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Heterozygous POLE or POLD1 germline pathogenic variants (PVs) cause polymerase proofreading associated polyposis (PPAP), a constitutional polymerase proofreading deficiency that typically presents with colorectal adenomas and carcinomas in adulthood. Constitutional mismatch-repair deficiency (CMMRD), caused by germline bi-allelic PVs affecting one of four MMR genes, results in a high propensity for the hematological, brain, intestinal tract, and other malignancies in childhood. Nonmalignant clinical features, such as skin pigmentation alterations, are found in nearly all CMMRD patients and are important diagnostic markers. Here, we excluded CMMRD in three cancer patients with highly suspect clinical phenotypes but identified in each a constitutional heterozygous POLE PV. These, and two additional POLE PVs identified in published CMMRD-like patients, have not previously been reported as germline PVs despite all being well-known somatic mutations in hyper-mutated tumors. Together, these five cases show that specific POLE PVs may have a stronger "mutator" effect than known PPAP-associated POLE PVs and may cause a CMMRD-like phenotype distinct from PPAP. The common underlying mechanism, that is, a constitutional replication error repair defect, and a similar tumor spectrum provide a good rationale for monitoring these patients with a severe constitutional polymerase proofreading deficiency according to protocols proposed for CMMRD., (© 2021 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Uncovering frequent motives of action by which variants impair 3' splice site (3'ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini-gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722-11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG-exclusion zone (AGEZ) between the authentic 3'ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3'ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single-nucleotide variants upstream of positions -3 interrupt the AGEZ. Of these AGEZ-interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3'ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3'ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position -3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice-neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing., (© 2020 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population., (© 2019 The Authors. Human Mutation Published by Wiley Periodicals, Inc.)

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome., (© 2019 The Authors. Human Mutation Published by Wiley Periodicals, Inc.)

Targeted next-generation-sequencing (NGS) panels have largely replaced Sanger sequencing in clinical diagnostics. They allow for the detection of copy-number variations (CNVs) in addition to single-nucleotide variants and small insertions/deletions. However, existing computational CNV detection methods have shortcomings regarding accuracy, quality control (QC), incidental findings, and user-friendliness. We developed panelcn.MOPS, a novel pipeline for detecting CNVs in targeted NGS panel data. Using data from 180 samples, we compared panelcn.MOPS with five state-of-the-art methods. With panelcn.MOPS leading the field, most methods achieved comparably high accuracy. panelcn.MOPS reliably detected CNVs ranging in size from part of a region of interest (ROI), to whole genes, which may comprise all ROIs investigated in a given sample. The latter is enabled by analyzing reads from all ROIs of the panel, but presenting results exclusively for user-selected genes, thus avoiding incidental findings. Additionally, panelcn.MOPS offers QC criteria not only for samples, but also for individual ROIs within a sample, which increases the confidence in called CNVs. panelcn.MOPS is freely available both as R package and standalone software with graphical user interface that is easy to use for clinical geneticists without any programming experience. panelcn.MOPS combines high sensitivity and specificity with user-friendliness rendering it highly suitable for routine clinical diagnostics., (© 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

3'EPCAM (Epithelial Cell Adhesion Molecule) genomic rearrangements can be a cause of mismatch repair deficiency in rare Lynch syndrome families. 3'EPCAM deletions include the polyadenylation signal and might result in promoter hypermethylation of the centromeric MSH2 gene in cis. A somatic rearrangement in trans affecting MSH2 is responsible for the final mismatch repair deficiency in the corresponding tumors but the mechanisms are not well documented. In this report two germline 3'EPCAM deletions are described together with the corresponding somatic mutations in the patient's colorectal tumors. Mutation and breakpoint analysis resulted in the identification of one novel (c.556-531&#95;*872del) and one known EPCAM deletion (c.859-689&#95;*14697del). Both deletions resulted from Alu mediated homologous recombination causing aberrant EPCAM-MSH2 fusion transcripts. The colorectal tumors of the deletion carriers were MSI-high. Strong hypermethylation of the MSH2 promoter was measured. Analysis of somatic genomic rearrangements showed a 4 Mb deletion including the EPCAM, MSH2 and MSH6 genes in one tumor and copy neutral loss of heterozygosity in the EPCAM-MSH2 region in the other tumor. This indicates that hemi- and homozygous hypermethylation of the MSH2 promoter and hence complete silencing of MSH2 expression was responsible for the mismatch repair deficiency in both colorectal tumors., (Copyright © 2013 Wiley Periodicals, Inc.)

