Identification of CUX1 as the recurrent chromosomal band 7q22 target gene in human uterine leiomyoma.

Uterine leiomyomas are benign solid tumors of mesenchymal origin which occur with an estimated incidence of up to 77% of all women of reproductive age. The majority of these tumors remains symptomless, but in about a quarter of cases they cause leiomyoma-associated symptoms including chronic pelvic pain, menorrhagia-induced anemia, and impaired fertility. As a consequence, they are the most common indication for pre-menopausal hysterectomy in the USA and Japan and annually translate into a multibillion dollar healthcare problem. Approximately 40% of these neoplasms present with recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 and/or 10q22. Using positional cloning strategies, we and others previously identified HMGA1, HMGA2, RAD51L1, MORF, and, more recently, NCOA1 as primary target (fusion) genes associated with tumor initiation in four of these distinct cytogenetic subgroups. Despite the fact that the del(7)(q22) subgroup is the largest among leiomyomas, and was first described more than twenty years ago, the 7q22 leiomyoma target gene still awaits unequivocal identification. We here describe a positional cloning effort from two independent uterine leiomyomas, containing respectively a pericentric and a paracentric chromosomal inversion, both affecting band 7q22. We found that both chromosomal inversions target the cut-like homeobox 1 (CUX1) gene on chromosomal band 7q22.1 in a way which is functionally equivalent to the more frequently observed del(7q) cases, and which is compatible with a mono-allelic knock-out scenario, similar as was previously described for the cytogenetic subgroup showing chromosome 14q involvement.

Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene.

Next-generation sequencing is a straightforward tool for the identification of disease genes in extended genomic regions. Autozygosity mapping was performed on a five-generation inbred Italian family with three siblings affected with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]), a rare autosomal-recessive genodermatosis characterised by poikiloderma, pachyonychia, and chronic neutropenia. The siblings were initially diagnosed as affected with Rothmund-Thomson syndrome (RTS [MIM #268400]), with which PN shows phenotypic overlap. Linkage analysis on all living subjects of the family identified a large 16q region inherited identically by descent (IBD) in all affected family members. Deep sequencing of this 3.4 Mb region previously enriched with array capture revealed a homozygous c.504-2 A>C mismatch in all affected siblings. The mutation destroys the invariant AG acceptor site of intron 4 of the evolutionarily conserved C16orf57 gene. Two distinct deleterious mutations (c.502A>G and c.666_676+1del12) identified in an unrelated PN patient confirmed that the C16orf57 gene is responsible for PN. The function of the predicted C16orf57 gene is unknown, but its product has been shown to be interconnected to RECQL4 protein via SMAD4 proteins. The unravelled clinical and genetic identity of PN allows patients to undergo genetic testing and follow-up.

Mutations in a new member of the chromodomain gene family cause CHARGE syndrome.

CHARGE syndrome is a common cause of congenital anomalies affecting several tissues in a nonrandom fashion. We report a 2.3-Mb de novo overlapping microdeletion on chromosome 8q12 identified by array comparative genomic hybridization in two individuals with CHARGE syndrome. Sequence analysis of genes located in this region detected mutations in the gene CHD7 in 10 of 17 individuals with CHARGE syndrome without microdeletions, accounting for the disease in most affected individuals.

Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation and several other cytogenetic aberrations, including heterozygous loss of chromosomal arms 1p, 6q, 11q and 13q and/or gains of 3q and 8q. The common intervals of chromosomal imbalance have been narrowed down using array-comparative genomic hybridization (CGH). However, the chromosomal intervals still contain many genes potentially involved in MCL pathogeny. Combined analysis of tiling-resolution array-CGH with gene expression profiling on 11 MCL tumours enabled the identification of genomic alterations and their corresponding gene expression profiles. Only subsets of genes located within given cytogenetic anomaly-intervals showed a concomitant change in mRNA expression level. The genes that showed consistent correlation between DNA copy number and RNA expression levels are likely to be important in MCL pathology. Besides several 'anonymous genes', we also identified various fully annotated genes, whose gene products are involved in cyclic adenosine monophosphate-regulated pathways (PRKACB), DNA damage repair, maintenance of chromosome stability and prevention of rereplication (ATM, ERCC5, FBXO5), energy metabolism (such as genes that are involved in the synthesis of proteins encoded by the mitochondrial genome) and signal transduction (ARHGAP29). Deregulation of these gene products may interfere with the signalling pathways that are involved in MCL tumour development and maintenance.

