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W E Highsmith

Publications and source records attributed to W E Highsmith.

35 records · Page 2Linked to original sources
PubMed1994

A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations.

BACKGROUND: Many patients with chronic pulmonary disease similar to that seen in cystic fibrosis have normal (or nondiagnostic) sweat chloride values. It has been difficult to make the diagnosis of cystic fibrosis in these patients because no associated mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been identified. METHODS: We evaluated 23 patients with pulmonary disease characteristic of cystic fibrosis but with sweat chloride concentrations in the normal range. Mutations in the CFTR gene were sought by direct sequencing of polymerase chain reaction-amplified nasal epithelial messenger RNA and by testing the functioning of affected epithelium. RESULTS: A cytidine phosphate guanosine dinucleotide C-to-T point mutation in intron 19 of the CFTR gene, termed 3849 + 10 kb C to T, was identified in 13 patients from eight unrelated families. This mutation was found in patients from three different ethnic groups with three different extended haplotypes. The mutation leads to the creation of a partially active splice site in intron 19 and to the insertion into most CFTR transcripts of a new 84-base-pair "exon," containing an in-frame stop codon, between exons 19 and 20. Normally spliced transcripts were also detected at a level approximately 8 percent of that found in normal subjects. This mutation is associated with abnormal nasal epithelial and sweat acinar epithelial function. CONCLUSIONS: We have identified a point mutation in intron 19 of CFTR and abnormal epithelial function in patients who have cystic fibrosis-like lung disease but normal sweat chloride values. The identification of this mutation indicates that this syndrome is a form of cystic fibrosis. Screening for the mutation should prove diagnostically useful in this population of patients.

Adolescent↗
PubMed1993

Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis.

We have developed a method for processing and detecting fungi in clinical specimens using the polymerase chain reaction (PCR) methodology. This PCR amplification of a segment of a ribosomal DNA gene results in a 310 bp product. The gene sequences used as amplimers have been highly conserved throughout the fungal kingdom and positive PCR results have been obtained for all genera and species of fungi tested (n = 42). Neither human nor a variety of pathogenic bacteria (n = 24) gave an amplified product. This PCR method can detect as few as 15 cells of Candida albicans in clinical specimens following a simple processing procedure. The sensitivity of the PCR for detecting other fungi remains to be determined. Analysis of the 310 bp amplified product, following digestion with the restriction enzyme HaeIII, can be used to further characterize the identity of the fungus involved into the following five groups: (i) Candida species and closely related yeasts; (ii) Cryptococci and Trichosporon species; (iii) Aspergilli and clinically related septate molds; (iv) the Zygomycetes; and (v) the dimorphic fungi.

Base Sequence↗
PubMed1992

Nontuberculous mycobacteria in adult patients with cystic fibrosis.

Because patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual microorganisms, we screened the sputum of adult CF patients for mycobacterial organisms. Acid-fast bacilli (AFB) smears and mycobacterial culture were performed on 297 sputum specimens from 87 patients. Cultures for mycobacteria were frequently overgrown with other bacteria; 22.6 percent of cultures were contaminated. Despite this limitation of mycobacterial culture, 17 patients had at least one positive culture for a Mycobacterium other than tuberculosis (MOTT). Eleven patients were positive for Mycobacterium avium-intracellulare (MAI), two for MAI and M chelonei, three for M chelonei, and one for M fortuitum. None was positive for M tuberculosis. Patients with CF with MOTT were similar to patients with CF without MOTT; only a slightly different (older) age distribution was recognized. The clinical significance of MOTT was difficult to determine in any individual patient, but patients with positive AFB smears appeared more likely to suffer pathogenic effects. We conclude that MOTT is frequently recovered from adult CF patients in the southeastern United States. A specific risk factor for colonization and/or pathogenic infection in this patient group was not evident. The general prevalence and clinical pathogenesis in CF patients in the United States remains to be determined.

Adolescent↗
PubMed1991

Detecting multiple cystic fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis.

The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.

Base Sequence↗
PubMed1990

Molecular probe protocol for determining carrier status in Duchenne and Becker muscular dystrophies.

By use of cDNA probes, molecular deletions were identified in 66.6% of 42 patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Owing to this high deletion rate, a new strategy for detecting DMD/BMD carriers is feasible in which the polymerase chain reaction is used as an initial screen for detecting the deletions occurring in specific deletion-prone exons. Because the deletions do not occur randomly, specific cDNA probes are utilized first with Southern blot analysis. Identification of a deletion permits direct analysis for DMD carrier status and removes the inherent limitations of the conventional restriction fragment length polymorphism technique. Carrier status is determined by scanning the autoradiographs with a densitometric spectrophotometer or by detection of a junction fragment.

Bacterial Proteins↗
PubMed1990

Frequency of the delta Phe508 mutation and correlation with XV.2c/KM-19 haplotypes in an American population of cystic fibrosis patients: results of a collaborative study.

