th centile, 912 (90.8%) had no identifiable mutation. 23 neonates were homozygotes or compound heterozygotes; 69 carried one identifiable mutation, of whom six had positive sweat tests. Median age at clinical assessment for the 29 neonates with cystic fibrosis was 3 weeks; six had meconium ileus and two had affected siblings. 63 neonates were identified as carriers of a cystic fibrosis mutation. Extra laboratory costs for measuring immunoreactive trypsinogen and direct gene analysis were $A1.50 per neonate screened. CONCLUSION--This strategy results in early and accurate diagnosis of cystic fibrosis and performs better than screening strategies based on immunoreactive trypsinogen measurement alone.     

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.     

Immunochemical studies of the plasma and cultured fibroblast media fractions containing the cystic fibrosis ciliary inhibitor.

The cystic fibrosis ciliary inhibitor (CFCI) has been fractionated from plasma of cystic fibrosis (CF) homozygotes and from the media of cultured fibroblasts derived from CF homozygotes. Plasma and fibroblast media from normal controls have been fractionated in an identical manner. Fractions from plasma and fibroblast culture media that demonstrate ciliary inhibitory activity contain several proteins in a molecular weight range of approximately 5,000-11,000. These proteins have been partially characterized by immunochemical analysis with antisera to 33 human serum proteins. Immunological determinants of albumin, C3 (but not C3a), C4, C5, alpha1-lipoprotein, beta-lipoprotein, beta2-microglobulin and immunoglobulin light chains have been detected by hemagglutination in fractions of CF plasma that inhibited ciliary activity and in analogous fractions from normal sera. None of the proteins were detected in media of cultured fibroblasts from either genotype. Since the same proteins and protein fragments were identified in both CF and normal plasma fractions, and were not detected in CF fibroblast media, it appears that none of these proteins can be identified as the CFCI. Identification of these proteins will permit further purification of the CFCI by immunochemical methods.     

Serum Binding of Calcium and the Red Cell Membrane in Cystic Fibrosis

THERE is evidence for a factor in the serum of cystic fibrosis patients which may play a role in the pathological manifestation of the genetic syndrome: serum of patients with cystic fibrosis alters the cilia movement of rabbit trachea in vitro¹ and of oyster cilia². This activity is apparently associated with the immunoglobulin fraction^3,4. Balfe et al.⁵ reported a modification of sodium flux in red cells obtained from patients with cystic fibrosis. In the course of searching for evidence of interaction of serum with red cells in cystic fibrosis, we found that there is a substantial increase in serum calcium binding in patients with cystic fibrosis. This seems to be associated with a modified membrane protein electrophoretic pattern of cystic fibrosis red cells in specified experimental conditions.     

Cystic fibrosis: the ciliary inhibitor is a small polypeptide associated with immunoglobulin G.

A small polypeptide within the molecular weight range of 6,000 to 11,000 was found in immunoglobulin fractions from sera of cystic fibrosis homozygotes and heterozygotes. This factor appears to be responsible for interfering with ciliary activity in oyster gills. Since it can be dissociated from IgG without reductive cleavage it must be bound in a non-covalent manner. After its dissociation the IgG fractions from cystic fibrosis sera no longer inhibited ciliary activity. This finding explains the differences previously observed in the molecular weights of the ciliary inhibitor synthesized by cultured cells and that of sera from cystic fibrosis genotypes.     

Characterization of the cell kinetics and growth properties of cystic fibrosis diploid fibroblasts in vitro.

The population growth kinetics of human diploid skin fibroblasts derived from cystic fibrosis homozygotes and heterozygotes and from normal subjects were investigated. Our data suggest the following: 1. Population doubling times increase with time in culture, with no significant differences observed among the three genotypes tested, when data were compared at the same subculture times or phases of growth. 2. The fraction of dividing cells in a population decreased with the duration in which the cells were in culture. No significant differences were obtained for cells derived from the three genotypes. 3. Cell cycle times were very similar (18-20 hr) when comparing the normal and cystic fibrosis lines or when comparing cystic fibrosis lines in phases 1 and 2 of growth. 4. No significant variations in population doubling times or growth fractions could be attributed to age or sex of the biopsy donor. 5. Variability in growth fractions and doubling times was minimal through the eighth subculture period but was very great in older cultures (tenth subculture). 6. Changes in growth fractions and doubling times appear to be due to the possibility of "aging" of human diploid fibroblasts in culture rather than to the presence or absence of genes for cystic fibrosis. 7. It is strongly indicated that differences in cell kinetic parameters cannot be used as the basis for differentiation between cells derived from normal or cystic fibrosis genotypes.     

Quantitative variation in cystic fibrosis-associated proteins in cystic fibrosis patients,carriers, and controls

Summary Serum samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls have been examined by isoelectric focusing (IEF). Our results suggest that cystic fibrosis protein (CFP) is a normal serum protein exhibiting quantitative variation primarily dependent on possession of the CF allele. It is concluded that detection of CFP by IEF is an inappropriate screening test for the CF gene due to lack of specificity.     

Amylase polymorphism: studies of sera and duodenal aspirates in normal individuals and in cystic fibrosis.

