Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

Unraveling the molecular interactions between α7 nicotinic receptor and a RIC3 variant associated with backward speech.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

Congenital myasthenic syndromes: increasingly complex.

PURPOSE OF REVIEW: Congenital myasthenia syndromes (CMS) are treatable, inherited disorders affecting neuromuscular transmission. We highlight that the involvement of an increasing number of proteins is making the understanding of the disease mechanisms and potential treatments progressively more complex. RECENT FINDINGS: Although early studies identified mutations of proteins directly involved in synaptic transmission at the neuromuscular junction, recently, next-generation sequencing has facilitated the identification of many novel mutations in genes that encode proteins that have a far wider expression profile, some even ubiquitously expressed, but whose defective function leads to impaired neuromuscular transmission. Unsurprisingly, mutations in these genes often causes a wider phenotypic disease spectrum where defective neuromuscular transmission forms only one component. This has implications for the management of CMS patients. SUMMARY: Given the widening nonneuromuscular junction phenotypes in the newly identified forms of CMS, new therapies need to include disease-modifying approaches that address not only neuromuscular weakness but also the multisystem involvement. Whilst the current treatments for CMS are highly effective for many subtypes there remains, in a proportion of CMS patients, an unmet need for more efficacious therapies.

DNA-Templated Anchoring of Proteins for Programmable Cell Functionalization and Immunological Response.

Membrane protein engineering exhibits great potential for cell functionalization. Although genetic strategies are sophisticated for membrane protein engineering, there still exist some issues, including transgene insertional mutagenesis, laborious, complicated procedures, and low tunability. Herein, we report a DNA-templated anchoring of exogenous proteins on living cell membranes to realize programmable functionalization of living cells. Using DNA as a scaffold, the model cell membranes are readily modified with proteins, on which the density and ratio of proteins as well as their interactions can be precisely controlled through predictable DNA hybridization. Then, the natural killer (NK) cells were engineered to gain the ability to eliminate the immune checkpoint signaling at the NK-tumor synapse, which remarkably promoted NK cell activation in immunotherapy. Given the versatile functions of exogenous proteins and flexible designs of programmable DNA, this method has the potential to facilitate membrane-protein-based cell engineering and therapy.

The Involvement of Ascorbic Acid in Cancer Treatment.

Vitamin C (VC), also known as ascorbic acid, plays a crucial role as a water-soluble nutrient within the human body, contributing to a variety of metabolic processes. Research findings suggest that increased doses of VC demonstrate potential anti-tumor capabilities. This review delves into the mechanisms of VC absorption and its implications for cancer management. Building upon these foundational insights, we explore modern delivery systems for VC, evaluating its use in diverse cancer treatment methods. These include starvation therapy, chemodynamic therapy (CDT), photothermal/photodynamic therapy (PTT/PDT), electrothermal therapy, immunotherapy, cellular reprogramming, chemotherapy, radiotherapy, and various combination therapies.

FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification.

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

Exploring the Trans-Cleavage Activity of CRISPR/Cas12a on Gold Nanoparticles for Stable and Sensitive Biosensing.

Taking advantage of the excellent trans-cleavage activity, CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics. However, the single-stranded DNA reporter of the current CRISPR-Dx suffers from poor stability and limited sensitivity, which make their application in complex biological environments difficult. Herein, we, for the first time, explore the trans-cleavage activity of CRISPR/Cas12a toward the substrate on gold nanoparticles and apply the new phenomenon to develop a spherical nucleic acid (SNA) reporter for stable and sensitive CRISPR-Dx biosensing. By anchoring the DNA substrate on gold nanoparticles, we discovered different trans-cleavage activities of different types of the Cas12a system (e.g., LbCas12a and AsCas12a) on a nanoparticle surface. The further study suggests that the trans-cleavage activity of LbCas12a on the nanoparticle surface is highly dependent on the density and length of DNA strands. Based on these interesting discoveries, we furthermore develop SNA reporter-based fluorescent CRISPR-Dx for stable and sensitive biosensing application. Compared to traditional ssDNA reporters, the SNA reporter exhibits improved stability, which enables the stable application in a complex serum environment. In addition, the SNA reporter system with tunable density exhibits high sensitivity with a detection limit of 10 fM, which is about 2 orders of magnitude lower than that of the ssDNA reporter system. Finally, the practical application of SNA reporter-based CRISPR-Dx in clinical serum was successfully achieved. These results indicate their significant potential in future research on biology science and medical diagnoses.

MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality.

RNA-seq has been widely used to reveal the molecular mechanism of variants of life process. We have developed an alternative method, MustSeq, which generates multiple second strands along a single 1st strand cDNA by random-priming initiation, immediately after reverse transcription for each RNA extract using sample-barcoded poly-dT primers, then 3' ends-enriching PCR is applied to construct the library. Unlike the conventional RNA seq, MustSeq avoids procedures such as mRNA isolation, fragmentation and RNA 5'-end capture, enables early pooling of multiple samples, and requires only one twentieth of sequencing reads of full-length sequencing. We demonstrate the power and features of MustSeq comparing with TruSeq and NEBNext RNA-seq, two conventional full-length methods and QuantSeq, an industrial 3' end method. In cancer cell lines, the reads distribution of CDS-exon as well as genes, lncRNAs and GO terms detected by MustSeq are closer than QuantSeq to TruSeq. In mouse hepatocarcinoma and healthy livers, MustSeq enriches the same pathways as by NEBNext, and reveals the molecular profile of carcinogenesis. Overall MustSeq is a robust and accurate RNA-seq method allowing efficient library construction, sequencing and analysis, particularly valuable for analysis of differentially expressed genes with a large number of samples. MustSeq will greatly accelerate the application of bulk RNA-seq on different fields, and potentially applicable for single cell RNA-seq.

Morphological differences between two types of Bruch's membrane defects in pathologic myopia.

PURPOSE: To investigate morphological differences between two types of Bruch's membrane (BM) defects-patchy atrophy (PA) and CNV-related macular atrophy (CNV-MA) METHODS: Eyes presenting with PA or CNV-MA were included. Scleral thickness (ST), choroidal thickness (CT), and scleral morphological characteristics were obtained by swept-source optical coherence tomography (SS-OCT). Fundus photographs were performed to measure the size of PA and CNV-MA lesions. RESULTS: Among a total of 167 eyes evaluated, 106 eyes had PA and 61 eyes had CNV-MA. In addition, dome-shaped macula (DSM) was identified in 20 (18.87%) and 10 (16.39%) eyes among PA and CNV-MA, respectively. The eyes of CNV-MA without DSM showed a thicker subfoveal ST (278.61 ± 56.17 vs 231.58 ± 66.09 mm, P < 0.001), a thinner subfoveal CT, and a higher rate of scleral perforating vessels (70.6% vs 50.0%, P = 0.021) when compared with those of PA without DSM. The size of PA/CNV-MA lesions was associated with CT in eyes without DSM. However, it was only associated with bulge height in eyes with DSM (r = 0.5, P = 0.013). CONCLUSIONS: The eyes with CNV-MA had a thicker sclera than those with PA, which add another evidence to indicate the absence of the progressive relationship between two types of BM defects. The enlargement of lesions in BM defects between eyes with and without DSM may be caused by different mechanical forces. SS-OCT, which focuses on scleral and choroid morphology, may be necessary for more accurate classification of pathologic myopia.

C/EBPß mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes.

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPß gene. Notably, C/EBPß also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPß and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPß alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPß binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPß. C/EBPß binds directly to the promoter of the musclin gene and upregulates its expression.

Genomic Characterization of Esophageal Squamous Cell Carcinoma Reveals Critical Genes Underlying Tumorigenesis and Poor Prognosis.

The genetic mechanisms underlying the poor prognosis of esophageal squamous cell carcinoma (ESCC) are not well understood. Here, we report somatic mutations found in ESCC from sequencing 10 whole-genome and 57 whole-exome matched tumor-normal sample pairs. Among the identified genes, we characterized mutations in VANGL1 and showed that they accelerated cell growth in vitro. We also found that five other genes, including three coding genes (SHANK2, MYBL2, FADD) and two non-coding genes (miR-4707-5p, PCAT1), were involved in somatic copy-number alterations (SCNAs) or structural variants (SVs). A survival analysis based on the expression profiles of 321 individuals with ESCC indicated that these genes were significantly associated with poorer survival. Subsequently, we performed functional studies, which showed that miR-4707-5p and MYBL2 promoted proliferation and metastasis. Together, our results shed light on somatic mutations and genomic events that contribute to ESCC tumorigenesis and prognosis and might suggest therapeutic targets.

Mechanisms of titanium dioxide nanoparticle-induced oxidative stress and modulation of plasma glucose in mice.

