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DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reactionor high-throughput RNA sequencingmethods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the chromosome 4q telomere allowing ectopic expression of theDUX4gene in skeletal muscle. Genetic analysis is difficult due to the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding theDUX4transcriptional start site. Within theDUX4exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.

The major determinant of disease severity in patients with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether their dystrophin gene (DMD) mutation disrupts the mRNA reading frame or allows expression of a partially functional protein. However, even in the complete absence of dystrophin, variability in disease severity is observed, and candidate gene studies have implicated several genes as possible modifiers. Our previous genome-wide association study (GWAS) for age at loss of ambulation (LOA) in DMD provided confirmation for the role of genetic modifiers of TGF-β signaling in disease progression. Here we present the largest genome-wide search to date for loci influencing disease severity in DMD patients. Availability of subjects for such studies is still quite limited, leading to modest sample sizes, which present a challenge for GWAS design. We have therefore taken special steps to minimize heterogeneity within our dataset at the DMD locus itself, taking a novel and conservative approach to mutation classification to effectively exclude the possibility of residual dystrophin expression. We have also utilized statistical methods that are well adapted to smaller sample sizes, including the use of a novel linear regression-like residual for time to ambulatory loss and the application of evidential statistics for the GWAS approach. Based on the resulting sample size of N = 419 patients, we have identified multiple potential candidate genetic modifier loci. In a companion paper to this one, we use a systematic bioinformatic pipeline to implicate specific genes within these loci as potential DMD modifiers.

Although the major determinant of disease severity in patients with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether their dystrophin gene (DMD) mutation disrupts the mRNA reading frame or allows expression of a partially functional protein, other genes have been proposed or demonstrated to modify the severity of disease progression. In a companion paper to this one, we describe our novel approaches to genome-wide association study (GWAS) of loss of ambulation (LOA) in the largest genome-wide search to date for loci influencing disease severity in DMD patients. Candidate regulatory SNPs that modify disease progression were identified using an evidential statistical paradigm and here we present a uniform application of recent functional genomic datasets to explore the potential functional impact of the top six candidate regions with PPLD scores of ≥0.4. The results of this analysis of the largest DMD GWAS survey to date elucidate recurrent and potentially new pathways for intervention in the dystrophinopathies.

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of non-coding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of DMD exon 2. We assessed the specificity of splice variation induced by AAV9.U7-ACCA delivery in the Dmd exon 2 duplication (Dup2) mouse model via an unbiased RNA-seq approach. Treatment-related effects on pre-mRNA splicing were quantified using local splicing variation (LSV) analysis. Filtering the transcriptome for differences in treatment-related splicing resulted in only 16 candidate off-target LSVs. Only a single candidate off-target LSV was found in both tissues and occurred at a known variable cassette exon. In contrast, four LSVs represented significant on-target correction of Dmd exon 2 splicing and transcriptome analysis showed correction of known dystrophin-deficient gene dysregulation. We conclude that the absence of off-target splicing induced by treatment with the U7-ACCA vector supports the continued clinical development of this approach.

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of noncoding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of

Significance Genome reduction is commonly observed in bacteria of several phyla engaging in obligate nutritional symbioses with insects. In Actinobacteria, however, little is known about the process of genome evolution, despite their importance as prolific producers of antibiotics and their increasingly recognized role as defensive partners of insects and other organisms. Here, we show that “Streptomyces philanthi,” a defensive symbiont of digger wasps, has a G+C-enriched genome in the early stages of erosion, with inactivating mutations in a large proportion of genes, causing dependency on its hosts for certain nutrients, which was validated in axenic symbiont cultures. Additionally, overexpressed catabolic and biosynthetic pathways of the bacteria inside the host indicate host–symbiont metabolic integration for streamlining and control of antibiotic production., Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but “Streptomyces philanthi” also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that—compared to in vitro symbiont cultures—“S. philanthi” in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host–symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner.

Background Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet‐primed PCR (TP‐PCR) is technically challenging and cost prohibitive for population surveys. Methods Here, we present a high throughput, low‐cost screening tool for CTG repeat expansions using TP‐PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. Results We determined that multimodal melt profiles from the TP‐PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri‐modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. Conclusion We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large‐scale testing programs such as population studies and newborn screening programs., Myotonic dystrophy type 1 is one of the most common forms of muscular dystrophy, caused by a CTG repeat expansion in the DMPK gene. Here, we present a high throughput, low‐cost screening tool to detect an expansion of the CTG repeat using TP‐PCR followed by melt curve analysis with saturating concentrations of SYBR GreenER dye. We demonstrate that accurate and robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots.

Objective:To determine whether the genetic prevalence of the CTG expansion in the DMPK gene associated with myotonic dystrophy (DM1) in an unbiased cohort is higher than previously reported population estimates, ranging from 5-20 per 100,000 individuals.Methods:This study used a cross-sectional cohort of de-identified dried blood spots (DBS) from the newborn screening program in the state of New York, taken from consecutive births from 2013-2014. Blood spots were screened for the CTG repeat expansion in the DMPK gene using triplet-repeat primed PCR and melt curve analysis. Melt curve morphology was assessed by four blinded reviewers to identify samples with possible CTG expansion. Expansion of the CTG repeat was validated by PCR fragment sizing using capillary electrophoresis for samples classified as positive or premutation to confirm the result. Prevalence was calculated as the number of samples with CTG repeat size ≥50 repeats compared to the overall cohort.Results:Out of 50,382 consecutive births, there were 24 with a CTG repeat expansion ≥50, consistent with a diagnosis of DM1. This represents a significantly higher DM1 prevalence of 4.76 per 10,000 births (95%CI 2.86, 6.67) or 1 in every 2,100 births. There were an additional 96 samples (19.1 per 10,000 or 1 in 525 births) with a CTG expansion in the DMPK gene in the premutation range (CTG)35-49.Conclusions:The prevalence of individuals with CTG repeat expansions in DMPK is up to five times higher than previous reported estimates. This suggests DM1, with multisystemic manifestations, is likely underdiagnosed in practice.

