Single Nucleotide Polymorphism Microarray Research Articles

Genome-Wide Genotyping of Acute Myeloid Leukemia with t(9;11) Reveals New Recurrent Genomic Alterations,

Abstract 3546Rearrangements of the mixed lineage leukemia (MLL) gene are associated with the development of acute leukemia, and a variety of translocation partners have been described to date. In acute myeloid leukemia (AML), the translocation t(9;11)(p22;q23), resulting in the MLLT3-MLL fusion gene, is the most common genetic event involving MLL. The translocation t(9;11) can occur de novo, or as a consequence of previous chemotherapy (t-AML). Both types exhibit significant biological and clinical heterogeneity, and cooperating genetic events have been implicated underlying these heterogeneous phenotypes. To identify additional genomic abnormalities in AML with t(9;11), we performed high-resolution, genome-wide analysis of DNA copy number alterations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 34 AMLs with t(9;11) [de novo AML, n=22; t-AML, n=12]. Samples were also analyzed for AML-associated mutations: FLT3 [internal tandem duplication (ITD; 2/33); tyrosine kinase domain (TKD; 2/26)], NPM1 (0/28), CEBPA (0/23), IDH1 (0/28), IDH 2 (0/28), DNMT3A (0/19), NRAS (0/6); and deregulated expression of EVI1 (8/16). Control DNA from remission bone marrow or peripheral blood was available for paired analysis in 12 (33%) cases. Data were processed using reference alignment, dChipSNP, and circular binary segmentation. Paired analysis revealed a mean of 1.9 somatic CNAs per case (range: 0–12); 45% of cases lacked any CNAs. Deletions were more common than gains (1.73 losses/case vs. 0.25 gains/case; p =0.04). There were no significant differences in the mean number of CNAs between de novo and therapy-related cases (de novo AML: 1.0, range: 0–2; t-AML: 2.7, range: 0–12; p =0.93). Recurrent deletions were detected at chromosomal bands 7q36.1–36.2 (n=2) and at the chromosomal translocation breakpoint at 11q23 (n=2). The del(7q36.1–36.2) overlapped with a minimally deleted region at 7q36.1 that we previously identified in 8% of core-binding factor AML containing only 4 genes (PRKAG2, GALNT11, GALNTL5 and MLL3). The only gene contained in both regions was MLL3, a member of the mixed-lineage leukemia gene family. The most recurrent CNA was trisomy 8 (n=5), also detected by conventional cytogenetics in all 5 cases. Novel recurrent focal gains were identified at 9p22.1 (n=2; size: 341 Kb) and at 13q21.33-q22.1 (n=2; size: 1021 Kb) with each region containing genes potentially involved in cancer pathogenesis (ACER2 in 9p; KLF5 in 13q). Analysis of CN-LOH revealed no such lesion in any of the cases. In summary, our data provide a comprehensive survey of CNAs in a well characterized cohort of AMLs with t(9;11). These data demonstrate a very low occurrence of CNAs, with no significant differences between de novo and therapy-related cases and complete absence of CN-LOH. Interestingly, a number of novel recurrent secondary genetic alterations were identified. Determining the functional role of these lesions in leukemogenesis and drug resistance should provide new insights into t(9;11)-bearing AMLs. Disclosures:No relevant conflicts of interest to declare.

Reply: PGD and aneuploidy screening for 24 chromosomes by genome-wide SNP analysis: a responsible path towards greater utility

There are probably more areas of agreement than disagreement between Professor Handyside and us (Bisignano et al., 2011; Handyside, 2011). We certainly agree with him that accurate validation is of paramount importance and that a consensus on the best way to achieve this is urgently required. At this point in time, our opinion is that array comparative genomic hybridization (CGH) has the most well-defined accuracy rate of the new comprehensive chromosome screening methods. The array-CGH approach has been evaluated in several independent laboratories, using various strategies and validated with several different cytogenetic techniques. These studies, which have been published in the literature following peer review, have all provided reassuring results concerning accuracy. Professor Handyside suggests that we were dismissive of the value of single-nucleotide polymorphism (SNP) data, especially in regard to its potential for detecting uniparental disomy (UPD) and providing information on parental origin of abnormalities. It is unfortunate if our article came across this way. We do see the potential merits in SNP analysis and have actually explored the use of SNP microarrays in our own laboratories. Our point is that to attempt to use this information clinically is premature. The accuracy of the data on parental origin is not clear, particularly when applied at the cleavage stage, where chromosomal mosaicism is common. Similarly, it is unclear whether UPD detected at the cleavage stage affect the entire embryo or whether they are typically present in a mosaic form. The necessary studies to determine the accuracy of parental origin and UPD