Mapping of constitutional translocation breakpoints in renal cell cancer patients: identification of KCNIP4 as a candidate gene.

Our group and others had previously developed a high throughput procedure to map translocation breakpoints using chromosome flow sorting in conjunction with microarray-based comparative genomic hybridization (arrayCGH). Here we applied both conventional positional cloning and integrated arrayCGH procedures to the mapping of constitutional chromosome anomalies in four patients with renal cell cancer (RCC), three with a chromosome 3 translocation, and one with an insertion involving chromosome 3. In one of these patients, who was carrying a t(3;4)(p13;p15), the KCNIP4 gene was found to be disrupted. KCNIP4 belongs to a family of potassium channel-interacting proteins and is highly expressed in normal kidney cells. In addition, KCNIP4 splice variants have specifically been encountered in RCC.

Microarray analyses reveal strong influence of DNA copy number alterations on the transcriptional patterns in pancreatic cancer: implications for the interpretation of genomic amplifications.

DNA copy number alterations are believed to play a major role in the development and progression of human neoplasms. Although most of these genomic imbalances have been associated with dysregulation of individual genes, their large-scale transcriptional consequences remain unclear. Pancreatic carcinomas frequently display gene copy number variation of entire chromosomes as well as of chromosomal subregions. These changes range from homozygous deletions to high-level amplifications and are believed to constitute key genetic alterations in the cellular transformation of this tumor type. To investigate the transcriptional consequences of the most drastic genomic changes, that is, genomic amplifications, and to analyse the genome-wide transcriptional effects of DNA copy number changes, we performed expression profiling of 29 pancreatic carcinoma cell lines and compared the results with matching genomic profiling data. We show that a strong association between DNA copy numbers and mRNA expression levels is present in pancreatic cancer, and demonstrate that as much as 60% of the genes within highly amplified genomic regions display associated overexpression. Consequently, we identified 67 recurrently overexpressed genes located in seven precisely mapped commonly amplified regions. The presented findings indicate that more than one putative target gene may be of importance in most pancreatic cancer amplicons.

Regulation of the MiTF/TFE bHLH-LZ transcription factors through restricted spatial expression and alternative splicing of functional domains.

The MiTF/TFE (MiT) family of basic helix-loop-helix leucine zipper transcription factors is composed of four closely related members, MiTF, TFE3, TFEB and TFEC, which can bind target DNA both as homo- or heterodimers. Using real-time RT-PCR, we have analyzed the relative expression levels of the four members in a broad range of human tissues, and found that their ratio of expression is tissue-dependent. We found that, similar to the MiTF gene, the genes for TFEB and TFEC contain multiple alternative first exons with restricted and differential tissue distributions. Seven alternative 5' exons were identified in the TFEB gene, of which three displayed specific expression in placenta and brain, respectively. A novel TFEC transcript (TFEC-C) encodes an N-terminally truncated TFEC isoform lacking the acidic activation domain (AAD), and is exclusively expressed in kidney and small intestine. Furthermore, we observed that a considerable proportion of the TFEC transcripts splice out protein-coding exons, resulting in transcription factor isoforms lacking one or more functional domains, primarily the basic region and/or the AAD. These isoforms were always co-expressed with the intact transcription factors and may act as negative regulators of MiTF/TFE proteins. Our data reveal that multiple levels of regulation exist for the MiTF/TFE family of transcription factors, which indicates how these transcription factors may participate in various cellular processes in different tissues.

