Background

Acute lymphoblastic leukemia (ALL) diagnosed within the first month of life is classified as congenital ALL and has a significantly worse outcome than ALL diagnosed in older children. This suggests that congenital ALL is a biologically different disease, and thus may be caused by a distinct set of mutations. To understand the somatic and germline mutations contributing to congenital ALL, the protein-coding regions in the genome were captured and whole-exome sequencing was employed for the identification of single-nucleotide variants and small insertion and deletions in the germlines as well as the primary tumors of four patients with congenital ALL.

Methods

Exome sequencing was performed on Illumina GAIIx or HiSeq 2000 (Illumina, San Diego, California). Reads were aligned to the human reference genome and the Genome Analysis Toolkit was used for variant calling. An in-house developed Ensembl-based variant annotator was used to richly annotate each variant.

Results

There were 1–3 somatic, protein-damaging mutations per ALL, including a novel mutation in Sonic Hedgehog. Additionally, there were many germline mutations in genes known to be associated with cancer predisposition, as well as genes involved in DNA repair.

Conclusion

This study is the first to comprehensively characterize the germline and somatic mutational profile of all protein-coding genes patients with congenital ALL. These findings identify potentially important therapeutic targets, as well as insight into possible cancer predisposition genes.

Background

Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity.

Methodology/Principal Findings

A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3.

Conclusion/Significance

Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients. Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi. In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively. In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi. These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib. In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.

The brachyolmias constitute a clinically and genetically heterogeneous group of skeletal dysplasias characterized by a short trunk, scoliosis and mild short stature1. Here, we identify a locus for an autosomal dominant form of brachyolmia on chromosome 12q24.1–12q24.2. Among the genes in the genetic interval, we selected TRPV4, which encodes a calcium permeable cation channel of the transient receptor potential (TRP) vanilloid family, as a candidate gene because of its cartilage-selective gene expression pattern. In two families with the phenotype, we identified point mutations in TRPV4 that encoded R616Q and V620I substitutions, respectively. Patch clamp studies of transfected HEK cells showed that both mutations resulted in a dramatic gain of function characterized by increased constitutive activity and elevated channel activation by either mechano-stimulation or agonist stimulation by arachidonic acid or the TRPV4-specific agonist 4α-phorbol 12,13-didecanoate (4αPDD). This study thus defines a previously unknown mechanism, activation of a calcium-permeable TRP ion channel, in skeletal dysplasia pathogenesis.

Motivation: The ability to detect copy-number variation (CNV) and loss of heterozygosity (LOH) from exome sequencing data extends the utility of this powerful approach that has mainly been used for point or small insertion/deletion detection.

Results: We present ExomeCNV, a statistical method to detect CNV and LOH using depth-of-coverage and B-allele frequencies, from mapped short sequence reads, and we assess both the method's power and the effects of confounding variables. We apply our method to a cancer exome resequencing dataset. As expected, accuracy and resolution are dependent on depth-of-coverage and capture probe design.

Availability: CRAN package ‘ExomeCNV’.

Contact: fsathira@fas.harvard.edu; snelson@ucla.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Background

The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases.

Methods

We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis.

Results

Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified.

Conclusions

Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.

Background: Genetic alteration of muscle cell glycosylation in muscular dystrophy models has ameliorated disease.

Results: A high throughput screen identified a small molecule, lobeline, which altered muscle cell glycosylation and improved laminin binding.

Conclusion: Lobeline increased abundance of sarcolemmal glycoproteins and increased laminin binding in an N-glycan-dependent manner.

Significance: A novel approach revealed an unexpected role for N-glycans in muscle cell function.

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.

Establishing the functional roles of genetic variants remains a significant challenge in the post-genomic era. Here, we present a method, allele-specific alternative mRNA processing (ASARP), to identify genetically influenced mRNA processing events using transcriptome sequencing (RNA-Seq) data. The method examines RNA-Seq data at both single-nucleotide and whole-gene/isoform levels to identify allele-specific expression (ASE) and existence of allele-specific regulation of mRNA processing. We applied the methods to data obtained from the human glioblastoma cell line U87MG and primary breast cancer tissues and found that 26–45% of all genes with sufficient read coverage demonstrated ASE, with significant overlap between the two cell types. Our methods predicted potential mechanisms underlying ASE due to regulations affecting either whole-gene-level expression or alternative mRNA processing, including alternative splicing, alternative polyadenylation and alternative transcriptional initiation. Allele-specific alternative splicing and alternative polyadenylation may explain ASE in hundreds of genes in each cell type. Reporter studies following these predictions identified the causal single nucleotide variants (SNVs) for several allele-specific alternative splicing events. Finally, many genes identified in our study were also reported as disease/phenotype-associated genes in genome-wide association studies. Future applications of our approach may provide ample insights for a better understanding of the genetic basis of gene regulation underlying phenotypic diversity and disease mechanisms.

