Sequences of primers used in this study.

<p>Sequences of primers used in this study.</p

BAC-derived Human HIP14 is not sufficient to rescue body weight in the <i>Hip14</i>−/− mice.

<p><i>Hip14−/−</i> mice demonstrate reduced body weight as early as 2 weeks of age (data not shown). <b>a.</b> Body weight is decreased in <i>Hip14−/−</i> females (89.2% of WT) at 3 months of age, and this is partially rescued (93.8% of WT) in BAC mice (WT 23.47±0.76, <i>Hip14</i>−/− 20.94±0.23 g, BAC 22.02±0.44 g). At 6 months, a similar partial rescue is observed (WT 30.00±1.74, <i>Hip14</i>−/− 24.75±0.49 g and 82.5% of WT, BAC 26.29±0.69 g and 87.6% of WT). Repeated measures ANOVA reveals a significant effect of genotype in females alone (F(2,27) = 7.53, p = 0.0025). Bonferroni post-tests reveal that WT differs significantly from both <i>Hip14</i>−/− (p<0.0001) and BAC (p<0.01). n = 10, 10, and 9 for WT, <i>Hip14</i>−/−, and BAC respectively. <b>b.</b> In males, <i>Hip14−/−</i> body weight is similarly decreased at 3 months (86.7% of WT) with partial rescue (91.7% of WT) in BAC (WT 28.79±0.56, <i>Hip14</i>−/− 24.95±0.57 g, BAC 26.39±0.47 g). By 6 months, partial rescue is further apparent (WT 33.91±0.71, <i>Hip14</i>−/− 28.30±0.70 g and 83.45% of WT, BAC 30.78±0.44 g and 90.75% of WT). A highly significant effect of genotype is present in males (F(2,37) = 26.81, p<0.0001). Bonferroni post-tests reveal a highly significant difference between WT and <i>Hip14</i>−/− at 3 and 6 months of age (p<0.0001), and a significant, but less robust, difference between WT and BAC mice at both ages (3 months p<0.05, 6 months p<0.01). However, BAC mice are also significantly different from <i>Hip14−/−</i> at both 3 (p<0.05) and 6 months (p<0.0001). n = 15, 10, and 8 for WT, <i>Hip14</i>−/−, and BAC respectively.</p

HIP14 mRNA and protein expression in <i>Hip14−/−</i> and <i>HIP14</i> BAC mice.

<p>Human <i>HIP14</i> mRNA (<b>a</b>) is expressed in BAC mice (p<0.0001) and no signal is detected in WT or <i>Hip14</i>−/− littermates (ANOVA p<0.0001, n = 6). Murine <i>Hip14</i> mRNA transcript (<b>b</b>) is significantly reduced in <i>Hip14−/−</i> mice (p<0.0001) and is not altered in the presence of human <i>HIP14</i> (ANOVA p<0.0001, n = 6). Primers for mouse actin were used as an endogenous control. HIP14 protein expression in striatum (<b>c</b>) and cortex (<b>d</b>) was assessed by western blot using an in-house HIP14 polyclonal antibody. Beta-tubulin was used as a loading control. Striatum and cortex both n = 5. Post-hoc Tukey tests: ***p<0.0001.</p

HIP14 rescues the neuropathological deficits seen in the <i>Hip14</i>−/− mouse.

<p>Mice aged 1 month were perfused with 4% PFA and sectioned using a cryostat, stained with anti-NeuN, mounted and volumes and neuronal counts were measured using Stereo investigator. <b>a.</b> Total brain weight shows a trend to rescue in mice aged 1 month, although one-way ANOVA analysis is not significant (WT: 321.5±3.78, <i>Hip14−/−:</i> 309.8±3.74, BAC: 319.2±5.32 g, ANOVA p = 0.14). Pairwise t-tests reveal that <i>Hip14−/−</i> whole brain weight is significantly decreased compared to WT (p = 0.04) and that BAC do not differ from WT (p = 0.7). <b>b.</b> Cerebellum weight is significantly decreased in <i>Hip14</i>−/− mice and is rescued to WT levels in BAC mice at 1 month (WT: 51.41±0.82, <i>Hip14−/−:</i> 46.45±0.83, BAC: 50.34±1.38 g, ANOVA p = 0.0038). Pairwise t-test analysis similarly reveals that <i>Hip14−/−</i> cerebellum is significantly decreased compared to WT (p = 0.0003) and that BAC do not differ from WT (p = 0.5). <b>c.</b> Forebrain weight shows a similar trend to rescue at 1 month of age (WT: 270.1±3.15, <i>Hip14−/−:</i> 263.4±3.06, BAC: 268.9±4.27 g, ANOVA p = 0.36). <b>d.</b> Striatal volume is significantly rescued in BAC mice at 1 month of age (WT:10.4±0.3, <i>Hip14</i>−/−: 8.8±0.4 mm³, BAC: 10.3±0.2 mm³; ANOVA p = 0.002). <b>e.</b> Striatal neuron count is likewise rescued by the human <i>HIP14</i> BAC at 1 month of age (WT:2.0±0.1, <i>Hip14</i>−/−: 1.7±0.08, BAC: 2.1±0.03 million cells; ANOVA p = 0.004). <b>f.</b> Finally, cortical volume loss observed in <i>Hip14−/−</i> mice is also rescued by human HIP14 at 1 month (WT:26.86±0.52, <i>Hip14</i>−/−: 23.66±0.75, BAC: 26.22±0.60 mm³; ANOVA p = 0.003). Similar observations are made at 3 months of age (data not shown). 1 month n = 12, 12, and 11 for WT, <i>Hip14</i>−/−, and BAC respectively. * p<0.05, **p<0.01, ***p<0.0001.</p

