Evaluation of Second-Generation Sequencing of 19 Dilated Cardiomyopathy Genes for Clinical Applications

Medical sequencing for diseases with locus and allelic heterogeneities has been limited by the high cost and low throughput of traditional sequencing technologies. “Second-generation” sequencing (SGS) technologies allow the parallel processing of a large number of genes and, therefore, offer great promise for medical sequencing; however, their use in clinical laboratories is still in its infancy. Our laboratory offers clinical resequencing for dilated cardiomyopathy (DCM) using an array-based platform that interrogates 19 of more than 30 genes known to cause DCM. We explored both the feasibility and cost effectiveness of using PCR amplification followed by SGS technology for sequencing these 19 genes in a set of five samples enriched for known sequence alterations (109 unique substitutions and 27 insertions and deletions). While the analytical sensitivity for substitutions was comparable to that of the DCM array (98%), SGS technology performed better than the DCM array for insertions and deletions (90.6% versus 58%). Overall, SGS performed substantially better than did the current array-based testing platform; however, the operational cost and projected turnaround time do not meet our current standards. Therefore, efficient capture methods and/or sample pooling strategies that shorten the turnaround time and decrease reagent and labor costs are needed before implementing this platform into routine clinical applications.Genetic testing for disorders with locus and allelic heterogeneity has been a challenge due to the high cost of sequencing entire coding regions of numerous genes. Classically, medical sequencing has used capillary-based “Sanger” sequencing technology and this has remained the gold standard for three decades. However, this method is expensive and has low throughput. It was not until novel technology platforms emerged that comprehensive testing became within reach. One such technology is array-based sequencing, which drastically increased the number of genes that could be analyzed simultaneously.^{1,2,3,4,5,6,7} We previously developed an array-based resequencing test for dilated cardiomyopathy (DCM), that doubled the number of genes analyzed in parallel while reducing test cost and turnaround time.⁶ However, this technology has two major drawbacks, particularly in a clinical setting. First, resequencing arrays have a poor detection rate for insertions and deletions (in/dels).^8,9 Second, the somewhat static nature of the chip design makes it time-consuming and impractical to add new content, especially in a disease area like DCM where genes are being discovered at a rapid pace. As such, novel technologies are needed to provide comprehensive sequencing of all DCM genes with high analytical sensitivity and with the goal of further reducing the cost of diagnostic testing.Second-generation sequencing (SGS) technologies, commonly referred to as “next-generation sequencing,” are based on massive parallel sequencing of millions of DNA templates through cycles of enzymatic treatment and image-based data acquisition. Several platforms have been developed in the last few years based on different biochemistries and cluster generation.^10,11,12 The most commonly used platforms include the Illumina Genome Analyzer (GAII; Solexa Technology),¹³ Roche Applied Sciences (454 sequencing),¹⁴ and ABI-Applied Biosystems (SOLiD platform).¹⁵ These technologies have been adopted for a wide variety of research applications^10,11,16 and have now matured sufficiently to be considered as robust enough for clinical applications. For example, SGS has recently been applied to resequencing the NF1 locus as well as the mitochondrial and small-cell lung cancer genomes.^17,18,19 In addition, whole exome resequencing has been conducted to discover genes underlying rare monogenic diseases.^20,21,22,23The specific advantages offered by SGS are twofold: the low cost per base and the ability to sequence millions of reads in parallel, allowing for simultaneous analysis of a large number of genes. However, these are offset by two major disadvantages: shorter reads and reduced accuracy as compared to Sanger sequencing.¹¹ Despite their disadvantages, improvements in these technologies promise to meet the technical requirements and strict quality standards of clinical diagnostics including analytical sensitivity, reproducibility and cost effectiveness.Several methodological approaches to capturing target gene regions have evolved to complement the higher capacity and throughput of novel sequencing technologies. These methods involve constructing and enriching a DNA “library” and use both PCR and/or hybridization as the mode of target selection. The classic PCR approach to library generation requires amplification of target regions, pooling, concatenation, shearing and ligation of adaptors and sequencing primers. Secondary droplet-based microfluidic technologies have evolved to facilitate high-throughput PCR in picoliter droplets.²⁴ With hybridization-based methods, libraries are constructed by shearing total gDNA followed by adaptor ligation and hybridization to oligonucleotides that are complementary to the desired target. Hybridization can be performed either on a solid surface array, on a filter, or by hybridization in solution^21,25,26,27 A third general approach to target selection uses molecular inversion probes. Molecular inversion probes consist of two primers linked together by a backbone and, similar to PCR, bind to specific target DNA. This is followed by gap filling, ligation, and enrichment steps.^28,29Important considerations in choosing a method include total amount of starting DNA, the size of the target region and the types of sequence alteration under investigation. Technical parameters such as target specificity, uniformity, and completeness of coverage also vary with each methodology.¹² Although each method has advantages or disadvantages depending on the application, we selected PCR, a well-established and robust targeted amplification method for this study.Leveraging on our array-based resequencing test for DCM,⁶ we evaluated the suitability of targeted PCR followed by Illumina GAII resequencing for a clinical testing environment. We used a number of samples enriched for a large number of substitutions as well as insertions and deletions in DCM genes to assess the analytical performance parameters. We also evaluated the cost and turnaround time of such a test and compared it to our current, array-based resequencing test.     