Genome-wide array-based comparative genomic hybridization reveals multiple amplification targets and novel homozygous deletions in pancreatic carcinoma cell lines.

Pancreatic carcinomas display highly complex chromosomal abnormalities, including many structural and numerical aberrations. There is ample evidence indicating that some of these abnormalities, such as recurrent amplifications and homozygous deletions, contribute to tumorigenesis by altering expression levels of critical oncogenes and tumor suppressor genes. To increase the understanding of gene copy number changes in pancreatic carcinomas and to identify key amplification/deletion targets, we applied genome-wide array-based comparative genomic hybridization to 31 pancreatic carcinoma cell lines. Two different microarrays were used, one containing 3,565 fluorescence in situ hybridization-verified bacterial artificial chromosome clones and one containing 25,468 cDNA clones representing 17,494 UniGene clusters. Overall, the analyses revealed a high genomic complexity, with several copy number changes detected in each case. Specifically, 60 amplicons at 32 different locations were identified, most frequently located within 8q (8 cases), 12p (7 cases), 7q (5 cases), 18q (5 cases), 19q (5 cases), 6p (4 cases), and 8p (4 cases). Amplifications of 8q and 12p were mainly clustered at 8q23-24 and 12p11-12, respectively, whereas amplifications on other chromosome arms were more dispersed. Furthermore, our analyses identified several novel homozygously deleted segments located to 9p24, 9p21, 9q32, 10p12, 10q22, 12q24, and 18q23. The individual complexity and aberration patterns varied substantially among cases, i.e., some cell lines were characterized mainly by high-level amplifications, whereas others showed primarily whole-arm imbalances and homozygous deletions. The described amplification and deletion targets are likely to contain genes important in pancreatic tumorigenesis.

12p-amplicon structure analysis in testicular germ cell tumors of adolescents and adults by array CGH.

All invasive testicular germ cell tumors of adolescents and adults (TGCTs), that is, seminomas and nonseminomas, show gain of 12p sequences, mostly as isochromosomes. Although several candidate genes have been suggested, the relevant gene(s) have not been identified yet. About 10% of testicular seminomas, however, show a more restricted amplification of the 12p11.2-p12.1 region, in which the various amplicons show an apparent overlap, allowing for the shortest region of amplification overlap approach, aiming at the identification of pathogenetically relevant sequences residing in this region. Here we report on a high-resolution 12p-amplicon architecture analysis using microarray-based comparative genomic hybridization, the results of which were subsequently confirmed by fluorescent in situ hybridization studies. The 12p-specific microarray contained 63 positionally selected BAC clones, which are more or less evenly distributed over the short arm of chromosome 12 (average spacing: less than 500 Kb), including 20 clones within the region of amplification. Out of a series of 17 seminomas, seven seminomas showed amplification of the whole amplicon region, of which three showed a dip in T/R value in the center of the amplified area. A more complex amplification pattern was found in the other 10 seminomas: three showed predominant amplification at the centromeric border; one mainly at the telomeric border; six showed a balanced amplification of both the centromeric and telomeric regions. The only nonseminoma investigated showed a structure in which the centromeric border was only amplified. These data support a mechanistic model in which at least two 12p genes, situated at the border regions of the amplicon, are positional candidates capable of actively supporting tumor progression in TGCTs.

Chromosome 3 translocations and familial renal cell cancer.

