Maria Guida Boavida

Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors.

Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that amplification by RT – PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identified in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel effector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.

Quality Assessment of Genetic Markers Used for Therapy Stratification

PURPOSE Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences

The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no “shadow bands”. These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations.

Translocation (8;17;15;21)(q22;q23;q15;q22) in acute myeloid leukemia (M2) : A four-way variant of t(8;21)

We report the results of cytogenetic, fluorescence in situ hybridization (FISH) and molecular analyses in a 15-year-old boy diagnosed with acute myeloid leukemia subtype M2 (AML-M2). Cytogenetic and FISH analyses, the latter with whole chromosome painting probes, revealed a complex translocation involving four chromosomes: t(8;17;15;21)(q22;q23;q15;q22). The observation of breakpoints at 8q22 and 21q22 suggested a rearrangement of the ETO and AML1 genes, respectively. Using a dual-color FISH test with ETO and AML1 probes, we demonstrated an AML1/ETO fusion signal on the derivative chromosome 8. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of AML1/ETO fusion transcripts identical to those found in classical t(8;21). The present case highlights the relevant role of the rearranged chromosome 8, which encodes the AML1/ETO fusion product in the pathogenesis of AML-M2.

Loss of Heterozygosity at Chromosome 9p21 in Primary Neuroblastomas: Evidence for Two Deleted Regions

The genes responsible for the development of neuroblastoma following in vivo deletion or mutation are largely unknown. We have performed loss of heterozygosity studies on a series of 24 Portuguese primary neuroblastomas using 6 polymorphic markers located at chromosome 9p21 spanning the p16/MTS1/CDKN2, p15/MTS2/CDKN2B, and the interferon alpha and beta genes. Loss of heterozygosity was observed in 4 of the 24 tumors (17%), a somewhat lower percentage than a previous study that identified patients by a mass screening program. A correlation was also observed between 9p21 LOH and 1p36 LOH in our group of tumors. Two distinct regions of 9p21 deletion were observed: one located in the region adjacent to the markers D9S162 and D9S1747 and a second located centromerically of the p16 gene near the D9S171 marker. The latter region is exclusive of the p16 gene. This result suggests the presence of at least one other tumor suppressor gene at 9p21, apart from the p16 and p15 genes, which may be of importance to the development of neuroblastoma.

Dose dependence of radiation-induced micronuclei in cytokinesis-blocked human lymphocytes

Following selection of appropriate culture conditions, various experiments were conducted to evaluate the suitability of the micronucleus assay in cytokinesis-blocked lymphocytes for biological dosimetry purposes. A dose-effect relationship was determined, based on the frequency of micronuclei induced by various doses of 60Co gamma-rays. The data were best fitted to a linear-quadratic model. To validate the system, an attempt was made to estimate unknown dose levels from the yield of micronuclei, by inverting the derived dose-response function. It was concluded that the assay provides a valid approach for dose assessment. The size of radiation-induced micronuclei was measured in relation to the dose. A significant difference in the proportion of large micronuclei between high and low doses was observed. The chromosomal composition of micronuclei, detected by immunofluorescent staining of kinetochores, showed that only a small proportion of micronuclei contains kinetochore. The possible contribution of various mechanisms for the formation of large radiation-induced micronuclei is discussed.

Increased levels of sister chromatid exchanges in military aircraft pilots.

Sister chromatid exchanges (SCEs) were scored in lymphocytes of nine high-performance pilots of alphajet aircrafts and of ten control individuals from the same air base. Statistical analysis of the mean SCE count per cell in the total number of cells analyzed as well as in those having 12 or more SCEs (high-frequency cells, HFCs) revealed a significant difference between pilots and controls, after adjusting for the effect of smoking. Analysis of the cell cycle kinetic data (replication and mitotic indices) revealed no significant differences either between pilots and controls or between smokers and nonsmokers. Previously, we reported an increase in the SCE levels in workers of the aeronautical industry exposed to noise and whole-body vibration. The present results corroborate those findings and indicate that noise and whole-body vibration may cause genotoxic effects in man.

Insertion of a short Alu sequence into the hMSH2 gene following a double cross over next to sequences with chi homology.

Alu repeat sequences and other multiple copy repetitive elements are present throughout the human genome and are active in promoting recombination. It is believed that reverse transcription of transcribed Alu repeats followed by chromosomal integration has been responsible for the wide dispersion and high copy number of these sequences. During studies on the hMSH2 gene we have used RT-PCR to amplify from peripheral blood lymphocytes a cDNA species in which 553 base pairs of hMSH2 cDNA have been deleted to be replaced by a short 36 base pair Alu sequence as a result of a genomic insertion/deletion event. The 36 base pair Alu insert is homologous to a 26 base pair Alu sequence previously implicated in the promotion of recombination and contains the GCTGG motif which is part of the prokaryotic chi sequence. A second chi-like sequence is also located within the deleted hMSH2 region. Both chi-like sequences are located within 4 bp of the two 4-bp regions of cross over containing the insertion/deletion breakpoints. This suggest that a double recombination event has occurred, providing direct evidence for the recombinogenic activity of this Alu element. Furthermore, it suggests that chi-like sequences may define recombination hotspots as in prokaryotes.

Pathological exon skipping in an HNPCC proband with MLH1 splice acceptor site mutation

One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T‐to‐A at position −11 of intron 1 splice acceptor) and investigated its functional consequences by RT‐PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full‐length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild‐type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.

Molecular characterization of a 7p15–21 homozygous deletion in a Wilms tumor

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) at band 7p15–21 in Wilms tumor. We previously reported that the minimal common region of LOH was located between markers D7S517 and D7S503 in bands 7p15–21. We also reported the identification of one Wilms tumor (GOS44) bearing a homozygous, interstitial deletion at a locus within this region. Homogeneous primary cell cultures have been derived from this tumor and have been used for all the subsequent analyses. Using PCR and a panel of STS markers mapping between D7S517 and D7S503, the physical boundaries of the homozygous deletion were determined to be between D7S638 and D7S644. The deleted region spans approximately 3 Mbp of genomic sequence and includes seven known genes (KIAA0744, KIAA0713, AHR, AGR2, NET6, HSPC028, and DGKB.) as well as five predicted genes with similarities to genes of known function (LOC‐91802, ‐116364, ‐96009, ‐92511, and ‐92512). The proximal breakpoint was found to lie between exon 6 and exon 7 of KIAA0744, and the distal breakpoint lay between exon 17 and exon 18 of DGKB. It is unlikely that a functional fusion gene product was generated as a consequence of the fusion between these two genes, because they are oriented in opposite directions on the chromosome. This is the only reported homozygous deletion recorded so far in Wilms tumor, and it provides the means to identify the tumor‐suppressor gene located in this deletion.