Breakpoints In Intron Research Articles

Absence of cutaneous neurofibromas in an NF1 patient with an atypical deletion partially overlapping the common 1.4 Mb microdeleted region

The majority of neurofibromatosis type 1 (NF1) microdeletions in 17q11.2 span approximately 1.4 Mb and have breakpoints that lie within the proximal and distal NF1-low copy repeats, termed NF1-REPs. Less frequent are patients with atypical deletions and non-recurring breakpoints. NF1 patients with gross deletions have been reported to manifest a more severe clinical phenotype than NF1 patients with intragenic mutations, and display early onset and extensive growth of neurofibromas. It has been suggested that the deletion of a neighboring gene or genes in addition to the NF1 gene may modify the expression of the disease, particularly with regard to the high burden of cutaneous neurofibromas. Thus, atypical deletions partially overlapping with the common 1.4 Mb microdeletion interval could prove useful in identifying possible genetic modifiers in the NF1 gene region whose haploinsufficiency might promote neurofibroma growth. Here we report a 20-year-old female who has an atypical deletion with a proximal breakpoint in NF1 intron 21 and a distal deletion breakpoint in the ACCN1 gene. The deletion spans 2.7 Mb and was mediated by an intrachromosomal non-homology-driven mechanism, for example, non-homologous end-joining (NHEJ). Remarkably, this patient did not exhibit cutaneous neurofibromas. However, genotype-phenotype comparisons in this and other previously reported patients with atypical deletions partially overlapping the commonly deleted 1.4 Mb interval do not identify a specific deleted region that is associated with increased neurofibroma growth.

Disruption of ETV6 in intron 2 results in upregulatory and insertional events in childhood acute lymphoblastic leukaemia

We describe four cases of childhood B-cell progenitor acute lymphoblastic leukaemia (BCP-ALL) and one of T-cell (T-ALL) with unexpected numbers of interphase signals for ETV6 with an ETV6-RUNX1 fusion probe. Three fusion negative cases each had a telomeric part of 12p terminating within intron 2 of ETV6, attached to sequences from 5q, 7p and 7q, respectively. Two fusion positive cases, with partial insertions of ETV6 into chromosome 21, also had a breakpoint in intron 2. Fluorescence in situ hybridisation (FISH), array comparative genomic hybridization (aCGH) and Molecular Copy-Number Counting (MCC) results were concordant for the T-cell case. Sequences downstream of TLX3 on chromosome 5 were deleted, leaving the intact gene closely apposed to the first two exons of ETV6 and its upstream promoter. qRT-PCR showed a significant upregulation of TLX3. In this study we provide the first incontrovertible evidence that the upstream promoter of ETV6 attached to the first two exons of the gene was responsible for the ectopic expression of a proto-oncogene that became abnormally close as the result of deletion and translocation. We have also shown breakpoints in intron 2 of ETV6 in two cases of insertion with ETV6-RUNX1 fusion.

A common 253-kb deletion involving VWF and TMEM16B in German and Italian patients with severe von Willebrand disease type 3.

Severe von Willebrand disease (VWD) type 3 is caused by large deletions, insertions, small truncating mutations, splice site mutations and missense mutations of the VWF gene, respectively. Large deletions have been regarded as being a rare cause of VWD type 3. Complete gene deletions have only been identified in Italian and German patients to date. However, their extent and breakpoints have not been determined yet. To identify the breakpoints of complete VWF deletions in patients with VWD type 3. Five index patients with large deletions from two unrelated German and three Italian families were investigated by polymerase chain reaction (PCR) and primer walking. Haplotypes were composed of eight deletion flanking markers. After initial characterization of a homozygous 253,246 bp deletion (Delta253 k) in a German patient, with the centromeric breakpoint located between CD9 and VWF and the telomeric breakpoint in intron 3 of TMEM16B, respectively, we identified the same Delta253 k in an additional two homozygous and two compound-heterozygous patients, and in their heterozygous parents. All patients share the same deletion-associated marker haplotype. The genomic structure of the breakpoint regions favors DNA double-strand breaks followed by non-homologous end-joining repair as an underlying molecular mechanism rather than a homologous recombination event. Our results suggest a single genetic origin of Delta253 k. Homozygosity for Delta253 k in non-consanguineous families from two different countries may indicate a higher incidence of large deletions in VWD type 3 than previously thought. The availability of a Delta253k-specific assay allows simple and rapid detection of even heterozygous patients and carriers.

