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Biomedical subjects

W W Grody

Publications and source records attributed to W W Grody.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular temporal bone pathology: III. Genotyping of the deltaF508 deletion in the DNA of patients with cystic fibrosis.

Genomic DNA from a single celloidin-embedded archival temporal bone section was used to identify a specific genetic mutation. The polymerase chain reaction was used to amplify and detect the deltaF508 deletion, a common molecular genetic defect in cystic fibrosis. This mutation, present in more than 70% of white patients and carriers with cystic fibrosis, results in the deletion of codon 508, which specifies the amino acid phenylalanine of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. When this technique was applied to archival specimens from four patients with cystic fibrosis, all expressed the carrier state of this defective gene. These data demonstrate the feasibility of identifying genetic mutations in archival temporal bone specimens.

Alleles↗
PubMed1998

Molecular temporal bone pathology: IV. Analysis of DNA template length using mitochondrial PCR primers.

The focus of this study was to identify, via molecular biology techniques, the length of the DNA templates present in individual archival celloidin-embedded human temporal bone sections. Earlier studies have suggested that the maximum template length present in these tissues is on the order of 471 base pair (bp). Polymerase chain reaction (PCR) amplification of 92 bp, 121 bp, 471 bp, and 609 bp regions of mitochondrial DNA (mtDNA), extracted from single archival celloidin-embedded human temporal bone sections, was used to assess the length of the template DNA extracted. These data are crucial to determine the limits of applying PCR technology to amplify specific genomic DNA targets located within the human inner ear. The results described should be of value to those investigators extracting DNA from archival individual human temporal bone sections for polymerase chain reaction assays of specific genetic alterations or infectious agents associated with temporal bone pathologies.

DNA Primers↗
PubMed1997

Prevalence of the factor V-Leiden mutation in four distinct American ethnic populations.

Resistance to activated protein C (APC) is the most common risk factor for venous thromboembolism, a major cause of morbidity and mortality with an incidence of about 1/1,000 per year. The Arg 506 to Gln mutation in exon 10 of the coagulation factor V gene (factor V-Leiden) has been found to be responsible for over 90% of the APC resistance cases and is an autosomal dominant trait. Initial studies have suggested that this mutation is restricted to individuals of European Caucasian extraction with an average allele frequency in European and American Caucasians of 4.4%, making it one of the most common monogenic disorders in the Caucasian population. A limited number of other ethnic populations have been tested and the mutation has been found only rarely. In our multiethnic survey of 602 individuals, Hispanic-Americans had the highest observed frequency of the factor V-Leiden mutant allele, 1.65%, while African-Americans had a somewhat lower frequency, 0.87%. No factor V-Leiden mutations were found in 191 Asian-Americans or 54 Native-Americans tested. These results indicate that the factor V-Leiden mutation segregates in populations with significant Caucasian admixture and is rare in genetically distant non-European groups. This ethnic stratification may be important in developing cost-effective selective screening programs to identify individuals at risk for thromboembolism and offer prophylactic therapy.

Alleles↗
PubMed1997

Donor origin of a posttransplant liver allograft malignancy identified by fluorescence in situ hybridization for the Y chromosome and DNA genotyping.

Posttransplantation malignancy in the allograft is a rare complication of orthotopic liver transplantation. In the described case, an abnormal T-tube cholangiogram, performed 6 months after orthotopic liver transplantation between a male donor and a female recipient, prompted needle liver biopsy. A moderately differentiated adenocarcinoma was found. Fluorescence in situ hybridization for the Y chromosome indicated male origin of malignancy. Donor-related disease was confirmed by comparative DNA analysis of genomic sequences from the donor liver, associated tumor, and recipient peripheral blood. Results of these investigations qualified the recipient for a second liver transplant.

Adenocarcinoma↗
PubMed1997

Demonstration of cytoplasmic tyrosinase mRNA in tissue-cultured cells by reverse transcription (RT) in situ polymerase chain reaction (PCR) and RT PCR in situ hybridization.

To evaluate the specificity and applicability to the study of human tumor cells of the reverse transcription (RT) in situ PCR and RT polymerase chain reaction (PCR) in situ hybridization techniques, we examined five melanoma cell lines and five nonmelanoma lines for tyrosinase mRNA using primers specific for tyrosinase. Each procedural step was optimized and minutely controlled, and results from the in situ techniques and solution-phase RT-PCR were compared. All melanoma lines showed a specific pattern of perinuclear cytoplasmic reaction not seen in nonmelanoma lines. There was exact agreement between the results from the RT in situ PCR and RT-PCR in situ hybridization techniques and those from solution-phase RT-PCR. Ribonuclease digestion abolished cytoplasmic staining, as did omission of the reverse transcriptase step. Nuclear staining was seen in melanoma and nonmelanoma lines, apparently as a result of DNA synthesis from repair-replication and mispriming or nonspecific amplification. Neither high concentrations of deoxyribonuclease nor long incubation periods abolished this effect completely. Demonstration of cytoplasmic mRNA by RT in situ PCR and RT-PCR in situ hybridization specifically identifies cells of melanocytic lineage.