Purpose

To assess the feasibility, safety, and toxicity of autologous tumor lysate-pulsed dendritic cell (DC) vaccination and toll-like receptor (TLR) agonists in patients with newly diagnosed and recurrent glioblastoma. Clinical and immune responses were monitored and correlated with tumor gene expression profiles.

Experimental Design

Twenty-three patients with glioblastoma (WHO grade IV) were enrolled in this dose-escalation study and received three biweekly injections of glioma lysate-pulsed DCs followed by booster vaccinations with either imiquimod or poly-ICLC adjuvant every three months until tumor progression. Gene expression profiling, IHC, FACS, and cytokine bead arrays were performed on patient tumors and PBMC.

Results

DC vaccinations are safe and not associated with any dose-limiting toxicity. The median overall survival from the time of initial surgical diagnosis of glioblastoma was 31.4 months, with a one-, two-, and three-year survival rate of 91%, 55% and 47%, respectively. Patients whose tumors had mesenchymal gene expression signatures exhibited increased survival following DC vaccination compared to historical controls of the same genetic subtype. Tumor samples with a mesenchymal gene expression signature had a higher number of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) compared with glioblastomas of other gene expression signatures (p = 0.006).

Conclusion

Autologous tumor lysate-pulsed DC vaccination in conjunction with TLR agonists is safe as adjuvant therapy in newly diagnosed and recurrent glioblastoma patients. Our results suggest that the mesenchymal gene expression profile may identify an immunogenic subgroup of glioblastoma that may be more responsive to immune-based therapies.

Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in ~7% of human malignancies and ~60% of melanomas1. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses2. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways3–5. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistantsubsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

BACKGROUND

Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease.

METHODS

We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation.

RESULTS

Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied.

CONCLUSIONS

GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations.

Chromosome 17q11-q21 is a region of the genome likely to harbor susceptibility to autism (MIM[209850]) based on prior evidence of linkage to the disorder. This linkage is specific to multiplex pedigrees containing only male probands (MO) within the Autism Genetic Resource Exchange (AGRE). Previously, Stone et al.1 completed a high-density SNP association study of 13.7Mb within this interval, but common variant association was not sufficient to account for the linkage signal. Here we extend this SNP-based association study to complete the coverage of the 2 LOD support interval around the chromosome 17q linkage peak by testing the majority of common alleles in 284 MO trios.

CONCLUSIONS

Markers within an interval containing the gene CACNA1G were found to be associated with Autism Spectrum Disorder at a locally significant level (p = 1.9 × 10-5). While establishing CACNA1G as a novel candidate for autism, these alleles do not contribute sufficient genetic effect to explain the observed linkage, indicating there is substantial genetic heterogeneity despite the clear linkage signal. The region thus likely harbors a combination of multiple common and rare alleles contributing to the genetic risk. These data, along with previous studies of Chromosomes 5 and 7q3, suggest few if any major common risk alleles account for ASD risk under major linkage peaks in the AGRE sample. This provides important evidence for strategies to identify ASD genes, suggesting they should focus on identifying rare variants and common variants of small effect.

We previously reported that specific oxysterols stimulate osteogenic differentiation of pluripotent bone marrow stromal cells (MSCs) through activation of hedgehog (Hh) signaling and may serve as potential future therapies for intervention in osteopenia and osteoporosis. In this study we report that the osteogenic oxysterol 20(S)-hydroxycholesterol (20S) induces the expression of genes associated with Notch signaling. Using M2-10B4 (M2) MSCs, we found that 20S significantly induced HES-1, HEY-1, and HEY-2 mRNA expression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not. Similar observations were made when M2 cells were treated with sonic hedgehog (Shh), and the specific Hh pathway inhibitor cyclopamine blocked 20S-induced Notch target gene expression. 20S did not induce Notch target genes in Smo−/− mouse embryonic fibroblasts, further confirming the role of Hh signaling in 20S-induced expression of Notch target genes. Despite the inability of liver X-receptor (LXR) synthetic ligand TO901317 to induce Notch target genes in M2 cells, LXR knockdown studies using siRNA showed inhibition of 20S-induced HEY-1 but not HES-1 expression, suggesting the partial role of LXR signaling in MSC responses to 20S. Moreover, 20S-induced Notch target gene expression was independent of canonical Notch signaling because neither 20S nor Shh induced CBF1 luciferase reporter activity or NICD protein accumulation in the nucleus, which are hallmarks of canonical Notch signaling activation. Finally, HES-1 and HEY-1 siRNA transfection significantly inhibited 20S-induced osteogenic genes, suggesting that the pro-osteogenic effects of 20S are regulated in part by HES-1 and HEY-1. © 2010 American Society for Bone and Mineral Research

Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50–100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a “Phenotype Sequencing” approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110–$340.

