Quantitative real-time PCR for gene dosage determinations in microdeletion genotypes

Covault, Jonathan; Abreu, Christine; Kranzler, Henry R.; Oncken, Cheryl

doi:10.2144/03353dd02

Cited by 21 publications

(14 citation statements)

References 12 publications

(10 reference statements)

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“…The TaqMan Drug Metabolism SNP Genotyping Assay for GSTM1 (assay ID: C4420299720) was used to determine glutathione S‐transferase mu 1 (GSTM1) gene deletion and allelic variance, using the AB StepOnePlus instrument. Data were quantified relative to a two‐copy gene control, a region in IVS10 of the breast cancer 1, early onset (BRCA1, NM_007294) gene …”

Section: Methodsmentioning

confidence: 99%

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Bioavailability of Glucoraphanin and Sulforaphane from High‐Glucoraphanin Broccoli

Sivapalan

Melchini

Saha

et al. 2018

Molecular Nutrition Food Res

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ScopeBroccoli accumulates 4‐methylsulphinylbutyl glucosinolate (glucoraphanin) which is hydrolyzed to the isothiocyanate sulforaphane. Through the introgression of novel alleles of the Myb28 transcription factor from Brassica villosa, broccoli genotypes have been developed that have enhanced levels of glucoraphanin. This study seeks to quantify the exposure of human tissues to glucoraphanin and sulforaphane following consumption of broccoli with contrasting Myb28 genotypes.Methods and resultsTen participants are recruited into a three‐phase, double‐blinded, randomized crossover trial (NCT02300324), with each phase comprising consumption of 300 g of a soup made from broccoli of one of three Myb28 genotypes (Myb28B/B, Myb28B/V, Myb28V/V). Plant myrosinases are intentionally denatured during soup manufacture. Threefold and fivefold higher levels of sulforaphane occur in the circulation following consumption of Myb28V/B and Myb28V/V broccoli soups, respectively. The percentage of sulforaphane excreted in 24 h relative to the amount of glucoraphanin consumed varies among volunteers from 2 to 15%, but does not depend on the broccoli genotype.ConclusionThis is the first study to report the bioavailability of glucoraphanin and sulforaphane from soups made with novel broccoli varieties. The presence of one or two Myb28V alleles results in enhanced delivery of sulforaphane to the systemic circulation.

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Section: Methodsmentioning

confidence: 99%

“…Data were quantified relative to a two-copy gene control, a region in IVS10 of the breast cancer 1, early onset (BRCA1, NM 007294) gene. [14]…”

Section: Genotypingmentioning

confidence: 99%

Bioavailability of Glucoraphanin and Sulforaphane from High‐Glucoraphanin Broccoli

Sivapalan

Melchini

Saha

et al. 2018

Molecular Nutrition Food Res

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“…GSTM1 (NM_000561) genotype was determined using a real-time PCR procedure based on Covault and colleagues, using gene specific primers and probe and quantified relative to a two-copy gene control, a region in IVS10 of the breast cancer 1, early onset ( BRCA1 , NM_007294) gene [21]. Primers and probes were designed using Applied Biosystems Primer Express (http://www.appliedbiosystems.com/) and are given with PCR conditions in Table S1.…”

Section: Methodsmentioning

confidence: 99%

Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate

et al. 2008

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BackgroundEpidemiological studies suggest that people who consume more than one portion of cruciferous vegetables per week are at lower risk of both the incidence of prostate cancer and of developing aggressive prostate cancer but there is little understanding of the underlying mechanisms. In this study, we quantify and interpret changes in global gene expression patterns in the human prostate gland before, during and after a 12 month broccoli-rich diet.Methods and FindingsVolunteers were randomly assigned to either a broccoli-rich or a pea-rich diet. After six months there were no differences in gene expression between glutathione S-transferase mu 1 (GSTM1) positive and null individuals on the pea-rich diet but significant differences between GSTM1 genotypes on the broccoli-rich diet, associated with transforming growth factor beta 1 (TGFβ1) and epidermal growth factor (EGF) signalling pathways. Comparison of biopsies obtained pre and post intervention revealed more changes in gene expression occurred in individuals on a broccoli-rich diet than in those on a pea-rich diet. While there were changes in androgen signalling, regardless of diet, men on the broccoli diet had additional changes to mRNA processing, and TGFβ1, EGF and insulin signalling. We also provide evidence that sulforaphane (the isothiocyanate derived from 4-methylsuphinylbutyl glucosinolate that accumulates in broccoli) chemically interacts with TGFβ1, EGF and insulin peptides to form thioureas, and enhances TGFβ1/Smad-mediated transcription.ConclusionsThese findings suggest that consuming broccoli interacts with GSTM1 genotype to result in complex changes to signalling pathways associated with inflammation and carcinogenesis in the prostate. We propose that these changes may be mediated through the chemical interaction of isothiocyanates with signalling peptides in the plasma. This study provides, for the first time, experimental evidence obtained in humans to support observational studies that diets rich in cruciferous vegetables may reduce the risk of prostate cancer and other chronic disease.Trial RegistrationClinicalTrials.gov NCT00535977

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“…GSTM1 and GSTT1 genotypes were determined by a multiplex polymerase chain reaction (PCR) using genomic DNA as previously described (32). Genotyping was performed by the 5’ nuclease assay (TaqMan) using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the constitutively present gene BRCA1 as an internal positive control.…”

Section: Methodsmentioning

confidence: 99%

Metabolism of isothiocyanates in individuals with positive and null GSTT1 and M1 genotypes after drinking watercress juice

et al. 2010

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Background & Aims-Isothiocyanates (ITCs) derived from cruciferous vegetables have been shown to be promising agents against cancer in human cell culture, animal models, and in epidemiological studies. Several epidemiological studies have demonstrated an inverse relationship between intake of dietary isothiocyanates and the risk of cancers, particularly lung, colon, and breast. More importantly, the protective effects of dietary ITCs appear to be influenced by glutathione S-transferase (GST) genotype; specifically, individuals with glutathione Stransferase theta 1 (GSTT1) and glutathione S-transferase Mu 1 (GSTM1) null are better protected than those with GSTT1 and M1 positive. Although majority of studies, especially those conducted in populations exposed to ITCs rich diet, demonstrated such effects there are a few studies showed opposite or no association. While evidence for the interactions of dietary ITCs with GST genes is relatively strong, the reasons for the differential effects remain unclear. In this study, we examined one possible mechanism whether subjects with the null genotypes excrete ITCs at a slower rate than those with the positive genotypes after drinking watercress juice, a rich source of ITCs.

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Quantitative real-time PCR for gene dosage determinations in microdeletion genotypes

Cited by 21 publications

References 12 publications

Bioavailability of Glucoraphanin and Sulforaphane from High‐Glucoraphanin Broccoli

Bioavailability of Glucoraphanin and Sulforaphane from High‐Glucoraphanin Broccoli

Broccoli Consumption Interacts with GSTM1 to Perturb Oncogenic Signalling Pathways in the Prostate

Metabolism of isothiocyanates in individuals with positive and null GSTT1 and M1 genotypes after drinking watercress juice

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