Microarray-based Comparative Genomic Hybridization Research Articles

Abstract 3829: Genomic index is a strong predictor of metastatic outcome in intermediate gastrointestinal stromal tumors and an inclusion criteria for imatinib adjuvant therapy

Abstract Gastrointestinal stroma tumors (GISTs) harboring a cKIT / PDGFRA activating mutation in 90% of cases benefit from the Imatinib mesylate (Glivec®) targeted therapy. Patients eligibility to imatinib adjuvant therapy is based, in Europe, on histological grading of tumor aggressiveness and no standard is currently available for tumor classified as intermediate risk (40% of patients). We recently validated that genomic index (GI), a measure of the number and type of genomic copy number alterations, constitutes a strong predictor of clinical outcome in GIST, making it a possible prognostic factor for the intermediate GIST subgroup. To definitively clarify whether this genomic grading system would permit to categorize intermediate-risk patients into good and poor prognosis, we selected a cohort of 82 intermediate patients based on the Armed Forces Institute of Pathology (AFIP) classification and performed genomic profiling from formalin-fixed, paraffin-embedded (FFPE) samples using a microarray-based comparative genomic hybridization (array CGH) approach. Data revealed that even if studied samples generally harbored a combination of the typical genetic aberrations found in GIST, i.e. 1p, 14q, 22q deletions and frequently lost CDKN2A locus on chromosome 9, they profoundly differed from each other on the total number of genomic changes and GI value, ranging, in the whole series, from 0 to 37 and 0 to 115.6, respectively. More interestingly, Kaplan-Meier analyses of metastatic-free survival unveiled that stratification of the tumors by the GI value at a cutoff of 10 (GI1<10 and GI2>10) separated the good (GI1) from the poor (GI2) prognosis patients (p<10-3), undoubtedly proven that metastatic-risk in GIST intermediate patients is strongly associated with high GI values and genome complexity. In conclusion, GI is validated here as a robust marker to predict the clinical outcome of patients diagnosed with GIST and classified as intermediate by the current histologic method for risk assessment. Applicable in numerous Pathology Laboratories already using aCGH with FFPE samples for routine diagnostic tests, this assay currently stands as the best tool to determine which intermediate GIST-patients are likely to benefit from imatinib therapy and constitutes as such a novel advance in the clinical management of GISTs. Citation Format: Lydia Lartigue, Pauline Lagarde, Céline Brulard, Agnès Neuville, Piotr Rutkowski, Paolo Dei Tos, Eva Waldermann, Maria Debiec-Rychter, Antoine Italiano, Jean-Michel Coindre, Frédéric Chibon. Genomic index is a strong predictor of metastatic outcome in intermediate gastrointestinal stromal tumors and an inclusion criteria for imatinib adjuvant therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2014-3829

Abstract 5176: Implications of clonality relationship and CD133 expression between cancer synchronous/preceding adenomas and their colorectal adenocarcinoma pairs

Abstract Background: It is thought that each cell only generates a unifocal lineage that is independent from one another in colorectal adenoma-carcinoma progression. This study compared the genome-wide copy number aberration profiles of adenoma and primary colorectal carcinomas (CRCs) derived from the same patient to trace their clonality relationship. The CD133 positive cells, putative colorectal cancer stem cells, are capable of tumor initiation and metastasis. Therefore, we also investigated the use of CD133 expression as a potential marker for prediction of subsequent risk of colorectal cancer. Methods: Microarray-based comparative genomic hybridization (aCGH) experiments were performed on 18 cancer-synchronous polyps (CSP) and 9 cancer-preceding polyps (CPP), together with their corresponding cancers, and 16 cases of incidental polyps (IP). Genome-wide copy number imbalances revealed by aCGH were analyzed to determine the clonal relationship between the paired adenomas and carcinomas. CD133 expression in cancerous tissue was evaluated by immunohistochemistry (IHC) staining. Results: There were more CGH lesions in CRC than CSP/CPP, and least in IP. Clonal relationship determined by tissues' aCGH profiles showed that 50 percent of CSP and 67 percent of CPP were clonally related to the concurrent or subsequent cancers, respectively. The expression level of stem cell marker CD133 was significantly higher in CSP/CPP than in IP (P<0.0001), and even higher CD133 staining were observed in CSP/CPP that were clonally related to the corresponding carcinomas than CSP/CPP that were unrelated (P<0.05). Conclusions: In the present study more than half of the polyp-carcinoma pairs share highly similar pattern of numerical chromosomal aberrations implying that the pairs might descent from the same monoclonal cell origin. The high expression of stem cell marker CD133 in colorectal adenomas is predictive of high cancer risk for polyp-bearing patients. Citation Format: Chih-Yung Yang, Ju-Yu Tseng, Chi-Hung Lin. Implications of clonality relationship and CD133 expression between cancer synchronous/preceding adenomas and their colorectal adenocarcinoma pairs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5176. doi:10.1158/1538-7445.AM2014-5176

