Cytogenetic Microarray Analysis Research Articles

Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing

Unlabelled Box BackgroundNo F8 genetic abnormality is detected in about 2% of severe hemophilia A patients using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. ObjectiveTo characterize, in a genetically unresolved severe hemophilia A patient, a new Xq28 rearrangement disrupting F8 using comprehensive molecular techniques including nanopore sequencing. ResultsLong‐range polymerase chain reaction (PCR) performed throughout F8 identified a nonamplifiable region in intron 25 indicating the presence of a genomic rearrangement. F8messanger ribonucleic acid (mRNA) analysis including 3′rapid amplification of complementary deoxyribonucleic acid (cDNA) ends and nanopore sequencing found the presence of a F8 fusion transcript in which F8 exon 26 was replaced by a 742‐bp pseudoexon corresponding to a noncoding region located at the beginning of the long arm of chromosome X (Xq12; chrX: 66 310 352‐66 311 093, GRCh37/hg19). Cytogenetic microarray analysis found the presence of a Xq11.1q12 gain of 3.8 Mb. The PCR amplification of junction fragments and fluorescent in situ hybridization (FISH) analysis found that the Xq11q12 duplicated region was inserted in the F8 intron 25 genomic region. ConclusionWe characterized a novel genomic rearrangement in which a 3.8‐Mb Xq11.1q12 gain inserted in the F8 intron 25 led to an aberrant fusion transcript in a patient with severe hemophilia A (HA), using comprehensive molecular techniques. This study highlights the value of single‐molecule long‐read sequencing technologies for molecular diagnosis of HA especially when conventional genetic approaches have failed.

Detecting Clonal Evolution in CLL Patients Treated with Targeted Therapies Versus Chemotherapy Utilizing Cytogenetic Microarray Analysis

BackgroundChronic lymphocytic leukemia (CLL) is a common B-cell lymphoproliferative neoplasm. Although not required for the diagnosis of CLL, important prognostic information is obtained with fluorescence in-situ hybridization (FISH) testing for chromosomal aberrations including del(11q), trisomy 12, del(13q), and del(17p). Abnormalities involving TP53, the ATM gene, and cases constituting complex cytogenetics (3 or more abnormalities), among others, have been shown to impact patient survival and response to traditional chemoimmunotherapy (CIT). Cytogenetic microarray analysis (CMA) is a laboratory technique used to detect unbalanced chromosomal abnormalities. CMA allows for comparison of hundreds of discrete loci within a chromosome and has been validated in CLL, with greater sensitivity in detecting genomic alterations compared to FISH. The acquisition of new genomic alterations is termed “clonal evolution” and has been shown to have a detrimental effect on survival in patients with CLL (Shanafelt TD, et al. J Clin Oncol. 2006). CIT results in a higher proportion of complete responses (CR) and eradication of CLL clones compared to targeted therapies. While targeted therapies may successfully suppress CLL clones, a CR is rare with single-agent treatment (Byrd JC, et al. Blood. 2015). CMA may be a valuable tool in monitoring clonal evolution throughout a patient's disease course and may have a role in evaluation of patients on suppressive therapies due to the relatively high risk of clonal emergence.MethodsThis was a single-center retrospective study. Patients with pathologically-confirmed CLL were analyzed utilizing data obtained from the electronic medical record. Data was extracted from the CMA reports and clinic documentation. To be included in the study, at least two serial CMA evaluations were required, separated by at least four weeks. In the interval between CMA results, subsequent systemic treatment was recorded, if any. Treatments were categorized as ibrutinib/idelalisib (“I”), any cytotoxic chemotherapy (“C”), and no treatment/observation (“N”). Change in the number of abnormalities detected by CMA was calculated for each interval and evaluated as both continuous and categorical (increase/no change/decrease). Patients with over three abnormalities were deemed to have complex disease.Genomic DNA of bone marrow or peripheral blood was extracted using Qiagen's Gentra Puregene kit. Chromosome microarray assay was performed using ThermoFisher Affymetrix CytoScan HD according to the manufacturer's protocol. Copy number and allele analysis was performed using Affymetrix Chromosome Analysis Suite using the default settings.ResultsThere were 34 patients who met inclusion criteria, each with 2-4 CMA results, for a total of 81 CMA results. Forty-seven treatment intervals were analyzed. The median age was 61 years, and 64% of patients were male. Based on the first CMA result, the median number of detected abnormalities was 2 and mean was 2.6; 13 patients (38%) had complex disease. Complexity was not associated with a clinically significant change in the number of chromosomal abnormalities regardless of what treatment was received, if any (p=0.14).Overall, 18% of patients (6/34) had a decrease in the number of abnormalities detected by CMA, while 21% (7/34) had an increase. Analyzing the data by treatment group, the mean change in number of abnormalities was -0.5, -0.6 and +0.3, in groups I, C and N, respectively (p=0.26) (Figure 1). Among the patients in group I, 23% (3/13) had a decrease in the number of abnormalities, while 23% demonstrated an increase. For those in group C, 25% (2/8) and 25% experienced increase and decrease, respectively. For patients in group N, 14% (3/21) had an increase and 0% had a decrease in CMA abnormalities. Among the two patients who experienced Richter's transformation, the complexity of their chromosomal abnormalities increased over time and preceded clinical emergence of transformation.DiscussionCMA can sensitively identify the emergence or clearance of CLL clones and help detect clonal evolution throughout periods of treatment or observation. Though a relatively small number of patients had serial CMA data for evaluation, these data suggest that CMA should be prospectively analyzed in patients with CLL and may be used as a dynamic clinical tool to potentially modify treatments in patients with sub-clinical evidence of treatment failure. DisclosuresBarta:Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding.