MIC gene polymorphism and haplotype diversity in rhesus macaques

Rhesus macaques (Macaca mulatta) mainly originating from India were analysed for their major histocompatibility complex class I-related (MIC) gene repertoire. Thus far, three distinct genes, designated MIC1, MIC2 and MIC3, have been identified in the rhesus macaque. In addition, an MICD pseudogene has been described mapping apart from the other loci in a telomeric direction. Genomic comparisons and the presence of a characteristic microsatellite in exon 5 suggest that the MIC1 gene is the equivalent of the human MICA gene. Hence, the MIC2 gene, lacking the microsatellite - as do humans -, is considered to be the equivalent of human MICB. The MIC3 gene, a hybrid of MICA and MICB, seems to be generated by a crossing-over event with one breakpoint in intron 3 and accordingly is named MICA/B. Apart from their human counterparts, MICA, MICB and MICA/B cluster in separate branches in the phylogenetic tree, confirming the hybrid character of the MICA/B gene. Population analyses have shown that the various genes display polymorphism, and six MICA, five MICB and three MICA/B alleles have been identified. In the panel of homozygous typing cells, two distinct haplotype configurations have been defined by segregation analyses. Each haplotype comprises an MICB gene in conjunction with either an MICA or an MICA/B gene. Furthermore, the presence of a polymorphic microsatellite in the MICA and MICA/B alleles facilitates speedy and accurate haplotyping.

Characterization of Gene Rearrangements Leading to Activation of MDR-1

Expression of the MDR-1/P-glycoprotein gene confers drug resistance both in vitro and in vivo. We previously reported that gene rearrangements resulting in a hybrid MDR-1 transcript represent a common mechanism for acquired activation of MDR-1/P-glycoprotein. We have identified hybrid MDR-1 transcripts in nine MDR-1-overexpressing cell lines and two patients with relapsed ALL. We characterize these rearrangements as follows. 1) Non-MDR-1 sequences in the hybrid MDR-1 transcripts are expressed in unselected cell lines, showing that these sequences are constitutively expressed. 2) The rearrangements occur randomly and involve partner genes (sequences) on chromosome 7 and on chromosomes other than 7. Breakpoints have been characterized in six cell lines. In one, the rearrangement occurred within intron 2 of MDR-1; in the other five, the rearrangement occurred 24 to >96 kb 5' of the normal start of transcription of MDR-1. In one cell line, homologous recombination involving an Alu repeat was observed. However, in the remaining five cell lines, nonhomologous recombination was observed. 3) The rearrangements arise during drug selection. The acquired rearrangements are not detected in parental cells. 4) Five of the six active promoters that captured MDR-1 controlled MDR-1 from a distance of 29 to more than 110 kb 5' to MDR-1. Transcription was initiated in an antegrade or retrograde direction. We conclude that drug selection with natural products targeting DNA or microtubules leads to DNA damage, nonhomologous recombination, and acquired drug resistance, wherein MDR-1 expression is driven by a random but constitutively active promoter.

High BCL6 expression predicts better prognosis, independent of BCL6 translocation status, translocation partner, or BCL6-deregulating mutations, in gastric lymphoma

AbstractTo investigate the role of BCL6 in the pathogenesis of gastric lymphoma, we analyzed the BCL6 promoter region for BCL6 translocations, somatic hypermutations, and deregulating mutations in 43 gastric lymphomas, including 4 extranodal marginal-zone B-cell lymphomas of mucosa-associated lymphoid tissues (MALT lymphomas), 33 diffuse large B-cell lymphomas (DLBCLs), and 6 composite DLBCLs with residual MALT lymphoma (DLCLMLs). BCL6 promoter substitutions by immunoglobulin (Ig) and non-Ig translocation partners, resulting in its deregulation, were frequently involved in DLBCL (36.4%) and DLCLML (50%). Two novel BCL6 translocation partner genes, 28S rRNA and DMRT1, and a new BCL6 translocation breakpoint in intron 2 were also identified. Deregulating mutations were found only in DLBCL (24.2%), which correlated significantly with high BCL6 protein expression. Significantly, high BCL6 expression correlated strongly with longer overall survival (OS), independent of mechanism in gastric DLBCL and DLCLML. Gastric DLBCLs were further subclassified into germinal center B-cell–like (GCB) and non-GCB subgroups immunohistochemically. High BCL6 expression was detected in all GCB cases, irrespective of BCL6 genetic alterations. In the non-GCB subgroup, BCL6-deregulating mutations correlated significantly with high BCL6 expression level. No significant correlation was found between the BCL6 expression level and OS in the non-GCB subgroup, which had significantly poorer prognosis than the GCB subgroup.