PTEN Protein Loss by Immunostaining: Analytic Validation and Prognostic Indicator for a High Risk Surgical Cohort of Prostate Cancer Patients

Analytically validated assays to interrogate biomarker status in clinical samples are crucial for personalized medicine. PTEN is a tumor suppressor commonly inactivated in prostate cancer that has been mechanistically linked to disease aggressiveness. Though deletion of PTEN, as detected by cumbersome FISH spot counting assays, is associated with poor prognosis, few studies have validated immunohistochemistry (IHC) assays to determine whether loss of PTEN protein is associated with unfavorable disease. PTEN IHC was validated by employing formalin fixed and paraffin-embedded isogenic human cell lines containing or lacking intact PTEN alleles. PTEN IHC was 100% sensitive and 97.8% specific for detecting genomic alterations in 58 additional cell lines. PTEN protein loss was then assessed on 376 prostate tumor samples, and PTEN FISH or high resolution single nucleotide polymorphism microarray analysis was done on a subset of these cases. PTEN protein loss, as assessed as a dichotomous IHC variable, was highly reproducible, correlated strongly with adverse pathologic features (e.g., Gleason score and pathologic stage), detected between 75% and 86% of cases with PTEN genomic loss, and was found at times in the absence of apparent genomic loss. In a cohort of 217 high risk surgically treated patients, PTEN protein loss was associated with decreased time to metastasis. These studies validate a simple method to interrogate PTEN status in clinical specimens and support the utility of this test in future multicenter studies, clinical trials, and ultimately perhaps for routine clinical care.

SNPpath: Characterizing cattle SNPs by enriched pathway terms

High-density single nucleotide polymorphism (SNP) microarrays have made large-scale genome-wide association studies (GWAS) and genomic selection (GS) feasible. Valuable insight into the genetic basis underlying complex polygenic traits will likely be gained by considering functionally related sets of genes simultaneously. SNPpath, a suite of computer-generated imagery-based web servers has been developed to automatically annotate and characterize cattle SNPs by enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms. The SNPpath allows users to navigate and analysis large SNP sets and is the only web server currently providing pathway annotations of cattle SNPs in National Center for Biotechnology Information's dbSNP database and three commercial platforms. Hence, we describe SNPpath and provide details of the query options, as well as biological examples of use. The SNPpath may be favorable for the analysis of combining SNP association analysis with pathway-driven gene set enrichment analysis and is freely available at http://klab.sjtu.edu.cn/SNPpath.

Abstract A69: Identification of prostate cancer genes in the African American Hereditary Prostate Cancer (AAHPC) study

Abstract Background: Prostate cancer (PCa) is a common malignancy and a leading cause of cancer death among African American men. The hereditary prostate cancer 1 gene (HPC1-1q24-25) was initially implicated as a PCa susceptibility locus in 1996, but was not replicated in the African American Hereditary Prostate Cancer (AAHPC) study, which recruited African American families with four or more affected family members (n=95 families). The objective of our study is to identify and confirm PCa genetic susceptibility loci in African Americans. Methods: Short tandem repeat polymorphism (STR) scans implicated several loci, but no specific family had a logarithm of odds score higher than 1.8. We sought to refine implicated loci through single nucleotide polymorphism microarray. Therefore, we devised a prioritization scoring system where families with multiple affected and unaffected brothers within a likely onset age bracket scored the highest. After Illumina 660K microarray run, Bayesian prior likelihoods will be coupled with family based statistics to analyze the data in 6 families (n=32). Results: Scoring criteria was effective at identifying families (n=31, 14 cases, 17 controls) most informative at identifying genes and SNPs associated PCa. A total of 51 SNPs in 13 genes were found to be associated with PCa Risk at the p<0.05 level including CTBP2 (4p16), HNF1B (17cen-q21.3), and PKHD1 (6p12.2). Conclusions: This study is a first step in identifying PCa genes that warrant further follow-up in the rest of the AAHPC families. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A69.

