L1Base 2: more retrotransposition-active LINE-1s, more mammalian genomes

LINE-1 (L1) insertions comprise as much as 17% of the human genome sequence, and similar proportions have been recorded for other mammalian species. Given the established role of L1 retrotransposons in shaping mammalian genomes, it becomes an important task to track and annotate the sources of this activity: full length elements, able to encode the cis and trans acting components of the retrotransposition machinery. The L1Base database (http://l1base.charite.de) contains annotated full-length sequences of LINE-1 transposons including putatively active L1s. For the new version of L1Base, a LINE-1 annotation tool, L1Xplorer, has been used to mine potentially active L1 retrotransposons from the reference genome sequences of 17 mammals. The current release of the human genome, GRCh38, contains 146 putatively active L1 elements or full length intact L1 elements (FLIs). The newest versions of the mouse, GRCm38 and the rat, Rnor_6.0, genomes contain 2811 and 492 FLIs, respectively. Most likely reflecting the current level of completeness of the genome project, the latest reference sequence of the common chimpanzee genome, PT 2.19, only contains 19 FLIs. Of note, the current assemblies of the dog, CF 3.1 and the sheep, OA 3.1, genomes contain 264 and 598 FLIs, respectively. Further developments in the new version of L1Base include an updated website with implementation of modern web server technologies. including a more responsive design for an improved user experience, as well as the addition of data sharing capabilities for L1Xplorer annotation

Exomes of 85 European individuals (CEU) as well as 88 African individuals (YRI) were filtered for rare compound heterozygous candidate variants.

<p>A) In average around 230 variants pass the filter in CEU exomes and 309 in YRI exomes. B) The potential compound heterozygotes are distributed over 31 genes in CEU individuals and 67 genes in YRI individuals. C) Altogether 1998 genes harbored potential compound heterozygous variants in the tested individuals and compound heterozygotes in 1066 genes occurred only in singular cases.</p

Illustration of mapping artifacts resulting in false positive variant detection.

<p>The illustrated sample carries a mutation in the maternal copy of a pseudogene of <i>NBPF10</i>. If the pseudogene is not included in the reference sequence, the reads originating from this pseudogene are mismapped. This may result in a false variant call. Indicative for false genotype calls are proportions of reads supporting the alternate allele that strongly deviate from 0.5 or 1.</p

The length of the coding sequence and the mean number of rare alleles per gene.

<p>In an average healthy individual from the 5000 exomes project there is more than one rare heterozygous variant in <i>MUC16</i> that has an allele frequency below 0.01 in the reference population. In contrast, the coding sequence of <i>PIGO</i> is much shorter and rare heterozygous variants occur in less than 8 out of 1000 exomes.</p

Compound Heterozygote Filtering Rules.

<p>If both parents of the index patient are unaffected it is not possible that one of the heterozygous disease causing mutations is present in a heterozygous state in both parents unless a recombination occurred between this variant and the second compound heterozygous mutation.</p

Filtering results for compound heterozyotes in a case study.

<p>With the filter settings for genotype frequency <0.01, effect on protein level (functional filter: missense, nonsense, stop loss, splice site, insertions or deletions), and compound heterozygous yields six variants in three genes. <i>MUC16</i> and <i>NBPR10</i> are both genes from large gene families known for their high variability and detection artifacts due to pseudogenes. The heterozygotes in <i>PIGO</i> remain as the likeliest candidates. The <i>Show</i> icon at the right end of the line links to an expert curated annotation database that indicates that the mutation in <i>PIGO</i> is causing Hyperphosphatasia with mental retardation syndrome and has been published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070151#pone.0070151-Krawitz1" target="_blank">[9]</a>. The gene view for <i>PIGO</i> lists all variant annotations for this gene and links to further knowledge bases. The length of the coding sequence of the longest transcript (max. CDS) and the mean number of rare heterozygous variant calls per exome (MRHC) are important parameters for the assessment of candidate genes.</p

AP1 and CTCF binding sites are overrepresented in Group 1 HOX-TF binding sites.

