Pharmacogenomics of coronary artery response to intravenous gamma globulin in kawasaki disease.

Kawasaki disease (KD) is a multisystem inflammatory illness of infants and young children that can result in acute vasculitis. The mechanism of coronary artery aneurysms (CAA) in KD despite intravenous gamma globulin (IVIG) treatment is not known. We performed a Whole Genome Sequencing (WGS) association analysis in a racially diverse cohort of KD patients treated with IVIG, both using AHA guidelines. We defined coronary aneurysm (CAA) (N = 234) as coronary z ≥ 2.5 and large coronary aneurysm (CAA/L) (N = 92) as z ≥ 5.0. We conducted logistic regression models to examine the association of genetic variants with CAA/L during acute KD and with persistence >6 weeks using an additive model between cases and 238 controls with no CAA. We adjusted for age, gender and three principal components of genetic ancestry. The top significant variants associated with CAA/L were in the intergenic regions (rs62154092 p < 6.32E-08 most significant). Variants in SMAT4, LOC100127, PTPRD, TCAF2 and KLRC2 were the most significant non-intergenic SNPs. Functional mapping and annotation (FUMA) analysis identified 12 genomic risk loci with eQTL or chromatin interactions mapped to 48 genes. Of these NDUFA5 has been implicated in KD CAA and MICU and ZMAT4 has potential functional implications. Genetic risk score using these 12 genomic risk loci yielded an area under the receiver operating characteristic curve (AUC) of 0.86. This pharmacogenomics study provides insights into the pathogenesis of CAA/L in IVIG-treated KD and shows that genomics can help define the cause of CAA/L to guide management and improve risk stratification of KD patients.

Novel functional insights into ischemic stroke biology provided by the first genome-wide association study of stroke in indigenous Africans.

BACKGROUND: African ancestry populations have the highest burden of stroke worldwide, yet the genetic basis of stroke in these populations is obscure. The Stroke Investigative Research and Educational Network (SIREN) is a multicenter study involving 16 sites in West Africa. We conducted the first-ever genome-wide association study (GWAS) of stroke in indigenous Africans. METHODS: Cases were consecutively recruited consenting adults (aged > 18 years) with neuroimaging-confirmed ischemic stroke. Stroke-free controls were ascertained using a locally validated Questionnaire for Verifying Stroke-Free Status. DNA genotyping with the H3Africa array was performed, and following initial quality control, GWAS datasets were imputed into the NIH Trans-Omics for Precision Medicine (TOPMed) release2 from BioData Catalyst. Furthermore, we performed fine-mapping, trans-ethnic meta-analysis, and in silico functional characterization to identify likely causal variants with a functional interpretation. RESULTS: We observed genome-wide significant (P-value < 5.0E-8) SNPs associations near AADACL2 and miRNA (MIR5186) genes in chromosome 3 after adjusting for hypertension, diabetes, dyslipidemia, and cardiac status in the base model as covariates. SNPs near the miRNA (MIR4458) gene in chromosome 5 were also associated with stroke (P-value < 1.0E-6). The putative genes near AADACL2, MIR5186, and MIR4458 genes were protective and novel. SNPs associations with stroke in chromosome 2 were more than 77 kb from the closest gene LINC01854 and SNPs in chromosome 7 were more than 116 kb to the closest gene LINC01446 (P-value < 1.0E-6). In addition, we observed SNPs in genes STXBP5-AS1 (chromosome 6), GALTN9 (chromosome 12), FANCA (chromosome 16), and DLGAP1 (chromosome 18) (P-value < 1.0E-6). Both genomic regions near genes AADACL2 and MIR4458 remained significant following fine mapping. CONCLUSIONS: Our findings identify potential roles of regulatory miRNA, intergenic non-coding DNA, and intronic non-coding RNA in the biology of ischemic stroke. These findings reveal new molecular targets that promise to help close the current gaps in accurate African ancestry-based genetic stroke's risk prediction and development of new targeted interventions to prevent or treat stroke.

Pharmacogenomics of Coronary Artery Response to Intravenous Gamma Globulin in Kawasaki Disease.