Constitutional mismatch repair deficiency (CMMR-D) due to biallelic germline mutations in one of four mismatch repair genes causes a childhood cancer syndrome characterized by a broad tumor spectrum including hematological malignancies, and brain and Lynch syndrome-associated tumors. Herein, we report three children who had in addition to CMMR-D-associated malignancies multiple pilomatricomas. These are benign skin tumors of hair matrical differentiation frequently associated with somatic activating mutations in the ß-catenin gene CTNNB1. In two of the children, the diagnosis of CMMR-D was confirmed by the identification of biallelic germline PMS2 mutations. In the third individual, we only found a heterozygous germline PMS2 mutation. In all nine pilomatricomas with basophilic cells, we detected CTNNB1 mutations. Our findings indicate that CTNNB1 is a target for mutations when mismatch repair is impaired due to biallelic PMS2 mutations. An elevated number of activating CTNNB1 alterations in hair matrix cells may explain the development of multiple pilomatricomas in CMMR-D patients. Of note, two of the children presented with multiple pilomatricomas and other nonmalignant features of CMMR-D before they developed malignancies. To offer surveillance programs to CMMR-D patients, it may be justified to suspect CMMR-D syndrome in individuals fulfilling multiple nonmalignant features of CMMR-D (including multiple pilomatricomas) and offer molecular testing in combination with interdisciplinary counseling., (Copyright © 2013 Wiley Periodicals, Inc.)

Kohlschütter-Tönz syndrome (KTS) is a rare autosomal recessive disorder characterized by amelogenesis imperfecta, psychomotor delay or regression and seizures starting early in childhood. KTS was established as a distinct clinical entity after the first report by Kohlschütter in 1974, and to date, only a total of 20 pedigrees have been reported. The genetic etiology of KTS remained elusive until recently when mutations in ROGDI were independently identified in three unrelated families and in five likely related Druze families. Herein, we report a clinical and genetic study of 10 KTS families. By using a combination of whole exome sequencing, linkage analysis, and Sanger sequencing, we identify novel homozygous or compound heterozygous ROGDI mutations in five families, all presenting with a typical KTS phenotype. The other families, mostly presenting with additional atypical features, were negative for ROGDI mutations, suggesting genetic heterogeneity of atypical forms of the disease., (© 2012 Wiley Periodicals, Inc.)

Legius syndrome presents as a mild neurofibromatosis type 1 (NF1) phenotype. Multiple café-au-lait spots and macrocephaly are present with or without axillary or inguinal freckling. Other typical NF1-associated features (Lisch nodules, bone abnormalities, neurofibromas, optic pathway gliomas, and malignant peripheral nerve sheath tumors) are systematically absent. Legius syndrome is caused by germline loss-of-function SPRED1 mutations, resulting in overactivation of the RAS-MAPK signal transduction cascade. The first families were identified in 2007. Here, we review all identified SPRED1 mutations and summarize molecular, clinical, and functional data. All mutations have been deposited in a database created using the Leiden Open Variation Database software and accessible at http://www.lovd.nl/SPRED1. At present, the database contains 89 different mutations identified in 146 unrelated probands, including 16 new variants described for the first time. The database contains a spectrum of mutations: 29 missense, 28 frameshift, 19 nonsense, eight copy number changes, two splicing, one silent, one in-frame deletion and a mutation affecting the initiation codon. Sixty-three mutations and deletions are definitely pathogenic or most likely pathogenic, eight SPRED1 mutations are probably benign rare variants, and 17 SPRED1 missense mutations are still unclassified and need further family and functional studies to help with the interpretation., (© 2012 Wiley Periodicals, Inc.)

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which--owing to extensive interparalog sequence exchange--closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples--all publicly available--and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5' breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations., (Copyright © 2012 Wiley Periodicals, Inc.)

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients., (© 2011 Wiley Periodicals, Inc.)

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties., (© 2011 Wiley Periodicals, Inc.)

