Biomedical subjects

Hany Ezzeldin

Publications and source records attributed to Hany Ezzeldin.

9 recordsLinked to original sources

PubMed2006

DPYD*2A mutation: the most common mutation associated with DPD deficiency.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) enzyme is responsible for the elimination of approximately 80% of administered dose of 5-FU. DPD deficiency has been associated with severe 5-FU toxicity. Syndrome of DPD deficiency manifests as diarrhea, stomatitis, mucositis, and neurotoxicity and in some cases death. This is a true pharmacogenetic syndrome, with symptoms being unrecognizable until exposure to the drug. PATIENTS AND METHODS: A 75-year-old patient with metastatic pancreatic adenocarcinoma developed grade 4 thrombocytopenia, grade 3 coagulopathy, and grade 3 neurologic toxicity with a fatal outcome following administration of 5-FU. Due to pancytopenia, DPD activity could not be determined in peripheral blood mononuclear cells (PBMC) using a previously described radioassay. Therefore, screening and genotypic analysis of homozygous and heterozygous, known and unknown sequence variants, in the DPYD gene were performed using DHPLC as previously described. All DPYD sequence variants identified by DHPLC were confirmed by DNA sequencing using a dideoxynucleotide chain termination method and capillary electrophoresis on an ABI 310 Automated DNA Sequencer. RESULTS: Genotyping analysis of the DPYD gene revealed the presence of the heterozygous mutation, IVS14 + 1 G > A, DPYD*2A. CONCLUSION: Genotypic analysis using DHPLC can be employed to screen DPD deficiency in a patient with severe neutropenia. The mutation IVS14 + 1 G > A, DPYD*2A, is the most common mutation associated with DPD deficiency. A G > A base change at the splice recognition sequence of intron 14, leads to exon skipping and results in a 165-bp deletion in the DPD mRNA. We have previously demonstrated that a homozygote DPYD*2A genotype results in complete deficiency while the heterozygous DPYD*2A genotype results in partial deficiency of DPD.

Adenocarcinoma↗

PubMed2006

Dihydropyrimidine dehydrogenase deficiency in an Indian population.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) deficiency is prevalent in 3-5% of the Caucasian population; however, the frequency of this pharmacogenetic syndrome in the Indian population and other racial and ethnic groups remains to be elucidated. PATIENTS AND METHODS: We describe an Indian patient who presented to clinic for the treatment of gastric adenocarcinoma with 5-flurouracil (5-FU) therapy who subsequently was diagnosed with DPD deficiency by using the peripheral blood mononuclear cell (PBMC) DPD radioassay. This observation prompted us to examine the data generated from healthy (cancer-free) Indian subjects who were enrolled in a large population study to determine the sensitivity and specificity of the uracil breath test (UraBT) in the detection of DPD deficiency. Thirteen Indian subjects performed the UraBT. UraBT results were confirmed by PBMC DPD radioassay. RESULTS: The Indian cancer patient demonstrated reduced DPD activity (0.11 nmol/min/mg protein) and severe 5-FU toxicities commonly associated with DPD deficiency. Of the 13 Indian subjects [ten men and three women; mean age, 26 years (range: 21-31 years)] enrolled in the UraBT, 12 Indian subjects demonstrated UraBT breath profiles and PBMC DPD activity within the normal range; one Indian subject demonstrated a reduced breath profile and partial DPD deficiency. CONCLUSIONS: DPD deficiency is a pharmacogenetic syndrome which is also present in the Indian population. If undiagnosed, the DPD deficiency can lead to death. Future epidemiological studies would be helpful to determine the prevalence of DPD deficiency among racial and ethnic groups, allowing for the optimization of 5-FU chemotherapy.

Adult↗

PubMed2004

Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography.

Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy. The aim of the current study was to develop a specific denaturing high-performance liquid chromatography (DHPLC) method for the rapid detection of these variations in the TSER in clinical samples. DHPLC analysis was validated in parallel with agarose gel electrophoresis (AGE), enzyme digestion, and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). The optimized DHPLC method resolved 100% of the known TSER variations, differentiated between homozygous and heterozygous genotypes, and allowed the qualitative and quantitative detection of AI, including LOH, in tumor samples. This DHPLC method was developed to permit the rapid, sensitive, and accurate identification of the TSER genotype (VNTRs, SNPs, and AI) in clinical protocols where response to flouropyrimidines may be correlated with TSER polymorphisms.

