High-density Comparative Genomic Hybridization Research Articles

Single-nucleotide polymorphisms and haplotypes of non-coding area in the CP gene are correlated with Parkinson's disease.

Our previous studies have demonstrated that ceruloplasmin (CP) dysmetabolism is correlated with Parkinson's disease (PD). However, the causes of decreased serum CP levels in PD patients remain to be clarified. This study aimed to explore the potential association between genetic variants of the CP gene and PD. Clinical features, serum CP levels, and the CP gene (both promoter and coding regions) were analyzed in 60 PD patients and 50 controls. A luciferase reporter system was used to investigate the function of promoter single-nucleotide polymorphisms (SNPs). High-density comparative genomic hybridization microarrays were also used to detect large-scale copy-number variations in CP and an additional 47 genes involved in PD and/or copper/iron metabolism. The frequencies of eight SNPs (one intronic SNP and seven promoter SNPs of the CP gene) and their haplotypes were significantly different between PD patients, especially those with lowered serum CP levels, and controls. However, the luciferase reporter system revealed no significant effect of the risk haplotype on promoter activity of the CP gene. Neither these SNPs nor their haplotypes were correlated with the Hoehn and Yahr staging of PD. The results of this study suggest that common genetic variants of CP are associated with PD and further investigation is needed to explore their functions in PD.

Four Copies of SNCA Responsible for Autosomal Dominant Parkinson's Disease in Two Italian Siblings.

Background. Parkinson's disease (PD) is mostly characterized by alpha-synuclein (SNCA) aggregation and loss of nigrostriatal dopamine-containing neurons. In this study a novel SNCA multiplication is described in two siblings affected by severe parkinsonism featuring early onset dyskinesia, psychiatric symptoms, and cognitive deterioration. Methods. SNCA dosage was performed using High-Density Comparative Genomic Hybridization Array (CGH-Array), Multiple Ligation Dependent Probe Amplification (MLPA), and Quantitative PCR (qPCR). Genetic analysis was associated with clinical evaluation. Results. Genetic analysis of siblings showed for the first time a 351 Kb triplication containing SNCA gene along with 6 exons of MMRN1 gene in 4q22.1 and a duplication of 1,29 Mb of a genomic region flanking the triplication. Conclusions. The identification of this family indicates a novel mechanism of SNCA gene multiplication, which confirms the genomic instability in this region and provides data on the genotype-phenotype correlation in PD patients.

Genome-wide analysis of copy number variation identifies candidate gene loci associated with the progression of non-alcoholic fatty liver disease.

Between 10 and 25% of individuals with non-alcoholic fatty liver disease (NAFLD) develop hepatic fibrosis leading to cirrhosis and hepatocellular carcinoma (HCC). To investigate the molecular basis of disease progression, we performed a genome-wide analysis of copy number variation (CNV) in a total of 49 patients with NAFLD [10 simple steatosis and 39 non-alcoholic steatohepatitis (NASH)] and 49 matched controls using high-density comparative genomic hybridization (CGH) microarrays. A total of 11 CNVs were found to be unique to individuals with simple steatosis, whilst 22 were common between simple steatosis and NASH, and 224 were unique to NASH. We postulated that these CNVs could be involved in the pathogenesis of NAFLD progression. After stringent filtering, we identified four rare and/or novel CNVs that may influence the pathogenesis of NASH. Two of these CNVs, located at 13q12.11 and 12q13.2 respectively, harbour the exportin 4 (XPO4) and phosphodiesterase 1B (PDE1B) genes which are already known to be involved in the etiology of liver cirrhosis and HCC. Cross-comparison of the genes located at these four CNV loci with genes already known to be associated with NAFLD yielded a set of genes associated with shared biological processes including cell death, the key process involved in ‘second hit’ hepatic injury. To our knowledge, this pilot study is the first to provide CNV information of potential relevance to the NAFLD spectrum. These data could prove invaluable in predicting patients at risk of developing NAFLD and more importantly, those who will subsequently progress to NASH.

