Comparative Genomic Hybridization Method Research Articles

Application of the FISH Method for Analyzing Chromosome Segregation Patterns in Preimplantation Embryos from Robertsonian Translocation Carriers

Robertsonian translocations (RTs) are very common balanced structural chromosome rearrangements in humans. Due to alterations in the chromosome segregation pattern and the formation of unbalanced gametes and embryos, the carriers of RTs have a significant risk of reproductive failure. For over 30 years, fluorescent in situ hybridization (FISH) has been used for preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) in carriers of RTs. The data obtained by performing FISH for PGT-SR can be useful for analyzing segregation patterns in preimplantation embryos. We analyzed chromosome segregation patterns in 964 embryos from 100 couples who were carriers of various types of RTs by performing FISH or using the array comparative genomic hybridization (aCGH) method. When the carriers were male, the predominant segregation mode was alternate (detected in 42.4% of embryos), whereas, when the carriers were female, alternate and adjacent types of segregation were detected in most embryos (32.3% and 26.6% of embryos, respectively). About 33% of balanced/euploid embryos in IVF cycles were formed when the carriers of RT were male, and about 24% of such embryos were formed when the carriers were female. No association was found between sperm parameters and malsegregation rates in male carriers of RT. In this study, we found that female and male RT carriers have different reproductive risks associated with chromosome segregation patterns and the rate of balanced/euploid embryos.

Rapid identification of mutations caused by fast neutron bombardment in Medicago truncatula

BackgroundFast neutron bombardment (FNB) is a very effective approach for mutagenesis and has been widely used in generating mutant libraries in many plant species. The main type of mutations of FNB mutants are deletions of DNA fragments ranging from few base pairs to several hundred kilobases, thus usually leading to the null mutation of genes. Despite its efficiency in mutagenesis, identification of the mutation sites is still challenging in many species. The traditional strategy of positional cloning is very effective in identifying the mutation but time-consuming. With the availability of genome sequences, the array-based comparative genomic hybridization (CGH) method has been developed to detect the mutation sites by comparing the signal intensities of probes between wild-type and mutant plants. Though CGH method is effective in detecting copy number variations (CNVs), the resolution and coverage of CGH probes are not adequate to identify mutations other than CNVs.ResultsWe report a new strategy and pipeline to sensitively identify the mutation sites of FNB mutants by combining deep-coverage whole-genome sequencing (WGS), polymorphism calling, and customized filtering in Medicago truncatula. Initially, we performed a bulked sequencing for a FNB white nodule (wn) mutant and its wild-type like plants derived from a backcross population. Following polymorphism calling and filtering, validation by manual check and Sanger sequencing, we identified that SymCRK is the causative gene of white nodule mutant. We also sequenced an individual FNB mutant yellow leaves 1 (yl1) and wild-type plant. We identified that ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN 1 (EGY1) is the candidate gene for M. truncatula yl1 mutant.ConclusionOur results demonstrated that the method reported here is rather robust in identifying the mutation sites for FNB mutants.

Development and Validation of a 34-Gene Inherited Cancer Predisposition Panel Using Next-Generation Sequencing.

The use of genetic testing to identify individuals with hereditary cancer syndromes has been widely adopted by clinicians for management of inherited cancer risk. The objective of this study was to develop and validate a 34-gene inherited cancer predisposition panel using targeted capture-based next-generation sequencing (NGS). The panel incorporates genes underlying well-characterized cancer syndromes, such as BRCA1 and BRCA2 (BRCA1/2), along with more recently discovered genes associated with increased cancer risk. We performed a validation study on 133 unique specimens, including 33 with known variant status; known variants included single nucleotide variants (SNVs) and small insertions and deletions (Indels), as well as copy-number variants (CNVs). The analytical validation study achieved 100% sensitivity and specificity for SNVs and small Indels, with 100% sensitivity and 98.0% specificity for CNVs using in-house developed CNV flagging algorithm. We employed a microarray comparative genomic hybridization (aCGH) method for all specimens that the algorithm flags as CNV-positive for confirmation. In combination with aCGH confirmation, CNV detection specificity improved to 100%. We additionally report results of the first 500 consecutive specimens submitted for clinical testing with the 34-gene panel, identifying 53 deleterious variants in 13 genes in 49 individuals. Half of the detected pathogenic/likely pathogenic variants were found in BRCA1 (23%), BRCA2 (23%), or the Lynch syndrome-associated genes PMS2 (4%) and MLH1 (2%). The other half were detected in 9 other genes: MUTYH (17%), CHEK2 (15%), ATM (4%), PALB2 (4%), BARD1 (2%), CDH1 (2%), CDKN2A (2%), RAD51C (2%), and RET (2%). Our validation studies and initial clinical data demonstrate that a 34-gene inherited cancer predisposition panel can provide clinically significant information for cancer risk assessment.

