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- Mantripragada, K. K., et al.
(författare)
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DNA copy-number analysis of the 22q11 deletion-syndrome region using array-CGH with genomic and PCR-based targets
- 2004
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Ingår i: International Journal of Molecular Medicine. - 1107-3756 .- 1791-244X. ; 13:2, s. 273-279
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Tidskriftsartikel (refereegranskat)abstract
- Deletions and duplications of genomic segments commonly cause developmental disorders. The resolution and efficiency in diagnosing such gene-dosage alterations can be drastically increased using microarray-based comparative genomic hybridization (array-CGH). However, array-CGH currently relies on spotting genomic clones as targets, which confers severe limitations to the approach including resolution of analysis and reliable gene-dosage assessment of regions with high content of redundant sequences. To improve the methodology for analysis, we compared the use of genomic clones, repeat-free pools of amplified genomic DNA and cDNAs (single and pooled) as targets on the array. For this purpose, we chose q11.2 locus on chromosome 22 as a testing ground. Microdeletions at 22q11 cause birth defects collectively described as the DiGeorge/velocardiofacial syndrome. The majority of patients present 3 Mb typical deletions. Here, we report the construction of a gene-dosage array, covering 6 Mb of 22q11 and including the typically deleted region. We hybridized DNA from six DiGeorge syndrome patients to the array, and show that as little as 11.5 kb non-redundant, repeat-free PCR-generated sequence can be used for reliable detection of hemizygous deletions. By extrapolation, this would allow analysis of the genome with an average resolution of 25 kb. In the case of cDNAs our results indicate that 3.5 kb sequence is necessary for accurate identification of haploid/diploid dosage alterations. Thus, for regions rich in redundant sequences and repeats, such as 22q11, a specifically tailored array-CGH approach is good for gene copy number profiling.
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| 2. |
- Mantripragada, K K, et al.
(författare)
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Identification of novel deletion breakpoints bordered by segmental duplications in the NF1 locus using high resolution array-CGH.
- 2006
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Ingår i: Journal of medical genetics. - : BMJ. - 1468-6244 .- 0022-2593. ; 43:1, s. 28-38
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Segmental duplications flanking the neurofibromatosis type 1 (NF1) gene locus on 17q11 mediate most gene deletions in NF1 patients. However, the large size of the gene and the complexity of the locus architecture pose difficulties in deletion analysis. We report the construction and application of the first NF1 locus specific microarray, covering 2.24 Mb of 17q11, using a non-redundant approach for array design. The average resolution of analysis for the array is approximately 12 kb per measurement point with an increased average resolution of 6.4 kb for the NF1 gene. METHODS: We performed a comprehensive array-CGH analysis of 161 NF1 derived samples and identified heterozygous deletions of various sizes in 39 cases. The typical deletion was identified in 26 cases, whereas 13 samples showed atypical deletion profiles. RESULTS: The size of the atypical deletions, contained within the segment covered by the array, ranged from 6 kb to 1.6 Mb and their breakpoints could be accurately determined. Moreover, 10 atypical deletions were observed to share a common breakpoint either on the proximal or distal end of the deletion. The deletions identified by array-CGH were independently confirmed using multiplex ligation-dependent probe amplification. Bioinformatic analysis of the entire locus identified 33 segmental duplications. CONCLUSIONS: We show that at least one of these segmental duplications, which borders the proximal breakpoint located within the NF1 intron 1 in five atypical deletions, might represent a novel hot spot for deletions. Our array constitutes a novel and reliable tool offering significantly improved diagnostics for this common disorder.
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| 4. |
- Pemmasani, S. P., et al.
(författare)
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Effect of microstructure and phase constitution on mechanical properties of Ti1-xAlxN coatings
- 2014
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Ingår i: Applied Surface Science. - : Elsevier. - 0169-4332 .- 1873-5584. ; 313, s. 936-946
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Tidskriftsartikel (refereegranskat)abstract
- Monolithic TiAlN coatings with varying Al content in the range 0-65 at.% were deposited by cathodic arc evaporation. The variation in mechanical properties was studied by nanoindentation and scratch testing, and correlated with the phase constitution, grain size and residual stress. The hardness was found to be nearly stable up to Al content of 53% followed by a large drop at 65%. Depending on the stoichiometry, phase constitution and microstructure of the Ti1-xAlxN coatings, the mechanical property measurements were observed to reveal distinct trends at particular Al contents-ranging from a large scatter to clustering of data around specific values. Focused Ion Beam milling and Transmission Electron Microscopy studies showed a gradual change in microstructure, from large columnar grains in TiN to finer columns at intermediate Al content and near equiaxed, ultrafine grains with a nanocomposite structure in case of Ti0.35Al0.65N. Scratch studies revealed the deformation modes to vary with Al content, with the ductile failure modes at low Al content giving way to brittle failure at the highest Al content. Toughness studies showed a gradual increase in toughness with Al%, with the maximum seen at 53% and a moderate drop seen at 65%. The toughness shows a close dependence on the mechanical properties, phase constitution and microstructure. The study outlines the role of Al content on the microstructure of PVD TiAlN coatings and highlights the advantage of a cubic, nanocomposite structure for enhancing the toughness of these coatings. © 2014 Elsevier B.V. All rights reserved.
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| 7. |
- Benetkiewicz, Magdalena, et al.
(författare)
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High-resolution gene copy number and expression profiling of human chromosome 22 in ovarian carcinomas
- 2005
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Ingår i: Genes, Chromosomes and Cancer. - : John Wiley & Sons. - 1045-2257 .- 1098-2264. ; 42:3, s. 228-237
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Tidskriftsartikel (refereegranskat)abstract
- Previous low-resolution studies of chromosome 22 in ovarian carcinoma have suggested its involvement in the development of the disease. We report a high-resolution analysis of DNA copy number and gene expression of 22q in 18 ovarian carcinomas using a 22q-specific genomic microarray. We identified aberrations in 67% of the studied tumors, which displayed 3 distinct gene copy number profiles. The majority of the cases (11 of 18) demonstrated heterozygous terminal deletions of various sizes, the smallest of which was 3.5 Mb. The second profile, detected in 3 tumors, revealed the coexistence of heterozygous deletions and different patterns of low-copy-number gain that involved the proximal half of 22q. The latter finding has not been reported previously in ovarian carcinoma. One case displayed a continuous deletion encompassing the entire 22q, consistent with monosomy 22. Furthermore, we compared the results with the available data on these tumors by using cDNA microarrays to define the degree of correlation between abnormalities at the DNA level and variation in mRNA expression. By a comparison with the expression data, we were able to identify 21 deleted genes showing low mRNA levels and 12 amplified genes displaying elevated gene expression, several of which play roles in cell cycle control and the induction of apoptosis. Our results indicated significant correlation between DNA copy number aberrations and variation in mRNA expression. We also identified several regions and candidate genes on 22q that should be studied further to determine their role in the development of ovarian cancer.
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| 8. |
- Buckley, Patrick G, et al.
(författare)
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A full-coverage, high-resolution human chromosome 22 genomic microarrayfor clinical and research applications
- 2002
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 11:25, s. 3221-3229
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Tidskriftsartikel (refereegranskat)abstract
- We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.
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