Gene networks for disorders of social behavior provide the mechanisms critical for identifying therapeutic targets and biomarkers. Large behavioral phenotypic effects of small human deletions make the positive sociality of Williams syndrome (WS) ideal for determining transcriptional networks for social dysfunction currently based on DNA variations for disorders such as autistic spectrum disorder (ASD) and schizophrenia (SCHZ). Consensus on WS networks has been elusive due to the need for larger cohort size, sensitive genome-wide detection and analytic tools. We report a core set of WS network perturbations in a cohort of 58 individuals (34 with typical, 6 atypical deletions and 18 controls). Genome-wide exon-level expression arrays robustly detected changes in differentially expressed gene (DEG) transcripts from WS deleted genes that ranked in the top 11 of 12 122 transcripts, validated by quantitative reverse transcription PCR, RNASeq and western blots. WS DEG’s were strictly dosed in the full but not the atypical deletions that revealed a breakpoint position effect on non-deleted CLIP2, a caveat for current phenotypic mapping based on copy number variants. Network analyses tested the top WS DEG’s role in the dendritic spine, employing GeneMANIA to harmonize WS DEGs with comparable query gene-sets. The results indicate perturbed actin cytoskeletal signaling analogous to the excitatory dendritic spines. Independent protein–protein interaction analyses of top WS DEGs generated a 100-node graph annotated topologically revealing three interacting pathways, MAPK, IGF1-PI3K-AKT-mTOR/insulin and actin signaling at the synapse. The results indicate striking similarity of WS transcriptional networks to genome-wide association study-based ASD and SCHZ risk suggesting common network dysfunction for these disorders of divergent sociality.

Eosinophilia (EOS) is an important component of airway inflammation and hyperresponsiveness in allergic reactions including those leading to asthma. Although cigarette smoking (CS) is a significant contributor to long-term adverse outcomes in these lung disorders, there are also the curious reports of its ability to produce acute suppression of inflammatory responses including EOS through poorly understood mechanisms. One possibility is that proinflammatory processes are suppressed by nicotine in CS acting through nicotinic receptor α7 (α7). Here we addressed the role of α7 in modulating EOS with two mouse models of an allergic response: house dust mites (HDM; Dermatophagoides sp.) and ovalbumin (OVA). The influence of α7 on EOS was experimentally resolved in wild-type mice or in mice in which a point mutation of the α7 receptor (α7E260A:G) selectively restricts normal signaling of cellular responses. RNA analysis of alveolar macrophages and the distal lung epithelium indicates that normal α7 function robustly impacts gene expression in the epithelium to HDM and OVA but to different degrees. Notable was allergen-specific α7 modulation of Ccl11 and Ccl24 (eotaxins) expression, which was enhanced in HDM but suppressed in OVA EOS. CS suppressed EOS induced by both OVA and HDM, as well as the inflammatory genes involved, regardless of α7 genotype. These results suggest that EOS in response to HDM or OVA is through signaling pathways that are modulated in a cell-specific manner by α7 and are distinct from CS suppression.

OBJECTIVE Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously, we reported that coding variants in latent transforming growth factor beta (TGFβ)-binding protein 4 (LTBP4) were associated with reduced TGFβ signaling and prolonged ambulation (p = 1.0 × 10-3 ) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole-genome association in the original cohort. METHODS We performed a genome-wide association study (GWAS) for single-nucleotide polymorphisms (SNPs) influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine-map association signals above the threshold for genome-wide significance. RESULTS Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p = 6.6 × 10-9 ) and rs710160 (chr19, p = 4.7 × 10-8 ). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, whereas the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFβ signaling by direct binding to LTBP4 and an inhibitor of proangiogenic nitric oxide signaling. INTERPRETATION Together with previous evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy. Ann Neurol 2018;84:234-245.

