Report card on molecular genetic testing: room for improvement?
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Clinical Laboratory Techniques↗
Biomedical subjects
W W Grody
Publications and source records attributed to W W Grody.
Immunohistochemical evaluation of K-ras, p53, and HER-2/neu expression in hyperplastic, dysplastic, and carcinomatous lesions of the pancreas: evidence for multistep carcinogenesis.
The pathobiology of precursor lesions leading to invasive pancreatic adenocarcinoma remains a controversial area, but knowledge of the mechanisms of tumorigenesis may lead to possibly earlier detection, prevention, and treatment in the future. We hypothesize that ductal hyperplasia and dysplasia of the pancreas represent precursor lesions and are part of a continuous developmental spectrum evolving into ductal adenocarcinoma of the pancreas. To further define this sequence, we studied the immunohistochemical markers HER-2/neu, K-ras, and p53 in 15 adenocarcinomas and 15 nonmalignant specimens of the pancreas. The 15 nonmalignant specimens of the pancreas included both normal pancreas and chronic pancreatitis. Overall, HER-2/neu was positive in normal ducts, ductal hyperplasia, dysplasia, and carcinoma cells in 0 of 30, 11 of 20 (55%), 10 of 15 (67%), and 12 of 15 (80%), respectively, with progressive increase in the intensity of staining; p53 was positive in 1 of 30 (3%), 0 of 20, 3 of 15 (20%), and 13 of 15 (80%), respectively, and K-ras was positive in 1 of 30 (3%), 6 of 20 (30%), 11 of 15 (73%), and 8 of 15% (53%), respectively. These data support the hypothesis that ductal hyperplasia and dysplasia of the pancreas represent precursor lesions, and, in a fashion similar to that in colorectal tumorigenesis, pancreatic cancer seems to accumulate progressive genetic alterations.
Adenocarcinoma↗
Recommended policies for uses of human tissue in research, education, and quality control. Ad Hoc Committee on Stored Tissue, College of American Pathologists.
As recipients of tissue and medical specimens, pathologists and other medical specialists regard themselves as stewards of patient tissues and consider it their duty to protect the best interests of both the individual patient and the public. The stewardship of slides, blocks, and other materials includes providing, under appropriate circumstances, patient materials for research, education, and quality control. The decision to provide human tissue for such purposes should be based on the specific (ie, direct patient care) and general (ie, furthering medical knowledge) interests of the patient and of society. The same standards of responsibility should apply to all medical professionals who receive and use specimens. This document proposes specific recommendations whereby both interests can be fostered safely, ethically, and reasonably.
Biological Specimen Banks↗
Cystic fibrosis: low frequency of DF508 mutation in 2 population samples from Rio de Janeiro, Brazil.
Blood samples from 44 unrelated cystic fibrosis (CF) patients from Rio de Janeiro, Brazil, were analyzed for the 8 European CF mutations. Six homozygous and 15 heterozygous carriers of the DF508 mutation were found, corresponding to 47.7% of CF patients (allele frequency 0.3068). The G542X and G551D mutations were also observed with allele frequencies of 0.0227 and 0.0114, respectively. An analysis of the DF508 mutation in 291 randomly chosen, healthy individuals was performed, and only 3 heterozygous carriers were identified. These results show that the frequency of the DF508 allele in Rio de Janeiro is much lower than the world average; this may be due to the extremely heterogeneous ethnic admixture of the study population. By combining the results of these 2 different samples (CF patients and random population) and admixture data from Rio de Janeiro, we can estimate the CF incidence in this population to be 1:3542 individuals. However, taking into account the Rio de Janeiro ethnic admixture, we can find an estimate of 1:6902 individuals.
Brazil↗
Cloning and characterization of the mouse and rat type II arginase genes.
Two forms of arginase, both catalyzing the hydrolysis of arginine to ornithine and urea, are found in animals ranging from amphibians to mammals. In humans, inherited deficiency of hepatic or type I arginase results in hyperargininemia, a syndrome characterized by periodic episodes of hyperammonemia, spasticity, and neurological deterioration. In these patients, a second extrahepatic or type II arginase activity is significantly increased, an induction that may partially compensate for the lack of AI activity and apparently mitigates some of the clinical effects of the condition. Cloning and characterization of the human AII cDNA was recently accomplished. The cloning, sequencing, and partial characterization of the mouse and rat AII cDNAs are reported herein. The DNA sequences predicted polypeptides of 354 amino acids, including a N-terminal mitochondrial import signal. Sequence homology to the human type II arginase, arginase activity data, and immunoprecipitation with an anti-AII antibody confirm the identity of these cloned genes as rodent extrahepatic type II arginases.
Amino Acid Sequence↗
The human arginases and arginase deficiency.
Arginase is the final enzyme in the urea cycle. Its deficiency is the least frequently described disorder of this cycle. It results primarily in elevated blood arginine, and less frequently in either persistent or acute elevations in blood ammonia. This appears to be due to a second arginase locus, expressed primarily in the kidney, which can be recruited to compensate, in part, for the deficiency of liver arginase. The liver arginase gene structure permitted study of the molecular pathology of patients with the disorder and the results of these studies and the inferences about the protein structure are presented. The conserved regions among all arginases allowed the cloning of AII, the second arginase isoform. It has been localized to the mitochondrion and is thought to be involved in ornithine biosynthesis. It shares the major conserved protein sequences, and structural features of liver arginase gene are also conserved. When AI and AII from various species are compared, it appears that the two diverged some time prior to the evolution of amphibians. The evidence for the role of AII in nitric oxide and polyamine metabolism is presented and this appears consonant with the data on the tissue distribution.
Amino Acid Metabolism, Inborn Errors↗
Diversity of cystic fibrosis mutation-screening practices.
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Cystic Fibrosis↗
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↗
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↗
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↗
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↗
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↗
Those elusive molecular diagnostics CPT codes.
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American Medical Association↗
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↗
Diagnostic molecular genetics: current applications and future technologies.
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DNA↗
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↗
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↗
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↗