MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences.

The sequence of chromosome 21 was a turning point for the understanding of Down syndrome. Comparative genomics is beginning to identify the functional components of the chromosome and that in turn will set the stage for the functional characterization of the sequences. Animal models combined with genome-wide analytical methods have proved indispensable for unravelling the mysteries of gene dosage imbalance.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12–13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

Down syndrome (DS) is one of the most frequent congenital birth defects, and the most common genetic cause of mental retardation. In most cases, DS results from the presence of an extra copy of chromosome 21. DS has a complex phenotype, and a major goal of DS research is to identify genotype -phenotype correlations. Cases of partial trisomy 21 and other HSA21 rearrangements associated with DS features could identify genomic regions associated with specific phenotypes. We have developed a BAC array spanning HSA21q and used array comparative genome hybridization (aCGH) to enable high-resolution mapping of pathogenic partial aneuploidies and unbalanced translocations involving HSA21. We report the identification and mapping of 30 pathogenic chromosomal aberrations of HSA21 consisting of 19 partial trisomies and 11 partial monosomies for different segments of HSA21. The breakpoints have been mapped to within B85 kb. The majority of the breakpoints (26 of 30) for the partial aneuploidies map within a 10-Mb region. Our data argue against a single DS critical region. We identify susceptibility regions for 25 phenotypes for DS and 27 regions for monosomy 21. However, most of these regions are still broad, and more cases are needed to narrow down the phenotypic maps to a reasonable number of candidate genomic elements per phenotype.

OBJECTIVE: In this study we used national data to determine changes in the prevalence of hospital admissions for medically complex children over a 15-year period. PATIENTS AND METHODS: Data from the Nationwide Inpatient Sample, a component of the Healthcare Cost and Utilization Project, was analyzed in 3-year increments from 1991 to 2005 to determine national trends in rates of hospitalization of children aged 8 days to 4 years with chronic conditions. Discharge diagnoses from the Nationwide Inpatient Sample were grouped into 9 categories of complex chronic conditions (CCCs). Hospitalization rates for each of the 9 CCC categories were studied both individually and in combination. Trends of children hospitalized with 2 specific disorders, cerebral palsy (CP) and bronchopulmonary dysplasia, with additional diagnoses in more than 1 CCC category were also examined. RESULTS: Hospitalization rates of children with diagnoses in more than 1 CCC category increased from 83.7 per 100 000 (1991–1993) to 166 per 100 000 (2003–2005) (P[r] < .001). The hospitalization rate of children with CP plus more than 1 CCC diagnosis increased from 7.1 to 10.4 per 100 000 (P = .002), whereas the hospitalization rates of children with bronchopulmonary dysplasia plus more than 1 CCC diagnosis increased from 9.8 to 23.9 per 100 000 (P < .001). CONCLUSIONS: Consistent increases in hospitalization rates were noted among children with diagnoses in multiple CCC categories, whereas hospitalization rates of children with CP alone have remained stable. The relative medical complexity of hospitalized pediatric patients has increased over the past 15 years.

The sequence of the tandem repeat sequence (D4Z4) associated with facioscapulohumeral muscular dystrophy (FSHD) has been determined: each copy of the 3.3 kb repeat contains two homeoboxes and two previously described repetitive sequences, LSau and a GC-rich low copy repeat designated hhspm3. By Southern blotting, FISH and isolation of cDNA and genomic clones we show that there are repeat sequences similar to D4Z4 at other locations in the human genome. Southern blot analysis of primate genomic DNA indicates that the copy number of D4Z4-like repeats has increased markedly within the last 25 million years. Two cDNA clones were isolated and found to contain stop codons and frameshifts within the homeodomains. An STS was produced to the cDNAs and analysis of a somatic cell hybrid panel suggests they map to chromosome 14. No cDNA clones mapping to the chromosome 4q35 D4Z4 repeats have been identified, although the possibility that they encode a protein cannot be ruled out. Although D4Z4 may not encode a protein, there is an association between deletions within this locus and FSHD. The D4Z4 repeats contain LSau repeats and are adjacent to 68 bp Sau3A repeats. Both of these sequences are associated with heterochromatic regions of DNA, regions known to be involved in the phenomenon of position effect variegation. We postulate that deletion of D4Z4 sequences could produce a position effect.

The production of mature germ cells capable of generating totipotent zygotes is a highly specialized and sexually dimorphic process. The transition from diploid primordial germ cell to haploid spermatozoa requires genome-wide reprogramming of DNA methylation, stage-and testis-specific gene expression, mitotic and meiotic division, and the histone-protamine transition, all requiring unique epigenetic control. Dnmt3L, a DNA methyltransferase regulator, is expressed during gametogenesis, and its deletion results in sterility. We found that during spermatogenesis, Dnmt3L contributes to the acquisition of DNA methylation at paternally imprinted regions, unique nonpericentric heterochromatic sequences, and interspersed repeats, including autonomous transposable elements. We observed retrotransposition of an LTR-ERV1 element in the DNA from Dnmt3L ؊/؊ germ cells, presumably as a result of hypomethylation. Later in development, in Dnmt3L ؊/؊ meiotic spermatocytes, we detected abnormalities in the status of biochemical markers of heterochromatin, implying aberrant chromatin packaging. Coincidentally, homologous chromosomes fail to align and form synaptonemal complexes, spermatogenesis arrests, and spermatocytes are lost by apoptosis and sloughing. Because Dnmt3L expression is restricted to gonocytes, the presence of defects in later stages reveals a mechanism whereby early genome reprogramming is linked inextricably to changes in chromatin structure required for completion of spermatogenesis.epigenetics ͉ meiosis ͉ histone modification ͉ heterochromatin ͉ DNA methylation

Stormorken syndrome is a rare autosomal-dominant disease with mild bleeding tendency, thrombocytopathy, thrombocytopenia, mild anemia, asplenia, tubular aggregate myopathy, miosis, headache, and ichthyosis. A heterozygous missense mutation in STIM1 exon 7 (c.910C>T; p.Arg304Trp) (NM_003156.3) was found to segregate with the disease in six Stormorken syndrome patients in four families. Upon sensing Ca(2+) depletion in the endoplasmic reticulum lumen, STIM1 undergoes a conformational change enabling it to interact with and open ORAI1, a Ca(2+) release-activated Ca(2+) channel located in the plasma membrane. The STIM1 mutation found in Stormorken syndrome patients is located in the coiled-coil 1 domain, which might play a role in keeping STIM1 inactive. In agreement with a possible gain-of-function mutation in STIM1, blood platelets from patients were in a preactivated state with high exposure of aminophospholipids on the outer surface of the plasma membrane. Resting Ca(2+) levels were elevated in platelets from the patients compared with controls, and store-operated Ca(2+) entry was markedly attenuated, further supporting constitutive activity of STIM1 and ORAI1. Thus, our data are compatible with a near-maximal activation of STIM1 in Stormorken syndrome patients. We conclude that the heterozygous mutation c.910C>T causes the complex phenotype that defines this syndrome.