SNP Microarray Analysis Research Articles

An Accumulation of Cytogenetic and Molecular Genetic Events Characterizes the Progression From MDS to Secondary AML: An Analysis of Paired Samples in 38 Patients Analyzed by Cytogenetics, Mutation Analysis and SNP Microarrays.

Abstract 1748Poster Board I-774A hallmark of myelodysplastic syndromes (MDS) is their progression to acute myeloid leukemia (AML). Even though the pathogenetic mechanisms underlying both MDS and de novo AML have broadly been studied on a genetic level, the molecular factors that determine the progression from MDS to secondary AML (s-AML) are largely unknown. In order to gain further insights into the underlying genetic events leading to progression from MDS to AML, we analyzed a total of 38 patients at both their MDS and s-AML disease state focusing on cytogenetics and mutations in NPM1, MLL, NRAS, RUNX1 and FLT3. Additionally, we performed genome-wide SNP microarray analyses (Affymetrix SNP Array 6.0) at both timepoints of investigation in a subset of 34/38 patients. The cohort comprised 38 MDS patients (male/female: 25/13; median age at diagnosis 69.5/range 29.2-82.9) representing WHO categories RARS (n=2), RCMD (n=3), RAEB-1 (n=11), RAEB-2 (n=8), MDS unclassifiable (n=9), CMML (n=3) and MDS/MPN overlap (n=2). Most patients presented in MDS phase with a normal karyotype (n=27; 71%) or showed a single cytogenetic abnormality (n=8). One patient had two alterations and two patients showed a complex karyotype. Molecular genetic analysis revealed the presence of MLL-PTD (n=2), NRAS (n=2) and RUNX1 (n=10, 26.3%) mutations, with mutations in RUNX1 being by far the most common molecular mutation detected in our MDS cohort. NPM1 and FLT3-ITD mutations were not seen at that stage. The median time between analysis of MDS and the diagnosis of s-AML was 269 days (range 40 to 1044). Progression to s-AML was defined by blasts in the bone marrow or peripheral blood >20% and was accompanied by accumulations of either cytogenetic or molecular genetic events. 10/38 patients showed increased cytogenetic complexity with gains of one to 10 aberrations in the course of disease evolution (median number of gained anomalies: 1). Three patients acquired trisomy 8 during disease progression. Moreover, individually different chromosomal losses (n=1), insertions (n=1), translocations (n=3) and further chromosomal gains (n=8) were detected. 16 additional molecular alterations were observed in 15/38 patients involving genes NPM1 (5/16), MLL (3/16), NRAS (3/16), RUNX1 (3/16) and FLT3-ITD (2/16). Of note, these patients exclusively showed either cytogenetic or molecular genetic progression. Only one patient acquired both cytogenetic abnormalities (del(5q) and del(7q)) and a new mutation in RUNX1. 12/38 patients did neither show cytogenetic progression nor did they gain mutations in the genes analyzed. The latter patients, however, showed a trend towards a better overall survival from time of diagnosis of s-AML when compared to the patients with progression markers (median overall survival: not reached vs 7.9 months, % surviving at 1 year: 87.5 vs. 49.7%, p=0.09). SNP microarray analyses revealed the presence of UPD (uniparental disomy) in 11/34 paired samples including chromosomal regions 2p, 4q (n=2), 7q, 11q, 12p, 13q, 17q, 19p, 19q and 21q. While 9/11 patients showed the same UPD at both their MDS and s-AML stages, UPD21q (upd(21)(q11.2-qter)) in one and UPD17q (upd(17)(q21.2-qter)) as well as UPD19q (upd(19q13.42-qter)) in another patient were acquired during disease progression. Interestingly, UPD21q acquired during disease progression coincided with a shift from heterozygous to a homozygous RUNX1 mutation suggesting a role for acquired UPD as a mechanism leading to copy-neutral loss of heterozygosity of RUNX1. UPD(4q) came along with a mutation in TET2, which is located on 4q. This mutation was already present at the MDS stage. In conclusion, our data provide evidence for a multistep pathogenetic mechanism in the progression from MDS to s-AML. Patients can be grouped in two categories; they either acquire cytogenetic aberrations or present a higher number of molecular genetic mutations. While RUNX1 mutations already occurred at a high frequency at the MDS stage, mutations in NPM1 and FLT3 were seen only after progression to s-AML. DisclosuresFlach:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kazak:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

AML with CBFB-MYH11 Rearrangement Is Characterized by RAS Pathway Alterations in 92% of Cases and Demonstrates a High Frequency of NF1 Deletions.

