Array-based Copy Number Analysis Research Articles

A homozygous AP3D1 missense variant in patients with sensorineural hearing loss as the leading manifestation

Loss-of-function variants in AP3D1 have been linked to Hermansky–Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein–protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

Molecular characterization of Burkitt lymphoma in the breast or ovary

Breast and ovary have been described as rare but typical sites of presentation of Burkitt lymphoma (BL) in females, particularly after puberty. We revised a historic series of 44 lymphomas of the breast or the ovary in women diagnosed between 1973 and 2014 as BL. Fluorescence in situ hybridization (FISH) was applied to all, and array-based copy number analysis as well as expression profiling to a subset of those cases. Of the 42 cases evaluable for FISH, 19 cases showed an IG-MYC translocation but only 9 of those fulfilled the criteria of the current WHO classification for the diagnosis of BL. Those nine cases resembled BL of other sites with regard to molecular features. Our findings along with literature data suggest that breast and ovarian BL (1) seem to be rarer than hitherto assumed, (2) share typical molecular features with other BL, and (3) predominantly affect women in the fertile age.

Abstract IA07: Molecular classification of T-cell lymphomas

Abstract Peripheral T-cell lymphomas (PTCLs) represent a clinically, histologically, and molecularly heterogeneous group of non-Hodgkin lymphomas derived from mature post-thymic T cells. Among them, the most common in Western countries is PTCL, not otherwise specified (NOS), accounting for approximately 30% of all PTCLs. Combining whole-exome and deep targeted-capture sequencing of 133 cases, we delineated the entire picture of genetic alterations in PTCL, NOS. Of note is the identification of a previously undescribed molecular subtype characterized by TP53 and/or CDKN2A mutations and deletions in PTCL, NOS without showing a T follicular helper cell phenotype. This subtype exhibited different prognosis and unique genetic features, including extensive chromosomal instability, which preferentially affected molecules involved in immune escape and transcriptional regulation. Among PTCLs, the most common entity in Japan is adult T-cell leukemia/lymphoma (ATL), which is an aggressive peripheral T-cell lymphoma associated with human T-cell leukemia virus type-1 (HTLV-1) infection. We previously carried out an integrated molecular study, in which whole-exome, transcriptome, and targeted resequencing, as well as array-based copy number analysis, were performed. We found recurrent genetic alterations in T-cell receptor/NF-κB signaling, T-cell trafficking, and other T cell-related pathways as well as immunosurveillance. Although our previous study discovered many driver mutations and copy number alterations, the whole-genome landscape of ATL still remains elusive. To this end, we have recently performed whole-genome sequencing (WGS) of 155 ATL samples, with a median depth of 95-fold for tumor samples. WGS presented a substantially different overview of driver alterations compared with WES: most driver genes were affected by more than one classes of somatic alterations, including structural variations (SV) and noncoding mutations. We integrated these genetic drivers using non-negative factorization clustering and identified several molecular subsets with distinct clinical features, including a leukemic subtype characterized by PLCG1 and STAT3 mutations. In addition, we newly identified several putative driver SVs and noncoding mutations (such as NOTCH1 truncations and mutations involving NFKBIZ 3′-untraslated region). Several types of SVs were linked to distinct alterations, suggesting their etiology, including associations of TP53 disruption with inversions and translocations, and inactivation of immune-related molecules with deletions. Our WGS analysis not only identifies novel somatic alterations, but also extends the overview of ATL genome, which can lead to future improvement of patient management. Taken together, our findings provide novel insights into genetic and molecular heterogeneity in PTCLs, which should help to devise a novel molecular classification and to exploit a new therapeutic strategy for these malignancies. Citation Format: Keisuke Kataoka. Molecular classification of T-cell lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA07.

