Minor Variant Research Articles

Correction: Characterization of intra- and inter-host norovirus P2 genetic variability in linked individuals by amplicon sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0201850.].

Potential Markers of Autoimmune Diseases, Alleles rs115662534(T) and rs548231435(C), Disrupt the Binding of Transcription Factors STAT1 and EBF1 to the Regulatory Elements of Human CD40 Gene.

CD40 receptor is expressed on B lymphocytes and other professional antigen-presenting cells. The binding of CD40 to its ligand CD154 on the surface of T helper cells plays an important role in the activation of B lymphocytes required for production of antibodies, in particular, against autoantigens. Association of several single nucleotide polymorphisms (SNPs) located in the non-coding areas of human CD40 locus with the elevated risk of autoimmune diseases has been demonstrated. The most studied of these SNPs is rs4810485 located in the first intron of the CD40 gene. Expression of the CD40 gene in B lymphocytes of donors homozygous for the common allelic variant of this polymorphism (G) is higher than in B cells from donors carrying the minor (T) variant. We investigated the enhancer activity of this fragment of the CD40 locus in human B cell lines and showed that it is independent on the rs4810485 alleles. However, the minor allelic variants of the rs4810485-linked SNPs rs548231435 and rs115662534 were associated with a significant decrease in the activity of the CD40 promoter due to the impairments in the binding of EBF1 and STAT1 transcription factors, respectively.

Clinical outcome in anti-neutrophil cytoplasmic antibody-associated vasculitis and gene variants of 11β-hydroxysteroid dehydrogenase type 1 and the glucocorticoid receptor.

We aimed to investigate whether five potential functional haplotypes of the glucocorticoid receptor (GR) gene and a single-nucleotide polymorphism of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) are associated with clinical outcome in ANCA-associated vasculitis. Patients diagnosed with ANCA-associated vasculitis (n = 241) were genotyped for five polymorphisms of the GR gene and one polymorphism of the HSD11B1 gene. GR gene haplotypes were predicted based on genotyping results. Relapse-free survival, mortality, renal survival, metabolic adverse events and infections were compared between carriers and non-carriers of GR haplotypes and the HSD11B1 genotype. Carriers of haplotype 4 (ER22/23EK + 9β+TthIII1) of GR had a significantly higher 5-year mortality risk [hazard ratio (HR) 4.5 (95% CI 1.6, 12.8)] and had a higher risk of developing end-stage renal disease [HR 7.4 (95% CI 1.9, 28.7)]. Carriers of a minor variant of HSD11B1 more frequently experienced relapse [HR 2.5 (95% CI 1.5, 4.1)] except if they also carried haplotype 1 (BclI) of GR. Homozygous carriers of haplotype 1 had a higher risk of developing dyslipidaemia [HR 4.1 (95% CI 1.8, 9.6)]. The occurrence of infections did not differ between GR haplotypes and HSD11B1 genotypes. Haplotypes 1 and 4 of GR and a polymorphism of the HSD11B1 gene were associated with clinically relevant inflammatory and metabolic outcomes in ANCA-associated vasculitis.

Altered ErbB4 splicing and cortical parvalbumin interneuron dysfunction in schizophrenia and mood disorders.

Working memory requires the activity of parvalbumin (PV) interneurons in the dorsolateral prefrontal cortex (DLPFC). Impaired working memory and lower PV expression in the DLPFC are reported in schizophrenia and to a lesser degree in mood disorders. We previously proposed that activity-dependent PV expression is lower in schizophrenia due to a shift in the splicing of erb-b2 receptor tyrosine kinase 4 (ErbB4) transcripts from major to inactive minor variants that reduces excitatory drive to PV interneurons. Here, we tested the hypothesis that the degree of major-to-minor shift in ErbB4 splicing predicts the level of PV expression across schizophrenia and mood disorders. Levels of ErbB4 splice variants and PV mRNA were quantified by PCR in the DLPFC from 40 matched tetrads (N = 160 subjects) of schizophrenia, bipolar disorder (BD), major depressive disorder (MDD), and unaffected comparison subjects. Relative to unaffected comparison subjects, the magnitude of increases in minor variant levels and decreases in major variant levels was greatest in schizophrenia, intermediate in BD, and least in MDD. The same rank order was present for the magnitude of increases in the composite splicing score, which reflects the degree of major-to-minor shift across all ErbB4 splice loci, and for the magnitude of deficient PV expression. Finally, the composite splicing score negatively predicted PV expression across all subject groups. Together, these findings demonstrate a shared relationship between ErbB4 splicing and PV expression and suggest that scaling of the major-to-minor shift in ErbB4 splicing may influence the severity of deficient PV interneuron activity across diagnoses.