Allelic Imbalance↗

PubMed2004

Rapid identification of dihydropyrimidine dehydrogenase deficiency by using a novel 2-13C-uracil breath test.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD)-deficient cancer patients have been shown to develop severe toxicity after administration of 5-fluorouracil. Routine determination of DPD activity is limited by time-consuming and labor-intensive methods. The purpose of this study was to develop a simple and rapid 2-(13)C-uracil breath test, which could be applied in most clinical settings to detect DPD-deficient cancer patients. EXPERIMENTAL DESIGN: Fifty-eight individuals (50 "normal," 7 partially, and 1 profoundly DPD-deficient) ingested an aqueous solution of 2-(13)C-uracil (6 mg/kg). (13)CO(2) levels were determined in exhaled breath at various time intervals up to 180 min using IR spectroscopy (UBiT-IR(300)). DPD enzyme activity and DPYD genotype were determined by radioassay and denaturing high-performance liquid chromatography, respectively. RESULTS: The mean (+/-SE) C(max), T(max), delta over baseline values at 50 min (DOB(50)) and cumulative percentage of (13)C dose recovered (PDR) for normal, partially, and profoundly DPD-deficient individuals were 186.4 +/- 3.9, 117.1 +/- 9.8, and 3.6 DOB; 52 +/- 2, 100 +/- 18.4, and 120 min; 174.1 +/- 4.6, 89.6 +/- 11.6, and 0.9 DOB(50); and 53.8 +/- 1.0, 36.9 +/- 2.4, and <1 PDR, respectively. The differences between the normal and DPD-deficient individuals were highly significant (all Ps <0.001). CONCLUSIONS: We demonstrated statistically significant differences in the 2-(13)C-uracil breath test indices (C(max), T(max), DOB(50), and PDR) among healthy and DPD-deficient individuals. These data suggest that a single time-point determination (50 min) could rapidly identify DPD-deficient individuals with a less costly and time-consuming method that is applicable for most hospitals or physicians' offices.

Antimetabolites, Antineoplastic↗

PubMed2004

Dihydropyrimidine dehydrogenase deficiency, a pharmacogenetic syndrome associated with potentially life-threatening toxicity following 5-fluorouracil administration.

Dihydropyrimidine dehydrogenase (DPD) deficiency is a pharmacogenetic syndrome associated with potentially life-threatening toxicity following the administration of standard doses of 5-fluorouracil. This syndrome derives its importance from the fact that approximately 2 million patients receive the drug worldwide each year. Population studies have suggested that 4%-7% of the American population exhibit dose-limiting toxicity that might be associated with a genetic defect in the DPYD gene that encodes for the DPD enzyme. During the past several years it has become increasingly clear that genetics is a major determinant of the variability in drug response, accounting for the probability of drug efficacy and the likelihood of toxic drug reactions. This article briefly discusses the clinical presentation, laboratory diagnosis, pharmacokinetics, inheritance, and the clinical management options of DPD deficiency. The variability of DPD enzyme activity in population studies and the different DPYD alleles together with new phenotypic and genotypic methods of screening for DPD deficiency will also be reviewed.

Antimetabolites, Antineoplastic↗

PubMed2004

Dihydropyrimidine dehydrogenase deficiency: impact of pharmacogenetics on 5-fluorouracil therapy.

Through the use of pharmacogenetic studies, interindividual variability in response (efficacy and toxicity) to 5-fluorouracil (5-FU) chemotherapy has been linked to the rate-limiting enzyme in the drug's catabolic pathway, known as dihydropyrimidine dehydrogenase (DPD). This pharmacogenetic syndrome, known as "DPD deficiency," results in excessive amounts of 5-FU available to be anabolized to its active metabolites and is relatively undetectable by clinical observation prior to 5-FU administration. Extensive studies have associated both profound and partial deficiency in DPD activity with severe, unanticipated toxicity after 5-FU administration, while research on the molecular basis behind DPD deficiency has been linked to various sequence variants of the DPYD gene. Due to the widespread use of 5-FU, the severity of toxicity associated with DPD deficiency, and the prevalence of DPD deficiency in the population, extensive research is continually being performed to develop quick and accurate phenotypic and genotypic assays suitable for clinical settings that would allow clinicians to identify patients susceptible to adverse 5-FU reactions.

Antidotes↗

PubMed2003

Denaturing high performance liquid chromatography analysis of the DPYD gene in patients with lethal 5-fluorouracil toxicity.