Report on first 54 patients undergoing preimplantation genetic diagnosis for genetic disorders together with 24 chromosome aneuploidy screening using microarrays

Report on first 54 patients undergoing preimplantation genetic diagnosis for genetic disorders together with 24 chromosome aneuploidy screening using microarrays

23-chromosome single nucleotide polymorphism (SNP) microarray detects genomic aberrations that may be missed by comparative genomic hybridization (CGH) arrays in preimplantation genetic screening (PGS)

23-chromosome single nucleotide polymorphism (SNP) microarray detects genomic aberrations that may be missed by comparative genomic hybridization (CGH) arrays in preimplantation genetic screening (PGS)

23-chromosome single nucleotide polymorphism (SNP) microarray preimplantation genetic screening (PGS) on blastocysts, versus day-3 embryos, results in significantly higher clinical pregnancy rates

To determine the clinical pregnancy rate using SNP PGS in women primarily with recurrent pregnancy loss (RPL) undergoing in vitro fertilization (IVF) Retrospective. A retrospective review was conducted of all embryos that underwent PGS by 23 chromosome SNP microarrays from 1/1/2010 to 1/25/2011. Patients underwent standard in vitro fertilization (IVF) and PGS due to primarily a history of > 2 spontaneous miscarriages. Embryo biopsy was performed at either the cleavage or blastocyst stage. Sample DNA was amplified and analyzed using HumanCytoSNP-12 DNA beadchips and GenomeStudio and KaryoStudio software. Parental DNA was analyzed and compared to embryonic cells to determine the presence of benign copy number variations. Embryos derived from parents with known translocations or inversions were excluded from the study. Binomial confidence intervals for proportions were calculated by the modified Wald method. 2,976 embryos from 317 clinical IVF cycles were tested. 2,704 (90.8%) of these embryos, derived from 249 (78.5%) IVF cycles, were at Day-3 of development (D3 group) when biopsy was performed. Conversely, 272 (9.2%) of embryos, derived from 68 (21.5%) IVF cycles, were at the blastocyst stage (D5 group) when biopsy was performed. Aneuploidy was observed in 59% of the Day 3 embryos compared with only 41% of Day 5 embryos.The clinical pregnancy rates were significantly higher (86%, P<.05) in the D5 group compared to the D3 group (65%). The miscarriage rate in the D3 and D5 groups were 6% and 9% respectively. These miscarriage rates were not statistically different. PGS using 23-chromosome SNP microarrays is practical in improving clinical pregnancy rates in couples with RPL. In addition, PGS performed on blastocysts, versus Day-3 embryos, results in significantly higher clinical pregnancy rates in similar patient populations.

Chromosomal duplications (≥200 KILOBASES (KB)) are more common than deletions ≥200 KB in developing human embryos as identified by 23 chromosome single nucleotide polymorphism (SNP) microarray

Chromosomal duplications (≥200 KILOBASES (KB)) are more common than deletions ≥200 KB in developing human embryos as identified by 23 chromosome single nucleotide polymorphism (SNP) microarray

Determination of chromosomal segregation patterns in embryos of balanced translocation carriers by single nucleotide polymorphism (SNP) microarray technology

A variety of chromosomal segregation patterns occur during meiotic maturation in carriers of a balanced translocation. Accurate counseling regarding the probability of obtaining a balanced or normal embryo depends on the relative distribution of those patterns but little or no data exist based on direct assessment of entire cohorts of embryos. This study directly evaluates the embryos of individuals with balanced translocations to determine their segregation patterns.

