The phenotype of 15 cases with rare 8q24.13-q24.3 deletions-A new syndrome or still an enigma?

Diagnosis of rare copy number variants (CNVs) with scarce literature evidence poses a major challenge for interpretation of the clinical significance of chromosomal microarray analysis (CMA) results, especially in the prenatal setting. Bioinformatic tools can be used to assist in this issue; however, this prediction can be imprecise. Our objective was to describe the phenotype of the rare copy number losses encompassing the 8q24.13-q24.3 locus, and to find common features in terms of genomic coordinates, gene content, and clinical phenotypic characteristics. Appropriate cases were retrieved using local databases of two largest Israeli centers performing CMA analysis. In addition, literature and public databases search was performed. Local database search yielded seven new patients with del (8)(q24.13q24.3) (one of these with an additional copy number variant). Literature and public databases search yielded eight additional patients. The cases showed high phenotypic variability, ranging from asymptomatic adults and fetuses with normal ultrasound to patients with autism/developmental delay (6/11 postnatal cases, 54.5%). No clear association was noted between the specific disease-causing/high-pLI gene content of the described del (8)(q24.13q24.3) to neurodevelopmental disorders, except for a possibly relevant locus encompassing the KCNQ3 gene. We present the challenges in classification of rare variants with limited clinical information. In such cases, genotype-phenotype correlation must be assessed with extra-caution and possibly using additional methods to assist the classification, especially in the prenatal setting.

Succinylated Jeffamine ED-2003 coated polycarbonate chips for low-cost analytical microarrays.

Analytical microarrays feature great capabilities for simultaneous detection and quantification of multiple analytes in a single measurement. In this work, we present a rapid and simple method for bulk preparation of microarrays on polycarbonate sheets. Succinylated Jeffamine® ED-2003 was screen printed on polycarbonate sheets to create a polyfunctional shielding layer by baking at 100 °C. After microdispension of capture probes (antibodies, oligonucleotides, or small molecules) in a microarray format, chips were assembled with a flow cell from double-sided tape. It was shown that the shielding layer was firmly coated and suppressed unspecific binding of proteins. Universal applicability was demonstrated by transferring established flow-based chemiluminescence microarray measurement principles from glass slides to polycarbonate chips without loss of analytical performance. Higher chemiluminescence signals could be generated by performing heterogeneous asymmetric recombinase polymerase amplification on polycarbonate chips. Similar results could be shown for sandwich microarray immunoassays. Beyond that, lower inter- and intra-assay variances could be measured for the analysis of Legionella pneumophila Serogroup 1, strain Bellingham-1. Even surface regeneration of indirect competitive immunoassays was possible, achieving a limit of detection of 0.35 ng L-1 for enrofloxacin with polycarbonate microarray chips. Succinylated Jeffamine ED-2003 coated polycarbonate chips have great potential to replace microtiter plates by flow-based chemiluminescence microarrays for rapid analysis. Therefore, it helps analytical microarrays to advance into routine analysis and diagnostics. Graphical abstract ᅟ.

A microcosting and cost-consequence analysis of clinical genomic testing strategies in autism spectrum disorder.

PurposeWhole-exome (WES) and whole-genome sequencing (WGS) increase the diagnostic yield in autism spectrum disorder (ASD) compared to chromosomal microarray (CMA), but there have been no comprehensive cost analyses. The objective was to perform such an assessment of CMA, WES, and WGS and compare the incremental cost per additional positive finding in hypothetical testing scenarios.MethodsFive-year patient and program costs were estimated from an institutional perspective. WES and WGS estimates were based on HiSeq 2500 with an additional WGS estimate for HiSeq X platforms. Parameter uncertainty was assessed with probabilistic and deterministic sensitivity analysis.ResultsThe cost per ASD sample was CAD$1,655 (95% CI: 1,611; 1,699) for WES, CAD$2,851 (95% CI: 2,750; 2,956) for WGS on HiSeq X, and CAD$5,519 (95% CI: 5,244; 5,785) on HiSeq 2500, compared to CAD$744 (95% CI 714, 773) for CMA. The incremental cost was over CAD$25,000 per additional positive finding if CMA was replaced by newer technology.ConclusionWhile costs for WES and WGS remain high, future reductions in material and equipment costs, and increased understanding of newly discovered variants and variants of unknown significance will lead to improved value.

Establishment of a reborn MMV-microarray technology: realization of microbiome analysis and other hitherto inaccessible technologies.

BACKGROUND: With the accelerating development of bioscience, the problem of research cost has become important. We previously devised and developed a novel concept microarray with manageable volumes (MMV) using a soft gel. It demonstrated the great potential of the MMV technology with the examples of 1024-parallel-cell culture and PCR experiments. However, its full potential failed to be expressed, owing to the nature of the material used for the MMV chip. RESULTS: In the present study, by developing plastic-based MMVs and associated technologies, we introduced novel technologies such as C2D2P (in which the cells in each well are converted from DNA to protein in 1024-parallel), NGS-non-dependent microbiome analysis, and other powerful applications. CONCLUSIONS: The reborn MMV-microarray technology has proven to be highly efficient and cost-effective (with approximately 100-fold cost reduction) and enables us to realize hitherto unattainable technologies.

Chromosomal microarray impacts clinical management.

Chromosomal microarray analysis (CMA) is standard of care, first-tier clinical testing for detection of genomic copy number variation among patients with developmental disabilities. Although diagnostic yield is higher than traditional cytogenetic testing, management impact has not been well studied. We surveyed genetic services providers regarding CMA ordering practices and perceptions about reimbursement. Lack of insurance coverage because of perceived lack of clinical utility was cited among the most frequent reasons why CMA was not ordered when warranted. We compiled a list of genomic regions where haploinsufficiency or triplosensitivity cause genetic conditions with documented management recommendations, estimating that at least 146 conditions potentially diagnosable by CMA testing have published literature supporting specific clinical management implications. Comparison with an existing clinical CMA database to determine the proportion of cases involving these regions showed that CMA diagnoses associated with such recommendations are found in approximately 7% of all cases (n = 28,526). We conclude that CMA impacts clinical management at a rate similar to other genetic tests for which insurance coverage is more readily approved. The information presented here can be used to address barriers that continue to contribute to inequities in patient access and care in regard to CMA testing.

Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array.

BACKGROUND: Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. METHODS: We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera) that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS) regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies. RESULTS: A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26) and 97.7% (43/44), respectively. CONCLUSIONS: Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

Lensfree holographic imaging of antibody microarrays for high-throughput detection of leukocyte numbers and function.

Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.

Fabrication of microwell arrays based on two-dimensional ordered polystyrene microspheres for high-throughput single-cell analysis.

This paper describes a method of fabricating rounded bottom microwell arrays (MA) in poly(dimethylsiloxane) (PDMS) by molding a monolayer of ordered polystyrene (PS) microspheres. PS microspheres were self-assembled on a glass slide and partially melted mainly from the bottom at 240 °C to increase adhesive force with the substrate. The partially melted PS arrays were used as master to generate MA. Microwell sizes are tunable in the 10-20 µm range with rounded bottoms; such a 3D structure is not readily obtainable through conventional soft lithography. Both adherent and nonadherent cell types can be retained in the microwells with high efficiency. As a demonstration of the advantage of real-time cell screening with this MA, single cell enzyme kinetic analysis was also carried out on trapped single cells. The PDMS MA may find applications in high-throughput drug screening, guided formation of cell clusters, and multicellular communication.

Translational bioinformatics: coming of age.

The American Medical Informatics Association (AMIA) recently augmented the scope of its activities to encompass translational bioinformatics as a third major domain of informatics. The AMIA has defined translational bioinformatics as "... the development of storage, analytic, and interpretive methods to optimize the transformation of increasingly voluminous biomedical data into proactive, predictive, preventative, and participatory health." In this perspective, I will list eight reasons why this is an excellent time to be studying translational bioinformatics, including the significant increase in funding opportunities available for informatics from the United States National Institutes of Health, and the explosion of publicly-available data sets of molecular measurements. I end with the significant challenges we face in building a community of future investigators in Translational Bioinformatics.

Analytical validation of an ultra low-cost mobile phone microplate reader for infectious disease testing.

Most mobile health (mHealth) diagnostic devices for laboratory tests only analyze one sample at a time, which is not suitable for large volume serology testing, especially in low-resource settings with shortage of health professionals. In this study, we developed an ultra-low-cost clinically-accurate mobile phone microplate reader (mReader), and clinically validated this optical device for 12 infectious disease tests. The mReader optically reads 96 samples on a microplate at one time. 771 de-identified patient samples were tested for 12 serology assays for bacterial/viral infections. The mReader and the clinical instrument blindly read and analyzed all tests in parallel. The analytical accuracy and the diagnostic performance of the mReader were evaluated across the clinical reportable categories by comparison with clinical laboratorial testing results. The mReader exhibited 97.59-99.90% analytical accuracy and <5% coefficient of variation (CV). The positive percent agreement (PPA) in all 12 tests achieved 100%, negative percent agreement (NPA) was higher than 83% except for one test (42.86%), and overall percent agreement (OPA) ranged 89.33-100%. We envision the mReader can benefit underserved areas/populations and low-resource settings in rural clinics/hospitals at a low cost (~$50 USD) with clinical-level analytical quality. It has the potential to improve health access, speed up healthcare delivery, and reduce health disparities and education disparities by providing access to a low-cost spectrophotometer.

Cost Effectiveness of Karyotyping, Chromosomal Microarray Analysis, and Targeted Next-Generation Sequencing of Patients with Unexplained Global Developmental Delay or Intellectual Disability.