Identification of NTRK3 Fusions in Childhood Melanocytic Neoplasms

Spitzoid neoplasms are a distinct group of melanocytic tumors. Genetically, they lack mutations in common melanoma-associated oncogenes. Recent studies have shown that spitzoid tumors may contain a variety of kinase fusions, including ROS1, NTRK1, ALK, BRAF, and RET fusions. We report herein the discovery of recurrent NTRK3 gene rearrangements in childhood melanocytic neoplasms with spitzoid and/or atypical features, based on genome-wide copy number analysis by single-nucleotide polymorphism array, which showed intragenic copy number changes in NTRK3. Break-apart fluorescence in situ hybridization confirmed the presence of NTRK3 rearrangement, and a novel MYO5A-NTRK3 transcript, representing an in-frame fusion of MYO5A exon 32 to NTRK3 exon 12, was identified using a rapid amplification of cDNA ends-based anchored multiplex PCR assay followed by next-generation sequencing. The predicted MYO5A-NTRK3 fusion protein consists of several N-terminal coiled-coil protein dimerization motifs encoded by MYO5A and C-terminal tyrosine kinase domain encoded by NTRK3, which is consistent with the prototypical structure of TRK oncogenic fusions. Our study also demonstrates how array-based copy number analysis can be useful in discovering gene fusions associated with unbalanced genomic aberrations flanking the fusion points. Our findings add another potentially targetable kinase fusion to the list of oncogenic fusions in melanocytic tumors.

Mitochondrial DNA content in breast cancer: Impact on in vitro and in vivo phenotype and patient prognosis.

Reduced mitochondrial DNA (mtDNA) content in breast cancer cell lines has been associated with transition towards a mesenchymal phenotype, but its clinical consequences concerning breast cancer dissemination remain unidentified. Here, we aimed to clarify the link between mtDNA content and a mesenchymal phenotype and its relation to prognosis of breast cancer patients. We analyzed mtDNA content in 42 breast cancer cell lines and 207 primary breast tumor specimens using a combination of quantitative PCR and array-based copy number analysis. By associating mtDNA content with expression levels of genes involved in epithelial-to-mesenchymal transition (EMT) and with the intrinsic breast cancer subtypes, we could not identify a relation between low mtDNA content and mesenchymal properties in the breast cancer cell lines or in the primary breast tumors. In addition, we explored the relation between mtDNA content and prognosis in our cohort of primary breast tumor specimens that originated from patients with lymph node-negative disease who did not receive any (neo)adjuvant systemic therapy. When patients were divided based on the tumor quartile levels of mtDNA content, those in the lowest quarter (≤ 350 mtDNA molecules per cell) showed a poorer 10-year distant metastasis-free survival than patients with > 350 mtDNA molecules per cell (HR 0.50 [95% CI 0.29–0.87], P = 0.015). The poor prognosis was independent of established clinicopathological markers (HR 0.54 [95% CI 0.30–0.97], P = 0.038). We conclude that, despite a lack of evidence between mtDNA content and EMT, low mtDNA content might provide meaningful prognostic value for distant metastasis in breast cancer.

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia.

BackgroundWe present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL.ResultsWe used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (‘other’ subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5.ConclusionsOur findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0039-z) contains supplementary material, which is available to authorized users.

DNA Methylation-Based Subtype Prediction for Pediatric Acute Lymphoblastic Leukemia (ALL)

We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. We used the methylation status of ~450,000 CpG sites from 546 well-characterized patients with T-ALL or B-cell precursor ALL with the cytogenetic subtypes high hyperdiploidy (HeH), t(12;21)(p13;q22)[ETV6/RUNX1], t(1;19)(q23;p13.3)[TCF3/PBX1], 11q23/MLL-rearrangements, t(9;22)(q34;q11)[BCR/ABL1], dic(9;20)(p13.2;q11.2), and intrachromosomal amplification of chromosome 21 iAMP21[RUNX1 x >3], to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived consisting of only 246 CpG sites that accurately predicted the subtype of >96% of the patients with known subtype. The mean sensitivity and specificity across the subtypes was 0.90 and 0.99, respectively. We then used the DNA methylation classifier to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (“other” subtype). Nearly half (n=106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative PCR and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5. The CpG sites constituting the classifiers highlight relevant biological characteristics of otherwise unclassified ALL patients and provide clues to the origin and development of leukemic transformation. Together with response to induction therapy, genetic aberrations are among the most important prognostic factors in pediatric ALL. Our findings indicate that DNA methylation profiling can help clarify some of the heterogeneity in cytogenetically undefined ALL patient groups and can potentially be implemented for diagnostics of ALL and other types of hematological cancers. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2223: Genome-wide analysis of copy number alterations and gene mutations in testicular germ cell cancer

