High-resolution Array Comparative Genomic Hybridization Research Articles

(058) Evaluation of Pathway Expression Following Down-Regulation of NELL1, a Potential Gene of Interest in Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we report on pathway analysis following up- and down-regulation of NELL1 using CRISPR modification in an in vitro setting. Objective This study aimed to investigate the influence of NELL1 modification on downstream gene regulation, focusing on pathways that were significantly regulated following NELL1 down-regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. Gene regulation was assessed using qRT-PCR. Results For down-regulation of NELL1, PCR analysis confirmed a 95% reduction in expression. Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 5,000 differentially expressed genes between, with significant changes in multiple pathways, including the HIF-1α pathway, which promotes keloid development by activating the TGF-β/Smad pathways. Additionally, we identified pathways related to ECM-receptor interaction, inflammation, apoptosis, and cell cycle regulation that are mediated by TGF-β/Smad signaling, which plays a crucial role in fibrosis. Our analysis also demonstrated upregulation of the PI3K-Akt pathway, which is involved in regulating type I collagen expression in response to TGF-β1. These results highlight the significance of NELL1 in fibrotic regulation and suggest its potential involvement in the development of PD. Conclusions We have created guides for and successfully down-regulated the expression of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. The identified modified pathways, including HIF-1α-mediated, TGF-β/Smad and PI3K-Akt, provide valuable insights into the potential molecular mechanisms underlying NELL1's role in regulation of fibrosis, and further investigation of these pathways may reveal potential therapeutic targets for fibrosis-related disorders. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Contraline, Techfields Inc, Alto Neurosciences, Curebase.

(153) Investigating the Role of NELL1 as a Gene of Interest in Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we describe the creation of lentiviral gRNA vectors for the up- and down-regulation of NELL1 in an in vitro setting, as well as PCR results for NELL1 regulation and preliminary results from RNA sequencing following CRISPR modification. Objective To determine the influence of NELL1 on extracellular matrix (ECM) deposition and downstream gene regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. For upregulation, gRNAs were delivered to HEK293 cells expressing the dCas9-VPR system adapted from Streptococcus pyogenes. Gene regulation via these systems was assessed using qRT-PCR. ECM deposition was evaluated using primary antibodies again Collagen I, Collagen III, and Elastin, and the appropriate secondary antibodies. RNA sequencing and analysis was performed by the High-Throughput Genomics and Bioinformatic Analysis core at the University of Utah. Results For up-regulation, four vectors were tested, with the most effective vector demonstrating a 40-fold increase in NELL1 expression via PCR testing. For down-regulation, three vectors were tested, with the most effective demonstrating a 95% reduction in expression via PCR testing (Figure 1). Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 10,000 differentially expressed genes between up-regulated and non-target cell lines, with significant changes in multiple pathways, including TNF-α signaling. Similarly, over 5,000 differentially expressed genes were found between down-regulated and non-target cell lines, with significant changes in inflammatory and G2-M checkpoint pathways, among others. Ongoing experiments are also testing the amount of extra-cellular matrix (ECM) deposited by up- and down-regulated cells and their non-target controls, in order to determine if changes in NELL1 expression influence ECM deposition. Conclusions We have demonstrated the creation of effective guides for the up- and down-regulation of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. Ongoing experiments will determine if changes in NELL1 expression also affect ECM deposition. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Vault Health.

Identification of Extremely Rare Pathogenic CNVs by Array CGH in Saudi Children with Developmental Delay, Congenital Malformations, and Intellectual Disability

Chromosomal imbalance is implicated in developmental delay (DD), congenital malformations (CM), and intellectual disability (ID), and, thus, precise identification of copy number variations (CNVs) is essential. We therefore aimed to investigate the genetic heterogeneity in Saudi children with DD/CM/ID. High-resolution array comparative genomic hybridization (array CGH) was used to detect disease-associated CNVs in 63 patients. Quantitative PCR was done to confirm the detected CNVs. Giemsa banding-based karyotyping was also performed. Array CGH identified chromosomal abnormalities in 24 patients; distinct pathogenic and/or variants of uncertain significance CNVs were found in 19 patients, and aneuploidy was found in 5 patients including 47,XXY (n = 2), 45,X (n = 2) and a patient with trisomy 18 who carried a balanced Robertsonian translocation. CNVs including 9p24p13, 16p13p11, 18p11 had gains/duplications and CNVs, including 3p23p14, 10q26, 11p15, 11q24q25, 13q21.1q32.1, 16p13.3p11.2, and 20q11.1q13.2, had losses/deletions only, while CNVs including 8q24, 11q12, 15q25q26, 16q21q23, and 22q11q13 were found with both gains or losses in different individuals. In contrast, standard karyotyping detected chromosomal abnormalities in ten patients. The diagnosis rate of array CGH (28%, 18/63 patients) was around two-fold higher than that of conventional karyotyping (15.87%, 10/63 patients). We herein report, for the first time, the extremely rare pathogenic CNVs in Saudi children with DD/CM/ID. The reported prevalence of CNVs in Saudi Arabia adds value to clinical cytogenetics.