Control of Artifactual Variation in Reported Intersample Relatedness during Clinical Use of a Mycobacterium tuberculosis Sequencing Pipeline.

Contact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artifactual variation between Mycobacterium tuberculosis isolates during routine next-generation sequencing of Mycobacterium spp., we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted and sequenced, reads were mapped, and consensus sequences were determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of nonmycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of nonmycobacterial bacterial DNA, we found significant increases in minor variant frequencies, of more than 1.5-fold, in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high-variation regions strongly influenced by the amount of nonmycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we demonstrated an approach identifying critical genomic regions contributing to clinically relevant artifactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multistep laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

Genetic Variant rs755622 Regulates Expression of the Multiple Sclerosis Severity Modifier D-Dopachrome Tautomerase in a Sex-Specific Way.

Multiple sclerosis (MS) is a sex-specific autoimmune disease involving central nervous system. Previous studies determined that macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (DDT) sex-specifically affect MS progression. Moreover, other studies reported that rs755622 polymorphism in promoter region of MIF gene is associated with risk of MS and affects the promoter activity to regulate MIF expression in a sex-specific way. Given that MIF and DDT share a part of promoter sequence, we surmise that rs755622 can also regulate DDT expression in a sex-specific way. However, this has not yet been studied. Here, we used five large-scale expression quantitative trait loci (eQTLs) and two RNA-seq datasets from brain and blood to assess the potential influence of rs755622 variant on expression of DDT in different genders by the linear regression and differential expression analysis. The results show that the minor allele frequency of rs755622 and expression of DDT are significantly increased in males for MS subjects and this minor allele variant can significantly upregulate DDT expression for males but not females, which suggests that the regulation of DDT expression level by rs755622 can affect MS progression in males. These findings further support and expand conclusions of previous studies and may help to better understand the mechanisms of MS.

Analyzing 74,248 Samples Confirms the Association Between CLU rs11136000 Polymorphism and Alzheimer\u2019s Disease in Caucasian But Not Chinese population

Clusterin (CLU) is considered one of the most important roles for pathogenesis of Alzheimer’s Disease (AD). The early genome-wide association studies (GWAS) identified the CLU rs11136000 polymorphism is significantly associated with AD in Caucasian. However, the subsequent studies are unable to replicate these findings in different populations. Although two independent meta-analyses show evidence to support significant association in Asian and Caucasian populations by integrating the data from 18 and 25 related GWAS studies, respectively, many of the following 18 studies also reported the inconsistent results. Moreover, there are six missed and a misclassified GWAS studies in the two meta-analyses. Therefore, we suspected that the small-scale and incompletion or heterogeneity of the samples maybe lead to different results of these studies. In this study, large-scale samples from 50 related GWAS studies (28,464 AD cases and 45,784 controls) were selected afresh from seven authoritative sources to reevaluate the effect of rs11136000 polymorphism to AD risk. Similarly, we identified that the minor allele variant of rs11136000 significantly decrease AD risk in Caucasian ethnicity using the allele, dominant and recessive model. Different from the results of the previous studies, however, the results showed a negligible or no association in Asian and Chinese populations. Collectively, our analysis suggests that, for Asian and Chinese populations, the variant of rs11136000 may be irrelevant to AD risk. We believe that these findings can help to improve the understanding of the AD’s pathogenesis.

