Fine-mapping of genes determining extrafusal fiber properties in murine soleus muscle.

Muscle fiber cross-sectional area (CSA) and proportion of different fiber types are important determinants of muscle function and overall metabolism. Genetic variation plays a substantial role in phenotypic variation of these traits; however, the underlying genes remain poorly understood. This study aimed to map quantitative trait loci (QTL) affecting differences in soleus muscle fiber traits between the LG/J and SM/J mouse strains. Fiber number, CSA, and proportion of oxidative type I fibers were assessed in the soleus of 334 genotyped female and male mice of the F34 generation of advanced intercross lines (AIL) derived from the LG/J and SM/J strains. To increase the QTL detection power, these data were combined with 94 soleus samples from the F2 intercross of the same strains. Transcriptome of the soleus muscle of LG/J and SM/J females was analyzed by microarray. Genome-wide association analysis mapped four QTL (genome-wide P < 0.05) affecting the properties of muscle fibers to chromosome 2, 3, 4, and 11. A 1.5-LOD QTL support interval ranged between 2.36 and 4.67 Mb. On the basis of the genomic sequence information and functional and transcriptome data, we identified candidate genes for each of these QTL. The combination of analyses in F2 and F34 AIL populations with transcriptome and genomic sequence data in the parental strains is an effective strategy for refining QTL and nomination of the candidate genes.

Known variant DPYD alleles do not explain DPD deficiency in cancer patients.

Dihydropyrimidine dehydrogenase (DPD) degrades over 80% of administered 5-fluorouracil (5FU), thereby regulating the efficacy of this commonly used anticancer agent. DPD activity is highly variable (8-21-fold) and individuals with reduced activity have a high risk of 5FU toxicity. DPYD encodes DPD protein and 13 different mutations have been reported in DPD-deficient subjects. However, the contribution of these variant genotypes to polymorphic DPD activity in vivo is not clear. The previously described DPYD mutations are contained in 10 exons. These 10 exons were sequenced in a cohort of cancer patients with reduced (n = 23) or normal (n = 14) DPD activity to determine the contribution of each variant allele to low DPD activity in vivo. Eight of the 13 previously defined DPYD mutations (G62A, delta TCAT295-298, C703T, G1003T, G1156T, delta C1897, G2657A, and G2983T) were not detected. A previously defined exon 13 mutation (G1601A) was detected in three individuals with reduced DPD activity. An exon 14 splice donor site mutation (intron14 G1A) was detected in a normal DPD activity individual. It was demonstrated that T85C, A1627G and G2194A are common polymorphisms. A novel exonic mutation (T1679G) was detected in a patient with reduced DPD activity and 5FU toxicity. In addition, three novel common polymorphisms were detected in introns 10 and 13. Only three patients did not have any mutations and 30 had multiple DPYD mutations in the regions examined. However, only 17% (4/23) of the patients with a low DPD phenotype have a molecular basis for reduced activity. Although novel DPYD variants have been identified in this study, the 17 DPYD mutations now described do not entirely explain polymorphic DPD activity and toxic response to 5FU. These data emphasize the complex nature of the molecular mechanisms controlling polymorphic DPD activity in vivo.

The frequency and distribution of thiopurine methyltransferase alleles in Caucasian and Asian populations.

Thiopurine methyltransferase metabolizes 6-mercaptopurine, thioguanine and azathioprine, thereby regulating cytotoxicity and clinical response to these thiopurine drugs. In healthy Caucasian populations, 89-94% of individuals have high thiopurine methyltransferase activity, 6-11% intermediate and 0.3% low, resulting from genetic polymorphism. Four variant thiopurine methyltransferase alleles were detected in over 80% of individuals with low or intermediate thiopurine methyltransferase activity. The wild-type allele is defined as TPMT*1 and the mutant alleles are TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3B (A719G). The frequency of these alleles in different ethnic groups is not well defined. In this study, DNA from 199 British Caucasian, 99 British South West Asian and 192 Chinese individuals was analysed for the presence of these variant alleles using polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction based assays. The frequency of individuals with a variant thiopurine methyltransferase genotype was: Caucasians 10.1% (20/199), South West Asians 2.0% (2/99) and Chinese 4.7% (9/192). Two TPMT*2 heterozygotes were identified in the Caucasian population, but this allele was not found in the two Asian populations. TPMT*3A was the only mutant allele found in the South West Asians (two heterozygotes). This was also the most common mutant allele in the Caucasians (16 heterozygotes and one homozygote) but was not found in the Chinese. All mutant alleles identified in the Chinese population were TPMT*3C (nine heterozygotes). This allele was found at a low frequency in the Caucasians (one heterozygote). This suggests that A719G is the oldest mutation, with G460A being acquired later to form the TPMT*3A allele in the Caucasian and South West Asian populations. TPMT*2 appears to be a more recent allele, which has only been detected in Caucasians to date. These ethnic differences may be important in the clinical use of thiopurine drugs.