Association between chromosomal translocation imbalances and aneuploidies in PGT-SR in reciprocal translocation carriers

Introduction Reciprocal translocation carriers are known to be at high risk of conceiving genetically unbalanced embryos, which may result in miscarriage or an affected neonate. Preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR), a method for embryonic chromosomal analysis, can detect not only translocation chromosome imbalance but also aneuploidy via comprehensive chromosomal analysis with whole-genome amplification (WGA), thereby potentially decreasing miscarriage rate. In this study, we aimed to investigate the relationship between chromosomal abnormalities derived from translocation chromosomes and aneuploidies and the frequency of chromosomal abnormalities among stimulation cycles in reciprocal translocation carriers receiving multiple PGT-SR cycles. Material and Method Between 2013 and 2017, we assessed 20 cycles of 8 couples (reciprocal translocation carriers in 5 females and 3 males; mean women's age, 35.1 years) who received 2 or more PGT-SR cycles at our clinic.Ovarian stimulation was primarily controlled, and when chromosomally normal embryos could not be obtained, a different protocol for ovarian stimulation was adapted. All oocytes were fertilized via intracytoplasmic sperm injection (ICSI). All embryos were cultured to the blastocyst stage. Approximately 5-10 trophectoderm (TE) cells were subjected to a biopsy using a laser system. Chromosomal analysis was performed using the microarray-based comparative genomic hybridization method or next-generation sequencing with whole-genome amplification. Result In all 20 cycles, the number of oocytes retrieved was 432 and the number of blastocysts subjected to biopsy was 90. Among 88 analyzable blastocysts, 19 had normal chromosomes (21.6%) and 69 had abnormal chromosomes (78.4%). Fifty-six embryos (81.2%) contained chromosomal imbalances derived from reciprocal translocation chromosomes and 37 embryos (53.6%) had aneuploidy, 24 embryos (34.8%) had both types of abnormalities. The incidence of chromosomal translocation-derived chromosomal imbalance was 60.0% (12/20) for male carriers and 64.7% (44/68) for female carriers, with no significant difference.The incidence of chromosomally normal embryos per cycle was 0–67%. Normal embryos were not obtained in 9 cycles (45%); however, they were obtained at different stimulation cycles. In 9 PGT-SR cycles with three or more embryos of female carriers subjected to biopsy, there was no correlation between the incidence of the abnormality due to chromosome translocation and aneuploidy. Conclusion There is no association between the incidence of translocation chromosome imbalance and aneuploidy. However, approximately 80% of embryos with chromosomal translocation-derived chromosomal imbalance include aneuploidy. The incidence of chromosomal aberrations in embryos has a large difference among the PGT-SR cycles even in the same carrier. In translocation carriers without normal embryos, performing different ovarian stimulation methods enhances the possibility of obtaining normal embryos.

Methotrexate-induced senescence of human colon cancer cells depends on p53 acetylation, but not genomic aberrations.