Protective C allele of the single-nucleotide polymorphism rs1335532 is associated with strong binding of Ascl2 transcription factor and elevated CD58 expression in B-cells

CD58 is expressed on the surface of antigen-presenting cells, including B-cells, and provides co-stimulation to regulatory T-cells (Treg) through CD2 receptor binding. Tregs appear to be essential suppressors of tissue-specific autoimmune responses. Thereby, CD58 plays protective role in multiple sclerosis (MS) and CD58 was identified among several loci associated with MS susceptibility. Minor (C) variant of the single-nucleotide polymorphism (SNP) rs1335532 is associated with lower MS risk according to genome-wide association studies (GWAS) and its presence correlates with higher CD58 mRNA levels in MS patients. We found that genomic region containing rs1335532 has enhancer properties and can significantly boost the CD58 promoter activity in lymphoblast cells. Using bioinformatics and pull-down assay we found that the protective (C) rs1335532 allele created functional binding site for ASCL2 transcription factor, a target of the Wnt signaling pathway. Both in B-lymphoblastoid cell lines and in primary B-cells, as well as in a monocytic cell line, activation of Wnt signaling resulted in an increased CD58 promoter activity in the presence of the protective but not the risk allele of rs1335532, whereas ASCL2 knockdown abrogated this effect. In summary, our results suggest that ASCL2 mediates the protective function of rs1335532 minor (C) allele in MS.

Abstract 5573: Highly multiplexed diagnostic test for the detection of extended EGFR del19 variants in patients with NSCLC

Abstract The detection of nucleic acids in circulation has become an increasingly important part of the clinical workup and testing for cancer patients, particularly those with non-small cell lung cancer (NSCLC). The utilization of blood-based PCR platforms for detection of somatic variant mutations addresses several limitations of previously established molecular testing approaches such as an inability to access tissue, insufficient tissue quantity or quality, and slow turnaround time for results. However, the number of variants detected may be limited by use of some rapid PCR technologies. We have focused in this study on the analytic and clinical development of a highly multiplexed assay for the detection of EGFR del19 while maintaining a rapid turn-around time. The test system includes the prospective collection of whole blood into a cfDNA BCT and the reproducible detection of del19 variants out of plasma using ddPCR-based technology. Analytic specificity and sensitivity was examined using in vitro DNA designed to mimic the EGFR del19 variants in the assay. Analytic sensitivity was performed and demonstrates that the theoretical limit of detection of the test was below 0.02% minor variant frequency when using high concentrations of synthetic standards. Precision studies were conducted with both analytic and clinical samples at high, medium and low mutation frequencies, over three consecutive days, three runs in one day and with two operators. All test parameters were successfully met. Normal healthy donor (n = 11) and reference ddPCR positive and negative NSCLC donor (n = 80) samples were tested. EGFR del19 variants were not identified in the plasma from the normal healthy donors or the EGFR del19 reference negative NSCLC donors. All reference positive cases were accurately detected above our required threshold of 2 variant copies with detection ranging from 14 to 1584. For all donor specimens the wild-type copies were well above the predefined clinical QC threshold of 300, ranging from 380 to 760 copies. Of note, no negative reference samples contained more than two mutant copies. We conclude that the developed test is highly suited for the sensitive and reproducible detection of multiple EGFR del19 variants from blood specimens in the clinical laboratory. Citation Format: Hestia S. Mellert, Leisa Jackson, Kristin Alexander, Amanda Weaver, Dianna Maar, Dawne Shelton, Gary Pestano. Highly multiplexed diagnostic test for the detection of extended EGFR del19 variants in patients with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5573.

PO-317 Improved performance and workflow using sanger sequencing for low-level somatic mutation detection from low amount of formalin-fixed paraffin-embedded (FFPE) DNA