Background: Kawasaki disease (KD) is a multisystem inflammatory illness of infants and young children that can result in acute vasculitis. The pathological walls of afflicted coronary arteries show propensity for forming thrombosis and aneurysms. The mechanism of coronary artery aneurysms (CAA) despite intravenous gamma globulin (IVIG) treatment is not known. Methods: We performed a Whole Genome Sequencing (WGS) association analysis in a racially diverse cohort of KD patients treated with IVIG, both using AHA guidelines. We defined coronary aneurysm (CAA) (N = 234) as coronary z>2.5 and large coronary aneurysm (CAA/L) (N = 92) as z>5.0. We conducted logistic regression models to examine the association of genetic variants with CAA/L during acute KD and with persistence >6 weeks using an additive model between cases and 238 controls with no CAA. We adjusted for age, gender and three principal components of genetic ancestry. We performed functional mapping and annotation (FUMA) analysis and further assessed the predictive risk score of genomic risk loci using the area under the receiver operating characteristic curve (AUC). Results: The top significant variants associated with CAA/L were in the intergenic regions (rs62154092 p<6.32E-08 most significant). Variants in SMAT4, LOC100127 , PTPRD, TCAF2 and KLRC2 were the most significant non-intergenic SNPs. FUMA identified 12 genomic risk loci with eQTL or chromatin interactions mapped to 48 genes. Of these NDUFA5 has been implicated in KD CAA and MICU and ZMAT4 has potential functional implications. Genetic risk score using these 12 genomic risk loci yielded an AUC of 0.86. Conclusions: This pharmacogenomics study provides insights into the pathogenesis of CAA/L in IVIG-treated KD patients. We have identified multiple novel SNPs associated with CAA/L and related genes with potential functional implications. The study shows that genomics can help define the cause of CAA/L to guide management and improve risk stratification of KD patients.

RNA-seq analysis identifies transcriptomic profiles associated with anal cancer recurrence among people living with HIV.

BACKGROUND: Chemoradiation therapy (CRT) is the standard of care for squamous cell carcinoma of the anus (SCCA), the most common type of anal cancer. However, approximately one fourth of patients still relapse after CRT. METHODS: We used RNA-sequencing technology to characterize coding and non-coding transcripts in tumor tissues from CRT-treated SCCA patients and compare them between 9 non-recurrent and 3 recurrent cases. RNA was extracted from FFPE tissues. Library preparations for RNA-sequencing were created using SMARTer Stranded Total RNA-Seq Kit. All libraries were pooled and sequenced on a NovaSeq 6000. Function and pathway enrichment analysis was performed with Metascape and enrichment of gene ontology (GO) was performed with Gene Set Enrichment Analysis (GSEA). RESULTS: There were 449 differentially expressed genes (DEGs) observed (390 mRNA, 12 miRNA, 17 lincRNA and 18 snRNA) between the two groups. We identified a core of upregulated genes (IL4, CD40LG, ICAM2, HLA-I (HLA-A, HLA-C) and HLA-II (HLA-DQA1, HLA-DRB5) in the non-recurrent SCCA tissue enriching to the gene ontology term 'allograft rejection', which suggests a CD4+ T cell driven immune response. Conversely, in the recurrent tissues, keratin (KRT1, 10, 12, 20) and hedgehog signaling pathway (PTCH2) genes involved in 'Epidermis Development,', were significantly upregulated. We identified miR-4316, that inhibit tumor proliferation and migration by repressing vascular endothelial growth factors, as being upregulated in non-recurrent SCCA. On the contrary, lncRNA-SOX21-AS1, implicated in the progression of many other cancers, was also found to be more common in our recurrent compared to non-recurrent SCCA.Our study identified key host factors which may drive the recurrence of SCCA and warrants further studies to understand the mechanism and evaluate their potential use in personalized treatment.Key MessageOur study used RNA sequencing (RNA-seq) to identify pivotal factors in coding and non-coding transcripts which differentiate between patients at risk for recurrent anal cancer after treatment. There were 449 differentially expressed genes (390 mRNA, 12 miRNA, 17 lincRNA and 18 snRNA) between 9 non-recurrent and 3 recurrent squamous cell carcinoma of anus (SCCA) tissues. The enrichment of genes related to allograft rejection was observed in the non-recurrent SCCA tissues, while the enrichment of genes related to epidermis development was positively linked with recurrent SCCA tissues.

Cognitive Processing Speed Is Strongly Related to Driving Skills, Financial Abilities, and Other Instrumental Activities of Daily Living in Persons With Mild Cognitive Impairment and Mild Dementia.