Abstract 272AML with abnormal bone marrow eosinophils and with inv(16)(p13q22) or t(16;16)(p13;q22) leading to a CBFB-MYH11 fusion gene have been classified as distinct entity in the WHO classification and are associated with a favorable outcome. Knock-in mouse models demonstrated that CBFB-MYH11 alone is not sufficient to cause a leukemic phenotype and that additional aberrations are essential for the development of AML. To decipher accompanying genetic lesions in AML with CBFB-MYH11 rearrangements we performed SNP microarray analyses and mutation analysis of KIT, NRAS, KRAS, FLT3, CBL and JAK2 in 37 newly diagnosed patients with CBFB-MYH11+ AML. Recurring gains of whole chromosomes observed both by chromosome banding analysis and SNP microarray assays included chromosomes 8 (n=5) and 22 (n=6). In one additional case SNP microarray analysis revealed also a partial gain of 8q ranging from 8q24.13 to 8q24.3. Recurring genomic losses identified by SNP microarrays but not by conventional karyotyping were deletions of 7q36.1 to 7q36.3 (n=2), 16p13 (n=5), 17q11 (n=6), and 18q23 (n=2). All five 16p13 deletions included the 5' part of the MYH11 gene located in the breakpoint region of the CBFB-MYH11 translocation/inversion, and in 4/5 cases also the ABCC1 gene, encoding a protein with relevance in multidrug resistance. Interestingly, the NF1 (neurofibromin 1) gene, which negatively regulates the RAS pathway, was found to be deleted in all cases with 17q11 loss as depicted by SNP microarrays (6/37). Therefore, we performed further molecular analyses of genes known to be also involved in the RAS pathway. The following mutations were detected: FLT3-ITD (3/37), FLT3-TKD (3/36), KITD816 (7/37), KIT exon 8 (2/37), KRAS (5/37), NRAS (14/37), CBL splice mutations (6/37). No mutations were found in cases screened for JAK2V617F (0/37) and in PTPN11 (0/6). Overall, 46 alterations were observed in the analyzed genes. In 10 cases two of these candidates and in one case three genes were affected. In summary, CBFB-MYH11 rearrangements are very frequently accompanied by molecular alterations leading to an aberrant activation of the RAS pathway. In 34 (92%) of 37 patients mutations within the RAS genes (KRAS, NRAS) or alterations in genes regulating RAS (FLT3, KIT, CBL, NF1) were observed. NF1, which was found to be deleted in 16% of cases in our cohort, was identified as a new target gene in AML with CBFB-MYH11 rearrangement. We conclude that these new insights implicate alterations of the RAS pathway as the principal cooperating mutation in CBFB-MYH11+ AML, and suggest to further evaluate the use of drugs targeting the RAS pathway into treatment regimens for this AML subtype. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Mutations of TET2 and JAK2 but Not CBL Are Detectable in a High Portion of Patients with Refractory Anemia with Ring Sideroblasts and Thrombocytosis (RARS-T).