Abstract Introduction and Objective Testicular germ cell cancer (TGCC) is the most common solid malignancy among young adult males. Cure could be obtained by intensive chemotherapy combined with retroperitoneal lymph node dissection, but often with substantially compromised quality of life. As for the common genetic lesions, somatic mutations and amplifications of KIT are found in about 20%-30% of cases with TGCCs. However, the molecular pathogenesis of TGCC is still poorly understood. In this study, to obtain a better understanding of the genetic basis of TGCC and to identify druggable molecular targets, we performed whole exome sequencing as well as SNP array-based copy number analysis in TGCC. Methods All cases underwent high orchiectomy and were histologically diagnosed as TGCC. Genomic DNA was extracted from fresh frozen specimens of TGCC. Whole exome sequencing was performed for paired tumor/normal DNA from 10 TGCC patients, in which target exomes were captured using SureSelect Human All Exon V5 (Agilent Technologies) and subjected to massively parallel sequencing on the Illumina platform (HiSeq2000). Copy number variants were also interrogated in 40 TGCCs (25 seminomas and 15 non-seminomas) using Affymetrix 250K NspI array. Results TGCC genome was triploid in most cases with high level amplification in12p. Loss of heterozygosity (LOH) of chromosome 4 was frequently observed in both seminoma and non-seminoma, whereas 11q deletion was found predominantly in cases with seminoma. In addition, we identified recurrent focal amplifications involving 4q12 and 22q11, from which KIT and MAPK1 were identified, respectively. In whole exome sequencing, 15 somatic mutations were detected per sample on average, which was relatively lower than other solid malignancies. When combined the results of copy number analysis with those of whole-exome sequencing, genes involved in RAS signaling pathway and chromatin modification were frequently altered in TGCC. Conclusions Our comprehensive analyses revealed genomic aberrations of TGCC in terms of copy number alterations and gene mutations. Mutated/amplified KIT and MAPK1 and other RAS pathway gene could be potential targets of small molecule inhibitors for therapeutics. Citation Format: Yusuke Sato, Aiko Sato-Otsubo, Yasunobu Nagata, Kenichi Yoshida, Yuichi Shiraishi, Hiromichi Suzuki, Masashi Sanada, Haruki Kume, Satoru Miyano, Yukio Homma, Seishi Ogawa. Genome-wide analysis of copy number alterations and gene mutations in testicular germ cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2014-2223

Posterior amorphous corneal dystrophy is associated with a deletion of small leucine-rich proteoglycans on chromosome 12.

Posterior amorphous corneal dystrophy (PACD) is a rare, autosomal dominant disorder affecting the cornea and iris. Next-generation sequencing of the previously identified PACD linkage interval on chromosome 12q21.33 failed to yield a pathogenic mutation. However, array-based copy number analysis and qPCR were used to detect a hemizygous deletion in the PACD linkage interval containing 4 genes encoding small leucine-rich proteoglycans (SLRPs): KERA, LUM, DCN, and EPYC. Two other unrelated families with PACD also demonstrated deletion of these SLRPs, which play important roles in collagen fibrillogenesis and matrix assembly. Given that these genes are essential to the maintenance of corneal clarity and the observation that knockout murine models display corneal phenotypic similarities to PACD, we provide convincing evidence that PACD is associated with haploinsufficiency of these SLRPs.

Abstract 3184: Integrative analysis of clear cell renal cell carcinoma.