Prior genetic studies of the human pancreatic amylase (Amy2) locus have been directed principally to the electrophoretic analysis of serum and urine, on the assumption that these fluids receive negligible contributions from the salivary (Amy 1) locus. In support of that assumption was the observation that the isozyme bands were lacking in patients with cystic fibrosis and in a postpancreatectomy patient. We have examined the sera of 97 patients having cystic fibrosis and find normal levels of serum amylase. On electrophoresis, three-quarters of the cystic fibrosis patients have a pattern (F-pattern) not observed in normal sera. The pattern is characterized by the absence of Pa 1. Comparative electrophoresis and mixing experiments indicate that the F-pattern is of salivary origin and is unmasked in cystic fibrosis by the absence of a pancreatic contribution. The normal serum pattern is considered to be an admixture of salivary and pancreatic amylase. On the assumption that duodenal fluids might more closely reflect the pancreatic (Amy 2) locus, electrophoretic studies were performed on 148 normal individuals and 37 individuals with cystic fibrosis. Electrophoretic phenotypes in duodenal aspirates are more complex than previously reported in studies of urine and serum; presumably because of the higher concentrations of amylase in the aspirates. Comparative electrophoresis and mixing experiments indicate that the phenotypes observed in duodenal aspirates also reflect admixture of pancreatic and salivary amylase. This recognition of pancreatic and salivary admixture in sera fortunately does not alter our prior understanding of the genetics of the Amy 2 polymorphism. The extensive studies which led to the delineation of the Amy 2 polymorphism were essentially based on the presence or absence of a variant band which proves now to be outside the zone of admixture.     

Differential concanavalin A binding of cystic fibrosis and normal liver alpha-L-fucosidase.

A novel technique has been employed to demonstrate that α-L-fucosidase purified from cystic fibrosis and control livers exhibits differential binding to the lectin Concanavalin A. The concentration of α-CH₃-mannoside necessary to prevent 50% binding of α-L-fucosidase to Concanavalin A is considerably lower for the cystic fibrosis enzyme (13.5 vs. 33.3 mM). Comparable results were found when binding studies were done on crude supernatant α-L-fucosidase from 8 cystic fibrosis and 8 control livers (5.6 ± 0.4 mM and 13.2 ± 3.4 mM, respectively), without any overlap of values between the cystic fibrosis and control livers. These results suggest that comparative lectin binding studies on cystic fibrosis and normal glycoproteins from readily available tissues might result in an assay for detecting the cystic fibrosis genotype.     

Serum alpha - fetoprotein levels in patients with cystic fibrosis and their parents and siblings.

Patients with cystic fibrosis (C.F.) showed raised serum levels of alpha-fetoprotein (AFP). A moderate but significant increase in serum AFP was present in their parents and some siblings. There was no correlation between the clinical severity of the disease and serum AFP concentration. Samples from control groups with gluten-induced malabsorption and bronchiectasis had normal levels. Persistent synthesis of AFP may be an associated marker of C.F. genes, and estimation of serum AFP might help in detecting heterozygote carriers in families at risk.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".     

Cystic fibrosis: Demonstration of an abnormality in mucopolysaccharides in cultured lymphoid lines

Cultured lymphoid cells of both homozygotes and heterozygotes for cystic fibrosis could be distinguished from those of normals by (1) growth pattern, gross clumping, and (2) a relative increase in dermatan sulfate, with a normal total mucopolysaccharide content. Lines derived from the genetic mucopolysaccharidoses also had these characteristics, but their total mucopolysaccharide content was markedly increased. These observations support the hypothesis that the cellular disturbance in cystic fibrosis resides in those cellular regions whose functions would be altered by mucopolysaccharide composition.This research was made possible through a grant from The National Foundation-March of Dimes and, in part, supported by The Cystic Fibrosis Children's Fund and The National Cystic Fibrosis Research Foundation.Career Scientist of the Health Research Council of New York City (I-797).     

Detection of cystic fibrosis homozygotes and heterozygotes by serum isoelectrofocusing

Summary It is suggested that the majority of individuals with the cystic fibrosis (CF) gene have a unique serum protein (CFP) which can be demonstrated by means of isoelectrofocusing (IEF) in thin layer polyacrylamide gels. We have found the CFP in 90% of patients with cystic fibrosis, in approximately 80% of individuals heterozygous for the CF gene, and in 8% of normal control individuals. We conclude that CFP is a useful marker for the CF gene.     

The noninvasive biochemical diagnosis of gastrointestinal disease, with special reference to children

Invasive tests to diagnose patients with gastrointestinal disease are rapidly being replaced by procedures which enable organ function to be assessed by monitoring the product of a metabolic reaction in readily available materials such as breath, blood, and urine. Examples of these approaches that will be assessed in this review include the hydrogen breath test for lactase deficiency, radioactive carbon dioxide breath measurements to test for fat digestion and absorption, and tests of pancreatic function based upon synthetic substrates from which fluorescein or para-aminobenzoic acid can be liberated by pancreas-specific enzymes. Significant advances have been made in improving the organ sensitivity of enzyme determinations. The determination of amylase isoenzymes has been less useful than the measurement of immunoreactive trypsin; this latter enzyme is greatly elevated in the blood of neonates with cystic fibrosis, whereas serum levels are greatly depressed in cystic fibrosis patients with pancreatic insufficiency as well as in most patients with steatorrhea due to chronic pancreatitis. Many of these tests are now becoming standard procedures in the investigation of infants with gastrointestinal disease.     

Electron paramagnetic resonance spectroscopy as a monitor of the pathway of the thermal unfolding of ribonuclease A.

α-L-Fucosidase activity is elevated in skin fibroblasts from cystic fibrosis patients when compared to controls. The activities of nine other acid hydrolases including neuraminidase are similar in cystic fibrosis and control fibroblasts. The relationship of these results to the recent finding of a decreased activity of α-L-fucosidase in the serum of cystic fibrosis patients is discussed. It is proposed that an abnormal distribution of α-L-fucosidase is involved in the pathogenesis of this disease.