Abstract 1766Poster Board I-792Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) forms a provisional entity within the category of MDS/MPN-U in the 2008 WHO classification. Although the identification of the JAK2V617F mutation was an important first step in distinguishing this entity from other hematological diseases, further genetic characterization is necessary. We performed comprehensive cytogenetic and molecular genetic investigations including targeted analysis of JAK2V617F, TET2, MPLW515 and CBL, markers known to be altered in MPN, as well as genome-wide single nucleotide polymorphism microarray analysis (SNP-A) in 23 RARS-T patients who fulfilled WHO 2008 diagnostic criteria. The JAK2V617F mutation was detectable in 15 out of 19 analyzed patients (78.9%), four of which were homozygous. However, our patients neither carried a MPLW515 mutation nor mutations in exons 8 or 9 of CBL genes. These genes were recently described to be mainly mutated in myeloproliferative neoplasms. In addition, conventional cytogenetic analysis did not reveal any recurrent cytogenetic abnormalities in RARS-T patients. We also performed SNP microarray analysis in a subset of 10 RARS-T patients. Although we did neither observe recurrent chromosomal gains or losses nor recurring regions of UPD, one patient showed a deletion spanning a 1.3 Mb region on the long arm of chromosome 4 (start: 105,497,200 bp from pter; end: 106,825,780 bp from pter). The deleted region contained TET2, a gene recently found to be altered in many subtypes of myeloid malignancies. To further clarify the 4q24 deletion detected by SNP-A analysis we performed fluorescence in situ hybridization (FISH). 20 out of 100 analyzed interphase nuclei and three metaphases showed only one signal for the probe spanning the TET2 gene in this patient. Interphase FISH with the TET2 probe was performed in nine additional cases not analyzed by SNP arrays due to a lack of material, but no additional case showing a deletion was detected. In addition to FISH, we performed TET2 sequencing in 19/23 RARS-T patients. TET2 mutations were detected in 5/19 patients (26%), of which 3/5 also presented the JAK2V617F mutation, whereas the remaining 2/5 did neither show JAK2V617F nor MPL nor CBL mutations. The five patients showed 6 individually different TET2 mutations. Three were nonsense and two missense mutations. One patient displayed a frameshift mutation leading to a premature stop codon. In summary, RARS-T patients demonstrated a high frequency of both JAK2 and TET2 mutations. Together with the less common MPL mutations described by others RARS-T presents a variety of mutations that overlap with the spectrum of mutations seen in MPN and other myeloid malignancies. Thus, a combination of molecular markers including JAK2 and TET2 should be investigated to more precisely describe RARS-T as an independent disease entity. DisclosuresFlach:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

4.45 Mb microduplication in chromosome band 14q12 including FOXG1 in a girl with refractory epilepsy and intellectual impairment

Microdeletions at 14q12 that include FOXG1, or loss of function mutations in FOXG1, are associated with the congenital variant of Rett syndrome. By SNP microarray analysis we identified a corresponding microduplication at 14q12 in a nine year old girl with symptomatic generalised epilepsy, severe intellectual impairment, and minor dysmorphisms, but without microcephaly. The 14q12 microduplication comprised 4.45 Mb of DNA and included FOXG1. This is the first report of duplication involving FOXG1 and suggests a dosage sensitive role for FOXG1 in brain development.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

Background:Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using...

Identification of novel dyslexia candidate genes through the analysis of a chromosomal deletion

Dyslexia is the most common childhood learning disorder and it is a significantly heritable trait. At least nine chromosomal loci have been linked to dyslexia, and additional susceptibility loci on other chromosomes have been suggested. Within two of these loci, DYX1C1 (15q21) and ROBO1 (3p12) have recently been proposed as dyslexia candidate genes through the molecular analysis of translocation breakpoints in dyslexic individuals carrying balanced chromosomal translocations. Moreover, genetic association studies have indicated a cluster of five dyslexia candidate genes in another linkage region on chromosome 6p22, although there is currently no consensus about which of these five genes contributes to the genetic susceptibility for dyslexia. In this article, we report the identification of four new dyslexia candidate genes (PCNT, DIP2A, S100B, and PRMT2) on chromosome region 21q22.3 by FISH and SNP microarray analyses of a very small deletion in this region, which cosegregates with dyslexia in a father and his three sons.

Spectral karyotyping and SNP microarray analysis define uniparental disomy (UPD) as a novel mutational mechanism in MSI- and CSI-colorectal cancers ∗

Spectral karyotyping and SNP microarray analysis define uniparental disomy (UPD) as a novel mutational mechanism in MSI- and CSI-colorectal cancers ∗

Spectral Karyotyping and SNP Microarray Analysis Define Uniparental Disomy (UPD) as a Novel Mutational Mechanism in MSI- and CSI-Colorectal Cancers

Spectral Karyotyping and SNP Microarray Analysis Define Uniparental Disomy (UPD) as a Novel Mutational Mechanism in MSI- and CSI-Colorectal Cancers

Molecular and Transcriptional Characterization of the Novel 17p11.2-p12 Chromosome Amplification in Multiple Myeloma.

