Identification of SARS-CoV-2 variants using viral sequencing for the Centers for Disease Control and Prevention genomic surveillance program.

BACKGROUND: The Centers for Disease Control and Prevention contracted with laboratories to sequence the SARS-CoV-2 genome from positive samples across the United States to enable public health officials to investigate the impact of variants on disease severity as well as the effectiveness of vaccines and treatment. Herein we present the initial results correlating RT-PCR quality control metrics with sample collection and sequencing methods from full SARS-CoV-2 viral genomic sequencing of 24,441 positive patient samples between April and June 2021. METHODS: RT-PCR confirmed (N Gene Ct value < 30) positive patient samples, with nucleic acid extracted from saliva, nasopharyngeal and oropharyngeal swabs were selected for viral whole genome SARS-CoV-2 sequencing. Sequencing was performed using Illumina COVIDSeq™ protocol on either the NextSeq550 or NovaSeq6000 systems. Informatic variant calling, and lineage analysis were performed using DRAGEN COVID Lineage applications on Illumina's Basespace cloud analytical system. All sequence data and variant calls were uploaded to NCBI and GISAID. RESULTS: An association was observed between higher sequencing coverage, quality, and samples with a lower Ct value, with < 27 being optimal, across both sequencing platforms and sample collection methods. Both nasopharyngeal swabs and saliva samples were found to be optimal samples of choice for SARS-CoV-2 surveillance sequencing studies, both in terms of strain identification and sequencing depth of coverage, with NovaSeq 6000 providing higher coverage than the NextSeq 550. The most frequent variants identified were the B.1.617.2 Delta (India) and P.1 Gamma (Brazil) variants in the samples sequenced between April 2021 and June 2021. At the time of submission, the most common variant > 99% of positives sequenced was Omicron. CONCLUSION: These initial analyses highlight the importance of sequencing platform, sample collection methods, and RT-PCR Ct values in guiding surveillance efforts. These surveillance studies evaluating genetic changes of SARS-CoV-2 have been identified as critical by the CDC that can affect many aspects of public health including transmission, disease severity, diagnostics, therapeutics, and vaccines.

NF1 patient missense variants predict a role for ATM in modifying neurofibroma initiation.

In Neurofibromatosis type 1, NF1 gene mutations in Schwann cells (SC) drive benign plexiform neurofibroma (PNF), and no additional SC changes explain patient-to-patient variability in tumor number. Evidence from twin studies suggests that variable expressivity might be caused by unidentified modifier genes. Whole exome sequencing of SC and fibroblast DNA from the same resected PNFs confirmed biallelic SC NF1 mutations; non-NF1 somatic SC variants were variable and present at low read number. We identified frequent germline variants as possible neurofibroma modifier genes. Genes harboring variants were validated in two additional cohorts of NF1 patients and by variant burden test. Genes including CUBN, CELSR2, COL14A1, ATR and ATM also showed decreased gene expression in some neurofibromas. ATM-relevant DNA repair defects were also present in a subset of neurofibromas with ATM variants, and in some neurofibroma SC. Heterozygous ATM G2023R or homozygous S707P variants reduced ATM protein expression in heterologous cells. In mice, genetic Atm heterozygosity promoted Schwann cell precursor self-renewal and increased tumor formation in vivo, suggesting that ATM variants contribute to neurofibroma initiation. We identify germline variants, rare in the general population, overrepresented in NF1 patients with neurofibromas. ATM and other identified genes are candidate modifiers of PNF pathogenesis.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential.

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

Pharmacogenetics of antiepileptic drug efficacy in childhood absence epilepsy.

