Capture Panel Research Articles

G.P.273: Nemaline myopathy 8 and KLHL40 in diseased and normal skeletal muscle

Mutations in the gene ( KLHL40 ), encoding Kelch-like family member 40, were recently identified in patients presenting with severe autosomal recessive nemaline myopathy (NEM8). Using exome sequencing or targeted re-sequencing of 276 known neuromuscular disease genes through a TargetSeq (Life Technologies) capture panel, we have identified two additional cases with KLHL40 mutations. Both were from consanguineous kindreds. Case 1 was diagnosed with severe SMA and congenital fractures. We identified a homozygous nonsense mutation in KLHL40 (c.46C>T, p. Gln16*). Muscle biopsy identified variation in fibre size and miliary nemaline bodies by electron microscopy seen as very fine scattered granules upon Gomori trichrome staining. Case 2 presented with severe nemaline myopathy, the biopsy showing numerous tiny nemaline bodies. We identified a homozygous missense mutation in KLHL40 (c.931C>A, p. Arg311Ser). Neither of the variants was present in 1000genomes or EVS databases. These data further suggest that muscle biopsy may be useful to inform genetic testing and that KLHL40 should be considered in patients presenting with severe miliary nemaline myopathy. Nemaline myopathy should also be considered in the differential diagnosis of severe SMA, especially in patients presenting with congenital fractures and contractures. The normal biological function of KLHL40 is largely unknown and therefore the basis of the severe muscle disease resulting from mutations of KLHL40 is also unknown. In investigations of control and diseased mouse and patient muscle, we show that KLHL40 is more abundant in the mouse soleus and diaphragm (muscles that comprise oxidative type I and IIA myofibres) than in the EDL, gastrocnemius and quadriceps muscles (type II glycolytic predominant). In addition we show that KLHL40 is increased in dystrophic mouse ( mdx ) and regenerating patient muscle (including DMD), suggesting that it may serve as a novel marker of skeletal muscle regeneration.

G.P.18: Neurogenetic disease diagnostics by targeted capture and next generation sequencing

Two problems for molecular diagnosis of neurogenetic disorders are high levels of genetic heterogeneity and involvement of large genes, e.g. titin. These problems result in many patients not being diagnosed by current Sanger-sequencing based diagnostics. To offset these problems we have developed a TargetSeq (Life Technologies) capture panel consisting of 335 disease genes with mutations detectable by next generation sequencing (NGS). The genes were 276 neurogenetic and 59 cardiomyopathy genes, targeted together because of clinical overlap. The 335 panel genes were captured from pools of 16 bar-coded patient DNA samples and sequenced 16 patients at a time using Ion Proton™ (Life Technologies) sequencing. Variant analysis was by either custom Annovar or Cartagenia (Cartagenia, Inc.) based pipelines. Testing the panel identified 88% of the known small-scale mutations in 28 positive controls. Known mutations deliberately chosen in regions not sequenced well by NGS were not detected. Using the FishingCNV package, we identified 90% of CNV controls, including 100% of CMT1A/HNPP duplication/deletion controls. Although one single exon CNV was detected, not all small CNVs were detected. We have screened >300 molecularly undiagnosed probands, identifying mutations in 59 disease genes, many of which were not previously analysed by the diagnostic laboratory due to Sanger sequencing costs. Using the targeted panel is not ethically different to traditional sequential Sanger sequencing diagnostics. The panel needs updating regularly with new disease gene discoveries. We now receive samples from all round Australasia for analysis of the 335-gene panel, which is cheaper than Sanger.

Microsatellite instability detection by next generation sequencing.

Microsatellite instability (MSI) is a useful phenotype in cancer diagnosis and prognosis. Nevertheless, methods to detect MSI status from next generation DNA sequencing (NGS) data are underdeveloped. We developed an approach to detect the MSI phenotype using NGS (mSINGS). The method was used to evaluate mononucleotide microsatellite loci that were incidentally sequenced after targeted gene enrichment and could be applied to gene or exome capture panels designed for other purposes. For each microsatellite locus, the number of differently sized repeats in experimental samples were quantified and compared to a population of normal controls. Loci were considered unstable if the experimental number of repeats was statistically greater than in the control population. MSI status was determined by the fraction of unstable microsatellite loci. We examined data from 324 samples generated using targeted gene capture assays of 3 different sizes, ranging from a 0.85-Mb to a 44-Mb exome design and incorporating from 15 to 2957 microsatellite markers. When we compared mSING results to MSI-PCR as a gold standard for 108 cases, we found the approach to be both diagnostically sensitive (range of 96.4% to 100% across 3 panels) and specific (range of 97.2% to 100%) for determining MSI status. The fraction of unstable microsatellite markers calculated from sequencing data correlated with the number of unstable loci detected by conventional MSI-PCR testing. NGS data can enable highly accurate detection of MSI, even from limited capture designs. This novel approach offers several advantages over existing PCR-based methods.