Two siblings with 11qter deletion syndrome that had been rescued in their mother by uniparental disomy

Jacobsen syndrome refers to a congenital anomaly caused by deletion at 11q23.3-qter. We here describe two siblings with the same 11q23.3-qter deletion. Both parents were healthy with a normal karyotype. Cytogenetic microarray analysis revealed no mosaicism in either parent but the mother showed uniparental disomy encompassing the deleted region found in the two siblings. The pattern of X chromosome inactivation was almost completely skewed in the mother. These data suggested that the mother was a carrier of the 11q23.3-qter deletion but that this had been rescued by disomy formation during early embryogenesis except for her germinal cells.

Molecular cytogenetic characterization of five F8 complex rearrangements: utility for haemophilia A genetic counselling.

Genomic inversions are usually balanced, but unusual patterns have been described in haemophilia A (HA) patients for intron 22 (Inv22) and intron 1 (Inv1) inversions leading to the hypothesis of more complex rearrangements involving deletions or duplications. To characterize five abnormal patterns either in Southern blot and long-range PCR for Inv22 or in PCR for Inv1. All patients were studied using cytogenetic microarray analysis (CMA). In all cases, CMA analysis found that each inversion was associated with complex Xq28 rearrangement. In three patients, CMA analysis showed large duplication ranging from 230 to 1302 kb and encompassing a various number of contiguous genes among which RAB39B. RAB39B duplication is a strong candidate gene for X-linked intellectual disability (XLID). Surprisingly, none of the severe HA patients with RAB39B duplication reported in this study or in the literature exhibited XLID. We hypothesise that F8 complex rearrangement down regulated RAB39B expression. In the two remaining patients, CMA analysis found Xq28 large deletion (from 285 to 522 kb). Moyamoya syndrome was strongly suspected in one of them who carried BRCC3 deletion. Because several F8 neighbouring genes are associated with other pathologies such as XLID and cardiovascular disease, all HA patients where complex Xq28 rearrangement was suspected should be referred to a geneticist for possible utility of a pangenomic study. Such investigation should be carefully considered in genetic counselling in female carriers to assess the risk of transmitting severe HA with a "contiguous gene syndrome".