Microarray-based comparative genomic hybridization analysis in neonates with congenital anomalies: detection of chromosomal imbalances

ObjectiveTo identify chromosomal imbalances by whole-genome microarray-based comparative genomic hybridization (array-CGH) in DNA samples of neonates with congenital anomalies of unknown cause from a birth defects monitoring program at a public maternity hospital. MethodsA blind genomic analysis was performed retrospectively in 35 stored DNA samples of neonates born between July of 2011 and December of 2012. All potential DNA copy number variations detected (CNVs) were matched with those reported in public genomic databases, and their clinical significance was evaluated. ResultsOut of a total of 35 samples tested, 13 genomic imbalances were detected in 12/35 cases (34.3%). In 4/35 cases (11.4%), chromosomal imbalances could be defined as pathogenic; in 5/35 (14.3%) cases, DNA CNVs of uncertain clinical significance were identified; and in 4/35 cases (11.4%), normal variants were detected. Among the four cases with results considered causally related to the clinical findings, two of the four (50%) showed causative alterations already associated with well-defined microdeletion syndromes. In two of the four samples (50%), the chromosomal imbalances found, although predicted as pathogenic, had not been previously associated with recognized clinical entities. ConclusionsArray-CGH analysis allowed for a higher rate of detection of chromosomal anomalies, and this determination is especially valuable in neonates with congenital anomalies of unknown etiology, or in cases in which karyotype results cannot be obtained. Moreover, although the interpretation of the results must be refined, this method is a robust and precise tool that can be used in the first-line investigation of congenital anomalies, and should be considered for prospective/retrospective analyses of DNA samples by birth defect monitoring programs.

Our experience with the aetiological diagnosis of global developmental delay and intellectual disability: 2006–2010

IntroductionGlobal developmental delay (GDD) and intellectual disability (ID) are common reasons for consultation in paediatric neurology. Results from aetiological evaluations of children with GDD/ID vary greatly, and consequently, there is no universal consensus regarding which studies should be performed. Material and methodWe review our experience with determining aetiological diagnoses for children with GDD/ID who were monitored by the paediatric neurology unit over the 5-year period between 2006 and 2010. ResultsDuring the study period, 995 children with GDD/ID were monitored. An aetiological diagnosis was established for 309 patients (31%), but not in 686 (69%), despite completing numerous tests. A genetic cause was identified in 142 cases (46% of the total aetiologies established), broken down as 118 cases of genetic encephalopathy and 24 of metabolic hereditary diseases. Our data seem to indicate that diagnosis is easier when GDD/ID is associated with cerebral palsy, epilepsy, infantile spasms/West syndrome, or visual deficit, but more difficult in cases of autism spectrum disorders. Genetic studies provide an increasing number of aetiological diagnoses, and they are also becoming the first step in diagnostic studies. Array CGH (microarray-based comparative genomic hybridisation) is the genetic test with the highest diagnostic yield in children with unexplained GDD/ID. DiscussionThe cost-effectiveness of complementary studies seems to be low if there are no clinically suspected entities. However, even in the absence of treatment, aetiological diagnosis is always important in order to provide genetic counselling and possible prenatal diagnosis, resolve family (and doctors’) queries, and halt further diagnostic studies.

The genetic basis for inactivation of Wnt pathway in human osteosarcoma.