Purpose

To report three low-passage cell lines from primary choroidal melanoma with metastatic outcome, which were stable for cytogenetic patterns and expression profiles of the primary melanoma.

Methods

In patients with choroidal melanoma, transscleral fine needle aspiration biopsy (FNAB) was performed immediately before plaque placement for 125iodine brachytherapy or immediately after enucleation. Cells were examined for cytopathology, evaluated by fluorescence in-situ hybridization (FISH) for the centromere of chromosome 3, analyzed by 250K whole genome Mapping Array and U133 plus 2.0 Expression Array, and placed in cell culture. At passage 3, the cell lines were analyzed by Mapping Array and Expression Array.

Results

Three cell lines were propagated from primary choroidal melanomas in three patients who subsequently developed metastasis. Two cell lines were stable for the entire chromosomal aberration pattern of the respective primary tumor. In the third, necrotic material from the biopsy prevented further analysis, yet resulted in a stable cell line. Each cell line had chromosome 3 loss, 6q loss, 8p loss, multiple 8q gain, and 16q loss. Additionally, two cell lines had chromosome 6p gain. Two cell lines had RNA expression profiles similar to the respective primary tumors; the third cell line had a similar RNA expression profile relative to the other two cell lines.

Conclusions

FNAB of primary choroidal melanomas resulted in highly characterized, low-passage cell lines, which were stable for the cytogenetic patterns and expression profiles found in the primary tumor. These cell lines represent novel tools for the study of metastatic choroidal melanoma biology.

Background

Since the introduction of next-generation DNA sequencers the rapid increase in sequencer throughput, and associated drop in costs, has resulted in more than a dozen human genomes being resequenced over the last few years. These efforts are merely a prelude for a future in which genome resequencing will be commonplace for both biomedical research and clinical applications. The dramatic increase in sequencer output strains all facets of computational infrastructure, especially databases and query interfaces. The advent of cloud computing, and a variety of powerful tools designed to process petascale datasets, provide a compelling solution to these ever increasing demands.

Results

In this work, we present the SeqWare Query Engine which has been created using modern cloud computing technologies and designed to support databasing information from thousands of genomes. Our backend implementation was built using the highly scalable, NoSQL HBase database from the Hadoop project. We also created a web-based frontend that provides both a programmatic and interactive query interface and integrates with widely used genome browsers and tools. Using the query engine, users can load and query variants (SNVs, indels, translocations, etc) with a rich level of annotations including coverage and functional consequences. As a proof of concept we loaded several whole genome datasets including the U87MG cell line. We also used a glioblastoma multiforme tumor/normal pair to both profile performance and provide an example of using the Hadoop MapReduce framework within the query engine. This software is open source and freely available from the SeqWare project (http://seqware.sourceforge.net).

Conclusions

The SeqWare Query Engine provided an easy way to make the U87MG genome accessible to programmers and non-programmers alike. This enabled a faster and more open exploration of results, quicker tuning of parameters for heuristic variant calling filters, and a common data interface to simplify development of analytical tools. The range of data types supported, the ease of querying and integrating with existing tools, and the robust scalability of the underlying cloud-based technologies make SeqWare Query Engine a nature fit for storing and searching ever-growing genome sequence datasets.

During the past several years, we have achieved a deeper understanding of the etiology/pathophysiology of major depressive disorder (MDD). However, this improved understanding has not translated to improved treatment outcome. Treatment often results in symptomatic improvement, but not full recovery. Clinical approaches are largely trial-and-error, and when the first treatment does not result in recovery for the patient, there is little proven scientific basis for choosing the next. One approach to enhancing treatment outcomes in MDD has been the use of standardized sequential treatment algorithms and measurement-based care. Such treatment algorithms stand in contrast to the personalized medicine approach, in which biomarkers would guide decision making. Incorporation of biomarker measurements into treatment algorithms could speed recovery from MDD by shortening or eliminating lengthy and ineffective trials. Recent research results suggest several classes of physiologic biomarkers may be useful for predicting response. These include brain structural or functional findings, as well as genomic, proteomic, and metabolomic measures. Recent data indicate that such measures, at baseline or early in the course of treatment, may constitute useful predictors of treatment outcome. Once such biomarkers are validated, they could form the basis of new paradigms for antidepressant treatment selection.

A primary component of next-generation sequencing analysis is to align short reads to a reference genome, with each read aligned independently. However, reads that observe the same non-reference DNA sequence are highly correlated and can be used to better model the true variation in the target genome. A novel short-read micro re-aligner, SRMA, that leverages this correlation to better resolve a consensus of the underlying DNA sequence of the targeted genome is described here.

