Except for hereditary disease, genetic factors that contribute to the development of renal epithelial tumors are unknown. There is a possibility that the MDR1 encoded plasma membrane transporter P-glycoprotein (PGP) influences the risk of development of renal neoplasms. PGP is known to be involved in uptake, binding, transport, and distribution of xe- nobiotics. There is evidence that the MDR1 C3435T polymor- phism drives expression and modulates disease risk. In an explorational case-control study, constitutional genotype fre- quencies were established at MDR1 C3435T of 537 healthy con- trol subjects and compared with those of 212 patients with renal epithelial tumors. There were 179 clear cell renal cell carcinoma (CCRCC) and 33 tumors collectively assigned as non-CCRCC. In a second study, genotypes of another 150 healthy control subjects and 50 patients with three non- CCRCC types (26 papillary RCC, 11 chromophobe RCC, and 13 renal oncocytic adenoma) were compared. PCR-restriction fragment length polymorphism- based analysis of constitu- tional DNA, and statistical analysis were applied. PGP expres- sion was analyzed by quantitative immunohistochemistry. The explorational study showed a significant association between T allele frequency and the occurrence of tumors (P 0.007). When tumors were histopathologically distinguished into fre- quent CCRCC and less frequent non-CCRCC, both patient groups contributed to this effect with a seemingly strong in- fluence by the latter (P 0.0419). The second study estab- lished the T allele as a risk factor especially for non-CCRCC (P 0.0005) with the highest risk for homozygote TT allele

Mutations at splice sites or surrounding sequences have been reported to cause aberrant splicing. However, splicing errors can also occur without sequence alterations. We investigated three tumor suppressor genes for aberrant splicing in tumors. At a low frequency per exon it was found in five of seven of the investigated in-frame exons of the neurofibromatosis type 1 (NF1) gene, in two of three exons of the neurofibromatosis type 2 (NF2) gene, and in one of three exons of the tuberous sclerosis 2 gene. It was detectable in all of the human tumor tissues tested (NF1 neurofibroma, sporadic intramedullar neurinoma, sporadic meningiomas, NF2 schwannoma, NF2 meningioma, basalioma, and naevus) as well as in cultured tumor cell lines and cultured primary cells. Hence, our data show that aberrant splicing is a very common process. According to simulations of the secondary structures of the pre-mRNA, we suggest that aberrant splicing is attributable to the rare occurrence of alternative structures at the splice donor site, which are not recognized by the splice machinery. In HeLa cells, aberrant splicing is found to be increased at elevated temperatures and low pH in vitro, conditions often found in tumor tissues. In three tumor tissues tested for one NF1 exon, we found approximately twice the amount of aberrant transcript as in normal tissues. Therefore, we suggest that the increase in aberrant splicing caused by environmental factors represents an additional mechanism for the reduction of the amount of tumor suppressor mRNA in the absence of relevant mutations in the tumor.

Except for hereditary disease, genetic factors that contribute to the development of renal epithelial tumors are unknown. There is a possibility that the MDR1 encoded plasma membrane transporter P-glycoprotein (PGP) influences the risk of development of renal neoplasms. PGP is known to be involved in uptake, binding, transport, and distribution of xenobiotics. There is evidence that the MDR1(C3435T) polymorphism drives expression and modulates disease risk. In an explorational case-control study, constitutional genotype frequencies were established at MDR1(C3435T) of 537 healthy control subjects and compared with those of 212 patients with renal epithelial tumors. There were 179 clear cell renal cell carcinoma (CCRCC) and 33 tumors collectively assigned as non-CCRCC. In a second study, genotypes of another 150 healthy control subjects and 50 patients with three non-CCRCC types (26 papillary RCC, 11 chromophobe RCC, and 13 renal oncocytic adenoma) were compared. PCR-restriction fragment length polymorphism-based analysis of constitutional DNA, and statistical analysis were applied. PGP expression was analyzed by quantitative immunohistochemistry. The explorational study showed a significant association between T allele frequency and the occurrence of tumors (P = 0.007). When tumors were histopathologically distinguished into frequent CCRCC and less frequent non-CCRCC, both patient groups contributed to this effect with a seemingly strong influence by the latter (P = 0.0419). The second study established the T allele as a risk factor especially for non-CCRCC (P = 0.0005) with the highest risk for homozygote TT allele carriers (P < 0.0001). Independently, MDR1(C3435T) genotype associated variations in PGP expression were shown in normal renal parenchyma with a 1.5-fold difference of median values (TT, 1.9; CC, 2.8; P = 0.0065). The data provide evidence for PGP to influence the susceptibility to develop renal epithelial tumors by virtue of its MDR1(C3435T) polymorphism and changes in expression. Especially T and TT carriers are at risk for developing non-CCRCC, i.e., papillary and chromophobe RCC as well as oncocytic adenomas.