Prenatal origin of childhood AML occurs less frequently than in childhood ALL

BackgroundWhile there is enough convincing evidence in childhood acute lymphoblastic leukemia (ALL), the data on the pre-natal origin in childhood acute myeloid leukemia (AML) are less comprehensive. Our study aimed to screen Guthrie cards (neonatal blood spots) of non-infant childhood AML and ALL patients for the presence of their respective leukemic markers.MethodsWe analysed Guthrie cards of 12 ALL patients aged 2–6 years using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements (n = 15) and/or intronic breakpoints of TEL/AML1 fusion gene (n = 3). In AML patients (n = 13, age 1–14 years) PML/RARalpha (n = 4), CBFbeta/MYH11 (n = 3), AML1/ETO (n = 2), MLL/AF6 (n = 1), MLL/AF9 (n = 1) and MLL/AF10 (n = 1) fusion genes and/or internal tandem duplication of FLT3 gene (FLT3/ITD) (n = 2) were used as clonotypic markers. Assay sensitivity determined using serial dilutions of patient DNA into the DNA of a healthy donor allowed us to detect the pre-leukemic clone in Guthrie card providing 1–3 positive cells were present in the neonatal blood spot.ResultsIn 3 patients with ALL (25%) we reproducibly detected their leukemic markers (Ig/TCR n = 2; TEL/AML1 n = 1) in the Guthrie card. We did not find patient-specific molecular markers in any patient with AML.ConclusionIn the largest cohort examined so far we used identical approach for the backtracking of non-infant childhood ALL and AML. Our data suggest that either the prenatal origin of AML is less frequent or the load of pre-leukemic cells is significantly lower at birth in AML compared to ALL cases.

Intronic breakpoint definition and transcription analysis in DMD/BMD patients with deletion/duplication at the 5′ mutation hot spot of the dystrophin gene

Dystrophin mutations occurring at the 5′ end of the gene frequently behave as exceptions to the “frame rule,” their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5′ dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3–7, 3–6 and duplication of exons 2–4.Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3–7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3–7 deletion.Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2–4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5′ dystrophin mutations, including recoding mechanisms and unusual splicing choices.

Genetic mapping of putative Chrna7 and Luzp2 neuronal transcriptional enhancers due to impact of a transgene-insertion and 6.8 Mb deletion in a mouse model of Prader-Willi and Angelman syndromes

BackgroundPrader-Willi and Angelman syndrome (PWS and AS) patients typically have an ~5 Mb deletion of human chromosome 15q11-q13, of opposite parental origin. A mouse model of PWS and AS has a transgenic insertion-deletion (TgPWS/TgAS) of chromosome 7B/C subsequent to paternal or maternal inheritance, respectively. In this study, we define the deletion endpoints and examine the impact on expression of flanking genes.ResultsUsing molecular and cytological methods we demonstrate that 13 imprinted and 11 non-imprinted genes are included in the TgPWS/TgAS deletion. Normal expression levels were found in TgPWS brain for genes extending 9.1- or 5.6-Mb centromeric or telomeric of the deletion, respectively. Our molecular cytological studies map the proximal deletion breakpoint between the Luzp2 and Siglec-H loci, and we show that overall mRNA levels of Luzp2 in TgPWS and TgAS brain are significantly reduced by 17%. Intriguingly, 5' Chrna7 shows 1.7-fold decreased levels in TgPWS and TgAS brain whereas there is a ≥15-fold increase in expression in neonatal liver and spleen of these mouse models. By isolating a Chrna7-Tg fusion transcript from TgAS mice, we mapped the telomeric deletion breakpoint in Chrna7 intron 4.ConclusionBased on the extent of the deletion, TgPWS/TgAS mice are models for PWS/AS class I deletions. Other than for the first gene promoters immediately outside the deletion, since genes extending 5.6–9.1 Mb away from each end of the deletion show normal expression levels in TgPWS brain, this indicates that the transgene array does not induce silencing and there are no additional linked rearrangements. Using gene expression, non-coding conserved sequence (NCCS) and synteny data, we have genetically mapped a putative Luzp2 neuronal enhancer responsible for ~33% of allelic transcriptional activity. The Chrna7 results are explained by hypothesizing loss of an essential neuronal transcriptional enhancer required for ~80% of allelic Chrna7 promoter activity, while the Chrna7 promoter is upregulated in B lymphocytes by the transgene immunoglobulin enhancer. The mapping of a putative Chrna7 neuronal enhancer inside the deletion has significant implications for understanding the transcriptional regulation of this schizophrenia-susceptibility candidate gene.