The human MN blood group antigens are isoforms of glycophorin A (GPA) encoded by the gene, GYPA, and are the most abundant erythrocyte sialoglycoproteins. The distribution of MN antigens has been widely studied in human populations yet the evolutionary and/or demographic factors affecting population variation remain elusive. While the primary function of GPA is yet to be discovered, it serves as the major binding site for the 175-kD erythrocyte-binding antigen (EB-175) of the malarial parasite, Plasmodium falciparum, a major selective pressure in recent human history. More specifically, exon two of GYPA encodes the receptor-binding ligand to which P. falciparum binds. Accordingly, there has been keen interest in understanding what impact, if any, natural selection has had on the distribution of variation in GYPA and exon two in particular. To this end, we resequenced GYPA in individuals sampled from both P. falciparum endemic (sub-Saharan Africa and South India) and non-endemic (Europe and East Asia) regions of the world. Observed patterns of variation suggest that GYPA has been subject to balancing selection in populations living in malaria endemic areas and in Europeans, but no such evidence was found in samples from East Asia, Oceania, and the Americas. These results are consistent with malaria acting as a selective pressure on GYPA, but also suggest that another selective force has resulted in a similar pattern of variation in Europeans. Accordingly, GYPA has perhaps a more complex evolutionary history, wherein on a global scale, spatially varying selective pressures have governed its natural history.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously we reported that coding variants in latent transforming growth factor beta-binding protein 4 (LTBP4) were associated with reduced TGFβ signaling and prolonged ambulation (p-value = 1.0 × 10(−3)) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole genome association in the original cohort. METHODS: We performed a genome-wide association study (GWAS) for SNPs influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine map association signals above the threshold for genome-wide significance. RESULTS: Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p-value = 6.6 × 10(−9)) and rs710160 (chr19, p-value = 4.7 × 10(−8)). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, while the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFβ signaling via direct binding to LTBP4 and an inhibitor of pro-angiogenic nitric oxide signaling. INTERPRETATION: Together with prior evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy.

Objective Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion—exon 45 (Δ45)—may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. Methods Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. Results As expected, deletions of either exons 45 to 47 (Δ45–47) or exons 45 to 48 (Δ45–48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45–46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44–45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. Interpretation The observation that Δ45–46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46–48) are better potential therapies than skipping of exon 46 alone. Ann Neurol 2015 Ann Neurol 2015;77:668–674

Background Harboring foregut microbial communities is considered a key innovation that allows herbivorous mammals to colonize new ecological niches. However, the functions of these chambers have only been well studied at the molecular level in ruminants. Here, we investigate gene expression in the foregut chamber of herbivorous rodents and ask whether these gene expression patterns are consistent with results in ruminants. We compared gene expression in foregut tissues of two rodent species: Stephen’s woodrat (Neotoma stephensi), which harbors a dense foregut microbial community, and the lab rat (Rattus norvegicus), which lacks such a community. Results We found that woodrats have higher abundances of transcripts associated with smooth muscle processes, specifically a higher expression of the smoothelin-like 1 gene, which may assist in contractile properties of this tissue to retain food material in the foregut chamber. The expression of genes associated with keratinization and cornification exhibited a complex pattern of differences between the two species, suggesting distinct molecular mechanisms. Lab rats exhibited higher abundances of transcripts associated with immune function, likely to inhibit microbial growth in the foregut of this species. Conclusions Some of our results were consistent with previous findings in ruminants (high expression of facilitative glucose transporters, lower expression of B4galnt2), suggestive of possible convergent evolution, while other results were unclear, and perhaps represent novel host-microbe interactions in rodents. Overall, our results suggest that harboring a foregut microbiota is associated with changes to the functions and host-microbe interactions of the foregut tissues. Electronic supplementary material The online version of this article (10.1186/s12864-017-4101-z) contains supplementary material, which is available to authorized users.

Beware the laboratory report: discrepancy in variant classification on reproductive carrier screening

Background: Binge drinking is responsible for over half of all alcohol-related deaths and results in significant health and economic costs to individuals and society. Knowledge of genetic aspects of this behavior, particularly as it emerges in young adulthood, could lead to improved treatment and prevention programs. Methods: We have focused on the association of variation in neuronal nicotinic receptor subunit genes (CHRNs) in a cohort of 702 Hispanic and non-Hispanic White young adults who are part of the Social and Emotional Contexts of Adolescent Smoking Patterns (SECASP) study. Fifty-five single nucleotide polymorphisms (SNPs) covering the variation in 5 CHRNs (CHRNA4, CHRNB2, CHRNA2, CHRNB3A6, and CHRNA5A3B4 )w ere studied. Results: Frequency of binge drinking and other correlated alcohol consumption measures were significantly associated with SNPs in CHRNA4 (p-values ranged from 0.0003 to 0.02), but not with SNPs in other CHRNs. This association was independent of smoking status in our cohort. Conclusions: Variants in CHRNA4 may contribute to risk of binge drinking in young adults in this cohort. Results will need to be confirmed in independent samples.

BACKGROUND c3orf75 is a conserved open reading frame within the human genome and has recently been identified as the Elongator subunit, ELP6 [1]. The Elongator enzyme complex has diverse roles, including translational control, neuronal development, cell migration and tumorigenicity [2]. OBJECTIVE To identify genes expressed early in human eosinophil development. METHODS Eosinophilopoiesis was investigated by gene profiling of IL-5 stimulated CD34+ cells; ELP6 mRNA is upregulated. A monoclonal antibody was raised to the recombinant protein predicted by the open reading frame. RESULTS ELP6 transcripts are upregulated in a human tissue culture model of eosinophil development during gene profiling experiments. Transcripts are expressed in most tissue types, as shown by reverse-transcriptase PCR. Western blot experiments show that human ELP6 is a 30 kDa protein expressed in the bone marrow, as well as in many other tissues. Flow cytometry experiments of human bone marrow mononuclear cells show that ELP6 is expressed intracellularly, in developing and mature human neutrophils, eosinophils and monocytes. CONCLUSIONS ELP6 is expressed intracellularly in developing and mature granulocytes and monocytes but not in lymphocytes and erythrocytes.