Nonallelic homologous recombination (NAHR) is responsible for the recurrent rearrangements that give rise to genomic disorders. Although meiotic NAHR has been investigated in multiple contexts, much less is known about mitotic NAHR despite its importance for tumorigenesis. Because type-2 NF1 microdeletions frequently result from mitotic NAHR, they represent a good model in which to investigate the features of mitotic NAHR. We have used microsatellite analysis and SNP arrays to distinguish between the various alternative recombinational possibilities, thereby ascertaining that 17 of 18 type-2 NF1 deletions, with breakpoints in the SUZ12 gene and its highly homologous pseudogene, originated via intrachromosomal recombination. This high proportion of intrachromosomal NAHR causing somatic type-2 NF1 deletions contrasts with the interchromosomal origin of germline type-1 NF1 microdeletions, whose breakpoints are located within the NF1-REPs (low-copy repeats located adjacent to the SUZ12 sequences). Further, meiotic NAHR causing type-1 NF1 deletions occurs within recombination hotspots characterized by high GC-content and DNA duplex stability, whereas the type-2 breakpoints associated with the mitotic NAHR events investigated here do not cluster within hotspots and are located within regions of significantly lower GC-content and DNA stability. Our findings therefore point to fundamental mechanistic differences between the determinants of mitotic and meiotic NAHR., (Hum Mutat 31:1163-1173, 2010. © 2010 Wiley-Liss, Inc.)

Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1-REPs B and C. The breakpoints of these approximately 1.0-Mb ("type-3") NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109-bp, respectively, were identified within the LRRC37B-P paralogues of NF1-REPs B and C, and were found to contain sequences capable of non-B DNA formation. Both LCRs contain LRRC37-core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37-containing LCRs at 17q21.31 is known to have mediated the 970-kb polymorphic inversions of the MAPT-locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37-containing LCRs in the human genome.

Sequence exchange between PMS2 and its pseudogene PMS2CL, embedded in an inverted duplication on chromosome 7p22, has been reported to be an ongoing process that leads to functional PMS2 hybrid alleles containing PMS2- and PMS2CL-specific sequence variants at the 5'-and the 3'-end, respectively. The frequency of PMS2 hybrid alleles, their biological significance, and the mechanisms underlying their formation are largely unknown. Here we show that overall hybrid alleles account for one-third of 384 PMS2 alleles analyzed in individuals of different ethnic backgrounds. Depending on the population, 14-60% of hybrid alleles carry PMS2CL-specific sequences in exons 13-15, the remainder only in exon 15. We show that exons 13-15 hybrid alleles, named H1 hybrid alleles, constitute different haplotypes but trace back to a single ancient intrachromosomal recombination event with crossover. Taking advantage of an ancestral sequence variant specific for all H1 alleles we developed a simple gDNA-based polymerase chain reaction (PCR) assay that can be used to identify H1-allele carriers with high sensitivity and specificity (100 and 99%, respectively). Because H1 hybrid alleles harbor missense variant p.N775S of so far unknown functional significance, we assessed the H1-carrier frequency in 164 colorectal cancer patients. So far, we found no indication that the variant plays a major role with regard to cancer susceptibility., ((c) 2010 Wiley-Liss, Inc.)

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type-2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type-2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type-2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs >or=500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type-2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR-mediated somatic type-2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type-2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences., ((c) 2010 Wiley-Liss, Inc.)

AML/MDS patients carrying 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are characterized by a complex aberrant karyotype (CAK) frequently including deletions within 5q, 17p, and 7q, older age and fast progression of the disease with extremely poor prognosis. MLL has been shown to be overexpressed in cases with 11q amplification. However, in most of the cases, the amplified region is not restricted to the MLL locus. In this study, we investigated 19 patients with AML/MDS and MLL gain/amplification. By means of array CGH performed in 12 patients, we were able to delineate the minimal deleted regions within 5q and 17p and identified three independent regions 11q/I-III that were amplified in all cases. Gene expression profiles established in 15 cases were used to identify candidate genes within these regions. Notably, analysis of our data suggests a correlation of loss of 5q and 17p and expression of genes present in 11q23-25. Furthermore, we demonstrate that the gene expression signature can be used to discriminate AML/MDS with MLL amplification from several other types of AML.

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects., ((c) 2007 Wiley-Liss, Inc.)