ObjectiveWe investigated the effect of polymorphisms in the P‐glycoprotein (P‐gp) MDR1 gene on steady‐state pharmacokinetics of digoxin in Caucasians. According to earlier data, homozygous TT of the exon 26 complementary deoxyribonucleic acid (cDNA) 3435C>T polymorphism was associated with low P‐gp expression in the human intestine.MethodsEight healthy male homozygous carriers of the wild‐type exon‐26 3435C>T (CC), 8 heterozygous subjects (CT), and 8 homozygous mutant (TT) subjects were selected. Seven further MDR1 polymorphisms were determined. Digoxin was administered orally twice daily on the first two study days; on days 3 to 5, 0.25 mg was given in the morning. On day 5, kinetic parameters were analyzed for genotype‐phenotype and haplotype‐phenotype relationships.ResultsThe area under the plasma concentration‐time curve from time zero to 4 hours [AUC(0–4)] (P = .042) and Cmax (P = .043) values of digoxin were higher in subjects with the 3435TT genotype than in those with the 3435CC. No influence of other single nucleotide polymorphisms (SNPs) on digoxin parameters was detected. Comparison of genotypes deduced from SNPs 2677G>T (exon 21) and 3435C>T revealed significant differences for AUC(0–4) (P = .034) and Cmax (P = .039), which were substantiated by haplotype analysis. Haplotype 12 (2677G/3435T), which had a frequency of 13.3% in a randomly drawn Caucasian sample (n = 687), was associated (Mann‐Whitney test) with higher AUC(0–4) values (P = .009) than were found in noncarriers (mean ± SD, 5.7 ± 0.9 μg · h/L [n = 7] versus 4.8 ± 0.9 μg · h/L [n = 17]). Haplotype 11 (2677G/3435C) had lower AUC(0–4) values (P = .013) compared with those of noncarriers (mean ± SD, 4.7 ± 0.9 μg · h/L [n = 16] versus 5.6 ± 0.9 μg · h/L [n = 8]). Results of haplotype analysis match data of other MDR1 studies.ConclusionHaplotype 12 codes for high values of AUC(0–4) and Cmax of orally administered digoxin. Analysis of MDR1 haplotypes is superior to unphased SNP analysis to predict MDR1 phenotype.Clinical Pharmacology & Therapeutics (2002) 72, 584–594; doi: 10.1067/mcp.2002.129196

Transport by ATP-dependent efflux pumps, such as P-glycoprotein (PGP) and multi-drug resistance related proteins (MRPs), influences bioavailability and disposition of drugs. These efflux pumps serve as defence mechanisms and determine bioavailability and CNS concentrations of many drugs. However, despite the fact that substantial data have been accumulated on the structure, function and pharmacological role of ABC transporters and even though modification of PGP function is an important mechanism of drug interactions and adverse effects in humans, there is a striking lack of data on variability of the underlying genes. This review focuses on the human drug transporter proteins PGP (MDR1) and the multi-drug resistance proteins MRP1 and MRP2. An overview is provided of pharmacologically relevant genetic, structural and functional data as well as on hereditary polymorphisms, their phenotypical consequences and pharmacological implications.

Background: P-glycoprotein, the gene product of MDR1, confers multidrug resistance against antineoplastic agents but also plays an important role in the bioavailability of common drugs in medical treatment. Various polymorphisms in the MDR1 gene were recently identified. A silent mutation in exon 26 (C3435T) was correlated with intestinal P-glycoprotein expression and oral bioavailability of digoxin.Objective: We wanted to establish easy-to-use and cost-effective genotyping assays for the major known MDR1 single nucleotide polymorphisms and study the allelic frequency distribution of the single nucleotide polymorphisms in a large sample of volunteers.Methods: In this study, the distribution of the major MDRI alleles was determined in 461 white volunteers with the use of polymerase chain reaction and restriction fragment length polymorphism.Results: Five amino acid exchanges were found with allelic frequencies of 11.2% for Asn21Asp and 5.5% for Ser400Asn, Strikingly, in exon 21 three variants were discovered at the same locus: 2677G (56.4%), 2677T (41.6%), and 2677A (1.9%), coding for 893Ala, Ser, or Thr. A novel missense Gln1107Pro mutation was found in two cases (0.2%). The highest frequencies were observed for intronic and silent polymorphisms; C3435T occurred in 53.9% of the subjects heterozygously, and 28.6% of individuals were homozygous carriers of 3435T/T with functionally restrained P-glycoprotein.Conclusion: This study provides the first analysis of MDR1 variant genotype distribution in a large sample of white subjects. It gives a basis for large-scale clinical investigations on the functional role of MDR1 allelic variants for bioavailability of a substantial number of drugs.