Human colon cancer C85 cell response to methotrexate has been documented previously to take on a form of reversible premature senescence. Seeking genomic aberrations encompassing candidate genes whose functional impairment could determine such a response to the drug, an array Comparative Genomic Hybridization method was applied, complemented by expression microarray data set searching. In the C85 cell genome, only short aberrations were identified, classified as focal chromosomal aberrations. 62% of the aberrant regions, selected by referral to normal human colon epithelium, were not carrying any gene. Out of the genes, subject to aberrations, 50% were protein-coding ones. Expression of those that could serve a signaling or a growth-regulatory function was found to be either downregulated or unchanged during C85 cell progression into methotrexate-induced senescence. Lack of extensive chromosomal instability in C85 cells is hypothesized to be attributed to the presence of the wild-type tumor suppressor p53 protein. Although two p53 protein isoforms were detected in C85 cells, stabilization and acetylation of the full-length p53 isoform were shown to underpin progression of the cells into premature senescence upon methotrexate treatment.

A case report of de novo 11q triplication, duplication, and segmental area of absence of heterozygosity in an infant with dysmorphic features, failure to thrive, and developmental delay.

Background With recent advances in array comparative genomic hybridization (aCGH) methods, several, previously unrecognized pathogenic copy number variants (CNVs) have been recognized. Intrachromosomal triplications are rare and have been reported in a few genomic regions. In this report, we describe an infant with complex chromosomal rearrangement involving the long arm of chromosome 11 with concomitant triplication, duplication, and segmental area of absence of heterozygosity (AOH). Case Presentation: We report an infant who was presented with dysmorphic features, severe failure to thrive, developmental delay, dysgenesis of the corpus callosum, and intestinal obstruction. The aCGHshowed 19,930 megabases (Mb) triplication at 11q13.3q14.3, 346 kilobases(Kb) duplication at 11q14.3 and an area of AOH at 11q14.3-qter. Conclusion The occurrence of triplication along with AOH (most likely as a result of segmental uniparental isodisomy) is a rare, complex genomic rearrangement. It is suggested that these complex genomic rearrangements coupled with segmental uniparental isodisomy arise as a result of one-ended DNA break repair coupled with microhomology-mediated break-induced replication (MMBIR).

Genetic Analysis of Copy Number Variation in Large Chorangiomas.

Chorangioma (CA) is the most common nontrophoblastic, vascular tumor-like lesion of the placenta with a reported incidence of 0.5% to 1% in all examined placentas. The underlying molecular mechanisms of CAs are still poorly elucidated, and a systematic investigation of the genetic background of CAs has not previously been done. Tissue biopsies from 8 large (>40 mm) histologically confirmed CAs and 8 unaffected matched placenta controls, along with standard control DNA samples were analyzed for large genomic deletions and duplications using array comparative genomic hybridization (array-CGH) method. Array-CGH analysis revealed no rare or novel copy number variants in the CA samples compared with either standard control DNA or unaffected placenta DNA from the same individual. In this study, a systematic genetic investigation of 8 large CAs failed to demonstrate any large-scale pathogenic genetic changes. This lack of association might support a nongenetic, nontumorous origin of these lesions; however, additional genetic studies focusing on smaller genomic alterations are required to fully assess any possible genetic contribution.