Titanium dioxide nanoparticles (TiO2 NPs) are reported to increase plasma glucose levels in mice at specific doses. The production and accumulation of reactive oxygen species (ROS) is potentially the most important factor underlying the biological toxicity of TiO2 NPs but the underlying mechanisms are unclear at present. Data from genome-wide analyses showed that TiO2 NPs induce endoplasmic reticulum (ER) stress and ROS generation, leading to the inference that TiO2 NP-induced ER stress contributes to enhancement of ROS in mice. Resveratrol (Res) effectively relieved TiO2 NP-induced ER stress and ROS generation by ameliorating expression of a common set of activated genes for both processes, signifying that ER stress and ROS are closely related. TiO2 NP-induced ER stress occurred earlier than ROS generation. Upon treatment with 4-phenylbutyric acid to relieve ER stress, plasma glucose levels tended toward normal and TiO2 NP increased ROS production was inhibited. These results suggest that TiO2 NP-induced ER stress promotes the generation of ROS, in turn, triggering increased plasma glucose levels in mice. In addition, Res that displays the ability to reduce ER stress presents a dietary polyphenol antioxidant that can effectively prevent the toxicological effects of TiO2 NPs on plasma glucose metabolism.

knnAUC: an open-source R package for detecting nonlinear dependence between one continuous variable and one binary variable.

BACKGROUND: Testing the dependence of two variables is one of the fundamental tasks in statistics. In this work, we developed an open-source R package (knnAUC) for detecting nonlinear dependence between one continuous variable X and one binary dependent variables Y (0 or 1). RESULTS: We addressed this problem by using knnAUC (k-nearest neighbors AUC test, the R package is available at https://sourceforge.net/projects/knnauc/ ). In the knnAUC software framework, we first resampled a dataset to get the training and testing dataset according to the sample ratio (from 0 to 1), and then constructed a k-nearest neighbors algorithm classifier to get the yhat estimator (the probability of y = 1) of testy (the true label of testing dataset). Finally, we calculated the AUC (area under the curve of receiver operating characteristic) estimator and tested whether the AUC estimator is greater than 0.5. To evaluate the advantages of knnAUC compared to seven other popular methods, we performed extensive simulations to explore the relationships between eight different methods and compared the false positive rates and statistical power using both simulated and real datasets (Chronic hepatitis B datasets and kidney cancer RNA-seq datasets). CONCLUSIONS: We concluded that knnAUC is an efficient R package to test non-linear dependence between one continuous variable and one binary dependent variable especially in computational biology area.

Eosin Y-catalyzed, visible-light-promoted carbophosphinylation of allylic alcohols via a radical neophyl rearrangement.

A visible-light-promoted phosphinylation of allylic alcohols with concomitant 1,2-aryl migration is described. This transformation proceeds smoothly under metal-free and mild conditions by using an inexpensive organic dye, eosin Y, as the photocatalyst, affording various ß-aryl-γ-ketophosphine oxides in moderate to good yields. Mechanistic studies suggested that the 1,2-aryl migration proceeded through a radical (neophyl) rearrangement.

Self-reported executive function and hoarding in adults with obsessive-compulsive disorder.

BACKGROUND: Hoarding behavior may distinguish a clinically and possibly etiologically distinct subtype of obsessive-compulsive disorder (OCD). Little is known about the relationship between executive dysfunction and hoarding in individuals with OCD. METHODS: The study sample included 431 adults diagnosed with DSM-IV OCD. Participants were assessed by clinicians for Axis I disorders, personality disorders, indecision, and hoarding. Executive functioning domains were evaluated using a self-report instrument, the Behavior Rating Inventory of Executive Function-Adult Version (BRIEF-A). We compared scores on these domains in the 143 hoarding and 288 non-hoarding participants, separately in men and women. We used logistic regression to evaluate relationships between executive function scores and hoarding, and correlation and linear regression analyses to evaluate relationships between executive function scores and hoarding severity, in women. RESULTS: In men, the hoarding group had a significantly higher mean score than the non-hoarding group only on the shift dimension. In contrast, in women, the hoarding group had higher mean scores on the shift scale and all metacognition dimensions, i.e., those that assess the ability to systematically solve problems via planning and organization. The relationships in women between hoarding and scores on initiating tasks, planning/organizing, organization of materials, and the metacognition index were independent of other clinical features. Furthermore, the severity of hoarding in women correlated most strongly with metacognition dimensions. CONCLUSIONS: Self-reported deficits in planning and organization are associated with the occurrence and severity of hoarding in women, but not men, with OCD. This may have implications for elucidating the etiology of, and developing effective treatments for, hoarding in OCD.

