A Common Copy Number Variation (CNV) Polymorphism in the <i>CNTNAP4</i> Gene: Association with Aging in Females

<div><p>Background</p><p>Aging is a biological process strongly determined by genetics. However, only a few single nucleotide polymorphisms (SNPs) have been reported to be consistently associated with aging. While investigating whether copy number variations (CNVs) could fill this gap, we focused on CNVs that have not been studied in previous SNP-based searches <i>via</i> tagging SNPs.</p> <p>Methods</p><p>TaqMan qPCR assays were developed to quantify 20 common CNVs in 222 senior American Caucasians in order to reveal possible association with longevity. The replication study was comprised of 1283 community-dwelling senior European Caucasians. Replicated CNVs were further investigated for association with healthy aging and aging-related diseases, while association with longevity was additionally tested in <i>Caenorhabditis elegans</i>.</p> <p>Results</p><p>In the discovery study of ≥80 <i>vs</i>.<80 years old seniors, a homozygous intronic CNV deletion in the <i>CNTNAP4</i> gene was inversely associated with survival to the age of 80 (OR=0.51, 95%CI 0.29-0.87, p=0.015 before correction for multiple testing). After stratification by sex, association remained significant in females (OR=0.41, 95%CI 0.21-0.77, p=0.007), but not in males (OR=0.97, 95%CI 0.33-2.79, p=1). The finding was validated in a replication study (OR=0.66, 95%CI 0.48-0.90, p=0.011 for females). <i>CNTNAP4</i> association with longevity was supported by a marked 25% lifespan change in <i>C. elegans</i> after knocking down the ortholog gene. An inverse association of the CNV del/del variant with female healthy aging was observed (OR=0.39, 95%CI 0.19-0.76, p=0.006). A corresponding positive association with aging-related diseases was revealed for cognitive impairment (OR=2.17, 95%CI 1.11-4.22, p=0.024) and, in independent studies, for Alzheimer’s (OR=4.07, 95%CI 1.17-14.14, p=0.036) and Parkinson’s (OR=1.59, 95%CI 1.03-2.42, p=0.041) diseases.</p> <p>Conclusion</p><p>This is the first demonstration for association of the <i>CNTNAP4</i> gene and one of its intronic CNV polymorphisms with aging. Association with particular aging-related diseases awaits replication and independent validation.</p> </div

NRX-1 knock down increases longevity in <i>C. elegans</i>.

<p>A. Lifespan curves for N2 <i>C. elegans</i> worms (Bristol strain) in control conditions (filled squares) or subjected to <i>NRX-1</i> bacterial RNAi (hollow squares). B. Mean lifespan of control worms and worms subjected to <i>NRX-1</i> RNAi in three independent experiments. Statistical significance was evaluated by Student’s t-test using Excell^® software. Statistically significant differences from control are indicated by symbols * (p<0.05) and ** (p<0.01).</p

The cGAS/STING axis contributes to the production of pro-inflammatory cytokines during <i>L</i>. <i>pneumophila</i> infection.

<p>(A-F) WT, <i>Tmem173</i>^<i>-/-</i> <i>and cGas</i>^<i>-/-</i> BMDMs were infected for 6 h with <i>L</i>. <i>pneumophila</i> WT at MOI 10 and relative cytokine expression was determined by qRT-PCR. (G-J) Cytokine protein concentrations in whole lung homogenates from <i>L</i>. <i>pneumophila</i>-infected mice were quantified by sandwich ELISA. Data are shown as mean ± SEM. (A-F) Data representative of 3 to 4 independent experiments carried out in duplicates. (G-J) Data representative of 6 o 7 mice per group. Data were analyzed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p

Endogenous HAQ STING is strongly impaired in mounting a type I IFN and proinflammatory cytokine responses against <i>Legionella</i> infection or stimulation with DNA or CDNs.