Copy number variations of <italic>HLA-DRB5</italic> is associated with systemic lupus erythematosus risk in Chinese Han population

Systemic lupus erythematosus (SLE) is a polygenic, systemic, autoimmune disease. Copy number variants (CNVS) have been discovered to be associated with a number of complex disorders. We undertook the current study to explore the potential associations between genomic CNVS and SLE in Chinese Han population. In the discovery stage, seven SLE patients were examined with the high-density comparative genomic hybridization microarrays in the screening test for SLE associated CNVS. Then, in the validation stage, 135 SLE patients and 219 matched healthy subjects were investigated for the CNVS of gene HLA-DRB5 by AccuCopy™ technology. Quantitative polymerase chain reaction was carried out to determine the copy number (CN) and mRNA level of HLA-DRB5 in SLE patients. Although the mRNA level of HLA-DRB5 between the CN deletion group and the CN normal group in SLE patients was not statistically positive (P = 0.46), our results still showed more CN of HLA-DRB5 in SLE patients than in healthy controls (P = 3.98 × 10(-6)). Odds ratio for CN deletion was 0.38 (95% confidence interval (CI), 0.23-0.61, P = 7.79 × 10(-5)) and for CN duplication was 1.89 (95% CI, 0.56-7.66, P = 0.37), respectively. These findings indicated that CNVS of HLA-DRB5 was associated with the risk of SLE, and CN deletion appeared to be protective for SLE.

Association between copy number variations of HLA-DQA1 and ankylosing spondylitis in the Chinese Han population

Ankylosing spondylitis (AS) is a chronic inflammatory disease with complex genetic traits. Multiple sequence variations have been associated with AS, but explained only a proportion of heritability. The studies herein aimed to explore potential associations between genomic copy number variation (CNV) and AS of Han Chinese. Five AS patients were examined with the high-density comparative genomic hybridization (CGH) microarrays in the first screen test for AS associated CNVs. A total of 533 AS patients and 792 unrelated controls were examined in confirmation studies with the AccuCopy assays. A significant association was observed between the CNV of the HLA-DQA1 and AS. Comparing with controls, AS patients showed an aberrant copy number (CN), and significantly increased number of patients had more than 2 copies of the HLA-DQA1. Therefore, CNV of the HLA-DQA1 may play an important role in susceptibility to AS in Han Chinese population.

High-Resolution Survey in Familial Parkinson Disease Genes Reveals Multiple Independent Copy Number Variation Events in PARK2

A high density comparative genomic hybridization array was designed to evaluate CNVs in the genomic region of six familial PD genes in 181 PD cases and 67 controls. No CNV was found in PARK7, ATP13A2, PINK1, and LRRK2. Intronic-only CNVs were found in SNCA and PARK2 but were not associated with PD risk. A whole-gene duplication of SNCA was found in one case. The allele frequency of PARK2 exonic CNV is significantly higher in cases than in controls (P = 0.02), higher in early-onset (AAO ≤ 40) than in late-onset cases (P = 0.001), and higher in familial than in sporadic cases (P = 0.005). Except for single exon 2 duplications, all PARK2 exonic CNVs have different breakpoints, even when the same exon(s) were involved. In conclusion, except for SNCA and PARK2, CNVs are not a major contributing mechanism for the familial PD genes examined. The majority of PARK2 exonic CNVs are not recurrent.