Copy number variations analysis in a cohort of 47 fetuses and newborns with congenital diaphragmatic hernia.

The congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and lung hypoplasia, is a common and severe birth defect that affects around 1 in 4000 live births. However, the etiology of most cases of CDH remains unclear. The aim of this study was to perform a retrospective analysis of copy number variations (CNVs) using a high-resolution array comparative genomic hybridization (array-CGH) in a cohort of fetuses and newborns with CDH. Forty seven fetuses and newborns with either isolated or syndromic CDH were analyzed by oligonucleotide-based array-CGH Agilent 180K technique. A mean of 10.2 CNVs was detected by proband with a total number of 480 CNVs identified based on five categories: benign, likely benign, of uncertain signification, likely pathogenic, and pathogenic. Diagnostic performance was estimated at 19.15% (i.e., likely pathogenic and pathogenic CNVs) for both CDH types. We identified 11 potential candidate genes: COL25A1, DSEL, EYA1, FLNA, MECOM, NRXN1, RARB, SPATA13, TJP2, XIRP2, and ZFPM2. We suggest that COL25A1, DSEL, EYA1, FLNA, MECOM, NRXN1, RARB, SPATA13, TJP2, XIRP2, and ZFPM2 genes may be related to CDH occurrence. Thus, this study provides a possibility for new methods of a positive diagnosis.

Molecular and clinical insights into complex genomic rearrangements related to MECP2 duplication syndrome

MECP2 duplication syndrome (MDS) is caused by copy number variation (CNV) spanning the MECP2 gene at Xq28 and is a major cause of intellectual disability (ID) in males. Herein, we describe two unrelated males harboring non-recurrent complex Xq28 rearrangements associated with MDS. Copy number gains were initially detected by quantitative real-time polymerase chain reaction and further delineated by high-resolution array comparative genomic hybridization, familial segregation, expression analysis and X-chromosome inactivation (XCI) evaluation in a carrier mother. SNVs within the rearrangements and/or fluorescent in situ hybridization (FISH) were used to assess the parental origin of the rearrangements. Patient 1 exhibited an intrachromosomal rearrangement, whose structure is consistent with a triplicated segment presumably embedded in an inverted orientation between two duplicated sequences (DUP-TRP/INV-DUP). The rearrangement was inherited from the carrier mother, who exhibits extreme XCI skewing and subtle psychiatric symptoms. Patient 2 presented a de novo (X;Y) unbalanced translocation resulting in duplication of Xq28 and deletion of Yp, originated in the paternal gametogenesis. Neurodevelopmental trajectory and non-neurological symptoms were consistent with previous reports, with the exception of cerebellar vermis hypoplasia in patient 2. Although both patients share the core MDS phenotype, patient 1 showed MECP2 transcript levels in blood similar to controls. Understanding the molecular mechanisms related to MDS is essential for designing targeted therapeutic strategies.

Identification of novel pathogenic copy number variations in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is a hereditary sensory-motor neuropathy characterized by a strong clinical and genetic heterogeneity. Over the past few years, with the occurrence of whole-exome sequencing (WES) or whole-genome sequencing (WGS), the molecular diagnosis rate has been improved by allowing the screening of more than 80 genes at one time. In CMT, except the recurrent PMP22 duplication accounting for about 60% of pathogenic variations, pathogenic copy number variations (CNVs) are rarely reported and only a few studies screening specifically CNVs have been performed. The aim of the present study was to screen for CNVs in the most prevalent genes associated with CMT in a cohort of 200 patients negative for the PMP22 duplication. CNVs were screened using the Exome Depth software on next generation sequencing (NGS) data obtained by targeted capture and sequencing of a panel of 81 CMT associated genes. Deleterious CNVs were identified in four patients (2%), in four genes: GDAP1, LRSAM1, GAN, and FGD4. All CNVs were confirmed by high-resolution oligonucleotide array Comparative Genomic Hybridization (aCGH) and/or quantitative PCR. By identifying four new CNVs in four different genes, we demonstrate that, although they are rare mutational events in CMT, CNVs might contribute significantly to mutational spectrum of Charcot-Marie-Tooth disease and should be searched in routine NGS diagnosis. This strategy increases the molecular diagnosis rate of patients with neuropathy.