Multiple myeloma (MM) is a fatal malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2–p12, an unstable chromosomal region characterized by a large number of low copy repeats which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2–p12 amplified region was detected in KMS-26 myeloma cell line by SNP microarray analysis. Further FISH mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary data base of myeloma cell lines, identified 12 significantly overexpressed genes in KMS–26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a data base including 11 monoclonal gammopathy of uncertain significance, 121 MM and 9 plasma cell leukaemia, showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in KMS–26 whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, as in the case of solid tumors, amplification/gain of the 17p11.2–p12 in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.

Molecular and transcriptional characterization of the novel 17p11.2‐p12 amplicon in multiple myeloma

Multiple myeloma (MM) is a malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2-p12, an unstable chromosomal region characterized by a large number of low-copy repeats, which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2-p12 amplified region was detected in the KMS-26 myeloma cell line by SNP microarray analysis. Further fluorescence in situ hybridization mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary database of myeloma cell lines identified 12 significantly overexpressed genes in the KMS-26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a database including 141 plasma cell dyscrasia primary tumors showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in the KMS-26, whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, similarly to solid tumors, amplification/gain of the 17p11.2-p12 region in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.

Single-blastomere whole-genome DNA fingerprinting results in unequivocal embryo identification – a powerful new clinical and diagnostic tool

Evaluation of the reproductive competence of individual embryos (EMB) has become the focus of many investigators in ART. Validation of any putative marker requires specific knowledge of whether the individual EMB manifesting that marker actually develops into a healthy child. Single EMB transfer is one way to evaluate these questions, but is compromised by significant cycle to cycle variability in oocyte quality, laboratory conditions, endometrial receptivity, and transfer quality. These variables could be eliminated by simultaneous transfer of more than 1 EMB in a single cycle with subsequent knowledge of which specific EMB ultimately delivered. This study seeks to validate a novel system for embryonic DNA fingerprinting which can reliably distinguish sibling EMBs from each other. Prospective, randomized, and blinded. Phase I: analysis was performed blinded on 20 single lymphocytes from 3 families, followed by an attempt to group the cells into individuals and siblings. Phase II: single blastomeres from 4 EMBs from each of 3 patients were studied blindly (47 blastomeres total), followed by an attempt to reassemble the individual EMBs and group siblings together. Amplification was done using a modification of the GenomePlex (Sigma-Aldrich) system, followed by 500K SNP microarray analysis (Affymetrix). Phase I: The SNP pattern from each cell was compared to the results from every other cell. It was possible to group siblings and reconstruct each family with 100% accuracy. There was no overlap in the 95% CIs of SNP similarity between the same individuals relative to siblings. Phase II: There was 100% accuracy in distinguishing which blastomeres were from the same, sibling, and unrelated EMBs. The figure shows the complete separation in SNP similarities between any two blastomeres from the same vs. sibling EMBs. These data demonstrate the first single blastomere DNA fingerprinting technology that results in unequivocal EMB identification. This method allows parallel analysis of whole genome aneuploidy and single gene disorders for PGD and makes it possible to evaluate the power of new diagnostics and interventions that improve IVF outcome.

Fractioned DNA Pooling: A New Cost-Effective Strategy for Fine Mapping of Quantitative Trait Loci

Selective DNA pooling (SDP) is a cost-effective means for an initial scan for linkage between marker and quantitative trait loci (QTL) in suitable populations. The method is based on scoring marker allele frequencies in DNA pools from the tails of the population trait distribution. Various analytical approaches have been proposed for QTL detection using data on multiple families with SDP analysis. This article presents a new experimental procedure, fractioned-pool design (FPD), aimed to increase the reliability of SDP mapping results, by "fractioning" the tails of the population distribution into independent subpools. FPD is a conceptual and structural modification of SDP that allows for the first time the use of permutation tests for QTL detection rather than relying on presumed asymptotic distributions of the test statistics. For situations of family and cross mapping design we propose a spectrum of new tools for QTL mapping in FPD that were previously possible only with individual genotyping. These include: joint analysis of multiple families and multiple markers across a chromosome, even when the marker loci are only partly shared among families; detection of families segregating (heterozygous) for the QTL; estimation of confidence intervals for the QTL position; and analysis of multiple-linked QTL. These new advantages are of special importance for pooling analysis with SNP chips. Combining SNP microarray analysis with DNA pooling can dramatically reduce the cost of screening large numbers of SNPs on large samples, making chip technology readily applicable for genomewide association mapping in humans and farm animals. This extension, however, will require additional, nontrivial, development of FPD analytical tools.