Background

DNA sequence comparison is a well-studied problem, in which two DNA sequences are compared using a weighted edit distance. Recent DNA sequencing technologies however observe an encoded form of the sequence, rather than each DNA base individually. The encoded DNA sequence may contain technical errors, and therefore encoded sequencing errors must be incorporated when comparing an encoded DNA sequence to a reference DNA sequence.

Results

Although two-base encoding is currently used in practice, many other encoding schemes are possible, whereby two ore more bases are encoded at a time. A generalized k-base encoding scheme is presented, whereby feasible higher order encodings are better able to differentiate errors in the encoded sequence from true DNA sequence variants. A generalized version of the previous two-base encoding DNA sequence comparison algorithm is used to compare a k-base encoded sequence to a DNA reference sequence. Finally, simulations are performed to evaluate the power, the false positive and false negative SNP discovery rates, and the performance time of k-base encoding compared to previous methods as well as to the standard DNA sequence comparison algorithm.

Conclusions

The novel generalized k-base encoding scheme and resulting local alignment algorithm permits the development of higher fidelity ligation-based next generation sequencing technology. This bioinformatic solution affords greater robustness to errors, as well as lower false SNP discovery rates, only at the cost of computational time.

Purpose of review

Duchenne muscular dystrophy is a progressive muscle degenerative disease caused by dystrophin mutations. The purpose of this review is to highlight two emerging therapies designed to repair the primary genetic defect, called `exon skipping' and `nonsense codon suppression'.

Recent findings

A drug, PTC124, was identified that suppresses nonsense codon translation termination. PTC124 can lead to restoration of some dystrophin expression in human Duchenne muscular dystrophy muscles with mutations resulting in premature stops. Two drugs developed for exon skipping, PRO051 and AVI-4658, result in the exclusion of exon 51 from mature mRNA. They can restore the translational reading frame to dystrophin transcripts from patients with a particular subset of dystrophin gene deletions and lead to some restoration of dystrophin expression in affected boys' muscle in vivo. Both approaches have concluded phase I trials with no serious adverse events.

Summary

These novel therapies that act to correct the primary genetic defect of dystrophin deficiency are among the first generation of therapies tailored to correct specific mutations in humans. Thus, they represent paradigm forming approaches to personalized medicine with the potential to lead to life changing treatment for those affected by Duchenne muscular dystrophy.

Purpose

To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina.

Methods

The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis.

Results

One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors.

Conclusions

ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.

Objectives

To describe a large multigenerational family with migraine-associated vertigo (MAV) combining a detailed phenotypic and genetic analysis.

Background

Migraine-associated vertigo is said to be highly prevalent in the general population and, like other migraine syndromes, its etiology is felt to have a strong genetic component. However, so far, there have been no reports of large families with MAV.

Methods

Detailed clinical study was conducted on a large multigenerational family with MAV. Genetic study using identical-by-descent analysis with dense single nucleotide polymorphism (SNP) arrays was performed to examine consistent inheritance pattern among the affecteds.

Results

Clinical features of MAV were variable although most had other migraine symptoms with at least some of their attacks. We did not find a region of the genome shared by all eight subjects with MAV indicating a polygenetic inheritance for MAV even in this single large family.

Conclusions

A region on 11q shared by most affected females may contain a susceptibility allele for MAV that is expressed exclusively or predominantly by women.

Background

In the post genome era, a major goal of biology is the identification of specific roles for individual genes. We report a new genomic tool for gene characterization, the UCLA Gene Expression Tool (UGET).

Results

Celsius, the largest co-normalized microarray dataset of Affymetrix based gene expression, was used to calculate the correlation between all possible gene pairs on all platforms, and generate stored indexes in a web searchable format. The size of Celsius makes UGET a powerful gene characterization tool. Using a small seed list of known cartilage-selective genes, UGET extended the list of known genes by identifying 32 new highly cartilage-selective genes. Of these, 7 of 10 tested were validated by qPCR including the novel cartilage-specific genes SDK2 and FLJ41170. In addition, we retrospectively tested UGET and other gene expression based prioritization tools to identify disease-causing genes within known linkage intervals. We first demonstrated this utility with UGET using genetically heterogeneous disorders such as Joubert syndrome, microcephaly, neuropsychiatric disorders and type 2 limb girdle muscular dystrophy (LGMD2) and then compared UGET to other gene expression based prioritization programs which use small but discrete and well annotated datasets. Finally, we observed a significantly higher gene correlation shared between genes in disease networks associated with similar complex or Mendelian disorders.

Discussion

UGET is an invaluable resource for a geneticist that permits the rapid inclusion of expression criteria from one to hundreds of genes in genomic intervals linked to disease. By using thousands of arrays UGET annotates and prioritizes genes better than other tools especially with rare tissue disorders or complex multi-tissue biological processes. This information can be critical in prioritization of candidate genes for sequence analysis.