Application of a target array Comparative Genomic Hybridization to prenatal diagnosis

<p>Abstract</p> <p>Background</p> <p>While conventional G-banded karyotyping still remains a gold standard in prenatal genetic diagnoses, the widespread adoption of array Comparative Genomic Hybridization (array CGH) technology for postnatal genetic diagnoses has led to increasing interest in the use of this same technology for prenatal diagnosis. We have investigated the value of our own designed DNA chip as a prenatal diagnostic tool for detecting submicroscopic deletions/duplications and chromosome aneuploidies.</p> <p>Methods</p> <p>We designed a target bacterial artificial chromosome (BAC)-based aCGH platform (MacArray™ M-chip), which specifically targets submicroscopic deletions/duplications for 26 known genetic syndromes of medical significance observed prenatally. To validate the DNA chip, we obtained genomic DNA from 132 reference materials generated from patients with 22 genetic diseases and 94 clinical amniocentesis samples obtained for karyotyping.</p> <p>Results</p> <p>In the 132 reference materials, all known genomic alterations were successfully identified. In the 94 clinical samples that were also subjected to conventional karyotyping, three cases of balanced chromosomal aberrations were not detected by aCGH. However, we identified eight cases of microdeletions in the Yq11.23 chromosomal region that were not found by conventional karyotyping. This region harbors the DAZ gene, and deletions may lead to non-obstructive spermatogenesis.</p> <p>Conclusions</p> <p>We have successfully designed and applied a BAC-based aCGH platform for prenatal diagnosis. This platform can be used in conjunction with conventional karyotyping and will provide rapid and accurate diagnoses for the targeted genomic regions while eliminating the need to interpret clinically-uncertain genomic regions.</p

17p12 deletion in breast cancer predicts resistance to neoadjuvant chemotherapy

Numerous studies have attempted to identify gene expression profiles which can be utilized to predict responses to neoadjuvant chemotherapy (NAC), but their findings are not clinically applicable at present. In the present study, we sought to determine DNA copy number alterations (CNAs) in breast cancer tissues which are associated with the response to NAC. Frozen tumor tissues from 63 breast cancer patients were obtained using core needle biopsy prior to NAC (3 cycles of docetaxel plus adriamycin) and were microdissected. Array comparative genomic hybridization (array CGH) with 4,045 bacterial artificial chromosome (BAC) probes was performed to identify the CNAs. Changes in tumor size in response to NAC were measured via magnetic resonance imaging. Fluorescence in situ hybridization (FISH) was conducted to verify array CGH results and for independent validation studies. CNAs at eight chromosomal loci encompassing 24 clones were correlated with changes in tumor size after NAC (p<0.05; t-test). Two CNAs were selected, 17p12 deletion and 17q21.32-33 gain, which were significantly associated with a smaller reduction in tumor size following NAC, via prioritization of the regions containing the candidate genes. In an independent validation set of samples from 39 patients, FISH assay further showed that the 17p12 deletion was markedly associated with smaller changes in tumor size (p=0.006), while the 17q21.32-33 gain was not significant (p=0.309). In conclusion, we successfully identified a 17p12 deletion in breast cancer tissue which can be applied in predicting tumor resistance to NAC

Gene expression defines natural changes in mammalian lifespan

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages

Predictive efficacy of low burden EGFR mutation detected by next-generation sequencing on response to EGFR tyrosine kinase inhibitors in non-small-cell lung carcinoma.

Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR) mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp and Ion Torrent Personal Genome Machine (PGM) techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC). We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%), PNA-LNA PCR clamp (27/57, 47.4%) and Ion Torrent PGM (26/57, 45.6%) detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003) than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195) or overall survival (34.39 vs. 44.10 months, P = 0.422) was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC

Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing

Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.Conrad DF, 2010, NATURE, V464, P704, DOI 10.1038/nature08516Kim JI, 2009, NATURE, V460, P1011, DOI 10.1038/nature08211Horowitz RE, 2008, NEW ENGL J MED, V359, P2393Shiffman D, 2008, PLOS ONE, V3, DOI 10.1371/journal.pone.0002895Kidd JM, 2008, NATURE, V453, P56, DOI 10.1038/nature06862Perry GH, 2008, AM J HUM GENET, V82, P685, DOI 10.1016/j.ajhg.2007.12.010Graham DSC, 2008, GENES IMMUN, V9, P93, DOI 10.1038/sj.gene.6364453Lohmueller KE, 2008, NATURE, V451, P994, DOI 10.1038/nature06611Korbel JO, 2007, SCIENCE, V318, P420, DOI 10.1126/science.1149504Hossain P, 2007, NEW ENGL J MED, V356, P213Larson MG, 2007, BMC MED GENET, V8, DOI 10.1186/1471-2350-8-S1-S5Redon R, 2006, NATURE, V444, P444, DOI 10.1038/nature05329Jee SH, 2006, NEW ENGL J MED, V355, P779Aitman TJ, 2006, NATURE, V439, P851, DOI 10.1038/nature04489Conrad DF, 2006, NAT GENET, V38, P75, DOI 10.1038/ng1697Wang YH, 2005, J LAB CLIN MED, V146, P321, DOI 10.1016/j.lab.2005.07.007Lindner I, 2005, MOL NUTR FOOD RES, V49, P972, DOI 10.1002/mnfr.200500087Tuzun E, 2005, NAT GENET, V37, P727, DOI 10.1038/ng1562Lee JW, 2005, ONCOGENE, V24, P1477, DOI 10.1038/sj.onc.1208304Iafrate AJ, 2004, NAT GENET, V36, P949, DOI 10.1038/ng1416Samuels Y, 2004, SCIENCE, V304, P554, DOI 10.1126/science.1096502Burchard EG, 2003, NEW ENGL J MED, V348, P1170COLIN Y, 1991, BLOOD, V78, P27474

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling