High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism

Groß, Eva; Seck, Katharina; Neubauer, Steffi; Mayr, Jutta; Hellebrand, Heide; Ratanaphan, Adisorn; Lutz, Veronika; Stockinger, Hubertus; Kiechle, Marion

doi:10.3892/ijo.22.2.325

Cited by 19 publications

(21 citation statements)

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“…Statistically significant difference between controls and the high-toxicity group was found in frequency of IVS14+1G>A carriers The DHPLC analysis performed in the low-toxicity and control groups revealed presence of five intronic variants flanking to analyzed exons (IVS9-51T>G, IVS10-15T>C, IVS12-11G>A, IVS13+39C>T, and IVS13+40A>G). All these variants of unknown significance were described previously in various populations and were not considered for further analysis in our study [12,24,25]. The strong association of IVS13+39C>T with c.1627A>G (I543V) reported in previous studies was also observed in our samples (data not shown) [16,26].…”

Section: Dpyd Alterations In the Group Of High-toxicity Patientssupporting

confidence: 65%

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy

Kleibl

Fidlerová²,

Kleiblová³

et al. 2009

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Alterations in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism contribute to the development of serious FPs-related toxicity. We performed mutation analysis of DPYD based on cDNA sequencing in 76 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 -27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment. Key words: dihydropyrimidine dehydrogenase gene [DPYD], fluoropyrimidines, 5-fluorouracil toxicity, mutation analysis, haplotypes, association study* Corresponding author † These authors contributed equally to this work.Fluoropyrimidines -5-fluorouracil (5-FU) and its derivates (e.g. capecitabine) -belong to the most frequently used anticancer drugs in treatment of solid cancers. Mechanism of action of 5-FU involves its anabolic conversions to 5-fluoropyrimidine nucleotides that exert profound inhibitory effect on thymidylate synthetase activity and interfere with RNA and DNA metabolism [1]. The development of severe toxicity is the critical complication of 5-FU-based therapy. It occurs in nearly one third of cases with progression to life-threatening complications in approximately 0.5% patients [2].About 80% of 5-FU is quickly ina...

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Section: Dpyd Alterations In the Group Of High-toxicity Patientssupporting

confidence: 65%

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy

Kleibl

Fidlerová²,

Kleiblová³

et al. 2009

neo

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“…The detection of DPYD sequence variants was carried out by DHPLC analysis (22). In brief, the mutation analysis was done on a Wave DNA fragment analysis system (Transgenomic, Omaha, NE) under partially denaturing conditions.…”

Section: Methodsmentioning

confidence: 99%

“…DNA was prepared from frozen EDTA-blood samples (10 mL) using standard techniques. The entire coding region of DPYD was amplified with 23 primer pairs corresponding to 23 exons and the exon-intron boundaries as previously described (22). Amplicons were generated with the Expand High Fidelity PCR System supplied by Roche GmbH (Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

Analysis of the DPYD Gene Implicated in 5-Fluorouracil Catabolism in a Cohort of Caucasian Individuals

Seck

Riemer

Kates

et al. 2005

Clinical Cancer Research

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Purpose: Complete or partial loss of dihydropyrimidine dehydrogenase (DPD) function has been described in cancer patients with intolerance to fluoropyrimidine drugs like 5-fluorouracil (5-FU) or Xeloda. The intention of this population study is to assess and to evaluate gene variations in the entire coding region of the dihydropyrimidine dehydrogenase gene (DPYD), which could be implicated in DPD malfunction. Experimental Design: A cohort of 157 individuals was genotyped by denaturing highperformance liquid chromatography; 100 of these genotypes were compared with functional studies on DPD activity and mRNA expression. Results: Twenty-three variants in coding and noncoding regions of the DPYD gene were detected, giving rise to15 common haplotypes with a frequency of >1%. Rare sequence alterations included a frameshift mutation (295-298delTCAT) and three novel point mutations, 1218G>A (Met Ser). DPD enzyme activity showed high variation in the analyzed population and correlated with DPD mRNA expression. In particular, the novel variants were not accompanied with decreased enzyme activity. However, a statistically significant deviation from the median DPD activity of the population was associated with the mutations 1601G>A (Ser Dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of pyrimidines. The homodimeric protein catalyzes the reduction of uracil and thymine in an NADPH-dependent manner and, furthermore, plays a critical role in the pharmacokinetics of fluoropyrimidine-based anticancer drugs. Eighty percent to 85% of administered standard doses of the common chemotherapeutic agent 5-fluorouracil (5-FU) are rapidly degraded by DPD to inactive compounds followed by excretion of a-fluoroh-alanine within 24 hours (1 -3). The rationally designed orally administered fluoropyrimidine drug Xeloda (capecitabine) is converted in situ into 5-FU due to intracellular thymidine phosphorylase activity. Capecitabine has been shown to be safer and more effective than 5-FU and has greater patient convenience (4, 5).

