An intronic single base exchange leads to a brown adipose tissue-specific loss of Ucp3 expression and an altered body mass trajectory.

Uncoupling protein 3 (Ucp3) is a transport protein of the inner mitochondrial membrane and presumably is implicated in the maintenance or tolerance of high lipid oxidation rates. Ucp3 is predominantly expressed in skeletal muscle and brown adipose tissue and is regulated by a transcription factor complex involving peroxisome proliferator-activated receptor-alpha, MyoD, and COUP transcription factor II. By analysis of a mutant Djungarian hamster model lacking Ucp3 transcription specifically in brown adipose tissue, we identified a putative transcription factor-binding site that confers tissue specificity. A naturally occurring intronic point mutation disrupting this site leads to brown adipose tissue-specific loss of Ucp3 expression and an altered body weight trajectory. Our findings provide insight into tissue-specific Ucp3 regulation and, for the first time, unambiguously demonstrate that changes in Ucp3 expression can interfere with body weight regulation.

Microsatellite analysis of free tumor DNA in urine, serum, and plasma of patients: a minimally invasive method for the detection of bladder cancer.

PURPOSE: Tumor cells may release DNA into circulation, which is subsequently carried as free DNA and enriched in blood and urine. The detection of tumors by microsatellite analysis of free DNA offers a possibility to establish a minimally invasive method for the detection of bladder cancer. EXPERIMENTAL DESIGN: We performed microsatellite analysis of free DNA of urine, serum, and plasma in comparison with DNA of lymphocytes and tumors of 40 patients with conspicuous bladder lesions. Six microsatellite markers were used for the detection of alterations on chromosomes 4, 9, and 17. RESULTS: Twenty-six of 36 bladder tumor tissue samples showed alterations. Microsatellite changes matching those in the tumor tissues were detected in at least one of the body fluids in 23 cases. CONCLUSIONS: The study indicates that simultaneous and multiple investigations of microsatellite markers on free DNA of urine and blood could have clinical relevance as a minimally invasive method for diagnosis and screening of bladder cancer.

Locking of 3' ends of single-stranded DNA templates for improved Pyrosequencing performance.

In Pyrosequencing, a DNA strand complementary to a single-stranded DNA (ssDNA) template is synthesized, whereby each incorporated nucleotide yields detectable light, and the light intensity is proportional to the incorporated nucleotides. Correct data interpretation (i.e., signal-to-noise ratio of light intensities) is hampered by artifacts due to the formation of secondary structures of single-stranded templates. Critical among these is the looping back of the template's nonbiotinylated 3' end to itself In the resulting structure, the 3' end functions as a primer, the extension of which results in background signals. We present two ways of preventing the self-priming of a template's 3' end: (i) the use of a modified oligonucleotide, called blOligo, which is complementary to the template's 3' end and (ii) the extension of the template's 3' end with a ddNMP. In contrast to unprotected 3' ends of ssDNA templates, causing inconsistent results, we show that protecting the 3' end of an ssDNA template using either blOligos or ddNMP enables the correct interpretation of signals and results in reliable quantification.

TP53 alterations as a potential diagnostic marker in superficial bladder carcinoma and in patients serum, plasma and urine samples.

Molecular markers are needed for better distinguishing of non-invasive papillary (pTa) and minimally invasive (pT1) bladder carcinomas and for identifying individual tumors with a high risk of recurrence or disease progression. First aim of our study was to evaluate TP53 microsatellite and mutation analysis as an effective concept for the characterization of superficial bladder tumors with different biological aggressiveness. Mutation screening in the TP53 hot spot region was performed in 55 microdissected superficial bladder tumor samples by direct genomic sequencing. PCR based LOH analysis was done with two markers at 17p13. Second, there is considerable interest in the development of non-invasive techniques that would detect recurrent bladder neoplasia. In order to evaluate TP53 alterations as a potential marker for a non-invasive diagnosis of recurrences or residuals and to determine whether tumor-specific DNA exhibiting LOH or sequences harbouring a mutation, can be detected in body fluids, mutation screening was performed in urine, plasma and serum of patients with a mutated primary tumor. LOH analysis with two markers at 17p was done in the corresponding urine and blood samples of 31 primary tumors. As seen from our results, TP53 inactivation by mutation seems to characterize higher malignant superficial bladder tumors which tend to recur and in which the probability is higher that the rezidives progress to muscle invasive growth pattern. Only in 2/8 cases, the TP53 mutation from the primary tumor could be re-detected in patients urine and blood. 17p microsatellite changes with at least one marker were found in 30/31 body fluids of the tumor patients (97%). Correlating the 17p status found in body fluids to the status of the primary tumor, the concordance is only about 52%. We conclude that TP53 genotyping as a non-invasive diagnostic tool in outpatient samples is of limited value for clinical practice.

