Early-onset parkinsonism (EOP) may be associated with different mutations in the parkin gene, including exon deletions and duplications. To test for gene dosage alterations, we developed a new method of quantitative duplex PCR using the fluorescence resonance energy transfer technique on the LightCycler (Roche Diagnostics). In 21 patients with EOP, three mutations (a single base pair substitution in exon 3 and small deletions in exon 9) were detected by conventional mutational screening (single-strand conformation polymorphism and sequence analysis), while alterations of gene dosage were found in seven patients. We identified heterozygous and compound heterozygous deletions of exons 2, 3, 5 and 7. The latter was also found in the homozygous state. In addition, two heterozygous duplications of exon 4 were observed. Remarkably, two patients carried more than two parkin mutations. This is the first study systematically screening all 12 exons of parkin by real-time, kinetic quantification and clearly shows that mutational analysis of the parkin gene should include gene dosage studies. Furthermore, our method of quantitative PCR is easily applicable to any other gene to be screened for deletions or duplications of whole exons.

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The aim of this study was to determine the presence of monoclonal myeloma precursor B cells in peripheral blood stem cell harvests and to investigate their role in the clinical outcome of multiple myeloma patients. A total of 39 multiple myeloma patients were treated with a sequential therapy including double high‐dose melphalan therapy followed by a double transplant procedure. The apheresis products for the second transplant were purged using a panel of four or five different mouse monoclonal antibodies against B‐cell antigens (CD10, CD19, CD20, CD22 and CD37). In 19/39 patients a tumour‐specific CDR III signal was identified in the diagnostic bone marrow. Gene scan analysis after CDR III PCR of the magnetic bead isolated B‐cell fraction from the apheresis products in these 19 patients revealed three different patterns: 32% of patients had a predominantly monoclonal B‐cell population; 63% of patients had an identifiable monoclonal signal within an oligoclonal B‐cell population. In only 1/19 patients were no monoclonal B cells identified in the B‐cell population of the apheresis product. A correlation between the clonal pattern and the clinical response after sequential chemotherapy was found. Patients with a predominance of monoclonal myeloma or myeloma precursor B cells had an early relapse or achieved a minimal response or a partial remission. Patients with an oligo‐ and/or polyclonal pattern achieved a high percentage of partial as well as complete remissions.

Serum neopterin concentrations were determined in 20,000 blood donations. For the 400 donations with neopterin concentrations above the 98 th percentile and another 1200 donations with neopterin concentrations in the lower range, results of human parvovirus (HPV) B19 tests were compared. Infectious specimens were identified by dot blot hybridization assay and polymerase chain reaction (PCR) that used the outer primers and detected 1 pg of HPV B19 DNA, corresponding to approximately 10(5) copies of the genome, in the specimens and by a nested PCR that detected 1-10 fg of DNA, corresponding to 10(2)-10(3) copies of the genome. Of 400 specimens with neopterin concentrations > or =12 nmol/L (98th percentile, current cutoff), 10 tested positive by dot blot hybridization assay (9 of these were confirmed by nested PCR). Among 1200 specimens with low neopterin concentrations (<12 nmol/L), no specimen containing HPV B19 DNA was detected. These findings suggest an association between elevated neopterin concentrations and HPV B19 infectivity.

Summary. This study investigated whether T-cell receptor excision circles (TRECs) are a prognostic marker for the outcome of myeloma patients undergoing a tandem autologous peripheral blood stem cell transplantation (PBSCT). Twenty-five patients were enrolled. Samples were obtained at study enrolment, after conventional therapy, between first and second transplantation and 3, 6, 12 and 24 months after the second PBSCT. TRECs were quantified using real-time polymerase chain reaction. A high variation in TREC levels was found at diagnosis (median TREC level 136/10 5 peripheral blood mononuclear cells (PBMCs); range 1-1729), suggesting individual differences in thymic output of naive T cells. Patients with more than 136 TRECs/ 10 5 P BMCs at diagnosis had a statistically significant better overall survival (P ¼ 0AE05) and event-free survival (P ¼ 0AE045), whereas low TREC levels correlated with a higher incidence of infectious complications. Median TREC values were lowest after the first PBSCT (52/10 5 PBMCs) and reached the baseline 12 months after the second transplantation. Patients with high TREC levels after the second PBSCT had a significantly higher probability of being in complete or partial remission 30 months after the second PBSCT. TREC levels were not correlated with b 2 -microglobulin and C-reactive protein levels at diagnosis. These data suggest that TRECs could be a relevant prognostic factor for patients who receive high-dose chemotherapy and autologous PBSCT.