OBJECTIVE: To determine whether common polymorphisms in CACNA1G, CACNA1H, CACNA1I, and ABCB1 are associated with differential short-term seizure outcome in childhood absence epilepsy (CAE). METHODS: Four hundred forty-six CAE children in a randomized double-blind trial of ethosuximide, lamotrigine, and valproate had short-term seizure outcome determined. Associations between polymorphisms (minor allele frequency ≥ 15%) in 4 genes and seizure outcomes were assessed. In vitro electrophysiology on transfected CACNA1H channels determined impact of 1 variant on T-type calcium channel responsiveness to ethosuximide. RESULTS: Eighty percent (357 of 446) of subjects had informative short-term seizure status (242 seizure free, 115 not seizure free). In ethosuximide subjects, 2 polymorphisms (CACNA1H rs61734410/P640L, CACNA1I rs3747178) appeared more commonly among not-seizure-free participants (p = 0.011, odds ratio [OR] = 2.63, 95% confidence limits [CL] = 1.25-5.56; p = 0.026, OR = 2.38, 95% CL = 1.11-5.00). In lamotrigine subjects, 1 ABCB1 missense polymorphism (rs2032582/S893A; p = 0.015, OR = 2.22, 95% CL = 1.16-4.17) was more common in not-seizure-free participants, and 2 CACNA1H polymorphisms (rs2753326, rs2753325) were more common in seizure-free participants (p = 0.038, OR = 0.52, 95% CL = 0.28-0.96). In valproate subjects, no common polymorphisms were associated with seizure status. In vitro electrophysiological studies showed no effect of the P640L polymorphism on channel physiology in the absence of ethosuximide. Ethosuximide's effect on rate of decay of CaV 3.2 was significantly less for P640L channel compared to wild-type channel. INTERPRETATION: Four T-type calcium channel variants and 1 ABCB1 transporter variant were associated with differential drug response in CAE. The in vivo P640L variant's ethosuximide effect was confirmed by in vitro electrophysiological studies. This suggests that genetic variation plays a role in differential CAE drug response. Ann Neurol 2017;81:444-453.

Neuronopathic Gaucher disease: dysregulated mRNAs and miRNAs in brain pathogenesis and effects of pharmacologic chaperone treatment in a mouse model.

Defective lysosomal acid ß-glucosidase (GCase) in Gaucher disease causes accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) that distress cellular functions. To study novel pathological mechanisms in neuronopathic Gaucher disease (nGD), a mouse model (4L;C*), an analogue to subacute human nGD, was investigated for global profiles of differentially expressed brain mRNAs (DEGs) and miRNAs (DEmiRs). 4L;C* mice displayed accumulation of GC and GS, activated microglial cells, reduced number of neurons and aberrant mitochondrial function in the brain followed by deterioration in motor function. DEGs and DEmiRs were characterized from sequencing of mRNA and miRNA from cerebral cortex, brain stem, midbrain and cerebellum of 4L;C* mice. Gene ontology enrichment and pathway analysis showed preferential mitochondrial dysfunction in midbrain and uniform inflammatory response and identified novel pathways, axonal guidance signaling, synaptic transmission, eIF2 and mammalian target of rapamycin (mTOR) signaling potentially involved in nGD. Similar analyses were performed with mice treated with isofagomine (IFG), a pharmacologic chaperone for GCase. IFG treatment did not alter the GS and GC accumulation significantly but attenuated the progression of the disease and altered numerous DEmiRs and target DEGs to their respective normal levels in inflammation, mitochondrial function and axonal guidance pathways, suggesting its regulation on miRNA and the associated mRNA that underlie the neurodegeneration in nGD. These analyses demonstrate that the neurodegenerative phenotype in 4L;C* mice was associated with dysregulation of brain mRNAs and miRNAs in axonal guidance, synaptic plasticity, mitochondria function, eIF2 and mTOR signaling and inflammation and provides new insights for the nGD pathological mechanism.

Genetically driven target tissue overexpression of CD40: a novel mechanism in autoimmune disease.

The CD40 gene, an important immune regulatory gene, is also expressed and functional on nonmyeloid-derived cells, many of which are targets for tissue-specific autoimmune diseases, including ß cells in type 1 diabetes, intestinal epithelial cells in Crohn's disease, and thyroid follicular cells in Graves' disease (GD). Whether target tissue CD40 expression plays a role in autoimmune disease etiology has yet to be determined. In this study, we show that target tissue overexpression of CD40 plays a key role in the etiology of autoimmunity. Using a murine model of GD, we demonstrated that thyroidal CD40 overexpression augmented the production of thyroid-specific Abs, resulting in more severe experimental autoimmune GD (EAGD), whereas deletion of thyroidal CD40 suppressed disease. Using transcriptome and immune-pathway analyses, we showed that in both EAGD mouse thyroids and human primary thyrocytes, CD40 mediates this effect by activating downstream cytokines and chemokines, most notably IL-6. To translate these findings into therapy, we blocked IL-6 during EAGD induction in the setting of thyroidal CD40 overexpression and showed decreased levels of thyroid stimulating hormone receptor-stimulating Abs and frequency of disease. We conclude that target tissue overexpression of CD40 plays a key role in the etiology of organ-specific autoimmune disease.