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer. The most frequent genetic event in the evolution of ccRCC is inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Recent studies have revealed frequent mutations of PBRM1 as well as other epigenetic regulators including BAP1, SETD2 and KDM5C, but their impact on therapeutics remains unclear. In this study, to obtain a better understanding of molecular pathogenesis of ccRCC, we performed an integrated genetic study of ccRCC including whole exome sequencing, copy number analysis as well as transcriptome and methylome analysis. A total of 106 paired specimens were analyzed by massively parallel sequencing of whole exomes (Agilent SureSelect, Illumina HiSeq2000) and SNP array-based copy number analysis (Affymetrix 250K array) as well as gene expression (Agilent Human Gene Expression 4x44k v2) and methylation (Illumina Infinium 450K) profiling and RNA sequencing (Illumina HiSeq2000). On average, 48.8 somatic mutations per sample were identified in whole exome analysis, in which VHL mutations were most frequent. PBRM1, BAP1 and SETD2 were also recurrently mutated and were further analyzed in 240 cases together with an additional 80 genes involved in chromatin regulation. PBRM1 mutations were found in 42% of the cases, while BAP1 and SETD2 were mutated in 12% and 10%, respectively. BAP1 mutations correlated with poor prognosis and SETD2 mutation was associated with the risk for metastatic diseases. Pathway analysis revealed frequent mutations of genes involved in mRNA processing. Among them, mutations of genes involved in 3’ splice site recognition, which were frequently mutated in MDS, were rare. Most of them were involved in release of intron, 3’-end processing or export to cytoplasm. In expression analysis, tumors were clustered into two clusters known as ccA and ccB, in which the ccA type was characterized by overexpressed genes involved in angiogenesis, whereas expression of genes in cell cycle regulators were a prominent feature in the ccB type tumors. In methylome analysis, 15 samples were clustered into hypermethylated subtype where all cases were included in the ccB type and were associated with poor prognosis. Homeobox genes were differently methylated in hypermethylated subtype which indicate deregulation of polycomb mediated gene silencing induce high-grade ccRCC. RUNX1 and SRPX2 were differently methylated between ccA type and ccB type, which may affect the differences of expression profile between two subtypes. In RNA sequencing, known fusion gene involving TFE3 and NONO was detected in single case. In total, 34 fusion genes were detected in 23 samples, although no recurrent fusion genes have been identified. Our results indicate that genomic analyses are useful for classification, prognostic prediction and development of treatment strategy in ccRCC. Citation Format: Yusuke Sato, Shigekatsu Maekawa, Yusuke Okuno, Yuichi Shiraishi, Aiko Sato, Genta Nagae, Teppei Shimamura, Yasunobu Nagata, Kenichi Yoshida, Masashi Sanada, Haruki Kume, Hiroyuki Aburatani, Satoru Miyano, Yukio Homma, Seishi Ogawa. Integrative analysis of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3184. doi:10.1158/1538-7445.AM2013-3184

Women’s experiences receiving abnormal prenatal chromosomal microarray testing results

Genomic microarrays can detect copy-number variants not detectable by conventional cytogenetics. This technology is diffusing rapidly into prenatal settings even though the clinical implications of many copy-number variants are currently unknown. We conducted a qualitative pilot study to explore the experiences of women receiving abnormal results from prenatal microarray testing performed in a research setting. Participants were a subset of women participating in a multicenter prospective study "Prenatal Cytogenetic Diagnosis by Array-based Copy Number Analysis." Telephone interviews were conducted with 23 women receiving abnormal prenatal microarray results. We found that five key elements dominated the experiences of women who had received abnormal prenatal microarray results: an offer too good to pass up, blindsided by the results, uncertainty and unquantifiable risks, need for support, and toxic knowledge. As prenatal microarray testing is increasingly used, uncertain findings will be common, resulting in greater need for careful pre- and posttest counseling, and more education of and resources for providers so they can adequately support the women who are undergoing testing.

