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The SDHA, SDHB, SDHC, and SDHD genes code for subunits of succinate dehydrogenase (SDH), which forms part of the mitochondrial respiratory chain. Germline mutations in the genes encoding SDHB and SDHD have been reported in familial paragangliomas/pheochromocytomas and in apparently sporadic pheochromocytomas. SDHB and SDHD mutations are widely distributed along the genes with no apparent hot spots. SDHB mutations are often detected in malignant and extra-adrenal pheochromocytomas. SDHD mutations are also detected frequently in head and neck paragangliomas. We sequenced the entire coding regions of the SDHB and SDHD genes in 17 pheochromocytomas. Weidentified novel heterozygous G to A point mutations at the first base of intron 3 of the SDHB gene in a malignant extra-adrenal abdominal pheochromocytoma patient, and at the first base of codon 111 of the SDHD gene in an adrenal pheochromocytoma patient. Further, we confirmed the SDHD mutation by DHPLC. The prevalence of SDHB and SDHD mutations in pheochromocytomas we examined was 12% (2/17). Thus, we identified two novel SDH mutations in Japanese pheochromocytomas. Further studies will investigate the oncogenic potential of these mutations. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mutations in the genes encoding succinate dehydrogenase (SDH) have been associated with susceptibility to pheochromocytoma. However, few reports have examined the level of SDH mRNAs expression. In this study, we examined the level of expression of mRNAs encoding SDHB, SDHC, and SDHD in pheochromocytoma, pheochromocytoma subgroups, and normal adrenal gland, and compared the expression of these genes to the level of expression of related genes in the same tissues. The mean relative level of expression of SDHB, SDHC, SDHD and VHL mRNA was 28.7 ± 6.2%, 16.6 ± 4.8%, 214 ± 47.5% and 25.9 ± 8.2%, respectively, in pheochromocytoma tissues compared to normal adrenal gland. Furthermore, the mean relative level of the RET proto-oncogene mRNA was 707 ± 149% in pheochromocytoma compared to normal adrenal gland. The level of expression of the SDH genes was highly correlated in each individual sample ( P < 0.0001). The level of expression of the SDH mRNAs correlated with the level of VHL mRNA ( P < 0.0001), but not with the level of RET mRNA. The level of SDH mRNAs expression also correlated with the expression of phenylethanolamine N-methyl transferase (PNMT), an adrenaline synthesizing enzyme ( P < 0.01), which may explain the correlation between SDH expression and adrenaline content ( P < 0.05). The level of SDH mRNAs expression correlated strongly with the expression of VEGF mRNA ( P < 0.0001). In multiple endocrine neoplasia (MEN) 2a, the expression of the SDH genes and VHL mRNA was significantly higher than that observed in adrenal or extra-adrenal pheochromocytoma. The expression of the corticotropin-releasing hormone (CRH) mRNA was significantly higher in extra-adrenal pheochromocytoma than in adrenal pheochromocytoma or MEN2a. Thus, tumor-specific gene expression exists in pheochromocytoma, which may explain the characteristics of the tumor. [ABSTRACT FROM AUTHOR]

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans. [ABSTRACT FROM AUTHOR]

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping. [ABSTRACT FROM AUTHOR]