Type A Aortic Dissection, Apparent Mineralocorticoid Excess Syndrome, and Syndromic Aortic Root Dilatation

ABSTRACT Objective: To determine the cause of acute life-threatening aortic root dissection in a 36-year-old male with a long-standing history of apparent mineralocorticoid excess (AME) and hypertension since childhood. Methods: Cortisol metabolites were analyzed by urine tandem mass spectrometry. Screening for DNA variants was performed using the Marfan Syndrome and Related Aortopathies NextGen Sequencing Panel (Collagen Diagnostic Laboratory, University of Washington, Seattle). Cytogenetic microarray analysis (CombiMatrix, CA) was performed. Sequencing of the 11β-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) in a region of homozygosity (ROH) identified on microarray analysis was targeted using Sanger sequencing (Prevention Genetics) on an ABI 3730x1 capillary sequencer. Results: There was reduced urinary excretion of all 11-carboxyl containing steroids and high excretion of free cortisol and its metabolites, consistent with a diagnosis of AME. Microarray analysis revealed 4 ROHs. One ROH contained ...

Study of six patients with complete F9 deletion characterized by cytogenetic microarray: role of the SOX3 gene in intellectual disability

Background Large deletions encompassing both the complete F9 gene and contiguous genes have been detected in patients with severe hemophilia B (HB). Some of these patients present other clinical features, such as intellectual disability (ID). Objectives/Methods In this study, we characterized six unrelated large deletions encompassing F9, by cytogenetic microarray analysis (CMA), to investigate genotype/phenotype correlation. Results Five of the six patients included in this study presented with ID associated with HB. CMA showed that the six large deletions, ranging in size from approximately 933 kb to 9.19 Mb, were located within the Xq26.3 to Xq28 bands. In all cases, the complete deletion of F9 was associated with the loss of various neighboring genes (5-28 other genes). The smallest region of overlap for ID was a 1.26-Mb region encompassing seven OMIM genes (LOC389895, SOX3, LINC00632, CDR1, SPANXF1, LDOC1, SPANXC). SOX3, our candidate gene for ID, encodes an early transcription factor involved in pituitary development. All of the patients studied who had both HB and ID had deletion of the SOX3 gene. Conclusions All HB patients with an atypical phenotype, especially if complete deletion of F9 is suspected, should be referred to a geneticist for possible pangenomic assessment, because haploinsufficiency of genes flanking F9, such as SOX3 in particular, may result in a broader phenotype, including ID. Such assessment would be of particular value for the genetic counseling of female carriers with F9 deletions, as it would facilitate analysis of the risk of transmitting HB associated with ID.

A French Approach to Test Fetuses with Ultrasound Abnormalities Using a Customized Microarray as First-Tier Genetic Test

Cytogenetic microarray analysis is now the first-tier genetic test used in a postnatal clinical setting to explore genomic imbalances in individuals with developmental disability and/or birth defects. However, in a prenatal setting, this technique is not widely implemented, largely because the clinical impact of some copy number variants (CNVs) remains difficult to assess. This limitation is especially true in France where termination of pregnancy for medical reasons may be performed at any stage of gestation. During a period of 15 months, we investigated 382 fetuses presenting with ultrasound anomalies, using a customized microarray designed to avoid the detection of CNVs raising challenges for genetic counseling. After excluding common aneuploidies, 20/374 (5.3%) fetuses had a pathogenic CNV, among which 12/374 (3.2%) could have been detected by karyotyping, whereas 8/374 (2.1%) were cryptic. Within these 374 cases, 300 were ongoing pregnancies at the time of array comparative genomic hybridization (aCGH) testing. For these pregnancies, we detected 18/300 (6%) pathogenic CNVs, among which 6/300 (2%) were cryptic. Using this approach, only 2/300 (0.6%) of the detected CNVs raised difficulties for genetic counseling. This study confirms the added value of this strategy in a prenatal clinical setting to minimize ethical issues for genetic counseling while enhancing the detection of genomic imbalances.

Genomic alterations in abnormal neutrophils isolated from adult patients with systemic lupus erythematosus.