The cystic fibrosis (CF) gene has been recently cloned, and a deletion of 3 basepairs (bp) of DNA was found on most of the CF chromosomes. This deletion leads to the synthesis of a protein that lacks a phenylalanine residue at position 508. Using two polymerase chain reaction protocols to study the frequency of this mutation in a series of 192 CF patients, we found the mutation on 72% of affected chromosomes. We then used this value to calculate the predictive value of a negative test result in a population-based screening program for CF carrier status. Haplotype analysis with the polymorphic markers XV.2c and KM-19 on 239 CF chromosomes revealed that 90.7% of CF chromosomes with the deletion had a single haplotype. This haplotype was also associated with 60.4% of CF chromosomes with unknown mutations. These values can be used to calculate the probability of whether an individual from the general population is a carrier of any CF mutation.

Base Sequence↗
PubMed1990

A model for molecular screening of newborns: simultaneous detection of Duchenne/Becker muscular dystrophies and cystic fibrosis.

Gene mutations responsible for the majority of Duchenne/Becker muscular dystrophy (DMD/BMD) and cystic fibrosis (CF) chromosomes have been identified. We describe a DNA-based strategy, rather than the traditional biochemical assays, for screening newborns. DNA sequences spanning the CF mutation and several DMD/BMD deletion-prone exons are amplified simultaneously via a multiplex polymerase chain reaction. The gel is visually inspected for DMD/BMD deletions and then blotted and hybridized with allele-specific oligonucleotides to determine the presence or absence of the CF mutation. We determined that blood spots provide sufficient DNA for the molecular analysis, so the procedure can be used in screening programs of newborns.

Base Sequence↗
PubMed1990

Determination of carrier status in Duchenne and Becker muscular dystrophies by quantitative polymerase chain reaction and allele-specific oligonucleotides.

Detection of carriers of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), in the deletion cases, involves calculating gene dosage from Southern blots. We show that the analysis of dosage can also be made from the polymerase chain reaction (PCR) with use of allele-specific oligonucleotides (ASOs). The deletion-prone exons are amplified, transferred to a membrane, and hybridized with ASOs complementary to the exons; the autoradiographic bands are then quantified with a densitometer. After determining the quantitative conditions of the amplification reaction, we were able to identify deletions in a DMD/BMD carrier female. The determination of carrier status via PCR removes several of the technical limitations of Southern analysis and is also cost- and labor-effective.

Alleles↗
PubMed1989

Mild cystic fibrosis linked to chromosome 7q22 markers with an uncommon haplotype.

Cystic fibrosis is the commonest autosomal recessive genetic disorder among northern Europeans and their descendants. Recently, investigators have mapped the gene for cystic fibrosis to chromosome 7. We report the results of DNA linkage analysis in a consanguineous family with mild cystic fibrosis. The probes used in this study were pmet D. pmet H, XV-2c, KM.19, and pJ3.11. Linkage to the identified cystic fibrosis locus of 7q22 was established with a peak logarithm of the odds (lod) score of 3.00 at a recombination fraction theta =0.00 using the tightly linked marker KM.19. In addition, we found the D haplotype, which is not commonly associated with cystic fibrosis, to be segregating in this family. The D haplotype is composed of the 1.4-kb allele detectable by XV-2c and the 6.6-kb allele detectable by KM.19. The three patients with cystic fibrosis who had consanguineous parents were homozygous DD, were among the least severely affected, and had no pancreatic insufficiency. The five patients with unrelated parents were heterozygous for the D haplotype and the commoner B haplotype, except one patient who was homozygous DD. All affected persons with pancreatic insufficiency had the DB genotype. These DNA linkage studies provide additional evidence for the existence of a cystic fibrosis allele that is associated with mild disease.

Adult↗
PubMed1987

Improved enzymatic hydrolysis of conjugated bile acids.

The hydrolysis of conjugated bile acids by cholylglycine hydrolase (EC 3.5.1.24) using the standard procedure for a commercial enzyme preparation was found to be incomplete, as judged by the use of 24-14C-taurocholic acid as a tracer. A method is proposed that incorporates the nonionic detergent Triton X-100 into the reaction mixture to achieve almost complete hydrolysis. It is proposed that the observed enhancement of enzyme activity is due to the formation of micelles by the detergent.

Amidohydrolases↗
PubMed1983

Bile acid synthesis by cultured hepatocytes. Inhibition by mevinolin, but not by bile acids.