Ethnic differences in thiopurine methyltransferase pharmacogenetics: evidence for allele specificity in Caucasian and Kenyan individuals.

Thiopurine methyltransferase (TPMT) degrades 6-mercaptopurine, azathioprine and 6-thioguanine which are commonly used in the treatment of autoimmune diseases, leukaemia and organ transplantation. TPMT activity is polymorphic as a result of gene mutations. Heterozygous individuals have an increased risk of haematological toxicity after thiopurine medication, while homozygous mutant individuals suffer life threatening complications. Previous population studies have identified ethnic variations in both phenotype and genotype, but limited information is available within African populations. This study determined the frequency of common TPMT variant alleles in 101 Kenyan individuals and 199 Caucasians. The frequency of mutant alleles was similar between the Caucasian (10.1%) and Kenyan (10.9%) populations. However, all mutant alleles in the Kenyan population were TPMT*3C compared with 4.8% in Caucasians. In contrast TPMT*3A was the most common mutant allele in the Caucasian individuals. This study confirms ethnic differences in the predominant mutant TPMT allele and the findings will be useful for the development of polymerase chain reaction-based strategies to prevent toxicity with thiopurine medications.

Nomenclature for human DPYD alleles.

To standardize DPYD allele nomenclature and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that each distinct allele be designed by DPYD followed by an asterisk and an Arabic numeral. The number specifies the key mutation and, where appropriate, a letter following the number indicates an additional mutation on the mutant allele. Criteria for classification as a distinct allele are also presented.

Therapeutic opportunities from tumour biology in metastatic colon cancer.

Tumour metastasis is the major cause of morbidity and mortality from colorectal cancer. While improvements in quality of life and patient survival have been made over the past 10 years, the majority of patients with metastatic colorectal cancer will die from their disease. As knowledge of the biology of colon cancer and its invasion/metastasis programme evolve, this presents new therapeutic opportunities for pharmacological and genetic intervention. This review discusses the current approaches to metastatic colorectal cancer therapy, details genomic and biological variance between primary and metastatic tumours, and highlights approaches for harnessing these differences to improve therapy.

Inhibitory effect of fish oil N-3 polyunsaturated fatty acids on the expression of endothelial cell adhesion molecules.

A rapid, cost-effective and sensitive method of Northern analysis using specific oligonucleotide probes combined with chemiluminescence was used to detect adhesion molecule transcripts with as little as 50 ng of total RNA from activated primary human umbilical vein endothelial cells (HUVEC). Activation of cultured HUVEC with the inflammatory cytokine interleukin 1beta (IL-1beta) resulted in distinct time- and dose-dependent patterns of expression of ICAM-1, E-Selectin and VCAM-1 transcripts. The levels of all three transcripts were maximal after 2.5 hours of stimulation with IL-1beta. However, the upregulation of ICAM-1 mRNA levels in response to IL-1beta was much more stable than the transient induction of E-selectin and VCAM-1 transcripts. In addition, we have found that eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) omega3 fatty acids that have been reported to modulate the adhesive properties of cells involved in immune/inflammatory responses do not affect the expression of ICAM-1, VCAM-1 or E-selectin in resting HUVEC. However, EPA (65 microM) or DHA (65 microM) do attenuate the induction of each of these adhesion molecules in IL-1beta-activated HUVEC.

Ethnic variation in the thymidylate synthase enhancer region polymorphism among Caucasian and Asian populations.

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.

Thiopurine methyltransferase alleles in British and Ghanaian populations.

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercapto-purine, 6-thioguanine and azathioprine. TPMT activity is inherited as an autosomal co-dominant trait, and several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. Although ethnic differences in TPMT activity have been described, population frequency analysis of TPMT alleles has not been well defined in different ethnic groups. The frequency of four allelic variants of the TPMT gene, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C were compared in British Caucasian (n = 199) and Ghanaian (n = 217) populations using PCR-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 14.8% of Ghanaians (31 heterozygotes, one homozygote). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the Ghanaian samples analysed. In contrast, 10.1% of British subjects had variant alleles, consisting of TPMT*2 (n = 2), TPMT*3A (n = 17) and TPMT*3C (n = 1) alleles. The frequencies of mutant alleles in this study were 5.3 and 7.6% in British Caucasians and Ghanaians, respectively. Among Ghanaian tribes, Ewe subjects had a lower frequency of mutant alleles (5.9%) than Ga (13.2%) or Fanti (11.6%), although this did not reach statistical significance. This study provides the first analysis of TPMT mutant allele frequency in an African population and indicates that, unlike Caucasians, TPMT*3C is the most common allele in African subjects.

