Analysis of rare, exonic variation amongst subjects with autism spectrum disorders and population controls.

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.

Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders.

To characterize the role of rare complete human knockouts in autism spectrum disorders (ASDs), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a 2-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤ 5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudoautosomal regions on the X chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together, these results provide compelling evidence that rare autosomal and X chromosome complete gene knockouts are important inherited risk factors for ASD.

UNAC tetraloops: to what extent do they mimic GNRA tetraloops?

The structures of four small RNAs each containing a different version of the UNAC loop were determined in solution using NMR spectroscopy and restrained molecular dynamics. The UMAC tetraloops (where M is A or C) exhibited a typical GNRA fold including at least one hydrogen bond between the first U and fourth C. In contrast, UGAC and UUAC tetraloops have a different orientation of the first and fourth residues, such that they do not closely mimic the GNRA fold. Although the UMAC tetraloops are excellent structural mimics of the GNRA tetraloop backbone, sequence comparisons typically do not reveal co-variation between the two loop types. The limited covariation is attributed to differences in the location of potential hydrogen bond donors and acceptors as a result of the replacement of the terminal A of GNRA with C in the UMAC version. Thus, UMAC loops do not readily form the common GNRA tetraloop-receptor interaction. The loop at positions 863-866 in E. coli 16S ribosomal RNA appears to be a major exception. However, in this case the GNRA loop does not in fact engage in the usual base to backbone tertiary interactions. In summary, UMAC loops are not just an alternative sequence version of the GNRA loop family, but instead they expand the types of interactions, or lack thereof, that are possible. From a synthetic biology perspective their inclusion in an artificial RNA may allow the establishment of a stable loop structure while minimizing unwanted long range interactions or permitting alternative long-range interactions. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 617-628, 2012.

Patterns and rates of exonic de novo mutations in autism spectrum disorders.

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

Ribosome origins: the relative age of 23S rRNA Domains.

The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.

NMR structure and Mg2+ binding of an RNA segment that underlies the L7/L12 stalk in the E.coli 50S ribosomal subunit.

Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G*A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037-1043 and 1112-1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson-Crick/Watson-Crick (cis W.C.) G*A conformation for the tandem (G*A)2 in the analog and a minimally perturbed helical duplex stem. Mg2+ titration studies imply that the cis W.C. geometry of the tandem (G*A)2 probably allows O6 of G20 and N1 of A4 to coordinate with a Mg2+ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg2+ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg2+ coordination.

The application of cluster analysis in the intercomparison of loop structures in RNA.

We have developed a computational approach for the comparison and classification of RNA loop structures. Hairpin or interior loops identified in atomic resolution RNA structures were intercompared by conformational matching. The root-mean-square deviation (RMSD) values between all pairs of RNA fragments of interest, even if from different molecules, are calculated. Subsequently, cluster analysis is performed on the resulting matrix of RMSD distances using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis objectively reveals groups of folds that resemble one another. To demonstrate the utility of the approach, a comprehensive analysis of all the terminal hairpin tetraloops that have been observed in 15 RNA structures that have been determined by X-ray crystallography was undertaken. The method found major clusters corresponding to the well-known GNRA and UNCG types. In addition, two tetraloops with the unusual primary sequence UMAC (M is A or C) were successfully assigned to the GNRA cluster. Larger loop structures were also examined and the clustering results confirmed the occurrence of variations of the GNRA and UNCG tetraloops in these loops and provided a systematic means for locating them. Nineteen examples of larger loops that closely resemble either the GNRA or UNCG tetraloop were found in the large ribosomal RNAs. When the clustering approach was extended to include all structures in the SCOR database, novel relationships were detected including one between the ANYA motif and a less common folding of the GAAA tetraloop sequence.

RNA ligation and the origin of tRNA.

A straightforward origin of transfer RNA, (tRNA), is difficult to envision because of the apparently complex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structural motif have been identified in ribosomal RNA. These examples show that the long-range interactions between the T-loop and D-loops seen in tRNA are not an essential part of the motif but rather are facilitated by it. Thus, the core T-loop structure could already have existed in a small RNA prior to the emergence of the tRNA. The tRNA might then have arisen by expansion of an RNA that carried the motif. With this idea in mind, Di Giulio's earlier hypothesis that tRNA evolved by a simple duplication or ligation of a minihelix RNA was re-examined. It is shown that an essentially modern tRNA structure can in fact be generated by the ligation of two 38-nucleotide RNA minihelices of appropriate sequence. Although rare, such sequences occur with sufficient frequency, (1 in 3 x 10(7)), that they could be found in a standard in vitro RNA selection experiment. The results demonstrate that a series of RNA duplications, as previously proposed, can in principal account for the origin of tRNA. More generally, the results point out that RNA ligation can be a powerful driving force for increased complexity in the RNA World.

Frequent occurrence of the T-loop RNA folding motif in ribosomal RNAs.

Analysis of atomic resolution structures of the rRNAs within the context of the 50S and the 30S ribosomal subunits have revealed the presence of nine examples of a recurrent structural motif, first observed in the TpsiC loop of tRNAs. The key component of this T-loop motif is a UA trans Watson-Crick/Hoogsteen base pair stacked on a Watson-Crick pair on one side. This motif is stabilized by several noncanonical hydrogen bonds, facilitating RNA-RNA as well as RNA-protein interactions. In particular, the sugar edge of the purine on the 3' side of the pivotal uridine in the UA pair frequently forms a noncanonical base pair with a distant residue. The bulged-out bases, usually seen as part of the motif, also use their Watson-Crick edges to interact with nearby residues via base-specific hydrogen bonds. In certain occurrences, a backbone reversal is stabilized by specific hydrogen bonds as is observed in the U-turn motifs and the adenosine residue of the key UA pair interacts with a third base via its Watson-Crick edge, essentially generating a base triple.

NCIR: a database of non-canonical interactions in known RNA structures.

The secondary and tertiary structure of an RNA molecule typically includes a number of non-canonical base-base interactions. The known occurrences of these interactions are tabulated in the NCIR database, which can be accessed from http://prion.bchs.uh.edu/bp_type/. The number of examples is now over 1400, which is an increase of >700% since the database was first published. This dramatic increase reflects the addition of data from the recently published crystal structures of the 50S (2.4 A) and 30S (3.0 A) ribosomal subunits. In addition, non-canonical interactions observed in published crystal and NMR structures of tRNAs, group I introns, ribozymes, RNA aptamers and synthetic oligonucleotides are included. Properties associated with these interactions, such as sequence context, sugar pucker conformation, glycosidic angle conformation, melting temperature, chemical shift and free energy, are also reported when available. Out of the 29 anticipated pairs with at least two hydrogen bonds, 28 have been observed to date. In addition, several novel examples, not generally predicted, have also been encountered, bringing the total of such pairs to 36. Added to this list are a variety of single, bifurcated, triple and quadruple interactions. The most common non-canonical pairs are the sheared GA, GA imino, AU reverse Hoogsteen, and the GU and AC wobble pairs. The most frequent triple interaction connects N3 of an A with the amino of a G that is also involved in a standard Watson-Crick pair.