Patient Position Reproducibility in Fractionated Stereotactically Guided Conformal Radiotherapy Using the BrainLab? Mask System

Purpose: Dedicated mask systems nowadays allow the use of stereotactic radiotherapy in fractionated regimes, therefore combining the advantages of high precision radiotherapy with the biological benefit of fractionation. Therefore the knowledge of institution specific isocenter accuracy is essential for decision-making about margins to be allowed to form the planning target volume. Patients and Methods: Measurements of isocenter deviations during fractionated treatments were performed in 33 patients using the simulator Simulix-xy (Oldelft) in connection with the BrainLab^® angiographic localizer-box as well as port-films. In both cases repeated images were overlaid by use of anatomical landmarks with a methodical accuracy in the order of 0.5 mm. Results: Both methods yield random isocenter deviations of less then 2 mm (standard deviation) in all three directions and no significant systematic deviations. These values are in the order of the accuracy of the method, obtained by comparison of two independent investigators, as well as they are comparable with the literature. Conclusions: The accuracy of less than 2 mm indicates safety margins of 3-4 mm as sufficient for clinical routine to cover the target in 95.5% of all set-ups (2 SD). Ziel: Spezielle Maskensysteme erlauben heutzutage die Anwendung der stereotaktischen Strahlentherapie in fraktionierten Regimes und damit die Kombination der Vorteile der Hochpräzisionsbestrahlung mit dem biologischen Nutzen der Fraktionierung. Deshalb ist die Kenntnis der institutsspezifischen Genauigkeit der Isozentrumseinstellung eine notwendige Voraussetzung für die Entscheidung über die erforderlichen Sicherheitsabstände. Patienten und Methode: Die Messung der Isozentrumsgenauigkeit erfolgte bei 33 Patienten sowohl durch Simulatorkontrollen in Verbindung mit der BrainLab^®-Localizer-Box oder durch Verifikation mit Portfilmen. In beiden Fällen wurden die Filme anhand anatomischer Lankdmarken mit einer methodischen Sicherheit von unter 0,5 mm überlagert. Ergebnisse: Beide Methoden zeigten zufällige Isozentrumsabweichungen von weniger als 2 mm (Standardabweichung) in allen drei Raumebenen (Abbildung 1, Tabelle 2) und keine signifikanten systematischen Abweichungen. Damit liegen die Ergebnisse im Bereich der methodischen Genauigkeit, wie durch den Vergleich der Befunde zweier unabhängiger Untersucher gezeigt wird (Tabelle 1), und sind mit Literaturdaten gut vergleichbar. Schlussfolgerung: Die Genauigkeit von unter 2 mm zeigt, dass ein Sicherheitsabstand von 3-4 mm für die klinische Routine ausreichend ist, um bei 95,5% der Einstellung die sichere Erfassung des Targets zu garantieren.     

Patient Position Reproducibility in Fractionated Stereotactically Guided Conformal Radiotherapy Using the BrainLab® Mask System

PURPOSE: Dedicated mask systems nowadays allow the use of stereotactic radiotherapy in fractionated regimes, therefore combining the advantages of high precision radiotherapy with the biological benefit of fractionation. Therefore the knowledge of institution specific isocenter accuracy is essential for decision-making about margins to be allowed to form the planning target volume. PATIENTS AND METHOD: Measurements of isocenter deviations during fractionated treatments were performed in 33 patients using the simulator Simulix-xy (Oldelft) in connection with the BrainLab angiographic localizer-box as well as port-films. In both cases repeated images were overlaid by use of anatomical landmarks with a methodical accuracy in the order of 0.5 mm. RESULTS: Both methods yield random isocenter deviations of less then 2 mm (standard deviation) in all three directions and no significant systematic deviations. These values are in the order of the accuracy of the method, obtained by comparison of two independent investigators, as well as they are comparable with the literature. CONCLUSIONS: The accuracy of less than 2 mm indicates safety margins of 3-4 mm as sufficient for clinical routine to cover the target in 95.5% of all set-ups (2 SD).     

