Kangsheng Wang | PubMed Health

PubMed2006

Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma.

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens. Expression values were normalized to uninvolved ovarian epithelium. Gene expression in matched frozen and FPE tissues demonstrated significant concordance (r = 0.92, P < 0.0001). However, comparison of amplified and unamplified RNA from frozen OEA tissues revealed an altered molecular profile in amplified RNA. Amplification of RNA from FPE tissues was not successful. The expression of Desert hedgehog (DHH), Indian hedgehog (IHH), Hedge-hog interacting protein (HHIP), Wnt10B, Wnt9B, and Wnt inhibitory factor (WIF1) were tumor-specific with no detectable expression in normal ovarian epithelium. In addition, several genes were significantly (P < 0.025) down-regulated in OEA, including cyclin E2, Porcupine, c-Myc, and Axin 2 (4.8-, 3.6-, 2.9-, and 1.9-fold, respectively). TaqMan low-density array provides an effective multivariate technique for examining gene expression in RNA isolated from either snap-frozen or archival FPE tissues and can identify tumor-specific genes, possibly leading to novel treatments.

Animals↗

PubMed2002

Profound dihydropyrimidine dehydrogenase deficiency resulting from a novel compound heterozygote genotype.

A familial approach was used to elucidate the genetic determinants of profound and partial dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) deficiency in an Alabama family. In 1988, our laboratory diagnosed profound DPD deficiency in a breast cancer patient with grade IV toxicity after cyclophosphamide/methotrexate/5-fluorouracil chemotherapy (R. B. Diasio et al., J. Clin. Investig., 81: 47-51, 1988). We now report the genetic analysis of archived genomic DNA that reveals that the proband was a compound heterozygote for two different mutations, one in each allele: (a) a G to A mutation in the GT 5' splicing recognition sequence of intron 14, which results in a 165-bp deletion (corresponding to exon 14) in the DPD mRNA (DPYD*2A); and (b) a T1679G mutation (now designated DPYD*13), which results in a I560S substitution. Sequence analysis revealed segregation of both mutations with the son and the daughter each inheriting one mutation. Phenotype analysis (DPD enzyme activity) confirmed that both children were partially DPD deficient. Plasma uracil and DPD mRNA levels were found to be within normal limits in both children. We conclude that profound DPD deficiency in the proband resulted from a combination of two mutations (one mutation in each allele) and that heterozygosity for either mutation results in partial DPD deficiency. Lastly, we identified two variant alleles reported previously as being associated with DPD enzyme deficiency [T85C resulting in a C29R substitution (DPYD*9A) and A496G (M166V) in a family member with normal DPD enzyme activity]. These data suggest that both variant alleles are unrelated to DPD deficiency and emphasize the need to perform detailed familial genotypic and phenotypic analysis while characterizing this pharmacogenetic syndrome.

Adult↗