GEFT aberrant expression in soft tissue sarcomas

Background: Guanine nucleotide exchange factor T (GEFT) exhibits high amplification level using high-resolution array comparative genomic hybridization in rhabdomyosarcoma. The overexpression rate of GEFT protein is higher in rhabdomyosarcoma than in normal striated muscle tissues. This study evaluated the aberrant expression of GEFT in multiple subtypes of soft tissue sarcoma (STS) and compared the differences in clinical pathology, histological feature and expression levels of GEFT protein and mRNA between chromosomal translocation-associated sarcomas (CTAS) and non-chromosomal translocation-associated sarcomas (NCTAS). Methods: GEFT protein expression was detected using immunohistochemistry (IHC) and tissue microarrays. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression of GEFT mRNA. Results: The rates of GEFT positive expression (196/219, 89.50%) and overexpression (113/219, 51.60%) were higher in multiple subtypes of STS than in normal striated muscle tissues. The rates of GEFT positive expression and overexpression in all subtypes of STS detected were significantly higher than that in the controls. No difference of GEFT expression was detected between CTAS and NCTAS. Conclusions: The abnormal expression of GEFT exists in various subtypes of STS, which may play an important role in tumorigenesis of STS.

GEFT aberrant expression in soft tissue sarcomas.

BackgroundGuanine nucleotide exchange factor T (GEFT) exhibits high amplification level using high-resolution array comparative genomic hybridization in rhabdomyosarcoma. The overexpression rate of GEFT protein is higher in rhabdomyosarcoma than in normal striated muscle tissues. This study evaluated the aberrant expression of GEFT in multiple subtypes of soft tissue sarcoma (STS) and compared the differences in clinical pathology, histological feature and expression levels of GEFT protein and mRNA between chromosomal translocation-associated sarcomas (CTAS) and non-chromosomal translocation-associated sarcomas (NCTAS).MethodsGEFT protein expression was detected using immunohistochemistry (IHC) and tissue microarrays. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression of GEFT mRNA.ResultsThe rates of GEFT positive expression (196/219, 89.50%) and overexpression (113/219, 51.60%) were higher in multiple subtypes of STS than in normal striated muscle tissues. The rates of GEFT positive expression and overexpression in all subtypes of STS detected were significantly higher than that in the controls. No difference of GEFT expression was detected between CTAS and NCTAS.ConclusionsThe abnormal expression of GEFT exists in various subtypes of STS, which may play an important role in tumorigenesis of STS.

Genomic changes of chromosomes 8p23.1 and 1q21: Novel mutations in malignant mesothelioma

IntroductionMalignant mesothelioma is an aggressive malignancy of the thoracic cavity caused by prior asbestos exposure. In the peritoneum the mesothelioma is an extremely rare condition. In the present preliminary study, high-resolution array-comparative genomic hybridization (a-CGH) was performed to identify genetic imbalances in a series of malignant peritoneal mesothelioma cases. Materials and methodsBetween 1990 and 2008, among the cases recorded in the Apulia Mesothelioma Register, we found 22 peritoneal mesothelioma cases. CGH-array was performed on samples from all patients. ResultsThe CGH-array analysis revealed multiple chromosomal imbalances. Interestingly, deletion at 8p23.1 was observed in 12 cases. Furthermore, another novel deletion at 1q21 was present in 11. Often, 1q21 and 8p23.1 losses were present in the same patient (7 cases). Losses of BAP1 and CDKN2A loci were not detected. DiscussionThe region at 8p23.1 contains the beta-defensin gene cluster (DEF) and 1q21 contains ubiquitin conjugating enzyme E2 (UBE2Q1). We hypotesized that the loss of function of ubiquitination, as well as of the defensins, could play an important role in the initial development and subsequent progression of mesothelioma.

Copy number variations in a population with prune belly syndrome.

