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To establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and T-cell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for routine diagnostics. All samples were studied using the BIOMED-2 multiplex polymerase chain reaction (PCR) protocol. In 176 samples, Southern blot (SB) data were also available, and the two types of molecular results were compared. Results of PCR and SB analysis of both T-cell receptor and immunoglobulin loci were concordant in 85% of samples. For discordant results, PCR results were more consistent with the final diagnosis in 73% of samples. No false-negative results were obtained by PCR analysis. In contrast, SB analysis failed to detect clonality in a relatively high number of samples, mainly in cases of low tumor burden. We conclude that the novel BIOMED-2 multiplex PCR strategy is of great value in diagnosing patients with suspected B- and T-cell proliferations. Because of its higher speed, efficiency, and sensitivity, it can reliably replace SB analysis in clonality diagnostics in a routine laboratory setting. Just as with SB results, PCR results should always be interpreted in the context of clinical, immunophenotypical, and histopathological data.

In addition to the classical Vkappa-Jkappa, Vkappa-kappa deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving Jkappa recombination signal sequence (RSS) or intronRSS elements can occur in the Igkappa (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JkappaRSS-intronRSS, Vkappa-intronRSS, and JkappaRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JkappaRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2-1.0 vs < 0.03). The initial JkappaRSS-intronRSS signal joint formation (except for Jkappa1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical Ckappa deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction and dermatological abnormalities. Inactivating mutations to either of the TSC1 and TSC2 tumour suppressor genes are responsible for the disease. TSC1 and TSC2 encode two large novel proteins called hamartin and tuberin, respectively. Hamartin and tuberin interact directly with each other and it has been reported that tuberin may act as a chaperone, preventing hamartin self-aggregation and maintaining the tuberin-hamartin complex in a soluble form. In this study, the ability of tuberin to act as a chaperone for hamartin was used to investigate the tuberin-hamartin interaction in more detail. A domain within tuberin necessary for the chaperone function was identified, and the effects of TSC2 missense mutations on the tuberin-hamartin interaction were investigated to allow specific residues within the central domain of tuberin that are important for the interaction with hamartin to be pin-pointed. In addition, the results confirm that phosphorylation may play an important role in the formation of the tuberin-hamartin complex. Although mutations that prevent tuberin tyrosine phosphorylation also inhibit tuberin-hamartin binding and the chaperone function, our results indicate that only hamartin is phosphorylated in the tuberin-hamartin complex.