Human HIP14 rescues behavioural alterations in the <i>Hip14−/−</i> mouse.

<p>Deficits in motor coordination and balance are rescued by BAC-derived human HIP14. <b>a.</b> Repeated measures ANOVA in combined genders reveals a significant effect of genotype F(2,300) = 600, p = 0.0035. Bonferroni post-hoc analysis reveals that BAC mice perform consistently better on the accelerating rotarod task than <i>Hip14</i>−/− mice (p<0.05 in trials 1,4; p<0.01 in trials 5,6) and superior to WT mice in later trials (p<0.05 trial 5). All other post-hoc comparisons are non-significant (p>0.05). n = 25, 20, and 17 for WT, <i>Hip14</i>−/−, and BAC respectively. (<b>b–j</b>) Human HIP14 normalizes the hyperactivity observed in <i>Hip14</i>−/− mice to WT levels. Spontaneous activity was assessed in Med Associates boxes at 3 and 6 months of age. Repeated measures ANOVA reveals a significant effect of genotype in ambulatory time (<b>b;</b> F(2,53) = 5.05, p = 0.0099) and counts (<b>e;</b> F(2,53) = 5.36, p = 0.0076), stereotypic time (<b>c</b>; F(2,53) = 19.70, p<0.0001) and counts (<b>f</b>; F(2,53) = 15.13, p<0.0001), distance traveled <b>(d;</b> F(2,53) = 3.60, p = 0.034), time resting (<b>g;</b>(2,53) = 4.99, p = 0.010), and ambulatory episodes (<b>j</b>; F(2,53) = 8.68, p = 0.0005). A significant effect was not seen in vertical counts <b>(h;</b> F(2,51) = 1.89, p = 0.1611<b>)</b>, and average velocity <b>(i;</b> F(2,54) = 0.68, p = 0.51<b>)</b>. The most robust effects were observed for stereotypic time (<b>c</b>) and counts (<b>f</b>). Single-housed mice were excluded from all analyses.*p<0.05, **p<0.01, ***p<0.0001.</p

A table summarizing the genotyping results for the 11 founders.

<p>A “+" sign indicates that a PCR product was detected. A “−" sign indicates that no PCR product was detected, suggesting that a truncated BAC construct was integrated in these lines.</p

Additional mutations in individuals with <i>LIPG</i> mutations and low HDLc.

<p>Additional mutations in individuals with <i>LIPG</i> mutations and low HDLc.</p

Representative segregation of mutations in families.

<p><b>A, B</b>) Segregation of (<b>A</b>) <i>LIPG</i> L130F-FS and (<b>B</b>) <i>GALNT2</i> D314A with elevated HDLc. For each individual, the individual ID, HDLc (in mmol/L) plus [HDLc percentile], and genotype are shown. Squares, Males; Circles, Females; Arrow, proband. Filled shape, HDLc≥90^th percentile; half-filled, HDLc between 80–89^th percentiles; empty shape, HDLc<80^th percentile. Slash = deceased. <b>C</b>) Percent of individuals in families with mutations at given HDLc percentiles or higher.</p

Demographics of unrelated probands with extreme HDLc.

a<p>, Average (SD);</p>b<p>, N (%);</p>c<p>, Average (95% confidence interval).</p

Mutations identified in probands with high or low HDLc.

<p>A) Overview of the study design. <b>B–D</b>) Predicted mutation effects on <b>B</b>) LIPG, <b>C</b>) CETP, and <b>D</b>) GALNT2 proteins. Red sequence changes are novel, blue are previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037437#pone.0037437-Edmondson1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037437#pone.0037437-deLemos1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037437#pone.0037437-Holleboom1" target="_blank">[28]</a>, and black is previously described in a subset of this cohort <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037437#pone.0037437-vanderSteeg1" target="_blank">[16]</a>.</p