Sequence divergence of Mus spretus and Mus musculus across a skin cancer susceptibility locus

<p>Abstract</p> <p>Background</p> <p><it>Mus spretus </it>diverged from <it>Mus musculus </it>over one million years ago. These mice are genetically and phenotypically divergent. Despite the value of utilizing <it>M. musculus </it>and <it>M. spretus </it>for quantitative trait locus (QTL) mapping, relatively little genomic information on <it>M. spretus </it>exists, and most of the available sequence and polymorphic data is for one strain of <it>M. spretus</it>, Spret/Ei. In previous work, we mapped fifteen loci for skin cancer susceptibility using four different <it>M. spretus </it>by <it>M. musculus </it>F1 backcrosses. One locus, <it>skin tumor susceptibility 5 </it>(<it>Skts5</it>) on chromosome 12, shows strong linkage in one cross.</p> <p>Results</p> <p>To identify potential candidate genes for <it>Skts5</it>, we sequenced 65 named and unnamed genes and coding elements mapping to the peak linkage area in outbred <it>spretus</it>, Spret/EiJ, FVB/NJ, and NIH/Ola. We identified polymorphisms in 62 of 65 genes including 122 amino acid substitutions. To look for polymorphisms consistent with the linkage data, we sequenced exons with amino acid polymorphisms in two additional <it>M. spretus </it>strains and one additional <it>M. musculus </it>strain generating 40.1 kb of sequence data. Eight candidate variants were identified that fit with the linkage data. To determine the degree of variation across <it>M. spretus</it>, we conducted phylogenetic analyses. The relatedness of the <it>M. spretus </it>strains at this locus is consistent with the proximity of region of ascertainment of the ancestral mice.</p> <p>Conclusion</p> <p>Our analyses suggest that, if <it>Skts5 </it>on chromosome 12 is representative of other regions in the genome, then published genomic data for Spret/EiJ are likely to be of high utility for genomic studies in other <it>M. spretus </it>strains.</p

Acoustic Features of Transfeminine Voices and Perceptions of Voice Femininity

The purpose of this study was to evaluate the relationships between acoustic measures of transfeminine voices and both self- and listener ratings of voice femininity. Connected speech samples were collected from 12 transfeminine individuals (M = 36.3 years, SD = 10.6 years) and a control group of five cisgender (cis) women and five cis men (M = 35.3 years, SD = 13.3 years). The acoustic measures of fundamental frequency (fo), fo variation, formant frequencies, and vocal intensity were calculated from these samples. Transfeminine speakers rated their own voices on a five-point scale of voice femininity. Twenty inexperienced listeners heard an excerpt of each speech sample and rated the voices on the same five-point scale of voice femininity. Spearman\u27s rank-order correlation coefficients were calculated to measure the relationships between the acoustic variables and ratings of voice femininity. Significant positive correlations were found between fo and both self-ratings (r = 0.712, P = 0.009) and listener ratings of voice femininity (r = 0.513, P \u3c 0.001). Significant positive correlations were found between intensity and both self-ratings (r = 0.584, P = 0.046) and listener ratings of voice femininity (r = 0.584, P = 0.046). No significant correlations were found between fo variation or formant frequencies and perceptual ratings of voice femininity. A Pearson\u27s chi-square test of independence showed that the distribution of self- and listener ratings differed significantly (χ2 = 9.668, P = 0.046). Self- and listener ratings were also shown to be strongly correlated (r = 0.912, P \u3c 0.001). This study provides further evidence to support the selection of training targets in voice feminization programs for transfeminine individuals and promotes the use of self-ratings of voice as an important outcome measure

The Impact of 3′UTR Variants on Differential Expression of Candidate Cancer Susceptibility Genes

<div><p>Variants in regulatory regions are predicted to play an important role in disease susceptibility of common diseases. Polymorphisms mapping to microRNA (miRNA) binding sites have been shown to disrupt the ability of miRNAs to target genes resulting in differential mRNA and protein expression. <i>Skin tumor susceptibility 5</i> (<i>Skts5</i>) was identified as a locus conferring susceptibility to chemically-induced skin cancer in NIH/Ola by SPRET/Outbred F1 backcrosses. To determine if polymorphisms between the strains which mapped to putative miRNA binding sites in the 3′ untranslated region (3′UTR) of genes at <i>Skts5</i> influenced expression, we conducted a systematic evaluation of 3′UTRs of candidate genes across this locus. Nine genes had polymorphisms in their 3′UTRs which fit the linkage data and eight of these contained polymorphisms suspected to interfere with or introduce miRNA binding. 3′UTRs of six genes, <i>Bcap29</i>, <i>Dgkb, Hbp1, Pik3cg, Twistnb</i>, and <i>Tspan13</i> differentially affected luciferase expression, but did not appear to be differentially regulated by the evaluated miRNAs predicted to bind to only one of the two isoforms. 3′UTRs from four additional genes chosen from the locus that fit less stringent criteria were evaluated. <i>Ifrd1</i> and <i>Etv1</i> showed differences and contained polymorphisms predicted to disrupt or create miRNA binding sites but showed no difference in regulation by the miRNAs tested. In summary, multiple 3′UTRs with putative functional variants between susceptible and resistant strains of mice influenced differential expression independent of predicted miRNA binding.</p> </div

