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In disease studies, family-based designs have become an attractive approach to analyzing next-generation sequencing (NGS) data for the identification of rare mutations enriched in families. Substantial research effort has been devoted to developing pipelines for automating sequence alignment, variant calling, and annotation. However, fewer pipelines have been designed specifically for disease studies. Most of the current analysis pipelines for family-based disease studies using NGS data focus on a specific function, such as identifying variants with Mendelian inheritance or identifying shared chromosomal regions among affected family members. Consequently, some other useful family-based analysis tools, such as imputation, linkage, and association tools, have yet to be integrated and automated. We developed FamPipe, a comprehensive analysis pipeline, which includes several family-specific analysis modules, including the identification of shared chromosomal regions among affected family members, prioritizing variants assuming a disease model, imputation of untyped variants, and linkage and association tests. We used simulation studies to compare properties of some modules implemented in FamPipe, and based on the results, we provided suggestions for the selection of modules to achieve an optimal analysis strategy. The pipeline is under the GNU GPL License and can be downloaded for free at . [ABSTRACT FROM AUTHOR]

Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer’s disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with strong genetic components. Several recent genome-wide association (GWA) studies in Caucasian samples have reported a number of gene regions and loci correlated with the risk of ASD—albeit with very little consensus across studies. Methods: A two-stage GWA study was employed to identify common genetic variants for ASD in the Taiwanese Han population. The discovery stage included 315 patients with ASD and 1,115 healthy controls, using the Affymetrix SNP array 6.0 platform for genotyping. Several gene regions were then selected for fine-mapping and top markers were examined in extended samples. Single marker, haplotype, gene-based, and pathway analyses were conducted for associations. Results: Seven SNPs had p-values ranging from 3.4~9.9*10−6, but none reached the genome-wide significant level. Five of them were mapped to three known genes (OR2M4, STYK1, and MNT) with significant empirical gene-based p-values in OR2M4 (p = 3.4*10−5) and MNT (p = 0.0008). Results of the fine-mapping study showed single-marker associations in the GLIS1 (rs12082358 and rs12080993) and NAALADL2 (rs3914502 and rs2222447) genes, and gene-based associations for the OR2M3-OR2T5 (olfactory receptor genes, p = 0.02), and GLIPR1/KRR1 gene regions (p = 0.015). Pathway analyses revealed important pathways for ASD, such as olfactory and G protein–coupled receptors signaling pathways. Conclusions: We reported Taiwanese Han specific susceptibility genes and variants for ASD. However, further replication in other Asian populations is warranted to validate our findings. Investigation in the biological functions of our reported genetic variants might also allow for better understanding on the underlying pathogenesis of autism. [ABSTRACT FROM AUTHOR]

GABRB3 encoding the β3 subunit of GABAA receptor has been implicated in multiple neuropsychiatric disorders, including substance abuse. Previous studies reported that SNPs at the 5′ regulatory region of GABRB3 could regulate GABRB3 gene expression and associated with childhood absence epilepsy (CAE). The study aimed to investigate whether SNPs at the 5′ regulatory region of GABRB3 were associated with heroin dependence in our population. We first re-sequenced 1.5 kb of the 5′regulatory region of GABRB3 gene to examine the SNP profile in the genomic DNA of 365 control subjects. Then, we conducted a case-control association analysis between 576 subjects with heroin dependence (549 males, 27 females) and 886 controls (472 males, 414 females) by genotyping the rs4906902 as a tag SNP. We also conducted a reporter gene assay to assess the promoter activity of two major haplotypes derived from SNPs at this region. We detected 3 common SNPs (rs4906902, rs8179184 and rs20317) at this region that had strong pair-wise linkage disequilibrium. The C allele of rs4906902 was found to be associated with increased risk of heroin dependence (odds ratio:1.27, p = 0.002). Two major haplotypes (C-A-G and T-G-C) derived from these 3 SNPs accounted for 99% of this sample, and reporter gene activity assay showed that haplotype C-A-G that contained the C allele of the tag SNP rs4906902 had higher activity than haplotype T-G-C. Our data suggest that GABRB3 might be associated with heroin dependence, and increased expression of GABRB3 might contribute to the pathogenesis of heroin dependence. [ABSTRACT FROM AUTHOR]