Prune Belly Syndrome (PBS) is a congenital multisystem myopathy with mild to lethal severity. While of uncertain etiology, 95% male predominance and familial occurrence suggest a genetic basis. As copy number variations (CNVs) can cause unexplained genetic disorders, we tested for novel CNVs in a large PBS population. We genotyped 21 unrelated PBS patients by high-resolution array comparative genomic hybridization (aCGH) and phenotyped using a novel PBS severity scoring system. Available parents were screened for detected CNV via quantitative PCR (qPCR). We additionally screened for recurrence of identified novel candidate CNVs on 106 PBS probands by qPCR. We identified 10 CNVs in 8 of 21 PBS patients tested (38%). Testing confirmed inheritance from an unaffected biological parent in six patients; parental samples were unavailable in two probands. One candidate CNV includes duplication of the X-chromosome AGTR2 gene, known to function in urinary tract development. Subsequent screening of the larger PBS cohort did not identify any recurrent CNVs. Presence of CNV did not correlate with PBS severity scoring. CNVs were uncommon in this large PBS population, but analysis of identified variants may inform disease pathogenesis and reveal targets for therapeutic intervention for this rare, severe disorder.

Chromosomal instability in women with primary ovarian insufficiency.

What is the prevalence of somatic chromosomal instability among women with idiopathic primary ovarian insufficiency (POI)? A subset of women with idiopathic POI may have functional impairment in DNA repair leading to chromosomal instability in their soma. The formation and repair of DNA double-strand breaks during meiotic recombination are fundamental processes of gametogenesis. Oocytes with compromised DNA integrity are susceptible to apoptosis which could trigger premature ovarian aging and accelerated wastage of the human follicle reserve. Genomewide association studies, as well as whole exome sequencing, have implicated multiple genes involved in DNA damage repair. However, the prevalence of defective DNA damage repair in the soma of women with POI is unknown. In total, 46 women with POI and 15 family members were evaluated for excessive mitomycin-C (MMC)-induced chromosome breakage. Healthy fertile females (n = 20) and two lymphoblastoid cell lines served as negative and as positive controls, respectively. We performed a pilot functional study utilizing MMC to assess chromosomal instability in the peripheral blood of participants. A high-resolution array comparative genomic hybridization (aCGH) was performed on 16 POI patients to identify copy number variations (CNVs) for a set of 341 targeted genes implicated in DNA repair. Array CGH revealed three POI patients (3/16, 18.8%) with pathogenic CNVs. Excessive chromosomal breakage suggestive of a constitutional deficiency in DNA repair was detected in one POI patient with the 16p12.3 duplication. In two patients with negative chromosome breakage analysis, aCGH detected a Xq28 deletion comprising the Centrin EF-hand Protein 2 (CETN2) and HAUS Augmin Like Complex Subunit 7 (HAUS7) genes essential for meiotic DNA repair, and a duplication in the 3p22.2 region comprising a part of the ATPase domain of the MutL Homolog 1 (MLH1) gene. Peripheral lymphocytes, used as a surrogate tissue to quantify induced chromosome damage, may not be representative of all the affected tissues. Another limitation pertains to the MMC assay which detects homologous repair pathway defects and does not test deficiencies in other DNA repair pathways. Our results provide evidence for functional impairment of DNA repair in idiopathic POI, which may predispose the patients to other DNA repair-related conditions such as accelerated aging and/or cancer susceptibility. Funding was provided by the National Institute of Child Health and Human Development. There were no competing interests to declare.

Application of high-resolution array comparative genomic hybridization in children with unknown syndromic microcephaly.

BackroundMicrocephaly can either be isolated or it may coexist with other neurological entities and/or multiple congenital anomalies, known as syndromic microcephaly. Although many syndromic cases can be classified based on the characteristic phenotype, some others remain uncertain and require further investigation. The present study describes the application of array-comparative genomic hybridization (array-CGH) as a diagnostic tool for the study of patients with clinically unknown syndromic microcephaly.MethodsFrom a cohort of 210 unrelated patients referred with syndromic microcephaly, we applied array-CGH analysis in 53 undiagnosed cases. In all the 53 cases except one, previous standard karyotype was negative. High-resolution 4 × 180K and 1 × 244K Agilent arrays were used in this study.ResultsIn 25 out of the 53 patients with microcephaly among other phenotypic anomalies, array-CGH revealed copy number variations (CNVs) ranging in size between 15 kb and 31.6 Mb. The identified CNVs were definitely causal for microcephaly in 11/53, probably causal in 7/53, and not causal for microcephaly in 7/53 patients. Genes potentially contributing to brain deficit were revealed in 16/53 patients.ConclusionsArray-CGH contributes to the elucidation of undefined syndromic microcephalic cases by permitting the discovery of novel microdeletions and/or microduplications. It also allows a more precise genotype-phenotype correlation by the accurate definition of the breakpoints in the deleted/duplicated regions.