Few studies have sought to identify specific genetic markers associated with cigarettes per day (CPD) during adolescence and young adulthood, the period of greatest vulnerability for the development of nicotine dependence.We used a longitudinal design to investigate the effect of neuronal nicotinic acetylcholine receptor (CHRN) subunit genes on CPD from 15 to 21 years of age in young smokers of European descent (N = 439, 59% female). The number of CPD typically smoked during the previous 30 days was self-reported. Single nucleotide polymorphisms (SNPs) from CHRN genes were genotyped using DNA extracted from saliva samples collected at the 5-year assessment. Mixed-model analyses of SNP effects were computed across age at the time of assessment using log-transformed CPD as the phenotype. Data from the 1000 Genomes Project were used to clarify the architecture of CHRN genes to inform SNP selection and interpretation of results.CPD was associated with a CHRNB3A6 region tagged by rs2304297, with CHRNA5A3B4 haplotype C (tagged by rs569207), and with the CHRNA2 SNP rs2271920, ps.004. The reliability of single-SNP associations was supported by the correspondence between a more extensive set of SNP signals and the underlying genetic architecture. The 3 signals identified in this study appear to make independent contributions to CPD, and their combined effect accounts for 5.5% of the variance in log-transformed CPD.Level of CPD during adolescence and young adulthood is associated with CHRNB3A6, CHRNA5A3B4, and CHRNA2.

A spectrum of dystrophin mutations causes Duchenne muscular dystrophy (DMD). The extreme genomic size of the DMD gene (2.2 Mb) leads to kilobase size deletions or duplications in approximately 70% of DMD patients, and the remaining 30% arise from point mutations that introduce premature stop codons or disrupt splicing1. DMD mutations typically completely ablate dystrophin protein expression, while the milder Becker muscular dystrophy (BMD) mutations are most often in-frame deletions that produce truncated, but partially functional dystrophin. DMD patients are clinically defined by loss of ambulation before the age of 12, whereas BMD patients often remain ambulatory beyond age 15, a distinction that generally correlates with the degree of protein expression. Those who lose ambulation between 12 and 15 years are described as intermediate muscular dystrophy (IMD). Many DMD boys now ambulate beyond the age of 12 reflecting their treatment with prednisone or deflazacort, which remain the only medications that alter the natural history of DMD, prolonging ambulation for up to 1 to more than 3 years2-5. Despite the complete absence of dystrophin, the DMD phenotype is associated with a range in rates of progression, supporting that genetic or environmental modifiers help determine clinical course. Animal models, particularly mice, can be used to map genetic modifiers since inbred strains and standardized environmental conditions reduce variation. Like DMD patients, muscular dystrophy patients with a founder mutation in the dystrophin associated protein, γ-sarcoglycan, (SGCG) lose ambulation over a wide age range, consistent with the presence of genetic modifiers6, 7. The Sgcg null mouse was used in a genomewide screen for modifiers of muscular dystrophy and identified the Ltbp4 gene encoding the latent TGFβ binding protein (LTBP) 4 as a modifier of muscular dystrophy8. Given the shared pathological mechanisms between loss of dystrophin and its associated proteins, the sarcoglycans, we reasoned that LTBP4 may modify DMD. LTBP4, a member of the fibrillin superfamily, binds to transforming growth factor (TGF)-β in the extracellular matrix where it regulates TGFβ activity9. We now examined human LTBP4 polymorphisms in 254 nonambulatory patients from the United Dystrophinopathy Project cohort. This United Dystrophinopathy cohort includes 900 DMD patients, most who still ambulate, identified from seven sites representing by far the largest uniformly characterized DMD population. We assessed the effect of LTBP4 genotype on the age at ambulatory loss, since it reflects disease progression, is not influenced by ascertainment bias, and is a milestone for which the month or age is recalled by most patients and/or families. A protective haplotype in LTBP4 was identified.

Cigarette smoking (CS) is a principal contributor to a spectrum of devastating lung diseases whose occurrence and severity may vary between individuals and not appear for decades after prolonged use. One explanation for the variability and delay in disease onset is that nicotine, the addictive component of CS, acts through the ionotropic nicotinic acetylcholine receptor (nAChR) alpha7 (α7) to modulate anti-inflammatory protection. In this study we measured the impact α7 signaling has on the mouse distal lung response to side-stream CS exposure for mice of the control genotype (α7G) and those in which the α7-receptor signaling mechanisms are restricted by point mutation (α7E260A:G). Flow cytometry results show that after CS there is an increase in a subset of CD11c (CD11chi) alveolar macrophages (AMs) and histology reveals an increase in these cells within the alveolar space in both genotypes although the α7E260A:G AMs tend to accumulate into large aggregates rather than more widely distributed solitary cells common to the α7G lung after CS. Changes to lung morphology with CS in both genotypes included increased tissue cavitation due to alveolar expansion and bronchial epithelium dysplasia in part associated with altered club cell morphology. RNA-Seq analysis revealed changes in epithelium gene expression after CS are largely independent of the α7-genotype. However, the α7E260A:G genotype did reveal some unique variations to transcript expression of gene sets associated with immune responsiveness and macrophage recruitment, hypoxia, genes encoding mitochondrial respiration complex I and extracellular fibrillary matrix proteins (including alterations to fibrotic deposits in the α7G proximal airway bronchioles after CS). These results suggest α7 has a central role in modulating the response to chronic CS that could include altering susceptibility to associated lung diseases including fibrosis and cancer.

