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Wayne W Grody

Publications and source records attributed to Wayne W Grody.

At least 19 recordsLinked to original sources
PubMed2006

Ornithine deficiency in the arginase double knockout mouse.

Knockout mouse models have been created to study the consequences of deficiencies in arginase AI and AII, both individually and combined. The AI knockout animals die by 14 days of age from hyperammonemia, while the AII knockout has no obvious phenotype. The double knockout (AI(-/-)/AII(-/-)) exhibits the phenotype of the AI-deficient mice, with the additional absence of AII not exacerbating the observed phenotype of the AI knockout animals. Plasma amino acid measurements in the double knockout have shown arginine levels increased roughly 100-fold and ornithine decreased roughly 10-fold as compared to wildtype. Liver ornithine levels were reduced to 2% of normal in the double knockout with arginine very highly elevated. Arginine and ornithine were also altered in other tissues in the double knockout mice, such as kidney, brain, and small intestine. This is the first demonstration that the fatal hyperammonemia in the AI knockout mouse is almost certainly due to ornithine deficiency, the amino acid needed to drive the urea cycle. Others have shown that the expression of ornithine aminotransferase (OAT) rapidly decreases in the intestine at the same age when the AI-deficient animals die, indicating that this enzyme is critical to the maintenance of ornithine homeostasis, at least at this early stage of mouse development. Although most human AI-deficient patients have no symptomatic hyperammonemia at birth, it is possible that clinically significant ornithine deficiency is already present.

Animals↗
PubMed2006

Monitoring standards for molecular genetic testing in the United Kingdom, the Netherlands, and Ireland.

Molecular genetic techniques have entered many areas of clinical practice. Public expectations from this technology are understandably high. To maintain confidence in this technology, laboratories must implement the highest standards of quality assurance (QA). External quality assessment (EQA) is recognized as an essential component of QA. The United Kingdom National External Quality Assessment Service (UKNEQAS) for Molecular Genetics, first set up in 1991, is currently the longest provider of EQA to molecular genetic testing laboratories in the UK, The Netherlands, and Ireland. Errors in the scheme are sporadic events. However, evidence from this and other EQA schemes suggests that a residual error rate persists, which should be taken into account in clinical practice. This EQA scheme has evolved from the respective scientific bodies of the constituent countries and retains a strong emphasis on collective peer review. It is essential that the steps taken to ensure quality in this rapidly expanding field are clear and transparent to participants and public alike. We describe the procedures developed and the governance imposed to monitor and improve analytical and reporting standards in participant laboratories and we compare our experiences with those of equivalent EQA services in the United States.

Cytogenetic Analysis↗
PubMed2006

The clinical significance of circulating tumor cells in the peripheral blood.

Tumors launch malignant cells into the circulation continuously. In early stages, the immune surveillance system eliminates these cells from the circulation, but at later times they may persist longer and be detected. The first recorded evidence of the presence of circulating tumor cells in the peripheral blood of cancer patients was documented in 1869. Now, modern molecular biologic and cell sorting techniques make their detection and characterization more practicable. This review will consider the methods currently available for their detection and characterization, and the clinical implications of their presence in various malignant conditions.

Animals↗
PubMed2006

Microelectronic array system for molecular diagnostic genotyping: Nanogen NanoChip 400 and molecular biology workstation.

Hundreds of gene mutations responsible for Mendelian disorders are currently tested in the clinical laboratory for pre- and postnatal diagnosis, carrier screening and presymptomatic testing. Since human genetic research is currently focused on determining the etiology of complex diseases, including heart disease, diabetes and neuropsychiatric traits, laboratorians will genotype increasing numbers of clinically relevant loci in the future. This will require accurate, high-throughput and cost-effective genotyping platforms, such as the DNA microarray. The Nanogen NanoChip platforms employ hybridization-based technology, using fluorescent detection and electronic control of the target or probe, to obtain clear genotype signal relative to background, and increased flexibility relative to similar chip-based single nucleotide polymorphism genotyping platforms. The scope of this review is intended to describe the operating principle, chips and instrumentation, analyte-specific reagents, published assay protocols, assay development, and clinical use of the NanoChip platforms. It is beyond the scope of this review to describe the use of NanoChip platforms in basic research, and to compare it against all available clinical single nucleotide polymorphism genotyping applications and platforms.

Computational Biology↗
PubMed2005

A novel method for creating artificial mutant samples for performance evaluation and quality control in clinical molecular genetics.

The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed2005

Developing a sustainable process to provide quality control materials for genetic testing.

PURPOSE: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. METHODS: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. RESULTS: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. CONCLUSIONS: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Centers for Disease Control and Prevention, U.S.↗
PubMed2004

Arginases I and II: do their functions overlap?