<p>(A-D) PBMCs from healthy volunteers (N = 4 for WT and N = 4 for HAQ) were isolated by density gradient centrifugation. 7 d after isolation cells were infected for 6 h with <i>L</i>. <i>pneumophila</i> at MOIs 10 and 50 or stimulated for the same period with 1 and 5 ug/ml 2´-3´cGAMP or either bacterial or synthetic DNA at a concentration of 0.2 or 1 ug/ml. RNA was isolated and the expression of <i>IFNB</i> (A), <i>IL1B</i> (B), <i>IL6</i> (C) and <i>TNFA</i> (D) was determined by qRT-PCR. Data are shown as the RQ of specified mRNAs. Data represent the mean ± SEM of 4 independent experiments carried out in triplicates. Differences were assessed with the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p

Type I IFN responses during <i>L</i>. <i>pneumophila</i> infection are mediated by the cGAS/STING pathway.

<p>(A-C) WT and <i>Tmem173</i>^-/- mouse BMDMs were left untreated or stimulated with 1 ug/ml <i>L</i>. <i>pneumophila</i> DNA (JR32 DNA) or 5 ug/ml 2´3-cGAMP (A) or were infected with <i>L</i>. <i>pneumophila</i> JR32 WT and 130b WT, or mutant strains deficient for <i>dotA</i> or <i>sdhA</i> at MOI 10 for 6 h (B, C). Expression of <i>Ifnb</i> (A, B) or <i>Irg1</i> (C) was measured by qRT-PCR. (D-G) WT and cGAS-deficient BMDMs were stimulated with <i>L</i>. <i>pneumophila</i> DNA or 2´3-cGAMP or infected with <i>L</i>. <i>pneumophila</i> JR32 WT, and expression of <i>Ifnb</i> and <i>Irg1</i> was quantified by qRT-PCR (D-F) or production of IP-10 was measured by ELISA (G). (H-N) WT, STING- and cGAS-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> JR32 WT or instilled with PBS as control (H-J). <i>Ifnb</i> and <i>Irg1</i> expression in the lungs was assessed 48 (H, I) or 144 h p.i. (K-N) by qRT-PCR, or IP-10 production was measured at 48 h (J). Data are represented as the relative quantification (RQ) of specified mRNAs. Data are shown as the mean + SEM of three to four independent experiments, measured in technical duplicates (Fig. 1A-G) or 6 to 7 mice per group (Fig. H-N). Analyses were performed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p

Distribution of <i>TMEM173/STING</i> HAQ and R232H in German patients and healthy controls.

<p>Distribution of <i>TMEM173/STING</i> HAQ and R232H in German patients and healthy controls.</p

Endogenous R232H STING is partly deficient in sensing bacterial CDN but responds normally to <i>Legionella</i> infection or stimulation with DNA.

<p>(A-D) PBMCs from healthy volunteers (N = 3 for WT and N = 3 for R232H) were isolated by density gradient centrifugation. 7 d after isolation cells were infected for 6 h with <i>L</i>. <i>pneumophila</i> at MOI 10 or stimulated for the same period with 1 ug/ml 2´-3´cGAMP, Rp-c-diAMPSS, cGMP or either bacterial DNA at a concentration of 1 ug/ml. RNA was isolated and the expression of <i>IFNB</i> (A), <i>IL1B</i> (B), and <i>IL6</i> (C) and <i>TNFA</i> (D) was determined by qRT-PCR. Data are shown as the RQ of specified mRNAs. Data represent the mean ± SEM of 3 independent experiments carried out in triplicates. Differences were assessed with the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p

<i>L</i>. <i>pneumophila</i> infection and stimulation with DNA or cGAMP induce weak cGAS-dependent type I IFN responses in THP-1 cells.

<p>WT THP-1 or cGAS-/- THP-1 clones A5 and B5 were allowed differentiation prior to stimulation with either cGAMP or synthetic DNA (A) or infection with two different strains of <i>L</i>. <i>pneumophila</i> (B). <i>IFNB</i> expression was determined by qRT-PCR. Data represent mean ± SEM of 2 independent experiments carried out in duplicates. Analyses were performed by employing the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p

STING contributes to the antibacterial defense in mice infected with <i>L</i>. <i>pneumophila</i>.

<p>WT, cGAS- and STING-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> WT and the bacterial loads in the lungs were assessed 144 h p.i. Data represent mean ± SEM of 6–13 mice per group. Comparisons were performed with the Mann-Whitney U Test. Comparisons with p < 0.05 were considered significant.</p