Evidence for the involvement of the proximal copy of the MAGEA9 gene in Xq28-linked CNV67 specific to spermatogenic failure.

Spermatogenic failure characterized by impaired sperm production is a common multifactorial disease with molecular and cytogenetic causes for its extreme phenotype that include azoospermia and severe oliogzoospermia. Recently, a high-resolution array-comparative genomic hybridization analysis of the X chromosome and a subsequent cohort study revealed three X-linked microdeletions (CNV64, CNV67, and CNV69) that were associated with decreased sperm production in a mixed group that included Spanish and Italian males. To confirm their spermatogenic effect, we examined the hemizygous deletions and copy dosage of the MAGE family member A9 (MAGEA9) gene, which is a potential X-linked candidate for the CNV67-related spermatogenic phenotype, to investigate their association with spermatogenic failure in 1722 Han males from southwest China. The individuals in this group consisted of 884 patients with idiopathic azoospermia/oliogzoospermia and 838 controls with normozoospermia. Our results showed that both CNV64 and CNV69 were more common in patients than in controls. Similar to that reported previously, the CNV67 was also identified as being specific to spermatogenic failure in our population, although it was rare. More importantly, the paralog ratio tests and sequence family variant analyses provided evidence that the CNV67 might cause a partial deletion of the proximal copy of the MAGEA9 and suggests that CNV67-related spermatogenic failure may be attributed to the functional defect of the Cancer/Testis gene. Our findings highlight the potential of the Xq-linked CNV67 to serve as a novel detection target in the etiological diagnosis of spermatogenic failure and male infertility, although its pathogenic mechanism remains to be elucidated.

Abstract A07: Detection of structural variants and recurrent breakpoint genes in colorectal adenoma-to-carcinoma progression

Abstract Background and Aim: The transition of pre-malignant colorectal adenomas (CRAs) into malignant colorectal carcinomas (CRCs) is accompanied by accumulation of somatic genetic alterations. The contribution of small nucleotide variants (SNVs, such as gene point mutations) and numerical DNA copy number aberrations (CNAs, such as gains and losses of large chromosomal segments) in tumor genomes has been studied extensively. In contrast, studies that systematically detect recurrent structural variants (SVs, such as chromosomal breaks) across large series of clinically well-defined samples are scarce. We recently developed “GeneBreak”, a computational method to identify genes that are recurrently affected by the genomic location of CNA-associated chromosomal breaks, and demonstrated high prevalence and clinical relevance of recurrent breakpoint genes in advanced CRCs (PLoS One 2015;10(9):e0138141). Using a similar approach, the present study aimed to determine what recurrent breakpoint genes contribute to colorectal adenoma-to-carcinoma progression. Methods: Differences in somatic CNA-associated gene breakpoint frequencies in 466 CRC and 118 CRA samples were examined using high-resolution array-comparative genomic hybridization (aCGH) profiles. Pearson's Chi-square statistic was applied to determine differences in gene breakpoint frequencies between CRC and CRA samples. Results: In total 21 recurrent breakpoint genes were more frequently affected in CRCs compared to CRAs (p<0.05). MACROD2 was affected by chromosomal breakpoints in 40% of CRCs while not being affected in CRAs. The frequencies of SVs in the other 20 recurrent breakpoint genes ranged from 5% to 29% in CRCs, and from 0% to 2% in CRAs. Conclusion: We identified 21 recurrent breakpoint genes that are frequently affected by SVs in CRCs but not in CRAs. Additional studies are needed to further elaborate the biological and clinical role of these 21 candidate driver genes for colorectal tumor progression. Citation Format: Evert van den Broek, Maurits J.J. Dijkstra, Quirinus J.M. Voorham, Beatriz Carvalho, Mark A. van de Wiel, Sanne Abeln, Gerrit A. Meijer, Remond J.A. Fijneman. Detection of structural variants and recurrent breakpoint genes in colorectal adenoma-to-carcinoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A07.