Genome-wide association analysis of juvenile idiopathic arthritis identifies a new susceptibility locus at chromosomal region 3q13.

OBJECTIVE: In a genome-wide association study of Caucasian patients with juvenile idiopathic arthritis (JIA), we have previously described findings limited to autoimmunity loci shared by JIA and other diseases. The present study was undertaken to identify novel JIA-predisposing loci using genome-wide approaches. METHODS: The discovery cohort consisted of Caucasian JIA cases (n = 814) and local controls (n = 658) genotyped on the Affymetrix Genome-Wide SNP 6.0 Array, along with 2,400 out-of-study controls. In a replication study, we genotyped 10 single-nucleotide polymorphisms (SNPs) in 1,744 cases and 7,010 controls from the US and Europe. RESULTS: Analysis within the discovery cohort provided evidence of associations at 3q13 within C3orf1 and near CD80 (rs4688011) (odds ratio [OR] 1.37, P = 1.88 × 10(-6) ) and at 10q21 near JMJD1C (rs647989 [OR 1.59, P = 6.1 × 10(-8) ], rs12411988 [OR 1.57, P = 1.16 × 10(-7) ], and rs10995450 [OR 1.31, P = 6.74 × 10(-5) ]). Meta-analysis provided further evidence of association for these 4 SNPs (P = 3.6 × 10(-7) for rs4688011, P = 4.33 × 10(-5) for rs6479891, P = 2.71 × 10(-5) for rs12411988, and P = 5.39 × 10(-5) for rs10995450). Gene expression data on 68 JIA cases and 23 local controls showed cis expression quantitative trait locus associations for C3orf1 SNP rs4688011 (P = 0.024 or P = 0.034, depending on the probe set) and JMJD1C SNPs rs6479891 and rs12411988 (P = 0.01 or P = 0.04, depending on the probe set and P = 0.008, respectively). Using a variance component liability model, it was estimated that common SNP variation accounts for approximately one-third of JIA susceptibility. CONCLUSION: Genetic association results and correlated gene expression findings provide evidence of JIA association at 3q13 and suggest novel genes as plausible candidates in disease pathology.

IFN-α mediates the development of autoimmunity both by direct tissue toxicity and through immune cell recruitment mechanisms.

IFN-α is known to play a key role in autoimmunity, but the mechanisms are uncertain. Although the induction of autoimmunity by IFN-α is consistent with primarily immunomodulatory effects, the high frequency of nonautoimmune inflammation suggests other mechanisms. We used thyroiditis as a model to dissect these possibilities. IFN-α treatment of cultured thyrocytes increased expression of thyroid differentiation markers, thyroglobulin, thyroid-stimulating hormone receptor, thyroid peroxidase, and sodium iodide transporter. RNAseq analysis demonstrated that pathways of Ag presentation, pattern recognition receptors, and cytokines/chemokines were also stimulated. These changes were associated with markedly increased nonapoptotic thyroid cell death, suggesting direct toxicity. To corroborate these in vitro findings, we created transgenic mice with thyroid-specific overexpression of IFN-α under control of the thyroglobulin promoter. Transgenic mice developed marked inflammatory thyroid destruction associated with immune cell infiltration of thyroid and surrounding tissues leading to profound hypothyroidism, findings consistent with our in vitro results. In addition, transgenic mice thyroids showed upregulation of pathways similar to those observed in cultured thyrocytes. In particular, expression of granzyme B, CXCL10, a subset of the tripartite motif-containing family, and other genes involved in recruitment of bystander cytotoxic immune responses were increased. Pathways associated with apoptosis and autophagy were not induced. Taken together, our data demonstrate that the induction of tissue inflammation and autoimmunity by IFN-α involves direct tissue toxic effects as well as provocation of destructive bystander immune responses.

Fucosyltransferase 2 non-secretor and low secretor status predicts severe outcomes in premature infants.

