Copy-number variants and polygenic risk for intelligence confer risk for autism spectrum disorder irrespective of their effects on cognitive ability

IntroductionRare copy number variants (CNVs) and polygenic risk for intelligence (PRS-IQ) both confer susceptibility for autism spectrum disorder (ASD) but have opposing effects on cognitive ability. The field has struggled to disentangle the effects of these two classes of genomic variants on cognitive ability from their effects on ASD susceptibility, in part because previous studies did not include controls with cognitive measures. We aim to investigate the impact of these genomic variants on ASD risk while adjusting for their known effects on cognitive ability.MethodsIn a cohort of 8,426 subjects with ASD and 169,804 controls with cognitive assessments, we found that rare coding CNVs and PRS-IQ increased ASD risk, even after adjusting for their effects on cognitive ability.ResultsBottom decile PRS-IQ and CNVs both decreased cognitive ability but had opposing effects on ASD risk. Models combining both classes of variants showed that the effects of rare CNVs and PRS-IQ on ASD risk and cognitive ability were largely additive, further suggesting that susceptibility for ASD is conferred independently from its effects on cognitive ability. Despite imparting mostly additive effects on ASD risk, rare CNVs and PRS-IQ showed opposing effects on core and associated features and developmental history among subjects with ASD.DiscussionOur findings suggest that cognitive ability itself may not be the factor driving the underlying liability for ASD conferred by these two classes of genomic variants. In other words, ASD risk and cognitive ability may be two distinct manifestations of CNVs and PRS-IQ. This study also highlights the challenge of understanding how genetic risk for ASD maps onto its dimensional traits

Identification of TMPRSS6 isoforms interacting partners.

A) BioVenn overlap of TMPRSS6 isoforms 2, 3 and 4 interacting partners (fold change vs mock ≥ 3) identified by mass spectrometry analysis of TMPRSS6 V5-tagged immunoprecipitation from transfected Hep3B cell membrane preparations. B) STRING interactome of 49 common protein partners.</p

TMPRSS6 isoforms 3 and 4 reduce isoform 2 activity.

A) TMPRSS6 isoforms representation adapted from our previous publication [1] licensed under CC BY 3.0. B) Expression of TMPRSS6 V5-tagged isoforms 2, 3 and 4 in transfected HEK293 cells assessed by western blotting against V5. C) Proteolytic activity was measured in the cell medium of HEK293 cells transfected with one or two TMPRSS6 V5-tagged isoforms. Boc-QAR-AMC (200 μM) fluorogenic substrate cleavage was monitored. Results are presented as relative activity over TMPRSS6 isoform 2 (TMPRSS6-2) activity. Statistical significance was assessed using one sample T test, *p < 0.0002 (n = 5).</p

TMPRSS6 cleaves TfR1.

A) TfR1-HA was transfected with one or two TMPRSS6-V5 tagged isoforms. Expression was detected in the cell lysate (CL) and cell media (CM) (n = 3). B) TfR1 cleavage quantification. Results are presented as TFR1 cleavage (%) relative to TFR1 cleavage by TMPRSS6 isoform 2. Statistical significance was assessed using one sample T test, *p < 0.02. The means ± SD are presented (n = 3).</p

Interacting protein list.

(TIF)</p

TfR1 cleavage controls.

TfR1-HA was transfected either with V5-tagged TMPRSS6 isoform 2 WT (active, TMPRSS6-2-WT-V5), catalytically inactivated TMPRSS6 isoform 2 (TMPRSS6-2-S762A-V5) or proprotein convertase 7 (PC7-V5). Expression was detected in the cell lysate (CL) and cell media (CM) (n = 3). (TIF)</p

TMPRSS6 isoforms engage in homo- and hetero-interactions.

HEK293 cells were co-transfected with V5- and HA-tagged TMPRSS6 isoforms. Homo- and hetero-interaction of TMPRSS6 isoforms were assessed by immunoprecipitation (IP) of cell lysate (CL) using an anti-V5 antibody. Isoform immunoprecipitation was detected using anti-V5 and anti-HA antibodies. Equal amount of CL was loaded on SDS-polyacrylamide gels and cell GAPDH was used as a loading control (n = 3).</p

Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function

Serine proteases do not induce an increase in phosphorylation of myosin regulatory light chain.

<p>Phosphorylation of MLC was assessed using phospho-specific antibodies (Ser19/Thr18) and western blotting <b>A</b>. A representative blot of phosphorylated and total MLC. <b>B</b>. Densitometry was performed with normalization to actin and the ratios of phosphorylated to total MLC were determined. n = 3–4; ns, not significant compared to control as analyzed by ANOVA with Dunnett’s posthoc test. <b>C</b>. MLC phosphorylation was also determined by Phos-tag gels. SCBN cells were plated on transwells and treated for 15 minutes apically in Krebs with 135 BAU/mL trypsin (T) or 1.5 BAU/mL matriptase (MT). Lysate was collected and phosphorylation of MLC assessed by Phos-tag gel electrophoresis. Protein with zero, one, or two phosphorylations (0p, 1p, 2p) are labeled. The positive control is SCBN cells treated with 3 μM of the phosphatase inhibitor calyculin A (CA) apically for 30 minutes. A blot with n = 3 is shown in <b>C</b>, while densitometry is shown in <b>D</b> and is the ratio of the phosphorylated band over the total MLC (all three bands combined), n = 6. No significant differences are seen as assed by ANOVA within each phosphorylated group.</p

EGF induces an increase in TER in SCBN cells similar to serine proteases.

<p><b>A</b>. SCBN cells were examined for mRNA expression of EGFR, ErbB2, ErbB3, and ErbB4 using RT-PCR. <b>B</b>. SCBN cells were mounted in Ussing chambers and treated with 50 or 100 ng/mL EGF and change in TER determined, n = 3 *p<0.05 ** p<0.01 compared to control by ANOVA with Dunnett’s post-hoc test. <b>C</b>. In Ussing chambers, SBTI was added apically to the cells 10 minutes post EGF treatment and representative tracing is shown. <b>D</b>. No significant differences were found when SBTI was added to cells treated with EGF, n = 3.</p