Objectives The collagen VI related muscular dystrophies (COL6-RD), Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are among the most common congenital muscular dystrophies and are characterized by distal joint laxity and a combination of distal and proximal joint contractures. Inheritance can be dominant negative (DN) or recessive depending on the type and location of the mutation. DN mutations allow incorporation of abnormal chains into secreted tetramers and are the most commonly identified mutation type in COL6-RD. Null alleles (nonsense, frameshift, and large deletions) do not allow incorporation of abnormal chains and act recessively. To better define the pathways disrupted by mutations in collagen VI, we have used a transcriptional profiling approach with RNA-Seq to identify differentially expressed genes in COL6-RD individuals from controls. Methods RNA-Seq allows precise detection of all expressed transcripts in a sample and provides a tool for quantification of expression data on a genomic scale. We have used RNA-Seq to identify differentially expressed genes in cultured dermal fibroblasts from 13 COL6-RD individuals (8 dominant negative and 5 null) and 6 controls. To better assess the transcriptional changes induced by abnormal collagen VI in the extracellular matrix (ECM); we compared transcriptional profiles from subjects with DN mutations and subjects with null mutations to transcriptional profiles from controls. Results Differentially expressed transcripts between COL6-RD and control fibroblasts include upregulation of ECM components and downregulation of factors controlling matrix remodeling and repair. DN and null samples are differentiated by downregulation of genes involved with DNA replication and repair in null samples. Conclusions Differentially expressed genes identified here may help identify new targets for development of therapies and biomarkers to assess the efficacy of treatments.

Objective: Creatine kinase (CK) levels are increased on dried blood spots in newborns related to the birthing process. As a marker for newborn screening, CK in Duchenne muscular dystrophy (DMD) results in false-positive testing. In this report, we introduce a 2-tier system using the dried blood spot to first assess CK with follow-up DMD gene testing. Methods: A fluorometric assay based upon the enzymatic transphosphorylation of adenosine diphosphate to adenosine triphosphate was used to measure CK activity. Preliminary studies established a population-based range of CK in newborns using 30,547 deidentified anonymous dried blood spot samples. Mutation analysis used genomic DNA extracted from the dried blood spot followed by whole genome amplification with assessment of single-/multiexon deletions/duplications in the DMD gene using multiplex ligation-dependent probe amplification. Results: DMD gene mutations (all exonic deletions) were found in 6 of 37,649 newborn male subjects, all of whom had CK levels >2,000U/l. In 3 newborns with CK >2,000U/l in whom DMD gene abnormalities were not found, we identified limb-girdle muscular dystrophy gene mutations affecting DYSF, SGCB, and FKRP. Interpretation: A 2-tier system of analysis for newborn screening for DMD has been established. This path for newborn screening fits our health care system, minimizes false-positive testing, and uses predetermined levels of CK on dried blood spots to predict DMD gene mutations. ANN NEUROL 2012;71:304–313

The use of hybridization-based methods for DMD mutation analysis is increasingly common. We report a case of Becker Muscular Dystrophy in which discrepant results between a PCR-based (SCAIP) and a comparative genomic hybridization assay incompletely characterized the mutation (an inversion of exons 23 and 24). These results demonstrate the limits of sensitivity and specificity of both tests, and highlight the need for more detailed analysis when intronic deletions are detected by comparative genome hybridization methods.

Nonsense mutations are usually predicted to function as null alleles due to premature termination of protein translation. However, nonsense mutations in the DMD gene, encoding the dystrophin protein, have been associated with both the severe Duchenne Muscular Dystrophy (DMD) and milder Becker Muscular Dystrophy (BMD) phenotypes. In a large survey, we identified 243 unique nonsense mutations in the DMD gene, and for 210 of these we could establish definitive phenotypes. We analyzed the reading frame predicted by exons flanking those in which nonsense mutations were found, and present evidence that nonsense mutations resulting in BMD likely do so by inducing exon skipping, confirming that exonic point mutations affecting exon definition have played a significant role in determining phenotype. We present a new model based on the combination of exon definition and intronic splicing regulatory elements for the selective association of BMD nonsense mutations with a subset of DMD exons prone to mutation-induced exon skipping.

Manifesting carriers of DMD gene mutations may present diagnostic challenges, particularly in the absence of a family history of dystrophinopathy. We review the clinical and genetic features in fifteen manifesting carriers identified among 860 subjects within the United Dystrophinopathy Project, a large clinical dystrophinopathy cohort whose members undergo comprehensive DMD mutation analysis. We defined manifesting carriers as females with significant weakness, excluding those with only myalgias/cramps. DNA extracted from peripheral blood was used to study X chromosome inactivation patterns. Among these manifesting carriers, age at symptom onset ranged from 2 to 47 years. Seven had no family history and eight had male relatives with Duchene muscular dystrophy (DMD). Clinical severity among the manifesting carriers varied from a DMD-like progression to a very mild Becker muscular dystrophy-like phenotype. Eight had exonic deletions or duplications and six had point mutations. One patient had two mutations (an exonic deletion and a splice site mutation), consistent with a heterozygous compound state. The X chromosome inactivation pattern was skewed toward nonrandom in four out of seven informative deletions or duplications but was random in all cases with nonsense mutations. We present the results of DMD mutation analysis in this manifesting carrier cohort, including the first example of a presumably compound heterozygous DMD mutation. Our results demonstrate that improved molecular diagnostic methods facilitate the identification of DMD mutations in manifesting carriers, and confirm the heterogeneity of mutational mechanisms as well as the wide spectrum of phenotypes.