OBJECTIVE: To investigate secretor gene fucosyltransferase 2 (FUT2) polymorphism and secretor phenotype in relation to outcomes of prematurity. STUDY DESIGN: Study infants were ≤32 weeks gestational age. Secretor genotype was determined from salivary DNA. Secretor phenotype was measured with H antigen, the carbohydrate produced by secretor gene enzymes, in saliva samples collected on day 9 ± 5. The optimal predictive cutoff point in salivary H values was identified with Classification and Regression Tree analysis. Study outcomes were death, necrotizing enterocolitis (NEC, Bell's stage II/III), and confirmed sepsis. RESULTS: There were 410 study infants, 26 deaths, 30 cases of NEC, and 96 cases of sepsis. Analyzed by genotype, 13% of 95 infants who were non-secretors, 5% of 203 infants who were heterozygotes, and 2% of 96 infants who were secretor dominant died (P = .01). Analyzed by phenotype, 15% of 135 infants with low secretor phenotype died, compared with 2% of 248 infants with high secretor phenotype (predictive value = 76%, P < .001). Low secretor phenotype was associated (P < .05) with NEC, and non-secretor genotype was associated (P = .05) with gram negative sepsis. Secretor status remained significant after controlling for multiple clinical factors. CONCLUSIONS: Secretor genotype and phenotype may provide strong predictive biomarkers of adverse outcomes in premature infants.

The susceptibility loci juvenile idiopathic arthritis shares with other autoimmune diseases extend to PTPN2, COG6, and ANGPT1.

OBJECTIVE: To test for associations between non-major histocompatibility complex susceptibility loci previously reported in autoimmune diseases and juvenile idiopathic arthritis (JIA). METHODS: Published autoimmune disease genome-wide association studies were reviewed, and 519 single-nucleotide polymorphisms (SNPs) were selected for association testing. The initial cohort included 809 JIA cases and 3,535 controls of non-Hispanic, European ancestry. Of the SNPs, 257 were successfully genotyped, while 168 were imputed with quality. Based on findings in the initial cohort, replication was sought for 21 SNPs in a second cohort of 1,015 JIA cases and 1,569 controls collected in the US and Germany. For the initial cohort, tests for association were adjusted for potential confounding effects of population structure by including principal components derived from a genome-wide association study as covariates in logistic regression models. Odds ratios (ORs) and 95% confidence intervals were calculated. RESULTS: Testing for association of previously reported autoimmune disease genetic associations in the initial cohort suggested associations with JIA in 13 distinct loci. Of these, 7 were validated in the replication cohort. Meta-analysis results for the replicating loci included PTPN22 (rs6679677 [OR 1.58, P = 1.98 × 10(-12) ], rs2476601 [OR 1.64, P = 1.90 × 10(-13) ], and rs2488457 [OR 1.32, P = 6.74 × 10(-8) ]), PTPN2 (rs1893217 [OR = 1.33, P = 1.60 × 10(-9) ] and rs7234029 [OR 1.35, P = 1.86 × 10(-10) ]), ADAD1-IL2-IL21 (rs17388568 [OR 1.24, P = 1.13 × 10(-6) ] and rs13143866 [OR 0.83, P = 1.95 × 10(-4) ]), STAT4 (rs3821236 [OR = 1.27, P = 2.36 × 10(-6) ] and rs7574865 [OR = 1.31, P = 2.21 × 10(-6) ]), C12orf30 (rs17696736 [OR = 1.19, P = 2.59 × 10(-5) ]), COG6 (rs7993214 [OR = 0.76, P = 1.10 × 10(-5) ]), and ANGPT1 (rs1010824 [OR = 0.79, P = 2.91 × 10(-4) ]). These polymorphisms have been reported in diseases such as rheumatoid arthritis, type 1 diabetes mellitus, Crohn's disease, and multiple sclerosis. CONCLUSION: General susceptibility loci for autoimmunity are shared across diseases, including JIA, suggesting the potential for common therapeutic targets and mechanisms.

A novel hereditary spastic paraplegia with dystonia linked to chromosome 2q24-2q31.

Spastic paraplegias (HSPs) and dystonias (DYTs) typically localize to different neuroanatomic systems. We report clinical and genetic data from large Ohio kindred with autosomal dominant (AD) HSP and DYT. Single and multipoint linkage using microsatellite and single nucleotide polymorphism array genotyping were performed on a large, multigenerational family with a novel, AD, highly penetrant neurological disease causing spasticity and DYT. Age of onset of spasticity and weakness is from the first year to the sixth decade, and age of onset of DYT from the first to third decade. There is no ataxia or apparent cognitive involvement. Neuroimaging and peripheral neurophysiology are normal. Generalized DYT improved markedly with deep brain stimulation in 1 child. The disease locus was mapped to a region on chromosome 2q 24-31, flanked by markers rs1424937-rs1559510, proximal to SPG13, in a region where there are no known HSP or DYT genes. A secondary analysis for candidate genes segregating with the DYT phenotype revealed two candidate regions with parametric lod scores above 2.0. On the basis of clinical presentation and linkage results, we conclude that this disease is a novel neurological disorder. Identifying the causative gene may elucidate an important pathway for pyramidal and extrapyramidal disorders.