Renal cell carcinomas (RCCs) occur in both sporadic and familial forms. In a subset of families the occurrence of RCCs co-segregates with the presence of constitutional chromosome 3 translocations. Previously, such co-segregation phenomena have been widely employed to identify candidate genes in various hereditary (cancer) syndromes. Here we survey the translocation 3-positive RCC families that have been reported to date and the subsequent identification of its respective candidate genes using positional cloning strategies. Based on allele segregation, loss of heterozygosity and mutation analyses of the tumors, a multi-step model for familial RCC development has been generated. This model is relevant for (i) understanding familial tumorigenesis and (ii) rational patient management. In addition, a high throughput microarray-based strategy is presented that will enable the rapid identification of novel positional candidate genes via a single step procedure. The functional consequences of the (fusion) genes that have been identified so far, the multi-step model and its consequences for clinical diagnosis, the identification of persons at risk and genetic counseling in RCC families are discussed.

Role of gain of 12p in germ cell tumour development.

Within the human testis, three entities of germ cell tumours are distinguished: the teratomas and yolk sac tumors of newborn and infants, the seminomas and nonseminomas of adolescents and young adults, referred to as testicular germ cell tumours (TGCT), and the spermatocytic seminomas. Characteristic chromosomal anomalies have been reported for each group, supporting their distinct pathogenesis. TGCT are the most common cancer in young adult men. The initiating pathogenetic event of these tumours occurs during embryonal development, affecting a primordial germ cell or gonocyte. Despite this intra-uterine initiation, the tumour will only be clinically manifest after puberty, with carcinoma in situ (IS) as the precursor. All invasive TGCT, both seminomas and nonseminomas, as well as CIS cells are aneuploid. The only consistent (structural) chromosomal abnormalities in invasive TGCT are gains of the short arm of chromosome 12, mostly due to isochromosome (i(12p)) formation. This suggests that an increase in copy number of a gene(s) on 12p is associated with the development of a clinically manifest TGCT. Despite the numerous (positional) candidate gene approaches that have been undertaken thus far, identification of a causative gene(s) has been hampered by the fact that most 12p gains involve rather large genomic intervals, containing unmanageable numbers of candidate genes. Several years ago, we initiated a search for 12p candidate genes using TGCT with a restricted 12p-amplification, cytogenetically identified as 12p11.2-p12.1. This approach is mainly based on identification of candidate genes mapped within the shortest region of overlap of amplification (SROA). In this review, data will be presented, which support the model that gain of 12p-sequences is associated with suppression of apoptosis and Sertoli cell-independence of CIS cells. So far, DAD-R is one of the most likely candidate genes involved in this process, possibly via N-glycosylation. Preliminary results on high through-put DNA- and cDNA array analyses of 12p-sequences will be presented.

Heterogeneity of structural abnormalities in the 7q31.3 approximately q34 region in myeloid malignancies.

Abnormalities in the long arm of chromosome 7 are a frequent chromosomal aberration in myeloid disorders. Most studies have focused on the analysis of del(7q), demonstrating the presence of several minimal deleted regions in 7q22 approximately q31. By contrast, few studies in myeloid disorders have been devoted to the analysis of translocations, either balanced or unbalanced, involving 7q. In this study, we used fluorescence in situ hybridization (FISH) to characterize the 7q31.3 approximately q34 region (markers D7S480-D7S2227) in patients with deletion or translocation of 7q. A total of 910 cases of myeloid disorders were studied by conventional cytogenetics. Fifty-eight (6%) patients had structural aberrations of 7q. FISH studies were carried out in the 27 patients with involvement of 7q31 approximately q34: 14 cases had an acute myelogenous leukemia and 13 cases had a myelodysplastic syndrome. FISH analysis revealed the existence of high complexity in the 7q31.3 approximately q34 region in patients with unbalanced translocations. No breakpoints in 7q31.3 approximately q34 were found in the cases with deletion or balanced translocation. Nevertheless, studies of unbalanced translocations showed several breakpoints in markers D7S480-D7S2227, which delineate a commonly altered region. The complexity of 7q rearrangements suggests that a synergy of different genetic factors, rather than the alteration of a single tumor suppressor gene, could be involved in the pathogenesis of del(7q) in myeloid disorders.

Ethnic variations in uterine leiomyoma biology are not caused by differences in myometrial estrogen receptor alpha levels.