Aliidiomarina soli sp. nov., isolated from saline-alkaline soil.

A Gram-stain-negative, motile, non-spore-forming bacterium, designated strain Y4G10-17T, was isolated from the saline-alkali farmland top soil, Inner Mongolia, northern China. Strain Y4G10-17T could grow at 4-45 °C (with 30 °C as the optimal temperature), pH 6.0-12.0 (optimal at pH 9.0) and in the presence of 1.0-12.0 % (w/v) NaCl (optimal at 4.0-6.0 %). Phylogenetic analysis based on the eight different copies of the 16S rRNA gene sequences revealed that strain Y4G10-17T shared the highest sequence similarity with Aliidiomarina maris CF12-14T, 97.93-98.66 %, and lower than 97.0 % sequence similarity with all other type strains. Its major cellular fatty acids contained iso-C15 : 0, iso-C17 : 0, summed feature 9 (iso-C17 : 1ω9c and/or C16 : 0 10-methyl), iso-C15 : 1 F, iso-C11 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c). Q-8 was the predominantubiquinone. The major polar lipids of strain Y4G10-17T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unknown lipids and one unknown aminolipid. The genomic DNA G+C content was 49.3 mol%. DNA-DNA hybridization revealed that strain Y4G10-17T showed 20.2±5 % genomic DNA relatedness with its close relative A. maris CF12-14T. Based on the phenotypic, phylogenetic and genotypic characteristics, strain Y4G10-17T represents a novel species within the genus Aliidiomarina, for which the name Aliidiomarina soli sp. nov. is proposed. The type strain is Y4G10-17T (=CGMCC 1.15759T=KCTC 52381T).

Ginsenoside Rg1 Promotes the Migration of Olfactory Ensheathing Cells via the PI3K/Akt Pathway to Repair Rat Spinal Cord Injury.

The aim of this study was to determine the effects of ginsenoside Rg1 on the migration of olfactory ensheathing cells (OECs) in vitro, and its influence on the therapeutic efficacy of OECs transplanted in vivo for the treatment of spinal cord injury (SCI). Primary cultured and purified OECs (prepared from rats) were treated with ginsenoside Rg1. The wound healing test indicated that ginsenoside Rg1 promoted the migration of OECs. Real-time RT-PCR demonstrated that ginsenoside Rg1 upregulated the expression of migration-related factors of OECs, including matrix metalloproteinases-2 (MMP-2), MMP-9, and neural cell adhesion molecule 1 (NCAM1). Moreover, Western blot analysis indicated that ginsenoside Rg1 significantly promoted the migration of OECs via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An SCI rat model was induced in vivo using a revised Allen's method. The Basso, Beattie, and Bresnahan (BBB) scores and histological analysis demonstrated that OECs, which were treated with ginsenoside Rg1, exhibited significant improvement in SCI compared with both the control group and the OEC group. Thus, ginsenoside Rg1 may represent a novel treatment target for SCI.

Parental bonding and hoarding in obsessive-compulsive disorder.

BACKGROUND: Hoarding behavior may indicate a clinically and possibly etiologically distinct subtype of obsessive-compulsive disorder (OCD). Empirical evidence supports a relationship between hoarding and emotional over-attachment to objects. However, little is known about the relationship between hoarding and parental attachment in OCD. METHOD: The study sample included 894 adults diagnosed with DSM-IV OCD who had participated in family and genetic studies of OCD. Participants were assessed for Axis I disorders, personality disorders, and general personality dimensions. The Parental Bonding Instrument (PBI) was used to assess dimensions of perceived parental rearing (care, overprotection, and control). We compared parental PBI scores in the 334 hoarding and 560 non-hoarding participants, separately in men and women. We used logistic regression to evaluate the relationship between parenting scores and hoarding in women, adjusting for other clinical features associated with hoarding. RESULTS: In men, there were no significant differences between hoarding and non-hoarding groups in maternal or paternal parenting scores. In women, the hoarding group had a lower mean score on maternal care (23.4 vs. 25.7, p<0.01); a higher mean score on maternal protection (9.4 vs. 7.7, p<0.001); and a higher mean score on maternal control (7.0 vs. 6.2, p<0.05), compared to the non-hoarding group. The magnitude of the relationships between maternal bonding dimensions and hoarding in women did not change after adjustment for other clinical features. Women who reported low maternal care/high maternal protection had significantly greater odds of hoarding compared to women with high maternal care/low maternal protection (OR=2.54, 95% CI=1.60-4.02, p<0.001). CONCLUSIONS: Perceived poor maternal care, maternal overprotection, and maternal overcontrol are associated with hoarding in women with OCD. Parenting dimensions are not related to hoarding in men. These findings provide further support for a hoarding subtype of OCD and for sex-specific differences in etiologic pathways for hoarding in OCD.