Comprehensive Assessment of Genetic Variants WithinTCF4in Fuchs' Endothelial Corneal Dystrophy

The single nucleotide variant (SNV), rs613872, in the transcription factor 4 (TCF4) gene was previously found to be strongly associated (P = 6 × 10(-26)) with Fuchs' endothelial corneal dystrophy (FECD). Subsequently, an intronic expansion of the repeating trinucleotides, TGC, was found to be even more predictive of disease. We performed comprehensive sequencing of the TCF4 gene region in order to identify the best marker for FECD within TCF4 and to identify other novel variants that may be associated with FECD. Leukocyte DNA was isolated from 68 subjects with FECD and 16 unaffected individuals. A custom capture panel was used to isolate the region surrounding the two previously validated markers of FECD. Sequencing of the TCF4 coding region, introns and flanking sequence, spanning 465 kb was performed at >1000× average coverage using the Illumina HiSequation 2000. TGC expansion (>50 repeats) was present in 46 (68%) FECD-affected subjects and one (6%) normal subject. A total of 1866 variants, including 1540 SNVs, were identified. Only two previously reported SNVs resided in the TCF4 coding region, neither of which segregated with disease. No variant, including TGC expansion, correlated perfectly with disease status. Trinucleotide repeat expansion was a better predictor of disease than any other variant. Complete sequencing of the TCF4 genomic region revealed no single causative variant for FECD. The intronic trinucleotide repeat expansion within TCF4 continues to be more strongly associated with FECD than any other genetic variant.

Targeted Cancer Exome Sequencing Discovers Novel Recurrent Mutations In MPN

The discovery of the JAK2V617F mutation in 2005 [Kralovics R, N Engl J Med 2005] represented a major breakthrough in the understanding of the molecular pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN). Nevertheless several observations suggest that the JAK2V617F mutation may not be the disease funding mutation, at least in most instances. Therefore, a great deal of effort is ongoing with the aim to identifying novel genetic lesions contributing to the disease pathogenesis. The two major theoretical and technical drawbacks to the identification of new somatic mutations are represented, respectively, by the huge number of genes potentially involved in tumorigenesis of MPN and by the availability of a “pure” germline control DNA. Buccal swabs and saliva have been generally considered as readily available sources of DNA of non-hematopoietic origin, but detection of the JAK2V617F mutation in at least some of these samples indeed suggested the presence of myeloid cell contamination [Levine RL, Cancer Cell 2005]. So, in order to discover novel mutations in MPN using upfront technologies based on next-generation sequencing (NGS) we designed a “cancer exome” capture panel of 2000 unique genes and microRNAs. This panel was used to capture libraries generated from genomic DNA extracted from granulocytes and in vitro expanded CD3+ T-lymphocytes as germline control, in a cohort of 20 MPN patients. These captured libraries were than massively sequenced using the Roche 454 FLX platform. DNA samples had been collected at the diagnosis of PV in 9 subjects and PMF in 6 subjects, while the remaining 5 DNA samples were from 5 of the 9 PV patients at the time they evolved to post-PV myelofibrosis.After extensive bioinformatics analysis and multiple control adjustments, we finally produced a list of 171 novel “true” somatic mutations occurring in genes and microRNAs coding regions of those MPN samples; some of these mutations have been already described in MPN, whereas novel variants represent the vast majority. Despite patients harbored different numbers of somatic mutations, spanning from four to twenty-one variants, only 22 genes appear recurrently mutated. It is worth of notice the acquisition of additional mutations and/or the occurrence of loss of some mutations at the time of disease evolution from PV to a post-PV Myelofibrosis in the five patients for whom samples were available at both disease phases. Some of them, either acquired (NTRK1, PRDM2, BRCA2 and BARD1) or lost (APC, CARS, MLL3 and FAT2) had been found also in another PV or PMF sample.To test the recurrence of these mutations, we screened a different cohort of 189 patients composed by PMF (91 samples), PV (50 patients) and post-PV Myelofibrosis (48 samples) by Ion AmpliSeq technology on an Ion Torrent PGM platform. Deep amplicon sequencing of granulocytes DNA achieved a sample median of 1000-fold coverage. Excluding JAK2, MPL, IDH2, ASXL1 known variants, for 7 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, NRAS) we demonstrated in MPN a global mutation frequency greater than the 3%. Whereas some new variants need functional validation to prove causal mechanisms, some other mutations have a well-known pathogenic role in solid cancers but here are described for the first time in MPN. Disclosures:No relevant conflicts of interest to declare.