Congenital afibrinogenaemia caused by uniparental isodisomy of chromosome 4 containing a novel 15-kb deletion involving fibrinogen Aalpha-chain gene.

Among rare inherited deficiencies of coagulation factors, congenital afibrinogenaemia is characterised by the lack of fibrinogen in plasma. In the last few years, several genetic defects underlying afibrinogenaemia (mostly point mutations) have been described in the fibrinogen gene cluster. In this study, the molecular basis responsible for afibrinogenaemia in a Thai proband was defined. Point mutation screening was accomplished by directly sequencing the three fibrinogen genes. The impossibility to amplify fibrinogen Aalpha-chain gene (FGA) exons 5 and 6 suggested the presence of a homozygous deletion. A specific long-range PCR assay enabled the identification of a novel 15-kb deletion, representing the largest afibrinogenaemia-causing deletion described so far. Direct sequencing of the deletion junction allowed mapping of the breakpoints in FGA intron 4 and in the intergenic region between Aalpha- and Bbeta-chain genes. Since the mutation was inherited only from the mother and nonpaternity was ruled out, a maternal uniparental disomy (UPD) was hypothesised. UPD test, carried out with markers covering the whole chromosome 4, revealed that maternal isodisomy was responsible for homozygosity of the 15-kb deletion in the proband. The apparently normal phenotype of the proband, except for afibrinogenaemia, suggests that UPD for chromosome 4 is clinically silent. This represents the first case of a documented complete isodisomy of chromosome 4 causing the phenotypic expression of a recessive disorder. In silico analyses of the regions surrounding the breakpoints suggested that the 15-kb deletion might have originated from an inappropriate repair of a double-strand break by the nonhomologous end joining mechanism.

Characteristic sequence motifs located at the genomic breakpoints of the translocation t(X;18) in synovial sarcomas.

The SYT-SSXI and SYT-SSX2 fusion genes, derived by reciprocal translocations t(X;18), are acquired genetic events strongly associated with the tumorigenesis of synovial sarcoma. In approaching the mechanisms underlying the formation of these fusion oncogenes, we have analysed the genomic sequences surrounding the SYT-SSX breakpoints in 10 tumors, two expressing SYT-SSXI and eight expressing SYT-SSX2 fusion transcripts. The breakpoints were found to be clustered in the 5' end of intron 10 of SYT, and in two cluster regions within intron 4 of SSX2, whereas the two breakpoints in SSX1 intron 4 were 0.5 kb apart. SYT intron 10 is abundant in repetitive regions with the interspersed repeats occupying 66% of the whole intron. Nine of the 10 breakpoints in intron 10 of SYT and six of the eight breakpoints in intron 4 of SSX2 were at or near repetitive regions. These findings suggest that repetitive regions may contribute to the distribution of genomic breakpoints. Several of the fusion sequences exhibited characteristic signs of nonhomologous end joining, including microhomologies at the end points as well as deletions. Sequences highly homologous (83-94%) to consensus topoisomerase II cleavage sites were identified at or near the breakpoints in all 10 tumors, suggesting a role of this enzyme in creating staggered ends at the breakpoint. Furthermore, sequences highly homologous to consensus Translin binding sequences were found at the breakpoints in two cases, and an Alu-Alu fusion and an insertion of a 206-bp LINE-1 element were found at the breakpoint in one case each. The demonstration of characteristic sequences at the SYT-SSX breakpoint regions is expected to improve our understanding of the molecular genetic mechanisms behind translocations in general, and of the SYT-SSX fusions in synovial sarcoma in particular.

ABO exon and intron analysis in individuals with the AweakB phenotype reveals a novel O1v-A2 hybrid allele that causes four missense mutations in the A transferase

BackgroundSince the cloning in 1990 of cDNA corresponding to mRNA transcribed at the blood-group ABO locus, polymorphisms due to ethnic and/or phenotypic variations have been reported. Some subgroups have been explained at the molecular level, but unresolved samples are frequently encountered in the reference laboratory.ResultsABO blood grouping discrepancies were investigated serologically and by ABO genotyping [duplex polymerase-chain-reaction (PCR) – restriction-fragment-length-polymorphism (RFLP) and PCR – allele-specific-primer (ASP) across intron 6] and DNA sequencing of the ABO gene and its proposed regulatory elements. Blood samples from five individuals living in Portugal, Switzerland, Sweden and the USA were analysed. These individuals were confirmed to be of Black ethnic origin and had the unusual AweakB phenotype but appeared to have the A2B genotype without previously reported mutations associated with weak A or B expression. Sequencing of this A allele (having 467C>T and 1061delC associated with the common A2 [A201] allele) revealed three mutations regularly encountered in the O1v [O02] allele: 106C>T (Val36Phe), 188G>A (Arg63His), 220C>T (Pro74Ser) in exons 3, 4 and 5, respectively. The additional presence of 46G>A (Ala16Thr) was noted, whilst 189C>T that normally accompanies 188G>A in O1v was missing, as were all O1v-related mutations in exons 6 and 7 (261delG, 297A>G, 646T>A, 681G>A, 771C>T and 829G>A). On screening other samples, 46G>A was absent, but two new O alleles were found, a Jordanian O1 and an African O1v allele having 188G>A but lacking 189C>T. Sequencing of introns 2, 3, 4 and 5 in common alleles (A1 [A101], A2, B [B101], O1, O1vand O2 [O03]) revealed 7, 12, 17 and 8 polymorphic positions, respectively, suggesting that alleles could be defined by intronic sequences. These polymorphic sites allowed definition of a breakpoint in intron 5 where the O1v-related sequence was fused with A2 to form the new hybrid. Intron 6 has previously been sequenced. Four new mutations were detected in the hybrid allele and these were subsequently also found in intron 6 of A2 alleles in other Black African samples.ConclusionsA novel O1v-A2 hybrid was defined by ABO exon/intron analysis in five unrelated individuals of African descent with the AweakB blood group phenotype.

Investigating the Mechanism of Chromosomal Deletion: Characterization of 39 Deletion Breakpoints in Introns 47 and 48 of the Human Dystrophin Gene

The region of the dystrophin gene containing introns 45–50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.

Investigating the Mechanism of Chromosomal Deletion: Characterization of 39 Deletion Breakpoints in Introns 47 and 48 of the Human Dystrophin Gene

The region of the dystrophin gene containing introns 45–50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.

Analysis of 22 deletion breakpoints in dystrophin intron 49.

Over 60% of Duchenne and Becker muscular dystrophies are caused by deletions spanning tens or hundreds of kilobases in the dystrophin gene. The molecular mechanisms underlying the loss of DNA at this genomic locus are not yet understood. By studying the distribution of deletion breakpoints at the genomic level, we have previously shown that intron 49 exhibits a higher relative density of breakpoints than most dystrophin introns. To determine whether the mechanisms leading to deletions in this intron preferentially involve specific sequence elements, we sublocalized 22 deletion endpoints along its length by a polymerase-chain-reaction-based approach and, in particular, analyzed the nucleotide sequences of five deletion junctions. Deletion breakpoints were homogeneously distributed throughout the intron length, and no extensive homology was observed between the sequences adjacent to each breakpoint. However, a short sequence able to curve the DNA molecule was found at or near three breakpoint junctions.

New cytogenetic variant, insertion (15;17)(q22;q12q21), in an adolescent with acute promyelocytic leukemia

We present the case of a 15-year-old female with acute promyelocytic leukemia and a new variant chromosome rearrangement identified as ins(15;17)(q22;q12q21) by conventional cytogenetic analysis. This finding was confirmed by fluorescence in situ hybridization using the PML-RARA DNA probe and whole chromosome paints 15 and 17. A typical PML-RARA fusion transcript consistent with a breakpoint in intron 3 of the PML gene and intron 2 of the RARA gene was identified by reverse transcription polymerase chain reaction.