An investigation of doubt in obsessive-compulsive disorder.

BACKGROUND: Clinicians have long considered doubt to be a fundamental characteristic of obsessive-compulsive disorder (OCD). However, the clinical relevance of doubt in OCD has not been addressed. METHODS: Participants included 1182 adults with OCD who had participated in family and genetic studies of OCD. We used a clinical measure of the severity of doubt, categorized as none, mild, moderate, severe, or extreme. We evaluated the relationship between doubt and OCD clinical features, Axis I disorders, personality and personality disorder dimensions, impairment, and treatment response. RESULTS: The severity of doubt was inversely related to the age at onset of OCD symptoms. Doubt was strongly related to the number of checking symptoms and, to a lesser extent, to the numbers of contamination/cleaning and hoarding symptoms. Doubt also was related to the lifetime prevalence of recurrent major depression and generalized anxiety disorder; to the numbers of avoidant, dependent, and obsessive-compulsive personality disorder traits; and to neuroticism and introversion. Moreover, doubt was strongly associated with global impairment and poor response to cognitive behavioral treatment (CBT), even adjusting for OCD severity and other correlates of doubt. CONCLUSIONS: Doubt is associated with important clinical features of OCD, including impairment and cognitive-behavioral treatment response.

Evidence for contribution of common genetic variants within chromosome 8p21.2-8p21.1 to restricted and repetitive behaviors in autism spectrum disorders.

BACKGROUND: Restricted and Repetitive Behaviors (RRB), one of the core symptom categories for Autism Spectrum Disorders (ASD), comprises heterogeneous groups of behaviors. Previous research indicates that there are two or more factors (subcategories) within the RRB domain. In an effort to identify common variants associated with RRB, we have carried out a genome-wide association study (GWAS) using the Autism Genetic Resource Exchange (AGRE) dataset (n = 1,335, all ASD probands of European ancestry) for each identified RRB subcategory, while allowing for comparisons of associated single nucleotide polymorphisms (SNPs) with associated SNPs in the same set of probands analyzed using all the RRB subcategories as phenotypes in a multivariate linear mixed model. The top ranked SNPs were then explored in an independent dataset. RESULTS: Using principal component analysis of item scores obtained from Autism Diagnostic Interview-Revised (ADI-R), two distinct subcategories within Restricted and Repetitive Behaviors were identified: Repetitive Sensory Motor (RSM) and Insistence on Sameness (IS). Quantitative RSM and IS scores were subsequently used as phenotypes in a GWAS using the AGRE ASD cohort. Although no associated SNPs with genome-wide significance (P < 5.0E-08) were detected when RSM or IS were analyzed independently, three SNPs approached genome-wide significance when RSM and IS were considered together using multivariate association analysis. These included the top IS-associated SNP, rs62503729 (P-value = 6.48E-08), which is located within chromosome 8p21.2-8p21.1, a locus previously linked to schizophrenia. Notably, all of the most significantly associated SNPs are located in close proximity to STMN4 and PTK2B, genes previously shown to function in neuron development. In addition, several of the top-ranked SNPs showed correlations with STMN4 mRNA expression in adult CEU (Caucasian and European descent) human prefrontal cortex. However, the association signals within chromosome 8p21.2-8p21.1 failed to replicate in an independent sample of 2,588 ASD probands; the insufficient sample size and between-study heterogeneity are possible explanations for the non-replication. CONCLUSIONS: Our analysis indicates that RRB in ASD can be represented by two distinct subcategories: RSM and IS. Subsequent univariate and multivariate genome-wide association studies of these RRB subcategories enabled the detection of associated SNPs at 8p21.2-8p21.1. Although these results did not replicate in an independent ASD dataset, genomic features of this region and pathway analysis suggest that common variants in 8p21.2-8p21.1 may contribute to RRB, particularly IS. Together, these observations warrant future studies to elucidate the possible contributions of common variants in 8p21.2-8p21.1 to the etiology of RSM and IS in ASD.