Epigenetic information is frequently erased near the start of each new generation (1). In some cases, however, epigenetic information can be transmitted from parent to progeny (epigenetic inheritance) (2). A particularly striking example of epigenetic inheritance is dsRNA-mediated gene silencing (RNAi) in C. elegans, which can be inherited for more than five generations (3–8). To understand this process we conducted a genetic screen for animals defective for transmitting RNAi silencing signals to future generations. This screen identified the gene heritable RNAi defective (hrde)-1. hrde-1 encodes an Argonaute (Ago) that associates with small interfering (si)RNAs in germ cells of the progeny of animals exposed to dsRNA. In nuclei of these germ cells, HRDE-1 engages the Nrde nuclear RNAi pathway to direct H3K9me3 at RNAi targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed siRNAs, which direct nuclear gene silencing in germ cells. In hrde-1 or nuclear RNAi deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Ago HRDE-1 directs gene-silencing events in germ cell nuclei, which drive multi-generational RNAi inheritance and promote immortality of the germ cell lineage. We propose that C. elegans uses the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans.

Copy number variants of the X-chromosome are a common cause of X-linked intellectual disability in males. Duplication of the Xq28 band has been known for over a decade to be the cause of the Lubs X-linked Mental Retardation Syndrome (OMIM 300620) in males and this duplication has been narrowed to a critical region containing only the genes MECP2 and IRAK1. In 2009, four families with a distal duplication of Xq28 not including MECP2 and mediated by low-copy repeats (LCRs) designated "K" and "L" were reported with intellectual disability and epilepsy. Duplication of a second more distal region has been described as the cause of the Int22h-1/Int22h-2 Mediated Xq28 Duplication Syndrome, characterized by intellectual disability, psychiatric problems, and recurrent infections. We report two additional families possessing the K/L-mediated Xq28 duplication with affected males having intellectual disability and epilepsy similar to the previously reported phenotype. To our knowledge, this is the second cohort of individuals to be reported with this duplication and therefore supports K/L-mediated Xq28 duplications as a distinct syndrome.

The CAGI-4 Hopkins clinical panel challenge was an attempt to assess state-of-the-art methods for clinical phenotype prediction from DNA sequence. Participants were provided with exonic sequences of 83 genes for 106 patients from the Johns Hopkins DNA Diagnostic Laboratory. Five groups participated in the challenge, predicting both the probability that each patient had each of the 14 possible classes of disease, as well as one or more causal variants. In cases where the Hopkins laboratory reported a variant, at least one predictor correctly identified the disease class in 36 of the 43 patients (84%). Even in cases where the Hopkins laboratory did not find a variant, at least one predictor correctly identified the class in 39 of the 63 patients (62%). Each prediction group correctly diagnosed at least one patient that was not successfully diagnosed by any other group. We discuss the causal variant predictions by different groups and their implications for further development of methods to assess variants of unknown significance. Our results suggest that clinically relevant variants may be missed when physicians order small panels targeted on a specific phenotype. We also quantify the false-positive rate of DNA-guided analysis in the absence of prior phenotypic indication.