OBJECTIVE: Uterine leiomyomas develop in women of reproductive age and regress after menopause, suggesting that they grow in a steroid hormone-dependent fashion. Furthermore, it is widely accepted that symptomatic uterine leiomyomas occur at a twofold to threefold higher frequency in black women than in white women. The present study was designed to investigate a possible physiologic role of racial differences in the myometrial estrogen receptor alpha in this phenomenon. METHODS: We compared the expression of the estrogen receptor and progesterone receptor in myometrium by ligand-binding assay and the estrogen receptor alpha by real-time polymerase chain reaction in women from different ethnic backgrounds who have uterine leiomyoma. RESULTS: Estrogen receptor and progesterone receptor concentrations and estrogen receptor alpha transcription levels were not statistically different between ethnic backgrounds. CONCLUSION: Neither on a transcriptional nor on a protein level were there statistically relevant differences in steroid hormone receptor levels. A causative role for these receptors in the ethnic variation of leiomyoma biology seems unlikely.

Multiplexed gene expression and fusion transcript analysis to detect ALK fusions in lung cancer.

Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration-approved companion diagnostic test (ie, fluorescence in situ hybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients. To explore alternative screening modalities for detecting ALK fusions, we designed a combination of two transcript-based assays to detect for presence or absence of ALK fusions using NanoString's nCounter technology. By using this combined gene expression and ALK fusion detection strategy, we developed a multiplexed assay with a quantitative scoring modality that is highly sensitive, reproducible, and capable of detecting low-abundant ALK fusion transcripts, even in samples with a low tumor cell content. In 66 archival NSCLC samples, our results were highly concordant to prior results obtained by fluorescence in situ hybridization and IHC. Our assay offers a cost-effective, easy-to-perform, high-throughput, and FFPE-compatible screening alternative for detection of ALK fusions.

Characterization of a recurrent t(1;2)(p36;p24) in human uterine leiomyoma.

Uterine leiomyomas are the most common neoplasms in women of reproductive age. Approximately 40% of these neoplasms show recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 or 10q22. Using positional cloning strategies, we and others had previously identified HMGA1, HMGA2, RAD51L1, and MYST4 (previously referred to as MORF); as primary target (fusion) genes associated with tumor development in three of these distinct cytogenetic subgroups. Here, we report the positional cloning of a single, recurrent, leiomyoma-associated anomaly, t(1;2)(p36;p24). Molecular characterization of the reciprocal breakpoint intervals showed that that AJAP1 (alias SHREW1) and NPHP4 flank the breakpoint on chromosome 1 and that ITSN2 and NCOA1 flank the breakpoint on chromosome 2. Detailed analysis of the breakpoint regions revealed that in this particular case the translocation was associated with a 27-bp deletion on chromosome 1 and a 136-bp duplication on chromosome 2. No breakpoint-spanning (fusion) genes were identified. In silico prediction of transcription factor binding sites, however, indicated the presence of several such sites in the respective breakpoint regions, and major changes therein as a result of the t(1;2)(p36;p24) under investigation. We postulate that transcriptional deregulation of one or more of these breakpoint-flanking genes may contribute to the development of human uterine leiomyomas.

The tumor suppressor gene FBXW7 is disrupted by a constitutional t(3;4)(q21;q31) in a patient with renal cell cancer.