Aberrant synaptic dysfunction is implicated in the pathogenesis of schizophrenia. The DLGAP2 gene encoding the SAP90/PSD-95-associated protein 2 (SAPAP2) located at the post-synaptic density of neuronal cells is involved in the neuronal synaptic function. This study aimed to investigate whether the DLGAP2 gene is associated with schizophrenia. We resequenced the putative promoter region and all the exons of the DLGAP2 gene in 523 patients with schizophrenia and 596 non-psychotic controls from Taiwan and conducted a case-control association analysis. We identified 19 known SNPs in this sample. Association analysis of 9 SNPs with minor allele frequency greater than 5% showed no association with schizophrenia. However, we found a haplotype (CCACCAACT) significantly associated with schizophrenia (odds ratio:2.5, p<0.001). We also detected 16 missense mutations and 1 amino acid-insertion mutation in this sample. Bioinformatic analysis showed some of these mutations were damaging or pathological to the protein function, but we did not find increased burden of these mutations in the patient group. Notably, we identified 5 private rare variants in 5 unrelated patients, respectively, including c.−69+9C>T, c.−69+13C>T, c.−69+47C>T, c.−69+55C>T at intron 1 and c.−32A>G at untranslated exon 2 of the DLGAP2 gene. These rare variants were not detected in 559 control subjects. Further reporter gene assay of these rare variants except c.−69+13C>T showed significantly elevated promoter activity than the wild type, suggesting increased DLGAP2 gene expression may contribute to the pathogenesis of schizophrenia. Our results indicate that DLGAP2 is a susceptible gene of schizophrenia. [ABSTRACT FROM AUTHOR]

Runs of homozygosity (ROH) may play a role in complex diseases. In the current study, we aimed to test if ROHs are linked to the risk of autism and related language impairment. We analyzed 546,080 SNPs in 315 Han Chinese affected with autism and 1,115 controls. ROH was defined as an extended homozygous haplotype spanning at least 500 kb. Relative extended haplotype homozygosity (REHH) for the trait-associated ROH region was calculated to search for the signature of selection sweeps. Totally, we identified 676 ROH regions. An ROH region on 11q22.3 was significantly associated with speech delay (corrected p = 1.73×10−8). This region contains the NPAT and ATM genes associated with ataxia telangiectasia characterized by language impairment; the CUL5 (culin 5) gene in the same region may modulate the neuronal migration process related to language functions. These three genes are highly expressed in the cerebellum. No evidence for recent positive selection was detected on the core haplotypes in this region. The same ROH region was also nominally significantly associated with speech delay in another independent sample (p = 0.037; combinatorial analysis Stouffer’s z trend = 0.0005). Taken together, our findings suggest that extended recessive loci on 11q22.3 may play a role in language impairment in autism. More research is warranted to investigate if these genes influence speech pathology by perturbing cerebellar functions. [ABSTRACT FROM AUTHOR]

GABRB3 encoding the β3 subunit of GABAA receptor has been implicated in multiple neuropsychiatric disorders, including substance abuse. Previous studies reported that SNPs at the 5' regulatory region of GABRB3 could regulate GABRB3 gene expression and associated with childhood absence epilepsy (CAE). The study aimed to investigate whether SNPs at the 5' regulatory region of GABRB3 were associated with heroin dependence in our population. We first re-sequenced 1.5 kb of the 5'regulatory region of GABRB3 gene to examine the SNP profile in the genomic DNA of 365 control subjects. Then, we conducted a case-control association analysis between 576 subjects with heroin dependence (549 males, 27 females) and 886 controls (472 males, 414 females) by genotyping the rs4906902 as a tag SNP. We also conducted a reporter gene assay to assess the promoter activity of two major haplotypes derived from SNPs at this region. We detected 3 common SNPs (rs4906902, rs8179184 and rs20317) at this region that had strong pair-wise linkage disequilibrium. The C allele of rs4906902 was found to be associated with increased risk of heroin dependence (odds ratio:1.27, p = 0.002). Two major haplotypes (C-A-G and T-G-C) derived from these 3 SNPs accounted for 99% of this sample, and reporter gene activity assay showed that haplotype C-A-G that contained the C allele of the tag SNP rs4906902 had higher activity than haplotype T-G-C. Our data suggest that GABRB3 might be associated with heroin dependence, and increased expression of GABRB3 might contribute to the pathogenesis of heroin dependence.

Aberrant synaptic dysfunction is implicated in the pathogenesis of schizophrenia. The DLGAP2 gene encoding the SAP90/PSD-95-associated protein 2 (SAPAP2) located at the post-synaptic density of neuronal cells is involved in the neuronal synaptic function. This study aimed to investigate whether the DLGAP2 gene is associated with schizophrenia. We resequenced the putative promoter region and all the exons of the DLGAP2 gene in 523 patients with schizophrenia and 596 non-psychotic controls from Taiwan and conducted a case-control association analysis. We identified 19 known SNPs in this sample. Association analysis of 9 SNPs with minor allele frequency greater than 5% showed no association with schizophrenia. However, we found a haplotype (CCACCAACT) significantly associated with schizophrenia (odds ratio:2.5, p<0.001). We also detected 16 missense mutations and 1 amino acid-insertion mutation in this sample. Bioinformatic analysis showed some of these mutations were damaging or pathological to the protein function, but we did not find increased burden of these mutations in the patient group. Notably, we identified 5 private rare variants in 5 unrelated patients, respectively, including c.-69+9C>T, c.-69+13C>T, c.-69+47C>T, c.-69+55C>T at intron 1 and c.-32A>G at untranslated exon 2 of the DLGAP2 gene. These rare variants were not detected in 559 control subjects. Further reporter gene assay of these rare variants except c.-69+13C>T showed significantly elevated promoter activity than the wild type, suggesting increased DLGAP2 gene expression may contribute to the pathogenesis of schizophrenia. Our results indicate that DLGAP2 is a susceptible gene of schizophrenia.

