High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects

A genetic investigation of sex bias in the prevalence of attention-deficit/hyperactivity disorder

Background Attention-deficit/hyperactivity disorder (ADHD) shows substantial heritability and is 2-7 times more common in males than females. We examined two putative genetic mechanisms underlying this sex bias: sex-specific heterogeneity and higher burden of risk in female cases. Methods We analyzed genome-wide autosomal common variants from the Psychiatric Genomics Consortium and iPSYCH Project (20,183 cases, 35,191 controls) and Swedish populationregister data (N=77,905 cases, N=1,874,637 population controls). Results Genetic correlation analyses using two methods suggested near complete sharing of common variant effects across sexes, with rg estimates close to 1. Analyses of population data, however, indicated that females with ADHD may be at especially high risk of certain comorbid developmental conditions (i.e. autism spectrum disorder and congenital malformations), potentially indicating some clinical and etiological heterogeneity. Polygenic risk score (PRS) analysis did not support a higher burden of ADHD common risk variants in female cases (OR=1.02 [0.98-1.06], p=0.28). In contrast, epidemiological sibling analyses revealed that the siblings of females with ADHD are at higher familial risk of ADHD than siblings of affected males (OR=1.14, [95% CI: 1.11-1.18], p=1.5E-15). Conclusions Overall, this study supports a greater familial burden of risk in females with ADHD and some clinical and etiological heterogeneity, based on epidemiological analyses. However, molecular genetic analyses suggest that autosomal common variants largely do not explain the sex bias in ADHD prevalence

Structural Characterization of the Saccharomyces cerevisiae THO Complex by Small-Angle X-Ray Scattering

The THO complex participates during eukaryotic mRNA biogenesis in coupling transcription to formation and nuclear export of translation-competent messenger ribonucleoprotein particles. In Saccharomyces cerevisiae, THO has been defined as a heteropentamer composed of the Tho2p, Hpr1p, Tex1p, Mft1p, and Thp2p subunits and the overall three-dimensional shape of the complex has been established by negative stain electron microscopy. Here, we use small-angle X-ray scattering measured for isolated THO components (Mft1p and Thp2p) as well as THO subcomplexes (Mft1p-Thp2p and Mft1p-Thp2p-Tho2p) to construct structural building blocks that allow positioning of each subunit within the complex. To accomplish this, the individual envelopes determined for Mft1p and Thp2p are first fitted inside those of the Mft1p-Thp2p and Mft1p-Thp2p-Tho2p complexes. Next, the ternary complex structure is placed in the context of the five-component electron microscopy structure. Our model reveals not only the position of each protein in the THO complex relative to each other, but also shows that the pentamer is likely somewhat larger than what was observed by electron microscopy

Docking of THO substructures.

<p><b>A.</b> Docking of the Mft1pΔC_232-392-Thp2p heterodimer (yellow) into the larger Mft1pΔC_336-392-Thp2p heterodimer (blue). The arrows indicate the proposed position of the Mft1p C-terminus. <b>B.</b> Docking of isolated Mft1pΔC_336-392 (green) and Thp2p (red) into the larger, heterodimeric Mft1pΔC_336-392-Thp2p envelope (blue). <b>C.</b> Docking of isolated Mft1pΔC_336-392 (green) and Thp2p (red) into the smaller, heterodimeric Mft1pΔC_232-392-Thp2p envelope (yellow). <b>D.</b> Docking of the larger, dimeric Mft1pΔC_336-392-Thp2p envelope (blue) into the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 envelope (purple). The arrows indicate the proposed position of the Tho2p C-terminus. Scale bars represent 100 Å.</p

Purification of THO subcomplexes.

<p><b>A.</b> Overview of the Tho2p, Mft1p and Thp2p constructs used relative to their full-length forms (boxed in grey). Blue boxes: Stretches of residues removed to obtain the construct directly below. Purple boxes: Strep II tag. <b>B.</b> THO complexes (THO1, 3, 4, and 5) were expressed and purified from <i>E. coli</i> in two steps and analysed by Coomassie-stained SDS-PAGE. The table shows for each construct the molecular weight of each protein and any associated tags (H = 6xHis, S = Strep II) as well as whether the protein is expressed (+/-). Positions on the gel for proteins confirmed by mass spectrometry are indicated with arrowheads and * indicates an <i>E. coli</i> protein contaminant. <b>C.</b> Purified THO subcomplexes analysed by Coomassie-stained SDS-PAGE: Heterodimeric THO4 (Mft1pΔC_270-392-Thp2p), heterodimeric THO3 (Mft1pΔC_336-392-Thp2p), and heterotrimeric THO3 (Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597). <b>D.</b> Overlay of gel filtration chromatograms obtained during isolation of the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 (Trimer) from the binary Mft1pΔC_336-392-Thp2p (Dimer). Between runs 1 (blue), 2, (red), and 3 (green), peak fractions were pooled, concentrated and re-applied to the column. Elution retention volumes are noted along with the positions of standards used for calibration: Blue Dextran (V₀, void volume, 2000 kDa), ferritin (440 kDa), aldolase (158 kDa), and conalbumin (75 kDa). Units on the y-axis are mAU absorption at 280 nm.</p

Envelopes of THO proteins and complexes obtained by SAXS.

<p>Molecular envelopes obtained using DAMMIF for THO proteins and subcomplexes. <b>A</b>. Mft1pΔC_336-392 (as isolated from the homodimer). <b>B</b>. Thp2p (as isolated from the homodimer). <b>C</b>. Mft1pΔC_232-392-Thp2p. <b>D</b>. Mft1pΔC_336-392-Thp2p. <b>E</b>. Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597. The proteins and complexes are each represented with an envelope shown in three perpendicular views as indicated. Scale bars represent 100 Å.</p

Placement of subunits in the THO complex.

<p><b>A.</b> Model showing the proposed location and orientation of the subunits Mft1p (green), Thp2p (red), and Tho2p (blue) within the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 THO complex (purple). The proposed location of the N and C terminal regions of Tho2p and Mft1p are indicated. Scale bars represent 100 Å. <b>B.</b> Comparison of the ternary Mft1pΔC_336-392-Thp2p-Tho2pΔC_1274-1597 SAXS envelope with the five-component EM reconstruction of the THO complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103470#pone.0103470-Pena1" target="_blank">[13]</a>. The positions of the proteins not part of our sample (Hpr1p and Tex1p) are indicated.</p

Comparison of sample types from variant calls—concordance rates.

<p>The concordance rates were calculated by pairwise comparison of variant calls before (upper panels) and after filtering (lower panels). The sample types compared were DBS_2x3.2 vs WB_ref in Pilot 1 <b>(A and B)</b>, DBS_2x3.2 vs WB_ref and WB_ref vs WB_ref replica in Pilot 2 <b>(C and D)</b> and DBS_2x1.6 vs WB_ref, DBS_2x3.2 vs WB_ref, DBS_2x3.2 vs DBS_2x1.6 and WB_ref vs WB_WGA_ref in Pilot 3 <b>(E and F)</b>. The rates have been presented per variant type: SNP, insertion, deletion and multiallelic calls, and comprise the averages of all comparisons made for a given sample pair, corresponding to the number of subjects in the pilot, i.e. Pilot 1 = 7, Pilot 2 = 8 and Pilot 3 = 7. Note that for comparisons using the DBS_2x1.6 sample type (see the fig), each individual replica was firstly compared to the WB_ref or DBS_2x3.2 sample types followed by the calculation of average values hereof, which were used in the figure.</p