Dihydropyrimidine dehydrogenase (DPD) enzyme deficiency is a pharmacogenetic syndrome with possible fatal outcome following 5-fluorouracil (5-FU) treatment. Several studies examining the molecular basis for DPD deficiency have identified over 30 sequence variations in the DPYD gene (which codes for the DPD enzyme). Our laboratory has recently developed and validated a denaturing high performance liquid chromatography method capable of identifying both known and unknown sequence variations in the DPYD gene. In the present study, we used this denaturing high performance liquid chromatography approach to examine the DPYD genotype of three patients who experienced lethal toxicity after administration of 5-FU. DPD enzyme activity could only be measured in one patient before death and demonstrated that lethal toxicity can occur in a partially DPD-deficient individual. Multiple heterozygous sequence variations (both known and unknown) were detected in all three patients including the novel variants 545T>A, M182K and 2329G>T, A777S. We conclude that (a) lethal toxicity can occur in partially DPD-deficient individuals after administration of 5-FU and is not exclusive to profoundly DPD-deficient individuals as suggested previously, (b) the complicated heterozygote genotype seen in these patients, combined with DPD deficiency being an autosomal codominant inherited syndrome, precludes the use of simple genotyping assays that identify only one or two mutations as a method for identifying DPD-deficient individuals; and (c) these multiple heterozygote genotypes (which are more difficult to accurately characterize) may be responsible for some of the conflicting reports which suggests a lack of correlation between phenotype and genotype.

Aged↗

PubMed2002

A high-throughput denaturing high-performance liquid chromatography method for the identification of variant alleles associated with dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). A pharmacogenetic syndrome has been described in which DPD-deficient patients are at risk for toxicity following administration of 5-FU. To date, there are at least 21 previously described mutations and/or polymorphisms that have been associated with DPD deficiency. In this study we describe the development of a highly specific, sensitive, inexpensive, and robust denaturing HPLC (DHPLC) method for rapidly identifying sequence variations (mutations and/or polymorphisms) in the gene (DPYD) that codes for the DPD enzyme. DHPLC conditions were optimized at three temperatures for analysis of the 23 exons of the DPYD gene using 25 amplicons representing the entire coding sequence, including all intron/exon boundaries (splice sites). Resolution of all 25 amplicons at the optimized temperature can be performed in 4.2 h. All 21 previously described sequence variations (mutations and/or polymorphisms) were prepared using site-directed mutagenesis from the wild-type DPYD gene, confirmed by sequence analysis, and subsequently resolved by DHPLC using the optimized conditions. These analyses generated reference chromatogram patterns for all known sequence variations previously encountered in DPD-deficient patients. In order to examine the utility and sensitivity of this approach, samples from patients with known sequence variations in the DPYD gene were analyzed. This DHPLC technique resolved 100% of the known DPYD sequence variations and differentiated between homozygous and heterozygous genotypes. We conclude that this DHPLC method is a highly specific and sensitive technique for rapidly detecting known sequence variations in the DPYD gene. In addition, this approach can be used to identify currently unrecognized unknown sequence variations in the DPYD gene and should be useful in future pharmacogenetic studies examining DPD deficiency.

Alleles↗

PubMed2002

Electroretinographic abnormalities in parents of patients with Leber congenital amaurosis who have heterozygous GUCY2D mutations.

BACKGROUND: Leber congenital amaurosis (LCA) is an infrequently encountered congenital form of retinitis pigmentosa with marked genetic and clinical heterogeneity. Thus far, 10 genes have been identified in this disorder since 1996. In the future, LCA may become treatable by gene and/or pharmacological intervention, and these therapies will likely be gene specific, giving major significance to rapid gene identification and gene-phenotype studies. OBJECTIVE: To test the hypothesis that parents of patients with LCA have identifiable electroretinographic and psychophysical changes. SUBJECTS, MATERIALS, AND METHODS: Complete eye examinations and electroretinographic studies were performed on 2 sets of parents whose offspring were diagnosed as having LCA and who were found to carry a mutation in 1 of the 10 LCA genes-GUCY2D. One set of parents also underwent static perimetry threshold measurements. RESULTS: We found that single flash-light-adapted a- and b-wave amplitudes, 30-Hz flicker, or both cone signals were significantly decreased in amplitude in 4 heterozygotes, while 2 parents showed delayed 30-Hz flicker implicit times. Electroretinographic rod-mediated signals were normal in 2 of the heterozygotes, but subnormal in 2. Static perimetry testing showed normal thresholds in the 2 heterozygotes tested. MAIN OUTCOME MEASURES: Single flash-light-adapted a- and b-wave amplitudes and implicit times, 30- or 32-Hz flicker amplitudes and implicit times, rod-mediated signals, and dark-adapted, rod-mediated thresholds. CONCLUSIONS: Some carrier parents of patients with LCA and a GUCY2D mutation develop measurable, cone and possibly rod abnormalities most consistent with a mild cone-rod dysfunction. This correlates well with the known retinal expression pattern of GUCY2D, which is considerably higher in cone compared with rod photoreceptor cells.

Adaptation, Ocular↗