Abstract 2092: Integrated genetic analysis of clear cell renal cell carcionoma

Abstract Renal cell carcinoma (RCC) is the most common form of adult kidney cancer and accounts for 2-3% of all adult malignancies, in which clear cell carcinoma accounts for more than 80% of the cases. As for the pathogenesis of clear cell RCC, inactivation of the VHL gene has been reported in 80% of clear cell RCC and more recently, frequent mutations of epigenetic regulators, including PBRM1, SETD2, KDM5C and UTX, have been demonstrated through high-throughput mutation studies, including PBRM1 has been demonstrated in ∼40% of the cases. Nevertheless, probably, our knowledge of the full spectrum of gene mutations in RCC is still incomplete. In this study, to obtain a better understanding of molecular pathogenesis of clear cell RCC, we performed an integrated genetic study of clear cell RCC, where a total of 93 paired specimens were analyzed by massively parallel sequencing of SureSelect (Agilent)-enriched whole exomes, SNP array-based copy number analysis (Affymetrix), as well as gene expression profiling (Agilent). In whole exome sequencing, 42 somatic mutations per sample were identified on average, which involved not only previously reported genes, but also a number of novel gene targets. Among these, mutations of genes involved in chromatin regulation or histone modification were preferentially found in advanced cases. To understand whole picture of gene mutations of epigenetic mechanism in clear cell RCC, mutation analysis of 85 genes involved in chromatin regulation or histone modification were performed in 180 cases using multiplexed barcode sequencing. A total of 201 somatic mutations were validated and 74% cases had at least one somatic mutation. PBRM1 mutations were found in 43% cases and SETD2 were mutated in 10% of cases. When comparing clinical picture with mutation status, SETD2 mutation was associated with the risk of metastasis, while PBRM1 mutations had no impact on prognosis. Our results showed that in clear cell RCC, multiple component of complexes involved in epigenetic regulation undergo gene mutations, confirming that deregulated epigenetic apparatus play important roles in pathogenesis of clear cell RCC. In this meeting, we will present the result of our integrated genetic analysis of RCC and discuss the genetic basis of RCC in terms of copy number alterations, gene mutations, as well as gene expression profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2092. doi:1538-7445.AM2012-2092

The Prognostic Significance of Various 13q14 Deletions in Chronic Lymphocytic Leukemia

To further our understanding of the biology and prognostic significance of various chromosomal 13q14 deletions in chronic lymphocytic leukemia (CLL). We analyzed data from SNP 6.0 arrays to define the anatomy of various 13q14 deletions in a cohort of 255 CLL patients and have correlated two subsets of 13q14 deletions (type I exclusive of RB1 and type II inclusive of RB1) with patient survival. Furthermore, we measured the expression of the 13q14-resident microRNAs by quantitative PCR (Q-PCR) in 242 CLL patients and subsequently assessed their prognostic significance. We sequenced all coding exons of RB1 in patients with monoallelic RB1 deletion and have sequenced the 13q14-resident miR locus in all patients. Large 13q14 (type II) deletions were detected in approximately 20% of all CLL patients and were associated with shortened survival. A strong association between 13q14 type II deletions and elevated genomic complexity, as measured through CLL-FISH or SNP 6.0 array profiling, was identified, suggesting that these lesions may contribute to CLL disease evolution through genomic destabilization. Sequence and copy number analysis of the RB1 gene identified a small CLL subset that is RB1 null. Finally, neither the expression levels of the 13q14-resident microRNAs nor the degree of 13q14 deletion, as measured through SNP 6.0 array-based copy number analysis, had significant prognostic importance. Our data suggest that the clinical course of CLL is accelerated in patients with large (type II) 13q14 deletions that span the RB1 gene, therefore justifying routine identification of 13q14 subtypes in CLL management.

Parental origin and mechanism of formation of a 46,X,der(X)(pter→q21.1::p11.4→pter)/45,X karyotype in a woman with mild Turner syndrome

To describe the parental origin and the mechanism of formation of a 46,X,der(X)(pter-->q21.1::p11.4-->pter)[23]/45,X[8] karyotype in a patient with mild Turner syndrome. Case report. A university hospital. A 23-year-old woman with normal height, gonadal dysgenesis, and mild Turner stigmata. Genotype-phenotype correlation, array-based copy number analysis, fluorescence in situ hybridization with locus-specific probes, and microsatellite marker-mediated haplotype analysis subsequent to whole genome amplification of microdissected chromosomes. Genotype-phenotype correlation, mechanism of formation, and parental origin. Formation in paternal meiosis by refolding in itself and unequal recombination between Xp and Xq were found as the most likely mechanism of formation. Formation of der(X) chromosomes in females can be more complex than previously thought. The nearly normal height of this patient could be explained by a combination of trisomy of the Xp-located SHOX gene and mosaicism with a 45,X cell line.

SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

BackgroundSET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene.ResultsOf 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ)-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.ConclusionCell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

High-resolution array copy number analyses for detection of deletion, gain, amplification and copy-neutral LOH in primary neuroblastoma tumors: Four cases of homozygous deletions of the CDKN2A gene

BackgroundNeuroblastoma is a very heterogeneous pediatric tumor of the sympathetic nervous system showing clinically significant patterns of genetic alterations. Favorable tumors usually have near-triploid karyotypes with few structural rearrangements. Aggressive stage 4 tumors often have near-diploid or near-tetraploid karyotypes and structural rearrangements. Whole genome approaches for analysis of genome-wide copy number have been used to analyze chromosomal abnormalities in tumor samples. We have used array-based copy number analysis using oligonucleotide single nucleotide polymorphisms (SNP) arrays to analyze the chromosomal structure of a large number of neuroblastoma tumors of different clinical and biological subsets.ResultsNinety-two neuroblastoma tumors were analyzed with 50 K and/or 250 K SNP arrays from Affymetrix, using CNAG3.0 software. Thirty percent of the tumors harbored 1p deletion, 22% deletion of 11q, 26% had MYCN amplification and 45% 17q gain. Most of the tumors with 1p deletion were found among those with MYCN amplification. Loss of 11q was most commonly seen in tumors without MYCN amplification. In the case of MYCN amplification, two types were identified. One type displayed simple continuous amplicons; the other type harbored more complex rearrangements. MYCN was the only common gene in all cases with amplification. Complex amplification on chromosome 12 was detected in two tumors and three different overlapping regions of amplification were identified. Two regions with homozygous deletions, four cases with CDKN2A deletions in 9p and one case with deletion on 3p (the gene RBMS3) were also detected in the tumors.ConclusionSNP arrays provide useful tools for high-resolution characterization of significant chromosomal rearrangements in neuroblastoma tumors. The mapping arrays from Affymetrix provide both copy number and allele-specific information at a resolution of 10–12 kb. Chromosome 9p, especially the gene CDKN2A, is subject to homozygous (four cases) and heterozygous deletions (five cases) in neuroblastoma tumors.

Array-Based DNA Diagnostics: Let the Revolution Begin

Advances in the fabrication of DNA microarrays as well as transformations in detection chemistries have vastly increased the throughput for genotyping, DNA sequencing, and array-based copy number analysis (ABCNA). Rapid changes in technology are not only affecting research but also revolutionizing DNA diagnostics. Here we focus on the application of high-throughput ABCNA and genotyping. Targeted and genome-wide ABCNA has led to the discovery of extensive DNA copy number variation in the population and the delineation of many previously unrecognized submicroscopic chromosomal aberrations (genomic disorders). High-throughput single-nucleotide polymorphism (SNP) genotyping is being widely applied in genome-wide association studies (GWASs) with recent successes in identification of common variants that confer risk for common adult diseases. Future applications of high-throughput genotyping and array-based DNA sequencing technology will undoubtedly involve research and diagnostic analyses of rare mutations and perhaps ultimately enable full individual genome sequencing.

Rapid array‐based genomic characterization of a subtle structural abnormality: A patient with psychosis and der(18)t(5;18)(p14.1;p11.23)

Array-based copy number analysis has recently emerged as a rapid means of mapping complex and/or subtle chromosomal abnormalities. We have compared two such techniques, using bacterial artificial chromosome (BAC) and single nucleotide polymorphism (SNP) arrays in the evaluation of a 45-year-old woman with dysmorphic features, mental retardation, psychosis, and an unbalanced derivative chromosome 18, (46,XX, der(18)t(18;?)(p12;?)). Both array-based methods demonstrated that the additional material on chromosome 18 was of 5p origin. The 5p duplication mapped telomeric to 25.320 Mb (BAC array) and 25.607 Mb (SNP array), corresponding to the band 5p14.1. Both BAC and SNP arrays also showed a deletion involving chromosome 18p extending telomeric from 8.437 Mb (BAC array) and 8.352 Mb (SNP array), corresponding to the band 18p11.23. Molecular cytogenetic mapping using fluorescence in situ hybridization (FISH) supported the array findings and further refined the breakpoint regions, confirming that the BAC and SNP chips were both useful in this regard. Both case reports and linkage analyses have implicated these chromosomal intervals in psychosis. The array-based experiments were completed over the course of several days. While these methods do not eliminate the requirement for traditional fine-mapping, they provide an efficient approach to identifying the origin and extent of deleted and duplicated material in chromosomal rearrangements.