Arginase, often perceived solely as the last of the now six enzymes of the urea cycle, exists in two forms and has a broad tissue distribution. A cytosolic form, AI, is highly expressed in the liver and is thought to be primarily involved in ureagenesis. A mitochondrial form, AII, has been thought to be more widely expressed and to be involved in the biosynthesis of polyamines, the amino acids ornithine, proline, and glutamate and in the inflammatory process, among others. This paper will address recent experiments that cast some doubt on the validity of these distinctions. Studies have now suggested that macrophages may express AI or AII in different experimental conditions, both in vivo and in vitro. In contrast, most studies, at least in cell culture, suggest that AII may be most highly expressed in cancers of a number of different types. Inhibition of arginase activity in vivo and in vitro has implicated this activity in maintaining ornithine levels for polyamine synthesis. In situ and "quantitative" PCR studies in mouse have demonstrated that AI and not AII is the predominant isoform expressed during development and in the majority of organs. Mouse knockout models for both AI and AII have been produced and are available to address their functions. Surprisingly, the AII knockout animal has no apparent phenotype except for some diminished fertility in homozygous males, consistent with the belief that AII, highly expressed in prostate, is important for sperm function in semen. The AI knockout animal has a more dramatic phenotype and dies at 10-12 days of life of hyperammonemia. The reason for the prolonged survival, as compared to other urea cycle knockout animals, may be due to the later occurrence of hypo-ornithinemia, a contention not yet proven. Transgenic manipulation of the AI knockout animal and breeding the AI and AII knockouts into single animals may address the ability of AII to rescue animals from some of the metabolic consequences of AI deficiency, as appears to happen in man. Newborn screening has given particular hope to patients affected by arginase (AI) deficiency. Increased arginine appears to be detectable by newborn screening with tandem mass spectrometry and the past years continue to demonstrate the therapeutic effectiveness of dietary management of the disorder, with patients treated from birth remaining normal and those treated late, ceasing to deteriorate and even improving in cognitive and physical functioning. Finally, prenatal diagnosis appears to be possible as was predicted, but never proven, some years ago.

Animals↗
PubMed2004

Prenatal screening for cystic fibrosis: past, present and future.

Prenatal screening for cystic fibrosis is reviewed. The disease, gene involved, molecular basis of disease, genotype/phenotype correlations and pilot trials are discussed, as well as historical perspectives, background and American College of Medical Genetics/American College of Obstetricians and Gynecologists recommendations. A number of complex challenges to the implementation of cystic fibrosis screening exist, including mutation testing of the cystic fibrosis transmembrane conductance regulator gene (CFTR), as well as laboratory and clinical issues. Current technologies for CFTR testing include reverse dot blots, amplification refractory mutation detection systems, oligonucleotide ligation assays, the Invader assay and NanoChip system. Emerging technologies are also considered, as well as quality assurance measures including analytical and clinical validation, reporting, residual risk calculations and prenatal diagnosis. An even greater challenge is clinical implementation, which focuses upon education and communication, choosing models, reporting, counseling and prenatal diagnosis.

Cystic Fibrosis↗
PubMed2003

Widespread expression of arginase I in mouse tissues. Biochemical and physiological implications.

Arginase I (AI), the fifth and final enzyme of the urea cycle, detoxifies ammonia as part of the urea cycle. In previous studies from others, AI was not found in extrahepatic tissues except in primate blood cells, and its roles outside the urea cycle have not been well recognized. In this study we undertook an extensive analysis of arginase expression in postnatal mouse tissues by in situ hybridization (ISH) and RT-PCR. We also compared arginase expression patterns with those of ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). We found that, outside of liver, AI was expressed in many tissues and cells such as the salivary gland, esophagus, stomach, pancreas, thymus, leukocytes, skin, preputial gland, uterus and sympathetic ganglia. The expression was much wider than that of arginase II, which was highly expressed only in the intestine and kidney. Several co-localization patterns of AI, ODC, and OAT have been found: (a) AI was co-localized with ODC alone in some tissues; (b) AI was co-localized with both OAT and ODC in a few tissues; (c) AI was not co-localized with OAT alone in any of the tissues examined; and (d) AI was not co-localized with either ODC or OAT in some tissues. In contrast, AII was not co-localized with either ODC or OAT alone in any of the tissues studied, and co-localization of AII with ODC and OAT was found only in the small intestine. The co-localization patterns of arginase, ODC, and OAT suggested that AI plays different roles in different tissues. The main roles of AI are regulation of arginine concentration by degrading arginine and production of ornithine for polyamine biosynthesis, but AI may not be the principal enzyme for regulating glutamate biosynthesis in tissues and cells.

Animals↗
PubMed2003

Cystic fibrosis mutations in Costa Rica.