Mutations in the collagen VI genes (COL6A1, COL6A2 and COL6A3) result in Ullrich congenital muscular dystrophy (CMD), Bethlem myopathy or phenotypes intermediate between Ullrich CMD and Bethlem myopathy. While Ullrich CMD can be caused by either recessively or dominantly acting mutations, Bethlem myopathy has thus far been described as an exclusively autosomal dominant condition. We report two adult siblings with classic Bethlem myopathy who are compound heterozygous for a single nucleotide deletion (exon 23; c.1770delG), leading to in-frame skipping of exon 23 on the maternal allele, and a missense mutation p.R830W in exon 28 on the paternal allele. The parents are carriers of the respective mutations and are clinically unaffected. The exon skipping mutation in exon 23 results in a chain incapable of heterotrimeric assembly, while p.R830W likely ameliorates the phenotype into the Bethlem range. Thus, autosomal recessive inheritance can also underlie Bethlem myopathy, supporting the notion that Ullrich CMD and Bethlem myopathy are part of a common clinical and genetic spectrum.

Introduction Previous research revealed significant associations between haplotypes in the CHRNA5-A3-B4 subunit cluster and scores on the Fagerstrom Test for Nicotine Dependence among individuals reporting daily smoking by age 17. The present study used subsamples of participants from that study to investigate associations between the CHRNA5-A3-B4 haplotypes and an array of phenotypes not analyzed previously (i.e., withdrawal severity, ability to stop smoking, and specific scales on the Wisconsin Inventory of Smoking Dependence Motives (WISDM-68) that reflect loss of control, strong craving, and heavy smoking. Methods Two cohorts of current or former smokers (N = 886) provided both self-report data and DNA samples. One sample (Wisconsin) comprised smokers making a quit smoking attempt, which permitted the assessment of withdrawal and relapse during the attempt. The other sample (Utah) comprised participants studied for risk factors for nicotine dependence and chronic obstructive pulmonary disease and included individuals originally recruited in the Lung Health Study. Results The CHRNA5-A3-B4 haplotypes were significantly associated with the targeted WISDM-68 scales (Tolerance, Craving, Loss of Control) in both samples of participants but only among individuals who began smoking early in life. The haplotypes were significantly associated with relapse likelihood and withdrawal severity, but these associations showed no evidence of an interaction with age at daily smoking. Discussion The CHRNA5-A3-B4 haplotypes are associated with a broad range of nicotine dependence phenotypes, but these associations are not consistently moderated by age at initial smoking.

Hepatic siderosis is common in patients with porphyria cutanea tarda (PCT). Mutations in the hereditary hemochromatosis (hh) gene (HFE) explain the siderosis in approximately 20% patients, suggesting that the remaining occurrences result from additional genetic and environmental factors. Two genes known to modify iron loading in hh are hepcidin (HAMP) and hemojuvelin (HJV). To determine if mutations in or expression of these genes influenced iron overload in PCT, we compared sequences of HAMP and HJV in 96 patients with PCT and 88 HFE C282Y homozygotes with marked hepatic iron overload. We also compared hepatic expression of these and other iron-related genes in a group of patients with PCT and hh. Two intronic polymorphisms in HJV were associated with elevated serum ferritin in HFE C282Y homozygotes. No exonic polymorphisms were identified. Sequencing of HAMP revealed exonic polymorphisms in 2 patients with PCT: heterozygosity for a G→A transition (G71D substitution) in one and heterozygosity for an A→G transition (K83R substitution) in the other. Hepatic HAMP expression in patients with PCT was significantly reduced, regardless of HFE genotype, when compared with patients with hh but without PCT with comparable iron overload. These data indicate that the hepatic siderosis associated with PCT likely results from dysregulated HAMP.

The murine model of Lyme disease provides a unique opportunity to study the localized host response to similar stimulus, Borrelia burgdorferi, in the joints of mice destined to develop severe arthritis (C3H) or mild disease (C57BL/6). Pathways associated with the response to infection and the development of Lyme arthritis were identified by global gene expression patterns using oligonucleotide microarrays. A robust induction of IFN-responsive genes was observed in severely arthritic C3H mice at 1 wk of infection, which was absent from mildly arthritic C57BL/6 mice. In contrast, infected C57BL/6 mice displayed a novel expression profile characterized by genes involved in epidermal differentiation and wound repair, which were decreased in the joints of C3H mice. These expression patterns were associated with disease state rather than inherent differences between C3H and C57BL/6 mice, because C57BL/6-IL-10−/− mice infected with B. burgdorferi develop more severe arthritis than C57BL/6 mice and displayed an early gene expression profile similar to C3H mice. Gene expression profiles at 2 and 4 wk postinfection revealed a common response of all strains that was likely to be important for the host defense to B. burgdorferi and mediated by NF-κB-dependent signaling. The gene expression profiles identified in this study add to the current understanding of the host response to B. burgdorferi and identify two novel pathways that may be involved in regulating the severity of Lyme arthritis.