3-Ureidopropionate contributes to the neuropathology of 3-ureidopropionase deficiency and severe propionic aciduria: a hypothesis.

3-Ureidopropionate (3-UPA) is a physiologic metabolite in pyrimidine degradation. Pathological accumulation of 3-UPA in body fluids is found in 3-ureidopropionase deficiency and severe forms of propionic aciduria. Both diseases clinically present with a severe neuropathology involving gray and white matter as well as with a dystonic dyskinetic movement disorder. To date nothing is known about the toxic nature of this metabolite. The aim of the present study was to elucidate whether 3-UPA may act as endogenous neurotoxin. Exposure of cultured chick neurons to 3-UPA induced a concentration- and time-dependent neurodegeneration. Neuronal damage was reduced by the antioxidant alpha-tocopherol and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. In contrast, the non-NMDA receptor antagonist CNQX, the metabotropic glutamate receptor antagonist L-AP3, and succinate showed no protective effect. Furthermore, 3-UPA elicited an increased production of reactive oxygen species followed by a delayed increase in intracellular calcium concentrations. Activity measurement of single respiratory chain complexes I-V revealed an inhibition of complex V activity, but not of the electron-transferring complexes I-IV by 3-UPA. In contrast, 3-UPA did not affect the mitochondrial beta-oxidation of fatty acids. In conclusion, our results provide strong evidence that 3-UPA acts as endogenous neurotoxin via inhibition of mitochondrial energy metabolism, resulting in the initiation of secondary, energy-dependent excitotoxic mechanisms.     

Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesis

The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases.     

Strain‐encoded cardiac MR during high‐dose dobutamine stress testing: Comparison to cine imaging and to myocardial tagging

Purpose

To investigate regional strain response during high‐dose dobutamine stress cardiac magnetic resonance imaging (DS‐CMR) using myocardial tagging and Strain‐Encoded MR (SENC).

Materials and Methods

Stress induced ischemia was assessed by wall motion analysis, by tagged CMR and by SENC in 65 patients with suspected or known CAD who underwent DS‐CMR in a clinical 1.5 Tesla scanner. Coronary angiography deemed as the standard reference for the presence or absence of CAD (≥50% diameter stenosis) in all patients.

Results

SENC and conventional tagging detected abnormal strain response in six and five additional patients, respectively, who were missed by cine images and proved to have CAD by angiography (P < 0.05 for SENC versus cine, P = 0.06 for tagging versus cine and p = NS for SENC versus tagging). On a per‐vessel level, wall motion analysis on cine images showed high specificity (95%) but moderate sensitivity (70%) for the detection of CAD. Tagging and SENC yielded significantly higher sensitivity of 81% and 89%, respectively (P < 0.05 for tagging and P < 0.01 for SENC versus wall motion analysis, and p = NS for SENC versus tagging), while specificity was equally high (96% and 94%, respectively, P = NS for all).

Conclusion

Both the direct color‐coded visualization of strain on CMR images and the generation of additional visual markers within the myocardium with tagged CMR represent useful adjuncts for DS‐CMR, which may provide incremental value for the detection of CAD in humans. J. Magn. Reson. Imaging 2009;29:1053–1061. © 2009 Wiley‐Liss, Inc.     

Polymorphisms in ABCG2, ABCC3 and CNT1 genes and their possible impact on chemotherapy outcome of lung cancer patients

The prognosis of lung cancer patients treated with chemotherapy is poor, motivating the search for predictive factors. Single nucleotide polymorphisms (SNPs) in membrane transporter genes could influence the pharmacokinetics of cytostatic drugs and therefore affect treatment outcome. We examined 6 SNPs with known or suspected phenotypic effect: ABCG2 G34A, C421A; ABCC3 C?211T, G3890A, C3942T and CNT1 G565A. For 349 Caucasian patients with primary lung cancer [161 small cell lung cancer (SCLC), 187 nonsmall cell lung cancer (NSCLC) and 1 mixed] receiving first‐line chemotherapy 3 different endpoints were analyzed: response after the 2nd cycle (R), progression‐free survival (PFS) and overall survival (OS). The prognostic value of the SNPs was analyzed using multivariable logistic regression, calculating odds ratios (ORs) when comparing genotype frequencies in responders and nonresponders after the 2nd cycle. Hazard ratios (HRs) for PFS and for OS were calculated using Cox regression methods. In all lung cancer patients, none of the investigated polymorphisms modified response statistically significant. The only significant result in the histological subpopulations was in SCLC patients carrying the ABCC3 ‐211T allele who showed significantly worsened PFS (HR: 1.79; 95% confidence interval (CI) 1.13–2.82). In an exploratory subgroup analysis significantly worse OS was seen for carriers of the ABCG2 421A‐allele treated with platinum‐based drugs (HR: 1.60; 95% CI 1.04–2.47; n = 256). In conclusion, this study prioritizes ABCC3 C‐211T and ABCG2 C421A as candidate transporter SNPs to be further investigated as possible predictors of the clinical outcome of chemotherapy in lung cancer patients. © 2008 Wiley‐Liss, Inc.     