Using polymerase chain reaction amplification of DNA in dried blood spots and a nonisotopic reverse dot blot hybridization method, we performed molecular genetic analysis for 6 and for 16 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 24 unrelated Costa Rican individuals with cystic fibrosis (CF). While many countries and ethnic groups have been surveyed for CF mutations since the cloning of CFTR, Costa Rica has not heretofore been studied. Moreover, Costa Rica represents an especially intriguing population because of its mixed European-African-Amerindian origins and the existence of a detailed historical record of the founding Spanish families. Thus, such a study may reveal not only the population frequencies of various mutant alleles in this country, but also something about their geographic migrations and ethnic founder effects. The most common CF mutation in Caucasians, deltaF508, was found in only 11 (23%) of the CF chromosomes studied, while the G542X mutation, relatively rare in the general population but more common in southern Europe, was observed in 12 (25%). None of the other mutations tested was found in any of the subjects. We failed to detect the second mutant allele in 17 subjects and could not detect either allele in 4 subjects. The high prevalence of the G542X mutation in our cohort, which exceeds that of both the general Caucasian population and the American Hispanic population, reflects the strong genetic influence of the original Spanish founding families of Costa Rica. These results highlight important differences in Costa Rican CF genotypes as compared both to other North American and European populations and to American Hispanics, raising important implications about isolated founder effects and strategies for population screening in that country.

Adolescent↗
PubMed2003

Ethical issues raised by genetic testing with oligonucleotide microarrays.

Because genes and alterations within them determine the identity, characteristics, and inheritance of every individual, the application of genetic science to humans has long been surrounded by apprehension, controversy, and real or perceived potential for abuse. Crude eugenics practices of the past now find a theoretical rebirth and transformation through the use of modern molecular genetic technologies for mutation detection, predictive and prenatal diagnosis, and, ultimately, gene replacement. The advent of oligonucleotide microarray analysis, in which hundreds or thousands of genes and mutations can be tested in parallel, offers tremendous promise for more accurate, sensitive, and efficient genetic testing. At the same time, however, this powerful technology dramatically increases the number and scope of ethical concerns accompanying each individual test request. This article considers the evolution and implications of these concerns, from the initial ordering of a microarray test by the physician to such issues as informed consent, privacy, confidentiality, clinical utility, discrimination, stigmatization, ethnic and population impact, and reimbursement.

Confidentiality↗
PubMed2002

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes.

Single nucleotide polymorphisms are implicated as having a significant role in regulating growth, development and, thereby, human health and disease. We have developed a method for identifying single nucleotide genetic alterations by combining hairpin-forming DNA probes and electrochemical detection of sandwich DNA hybridization. Incorporation of hairpin-forming competitor probes and the catalyzed reporter deposition amplification system further improves assay specificity by 7-fold and sensitivity by 100-fold. We have demonstrated that the system successfully identified the factor V Leiden mutations from human blood specimens.

DNA Probes↗
PubMed2002

Cloning and characterization of human agmatinase.

Arginine decarboxylase (ADC) and agmatinase are part of an operon in Escherichia coli, which constitutes the primary pathway of polyamine synthesis from arginine. This pathway is also known to exist in plants, but until recently, neither agmatine nor ADC, the enzyme that synthesizes it, nor agmatinase the enzyme that is responsible for conversion of agmatine to putrescine, were known to exist in man or other mammals. We describe here the cloning of the agmatinase gene and the tissue distribution of its transcription product. Human agmatinase contains 352 amino acid residues and has a calculated molecular weight of 37,688 kDa. It has 56% similarity to E. coli agmatinase and 42% similarity to human arginases I and II and shares highly conserved substrate-binding domains with these well-characterized enzymes.

Amino Acid Sequence↗
PubMed2002

Discordant pursuit asymmetry and esotropia in monozygous twins.

PURPOSE: To report the relationship between infantile esotropia and asymmetry of monocular pursuit tracking. DESIGN: Observational family case series. METHODS: A twin pair discordant for infantile esotropia was confirmed by DNA analysis to be monozygous at 99.95% probability. Digitally sampled electro-oculographic recordings of monocular horizontal pursuit eye movements were performed for the twins and their orthophoric father. RESULTS: Mean directional asymmetry for right and left eyes was 31% and 5% for the esotropic twin, 10% and 8% for the orthophoric twin, and 5% and 6% for the father. The asymmetry was statistically significant only for the deviating right eye of the esotropic twin (P = 0.0217). CONCLUSION: In this family, monocular pursuit asymmetry is probably not a heritable predisposition to infantile esotropia, but a consequence of early strabismus. Discordance of infantile esotropia and pursuit asymmetry in monozygous twins implicates environmental in addition to genetic causes.

DNA↗
PubMed2002

Arginase expression in mouse embryonic development.

We are using the model of the developing mouse embryo to elucidate the pattern of arginase expression in mammalian cells in normal animals and in arginase I (AI) deficiency during development by digoxigenin-labeled RNA in situ hybridization. Our goal is to understand the regulation of these isozymes, with the expectation that this knowledge will help patients suffering from AI deficiency. We found that AI mRNA was widely and strongly expressed in the normal developing mouse embryo; in contrast, a relatively strong AII mRNA signal was found only in the intestine. In the AI knockout mouse embryo, no AII overexpression was found. These results indicated that arginases are needed in mouse embryonic development and AI is the principal form required. The strong AI expression in the peripheral nervous system suggests that the pathogenesis of the neurological retardation in AI deficiency may be conditioned by AI deficiency in the nervous system during embryonic development.

Animals↗