Evidence that paternal expression of the epsilon-sarcoglycan gene accounts for reduced penetrance in myoclonus-dystonia

Myoclonus-dystonia (M-D) is a movement disorder characterized by rapid muscle contractions and sustained twisting and repetitive movements and has recently been associated with mutations in the epsilon-sarcoglycan gene (SGCE). The mode of inheritance is autosomal dominant with reduced penetrance upon maternal transmission, suggesting a putative maternal imprinting mechanism. We present an apparently sporadic M-D case and two patients from an M-D family with seemingly autosomal recessive inheritance. In both families, we detected an SGCE mutation that was inherited from the patients' clinically unaffected fathers in an autosomal dominant fashion. Whereas, in the first family, RNA expression studies revealed expression of only the mutated allele in affected individuals and expression of the normal allele exclusively in unaffected mutation carriers, the affected individual of the second family expressed both alleles. In addition, we identified differentially methylated regions in the promoter region of the SGCE gene as a characteristic feature of imprinted genes. Using a rare polymorphism in the promoter region in a family unaffected with M-D as a marker, we demonstrated methylation of the maternal allele, in keeping with maternal imprinting of the SGCE gene. Loss of imprinting in the patient with M-D who had biallelic expression of the SGCE gene was associated with partial loss of methylation at several CpG dinucleotides.     

Phage annealing proteins promote oligonucleotide-directed mutagenesis in <Emphasis Type="Italic">Escherichia coli</Emphasis> and mouse ES cells

Background

The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined.

Results

Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα) but only requires a phage annealing protein (RecT or Redβ). Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein.

Conclusion

Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

     

Linkage disequilibrium patterns and tagSNP transferability among European populations

The pattern of linkage disequilibrium (LD) is critical for association studies, in which disease-causing variants are identified by allelic association with adjacent markers. The aim of this study is to compare the LD patterns in several distinct European populations. We analyzed four genomic regions (in total, 749 kb) containing candidate genes for complex traits. Individuals were genotyped for markers that are evenly distributed at an average spacing of approximately 2-4 kb in eight population-based samples from ongoing epidemiological studies across Europe. The Centre d'Etude du Polymorphisme Humain (CEPH) trios of the HapMap project were included and were used as a reference population. In general, we observed a conservation of the LD patterns across European samples. Nevertheless, shifts in the positions of the boundaries of high-LD regions can be demonstrated between populations, when assessed by a novel procedure based on bootstrapping. Transferability of LD information among populations was also tested. In two of the analyzed gene regions, sets of tagging single-nucleotide polymorphisms (tagSNPs) selected from the HapMap CEPH trios performed surprisingly well in all local European samples. However, significant variation in the other two gene regions predicts a restricted applicability of CEPH-derived tagging markers. Simulations based on our data set show the extent to which further gain in tagSNP efficiency and transferability can be achieved by increased SNP density.     

A marker for the end of adolescence

Between childhood and adulthood, we go through puberty and adolescence. While the end of puberty is defined as the point of cessation of bone growth (epiphyseal closure; girls: 16 y; boys: 17.5 y), the end of adolescence (19 y) is defined less clearly, by a mixture of physical, psychological, social, and mental measures [1]. One conspicuous property of adolescence is the apparently unsaturable capacity to stay up late and to sleep in. Investigating ‘chronotypes’ we observed an abrupt change in the timing of sleep at around the age of 20 and propose this change as the first biological marker of the end of adolescence.     

High Abundance of Neotropical Drosophilids (Diptera: Drosophilidae) in Four Cultivated Areas of Central Brazil

The drosophilid assemblages of four cultivated areas (soy, bean, corn, and orange plantations) grown in the core of the Neotropical region were analyzed by comparing their abundances and compositions. The collections, which were gathered using 38 banana traps, captured 12,560 drosophilids, including nine Neotropical and six exotic species. Most of the flies were collected in the bean (43%) and soy (42%) fields. The composition and relative abundance of species also varied among cultivated areas, with orange orchards presenting the highest relative abundance of exotics due to the dominance of the Afrotropical Zaprionus indianus (Gupta). Crop plantations were dominated by a Neotropical species, Drosophila cardini (Sturtevant), which has been shown to be well adapted to dry and disturbed environments. We discuss the drosophilid assemblages of the cultivated areas, comparing them with assemblages from neighbor urban and natural environments. The low drosophilid richness found in this study is similar to the richness found in urban environments and lower than the drosophilid richness of forests, supporting a pattern already known for other taxa. The high abundance of drosophilids in cultivated areas, as well as the dominance of a Neotropical species (D. cardini) in the crop assemblages, was a surprising result.     

Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy with TLR4 agonists

Immunogenic cell death induced by anticancer chemotherapy is characterized by a series of molecular hallmarks that include the exodus of high-mobility group box 1 protein (HMGB1) from dying cells. HMGB1 is a nuclear nonhistone chromatin-binding protein. It is secreted at the late stages of cellular demise and engages Toll-like receptor4 (TLR4) on dendritic cells (DCs) to accelerate the processing of phagocytic cargo in the DC and to facilitate antigen presentation by DC to T cells. The absence of HMGB1 expression by dying tumor cells exposed to anthracyclines or oxaliplatin compromises DC-dependent T-cell priming by tumor-associated antigens. Here, we show that transplantable tumors exhibiting weak expression of nuclear HMGB1 respond to chemotherapy more effectively if the treatment is combined with the local or systemic administration of a highly purified and physiochemically defined and standardized lipopolysaccharide solution, which acts as a high-potency and exclusive TLR4 agonist, called Dendrophilin (DEN). The synergistic antitumor effects mediated by the combination of chemotherapy and immunotherapy relied upon the presence of the MyD88 (myeloid differentiation primary response gene) adapter of TLR4 (but not that of the TIR-domain-containing adapter-inducing interferon-β adapter), in line with the well-characterized action of DEN on the MyD88 signaling pathway. DEN and anthracyclines synergized to induce intratumoral accumulation of interferon-γ-producing CD4⁺ and CD8⁺ T lymphocytes. Moreover, DEN could restore the immunogenicity of dying tumor cells from which HMGB1 had been depleted by RNA interference. These findings underscore the potential clinical utility of combination regimens involving immunogenic chemotherapy and certain TLR4 agonists in advanced HMGB1-deficient cancers.     

Copy number variation and association over T-cell receptor genes—influence of DNA source

Genomic copy number variants (CNVs) are a common, heritable source of inter-individual differences in genomic sequence. Their influence on phenotypic variability and their involvement in the pathogenesis of several common diseases is well established and the object of many current studies. In the course of examining CNV association to various quantitative traits in a general population, we have detected a strong association of CNVs over the four TCR genes to lymphocyte and neutrophil numbers in blood. In a small replication series, we have further characterized the nature of these CNVs and found them not to be germline, but dependent on the origin of analysed DNA. Germline deletion and rearrangement around the T-cell receptor (TCR) genes naturally occurs in white blood cells. Blood DNA derived from persons with high lymphocyte counts generates variable intensity signals which behave like germline CNVs over these genes. As DNA containing a relative high proportion of these CNV-like events involving the TCR genes has the ability to influence genotype counts of SNPs in the regions of these genes, care should be taken in interpreting and replicating association signals on variants within these genes when blood-derived DNA is the only source of data.