IntroductionSanger sequencing is often used in oncology research applications for molecular profiling of cancers. Applied Biosystems Minor Variant Finder (MVF) Software now enables low-level variant detection in Sanger sequencing traces.RAS mutational testing is frequently performed by clinical researchers due to the strong correlation between RAS mutational profiles of colorectal cancers and their anti–epidermal growth factor receptor (EGFR) response.Material and methodsWe have improved and simplified the workflow for the extended RAS research assay. The ready-to-use plates are preloaded with primers for all the eight frequently studied hotspot regions of KRAS and NRAS genes thus only the PCR mix and templates need to be added. Minimised hands-on time, extra convenience and flexibility gained by using the SeqStudio Genetic Analyzer which utilises an all-in-one cartridge and provides simple plate setup with easy-to-use touch-screen interface and real-time/remote data monitoring.Results and discussionsThe performance of the assay on the SeqStudio instrument was tested in the laboratory of Dr. Quagliata (University of Basel, Switzerland) on twenty-two FFPE DNA samples derived from colon cancer biopsies. Variants (ranging from 5.25% to 91.1%) were successfully detected even from research samples where less than 1 ng of input DNA per reaction was used. Only limited amount of microdissected FFPE sections were available for 6 research samples where DNA concentrations were below 1 ng (down to 0.094 ng).ConclusionRAS mutations down to 5% level can be detected even from less than 1 ng FFPE DNA with simplified workflow, fast turnaround time and low cost per sample.

SH2B3 (lnk) Regulates Type 1 Diabetes Immune Phenotypes

Background: The type 1 diabetes (T1D)-associated risk gene SH2B Adaptor Protein 3 (SH2B3) encodes a phosphatase with specificity for Janus Kinase 2 (JAK2). We hypothesized that variations in SH2B3 would affect immune signaling pathways relevant to T1D autoimmunity. Here we report: 1) Occurrence of the SH2B3 risk variant among T1D patients, unaffected first-degree relatives and controls; 2) Prevalence of SH2B3 risk genotype in African Americans in the Southeastern U.S.; and 3) Alterations to immune composition associated with the SH2B3 risk variant. Approach and Results: 1) The University of Florida Diabetes Institute (UFDI) cohort (n=1946) was analyzed for genetic risk of T1D using a Taqman single nucleotide polymorphism (SNP) genotyping array with coverage of 32 SNPs. Two SNPs (rs3184504 and rs653178) corresponding to SH2B3 were in perfect linkage disequilibrium and will be described here as minor (T1D risk) or common variant. Among Caucasian subjects, the odds ratio (OR) for the minor variant among T1D subjects was 1.30 (1.06-1.59, p=1.42x10-2), similar to previous reports. Among African American subjects, the minor variant OR was elevated (2.93, 1.22-7.03, p=1.30x10-2), representing the most highly-associated non-HLA gene. Complete blood count (CBC, n=954) and flow cytometric human immunophenotyping (HIP, n=252) were performed on a subset of subjects. Individuals carrying the risk variant of SH2B3 had significantly more blood monocytes and neutrophils but similar numbers of lymphocytes compared to non-carriers. HIP revealed a significantly higher frequency of type 1 (CXCR3+) CD8+ T cells in subjects with the risk variant of SH2B3. Conclusions: The risk variant of SH2B3 is associated with both increased risk for T1D and seroconversion in Caucasian and African American subjects. Phenotypically, this element of T1D genetic risk presents as increased frequency of some myeloid cell populations and either expansion or polarization of CD8+ T cells bearing the canonical Th1 chemokine receptor CXCR3. Disclosure A. Camargo: None. D.J. Perry: None. T.M. Brusko: Stock/Shareholder; Self; OneVax, LLC. Advisory Panel; Self; Caladrius Biosciences, Inc.. Consultant; Self; Merck & Co., Inc., Sanofi-Aventis. M.A. Wallet: None.

Abstract 614: From Genotype to Phenotype: Molecular and Functional Characterization of the CVD-associated SNP rs11574 in ID3