Informatics-based molecular karyotyping of products of conception (POC) with maternal cell contamination (MCC) detection: report on 344 consecutive analyses

Cytogenetic analysis of POC using tissue culture has limitations including long time to results, culture failure, and MCC. The objective of this study is to report our initial experience with molecular karyotyping of POC using Single Nucleotide Polymorphism (SNP) microarray which determines number and parental origin of each chromosome.

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

The rate of de novo and inherited aneuploidy as determined by 23-chromosome single nucleotide polymorphism microarray (SNP) in embryos generated from parents with known chromosomal translocations

The rate of de novo and inherited aneuploidy as determined by 23-chromosome single nucleotide polymorphism microarray (SNP) in embryos generated from parents with known chromosomal translocations

Genome-wide copy number profiling using a 100K SNP array reveals novel disease-related genes BORIS and TSHZ1 in juvenile angiofibroma

Juvenile angiofibroma (JA) is a unique fibrovascular tumor, which is almost exclusively found in the posterior nasal cavity of adolescent males. Although histologically classified as benign, the tumor often shows an aggressive growth pattern and has been associated with chromosomal imbalances, amplification of oncogenes and epigenetic dysregulation. We present the first genome-wide profiling of JAs (n=14) with a 100K single nucleotide polymorphism (SNP) microarray. Among the 30 novel JA-specific amplifications detected on autosomal chromosomes with this technique, the genes encoding the cancer-testis antigen BORIS (brother of the regulator of imprinted sites) and the developmental regulator protein TSHZ1 (teashirt zinc finger homeobox 1) were selected for further analysis. Gains for both BORIS (20q13.3) and TSHZ1 (18q22.3) were confirmed by quantitative genomic PCR. Furthermore, quantitative RT-PCR revealed a significant up-regulation of BORIS (p<0.001) and TSHZ1 transcripts (p<0.05) for JAs compared to nasal mucosa. Following detection of BORIS and TSHZ1 proteins in western blots of JAs, subcellular localization was determined for both proteins in immunostaining of JA cryosections. In conclusion, genomic copy number profiling using an SNP microarray has been proven to be a suitable and reliable tool for identifying novel disease-related genes in JAs and newly implicates BORIS and TSHZ1 overexpression in the pathogenesis of JAs. Detection of BORIS in JAs is described with special regard to tumor proliferation and epigenetic dysregulation, and the finding of TSHZ1 amplifications is discussed with special respect to the hypothesis of JAs as malformations of the first branchial arch artery.

DNA microarray as a tool in establishing genetic relatedness—Current status and future prospects

In the past decades, microarray technology has definitely put an edge to the field of genetic research. Our aim was to determine whether single nucleotide polymorphism (SNP) microarrays could be used as a tool in establishing genetic relationships where current molecular genetic methods are not sufficient. We used the Genechip, Affymetrix GenomeWide SNP Array 6.0, which detects more than 900,000 SNP markers dispersed throughout the human genome. The intention was to find a good selection of SNP markers that could be used for statistical evaluation of relatedness in a forensic setting. We conducted pairwise comparisons in the R-package FEST as well as pedigree comparisons in Merlin. Our methods were applied on two separate families, where relationships as distant as 3rd cousins were known. In addition, a question about a possible common ancestry between the two families was tested. Relationships as distant as 2nd cousins could be readily distinguished both from unrelated and other, genetically, closer relationships. This was achieved with a selection of 5774 markers, where each pair of markers was separated by a genetic distance of at least 0.5cM (centiMorgan). When considering 3rd cousins, and more distant relationships, the number of markers needs to be extended, consequently decreasing the genetic distance between the markers. However, inclusion of a too large number of markers presents new challenges and our results imply that the use of too dense sets of markers always yields the highest probability for the genetically closest relationship hypothesis. Simulations confirm that this is most probably caused by the fact that the computational model assumes linkage equilibrium between markers, a problem that will be further evaluated. Our results do however suggest that SNP-data derived from microarrays are well suited for kinship determination provided linkage disequilibrium is properly accounted for.

Live birth outcome with trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray–based comprehensive chromosome screening in infertile patients

The combination of trophectoderm biopsy, blastocyst vitrification, and single-nucleotide polymorphism microarray-based technology for comprehensive chromosome screening results in high implantation and live birth rates that could contribute to the practical application of single embryo transfer for infertility patients.