BACKGROUND: Cognitive processing speed is important for performing everyday activities in persons with mild cognitive impairment (MCI). However, its role in daily function has not been examined while simultaneously accounting for contributions of Alzheimer's disease (AD) risk biomarkers. We examine the relationships of processing speed and genetic and neuroimaging biomarkers to composites of daily function, mobility, and driving. METHOD: We used baseline data from 103 participants on the MCI/mild dementia spectrum from the Applying Programs to Preserve Skills trial. Linear regression models examined relationships of processing speed, structural magnetic resonance imaging (MRI), and genetic risk alleles for AD to composites of performance-based instrumental activities of daily living (IADLs), community mobility, and on-road driving evaluations. RESULTS: In multivariable models, processing speed and the brain MRI neurodegeneration biomarker Spatial Pattern of Abnormality for Recognition of Early Alzheimer's disease (SPARE-AD) were significantly associated with functional and mobility composite performance. Better processing speed and younger age were associated with on-road driving ratings. Genetic risk markers, left hippocampal atrophy, and white matter lesion volumes were not significant correlates of these abilities. Processing speed had a strong positive association with IADL function (p < .001), mobility (p < .001), and driving (p = .002). CONCLUSIONS: Cognitive processing speed is strongly and consistently associated with critical daily functions in persons with MCI in models including genetic and neuroimaging biomarkers of AD risk. SPARE-AD scores also significantly correlate with IADL performance and mobility. Results highlight the central role of processing speed in everyday task performance among persons with MCI/mild dementia.

Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells.

The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

Establishment of Singleton-Merten syndrome pulp cells: evidence of mineralization dysregulation.

Singleton-Merten syndrome (SMS) is a rare disease with a phenotype of dental dysplasia. Currently, the underlying mechanism of this disease is unknown. In order to investigate the functional mechanism of the SMS tooth phenotypes, we isolated dental pulp tissue and established SMS primary pulp cells. These cells exhibited normal morphology and could be maintained in culture. Their ability to express alkaline phosphatase and mineralize was confirmed by in vitro staining. A comparative osteogenesis polymerase chain reaction array analysis was performed revealing 22 genes up-regulated and 8 genes down-regulated greater than 2-fold in SMS versus unaffected pulp cells. Down-regulated genes included ALP, IGF2, TGFBR2 and COL1A1. Collagen type I was reduced in SMS cells as shown by Western blot analysis. Furthermore, matrix metallopeptidase 13 was found to be dramatically increased in SMS pulp cells. Our findings suggest that dentin mineralization is dysregulated in SMS and may contribute to the root phenotype found in this disease.

Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(v)ß3/α(v)ß5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

Calcium/calmodulin-dependent kinase II regulates notch-1 signaling in prostate cancer cells.

Notch signaling is associated with prostate osteoblastic bone metastases and calcium/calmodulin-dependent kinase II (CaMKII) is associated with osteoblastogenesis of human mesenchymal stem cells. Here we show that prostate cancer cell lines C4-2B and PC3, both derived from bone metastases and express Notch-1, have all four isoforms of CaMKII (alpha, beta, gamma, delta). In contrast, prostate cancer cell lines LNcaP and DU145, which are not derived from bone metastases and lack the Notch-1 receptor, both lack the alpha isoform of CaMKII. In addition, DU145 cells also lack the beta-isoform. In C4-2B cells, inhibition of CaMKII by KN93 or gamma-secretase by L-685,458 inhibited the formation of the cleaved form of Notch-1 thus inhibiting Notch signaling. KN93 inhibited down stream Notch-1 signaling including Hes-1 gene expression, Hes-1 promoter activity, and c-Myc expression. In addition, both KN93 and L-685,458 inhibited proliferation and Matrigel invasion by C4-2B cells. The activity of gamma-secretase was unaffected by KN93 but markedly inhibited by L-685,458. Inhibition of the expression of alpha, beta, or gamma-isoform by siRNA did not affect Hes-1 gene expression, however when expression of one isoform was inhibited by siRNA, there were compensatory changes in the expression of the other isoforms. Over-expression of CaMKII-alpha increased Hes-1 expression, consistent with Notch-1 signaling being at least partially dependent upon CaMKII. This unique crosstalk between CaMKII and Notch-1 pathways provides new insight into Notch signaling and potentially provides new targets for pharmacotherapeutics.