Atherosclerosis is the primary pathological process of CVD and is strongly controlled by heritable factors; however, the mechanisms and pathways through which many of these genetic components regulate plaque development are poorly understood. Recently, a SNP in the coding region of the gene inhibitor of differentiation 3 ( ID3 ) at rs11574 was identified in multiple studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105 th amino acid of ID3 from an alanine to a threonine and is associated with an attenuated ability of ID3 to antagonize bHLH transcription factors and to prevent them from binding to DNA and activating transcription. The current study utilized co-immunoprecipitation to demonstrate that the minor allele of rs11574 specifically impairs the ability of ID3 to bind to and sequester E12 and no other member of the bHLH family. ChIP studies further demonstrated that the impaired regulatory ability of the ID3 minor allele variant increases E12 occupancy at promoter regions and enhances transcription of E12 target genes including smooth muscle alpha actin ( Acta2 ) and p21 in murine vascular smooth muscle cells (VSMCs). To study the role of this SNP in human cells in intact chromatin under endogenous regulation, we genome edited human 293T cells using CRISPR/Cas9 to produce the allelic variants of rs11574 in the ID3 gene (GG, AG, and AA). Edited cells showed significant changes in cellular proliferation, gene expression, and promoter occupancy depending upon genotype at rs11574. Cells containing the minor allele of rs11574 displayed significantly increased p21 expression and proliferated more slowly than cells containing the major allele of rs11574; whereas, cells heterozygote at rs11574 displayed an intermediate phenotype. These data implicate rs11574 in the regulation of cellular proliferation and mature VSMC marker expression, key phenotypes regulated in the processes of lesion development and formation.

Identification of immunological pathways altered by a pro-autoimmune genetic risk variant, PTPN22 1858C>T, that enhances the prevalence of Type 1 diabetes

Abstract Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) encodes lymphoid tyrosine phosphatase (Lyp) adaptor molecule that is involved in numerous biochemical pathways in hematopoietic cells. Studies have shown an association with the minor allelic variant of PTPN22, R620W, and increased incidence of autoimmune disorders such as type 1 diabetes (T1D). Approximately 10% of the North American population carry the R620W minor allele, which increases the risk of T1D by as much as 2–4-fold. The exact mechanisms by which this pro-autoimmune risk allele enhances the incidence of T1D is unknown. To address this gap of knowledge our lab introduced the murine ortholog of the human R620W allele, R619W, into the endogenous locus of Non-obese diabetic (NOD) mice that develop spontaneous T1D. NOD mice harboring this mutation (NOD 619W/W) demonstrated a similar phenotype seen in affected humans with accelerated onset of T1D and enhanced anti-islet autoantibodies (IAA). Clinical manifestation of T1D in humans is preceded by IAA in the serum. IAA affinity can often identify individuals at risk of developing diabetes. Germinal centers (GC) are lymphoid structures where affinity maturation occurs with the help of follicular helper T cells (Tfh) cells. An increase in circulating Tfh cells has been reported in newly diagnosed and pre-diabetic individuals, thus suggesting a role for GC reactivity in T1D. Similar to human observations, NOD 619W/W mice also show enhanced IAA and increased Tfh and GC B cell numbers before onset of T1D therefore, our lab is currently examining the immunological pathways altered by this pro-autoimmune risk alleles and their contributions to T1D.

Deep-Coverage MPS Analysis of Heteroplasmic Variants within the mtGenome Allows for Frequent Differentiation of Maternal Relatives.

Distinguishing between maternal relatives through mitochondrial (mt) DNA sequence analysis has been a longstanding desire of the forensic community. Using a deep-coverage, massively parallel sequencing (DCMPS) approach, we studied the pattern of mtDNA heteroplasmy across the mtgenomes of 39 mother-child pairs of European decent; haplogroups H, J, K, R, T, U, and X. Both shared and differentiating heteroplasmy were observed on a frequent basis in these closely related maternal relatives, with the minor variant often presented as 2–10% of the sequencing reads. A total of 17 pairs exhibited differentiating heteroplasmy (44%), with the majority of sites (76%, 16 of 21) occurring in the coding region, further illustrating the value of conducting sequence analysis on the entire mtgenome. A number of the sites of differentiating heteroplasmy resulted in non-synonymous changes in protein sequence (5 of 21), and to changes in transfer or ribosomal RNA sequences (5 of 21), highlighting the potentially deleterious nature of these heteroplasmic states. Shared heteroplasmy was observed in 12 of the 39 mother-child pairs (31%), with no duplicate sites of either differentiating or shared heteroplasmy observed; a single nucleotide position (16093) was duplicated between the data sets. Finally, rates of heteroplasmy in blood and buccal cells were compared, as it is known that rates can vary across tissue types, with similar observations in the current study. Our data support the view that differentiating heteroplasmy across the mtgenome can be used to frequently distinguish maternal relatives, and could be of interest to both the medical genetics and forensic communities.