Mature Cystic Ovarian Teratoma: A Study of 43 Congolese Cases

Background: The mature cystic ovarian teratoma (MCOT) is a common benign ovarian neoplasm in the world, most prevalent among women of childbearing age. It is a disease related to parental imprinting in which genome is exclusively maternal. The purpose of the study was to review the tumour features in Congolese patients in order to clarify its prevalence, and its histological forms and to report the eventual associated chromosomal anomalies described in literature. Methods: It was a retrospective study of MCOT seen in University Teaching Hospital of Brazzaville for a period of seven years, and five DNAs previously extracted from five MCOT. Two antibodies: DLK1 and TIMP2 were also studied by immunohistochemistry (IHC). The confirmed positive diagnosis was performed by Hematoxylin-Eosin method. The eventual presence of chromosomal anomalies was investigated by metaphasic Comparative Genomic Hybridization (mCGH) method. IHC analysis had permit to verify expression of paternal imprinted gene DLK1 expression compared to maternal imprinted gene TIMP2. Results: During the study period, a total of 215 cases of ovarian neoplasms have been seen, from which 20% (43/215 cases) with confidence interval (CI) 95% = [14.7% - 25.3%] were diagnosed MCOT. They represent 38.7% (43/111 cases) (IC 95% = [29.9% - 48.1%]) of all benign ovarian tumors and they account for 0.4% (43/10170) of all tumors diagnosed during the study period. The average age of patients having MCOT was 30.7 years with extreme ranging from 2 to 70 years. Cutaneous tissue was the most prevalent histological form. About IHC analysis, no staining was observed with mono-allelic paternal gene DLK1 contrasting with high TIMP2 staining of nuclear stem cells in the basal layer of the epidermis. Concerning mCGH, no profile revealed chromosomal rearrangements. Conclusion: The benign ovarian teratoma remains a common ovarian neoplasm in young Congolese women in reproductive age. The predominant skin tissue form signed the current called ovarian dermoid cyst. The CGH analysis in our study revealed that pathology is not associated with chromosomal anomalies, but some associations are described in literature.

Model Organisms Facilitate Rare Disease Diagnosis and Therapeutic Research.

Efforts to identify the genetic underpinnings of rare undiagnosed diseases increasingly involve the use of next-generation sequencing and comparative genomic hybridization methods. These efforts are limited by a lack of knowledge regarding gene function, and an inability to predict the impact of genetic variation on the encoded protein function. Diagnostic challenges posed by undiagnosed diseases have solutions in model organism research, which provides a wealth of detailed biological information. Model organism geneticists are by necessity experts in particular genes, gene families, specific organs, and biological functions. Here, we review the current state of research into undiagnosed diseases, highlighting large efforts in North America and internationally, including the Undiagnosed Diseases Network (UDN) (Supplemental Material, File S1) and UDN International (UDNI), the Centers for Mendelian Genomics (CMG), and the Canadian Rare Diseases Models and Mechanisms Network (RDMM). We discuss how merging human genetics with model organism research guides experimental studies to solve these medical mysteries, gain new insights into disease pathogenesis, and uncover new therapeutic strategies.

Genome-Wide Deletion Screening with the Array CGH Method in Mouse Offspring Derived from Irradiated Spermatogonia Indicates that Mutagenic Responses are Highly Variable among Genes.

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10-2/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10-5/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.

Role of Sirt3 in mitochondrial biogenesis and developmental competence of human in vitro matured oocytes.

Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)? Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes. Cytoplasmic immaturity in IVM oocytes may lead to reduced developmental competence. Mitochondrial dysfunction results in the accumulation of free radicals and leads to DNA mutations, protein damage, telomere shortening and apoptosis. SIRT3 (in the Sirtuin protein family) has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species scavenging proteins. In vivo matured metaphase II (IVO-MII) oocytes and IVM-MII oocytes were donated by 324 infertile patients undergoing assisted reproductive technology cycles (12 patients for 60 IVO oocytes, and 312 patients for 403 IVM oocytes). Five oocytes each in the germinal vesicle (GV), IVM and IVO groups were compared with respect to mRNA levels for Sirt1-7 mRNA, and five samples at each developmental stage were analysed for Sirt3 mRNA. IVM-MII oocytes were injected with in vitro transcribed mRNA (n = 59) or small interfering RNA (siRNA) (n = 78). In human and mouse, IVM, mRNA-injection IVM, and siRNA-injection IVM groups (n = 5 each) were analysed for mitochondrial DNA copy number and abundance of Sirt3 and Pgc1α (an inducer of mitochondrial biogenesis) mRNAs. Human blastocysts in the IVO (n = 12), IVM (n = 9), mRNA-injection IVM (n = 13) and siRNA-injection IVM (n = 6) groups were used to generate embryonic stem cells (ESCs). In addition, 587 IVO-MII and 1737 IVM-MII oocytes from 83 mice were collected to compare the preliminary human oocyte data with another species. mRNA abundance was analysed by single-cell real-time PCR. Karyotyping of human embryos was performed with an array comparative genomic hybridization method, and that of ESCs by cytogenetic analysis. The function of the Sirt3 gene was investigated using siRNA and in vitro transcribed mRNA injection. Markers of ESCs were identified using immunofluorescence. A retrospective analysis revealed a higher spontaneous abortion rate (P < 0.01) and decrease in high quality embryo rate (P < 0.01) in patients with IVM versus controlled ovarian stimulation (COS) cycles. A decrease in abundance of Sirt3 mRNA (P < 0.01) and mitochondrial biogenesis (P < 0.05) were identified in human IVM compared with IVO oocytes. The developmental potential of human IVM-MII oocytes to the blastocyst stage was significantly reduced when Sirt3 mRNA was inhibited by siRNA (P < 0.05 versus IVM-MII group) but could be up-regulated by injection of Sirt3 mRNAs. Compared with IVO-MII group, comparable generation efficiency of human ESCs can be obtained using blastocysts from IVM-MII oocytes with Sirt3 mRNA injection. Sirt3 mRNA was significantly increased in mouse zygotes after IVF (P < 0.001 versus MII oocytes) but gradually declined until the blastocyst stage. In mice, lower Sirt3 mRNA levels were observed IVM-MII oocytes and preimplantation embryos compared with in vivo controls, and mitochondrial biogenesis and the developmental efficiency from oocytes to blastocyst were affected by the abundance of Sirt3 mRNA in accordance with human. Therefore a similar role for Sirt3 mRNA in IVM-MII oocytes was observed in mouse and human. The couples in the study had a variety of different simple and complex factors causing infertility. Additional studies with a larger number of oocytes are required to confirm the present results owing to the limited number of human oocytes in the present study. To our knowledge, this is the first study investigating a role of the Sirt3 gene in mitochondrial biogenesis and the developmental competence of human IVM-MII oocytes. The observation may help to improve clinical application of the IVM procedure. This work was supported in part by the National Natural Science Foundation of Key Program (31230047), Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Foundation of General Program (31371521 and 81571400), Beijing Nova Program (xxjh2015011), and Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and the National Natural Science Foundation of Young Scholar (31501201). The authors have declared that no conflict of interest exists.

Angelman-Like Syndrome: A Genetic Approach to Diagnosis with Illustrative Cases.

Epigenetic abnormalities in 15q11-13 imprinted region and UBE3A mutation are the two major mechanisms for molecularly confirmed Angelman Syndrome. However, there is 10% of clinically diagnosed Angelman Syndrome remaining test negative. With the advancement of genomic technology like array comparative genomic hybridization and next generation sequencing methods, it is found that some patients of these test negative Angelman-like Syndromes actually have alternative diagnoses. Accurate molecular diagnosis is paramount for genetic counseling and subsequent management. Despite overlapping phenotypes between Angelman and Angelman-like Syndrome, there are some subtle but distinct features which could differentiate them clinically. It would provide important clue during the diagnostic process for clinicians.