Colonic Neoplasms↗
PubMed1997

Friedreich's ataxia GAA repeat expansion in patients with recessive or sporadic ataxia.

To explore the clinical heterogeneity associated with the Friedreich's ataxia (FRDA) expanded repeat and provide preliminary guidance for future gene testing in patients suspected of having FRDA, we tested patients with typical FRDA (group I), late-onset FRDA or FRDA with retained reflexes (group II), as well as those with early onset "non-Friedreich's" recessive or sporadic ataxia (group III). Eighty-seven percent of families in group I tested positive for the FRDA triplet repeat expansion. Thirty-six percent of families in group II demonstrated the FRDA expansion. Only one of 11 patients in group III had the FRDA expansion. Clinical criteria did not clearly distinguish between expansion-positive and expansion-negative individuals in groups I and II. Minimal criteria that were present in all the patients who tested positive were recessive or sporadic inheritance, progressive caudal-rostral gait and limb ataxia, and at least one of the following: dysarthria, Babinski sign, or cardiomyopathy. This study confirms recent findings that some patients in group II can carry the FRDA mutation. However, we did not observe the FRDA expansion in 64% of group II families or in 13% of families with typical FRDA (group I), suggesting other genetic or environmental causes for their ataxia.

Adolescent↗
PubMed1997

Attitudes toward genetic carrier screening for cystic fibrosis among pregnant women: the role of health beliefs and avoidant coping style.

In this study we examined the relations among psychosocial factors associated with pregnant women's attitudes toward genetic carrier testing for cystic fibrosis (CF). A sample of 511 pregnant women attending various health clinics for general prenatal care were educated about CF. Women's health beliefs, coping styles, and attitudes toward CF carrier screening were assessed. Results from structural equation modeling analyses indicated that women who perceived themselves as more likely to be carriers of the CF gene and who perceived greater benefits of screening were positively inclined toward genetic screening. Perceived barriers to screening were negatively associated with women's attitudes toward CF genetic testing. In addition, the findings suggest that some types of avoidant coping styles may indirectly influence one's decision to undergo genetic screening through perceptions of risk, benefits, and barriers. Given the advent of genetic screening options for many diseases, in this study we address some issue in women's attitudes toward prenatal screening that are relevant to a variety of genetic screening programs.

Adaptation, Psychological↗
PubMed1997

PCR-based screening for cystic fibrosis carrier mutations in an ethnically diverse pregnant population.

As the most common lethal autosomal recessive disorder in North America, cystic fibrosis (CF) is an obvious candidate for general population carrier screening. Although the identification of the causative gene has made detection of asymptomatic carriers possible, the extreme heterogeneity of its mutations has limited the sensitivity of the available DNA screening tests and has called into question their utility when they are applied to patients with no family history of the disease. The purpose of this study was to determine the technical feasibility, patient acceptance and understanding, and psychosocial impact of large-scale CF carrier screening in an ethnically diverse pregnant population. A total of 4,739 pregnant women attending prenatal clinics located in both an academic medical center and a large HMO were invited in person to participate. Of this group, 3,543 received CF instruction and assessments of knowledge and mood, and 3,192 underwent DNA testing for the six most common CF mutations, by means of a noninvasive PCR-based reverse-dot-blot method. Overall participation rates (ranging from 53% at the HMO to 77% at the academic center) and consent rates for DNA testing after CF instruction (>98%) exceeded those of most other American studies. The PCR-based screening method worked efficiently on large numbers of samples, and 55 carriers and one at-risk couple were identified. Understanding of residual risk, anxiety levels, and overall satisfaction with the program were acceptable across all ethnic groups. Our strategy of approaching a motivated pregnant population in person with a rapid and noninvasive testing method may provide a practical model for developing a larger CF screening program targeting appropriate high-risk groups at the national level, and may also serve as a paradigm for population-based screening of other genetically heterogeneous disorders in the future.

Academic Medical Centers↗
PubMed1996

Cloning and characterization of the human type II arginase gene.

There are two forms of arginase in humans, both catalyzing the hydrolysis of arginine to ornithine and urea. Recent studies in animal models and in Type I arginase-deficient patients suggest that Type II arginase is inducible and may play an important role in the regulation of extra-urea cycle arginine metabolism by modulating cellular arginine concentrations. We PCR amplified and cloned the human Type II arginase gene, the first nonliver arginase gene reported in mammals. While sequence homology to Type I arginase, arginase activity data, and immunoprecipitation with an anti-AII antibody confirm the identity of this gene, Northern blot analysis demonstrates its differential expression in the brain, prostate, and kidney. Type II arginase may be an important part of the arginine regulatory system affecting nitric oxide synthase, arginine decarboxylase, kyotorphin synthase, and arginine-glycine transaminase activities and polyamine and proline biosynthesis.