BackgroundOsteosarcoma is a highly genetically unstable tumor with poor prognosis. We performed microarray-based comparative genomic hybridization (aCGH), transcriptome sequencing (RNA-seq), and pathway analysis to gain a systemic view of the pathway alterations of osteosarcoma.MethodsaCGH experiments were carried out on 10 fresh osteosarcoma samples. The output data (Gene Expression Omnibus Series accession number GSE19180) were pooled with published aCGH raw data (GSE9654) to determine recurrent copy number changes. These were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify altered pathways in osteosarcoma. Transcriptome sequencing of six osteosarcomas was performed to detect the expression profile of Wnt signaling pathway genes. Protein expression of WNT1, β-catenin, c-myc, and cyclin D1 in the Wnt pathway was detected by immunohistochemistry (IHC) in an independent group of 46 osteosarcoma samples.ResultsKEGG pathway analysis identified frequent deletions of Wnt and other Wnt signaling pathway genes. At the mRNA level, transcriptome sequencing found reduced levels of mRNA expression of Wnt signaling pathway transcripts. While WNT1 protein expression was detected by IHC in 69.6% (32/46) of the osteosarcomas, no β-catenin protein was detected in the nucleus. β-catenin protein expression was, however, detected in the membrane and cytoplasm of 69.6% (32/46) of the osteosarcomas. c-myc protein expression was detected in only 47.8% (22/46) and cyclin D1 protein expression in 52.2% (24/46) of osteosarcoma samples. Kaplan-Meier survival analysis showed that WNT1-negative patients had a trend towards longer disease free survival than WNT1-positive patients. Interestingly, in WNT1-negative patients, those who were also cyclin D1-negative had significantly longer disease free survival than cyclin D1-positive patients. However, there was no significant association between any of the investigated proteins and overall survival of human osteosarcoma patients.ConclusionsFrequent deletions of Wnt and other Wnt signaling pathway genes suggest that the Wnt signaling pathway is genetically inactivated in human osteosarcoma.

New candidate loci identified by array-CGH in a cohort of 100 children presenting with syndromic obesity.

Syndromic obesity is defined by the association of obesity with one or more feature(s) including developmental delay, dysmorphic traits, and/or congenital malformations. Over 25 syndromic forms of obesity have been identified. However, most cases remain of unknown etiology. The aim of this study was to identify new candidate loci associated with syndromic obesity to find new candidate genes and to better understand molecular mechanisms involved in this pathology. We performed oligonucleotide microarray-based comparative genomic hybridization in a cohort of 100 children presenting with syndromic obesity of unknown etiology, after exhaustive clinical, biological, and molecular studies. Chromosomal copy number variations were detected in 42% of the children in our cohort, with 23% of patients with potentially pathogenic copy number variants. Our results support that chromosomal rearrangements are frequently associated with syndromic obesity with a variety of contributory genes having relevance to either obesity or developmental delay. A list of inherited or apparently de novo duplications and deletions including their enclosed genes and not previously linked to syndromic obesity was established. Proteins encoded by several of these genes are involved in lipid metabolism (ACOXL, MSMO1, MVD, and PDZK1) linked with nervous system function (BDH1 and LINGO2), neutral lipid storage (PLIN2), energy homeostasis and metabolic processes (CDH13, CNTNAP2, CPPED1, NDUFA4, PTGS2, and SOCS6).

Array-comparative genomic hybridization profiling of immunohistochemical subgroups of diffuse large B-cell lymphoma shows distinct genomic alterations.

Diffuse large B-cell lymphoma (DLBCL) displays striking heterogeneity at the clinical, genetic and molecular levels. Subtypes include germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL, according to microarray analysis, and germinal center type or non-germinal center type by immunohistochemistry. Although some reports have described genomic aberrations based upon microarray classification system, genomic aberrations based upon immunohistochemical classifications have rarely been reported. The present study aimed to ascertain the relationship between genomic aberrations and subtypes identified by immunohistochemistry, and to study the pathogenetic character of Chinese DLBCL. We conducted immunohistochemistry using antibodies against CD10, BCL6 and MUM1 in 59 samples of DLBCL from Chinese patients, and then performed microarray-based comparative genomic hybridization for each case. Characteristic genomic differences were found between GCB and non-GCB DLBCL from the array data. The GCB type was characterized by more gains at 7q (7q22.1, P < 0.05) and losses at 16q (P ≤ 0.05), while the non-GCB type was characterized by gains at 11q24.3 and 3q13.2 (P < 0.05). We found completely different mutations in BCL6+ and BCL6− non-GCB type DLBCL, whereby the BCL6− group had a higher number of gains at 1q and a loss at 14q32.13 (P ≤ 0.005), while the BCL6+ group showed a higher number of gains at 14q23.1 (P = 0.15) and losses at 6q (P = 0.07). The BCL6− group had a higher frequency of genomic imbalances compared to the BCL6+ group. In conclusion, the BCL6+ and BCL6− non-GCB type of DLBCL appear to have different mechanisms of pathogenesis.