Abstract 4861: Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes

Abstract Background: Next generation sequencing (NGS) has revealed that the genetic profiles of individual tumors are highly heterogeneous due to their subclonal composition. Tumor heterogeneity has profound clinical implications affecting differences in treatment response and therapeutic resistance. Methods: An estrogen receptor positive, Her2 normal breast cancer patient underwent definitive surgical treatment with modified radical mastectomy. Multiple samples were obtained from the primary cancer and metastatic lymph nodes. Flow cytometry based methods were used to isolate and classify distinct cell subpopulations according to their ploidy, and samples were processed for whole exome sequencing (WES). Comparative Genomic Hybridization (CGH) methods were used to identify somatic unique copy-number alterations (CNAs). WES-Single Nucleotide Variations (SNVs) were used to infer subclonal phylogenetic relationships using Maximum Parsimony methods and annotated with variation-cluster-barcodes (vc-barcodes) generated using a variation Bayesian mixture model (VBMM) approach that focuses on copy-number neutral sequence variations for subclone identification (SciClone). Our comparative phylogenetic-VBMM clustering method was used to identify CNA and/or SNV drivers that are key in differentiating subclonal populations at multiple tumor sites. Results: CNA-neutral WES-SNVs from 25 subclonal populations (isolated from 4 distinct sites) clustered into 6 major groups and were used to generate vc-barcodes. A phylogenetic reconstruction of subclonal architecture annotated with vc-barcode information expedited identification of key variations underlying the differentiation of subclones at distinct tumor sites. For example, we identified an amplification event involving the Anaplastic Lymphoma Kinase (ALK) gene that was more frequent in primary biopsy subclones (12/17, ∼71%) in comparison to metastatic subclones (2/6, ∼34%). As the ALK amplification is lost, we find that most of the metastatic subclones also acquire a predicted damaging mutation in this oncogene (4/6, ∼67%). In addition, we identified a potential driver metastatic cell lineage that carries the ALK amplification in the absence of the nonsynonymous mutation. Conclusions: We developed a hybrid clustering methodology and used it to reconstruct the subclonal architecture in primary and metastatic tumors from a single breast cancer patient. Our methods have identified several CNAs and/or SNVs underlying subclonal differentiation, as well as potential biomarkers of disease progression and indicators of emerging resistance. Application of these methods to larger cohorts and types of tumors should be conducted to ascertain more precise estimates of the predictive accuracy of our processes. Citation Format: Mia D. Champion, Princy Francis, Barbara A. Pockaj, Michael T. Barrett. Hybrid clustering methodologies to distinguish CNAs and/or SNVs that drive subclonal differentiation in samples from a breast cancer patient primary tumor and metastatic lymph nodes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4861. doi:10.1158/1538-7445.AM2015-4861

Identification of Sex Chromosomes by Means of Comparative Genomic Hybridization in a Lizard, Eremias multiocellata.

Eremias multiocellata is a viviparous lizard that is known to exhibit temperature-dependent sex determination (TSD). Conventional Giemsa staining under light microscope examination has identified the karyotype of this species to be 2 n=36 I+2 m, with no detectable heteromorphic sex chromosomes. However, a highly differentiated female-specific chromosome, W, which is homomorphic with the Z chromosome, is found in the present study by the high-resolution cytogenetic method of comparative genomic hybridization (CGH). The results show that E. multiocellata is a viviparous lizard with both TSD and ZW heterogametic sex chromosomes. Despite the fact that a different sex ratio of male offspring was found in two populations (separated by an altitude of 1400 m) in previous incubation experiments, we demonstrate, using genomic in situ hybridization (GISH), that there is no significant chromosomal loss or acquisition between the two populations. This suggests that temperature may play a more important role in lowland populations. These results most likely indicate that E. multiocellata is transitioning between the evolutionary processes of TSD and genotypic sex determination (GSD) systems, and also give clues to the effect of TSD versus GSD in this process.

In This Issue

see State-based surveillance for selected hemoglobinopathies Despite widespread newborn screening in the United States for sickle cell disease and thalassemia, there is no national system to monitor the health outcomes of individuals diagnosed with these disorders. To gain a better understanding of prevalence and treatment outcomes, the National Institutes of Health and the Centers for Disease Control and Prevention collaborated on a pilot surveillance system in six states, beginning in 2004. The Registry and Surveillance System for Hemoglobinopathies (RuSH) identified individuals using multiple data sources, including newborn screening records, emergency room records, and Medicaid claims. Researchers collected demographic data, clinical characteristics, and health care–utilization records. Although the program was designed to produce a standardized system, difficulties with access to records in some states made a standardized approach untenable. But it did become clear that creating a comprehensive picture required combining data from many sources because information for individuals found through one data source was not present in others. In addition, the various sources contained different types of information. The research team concluded that combining all these data yielded a more comprehensive picture of the number of individuals living with hemoglobinopathies, as well as how and where they receive health care. A follow-up project—the Public Health Research Epidemiology and Surveillance for Hemoglobinopathies (PHRESH), now being conducted in two of the RuSH states (California and Georgia)—is expected to validate and refine the data collected in RuSH. see Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing Copy-number variants (CNVs)—structural changes that result in deletion or duplication of chromosomal segments—are a significant contributor to inherited genetic disease. Currently, clinical testing for CNVs involves exon–targeted array comparative genomic hybridization or other methods involving stepwise probing of genomic regions of interest. But molecular diagnosis of human genetic diseases in clinical settings is rapidly moving toward massively parallel sequencing (MPS) technology. The ability to identify both single-nucleotide alterations and larger deletions and duplications using the same technology would greatly simplify and streamline the clinical workflow. Now, a team from Baylor College of Medicine, Houston, Texas, reports that it has taken a step in that direction using a method that analyzes copy number by comparing the number of sequencing reads of each exon of interest. Using previously collected clinical samples, the investigators correctly identified 11 samples with deletions. However, identification of duplications proved more problematic, with high false-positive rates. Similar to previous reports using sequence data, false-positive findings typically arose from areas of highly repetitive GC sequences. The authors concluded that, with further refinements to the methodology, point mutations and exonic deletions can be detected reliably using MPS.

MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

BackgroundEpithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis.MethodsWe identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach.Results17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3′-untranslated region of PTEN mRNA using a dual-luciferase reporter assay.ConclusionsMiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-799) contains supplementary material, which is available to authorized users.

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene, gene panel, or exome/genome sequencing.

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing-based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.

Preliminary Results on Using Array Comparative Genomic Hybridization of Colorectal Cancer Tissues

Recent advances in cytogenetics have provided an opportunity for improvement in the measurement of aneuploidy by comparative genomic hybridization methods. In this study, chromosomal number variation(CNV) detection for 12 human colorectal cancer DNA samples was tried. Total 82 CNV was found, 19 gains and 63 losses, which including 326 chromosomal number variation regions. These results lead us to future studies that will be required to ascertain the meaning of gains and losses of chromosomes.

Bayesian disease classification using copy number data.

DNA copy number variations (CNVs) have been shown to be associated with cancer development and progression. The detection of these CNVs has the potential to impact the basic knowledge and treatment of many types of cancers, and can play a role in the discovery and development of molecular-based personalized cancer therapies. One of the most common types of high-resolution chromosomal microarrays is array-based comparative genomic hybridization (aCGH) methods that assay DNA CNVs across the whole genomic landscape in a single experiment. In this article we propose methods to use aCGH profiles to predict disease states. We employ a Bayesian classification model and treat disease states as outcome, and aCGH profiles as covariates in order to identify significant regions of the genome associated with disease subclasses. We propose a principled two-stage method where we first make inferences on the underlying copy number states associated with the aCGH emissions based on hidden Markov model (HMM) formulations to account for serial dependencies in neighboring probes. Subsequently, we infer associations with disease outcomes, conditional on the copy number states, using Bayesian linear variable selection procedures. The selected probes and their effects are parameters that are useful for predicting the disease categories of any additional individuals on the basis of their aCGH profiles. Using simulated datasets, we investigate the method’s accuracy in detecting disease category. Our methodology is motivated by and applied to a breast cancer dataset consisting of aCGH profiles assayed on patients from multiple disease subtypes.

Chronic lymphocytic leukemia in a child: A challenging diagnosis in pediatric oncology practice

Chronic lymphocytic leukemia/lymphoma (CLL) is an extremely rare disease during childhood. We report a 16-year-old female who presented with lymphadenopathies and she was diagnosed as T cell lymphoblastic lymphoma. Her chemotherapy response was minimal and clinical findings were unusual. Therefore, her biopsy specimen was re-examined and diagnosis was changed to CLL. Chemotherapy protocol including fludarabine, cyclophosphamide, rituximab was administrated and good response was observed. In our patient deletion at 1q21.2 region that includes aryl hydrocarbon receptor nuclear translocator (ARNT) gene was detected via comparative genomic hybridization method. ARNT gene deletion may be a new mutation in chronic lymphocytic leukemia development.