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.

To evaluate whether alterations in the multidrug-resistance (MDR)-1 gene correlate with intestinal MDR-1 expression and uptake of orally administered P-glycoprotein (PGP) substrates, we analyzed the MDR-1 sequence in 21 volunteers whose PGP expression and function in the duodenum had been determined by Western blots and quantitative immunohistology ( n = 21) or by plasma concentrations after orally administered digoxin ( n = 8 + 14). We observed a significant correlation of a polymorphism in exon 26 (C3435T) of MDR-1 with expression levels and function of MDR-1. Individuals homozygous for this polymorphism had significantly lower duodenal MDR-1 expression and the highest digoxin plasma levels. Homozygosity for this variant was observed in 24% of our sample population ( n = 188). This polymorphism is expected to affect the absorption and tissue concentrations of numerous other substrates of MDR-1.

The EVI2B gene is one of three genes embedded in intron 27b of the neurofibromatosis type 1 (NF1; M. Recklinghausen) gene, which are transcribed in the direction opposite that of the NF1 gene. The function of EVI2B and its relation to NF1 symptoms is unknown. Here, the amounts of NF1 and EVI2B mRNA were investigated in detail in cells involved in NF1 manifestations as café-au-lait macules and neurofibromas. These investigations showed that aside from the NF1 gene, EVI2B is involved in melanocyte and keratinocyte differentiation. Whereas in NF1 melanocytes from café-au-lait macules, EVI2B expression was not altered, in fibroblast-like cells derived from neurofibromas, an increased level of EVI2B mRNA was found. We investigated whether this increase was attributable to an influence of NF1 gene expression on the expression of the EVI2B gene, as suggested by the fact that the EVI2B primary transcript is antisense to the NF1 primary transcript. Investigations of cells derived from patients with different amounts of NF1 pre-mRNA showed no correlation between the amount of NF1 pre-mRNA and the increased level of EVI2B mRNA.

Neurofibromatosis type 1 (NF1) is a common autosomal dominantly inherited disorder characterized by neurofibromas and café-au-lait macules. Most of the NF1 gene germline mutations result in a reduction in the level of neurofibromin. As shown recently, the neurofibromin level can be regulated posttranslationally through alteration of the protein half-life. This raises the question as to whether this type of regulation is also operating in cultured melanocytes of NF1 patients especially in melanocytes derived from café-au-lait macules. In melanocytes cultured without phorbol 12-myristate 13-acetate (PMA) the neurofibromin half-lives were 24 h (healthy controls, MC), 26 h (apparently healthy skin of NF1 patients, MNFS) and 25 h (café-au-lait macules of NF1 patients, MNFC). In PMA-stimulated cells the neurofibromin half-lives were 68 h (MC) and 73 h (MNFS) whereas it was 45 h in melanocytes derived from NF1 café-au-lait macules. The amount of NF1 mRNA was not altered under these culture conditions as shown by competitive RT-PCR. We speculate that this regulation is involved in the formation of some NF1 symptoms, for instance in the formation of café-au-lait macules.

The close association of neurofibromatosis type 1 (NF1) with gliomas raises the question of whether the NF1 gene may be involved in the pathogenesis of sporadic astrocytic brain tumors. However, no frequent mutations within NF1 have been described in these tumors. Recent data on a limited series of gliomas indicate that NF1 expression may even be increased, thereby questioning the role of NF1 as a tumor suppressor in astrocytomas. In the present study, we examined the expression of NF1 in a series of 96 tumors including astrocytomas, meningiomas and plexiform neurofibromas. NF1 RNA transcription levels were compared to those of the reference genes B2M , ACTB and GAPD . The expression of OMGP , which is interposed in the NF1 gene, served as an additional control. NF1 expression did not significantly diverge among different malignancy stages of astrocytomas. As expected, the plexiform neurofibromas showed only very low NF1 expression. A striking finding was the highly variable expression of those genes selected to serve as references. While B2M and ACTB exhibited comparable levels of expression within different grades of astrocytomas and meningiomas, GAPD showed an inverse pattern in these tumors. In conclusion, NF1 expression is strongly reduced in NF1-associated plexiform neurofibromas but not in astrocytic tumors. The significant differences between B2M , ACTB and GAPD transcript levels brings into question the common practice of defining gene expression as a ratio between the transcripts of interest and those of these reference genes.