We describe 94 pathogenic NF1 gene alterations in a cohort of 97 Austrian neurofibromatosis type 1 patients meeting the NIH criteria. All mutations were fully characterized at the genomic and mRNA levels. Over half of the patients carried novel mutations, and only a quarter carried recurrent minor-lesion mutations at 16 mutational warm spots. The remaining patients carried NF1 microdeletions (7%) and rare recurring mutations. Thirty-six of the mutations (38%) altered pre-mRNA splicing, and fall into five groups: exon skipping resulting from mutations at authentic splice sites (type I), cryptic exon inclusion caused by deep intronic mutations (type II), creation of de novo splice sites causing loss of exonic sequences (type III), activation of cryptic splice sites upon authentic splice-site disruption (type IV), and exonic sequence alterations causing exon skipping (type V). Extensive in silico analyses of 37 NF1 exons and surrounding intronic sequences suggested that the availability of a cryptic splice site combined with a strong natural upstream 3' splice site (3'ss)is the main determinant of cryptic splice-site activation upon 5' splice-site disruption. Furthermore, the exonic sequences downstream of exonic cryptic 5' splice sites (5'ss) resemble intronic more than exonic sequences with respect to exonic splicing enhancer and silencer density, helping to distinguish between exonic cryptic and pseudo 5'ss. This study provides valuable predictors for the splicing pathway used upon 5'ss mutation, and underscores the importance of using RNA-based techniques, together with methods to identify microdeletions and intragenic copy-number changes, for effective and reliable NF1 mutation detection.

Chromosome arm 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are rare but recurrent aberrations in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We have recently shown that in addition to the MLL core amplicon, independent sequences in 11q23-24 and/or 11q13.5 are coamplified within the same cytogenetic markers in 90% and 60% of patients, respectively. Here we further narrow down the minimal amplicon in 11q13.5 to 1.17 Mb by means of semi-quantitative PCR and FISH analyses. The newly defined amplicon contains seven genes, including the GRB2-associated binding protein 2 (GAB2). Using real-time RT-PCR we show a significant transcriptional upregulation of GAB2 in the patients who have GAB2 coamplified with MLL. Thus, the adaptor molecule GAB2 that has already been shown to enhance oncogenic signaling in other neoplasias appears as a novel target of 11q amplification in AML/MDS., ((c) 2006 Wiley-Liss, Inc.)

Neurofibromatosis type 1 (NF1), the most common tumor-predisposing disorder in humans, is caused by defects in the NF1 tumor-suppressor gene. Comprehensive mutation analysis applying RNA-based techniques complemented with FISH analysis achieves mutation detection rates of approximately 95% in NF1 patients. The majority of mutations are minor lesions, and approximately 5% are total gene deletions. We found 13 single- and/or multiexon deletions/duplications out of 1,050 detected mutations using our RNA-based approach in a cohort of 1,100 NF1 patients and confirmed these changes using multiplex ligation-dependent probe amplification (MLPA). With MLPA, we found another 12 novel multiexon deletion/duplications in 55 NF1 patients for whom analysis with multiple assays had not revealed a NF1 mutation, including 50 previously analyzed comprehensively. The extent of the 22 deletions and 3 duplications varied greatly, and there was no clustering of breakpoints. We also evaluated the sensitivity of MLPA in identifying deletions in a mosaic state. Furthermore, we tested whether the MLPA P122 NF1 area assay could distinguish between type I deletions, with breakpoints in low-copy repeats (NF1-LCRs), and type II deletions, caused by aberrant recombination between the JJAZ gene and its pseudogene. Our study showed that intragenic deletions and/or duplications represent only approximately 2% of all NF1 mutations. Although MLPA did not substantially increase the mutation detection rate in NF1 patients, it was a useful first step in a comprehensive mutation analysis scheme to quickly pinpoint patients with single- or multiexon deletions/duplications as well as patients with a total gene deletion who will not need full sequencing of the complete coding region.

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein., (Copyright 2004 Wiley-Liss, Inc.)

Amplification within chromosome arm 11q involving the mixed-lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix-based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700-kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23-24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes., (Copyright 2004 Wiley-Liss, Inc.)