A novel Y chromosome microdeletion potentially associated with defective spermatogenesis identified by custom array comparative genome hybridization

Male infertility is a major health problem worldwide. Oligospermia and azoospermia are the most common symptoms of this disorder. Despite recent advances, the aetiopathogenesis of defective spermatogenesis remains largely uncertain. The aim of this study is to discover unknown or novel chromosome aberrations associated with male reproductive failure. We developed a high-resolution custom array comparative genomic hybridization for initial screening of copy number variations in 10 patients with idiopathic oligozoospermia and azoospermia and eight normal fertile men. We found that deletions were mainly located in the deleted-in-azoospermia subregion and were confined to patients. More importantly, an interesting microdeletion of the Y chromosome designated as D01 was detected in four out of 10 patients with oligozoospermia and azoospermia. We validated this recurrent deletion in nine out of 100 additional infertile men using polymerase chain reaction assays, whereas, it was not present in 100 proven fertile controls(P = 0.002). Furthermore, a bioinformatics analysis demonstrated that the 5' terminal of D01 is situated proximal to several conserved transcription factor binding sites within the Y chromosome. Our study indicated that this newly identified Y chromosome deletion might be potentially associated with impaired spermatogenesis and it is worthy of further investigations in larger cohorts.

Genomic profiling of stage II and III colon cancers reveals APC mutations to be associated with survival in stage III colon cancer patients.

Tumor profiling of DNA alterations, i.e. gene point mutations, somatic copy number aberrations (CNAs) and structural variants (SVs), improves insight into the molecular pathology of cancer and clinical outcome. Here, associations between genomic aberrations and disease recurrence in stage II and III colon cancers were investigated. A series of 114 stage II and III microsatellite stable colon cancer samples were analyzed by high-resolution array-comparative genomic hybridization (array-CGH) to detect CNAs and CNA-associated chromosomal breakpoints (SVs). For 60 of these samples mutation status of APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS was determined using targeted massive parallel sequencing. Loss of chromosome 18q12.1-18q12.2 occurred more frequently in tumors that relapsed than in relapse-free tumors (p < 0.001; FDR = 0.13). In total, 267 genes were recurrently affected by SVs (FDR < 0.1). CNAs and SVs were not associated with disease-free survival (DFS). Mutations in APC and TP53 were associated with increased CNAs. APC mutations were associated with poor prognosis in (5-fluorouracil treated) stage III colon cancers (p = 0.005; HR = 4.1), an effect that was further enhanced by mutations in MAPK pathway (KRAS, NRAS, BRAF) genes. We conclude that among multiple genomic alterations in CRC, strongest associations with clinical outcome were observed for common mutations in APC.

A customized high-resolution array-comparative genomic hybridization to explore copy number variations in Parkinson’s disease

Parkinson’s disease (PD), the second most common progressive neurodegenerative disorder, was long believed to be a non-genetic sporadic syndrome. Today, only a small percentage of PD cases with genetic inheritance patterns are known, often complicated by reduced penetrance and variable expressivity. The few well-characterized Mendelian genes, together with a number of risk factors, contribute to the major sporadic forms of the disease, thus delineating an intricate genetic profile at the basis of this debilitating and incurable condition. Along with single nucleotide changes, gene-dosage abnormalities and copy number variations (CNVs) have emerged as significant disease-causing mutations in PD. However, due to their size variability and to the quantitative nature of the assay, CNV genotyping is particularly challenging. For this reason, innovative high-throughput platforms and bioinformatics algorithms are increasingly replacing classical CNV detection methods. Here, we report the design strategy, development, validation and implementation of NeuroArray, a customized exon-centric high-resolution array-based comparative genomic hybridization (aCGH) tailored to detect single/multi-exon deletions and duplications in a large panel of PD-related genes. This targeted design allows for a focused evaluation of structural imbalances in clinically relevant PD genes, combining exon-level resolution with genome-wide coverage. The NeuroArray platform may offer new insights in elucidating inherited potential or de novo structural alterations in PD patients and investigating new candidate genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s10048-016-0494-0) contains supplementary material, which is available to authorized users.

Nonrecurrent PMP22-RAI1 contiguous gene deletions arise from replication-based mechanisms and result in Smith-Magenis syndrome with evident peripheral neuropathy.