FBXW7 (alias CDC4) is a p53-dependent tumor suppressor gene that exhibits mutations or deletions in a variety of human tumors. Mutation or deletion of the FBXW7 gene has been associated with an increase in chromosomal instability and cell cycle progression. In addition, the FBXW7 protein has been found to act as a component of the ubiquitin proteasome system and to degrade several oncogenic proteins that function in cellular growth regulatory pathways. By using a rapid breakpoint cloning procedure in a case of renal cell cancer (RCC), we found that the FBXW7 gene was disrupted by a constitutional t(3;4)(q21;q31). Subsequent analysis of the tumor tissue revealed the presence of several anomalies, including loss of the derivative chromosome 3. Upon screening of a cohort of 29 independent primary RCCs, we identified one novel pathogenic mutation, suggesting that the FBXW7 gene may also play a role in the development of sporadic RCCs. In addition, we screened a cohort of 48 unrelated familial RCC cases with unknown etiology. Except for several known or benign sequence variants such as single nucleotide polymorphisms (SNPs), no additional pathogenic variants were found. Previous mouse models have suggested that the FBXW7 gene may play a role in the predisposition to tumor development. Here we report that disruption of this gene may predispose to the development of human RCC.

Identification of tumor-specific molecular signatures in intracranial ependymoma and association with clinical characteristics.

PURPOSE: To delineate clinically relevant molecular signatures of intracranial ependymoma. MATERIALS AND METHODS: We analyzed 24 primary intracranial ependymomas. For genomic profiling, microarray-based comparative genomic hybridization (CGH) was used and results were validated by fluorescent in situ hybridization and loss of heterozygosity mapping. We performed gene expression profiling using microarrays, real-time quantitative reverse transcriptase polymerase chain reaction, and methylation analysis of selected genes. We applied class comparison analyses to compare both genomic and expression profiling data with clinical characteristics. RESULTS: A variable number of genomic imbalances were detected by array CGH, revealing multiple regions of recurrent gain (including 2q23, 7p21, 12p, 13q21.1, and 20p12) and loss (including 5q31, 6q26, 7q36, 15q21.1, 16q24, 17p13.3, 19p13.2, and 22q13.3). An ependymoma-specific gene expression signature was characterized by the concurrent abnormal expression of developmental and differentiation pathways, including NOTCH and sonic hedgehog signaling. We identified specific differentially imbalanced genomic clones and gene expression signatures significantly associated with tumor location, patient age at disease onset, and retrospective risk for relapse. Integrated genomic and expression profiling allowed us to identify genes of which the expression is deregulated in intracranial ependymoma, such as overexpression of the putative proto-oncogene YAP1 (located at 11q22) and downregulation of the SULT4A1 gene (at 22q13.3). CONCLUSION: The present exploratory molecular profiling study allowed us to refine previously reported intervals of genomic imbalance, to identify novel restricted regions of gain and loss, and to identify molecular signatures correlating with various clinical variables. Validation of these results on independent data sets represents the next step before translation into the clinical setting.

Novel chromosomal imbalances in mantle cell lymphoma detected by genome-wide array-based comparative genomic hybridization.

Mantle cell lymphoma (MCL) is an aggressive, highly proliferative B-cell non-Hodgkin lymphoma, characterized by the specific t(11;14)(q13;q32) translocation. It is well established that this translocation alone is not sufficient to promote MCL development, but that additional genetic changes are essential for malignant transformation. We have identified such additional tumorigenic triggers in MCL tumors, by applying genome-wide array-based comparative genomic hybridization with an 800-kilobase (kb) resolution. This strategy, combined with a newly developed statistical approach, enabled us to confirm previously reported genomic alterations such as loss of 1p, 6q, 11q, 13q and gain of 3q and 8q, but it also facilitated the detection of novel recurrent genomic imbalances, such as gain of 4p12-13 and loss of 20p12.1-12.3, 20q12-13.2, 22q12.1-12.3, and 22q13.31-13.32. Genomic hotspot detection allowed for the identification of small genomic intervals that are frequently affected (57%-93%), resulting in interesting positional candidate genes such as KITLG, GPC5, and ING1. Finally, by assessing multiple biopsies from the same patient, we show that seemingly stable genomes do show subtle genomic changes over time. The follow-up of multiple biopsies of patients with MCL by high-resolution genomic profiling is expected to provide us with new clues regarding the relation between clinical outcome and in vivo cytogenetic evolution.