Thymidine phosphorylase and dihydropyrimidine dehydrogenase protein expression in colorectal cancer.

It is essential for actively proliferating cells to increase their rate of DNA synthesis to progress through the cell cycle. This is reflected in the increased uracil usage that is a common feature in solid tumours. Thymidine phosphorylase (TP) anabolises formation of pyrimidine nucleosides available for DNA synthesis, whereas dihydropyrimidine dehydrogenase (DPD) catabolises the degradation of pyrimidine bases, thereby reducing levels of uracil and thymine available for DNA synthesis. In addition, tissue levels of TP or DPD have been associated with the clinical efficacy of pyrimidine anti-metabolites commonly used in the treatment of colorectal cancer. There is little information, however, on the relative expression or degree of co-ordinated regulation of either protein in primary or metastatic colorectal cancer. DPD and TP protein levels were measured in 15 primary colorectal carcinomas, 10 colorectal liver metastases and 25 adjacent uninvolved tissues. DPD was reduced in 67% (10/15) of colorectal tumours (mean tumour/normal = 0.52) and in all liver metastases (mean tumour/normal = 0.41) compared with the corresponding normal tissue. In contrast, TP was increased in 80% (12/15) of colorectal tumours (mean tumour/normal = 18.91) and in all metastases (mean tumour/normal = 3.70). TP and DPD protein expression were highly variable in uninvolved and tumour tissues. The ratio of TP:DPD was higher in 87% of colorectal tumours and in all liver metastases compared with the adjacent uninvolved tissues. This suggests the presence of co-ordinated regulation of these pyrimidine metabolic enzymes and offers a strategy for optimising the use of pyrimidine-based chemotherapy.

Thiopurine methyltransferase genotype predicts therapy-limiting severe toxicity from azathioprine.

BACKGROUND: Substantial hematologic toxicity limits the use of azathioprine. OBJECTIVE: To evaluate 1) polymorphic inactivation of azathioprine by thiopurine methyltransferase and 2) clinical toxicity. DESIGN: Prospective cohort study. SETTING: Two rheumatology units. PATIENTS: 67 patients for whom azathioprine was prescribed as second-line therapy for rheumatic disease. MEASUREMENTS: Polymerase chain reaction-based assays were used to detect mutations in thiopurine methyltransferase. The primary end point was discontinuation of azathioprine therapy because of toxicity. RESULTS: Six of 67 patients (9%) were heterozygous for mutant thiopurine methyltransferase alleles. Five of the 6 patients discontinued therapy within 1 month of starting treatment because of low leukocyte counts. The sixth patient did not adhere to treatment. Patients with wild-type thiopurine methyltransferase alleles received therapy longer than did patients with mutant alleles (median duration of therapy, 39 weeks [range, 6 to 180 weeks] and 2 weeks [range, 2 to 4 weeks], respectively; P = 0.018). CONCLUSION: Analysis of thiopurine methyltransferase genotype is a quick way to identify patients at risk for acute toxicity from azathioprine.

Cloning and initial characterization of the human DPYD gene promoter.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the degradation of pyrimidine bases and pyrimidine-based antimetabolites. Reduced DPD activity is associated with toxicity to 5-fluorouracil (5FU) therapy in cancer patients and with neurological abnormalities in paediatric patients. Although variant DPYD alleles have been identified in DPD-deficient patients, they do not adequately explain polymorphic DPD activity or associated clinical phenotypes in vivo. DPD may be transcriptionally regulated as mRNA levels correlate with activity and are differentially regulated in human tissues. A 1.85 kb 5' flanking region of the human DPYD gene was cloned and has transcriptional activity in cultured cells. Analysis of this 5' flanking region in rhesus and cynomolgus monkeys demonstrated conservation (>96%) between humans and primates. Putative binding sites for ubiquitous and cell-specific factors were identified. A polymorphism that disrupts a putative gamma-interferon response element was identified in a cancer patient with reduced DPD activity and severe 5FU toxicity. Further insight into regulation of DPD expression may identify new avenues for the treatment of clinical problems associated with DPD deficiency.