We report the first genome-wide association study of a joint analysis using 795 Han Chinese individuals with treatment-refractory schizophrenia (TRS) and 806 controls. Three loci showed suggestive significant association with TRS were identified. These loci include: rs10218843 (P = 3.04 × 10(-7)) and rs11265461 (P = 1.94 × 10(-7)) are adjacent to signaling lymphocytic activation molecule family member 1 (SLAMF1); rs4699030 (P = 1.94 × 10(-6)) and rs230529 (P = 1.74 × 10(-7)) are located in the gene nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1); and rs13049286 (P = 3.05 × 10(-5)) and rs3827219 (P = 1.66 × 10(-5)) fall in receptor-interacting serine/threonine-protein kinase 4 (RIPK4). One isolated single nucleotide polymorphism (SNP), rs739617 (P = 3.87 × 10(-5)) was also identified to be associated with TRS. The -94delATTG allele (rs28362691) located in the promoter region of NFKB1 was identified by resequencing and was found to associate with TRS (P = 4.85 × 10(-6)). The promoter assay demonstrated that the -94delATTG allele had a significant lower promoter activity than the -94insATTG allele in the SH-SY5Y cells. This study suggests that rs28362691 in NFKB1 might be involved in the development of TRS.

Methamphetamine (meth) is a highly addictive psychostimulant that is among the most widely abused illicit drugs, with an estimated over 35 million users in the world. Several lines of evidence suggest that chronic meth abuse is a major factor for increased risk of infections with human immunodeficiency virus and possibly other pathogens, due to its immunosuppressive property. Influenza A virus infections frequently cause epidemics and pandemics of respiratory diseases among human populations. However, little is known about whether meth has the ability to enhance influenza A virus replication, thus increasing severity of influenza illness in meth abusers. Herein, we investigated the effects of meth on influenza A virus replication in human lung epithelial A549 cells. The cells were exposed to meth and infected with human influenza A/WSN/33 (H1N1) virus. The viral progenies were titrated by plaque assays, and the expression of viral proteins and cellular proteins involved in interferon responses was examined by Western blotting and immunofluorescence staining. We report the first evidence that meth significantly reduces, rather than increases, virus propagation and the susceptibility to influenza infection in the human lung epithelial cell line, consistent with a decrease in viral protein synthesis. These effects were apparently not caused by meth's effects on enhancing virus-induced interferon responses in the host cells, reducing viral biological activities, or reducing cell viability. Our results suggest that meth might not be a great risk factor for influenza A virus infection among meth abusers. Although the underlying mechanism responsible for the action of meth on attenuating virus replication requires further investigation, these findings prompt the study to examine whether other structurally similar compounds could be used as anti-influenza agents.

Hypofunction of N-methyl-D-aspartate (NMDA) receptor-mediated signal transduction has been implicated in the pathophysiology of schizophrenia. Post-synaptic density protein 95 (PSD95) plays a critical role in regulating the trafficking and activity of the NMDA receptor and altered expression of the PSD95 has been detected in the post-mortem brain of patients with schizophrenia. The study aimed to examine whether the DLG4 gene that encodes the PSD95 may confer genetic susceptibility to schizophrenia. We re-sequenced the core promoter, all the exons, and 3' untranslated regions (UTR) of the DLG4 gene in 588 Taiwanese schizophrenic patients and conducted an association study with 539 non-psychotic subjects. We did not detect any rare mutations at the protein-coding sequences of the DLG4 gene associated with schizophrenia. Nevertheless, we identified four polymorphic markers at the core promoter and 5' UTR and one single nucleotide polymorphism (SNP) at the 3'UTR of the DLG4 gene in this sample. Genetic analysis showed an association of a haplotype (C-D) derived from 2 polymorphic markers at the core promoter (odds ratio = 1.26, 95% confidence interval = 1.06-1.51, p = 0.01), and a borderline association of the T allele of the rs13331 at 3'UTR with schizophrenia (odds ratio = 1.19, 95% confidence interval = 0.99-1.43, p = 0.06). Further reporter gene assay showed that the C-D-C-C and the T allele of the rs13331 had significant lower activity than their counter parts. Our data indicate that the expression of the DLG4 gene is subject to regulation by the polymorphic markers at the core promoter region, 5' and 3'UTR of the gene, and is associated with the susceptibility of schizophrenia.

Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.