The ability to sense bitter taste is vital for detecting toxins in food. Phenylthiocarbamide (PTC) is unusual in that to us it tastes either very bitter, or almost tasteless, depending on an individual's genetics. In a classic Nature paper in 1939, R. A. Foster, E. B. Ford and J. S. Huxley showed that chimpanzees also have variable PTC sensitivity, which was thought to reflect a shared ancient genetic polymorphism maintained by natural selection. Now that the TAS2R38 locus responsible for human PTC sensitivity has been identified, Wooding et al. have revisited the comparison. TAS2R38 is also involved in chimpanzees but, surprisingly, the mutations responsible for the polymorphism differ in the two species. ‘Non-taster’ alleles seem to have evolved at least twice, independently, during hominid evolution. The cover photo by D. J. Cox was taken in Chattanooga Zoo, Tennessee, in March 2003; the chimp is gathering as much fruit as he can carry. It was reported over 65 years ago that chimpanzees, like humans, vary in taste sensitivity to the bitter compound phenylthiocarbamide (PTC)1. This was suggested to be the result of a shared balanced polymorphism, defining the first, and now classic, example of the effects of balancing selection in great apes. In humans, variable PTC sensitivity is largely controlled by the segregation of two common alleles at the TAS2R38 locus, which encode receptor variants with different ligand affinities2,3,4. Here we show that PTC taste sensitivity in chimpanzees is also controlled by two common alleles of TAS2R38; however, neither of these alleles is shared with humans. Instead, a mutation of the initiation codon results in the use of an alternative downstream start codon and production of a truncated receptor variant that fails to respond to PTC in vitro. Association testing of PTC sensitivity in a cohort of captive chimpanzees confirmed that chimpanzee TAS2R38 genotype accurately predicts taster status in vivo. Therefore, although Fisher et al.'s observations1 were accurate, their explanation was wrong. Humans and chimpanzees share variable taste sensitivity to bitter compounds mediated by PTC receptor variants, but the molecular basis of this variation has arisen twice, independently, in the two species.

We sequenced and analyzed the subtelomeric regions of 1356 macronuclear "nanochromosomes" of the spirotrichous ciliate Oxytricha trifallax. We show that the telomeres in this species have a length of 20 nt, with minor deviations; there is no correlation between telomere lengths at the two ends of the molecule. A search for open reading frames revealed that the 3' and 5' untranslated regions are short, with a median length of approximately 130 nt, and that surprisingly there are no detectable differences between sequences upstream and downstream of genes. Our results confirm a previously reported purine bias in the first approximately 80 nucleotides of the subtelomeric regions, but with this larger data set we curiously detected a 10 bp periodicity in the bias; we relate this finding to the possible regulatory and structural functions these regions must serve. Palindromic sequences in opposing subtelomeric regions, although present in most sequences, are not statistically significant.

Familial combined hyperlipidemia (FCHL) is the most common familial dyslipidemia, and is implicated in up to 20% of cases of premature coronary heart disease. Positive linkage to chromosome 1q was found in FCHL families participating in the NHLBI Family Heart Study (FHS), replicating linkage found in other studies. The HcB-19 mouse, which shares phenotypes with FCHL, was shown in other studies to have a nonsense mutation in the thioredoxin interacting protein gene (txnip). txnip is a gene on mouse chromosome 3 in a region syntenic with the 1q human FCHL linkage region. We re-sequenced the human homolog of mouse txnip in the FHS sample and identified nine single nucleotide polymorphisms (SNPs). We did not observe the nonsense mutation found in the HcB-19 mouse, and only three of the SNPs discovered were sufficiently polymorphic for analysis. No association between FCHL and the TXNIP gene was found. Within FCHL cases, presence of variants also did not significantly affect body mass index or levels of lipids, insulin, or glucose. Our results suggest that in this sample, TXNIP does not play a major role in FCHL or related traits, and is unlikely to account for the positive evidence of linkage in this region.

Bacteriophage T4 DNA topoisomerase gene 60 contains a 50 nucleotide untranslated region within the coding sequence of its mRNA. Translational bypass of this sequence by elongating ribosomes has been postulated for the mode of synthesis of an 18 kd polypeptide specified by the split coding segments. Ribosome bypass of the untranslated region also occurs when a segment of gene 60 is fused to lacZ and expressed in E. coli. The efficiency of bypass in these gene 60-lacZ fusions approaches 100%. Here, mutations that delete, insert, or substitute nucleotides from gene 60-lacZ fusions are examined. Essential features necessary for high level gap bypass emerging from this analysis are a cis-acting nascent peptide sequence, a short duplication bordering the gap, and a stop codon contained in a stem-loop structure at the 5' junction of the gap.

Ciliates, microbial eukaryotes, have been important experimental organisms for >100 years, contributing to the discovery and understanding of self-splicing RNA, telomere biochemistry and transcriptional regulation by histone modification. We have completed a pilot sequencing project of the Oxytricha trifallax macronuclear (MAC) genome. When complete, this genome sequence, along with the emerging Tetrahymena thermophila and Paramecium tetraurelia sequences, will provide an important resource for basic eukaryotic cell biology, molecular evolution and the related apicomplexan parasites, such as Plasmodium spp.