IntroductionPatients with systemic lupus erythematosus (SLE) have an abnormal population of neutrophils, called low-density granulocytes (LDGs), that express the surface markers of mature neutrophils, yet their nuclear morphology resembles an immature cell. Because a similar discrepancy in maturation status is observed in myelodysplasias, and disruption of neutrophil development is frequently associated with genomic alterations, genomic DNA isolated from autologous pairs of LDGs and normal-density neutrophils was compared for genomic changes.MethodsAlterations in copy number and losses of heterozygosity (LOH) were detected by cytogenetic microarray analysis. Microsatellite instability (MSI) was detected by capillary gel electrophoresis of fluorescently labeled PCR products.ResultsControl neutrophils and normal-density SLE neutrophils had similar levels of copy number variations, while the autologous SLE LDGs had an over twofold greater number of copy number alterations per genome. The additional copy number alterations found in LDGs were prevalent in six of the thirteen SLE patients, and occurred preferentially on chromosome 19, 17, 8, and X. These same SLE patients also displayed an increase in LOH. Several SLE patients had a common LOH on chromosome 5q that includes several cytokine genes and a DNA repair enzyme. In addition, three SLE patients displayed MSI. Two patients displayed MSI in greater than one marker, and one patient had MSI and increased copy number alterations. No correlations between genomic instability and immunosuppressive drugs, disease activity or disease manifestations were apparent.ConclusionsThe increased level of copy number alterations and LOH in the LDG samples relative to autologous normal-density SLE neutrophils suggests somatic alterations that are consistent with DNA strand break repair, while MSI suggests a replication error-prone status. Thus, the LDGs isolated have elevated levels of somatic alterations that are consistent with genetic damage or genomic instability. This suggests that the LDGs in adult SLE patients are derived from cell progenitors that are distinct from the autologous normal-density neutrophils, and may reflect a role for genomic instability in the disease.Electronic supplementary materialThe online version of this article (doi:10.1186/ar4681) contains supplementary material, which is available to authorized users.

Integration of microarray technology into prenatal diagnosis: counselling issues generated during the NICHD clinical trial

Cytogenetic microarray analysis (CMA) in prenatal testing detects chromosome abnormalities and new genetic syndromes that would be missed by conventional cytogenetics and has the potential to significantly enhance prenatal genetic evaluation. A large Eunice Kennedy Shriver National Institute Of Child Health and Human Development (NICHD)-sponsored multicentered trial to assess the role of CMA as a primary prenatal diagnostic tool has been completed, and results will soon be available. Integration of this technology into clinical care will require thoughtful changes in patient counseling. Here, we examine four cases, all ascertained in the NICHD prenatal microarray study, to illustrate the challenges and subtleties of genetic counseling required with prenatal CMA testing. Although the specifics of each case are distinct, the underlying genetic principles of uncertainty, variable expressivity, and lack of precise genotype-phenotype correlation are well known and already part of prenatal counseling. Counselor and practitioner education will need to include both the science of interpreting array findings as well as development of improved approaches to uncertainty. A team approach to interpretation will need to be developed, as will standardized guidelines by professional organizations and laboratories. Of equal import is additional research into patient attitudes and desires, and a better understanding of the full phenotypic spectrum of copy number variants discovered in utero.

A Novel 2.3 Mb Microduplication of 9q34.3 Inserted into 19q13.4 in a Patient with Learning Disabilities

Insertional translocations in which a duplicated region of one chromosome is inserted into another chromosome are very rare. We report a 16.5-year-old girl with a terminal duplication at 9q34.3 of paternal origin inserted into 19q13.4. Chromosomal analysis revealed the karyotype 46,XX,der(19)ins(19;9)(q13.4;q34.3q34.3)pat. Cytogenetic microarray analysis (CMA) identified a ~2.3Mb duplication of 9q34.3 → qter, which was confirmed by Fluorescence in situ hybridisation (FISH). The duplication at 9q34.3 is the smallest among the cases reported so far. The proband exhibits similar clinical features to those previously reported cases with larger duplication events.