Gistic Evaluation in Sezary Syndrome

AbstractAbstract 4814Sezary Syndrome (SS) is characterized by specific chromosomal abnormalities, however is not completely clarified yet which are the genetics hits associated to the initial phase or associated to disease progression. In this study, employing both high density Comparative Genomic Hybridization array (aCGH) and Single Nucleotide Polymorphism (SNP) array technologies, we elucidated the most frequent and significant chromosomal gain and loss regions, and new potentially relevant aberration for the disease progression onset.A total of 30 samples derived from 18 SS patients were analyzed on the Gene Chip Human Mapping 10K Array (Affymetrix). Genotype call and signal information performed by dChip SNP Software (http://www.dchip.org) provided us normalization and simultaneous measurement of Loss of Heterozygosity (LOH) and DNA Copy Number (CN) changes. We have further refined our analysis with a systematic method, Genomic Identification of Significant Targets in Cancer (GISTIC), able to identify regions that were significantly amplified o deleted, assigning a G-score that considers the amplitude of the aberration as well as the frequency of its occurrence across samples (http://genepattern.broadinstitute.org). Our data, generating by the integration of this two methods (see Table 1), revealed 19 significant focal event, including 10 amplification (10p, 17q, 8q, 6p, 4p, 1q, 18q, 21q, 3q, 1p) and 9 deletions (14q, 17p, 10p, 9p, 8p, 13q, 12p, 6q, 2p).In addition, new significant aberrant regions were identified, such as losses of 9p21.3, locus of an important tumor-suppressor gene CDKN2A (p16INK4a/p14ARF) and CDKN2B (p15INK4b). Recently, several study have reported the great influence of alteration in p16INK4a/p14ARF for prognosis of Diffuse large B-cell lymphoma (DLBL), primary cutaneous DLBCL leg-type, Blastic plasmacytoid dendritic cell neoplasm (BPDCN). Across our set of samples, 9p21.3 deletion occur in 8/30 samples from 6 patient, prevalently in heterozygosity state (Log2 ratio from −0.3484 to –0.7691). Two cases presented homozygous deletion (Log2 ratio from −1.2157 to –1.5342), and in four patients losses appeared only in a following phase of observation, suggesting a tumor progression event.Table 1Significant regions of chromosomal gainGenes in RegionSignificant regions of chromosomal lossGenes in RegionCytobandQ valueGISTIC peakCytobandQ valueGISTIC peak10p12.338.07E-1317765313-19264851814q11.21.85E-4021395737-22130391117q11.13.54E-0920147238-6203403556317p13.12.15E-101-150534862348q22.32.93E-0770887466-14627482631410p11.221.98E-0730280444-3266464186p25.30.00200621-126354969p21.30.0007482521283632-22089567114p15.320.007180317395812-2034058948p23.30.000763081-321773521791q23.30.039674158849705-159641626813q21.310.002829563681448-65120691018q22.30.19131-7611715325112p13.10.0254129304151-174141188521q22.30.19131-469443232236q24.10.029124139319577-14163596543q290.2191169050880-1995057401632p23.30.04340324636437-439108321261p36.310.116331-20338260239 Disclosures:No relevant conflicts of interest to declare.

Mechanisms of Genomic Instabilities Underlying Two Common Fragile-Site-Associated Loci, PARK2 and DMD, in Germ Cell and Cancer Cell Lines

Common fragile sites (CFSs) are specific chromosome regions that exhibit an increased frequency of breaks when cells are exposed to a DNA-replication inhibitor such as aphidicolin. PARK2 and DMD, the causative genes for autosomal-recessive juvenile Parkinsonism and Duchenne and Becker muscular dystrophy, respectively, are two very large genes that are located within aphidicolin-induced CFSs. Gross rearrangements within these two genes are frequently observed as the causative mutations for these diseases, and similar alterations within the large fragile sites that surround these genes are frequently observed in cancer cells. To elucidate the molecular mechanisms underlying this fragility, we performed a custom-designed high-density comparative genomic hybridization analysis to determine the junction sequences of approximately 500 breakpoints in germ cell lines and cancer cell lines involving PARK2 or DMD. The sequence signatures where these breakpoints occur share some similar features both in germ cell lines and in cancer cell lines. Detailed analyses of these structures revealed that microhomologies are predominantly involved in rearrangement processes. Furthermore, breakpoint-clustering regions coincide with the latest-replicating region and with large nuclear-lamina-associated domains and are flanked by the highest-flexibility peaks and R/G band boundaries, suggesting that factors affecting replication timing collectively contribute to the vulnerability for rearrangement in both germ cell and somatic cell lines.

Detection and differentiation of Clostridium botulinum type A strains using a focused DNA microarray