Hereditary neuropathy with liability to pressure palsies (HNPP) and Smith-Magenis syndrome (SMS) are genomic disorders associated with deletion copy number variants involving chromosome 17p12 and 17p11.2, respectively. Nonallelic homologous recombination (NAHR)-mediated recurrent deletions are responsible for the majority of HNPP and SMS cases; the rearrangement products encompass the key dosage-sensitive genes PMP22 and RAI1, respectively, and result in haploinsufficiency for these genes. Less frequently, nonrecurrent genomic rearrangements occur at this locus. Contiguous gene duplications encompassing both PMP22 and RAI1, i.e., PMP22-RAI1 duplications, have been investigated, and replication-based mechanisms rather than NAHR have been proposed for these rearrangements. In the current study, we report molecular and clinical characterizations of six subjects with the reciprocal phenomenon of deletions spanning both genes, i.e., PMP22-RAI1 deletions. Molecular studies utilizing high-resolution array comparative genomic hybridization and breakpoint junction sequencing identified mutational signatures that were suggestive of replication-based mechanisms. Systematic clinical studies revealed features consistent with SMS, including features of intellectual disability, speech and gross motor delays, behavioral problems and ocular abnormalities. Five out of six subjects presented clinical signs and/or objective electrophysiologic studies of peripheral neuropathy. Clinical profiling may improve the clinical management of this unique group of subjects, as the peripheral neuropathy can be more severe or of earlier onset as compared to SMS patients having the common recurrent deletion. Moreover, the current study, in combination with the previous report of PMP22-RAI1 duplications, contributes to the understanding of rare complex phenotypes involving multiple dosage-sensitive genes from a genetic mechanistic standpoint.

Genomic characterization of recurrent high-grade astroblastoma

Astroblastomas are rare primary brain tumors, diagnosed based on histologic features. Not currently assigned a WHO grade, they typically display indolent behavior, with occasional variants taking a more aggressive course. We characterized the immunohistochemical characteristics, copy number (high-resolution array comparative genomic hybridization, OncoCopy) and mutational profile (targeted next-generation exome sequencing, OncoPanel) of a cohort of seven biopsies from four patients to identify recurrent genomic events that may help distinguish astroblastomas from other more common high-grade gliomas. We found that tumor histology was variable across patients and between primary and recurrent tumor samples. No common molecular features were identified among the four tumors. Mutations commonly observed in astrocytic tumors (IDH1/2, TP53, ATRX, and PTEN) or ependymoma were not identified. However one case with rapid clinical progression displayed mutations more commonly associated with GBM (NF1(N1054H/K63)*, PIK3CA(R38H) and ERG(A403T)). Conversely, another case, originally classified as glioblastoma with nine-year survival before recurrence, lacked a GBM mutational profile. Other mutations frequently seen in lower grade gliomas (BCOR, BCORL1, ERBB3, MYB, ATM) were also present in several tumors. Copy number changes were variable across tumors. Our findings indicate that astroblastomas have variable growth patterns and morphologic features, posing significant challenges to accurate classification in the absence of diagnostically specific copy number alterations and molecular features. Their histopathologic overlap with glioblastoma will likely confound the observation of long-term GBM "survivors". Further genomic profiling is needed to determine whether these tumors represent a distinct entity and to guide management strategies.

Identification of 42 Genes Linked to Stage II Colorectal Cancer Metastatic Relapse.

Colorectal cancer (CRC) is one of the leading causes of cancer mortality. Metastasis remains the primary cause of CRC death. Predicting the possibility of metastatic relapse in early-stage CRC is of paramount importance to target therapy for patients who really need it and spare those with low-potential of metastasis. Ninety-six stage II CRC cases were stratified using high-resolution array comparative genomic hybridization (aCGH) data based on a predictive survival algorithm and supervised clustering. All genes included within the resultant copy number aberrations were each interrogated independently at mRNA level using CRC expression datasets available from public repositories, which included 1820 colon cancers, and 167 normal colon tissues. Reduced mRNA expression driven by copy number losses and increased expression driven by copy number gains revealed 42 altered transcripts (29 reduced and 13 increased transcripts) associated with metastatic relapse, short disease-free or overall survival, and/or epithelial to mesenchymal transition (EMT). Resultant genes were classified based on gene ontology (GO), which identified four functional enrichment groups involved in growth regulation, genomic integrity, metabolism, and signal transduction pathways. The identified 42 genes may be useful for predicting metastatic relapse in stage II CRC. Further studies are necessary to validate these findings.