Diversity of Genomic Breakpoints in TFG-ALK Translocations in Anaplastic Large Cell Lymphomas : Identification of a New TFG-ALK XL Chimeric Gene with Transforming Activity

Anaplastic large cell lymphomas are associated with chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene at 2p23 that result in the expression of novel chimeric ALK proteins with transforming properties. In most of these tumors, the t(2;5)(p23;q35) generates the NPM-ALK fusion gene. However, several studies have now demonstrated that genes other than NPM may be fused to the ALK gene. We have recently described two different ALK rearrangements involving the TRK-fused gene (TFG) in which the same portion of ALK was fused to different length fragments of the 5' TFG region. These two rearrangements encoded chimeric proteins of 85 kd (TFG-ALK(S)) and 97 kd (TFG-ALK(L)), respectively. In this study, we have identified a new ALK rearrangement in which the catalytic domain of ALK was fused to a larger fragment of the TFG gene (TFG-ALK(XL)), encoding for a fusion protein of 113 kd. Genomic analysis of these three TFG-ALK rearrangements revealed that the TFG breakpoints occur at introns 3, 4, and 5, respectively, whereas the ALK breakpoints always occur in the same intron. No homologous regions or known recombination sequences were found in these regions. Transfection experiments using NIH-3T3 fibroblasts showed a similar transforming efficiency of TFG-ALK variants compared with NPM-ALK. In addition, in common with NPM-ALK, the TFG-ALK proteins formed stable complexes with the signaling proteins Grb2, Shc, and PLC-gamma. In conclusion, these findings indicate that the TFG may use a variety of intronic breakpoints in ALK rearrangements generating fusion proteins of different molecular weights, but with similar transforming potential than NPM-ALK.

Molecular characterization of genomic AML1-ETO fusions in childhood leukemia.

T(8;21) AML1(CBFA2)-ETO(MTG8) is the most common chromosomal translocation in acute myeloid leukemia (AML) in both children and adults. We sought to understand the structure and gain insight into the fusion process between AML1 and ETO by sequencing genomic fusions in 17 primary childhood AMLs and two cell lines with t(8;21). Reciprocal translocations were sequenced for seven of the 19 samples. We assumed a null hypothesis that the translocation breakpoints would be evenly distributed along the intronic breakpoint cluster regions. Testing for multimodality via smoothed bootstrap statistical methods suggested, however, the presence of two separate cluster regions within both the AML1 and ETO breakpoint cluster regions. ETObreakpoints were predominantly located in intron 1B in a defined cluster 5' of exon 1A (scan statistic P value = 0.00001). All patients with available RNA expressed an AML1-ETO mRNA fusion between exon 5 of AML1 and exon 2 of ETO. Since the structural restraints for the fusion protein of AML1-ETO exclude exon 1A, we reason that ETO intron 1B harbors a structural feature with propensity for breakage and/or recombination. Chromosomal breakpoints displayed evidence of fusion by a non-homologous end joining process, with microhomologies and nontemplate nucleotides at some fusion junctions. Breakpoints in general displayed similar complexity of duplications, deletions, and insertions to other common pediatric leukemia translocations (TEL-AML1, MLL-AF4, PML-RARA, CBFB-MYH11) that we and others have analyzed.

Multi-exon deletions of the FBN1 gene in Marfan syndrome.

BackgroundMutations in the fibrillin -1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion.MethodsWe used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exonsResultsTwo novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5th LTBP (8-cysteine) domain and the adjacent 25th calcium-binding EGF-like (6-cysteine) domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44–46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening.ConclusionsTwo novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly.

A deletion/insertion mutation in the BRCA2 gene in a breast cancer family: a possible role of the Alu-polyA tail in the evolution of the deletion.

Patients with breast and/or ovarian cancer were screened for gross rearrangements in the BRCA2 gene by Southern hybridization, with exon 10 and a fragment of exon 11 used as probes. One breast cancer patient with a positive family history had a 6.2-kb deletion including exons 12 and 13. The deletion breakpoint in intron 11 was in the 3' polyA tail of an Alu element, where a track of approximately 60 adenine nucleotide residues was inserted. Expansion of the Alu-polyA tail may have resulted from polymerase slippage during replication, representing a novel mechanism in which Alu elements mediate deletion/insertion mutations.