The response by individuals to nicotine is likely to reflect the interaction of this compound with target nAChRs. However, resolving how different genetic backgrounds contribute to unique mouse strain-specific responses to this compound remains an important and unresolved issue. To examine this question in detail, expression of the nicotine acetylcholine receptor (nAChR) subunits α3, α4, α5, α7, β2, and β4 was measured in the dorsal hippocampus using immunohistochemistry in mouse strains or lines BALB/c, C3H/J, C57BL/6, CBA/J, DBA/2, Long Sleep (LS), Short Sleep (SS), and CF1. The nAChRs in all mice colocalized with glutamic acid decarboxylase (GAD)-positive interneurons that were subclassified into at least four groups based on nAChR subunit heterogeneity. A notable difference between mouse strains was the expression of nAChRs by astrocyte subpopulations in CA1 subregions whose numbers vary inversely with nAChR-immunostained neurons. This novel relationship also correlated with published parameters of strain sensitivity to nicotine. Attempts to identify the origin of this significant difference in nAChR expression among strains included comparison of the entire nAChRα4 gene sequence. Although multiple polymorphisms were identified, including two that changed nAChRα4 amino acid coding, none of these clearly correlate with strain-related differences in cell type-specific nAChR expression. These findings suggest that mouse strain-specific behavioral and physiological responses to nicotine are likely to be a reflection of a complex interplay between genetic factors that shape differences in expression and cellular architecture of this modulatory neurotransmitter system in the mammalian nervous system. J. Comp. Neurol. 468:334–346, 2004. © 2003 Wiley-Liss, Inc.

The ubiquitin ligase NEDD4L is a candidate gene for essential hypertension on both functional and genetic grounds. By targeting the epithelial sodium channel (ENaC) for degradation, NEDD4L is a significant determinant of sodium reabsorption in the distal nephron. Genetic linkage has been reported to a region of chromosome 18q harboring the gene, with phenotypes that include a rare orthostatic hypotension disorder, essential hypertension, and postural change in systolic blood pressure. A systematic search for genetic polymorphisms by resequencing exons and intron boundaries in 48 Caucasians yielded 38 variants. Among these, variant 13 is common, with either G (70%) or A (30%) as the last nucleotide of a putative exon 1. This mutation could affect the generation of a previously unrecognized splice isoform. In subsequent experiments, (1) we confirmed the presence of this putative isoform in both kidney and adrenals; (2) we established that variant 13-A leads to the systematic use of an alternative splice site, generating a transcript encoding a nonfunctional protein; and (3) we demonstrated differences in tissue-specific expression of the novel isoform relative to its previously reported counterpart. Variant 13-A precludes the formation of a transcript encoding a full-length Ca2+-dependent lipid-binding (C2) domain with very high evolutionary conservation among NEDD4L orthologs. A similar C2 domain in the paralogous NEDD4 gene plays a significant role in the transfer of its product to the apical membrane of epithelial cells. Differential function of NEDD4L isoforms could prove significant in blood pressure regulation through an effect on ENaC-dependent sodium reabsorption.

CYP1A2 is a cytochrome P450 gene that is involved in human physiological responses to a variety of drugs and toxins. To investigate the role of population history and natural selection in shaping genetic diversity in CYP1A2, we sequenced a 3.7-kb region 5' from CYP1A2 in a diverse collection of 113 individuals from three major continental regions of the Old World (Africa, Asia, and Europe). We also examined sequences in the 90-member National Institutes of Health DNA Polymorphism Discovery Resource (PDR). Eighteen single-nucleotide polymorphisms (SNPs) were found. Most of the high-frequency SNPs found in the Old World sample were also found in the PDR sample. However, six SNPs were detected in the Old World sample but not in the PDR sample, and two SNPs found in the PDR sample were not found in the Old World sample. Most pairs of SNPs were in complete linkage disequilibrium with one another, and there was no indication of a decline of disequilibrium with physical distance in this region. The average +/- SD nucleotide diversity in the Old World sample was 0.00043+/-0.00026. The African population had the highest level of nucleotide diversity and the lowest level of linkage disequilibrium. Two distinct haplotype clusters with broadly overlapping geographical distributions were present. Of the 17 haplotypes found in the Old World sample, 12 were found in the African sample, 8 were found in Indians, 5 were found in non-Indian Asians, and 5 were found in Europeans. Haplotypes found outside Africa were mostly a subset of those found within Africa. These patterns are all consistent with an African origin of modern humans. Seven SNPs were singletons, and the site-frequency spectrum showed a significant departure from neutral expectations, suggesting population expansion and/or natural selection. Comparison with outgroup species showed that four derived SNPs have achieved high (0.90) frequencies in human populations, a trend consistent with the action of positive natural selection. These patterns have a number of implications for disease-association studies in CYP1A2 and other genes.

Here we describe a high-throughput screen to isolate transcripts with spatially restricted patterns of expression in early embryos. Our approach utilizes robotic automation for rapid analysis of sequence-selected cDNAs in a whole-mount in situ hybridization assay. We determined the spatial distribution of a random collection of 778 different genes from an embryonic cDNA library and show that a significant fraction of these exhibit patterned profiles of expression. In addition, gene ontology studies revealed groups of gene products exhibiting shared expression patterns, providing new insights into the largely overlooked effector molecules that function in development. As described in this paper, automated hybridization to whole-mount embryos in situ proved to be straightforward and provided us with a very powerful method for the global survey of gene expression in early embryos. From the perspective of biological significance, our finding that many spatially restricted transcripts correspond to loci encoding novel transcripts that have not been previously identified in nearly saturating genetic screens for maternal effect and zygotic lethals is particularly notable.[Supplementary material available online at http://www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N. Brown]

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.