Amino Acid Sequence↗
PubMed1996

Loss of function mutations in conserved regions of the human arginase I gene.

We have utilized SSCP analysis to identify disease-causing mutations in a cohort with arginase deficiency. Each of the patient's mutations was reconstructed in vitro by site-directed mutagenesis to determine the effect of the mutations on enzyme activity. In addition we identified six areas of cross-species homology in the arginase protein, four containing conserved histidine residues thought to be important to Mn(2+)-dependent enzyme function. Mapping patient mutations in relationship to the conserved regions indicates that substitution mutations within the conserved regions and randomly occurring microdeletions and nonsense mutations have a significant effect on enzymatic function. In vitro mutagenesis was utilized to create nonpatient substitution mutations in the conserved histidine residues to verify their importance to arginase activity. As expected, replacement of histidine residues with other amino acids dramatically reduces arginase activity levels in our bacterial expression system.

Amino Acid Sequence↗
PubMed1996

Delivery of cytosolic liver arginase into the mitochondrial matrix space: a possible novel site for gene replacement therapy.

As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that constitute the urea cycle. A portion of this cycle takes place in the mitochondria, while the remainder is cytosolic. Liver arginase (L-arginine ureahydrolase, A1) is the fifth enzyme of the cycle, catalyzing the hydrolysis of arginine to ornithine and urea within the cytosol. Patients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spasticity. However, the hyperammonemic effects are not so catastrophic in arginase deficiency as compared to other urea cycle defects. Earlier studies have suggested that this is due to the mitigating effect of a second isozyme of arginase (AII) expressed predominantly in the kidney and localized within the mitochondria. In order to explore the curious dual evolution of these two isozymes, and the ways in which the intriguing, aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and competition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. Stability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incubation of the transfected cells with increasing amounts of arginine produced enhanced levels of mitochondrial AI activity, a substrate-induced effect that we have previously seen with native AII but never AI. Along with exploring the basic biological questions of regulation and subcellular localization in this unique dual-enzyme system, these results suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.

Arginase↗
PubMed1996

Comparative properties of arginases.

Arginase is a primordial enzyme, widely distributed in the biosphere and represented in all primary kingdoms. It plays a critical role in the hepatic metabolism of most higher organisms as a cardinal component of the urea cycle. Additionally, it occurs in numerous organisms and tissues where there is no functioning urea cycle. Many extrahepatic tissues have been shown to contain a second form of arginase, closely related to the hepatic enzyme but encoded by a distinct gene or genes and involved in a host of physiological roles. A variety of functions has been proposed for the "extrahepatic" arginases over the last three decades. In recent years, interest in arginase has been stimulated by a demonstrated involvement in the metabolism of the ubiquitous and multifaceted molecule nitric oxide. Molecular biology has begun to furnish new clues to the disparate functions of arginases in different environments and organisms. Comparative studies of arginase sequences are also beginning to elucidate the comparative evolution of arginases, their molecular structures and the nature of their catalytic mechanism. Further studies have sought to clarify the involvement of arginase in human disease. This review presents an outline of the current state of arginase research by giving a comparative overview of arginases and their associated properties.

Amino Acid Sequence↗
PubMed1996

Polymerase chain reaction-based K-ras mutation detection of pancreatic adenocarcinoma in routine cytology smears.

The cytologic diagnosis of pancreatic carcinoma is notoriously difficult, particularly in distinguishing benign atypia from well-differentiated adenocarcinoma. Mutation of codon 12 in the K-ras oncogene is frequently found with pancreatic cancers. Detection by polymerase chain reaction (PCR) followed by restriction endonuclease digestion can provide a powerful tool to improve and confirm diagnosis. The authors examined the utility of PCR-based detection in the diagnosis of pancreatic carcinoma using routinely obtained cytology smears that could be collected at most hospitals. Pancreatic cytology smears were collected retrospectively from 60 patients. DNA was extracted from the slides and amplified by PCR using mismatched primers that generated a Bst-N1 recognition site with the wild type codon 12 but not with the mutant allele. Results were compared with clinical follow-up. K-ras codon 12 mutations were observed in 44 of 46 (95.7%) cases of pancreatic cancer, but not in 12 benign cases nor in 2 cases of islet cell tumor. The amplification and digestion steps proved robust and sensitive, capable of detecting mutant K-ras alleles from cytology smears that contained only small foci of suspicious cells. Our results indicate that K-ras mutation analysis can be done reliably within 1 to 2 days from routine cytology slides without special handling, increasing the sensitivity of diagnosis in ambiguous cases while maintaining cost-effective and relatively noninvasive sampling strategy.

Adenocarcinoma↗