Uncommon Filaggrin Variants Are Associated with Persistent Atopic Dermatitis in African Americans

Atopic dermatitis (AD) is a common illness that has been associated with filaggrin gene (FLG) loss of function (LoF) variation. In African Americans, a group that commonly has AD and has not been well studied, FLG LoF variation is rarely found. Our objective was to use massively parallel sequencing to evaluate FLG LoF variation in children of African ancestry to evaluate the association between FLG LoF variation and AD and AD persistence. We studied 262 African American children with AD. Nine unique FLG exon 3 LoF variants were identified for an overall minor variant frequency of 6.30% (95% confidence interval [CI]= 4.37-8.73). The most common variants were p.R501X (1.72%, 95% CI= 0.79-3.24), p.S3316X (1.34%, 95% CI= 0.54-2.73), and p.R826X (0.95%, 95% CI= 0.31-2.2). Over an average follow-up period of 96.4 (95% CI= 92.0-100.8) months, African American children with FLG LoF were less likely to be symptom free (odds ratio= 0.36, 95% CI= 0.14-0.89, P= 0.027) compared with a FLG wild-type child. In contrast to previous reports, uncommon FLG LoF variants in African American children exist and are associated with AD and more persistent AD. In contrast to Europeans, no FLG LoF variants predominate in African American children. Properly determining FLG LoF status requires advanced sequencing techniques.

Combined annotation-dependent depletion score for BRCA1/2 variants in patients with breast and/or ovarian cancer.

Utility of combined annotation‐dependent depletion (CADD) score was recently reported to rank pathogenicity as C‐scores ranging 1‐99 for both confirmed deleterious mutation. Using C‐scores for BRCA1/2 variants, we tried to constitute the classification system for variant of uncertain significance (VUS), which had been a major problem of genetic testing for hereditary breast and/or ovarian cancer. We analyzed BRCA1/2 genes for 283 patients with breast and/or ovarian cancer. The deleterious mutation and missesne mutations, minor variant, and wild type of BRCA1 and ‐2 were 5, 27, 251 and 15, 85, 183, respectively. Meanwhile, the variants with C‐score ≥10 were involved in 19/283 (6.7%) in BRCA1 and 34/283 (12%) in BRCA2. All deleterious mutations were included in this group. Frequency of personal history and family history of ovarian cancer were significantly high, and frequency of serous adenocarcinoma of ovary and triple negative breast cancer was relatively high in the group with deleterious mutations. Similar findings were seen in patients with variants of C‐score ≥10. According to the C‐score and population frequency, we could define VUS for 11 patients out of 283 patients (3.9 CADD is useful to classify the variant of BRCA1/2 and selecting the patient who needs further segregation studies.

Association of Dopamine Beta-Hydroxylase Polymorphisms with Alzheimer’s Disease, Parkinson’s Disease and Schizophrenia: Evidence Based on Currently Available Loci