Microarray-based ultra-high resolution discovery of genomic deletion mutations

BackgroundOligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low.ResultsHere we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size.ConclusionsOur findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence.

Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast

Micropapillary carcinoma (MPC) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations (CNAs) distinct from that of grade- and oestrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray-based comparative genomic hybridization (aCGH) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs. Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC-NSTs, and recurrent mutations affecting mitogen-activated protein kinase family genes and NBPF10. RNA-sequencing analysis identified 17 high-confidence fusion genes, eight of which were validated and two of which were in-frame. No recurrent fusions were identified in an independent series of MPCs and IC-NSTs. Forced expression of in-frame fusion genes (SLC2A1–FAF1 and BCAS4–AURKA) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out-of-frame rearrangements was found in one MPC and in 13% of HER2-positive breast cancers, identified through a re-analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild-type CDK12 in a CDK12-null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities.

Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis

BackgroundThe MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism.ResultsA range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines.ConclusionsOur study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.

Insights into Common Microdeletion Syndromes

Microdeletion syndromes are due to submicroscopic chromosomal deletions and display a complex clinical and behavioral phenotype. This occurs because of an imbalance of normal dosage of genes that are present in that segment of chromosome. Many clinical characteristics of the well-known microdeletion syndromes are very specific and have been well defined. It is not always possible to detect these microdeletions by using the conventional or high resolution karyotyping. This necessitates the application of molecular cytogenetic techniques and the recent widespread usage and an in-depth knowledge of methods such as G-band karyotyping, multiples ligation-dependent probe amplification (MLPA), quantitative fluorescent PCR (QFPCR), sequencing and microarray-based comparative genomic hybridization (array CGH) had further led to identification of new microdeletions and sound description of identification of new microdeletion syndromes. This review appraises the common microdeletion syndromes focusing on the incidence rate, clinical manifestations and the mechanism involved therein.

Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

Head circumference alone at birth, is it practical?

Head circumference alone at birth, is it practical?

BACs-on-Beads technology: a reliable test for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples.

Use of array CGH for molecular characterization of genetic disorders

Background Microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that has been developed to screen entire genome for copy number variations (CNV) with resolution beyond the capacity of light microscope. ACMG has recommended that array CGH can be used as the first line investigation modality in cases of non-syndromic mental retardation. We present here three cases in which use of array CGH has provided an insight of genetic abnormality.

A Large Novel Deletion Downstream of PAX6 Gene in a Chinese Family with Ocular Coloboma

PurposeThe paired box gene 6 (PAX6) is an essential transcription factor for eye formation. Genetic alterations in PAX6 can lead to various ocular malformations including aniridia. The purpose of this study was to identify genetic defects as the underlying cause of familial ocular coloboma in a large Chinese family.MethodsAfter linkage analysis was carried out in this family, all exons of PAX6 in the proband were sequenced by the Sanger sequencing technique. Then the genome of the proband was evaluated by a microarray-based comparative genomic hybridization (aCGH). Quantitative real-time PCR was applied to verify the abnormal aCGH findings.ResultsAll patients presented bilateral partial coloboma of iris, severe congenital nystagmus, hyperpresbyopia and congenital posterior polar cataracts. Two-point linkage analysis in the autosomal dominant family showed loss of heterozygosity at the D11S914 locus. There was no pathogenic mutation in the exons of PAX6. The aCGH analysis revealed a 681 kb heterozygous deletion on chromosome 11p13. Quantitative real-time PCR verified the deletion in the patients and further confirmed this deletion cosegregation with the ocular coloboma phenotype in the family.ConclusionsThe 681 kb large deletion of chromosome 11p13 downstream of PAX6 is the genetic cause of the familial ocular coloboma in this large Chinese family. aCGH should be applied if there is a negative result for the mutation detection of PAX6 in patients with ocular coloboma.