Luciferase assays for 3′UTRs of SPRET/Outbred and NIH/Ola.

<p>Representative relative luciferase units normalized to mock for the pGL3 luciferase vector (dark gray), NIH 3′UTR (Black) and SPRET/Outbred 3′UTRs (light gray) for six genes are shown. P-values of differential expression between NIH/Ola and SPRET/Outbred are indicated. A. <i>Bcap29</i>; B. <i>Dgkb</i>; C. <i>Hbp1</i>; D. <i>Pik3cg</i>; E. <i>Twistnb</i>; F. <i>Tspan13</i>.</p

mRNA expression of candidate genes with luciferase expression differences.

<p>Quantitative PCR of seven genes evaluated for differential luciferase expression between SPRET/Outbred and NIH/Ola 3′UTR are shown. <i>Hprt</i>, <i>Ppia</i> and <i>L19</i> were used as loading controls; results normalizing to <i>Hprt</i> are shown. Percentage relative expression of <i>Hprt</i> for NIH/Ola and Spret/Outbred was normalized to NIH/Ola expression. P-values are indicated. A. <i>Bcap29</i>, B. <i>Dgkb</i>, C. <i>Hbp1</i>, D. <i>Meox2</i>, E. <i>Pik3cg</i>, F. <i>Tspan13</i>, G. <i>Twistnb</i>. Black bars, NIH/Ola; Gray bars, SPRET/Outbred.</p

Candidate microRNAs for genes showing differential luciferase expression.

<p>SPRET, SPRET/Outbred; miRNAs in bold are those that were evaluated, ^aFree energy binding of target 3′UTR by MicroInspector was greater than −22 kcal/Joule; ^bmiR expression, The miRNA is expressed in skin; ^cCancer, The miRNA is differentially expressed in cancer, ^dthe SNP is predicted to be in the miRNA seed region.</p

Luciferase and mRNA results of <i>Etv1</i> and <i>Ifrd1</i>.

<p>Representative relative luciferase units normalized to mock for the pGL3 luciferase vector (dark gray), NIH 3′UTR (Black) and SPRET/Outbred 3′UTRs (light gray) for A. Etv1 and B. Ifrd1 are shown. Representative experiments showing no effect of miRNA on luciferase expression for the predicted SPRET/Outbred target for C. <i>Etv1</i> and <i>miR-673</i> and D. <i>Ifrd1</i> with <i>miR-3064-5P</i>. ApGL3, pGL3 luciferase vector without insert; NIH, NIH/Ola 3′UTR; SPRET, SPRET/Outbred 3′UTR; NC, scrambled control miRNA, Dark Gray bars, pGL3 luciferase vector; Black bars, pGL3 vector with the NIH/Ola 3′UTR; Light gray bars, pGL3 vector containing the SPRET/Outbred 3′UTR.</p

3′UTR SNPs only in SPRET/Outbred.

<p>Position, nucleotide position in gene according to the Ensembl database, SPRET/O, SPRET/Outbred; -, no insertion or deletion; Ins, insertion; Del, deletion.</p

<i>Twistnb</i> expression under differential experimental conditions.

<p>Representative experiments showing non-specific effect of <i>miR-3074-5p</i> and/or <i>miR-691</i> on both isoforms of <i>Twistnb</i> are shown at a low dose transfection of miRNA precursors. A. <i>Twistnb</i> 3′UTRs at 24 hours post-transfection with <i>miR-3074-5p</i>, <i>miR-691</i> or both miRNAs. B. <i>Twistnb</i> 3′UTRs at 48 hours post-transfection with <i>miR-3074-5p</i>, <i>miR-691</i> or both miRNAs. pGL3, pGL3 luciferase vector without insert; NIH, NIH/Ola 3′UTR; SPRET, SPRET/Outbred 3′UTR; NC, scrambled control miRNA; Dark Gray bars, pGL3 luciferase vector; Black bars, pGL3 vector with the NIH/Ola 3′UTR; Light gray bars, pGL3 vector containing the SPRET/Outbred 3′UTR.</p