Background/Aims: The neuropathies Alzheimer’s disease (AD), Parkinson’s disease (PD), and schizophrenia (SCZ) have different pathological mechanisms but share some common neurodegenerative features, such as gradual loss of neuronal structure and function. Dopamine beta-hydroxylase (DBH), a gene located in the chromosomal region 9q34, plays a crucial role in the process of converting dopamine into norepinephrine (NE). Several case–control studies have reported this pathway in the pathogenesis of AD, PD and SCZ. However, the results are controversial. Methods: We conducted a meta-analysis to estimate the associations between polymorphisms in this gene and AD, PD and SCZ. Seven databases (PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wan Fang, SZ Gene and AD Gene) were searched to identify eligible studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to evaluate the associations of DBH variants with AD, PD and SCZ susceptibility. Results: A total of 41 studies involving 10506 cases and 15083 controls were included in our meta-analysis. The analysis results indicated that a lack of association (P > 0.05) was observed between most of the currently available DBH polymorphisms and the neurological diseases AD, PD and SCZ; however, the DBH rs1611131 (allelic model: OR = 0.889, 95% CI: 0.815 - 0.969; dominant model: OR = 0.868, 95% CI: 0.778 - 0.968), rs2283123 (allelic model: OR = 0.285, 95% CI: 0.095 - 0.862; dominant model: OR = 0.290, 95% CI: 0.094 -0.897) and rs2007153 (allelic model: OR = 2.196, 95% CI: 1.506 - 3.200; dominant model: OR = 2.985, 95% CI: 1.465 - 6.084; recessive model: OR = 2.729, 95% CI: 1.548 - 4.812) variants were shown to be significantly associated with the risk of AD (the former variant) and SCZ (the latter two variants). Conclusion: On the one hand, most DBH polymorphisms from the currently available loci showed no linkage to AD, PD or SCZ, indicating the lower possibility of these loci serving as genetic markers of the risks of diseases with neurodegenerative characteristics. On the other hand, the DBH rs2283123 and rs2007153 polymorphisms could have opposite effects on SCZ development in Caucasians and be more specific in Croatians, while the DBH rs1611131 minor variant might have a protective effect on AD risk in Caucasians; however, these results require further study.

Assessment of mitochondrial DNA heteroplasmy detected on commercial panel using MPS system with artificial mixture samples.

Mitochondrial DNA (mtDNA) heteroplasmy is a potential genetic marker for forensic mtDNA analysis as well as phylogenic studies. Frequency of mtDNA heteroplasmy has been investigated in different populations through massively parallel sequencing (MPS) analysis, revealing various levels of frequency based on different MPS systems. For accurate heteroplasmy identification, it is essential to explore reliable detection threshold on various MPS systems. In addition, software solutions and pipelines need to be evaluated to analyze data effectively. In this study, heteroplasmy analysis was conducted on a commercially available mtDNA analysis system developed for forensic caseworks with artificially mixed DNA samples known for ratios and variant positions for assessment. mtDNA heteroplasmy > 10% was detectable with Torrent Variant Caller (TVC) while lower levels were identified using GeneMarker® HTS specialized software for minor variant detection. This study implies that analytical parameters and tools need to be optimized and evaluated for low-level heteroplasmy identification. Automated system with simple and efficient workflow is needed for forensic caseworks.

Quaternion Invariant Extended Kalman Filtering for Spacecraft Attitude Estimation

The spacecraft attitude estimation is addressed in the framework of invariant Kalman filtering, which rests on invariance of the system dynamics and output map with respect to appropriate coordinate transformations. The available measurements are assumed to be the angular velocity from three-axis gyroscopes and vector measurements from attitude sensors. Two continuous-discrete quaternion filters are developed from output state errors defined in the inertial frame and the spacecraft body frame, respectively. The former is termed the right-invariant extended Kalman filter and the latter is termed the left-invariant extended Kalman filter. These two filters both respect the norm constraint of the attitude quaternion but stem from different invariance properties of the system dynamics. It is shown that the left-invariant extended Kalman filter bears much resemblance to, and thus can be viewed as, a minor variant of the conventional quaternion multiplicative extended Kalman filter. The right-invariant extended Kalman filter possesses less dependence on the estimated trajectory and, as a result, better robustness than the left-invariant extended Kalman filter and multiplicative extended Kalman filter. Extensive Monte Carlo simulations of spacecraft attitude determination implementations demonstrate the advantageous performance of the right-invariant extended Kalman filter as compared to the left-invariant extended Kalman filter, multiplicative extended Kalman filter, and some of their improved variants.

Cryptic Microheteroresistance Explains Mycobacterium tuberculosis Phenotypic Resistance.

Minority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays. To determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations. We used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group). Among 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n = 20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n = 18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n = 3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n = 53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (<5%) subpopulations (n = 3/66) across both groups. Cryptic minor variant mycobacterial subpopulations exist below the resolving capability of current drug susceptibility testing methodologies, and may explain an important proportion of false-negative resistance determinations.