Polymorphisms in the IBD5 locus are associated with Crohn disease in pediatric Ashkenazi Jewish patients.

OBJECTIVES: To analyze the IBD5 locus in a homogenous cohort of Ashkenazi Jewish (AJ) children with Crohn disease (CD). PATIENTS AND METHODS: A total of 83 AJ children with CD and 73 AJ healthy controls were studied. Genotyping for single nucleotide polymorphisms (SNPs) including OCTN1 (SLC22A4; 1672C-->T), OCTN2 (SLC22A5; 207G-->C), IGR2096, IGR2198, and IGR2230 genes was performed using the TaqMan system. NOD2/CARD15 variants also were typed using established methods. RESULTS: All IBD5 SNPs tested were in linkage disequilibrium (D'>0.8), and showed significant association with CD in our cohort of AJ children. The IGR2096 SNP, which is not located within the same linkage disequilibrium block as the OCTN1 and 2 SNPs, showed an even stronger association with CD (P = 0.017; odds ratio = 1.7). Patients with CD who had the OCTN1 susceptibility allele were more likely to carry 1 of the 3 NOD2/CARD15 SNPs tested (P = 0.01; odds ratio = 4.8). CONCLUSIONS: We have demonstrated a significant association between the IBD5 locus and CD in a homogenous cohort of pediatric AJ patients. Due to the tight linkage disequilibrium in the region, it is not possible to identify the causative IBD5 variant. Future functional studies will ultimately reveal the causative gene variant at this locus.

Multiple interactions between the alpha 2C- and beta1-adrenergic receptors influence heart failure survival.

BACKGROUND: Persistent stimulation of cardiac beta1-adrenergic receptors by endogenous norepinephrine promotes heart failure progression. Polymorphisms of this gene are known to alter receptor function or expression, as are polymorphisms of the alpha 2C-adrenergic receptor, which regulates norepinephrine release from cardiac presynaptic nerves. The purpose of this study was to investigate possible synergistic effects of polymorphisms of these two intronless genes (ADRB1 and ADRA2C, respectively) on the risk of death/transplant in heart failure patients. METHODS: Sixteen sequence variations in ADRA2C and 17 sequence variations in ADRB1 were genotyped in a longitudinal study of 655 white heart failure patients. Eleven sequence variations in each gene were polymorphic in the heart failure cohort. Cox proportional hazards modeling was used to identify polymorphisms and potential intra- or intergenic interactions that influenced risk of death or cardiac transplant. A leave-one-out cross-validation method was utilized for internal validation. RESULTS: Three polymorphisms in ADRA2C and five polymorphisms in ADRB1 were involved in eight cross-validated epistatic interactions identifying several two-locus genotype classes with significant relative risks ranging from 3.02 to 9.23. There was no evidence of intragenic epistasis. Combining high risk genotype classes across epistatic pairs to take into account linkage disequilibrium, the relative risk of death or transplant was 3.35 (1.82, 6.18) relative to all other genotype classes. CONCLUSION: Multiple polymorphisms act synergistically between the ADRA2C and ADRB1 genes to increase risk of death or cardiac transplant in heart failure patients.

Mutation of sodium channel SCN3A in a patient with cryptogenic pediatric partial epilepsy.

Mutations in the sodium channel genes SCN1A and SCN2A have been identified in monogenic childhood epilepsies, but SCN3A has not previously been investigated as a candidate gene for epilepsy. We screened a consecutive cohort of 18 children with cryptogenic partial epilepsy that was classified as pharmacoresistant because of nonresponse to carbamazepine or oxcarbazepine, antiepileptic drugs that bind sodium channels. The novel coding variant SCN3A-K354Q was identified in one patient and was not present in 295 neurological normal controls. Twelve novel SNPs were also detected. K354Q substitutes glutamine for an evolutionarily conserved lysine residue in the pore domain of SCN3A. Functional analysis of this mutation in the backbone of the closely related gene SCN5A demonstrated an increase in persistent current that is similar in magnitude to epileptogenic mutations of SCN1A and SCN2A. This observation of a potentially pathogenic mutation of SCN3A (Nav1.3) indicates that this gene should be further evaluated for its contribution to childhood epilepsy.

Molecular, phenotypic, and sample-associated data to describe pluripotent stem cell lines and derivatives.