Acrylamide exposure measured by food frequency questionnaire and hemoglobin adduct levels and prostate cancer risk in the Cancer of the Prostate in Sweden Study

Acrylamide, a probable human carcinogen, is formed during the cooking of many commonly consumed foods. Data are scant on whether dietary acrylamide represents an important cancer risk in humans. We studied the association between acrylamide and prostate cancer risk using 2 measures of acrylamide exposure: intake from a food frequency questionnaire (FFQ) and acrylamide adducts to hemoglobin. We also studied the correlation between these 2 exposure measures. We used data from the population‐based case‐control study Cancer of the Prostate in Sweden (CAPS). Dietary data was available for 1,499 cases and 1,118 controls. Hemoglobin adducts of acrylamide were measured in blood samples from a subset of 170 cases and 161 controls. We calculated odds ratios (ORs) for the risk of prostate cancer in high versus low quantiles of acrylamide exposure using logistic regression. The correlation between FFQ acrylamide intake and acrylamide adducts in non‐smokers was 0.25 (95% confidence interval: 0.14–0.35), adjusted for age, region, energy intake, and laboratory batch. Among controls the correlation was 0.35 (95% CI: 0.21–0.48); among cases it was 0.15 (95% CI: 0.00–0.30). The OR of prostate cancer for the highest versus lowest quartile of acrylamide adducts was 0.93 (95% CI: 0.47–1.85, p‐value for trend = 0.98). For FFQ acrylamide, the OR of prostate cancer for the highest versus lowest quintile was 0.97 (95% CI: 0.75–1.27, p trend = 0.67). No significant associations were found between acrylamide exposure and risk of prostate cancer by stage, grade, or PSA level. Acrylamide adducts to hemoglobin and FFQ‐measured acrylamide intake were moderately correlated. Neither measure of acrylamide exposure—hemoglobin adducts or FFQ—was associated with risk of prostate cancer. © 2008 Wiley‐Liss, Inc.     

Endogenous expression of the sodium iodide symporter mediates uptake of iodide in murine models of colorectal carcinoma

The sodium iodide symporter (NIS) mediates iodide uptake into the thyroid. Because of this mechanism, differentiated thyroid cancer is susceptible for radioiodine therapy. Functional NIS expression in extrathyroidal tumors has been reported mainly in breast cancer. We screened colorectal tumors for NIS expression and investigated the mechanisms regulating NIS activity. Cell lines were screened for iodide uptake in vitro and NIS expression was evaluated by real‐time RT‐PCR, immunocytochemistry and immunoblotting. Iodide and pertechnetate uptake were evaluated in allograft tumors by biodistribution studies and scintigraphy. Tumors of transgenic mouse models for colorectal cancer harboring mutations in the oncogenes KRAS, β‐catenin or the tumor‐suppressor gene adenomatous‐polyposis coli (APC) were screened for NIS expression by RT‐PCR. In vitro, functional NIS activity was detected in murine CMT93 rectal carcinoma cells and NIS expression was verified on mRNA and protein level. Inhibition of tyrosine kinases increased iodide uptake. Inhibition of tyrosine phosphatases decreased iodide uptake. In vivo, functional NIS expression was preserved in CMT93 tumors and tumor uptake could be enhanced by treatment of mice with tyrosine kinase inhibitors. In transgenic murine models of colorectal cancer, 14% of endogenous tumors expressed elevated levels of NIS mRNA. We conclude that NIS is functionally expressed in a subset of murine colorectal tumors and its activity is regulated by tyrosine phosphorylation. Therefore, with specific tyrosine kinase inhibition, these tumors might be susceptible for radioiodine treatment. Further studies are justified to identify the specific pathways regulating NIS activity and to transfer these findings to human cell lines and tissues. © 2009 UICC