BACKGROUND: Genetic diagnosis of unexplained global developmental delay and intellectual disability (GDD/ID) often ends the diagnostic odyssey and can lead to changes in clinical management. OBJECTIVE: The objective of this study was to investigate the cost effectiveness of testing scenarios involving several methods used to diagnose GDD/ID: karyotyping, chromosomal microarray analysis (CMA), and targeted next-generation sequencing (NGS). METHODS: We used decision-tree models to estimate the number of genetic diagnoses, the cost from a payers' perspective in the USA, and the incremental cost per additional genetic diagnosis. Model parameters were taken from peer-reviewed literature and governmental fee schedules. RESULTS: CMA testing results in more genetic diagnoses at an incremental cost of US $2692 per additional diagnosis compared with karyotyping, which has an average cost per diagnosis of US $11,033. Performing both tests sequentially results in the same number of diagnoses, but the total cost is less when CMA testing is done first and karyotyping second. Furthermore, when CMA testing yields a variant of unknown significance, additional genetic diagnoses can be obtained at an incremental cost of US $4220 by CMA testing of both parents, and when parents are not available or the patient had a normal CMA result, targeted NGS of the patient can add diagnoses at a further incremental cost of US $12,295. CONCLUSION: These results provide a cost effectiveness rationale for the use of CMA as the first-tier test for the genetic diagnosis of unexplained GDD/ID and further indicate that testing of both parents may be cost effective when a variant of unknown significance is detected in the patient.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma.

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 µL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Activity-based high-throughput profiling of metalloprotease inhibitors using small molecule microarrays.

We herein describe a high-throughput small molecule microarray (SMM) method that enables quick and cost-effective identification of potent inhibitors of metalloproteases in an activity-dependent manner, thereby offering a rapid means for inhibitor discovery and profiling.

High-throughput and automated diagnosis of antimicrobial resistance using a cost-effective cellphone-based micro-plate reader.

Routine antimicrobial susceptibility testing (AST) can prevent deaths due to bacteria and reduce the spread of multi-drug-resistance, but cannot be regularly performed in resource-limited-settings due to technological challenges, high-costs, and lack of trained professionals. We demonstrate an automated and cost-effective cellphone-based 96-well microtiter-plate (MTP) reader, capable of performing AST without the need for trained diagnosticians. Our system includes a 3D-printed smartphone attachment that holds and illuminates the MTP using a light-emitting-diode array. An inexpensive optical fiber-array enables the capture of the transmitted light of each well through the smartphone camera. A custom-designed application sends the captured image to a server to automatically determine well-turbidity, with results returned to the smartphone in ~1 minute. We tested this mobile-reader using MTPs prepared with 17 antibiotics targeting Gram-negative bacteria on clinical isolates of Klebsiella pneumoniae, containing highly-resistant antimicrobial profiles. Using 78 patient isolate test-plates, we demonstrated that our mobile-reader meets the FDA-defined AST criteria, with a well-turbidity detection accuracy of 98.21%, minimum-inhibitory-concentration accuracy of 95.12%, and a drug-susceptibility interpretation accuracy of 99.23%, with no very major errors. This mobile-reader could eliminate the need for trained diagnosticians to perform AST, reduce the cost-barrier for routine testing, and assist in spatio-temporal tracking of bacterial resistance.

Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites.

The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 µM, and a dynamic detection range of 0.3-15 µM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications.

Exploiting parallel R in the cloud with SPRINT.

BACKGROUND: Advances in DNA Microarray devices and next-generation massively parallel DNA sequencing platforms have led to an exponential growth in data availability but the arising opportunities require adequate computing resources. High Performance Computing (HPC) in the Cloud offers an affordable way of meeting this need. OBJECTIVES: Bioconductor, a popular tool for high-throughput genomic data analysis, is distributed as add-on modules for the R statistical programming language but R has no native capabilities for exploiting multi-processor architectures. SPRINT is an R package that enables easy access to HPC for genomics researchers. This paper investigates: setting up and running SPRINT-enabled genomic analyses on Amazon's Elastic Compute Cloud (EC2), the advantages of submitting applications to EC2 from different parts of the world and, if resource underutilization can improve application performance. METHODS: The SPRINT parallel implementations of correlation, permutation testing, partitioning around medoids and the multi-purpose papply have been benchmarked on data sets of various size on Amazon EC2. Jobs have been submitted from both the UK and Thailand to investigate monetary differences. RESULTS: It is possible to obtain good, scalable performance but the level of improvement is dependent upon the nature of the algorithm. Resource underutilization can further improve the time to result. End-user's location impacts on costs due to factors such as local taxation. CONCLUSIONS: Although not designed to satisfy HPC requirements, Amazon EC2 and cloud computing in general provides an interesting alternative and provides new possibilities for smaller organisations with limited funds.

Comparison of dkgB-linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica.

Two DNA-based methods were compared for the ability to assign serotype to 139 isolates of Salmonella enterica ssp. I. Intergenic sequence ribotyping (ISR) evaluated single nucleotide polymorphisms occurring in a 5S ribosomal gene region and flanking sequences bordering the gene dkgB. A DNA microarray hybridization method that assessed the presence and the absence of sets of genes was the second method. Serotype was assigned for 128 (92.1%) of submissions by the two DNA methods. ISR detected mixtures of serotypes within single colonies and it cost substantially less than Kauffmann-White serotyping and DNA microarray hybridization. Decreasing the cost of serotyping S. enterica while maintaining reliability may encourage routine testing and research.