<p>(A) <i>De novo</i> motif analysis of all Group 1 HOX-TF peaks (here, HOXA10 results) identifies overrepresented binding sites. A comparison of these motifs to known AP1 and CTCF motifs is shown below. (B) Centrimo analysis identifies the position of best binding site matches in all peak sequences. Blue and black lines indicate enrichment of the given HOXA10 or HOXD13 motif shown below, respectively. Yellow lines indicate enrichment for CTCF motif shown below. (C) The overlap of peaks containing a HOX (Group 1- blue, Group 2- black) or a CTCF (yellow) binding site. The red overlap indicates peaks containing a HOX and a CTCF binding site.</p

Viral expression of HOX-TFs in chicken micromass culture (chMM) modifies chondrogenic cell differentiation.

<p>(A) Individual HOX-TF expressing chMM cultures stained with Alcian blue (top) and Eosin (bottom). Alcian Blue staining of four biological replicates was quantified and compared to mock-infected chMM. Error bars indicate standard deviation from four replicates. (B) Hierarchical clustering of differentially regulated genes in the nine HOX-TF expressing cultures (all RNA-seq shown in replicates). The top 50 differentially regulated genes from each sample were selected (Criteria: p-Val ≤10e-5, base mean≥30, fold change≥2) and for each replicate, the log2-transformed fold changes relative to mock-infected cultures of these 205 genes were subjected to hierarchical clustering.</p

Gfi1-ko/ko mice kept under nonSPF conditions develop osteopenia.

<p><b>(A)</b> Trabecular bone of vertebrae was measured with microCT. Gfi1-ko/ko mice kept under nonSPF conditions developed severe osteopenia as indicated by 60% BV/TV reduction compared to Gfi1-wt/wt mice. Upon SPF breeding Gfi1-ko/ko mice show a 15% reduction in BV/TV. However, SPF+nonSPF conditions induced intermediate osteopenia characterized by approx. 30% BV/TV reduction. Please note Gfi1-ko/ko mice kept at nonSPF conditions develop cortical bone osteopenia in femur. <b>(B)</b> Representative bone sections stained with von Kossa/ Kernechtrot illustrate trabecular bone in vertebrae of mice kept under nonSPF, SPF and SPF+nonSPF conditions. <b>(C)</b> Quantification of bone tissue by histomorphometry revealed reduced BV/TV values in Gfi1-ko/ko vertebrae under nonSPF and SPF+nonSPF conditions compared to controls. Breeding within the SPF environment did not significantly affect bone mass of Gfi1-ko/ko mice. <b>(D)</b> Histomorphometric quantification of the osteoblast covered bone surface (Ob.S/BS) shows reduced but elevated values upon nonSPF and SPF+nonSPF breeding, respectively. SPF breeding did not affect Ob.S/BS between control and mutant mice. <b>(E)</b> Quantification of the osteoclast covered bone surface (Oc.S/BS) revealed diminished but raised values in Gfi1-ko/ko mice upon nonSPF and SPF+nonSPF breeding compared to their corresponding Gfi1-wt/wt controls, respectively. Gfi1-ko/ko mutants grown under SPF conditions showed elevated Oc.S/BS counts compared to Gfi1-wt/wt mice. For better comparability, Gfi1-wt/wt values were set to 1 and Gfi1-ko/ko values were relatively calculated for each breeding condition. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s008" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s009" target="_blank">S3</a> Tables for absolute values. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Housing conditions determine Gfi1-ko/ko mice body mass and survival.

<p><b>(A)</b> Average growth curves from control mice kept under nonSPF conditions indicate evolving development also beyond weaning (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). However, soon after weaning (P19) Gfi1-ko/ko mutants display significantly delayed growth. Development under SPF growth conditions did not affect growth of Gfi1-ko/ko mice (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). Upon SPF+nonSPF housing Gfi1-ko/ko mice demonstrated relative normal growth compared to controls (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3); however, Gfi1-ko/ko mice show significant body mass reduction. All curves show values of male mice. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s007" target="_blank">S1 Table</a> for health monitoring. <b>(B)</b> At SPF and SPF+nonSPF conditions Gfi1-ko/ko mice showed a mortality rate of approx. 9% and 6%, respectively. <b>(C)</b> Final body mass of controls and Gfi1-ko/ko mice was assessed at indicated time points for SPF and SPF+nonSPF conditions. Combined SPF+nonSPF breeding caused a body mass reduction of approx. 25% in male and female Gfi1-ko/ko mice. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p