A novel immunodeficiency disorder characterized by genetic amplification of interleukin 25

Many primary immunodeficiency disorders of differing etiologies have been well characterized, and much understanding of immunological processes has been gained by investigating the mechanisms of disease. Here, we have used a whole-genome approach, employing single-nucleotide polymorphism and gene expression microarrays, to provide insight into the molecular etiology of a novel immunodeficiency disorder. Using DNA copy number profiling, we define a hyperploid region on 14q11.2 in the immunodeficiency case associated with the interleukin (IL)-25 locus. This alteration was associated with significantly heightened expression of IL25 following T-cell activation. An associated dominant type 2 helper T cell bias in the immunodeficiency case provides a mechanistic explanation for recurrence of infections by pathogens met by Th1-driven responses. Furthermore, this highlights the capacity of IL25 to alter normal human immune responses.

Genome integrity of myeloproliferative neoplasms in chronic phase and during disease progression

AbstractPhiladelphia chromosome–negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with increased production of terminally differentiated cells. The disease course is generally chronic, but some patients show disease progression (secondary myelofibrosis or accelerated phase) and/or leukemic transformation. We investigated chromosomal aberrations in 408 MPN samples using high-resolution single-nucleotide polymorphism microarrays to identify disease-associated somatic lesions. Of 408 samples, 37.5% had a wild-type karyotype and 62.5% harbored at least 1 chromosomal aberration. We identified 25 recurrent aberrations that were found in 3 or more samples. An increased number of chromosomal lesions was significantly associated with patient age, as well as with disease progression and leukemic transformation, but no association was observed with MPN subtypes, Janus kinase 2 (JAK2) mutational status, or disease duration. Aberrations of chromosomes 1q and 9p were positively associated with disease progression to secondary myelofibrosis or accelerated phase. Changes of chromosomes 1q, 7q, 5q, 6p, 7p, 19q, 22q, and 3q were positively associated with post-MPN acute myeloid leukemia. We mapped commonly affected regions to single target genes on chromosomes 3p (forkhead box P1 [FOXP1]), 4q (tet oncogene family member 2 [TET2]), 7p (IKAROS family zinc finger 1 [IKZF1]), 7q (cut-like homeobox 1 [CUX1]), 12p (ets variant 6 [ETV6]), and 21q (runt-related transcription factor 1 [RUNX1]). Our data provide insight into the genetic complexity of MPNs and implicate new genes involved in disease progression.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

Genome-wide Association Study Using Pooled DNA to Identify Candidate Markers Mediating Susceptibility to Postoperative Nausea and Vomiting

A family history has been established as a risk factor for postoperative nausea and vomiting (PONV), but the identities of susceptibility genes remain unknown. The goal of this study was to identify the genetic loci that may contribute to PONV susceptibility in an adult population. The authors performed a genome-wide association study involving pooling of DNA obtained from 122 patients with severe PONV and 129 matched controls. Each pool was hybridized to a single nucleotide polymorphism (SNP) microarray, and probe intensity was used to predict allele frequency. Differences in allele frequency between SNP in the PONV and control groups were ranked after accounting for the pooling error. The highest ranking SNPs were selected for individual genotyping in the subjects from whom the DNA pool was comprised and in the new verification cohort consisting of 208 subjects (104 PONV patients and 104 controls). The authors identified 41 SNP targets showing substantial difference in allelic frequency between pools. These markers were first genotyped in the individual DNA samples from which the pools were comprised. The authors observed evidence for an association between PONV and 19 different loci in the genome. In the separate verification cohort, the association with PONV was observed for four SNPs. This association remained significant after correcting for multiple testing (P < 0.0023) for one SNP (rs2165870), which is located upstream of the promoter for the muscarinic acetylcholine receptor 3 subtype (CHRM3) gene. The authors performed the genome-wide association study for PONV using pooled DNA samples. Through individual genotyping, they confirmed association of at least one SNP that is predictive of PONV susceptibility.