Human galanin (GAL) and galanin 1 receptor (GALR1) variations are not involved in fat intake and early onset obesity.

The neuropeptide galanin (GAL) is involved in food intake and in fat ingestion. Presumably, these effects are conveyed via the galanin 1 receptor (GALR1). We screened the coding region of GAL (including 444 bp of its promoter region) and GALR1 for mutations using single-strand conformation polymorphism analysis and denaturing HPLC in up to 191 obese children and adolescents and 106 healthy underweight young adults (students). In GAL, we identified 3 novel single nucleotide polymorphisms (SNPs; silent: g.-419T-->C, g.-244G-->A; missense: g.47C-->T: Ala16Val) and one infrequent missense variation (c.253A-->G: Asn85Asp), and in GALR1 2 novel SNPs (silent: c.150C-->T, missense: c.793A-->T: Ile265Phe). To test for an association with obesity, we genotyped 7 SNPs (GAL: g.-244G-->A, g.47C-->T, rs7101947, rs1042577, rs3136540; GALR1: c.150C-->T, c.793A-->T) in up to 322 obese children and adolescents compared with up to 277 healthy underweight and normal weight young adults. Furthermore, we analyzed these SNPs with respect to potential effects on the percentage of energy consumed as fat in obese children and adolescents. Allele and genotype frequencies did not differ among the groups tested. In addition, we performed a pedigree transmission disequilibrium test (PDT) for one SNP (GAL: g.-244G-->A) in 610 (518 independent) obesity-trios (obese child or adolescent and both of its parents). However, the PDT for SNP GAL g.-244G-->A revealed no transmission disequilibrium. We conclude that the analyzed SNPs in GAL and GALR1 do not play a major role in early onset obesity or dietary fat intake in the obese children and adolescents of our study groups.

Analysis of genetic alterations in normal bladder urothelium.

OBJECTIVES: To determine whether normal mucosa had already acquired genetic changes, we analyzed the loss of heterozygosity (LOH) and chromosomal changes in normal urothelium from patients with bladder cancer and those without any history of bladder cancer. METHODS: Sixteen patients with bladder cancer and 15 patients with benign prostatic hyperplasia were included in this study. Tumor tissue, as well as macroscopically normal mucosa, was examined histopathologically. We performed comparative genomic hybridization according to standard protocols to detect chromosomal alterations. Furthermore, we analyzed LOH using four markers on chromosome 9 according to standard protocols for polymerase chain reaction. RESULTS: In 75% of tumor samples, LOH or shifts were detected at least with one marker on chromosome 9. Comparative genomic hybridization revealed chromosomal alterations in 12 (75%) of 16 tumors. The loss of chromosome 9 was seen frequently (56%). LOH was observed in normal mucosa in 5 of 16 patients with tumor and 1 of 15 patients with benign prostatic hyperplasia. Chromosomal alterations were also seen in the normal mucosa of 1 patient with tumor and 2 patients with benign prostatic hyperplasia (chromosomes 1, 2, 6, 14, and 17). CONCLUSIONS: Our results indicated that no general genetic instability is present in the bladder urothelium. Therefore, in most patients with bladder cancer, it seems that multifocality and recurrence are not caused by genetic instability of normal urothelial cells but develop owing to cell migration or intraluminal spread.