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“…19 The genotypes of ECRG2 STR were identified as TCA 3 /TCA 3 , TCA 4 /TCA 4 , and TCA 3 /TCA 4 , which were confirmed by DNA sequencing. In order to determine somatic stability of ECRG2 STR, the genotype of the STR in the DNA samples from blood, tumor and normal tissue adjacent to tumor from the same patient was detected at the same time.…”

Section: Discussionmentioning

confidence: 84%

Short tandem repeat polymorphism in a novel esophageal cancer‐related gene (ECRG2) implicates susceptibility to esophageal cancer in Chinese population

Yue

Tan

et al. 2003

Intl Journal of Cancer

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We have previously cloned and identified a novel esophageal cancer related gene 2 (ECRG2; GenBank Accession Number AF268198), which is down-regulated in esophageal squamous cell carcinoma (ESCC) and involved in the induction of the apoptosis in esophageal cancer cell lines. In the present study, we have found a short tandem repeat (STR) polymorphism in the noncoding region of the exon 4 of the ECRG2 gene by using PCR-denaturing high-performance liquid chromatography (DHPLC Esophageal cancer, with a 5-year survival rate Ͻ10%, is one of the most common fetal cancers worldwide and occurs at very high frequencies in certain areas of China, Iran, South Africa, Uruguay, France and Italy. 1 Fifty percent of esophageal cancer cases in the world occur in China. Among high-risk areas of esophageal squamous cell carcinoma (ESCC), Linxian County in Henan Province has the highest age-adjusted mortality rate of this cancer. 2 Epidemiological studies have revealed that the incidence of ESCC is associated with several factors, such as N-nitrosamines, which have been shown to be involved in the etiology of ESCC in Linxian. 3 However, studies in high-risk areas have demonstrated a strong tendency towards familial aggregation or clustering of cases within families, suggesting that genetic susceptibility factors may also play an important role in the etiology of ESCC.Previous studies have shown that several genetic abnormalities including amplification of C-myc, Int-2 and Hst, 4,5 mutation and/or deletion of p53 and Rb 2 and allelic deletion 6 have frequently occurred in human ESCC and esophageal cancer cell lines. However, the genetic events that lead to the development of esophageal cancer are not clear yet. In recent years, many studies of esophageal cancer are focused on the cloning and identification of novel esophageal cancer-related genes, which might play an important role in the carcinogenesis and development of the cancer. [7][8][9] Recently, we have cloned and identified a novel ESCC-related gene, ECRG2 (Genbank Accession Number AF 268198), from human normal esophageal epithelia. Firstly, we obtained an EST fragment by mRNA differential display techniques from 3 normal esophageal epithelia and 2 primary ESCC tissues from high incidence families in Linxian County and named it ECRG2. The RT-PCR detection results demonstrated that the expression of ECRG2 was down-regulated in ESCC as well as colon and brain tumors. 10 Secondly, we obtained the 569 bp full-length cDNA of the ECRG2 gene by SMARTTM RACE technique. We found that the gene contains 4 exons, spans about 3,540 bp on chromosome 5q32-33 and has a 258 bp open reading frame encoding an 85-amino acids polypeptide. 11 Analysis by bioinformatics has shown that 97% of the amino acid sequences of ECRG2 are homologous to a tumor associated KAZAL-type serine protease inhibitor that may play a central role in the protection of esophageal mucosa. 12 Thirdly, we studied the function of the ECRG2 gene by yeast 2-hybrid system and identified that the ECRG2 gene interacts directly ...

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High-throughput genotyping by DHPLC of the dihydropyrimidine dehydrogenase gene implicated in (fluoro)pyrimidine catabolism

Cited by 19 publications

References 0 publications

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy

Analysis of the DPYD Gene Implicated in 5-Fluorouracil Catabolism in a Cohort of Caucasian Individuals

Short tandem repeat polymorphism in a novel esophageal cancer‐related gene (ECRG2) implicates susceptibility to esophageal cancer in Chinese population

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