A focused oligonucleotide microarray featuring 62 probes targeting strain variable regions of the Clostridium botulinum strain ATCC 3502 genome sequence was developed to differentiate C. botulinum type A strains. The strain variable regions were selected from deletions identified among a panel of 10 type A strains compared to the strain ATCC 3502 genome sequence using high density comparative genomic hybridization microarrays. The focused microarray also featured specific probes for the detection of the neurotoxin genes of various serotypes (A–G), toxin gene cluster components (ha70 and orfX1), and fldB as a marker for proteolytic clostridia (Group I). Eight pairs of strains selected from separate type A botulism outbreaks were included in the 27 subtype A1–A4 strains examined in this study. Each outbreak related strain pair consisted of strains isolated from different sources (stool and food). Of the eight outbreak related strain pairs, six groups of strains with indistinguishable hybridization patterns were identified. Outbreak related strains were shown to have identical hybridization patterns. Strain pairs from three separate outbreaks involving strains harboring both the type A neurotoxin gene (bont/A) and an unexpressed type B neurotoxin gene (bont/B) shared the same probe hybridization profile. The focused microarray format provides a rapid approach for neurotoxin gene detection and preliminary determination of the relatedness of strains isolated from different sources.

G.P.7.04 High Density-comparative genomic hybridization (HD-CGH) array as a tool to detect deep intronic mutations in the dystrophin gene

Mutations in the dystrophin gene account for Duchenne (DMD) and Becker (BMD) muscular dystrophies, X-linked recessive hereditary myopathies. Due to the enormous size of the gene, achieving an exhaustive molecular diagnosis in these patients is challenging. The development of new techniques such as multiplex ligation-dependent probe amplification (MLPA) has simplified the detection of quantitative changes. The routinely investigated pathogenic changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing.

The complicated copy number alterations in chromosome 7 of a lung cancer cell line is explained by a model based on repeated breakage-fusion-bridge cycles

The drug-resistant lung cancer cell line PTX250, which has been previously established by exposure to an anti-cancer drug paclitaxel, has an increased copy number in the MDR1/ABCB1 locus region. In addition, the flanking regions also exhibit aberrant copy numbers, making the copy number profile of chromosome 7 complicated. In this study, we tested whether the breakage-fusion-bridge (BFB) cycle model can explain such copy number alterations. An analysis using fluorescence in situ hybridization (FISH) with a painting probe demonstrated that the aberrant chromosome, designated chromosome 7 amp, was derived from an intact chromosome 7. Using high-density comparative genomic hybridization arrays, we examined the copy number profile in detail and divided chromosome 7 amp into seven segments. Based on copy numbers of each segment, which were determined using interphase- and metaphase-FISH analysis, we constructed a formation model for the complicated copy number alteration. Six-time BFB cycles and the cycle-termination by healing of broken ends were presupposed in the model. Locations and orientations of the segments observed in chromosome 7 amp agreed well with those predicted from the model. Telomere addition was also cytogenetically confirmed. In all, it could be concluded that the complicated copy number alteration found in chromosome 7 amp is generated from the intact chromosome 7 by the repeated BFB cycles.

A stepwise framework for the normalization of array CGH data

BackgroundIn two-channel competitive genomic hybridization microarray experiments, the ratio of the two fluorescent signal intensities at each spot on the microarray is commonly used to infer the relative amounts of the test and reference sample DNA levels. This ratio may be influenced by systematic measurement effects from non-biological sources that can introduce biases in the estimated ratios. These biases should be removed before drawing conclusions about the relative levels of DNA. The performance of existing gene expression microarray normalization strategies has not been evaluated for removing systematic biases encountered in array-based comparative genomic hybridization (CGH), which aims to detect single copy gains and losses typically in samples with heterogeneous cell populations resulting in only slight shifts in signal ratios. The purpose of this work is to establish a framework for correcting the systematic sources of variation in high density CGH array images, while maintaining the true biological variations.ResultsAfter an investigation of the systematic variations in the data from two array CGH platforms, SMRT (Sub Mega base Resolution Tiling) BAC arrays and cDNA arrays of Pollack et al., we have developed a stepwise normalization framework integrating novel and existing normalization methods in order to reduce intensity, spatial, plate and background biases. We used stringent measures to quantify the performance of this stepwise normalization using data derived from 5 sets of experiments representing self-self hybridizations, replicated experiments, detection of single copy changes, array CGH experiments which mimic cell population heterogeneity, and array CGH experiments simulating different levels of gene amplifications and deletions. Our results demonstrate that the three-step normalization procedure provides significant improvement in the sensitivity of detection of single copy changes compared to conventional single step normalization approaches in both SMRT BAC array and cDNA array platforms.ConclusionThe proposed stepwise normalization framework preserves the minute copy number changes while removing the observed systematic biases.