Diagnosis of fetal submicroscopic chromosomal abnormalities in failed array CGH samples: copy number by sequencing as an alternative to microarrays for invasive fetal testing.

OBJECTIVES: Array comparative genomic hybridization (CGH) has become the technology of choice for high-resolution prenatal whole genome analysis. Limitations of microarrays are mainly related to the analog nature of the analysis, and poor-quality DNA can result in failed quality metrics with these platforms. We examined a cohort of abnormal fetuses with failed array CGH results using a next-generation sequencing algorithm, CNV-Seq. We assessed the ability of the platform to handle suboptimal prenatal samples and generate interpretable molecular karyotypes. METHODS: Nine samples obtained from abnormal fetuses and one from a normal control fetus were sequenced using an Illumina GAIIx. A segmentation algorithm for sequencing data was used to determine regional copy number data on the sequencing datasets. RESULTS: Phred quality scores were satisfactory for analysis of all samples. CNV-Seq identified both large- and small-scale abnormalities in the cohort, and normal results were obtained for fetuses for which microarray data were previously uninterpretable. No variants of uncertain significance were detected. Analysis of the digital sequencing datasets offered some advantages over array CGH output. CONCLUSIONS: Using next-generation sequencing for the detection of genomic copy number variants may be advantageous for poor-quality, invasively-acquired prenatal samples. CNV-Seq could become a potential alternative to array CGH in this setting.

Diagnosis of copy number variation by Illumina next generation sequencing is comparable in performance to oligonucleotide array comparative genomic hybridisation.

Array comparative genomic hybridisation (aCGH) profiling is currently the gold standard for genetic diagnosis of copy number. Next generation sequencing technologies provide an alternative and adaptable method of detecting copy number by comparing the number of sequence reads in non-overlapping windows between patient and control samples. Detection of copy number using the BlueGnome 8×60k oligonucleotide aCGH platform was compared with low resolution next generation sequencing using the Illumina GAIIx on 39 patients with developmental delay and/or learning difficulties who were referred to the Leeds Clinical Cytogenetics Laboratory. Sensitivity and workflow of the two platforms were compared. Customised copy number algorithms assessed sequence counts and detected changes in copy number. Imbalances detected on both platforms were compared. Of the thirty-nine patients analysed, all eleven imbalances detected by array CGH and confirmed by FISH or Q-PCR were also detected by CNV-seq. In addition, CNV-seq reported one purported pathogenic copy number variant that was not detected by array CGH. Non-pathogenic, unconfirmed copy number calls were detected by both platforms; however few were concordant between the two. CNV-seq offers an alternative to array CGH for copy number analysis with resolution and future costs comparable to conventional array CGH platforms and with less stringent sample requirements.

Combination treatment for acute ischemic stroke: A ray of Hope?

BACKGROUND: With the implementation of thrombolysis, a large number of distinct pharmacological agents are now under consideration for the treatment of acute ischemic stroke, with disappoiting early results. Because the processes that ultimately lead to ischemic cell death involve a variety of pathophysiologic pathways, it is likely that combinations of agents may be necessary to positively affect neurological outcome. We review the general strategies under consideration for reduction of ischemic injury in the central nervous system, the types of possible interactions between compounds, and the experimental evidence showing effective combination therapies. SUMMARY OF REVIEW: Reduction of ischemic injury has been attempted by the following pharmacologic mechanisms: thrombolysis, neuroprotection, and perfusion/reperfusion enhancers. There is experimental evidence that the combination of thrombolytic therapy with a neuroprotective agent is additive in some ischemic models, as is the combination of a thrombolytic with an agent that facilitates reperfusion (thromboxane A(2) receptor antagonist and neutrophil adhesion/activation inhibition). Combinations of neuroprotective agents such as glutamate antagonists and calcium channel antagonists may be additive, and other combinations of neuroprotective agents, such as a glutamate antagonist with a gamma-aminobutyric acid (GABA) agonist, have even shown synergism in a rat stroke model. It has also been suggested that lower doses of toxic drugs may be used together to yield a positive neurologic outcome. Successful demonstration of additive or synergistic effects of pharmacologic agents in ischemia will depend on (1) the model used (well below a maximal "ceiling effect"); (2) the timing of drug administration; (3) the doses of the drugs used; and (4) the primary neurologic endpoint used. (Infarction size requires prolonged survival.) CONCLUSIONS: It appears from preclinical studies that some combinations of pharmacotherapeutic agents may be beneficial in cerebral ischemia, but rigorous evaluation is needed before initiating clinical trials.