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Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission. © 2012 The American Society of Human Genetics.

New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 μg. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-μg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ∼0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures.

In biological terms, aging can be defined as cellular senescence, which results in a diminished ability to respond to stress, increased homeostatic imbalance and risk of diseases such as cancer, and eventually death. Research in a variety of organisms has revealed that many factors are involved in the aging process at the molecular level. These include telomere-shortening, accumulation of genetic mutations, oxidative stress, and molecular pathways altered by quantitative and qualitative changes in nutrition (for review, see Vijg and Campisi 2008). More recently, several small-scale profiling studies have found directional epigenetic perturbations associated with aging in mammals (Fraga et al. 2005; Bjornsson et al. 2008; Boks et al. 2009; Christensen et al. 2009). Epigenetic modifications, such as DNA methylation and post-translational modifications of histone proteins, are indispensable for many aspects of genome function, including gene expression. The perturbation of epigenetic landscapes during aging could potentially influence cellular functions, thereby impacting on the development of various aging-associated phenotypes and/or diseases, such as cancer. However, until now, we have lacked a genome-scale view of aging-associated epigenomic dynamics. Such information would potentially reveal key genomic regions/features or molecular pathways that are susceptible to aging-related epigenetic perturbations. Here, we report the first genome-scale study of epigenomic dynamics during normal human aging. Our data support a model in which aging-associated differentially methylated regions (aDMRs) that gain methylation with age (hyper-aDMRs) arise in precursor/stem cells preferentially at bivalent chromatin domain promoters. This same category of promoters is frequently hypermethylated in cancers and in vitro cell culture, pointing to a novel mechanistic link between aberrant hypermethylation in cancer, aging, and cell culture.

V(D)J genomic recombination joins single gene segments to encode an extensive repertoire of antigen receptor specificities in T and B lymphocytes. This process initiates with double-stranded breaks adjacent to conserved recombination signal sequences that contain either 12- or 23-nucleotide spacer regions. Only recombination between signal sequences with unequal spacers results in productive coding genes, a phenomenon known as the “12/23 rule.” Here we present two novel genomic tools that allow the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues using second-generation sequencing. Further, we provide strong evidence that the 12/23 rule of genomic recombination is frequently violated under physiological conditions, resulting in unanticipated hybrid recombinations in ∼10% of Tcra excision circles. Hence, we demonstrate that strict adherence to the 12/23 rule is intrinsic neither to recombination signal sequences nor to the catalytic process of recombination and propose that nonclassical excision circles are liberated during the formation of antigen receptor diversity.

Ps230 is the largest representative of a 10-member family of proteins found in allPlasmodiumspecies. The family is defined by partially conserved, cysteine-rich double domains that are ≈350 aa in length and have one to three predicted disulfide bridges in each half. InPlasmodium falciparum, the most dangerous human malaria, Pf12 is the smallest member of the family, comprising just one double domain. Pfs230, with 7 double domains, and Pfs48/45 and Pfs47, with 1.5 double domains each, are found on the gamete surfaces and are thus potential candidates for a transmission-blocking vaccine. Fold prediction analyses of the double domains in Pfs230 reveal structural resemblance to SAG1 (surface antigen 1), a surface protein with a double β-sandwich structure from another apicomplexan parasite,Toxoplasma gondii. Template-directed modeling onto SAG1 clearly establishes the structural link between SAG1 and Pfs230 and produces positions for the cysteines that accord with the disulfide-bonding arrangement predicted for the Pfs230 family in earlier work. A highly clustered region of polymorphisms within the second double domain in Pfs230 maps to one side of the sandwich surface. This observation suggests that this region may be functional and reinforces the validity of these molecular models for the core domains of the Pfs230 family of proteins.

Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is ∼50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14+ monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D–discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D–associated methylation variable positions (T1D–MVPs). We confirmed these T1D–MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D–discordant MZ pairs (P = 0.035). Then, to establish the temporal origins of the T1D–MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D–MVPs are enriched in singletons both before (P = 0.001) and at (P = 0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (P = 0.0023). Combined, these results suggest that T1D–MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease., Author Summary Type 1 diabetes (T1D) is a complex autoimmune disease affecting >30 million people worldwide. It is caused by a combination of genetic and non-genetic factors, leading to destruction of insulin-secreting cells. Although significant progress has recently been made in elucidating the genetics of T1D, the non-genetic component has remained poorly defined. Epigenetic modifications, such as methylation of DNA, are indispensable for genomic processes such as transcriptional regulation and are frequently perturbed in human disease. We therefore hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology, and we performed a genome-wide DNA methylation analysis of a specific subset of immune cells (monocytes) from monozygotic twins discordant for T1D. This revealed the presence of T1D–specific methylation variable positions (T1D–MVPs) in the T1D–affected co-twins. Since these T1D–MVPs were found in MZ twins, they cannot be due to genetic differences. Additional experiments revealed that some of these T1D–MVPs are found in individuals before T1D diagnosis, suggesting they arise very early in the process that leads to overt T1D and are not simply due to post-disease associated factors (e.g. medication or long-term metabolic changes). T1D–MVPs may thus potentially represent a previously unappreciated, and important, component of type 1 diabetes risk.