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing

BackgroundLeber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are inherited retinal diseases that cause early onset severe visual impairment. An accurate molecular diagnosis can refine the clinical diagnosis and...

Next-Generation Sequencing–Based Molecular Diagnosis of a Chinese Patient Cohort With Autosomal Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease; therefore, an accurate molecular diagnosis is essential for appropriate disease treatment and family planning. The prevalence of RP in China had been reported at 1 in 3800, resulting in an estimated total of 340,000 Chinese RP patients. However, genetic studies of Chinese RP patients have been very limited. To date, no comprehensive molecular diagnosis has been done for Chinese RP patients. With the emergence of next-generation sequencing (NGS), comprehensive molecular diagnosis of RP is now within reach. The purpose of this study was to perform the first NGS-based comprehensive molecular diagnosis for Chinese RP patients. Thirty-one well-characterized autosomal recessive RP (arRP) families were recruited. For each family, the DNA sample from one affected member was sequenced using our custom capture panel, which includes 163 retinal disease genes. Variants were called, filtered, and annotated by our in-house automatic pipeline. Twelve arRP families were successfully molecular diagnosed, achieving a diagnostic rate of approximately 40%. Interestingly, approximately 63% of the pathogenic mutations we identified are novel, which is higher than that observed in a similar study on European descent (45%). Moreover, the clinical diagnoses of two families were refined based on the pathogenic mutations identified in the patients. We conclude that comprehensive molecular diagnosis can be vital for an accurate clinical diagnosis of RP. Applying this tool on patients from different ethnic groups is essential for enhancing our knowledge of the global spectrum of RP disease-causing mutations.

California's Policy Approach to Develop Carbon Capture, Utilization and Sequestration as a Mitigation Technology

While California has been at the forefront in adopting an aggressive climate change mitigation policy, it has taken a more measured and tentative approach toward creating an enabling policy and a regulatory framework for carbon capture, utilization and sequestration (CCUS) technologies to contribute to greenhouse gas (GHG) reductions. In 2005, Governor's Executive Order S-3-05 required that California reduce GHG emissions to 1990 levels by 2020 and to 80% below 1990 levels by 2050. In 2006, State Assembly Bill 32 codified the 2020 goal into law. In 2006, the California Legislature required two California agencies, the California Energy Commission and the Department of Conservation, to produce a report recommending how the state could facilitate commercial adoption of geologic sequestration from industrial sources. In 2010, three state agencies, the Energy Commission, Public Utilities Commission, and Air Resources Board, convened the California Carbon Capture and Storage Review Panel to make recommendations on specific policy, institutional, and regulatory changes necessary for California to enable commercial-scale carbon capture and geologic storage projects. Since 2006, several legislative bills have been introduced to establish regulatory authority, liability, and address pore space ownership issues, but none have made it into law.To meet the state's aggressive targets, especially to 2050 goal, will nevertheless require widespread adoption of CCUS technologies, according to studies by the California Council on Science and Technology. California contributes 7.5% of the total GHG emissions in the USA, or 1.8% of global GHG emissions. Over half of this currently is from point sources, but that proportion will increase as the state pursues electrification of the transportation sector. Trajectories of future GHG emissions growth suggest mitigation technologies must be implemented at rates on the order of 10–20 million tonnes of GHGs removed per year.The cap-and-trade system recently adopted in California to address the GHG reduction mandates of Assembly Bill 32 would seem to encourage pursuit CCUS technology projects by industrial emitters, but uncertainties preclude developing viable business cases. Uncertainty includes a lack of data on the costs of capture and storage and the lack of cap-and-trade accounting protocols for CCUS technologies. Although these protocols are scheduled to be developed, they will lag the initialization of cap-and-trade in 2012.

ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing

Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits.

DEVELOPMENT OF A RECYCLING SPRAYER FOR EFFICIENT ORCHARD PESTICIDE APPLICATION

Pesticide runoff and drift continue to be major problems in orchard spray applications. A new orchard sprayer, the RECUPAIR (Patent Pending), was designed to capture most of the droplets that do not deposit on target trees. The RECUPAIR was developed jointly by Agriculture and Agri–Food Canada HRDC and Universite Laval and a prototype was built for citrus orchards at the University of Florida. The sprayer consists of an axial–flow fan, over–the–row air and liquid ducts, hydraulic nozzles, and a spray capture and re–circulating system. The spray is discharged horizontally from the far side of the sprayer and unused droplets are collected on a capture panel located on the sprayer chassis. Design features and functional mechanisms were evaluated and discussed. Preliminary results indicated that the sprayer could be effective in reducing pesticide wastage in citrus applications.