The use of induced pluripotent stem cells (iPSC) derived from independent patients and sources holds considerable promise to improve the understanding of development and disease. However, optimized use of iPSC depends on our ability to develop methods to efficiently qualify cell lines and protocols, monitor genetic stability, and evaluate self-renewal and differentiation potential. To accomplish these goals, 57 stem cell lines from 10 laboratories were differentiated to 7 different states, resulting in 248 analyzed samples. Cell lines were differentiated and characterized at a central laboratory using standardized cell culture methodologies, protocols, and metadata descriptors. Stem cell and derived differentiated lines were characterized using RNA-seq, miRNA-seq, copy number arrays, DNA methylation arrays, flow cytometry, and molecular histology. All materials, including raw data, metadata, analysis and processing code, and methodological and provenance documentation are publicly available for re-use and interactive exploration at https://www.synapse.org/pcbc. The goal is to provide data that can improve our ability to robustly and reproducibly use human pluripotent stem cells to understand development and disease.

Dissecting the Genetic Susceptibility to Graves' Disease in a Cohort of Patients of Italian Origin.

Graves' disease (GD) is an autoimmune oligogenic disorder with a strong hereditary component. Several GD susceptibility genes have been identified and confirmed during the last two decades. However, there are very few studies that evaluated susceptibility genes for GD in specific geographic subsets. Previously, we mapped a new locus on chromosome 3q that was unique to GD families of Italian origin. In the present study, we used association analysis of single-nucleotide polymorphism (SNPs) at the 3q locus in a cohort of GD patients of Italian origin in order to prioritize the best candidates among the known genes in this locus to choose the one(s) best supported by the association. DNA samples were genotyped using the Illumina GoldenGate genotyping assay analyzing 690 SNP in the linked 3q locus covering all 124 linkage disequilibrium blocks in this locus. Candidate non-HLA (human-leukocyte-antigen) genes previously reported to be associated with GD and/or other autoimmune disorders were analyzed separately. Three SNPs in the 3q locus showed a nominal association (p < 0.05): rs13097181, rs763313, and rs6792646. Albeit these could not be further validated by multiple comparison correction, we were prioritizing candidate genes at a locus already known to harbor a GD-related gene, not hypothesis testing. Moreover, we found significant associations with the thyroid-stimulating hormone receptor (TSHR) gene, the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene, and the thyroglobulin (TG) gene. In conclusion, we identified three SNPs on chromosome 3q that may map a new GD susceptibility gene in this region which is unique to the Italian population. Furthermore, we confirmed that the TSHR, the CTLA-4, and the TG genes are associated with GD in Italians. Our findings highlight the influence of ethnicity and geographic variations on the genetic susceptibility to GD.

Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community.

Clinical Impact and Cost-Effectiveness of Whole Exome Sequencing as a Diagnostic Tool: A Pediatric Center's Experience.

BACKGROUND: There are limited reports of the use of whole exome sequencing (WES) as a clinical diagnostic tool. Moreover, there are no reports addressing the cost burden associated with genetic tests performed prior to WES. OBJECTIVE: We demonstrate the performance characteristics of WES in a pediatric setting by describing our patient cohort, calculating the diagnostic yield, and detailing the patients for whom clinical management was altered. Moreover, we examined the potential cost-effectiveness of WES by examining the cost burden of diagnostic workups. METHODS: To determine the clinical utility of our hospital's clinical WES, we performed a retrospective review of the first 40 cases. We utilized dual bioinformatics analyses pipelines based on commercially available software and in-house tools. RESULTS: Of the first 40 clinical cases, we identified genetic defects in 12 (30%) patients, of which 47% of the mutations were previously unreported in the literature. Among the 12 patients with positive findings, seven have autosomal dominant disease and five have autosomal recessive disease. Ninety percent of the cohort opted to receive secondary findings and of those, secondary medical actionable results were returned in three cases. Among these positive cases, there are a number of novel mutations that are being reported here. The diagnostic workup included a significant number of genetic tests with microarray and single-gene sequencing being the most popular tests. Significantly, genetic diagnosis from WES led to altered patient medical management in positive cases. CONCLUSION: We demonstrate the clinical utility of WES by establishing the clinical diagnostic rate and its impact on medical management in a large pediatric center. The cost-effectiveness of WES was demonstrated by ending the diagnostic odyssey in positive cases. Also, in some cases it may be most cost-effective to directly perform WES. WES provides a unique glimpse into the complexity of genetic disorders.