Nuestra experiencia en el diagnóstico etiológico del retraso global del desarrollo y discapacidad intelectual: 2006-2010

IntroductionGlobal developmental delay (GDD) and intellectual disability (ID) are common reasons for consultation in paediatric neurology. Results from aetiological evaluations of children with GDD/ID vary greatly, and consequently, there is no universal consensus regarding which studies should be performed. Material and methodWe review our experience with determining aetiological diagnoses for children with GDD/ID who were monitored by the paediatric neurology unit over the 5-year period between 2006 and 2010. ResultsDuring the study period, 995 children with GDD/ID were monitored. An aetiological diagnosis was established for 309 patients (31%), but not in 686 (69%), despite completing numerous tests. A genetic cause was identified in 142 cases (46% of the total aetiologies established), broken down as 118 cases of genetic encephalopathy and 24 of metabolic hereditary diseases. Our data seem to indicate that diagnosis is easier when GDD/ID is associated with cerebral palsy, epilepsy, infantile spasms/West syndrome, or visual deficit, but more difficult in cases of autism spectrum disorders. Genetic studies provide an increasing number of aetiological diagnoses, and they are also becoming the first step in diagnostic studies. Array CGH (microarray-based comparative genomic hybridisation) is the genetic test with the highest diagnostic yield in children with unexplained GDD/ID. DiscussionThe cost-effectiveness of complementary studies seems to be low if there are no clinically suspected entities. However, even in the absence of treatment, aetiological diagnosis is always important in order to provide genetic counselling and possible prenatal diagnosis, resolve family (and doctors’) queries, and halt further diagnostic studies.

Different loss of material in recurrent chromosome 20 interstitial deletions in Shwachman-Diamond syndrome and in myeloid neoplasms

BackgroundAn interstitial deletion of the long arms of chromosome 20, del(20)(q), is frequent in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN), and it is recurrent in the BM of patients with Shwachman-Diamond syndrome (SDS), who have a 30-40% risk of developing MDS and AML.ResultsWe report the results obtained by microarray-based comparative genomic hybridization (a-CGH) in six patients with SDS, and we compare the loss of chromosome 20 material with one patient with MDS, and with data on 92 informative patients with MDS/AML/MPN and del(20)(q) collected from the literature.ConclusionsThe chromosome material lost in MDS/AML/MPN is highly variable with no identifiable common deleted regions, whereas in SDS the loss is more uniform: in 3/6 patients it was almost identical, and the breakpoints that we defined are probably common to most patients from the literature. In some SDS patients less material may be lost, due to different distal breakpoints, but the proximal breakpoint is in the same region, always leading to the loss of the EIF6 gene, an event which was related to a lower risk of MDS/AML in comparison with other patients.

Pre-Descemet Corneal Dystrophy and X-Linked Ichthyosis Associated With Deletion of Xp22.31 Containing the STS Gene

To report the association of X-linked ichthyosis and pre-Descemet corneal dystrophy with a deletion of the steroid sulfatase gene (STS) detected with microarray-based comparative genomic hybridization (aCGH). A slit-lamp biomicroscopic examination and cutaneous examination were performed, after which a saliva sample was collected as a source of genomic DNA. Polymerase chain reaction amplification of each of the 10 exons of STS was performed, as was aCGH on genomic DNA to detect copy number variation. The slit-lamp examination revealed punctate opacities in the posterior corneal stroma of each eye. The cutaneous examination demonstrated scaling and flaking skin of the arms and legs. Polymerase chain reaction amplification using primers designed to amplify each of the 10 exons of STS failed to produce any amplicons. Subsequently, aCGH performed on genomic DNA revealed a microdeletion in the Xp22.31 cytoband of approximately 1.7 megabases, containing STS. The identification of a microdeletion within Xp22.3 containing STS with aCGH in an individual with suspected pre-Descemet corneal dystrophy and X-linked ichthyosis demonstrates the clinical utility of copy number variation analysis in confirming a presumptive clinical diagnosis.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development.