The ability of cultured hepatocytes to maintain a constant rate of bile acid synthesis and secretion in a chemically defined serum-free culture medium allowed us to examine the direct effects of bile acids on their synthesis. Mass quantitation of bile acids by gas liquid chromatography showed that adding taurochenodeoxycholate at concentrations which were from 2 to 20 times the concentration of bile acids found in rat portal blood increased rather than decreased the secretion of cholic acid. Using a more direct approach to measure relative rates of bile acid synthesis and secretion, we determined the rate of de novo 14C-bile acid synthesis. Adding taurocholate at concentrations ranging from 2 to 60 times the concentration found in rat portal blood did not inhibit 14C-bile acid synthesis. Furthermore, free and conjugated di- and tri-hydroxy bile acids did not inhibit bile acid synthesis. In contrast, mevinolin, a potent inhibitor of cholesterol biosynthesis, inhibited 14C-bile acid synthesis by 63%. Since previous demonstration of bile acid negative feedback regulation was achieved in vivo by infusing taurocholate into the intestines of bile-diverted rats, we examined the possibility that bile acid synthesis must be induced in order for bile acids to inhibit their synthesis. Hepatocytes obtained from bile-diverted rats exhibited a 5-fold increase in the synthesis and secretion of 14C-bile acids. However, taurocholate did not inhibit bile acid synthesis by cells from bile-diverted rats. These data show that bile acids do not interact directly with the hepatocyte to inhibit their synthesis.

Animals↗
PubMedDate not supplied

Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T carriers: results from a collaborative study.

PURPOSE: We expect that the mutation panel currently recommended for preconception/prenatal CF carrier screening will be modified as new information is learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. One such example is the I148T mutation, originally described as a severe CF mutation. After implementation of CF population-based carrier screening, we learned that I148T exists as a complex allele with 3199del6 in patients with clinical CF, whereas asymptomatic compound heterozygotes for I148T and a second severe CF mutation were negative for 3199del6. METHODS: We performed reflex testing for 3199del6 on 663 unrelated specimens, including I148T heterozygotes, compound heterozygotes, and a homozygous individual. RESULTS: Less than 1% of I148T carriers were also positive for 3199del6. Excluding subjects tested because of a suspected or known CF diagnosis or positive family history, 0.6% of I148T-positive individuals were also positive for 3199del6. We identified 1 I148T homozygote and 6 unrelated compound heterozygous individuals with I148T and a second CF variant (2 of whom also carried 3199del6). In addition, one fetus with echogenic bowel and one infertile male were heterozygous for I148T (3199del6 negative). CONCLUSIONS: Reflex testing for 3199del6 should be considered whenever I148T is identified. Reflex testing is of particular importance for any symptomatic patient or whenever one member of a couple carries a deleterious CF mutation and the other member is an I148T heterozygote. Further population data are required to determine if I148T, in the absence of 3199del6, is associated with mild or atypical CF or male infertility.

Cystic Fibrosis↗
PubMedDate not supplied

Optimization, assessment, and proposed use of a direct nested reverse transcription-polymerase chain reaction protocol for the detection of hepatitis C virus.

OBJECTIVES: To compare the performance of reverse transcription followed by the polymerase chain reaction (RT-PCR), without RNA purification, with the performance of classic protocols. STUDY DESIGN/METHODS: Direct and classic techniques were used to test three groups of samples: six hepatitis C virus (HCV) seroconversion panels (n = 90), a HCV RNA reference panel (n = 26), and serial dilutions of four HCV-positive sera (n = 24). These methods were then applied sequentially through a clinical diagnostic algorithm to test 268 samples from high-risk patients. RESULTS: For the three groups of samples, we found a 94% concordance between direct and purified RT-PCR methods. For the detection of HCV RNA in clinical samples, sensitivity was maximized and cost minimized using both protocols according to the proposed algorithm. CONCLUSIONS: The direct PCR method is reliable, sensitive, and can result in time and cost savings. The suggested testing algorithm can enhance sensitivity and time savings for populations with a high prevalence of infection.

Algorithms↗
PubMedDate not supplied

Neonatal screening for biotinidase deficiency.

Children with juvenile-onset multiple carboxylase deficiency lack biotinidase activity (biotinamide amidohydrolase, EC 3.5.1.12) in the liver and other tissues. Hence, little free biotin is metabolically available, resulting in seizures, acidosis, and serious neurological damage. As the absence of hepatic biotinidase activity is reflected in serum, assessment of biotinidase status can easily be made from a blood sample. A convenient qualitative procedure for screening infants has been employed in order to estimate serum levels of biotinidase in as little as 10 microliters of sample. This colorimetric procedure detects the formation of free p-aminobenzoate cleaved from the substrate, N-biotinyl-p-aminobenzoate at pH 6.0. The assay is easily performed and has a low incidence of false positive results. A kinetic assay for serum biotinidase has also been developed using biotinyl-p-nitroanilide (BpNA) as substrate. When 50 microliters of biotinidase positive serum was incubated with 0.2 mM BpNA in phosphate buffer at pH 6.0, an increase in absorbance was observed at 405 nm. The rate of change in absorbance was followed kinetically on the Roche Cobas BIO analyzer at 37 degrees C. Monitoring the increase in absorbance of para-nitroanilide every 60 seconds over 30 minutes demonstrated linearity from 10 to 30 minutes. In comparing results from this kinetic assay on 48 randomly selected sera with those obtained using a colorimetric procedure, a correlation coefficient of 0.85 was obtained. Several false positive results were observed in clearly lipemic sera.

4-Aminobenzoic Acid↗