There is evidence that 8q amplification is associated with poor prognosis in hepatoblastoma. A previous comparative genomic hybridization analysis identified a critical region in chromosomal bands 8q11.2-q13. Using restriction landmark genomic scanning in combination with a virtual genome scan, we showed that this region is delineated by sequences within contig NT&#95;008183 of chromosomal subbands 8q11.22-q11.23. A real-time PCR-based genomic copy number assay of 20 hepatoblastomas revealed gain or amplification in this critical chromosomal region in eight tumors. The expression of four genes and expressed sequence tags (ESTs) within this newly defined region was assayed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in four tumors with and six tumors without gain or amplification. The PLAG1 oncogene was found to be highly expressed in all but one tumor compared to normal liver tissue. Furthermore, quantitative RT-PCR revealed that the expression level of the developmentally regulated transcription factor PLAG1 was 3-12 times greater in hepatoblastoma tumors and cell lines compared to age-matched normal liver and comparable to the expression in fetal liver tissue. PLAG1 has been shown be a transcriptional activator of IGF2 in other tumor types. Using luciferase reporter assays, we demonstrated that PLAG1 transactivates transcription from the embryonic IGF2 promoter P3, also in hepatoblastoma cell lines. Thus, our results provide evidence that PLAG1 overexpression may be responsible for the frequently observed up-regulation of IGF2 in hepatoblastoma and therefore may be implicated in the molecular pathogenesis of this childhood neoplasia., (Copyright 2003 Wiley-Liss, Inc.)

Restriction landmark genome scanning (RLGS) allows comparative analysis of several thousand DNA fragments in the genome and provides a means to identify CpG islands that are altered in tumor cells as a result of amplification, deletion, or methylation changes. We have developed a novel informatics tool, designated virtual genome scan (VGS), that makes it possible to predict automatically the sequence of fragments in RLGS patterns by matching to the human genome sequence. A combination of RLGS and VGS was utilized to identify changes of chromosome 1-derived fragments in neuroblastoma. A NotI-EcoRV fragment was found to be absent frequently in neuroblastoma cell line RLGS patterns. Sequence prediction by VGS as well as cloning of the fragment showed that it contained a CpG island that is part of the human orthologue of the hamster homeobox gene Alx3. Expression analysis in a panel of human and mouse tissues showed predominant expression of ALX3 in brain tissue. Methylation-sensitive sequence analysis of the promoter region in neuroblastoma cell lines indicated that methylation of specific sequences correlated with repression of the ALX3 gene. Expression was re-induced after treatment with the methylation inhibitor 5-aza-2'-deoxycytidine. Promoter methylation analysis of ALX3 in primary neuroblastoma tumors, using methylation-sensitive polymerase chain reaction, found preferential ALX3 methylation in advanced-stage tumors. The VGS approach we have implemented in combination with RLGS is useful for the identification of genomic CpG island-related methylation changes or deletions in cancer., (Copyright 2002 Wiley-Liss, Inc.)

Background: Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours., Procedure: We have implemented an approach for the two-dimensional separation of human genomic restriction fragments to detect and isolate as yet unknown amplified sequences in the MYCN amplicon in neuroblastoma. Using this approach we have recently cloned a novel gene referred to as NAG that is frequently coamplified with MYCN in neuroblastoma., Results and Conclusions: We report here the identification and cloning of two additional CpG islands that are amplified in neuroblastoma. One contains a sequence that is identical to the first intron of DDX1. The other represents a novel CpG island that is associated with an as yet unidentified gene. We show that the novel CpG island is located in close proximity to the MYCN locus on chromosome 2 and is as frequently coamplified with MYCN in neuroblastoma as NAG and DDX1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) provides improved efficiency in detecting NF1 mutations which are dispersed throughout the gene which spans 350 kilobases of genomic DNA. We have applied the PTT and subsequent sequence analysis of cloned cDNA to identify mutations in NF1 patients. We report here the identification of two novel (W336X and Q315X), and one recurrent (R304X) mutation located in exon 7 and show that all three premature termination codons lead to skipping of exon 7 in a proportion of the transcripts derived from the mutated allele. Possible mutation-induced alterations of the RNA secondary structure and their impact on skipping of exon 7 of the NF1 gene are explored and discussed., (Copyright Wiley-Liss, Inc.)

Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer-based approach for the analysis of two-dimensional separations of human genomic restriction fragments. Through the use of methylation-sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two-dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor.