Evaluation of a target region capture sequencing platform using monogenic diabetes as a study-model

Gao, Rui; Liu, Yanxia; Gjesing, Anette P.; Hollensted, Mette; Wan, Xuejin; He, Shuwen; Pedersen, Oluf; Yi, Xin; Wang, Jun; Hansen, Torben

doi:10.1186/1471-2156-15-13

Cited by 56 publications

(44 citation statements)

References 29 publications

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“…Finally, we found that the 5= HIV integration junction does not always occur at the exact terminus of the HIV LTR and that both truncated U3 regions and nef additions appear to be present. Targeted probe-based enrichment has been used for sequence enrichment of HIV-1-derived lentiviral vectors (36), other viruses, such as human herpesviruses (37), and Merkel cell polyomavirus (38), as well as the detection of human genetic mutations (39,40). While Ustek et al were able to enrich deep sequencing libraries for the targeted lentiviral vector, over 90% of fragments that were pulled down were not complementary to the target region (36).…”

Section: Discussionmentioning

confidence: 99%

HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay

Sunshine

Kirchner

Amr

et al. 2016

J Virol

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Antiretroviral therapy (ART) is successful in the suppression of HIV but cannot target and eradicate the latent proviral reservoir. The location of retroviral integration into the human genome is thought to play a role in the clonal expansion of infected cells and HIV persistence. We developed a high-throughput targeted sequence capture assay that uses a pool of HIV-specific probes to enrich Illumina libraries prior to deep sequencing. Using an expanded clonal population of ACH-2 cells, we demonstrate that this sequence capture assay has an extremely low false-positive rate. This assay assessed four cellular models commonly used to study HIV latency and latency-reversing agents: ACH-2 cells, J-Lat cells, the Bcl-2-transduced primary CD4؉ model, and the cultured T CM (central memory) CD4؉ model. HIV integration site characteristics and genes were compared between these cellular models and to previously reported patient data sets. Across these cellular models, there were significant differences in integration site characteristics, including orientation relative to that of the host gene, the proportion of clonally expanded sites, and the proportion located within genic regions and exons. Despite a greater diversity of minority integration sites than expected in ACH-2 cells, their integration site characteristics consistently differed from those of the other models and from the patient samples. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. These findings show that integration site differences exist among the commonly used cellular models of HIV latency and in comparison to integration sites found in patient samples. IMPORTANCEDespite the success of ART, currently there is no successful therapy to eradicate integrated proviruses. Cellular models of HIV latency are used to test the efficacy of latency-reversing agents, but it is unclear how well these models reflect HIV integration into the human genome in vivo. We have developed a novel probe-based sequence enrichment assay to sequence and analyze integrated HIV. We compared HIV integration site characteristics between four cellular models and to previously described patient data sets. Significant differences were detected in the distribution of HIV integration sites between cellular models of HIV latency and compared to data sets from patient samples. The results from this study have implications for how well these cellular models of HIV infection truly reflect HIV integration in vivo and their applicability in drug discovery for novel latency-reversing agents.

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Section: Discussionmentioning

confidence: 99%

HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay

Sunshine

Kirchner

Amr

et al. 2016

J Virol

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“…Sequencing of GPRC6A-The coding regions and intronexon boundaries of GPRC6A was examined using targeted region capture and next generation sequencing as described (40). The coding regions were covered with a minimum mean depth of 38 in 99.9% of all individuals, and the median depth per individual was 196.…”

Section: Methodsmentioning

confidence: 99%

Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

Jørgensen

Have

Underwood

et al. 2017

Journal of Biological Chemistry

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Edited by Henrik G. DohlmanGPRC6A is a G protein-coupled receptor activated by Lamino acids, which, based on analyses of knock-out mice, has been suggested to have physiological functions in metabolism and testicular function. The human ortholog is, however, mostly retained intracellularly in contrast to the cell surface-expressed murine and goldfish orthologs. The latter orthologs are G q -coupled and lead to intracellular accumulation of inositol phosphates and calcium release. In the present study we cloned the bonobo chimpanzee GPRC6A receptor, which is 99% identical to the human receptor, and show that it is cell surface-expressed and functional. By analyses of chimeric human/mouse and human/bonobo receptors, bonobo receptor mutants, and the single nucleotide polymorphism database at NCBI, we identify an insertion/deletion variation in the third intracellular loop responsible for the intracellular retention and lack of function of the human ortholog. Genetic analyses of the 1000 genome database and the Inter99 cohort of 6,000 Danes establish the distribution of genotypes among ethnic groups, showing that the cell surface-expressed and functional variant is much more prevalent in the African population than in European and Asian populations and that this variant is partly linked with a stop codon early in the receptor sequence (rs6907580, amino acid position 57). In conclusion, our data solve a more than decade-old question of why the cloned human GPRC6A receptor is not cell surface-expressed and functional and provide a genetic framework to study human phenotypic traits in large genome sequencing projects linked with physiological measurement and biomarkers.Communication between the exterior and interior of cells is essential for cellular survival. A pivotal class of proteins, specialized to carry out such signal transduction, is the G proteincoupled receptors (GPCRs), 2 a family that includes ϳ800 subtypes in humans. The GPCR class C, group 6, member A (GPRC6A) belongs to the non-olfactory GPCRs as part of the class C receptors. Human GPRC6A (h6A) was cloned from a human kidney cDNA library in 2004 (1). Cloning and deorphanization of the mouse (2, 3) and rat (4) GPRC6A orthologs rapidly followed. These studies, together with recent evidence (5) support the existence of GPRC6A as a dimer on the cell surface. However, surprisingly, h6A has been shown to be retained intracellularly and thus does not respond to agonists, which contrasts findings for the mouse, rat, and goldfish orthologs (2, 3, 6). To deorphanize h6A, we generated chimeras between the human and goldfish GPRC6A orthologs. We found that a fusion of the human large extracellular amino-terminal domain (ATD) to the 7-transmembrane (7TM) and C-terminal domains of the orthologous goldfish 5.24 receptor allowed efficient surface expression of the chimera and thereby a way to

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“…Genetic testing of all causes of neonatal diabetes is now predominantly by targeted panels (e.g. (23,25,26)) occurring immediately after the diagnosis of diabetes with in the first 6 months of life (22). Targeted sequencing should include LRBA as in all 4 probands with neonatal diabetes this was the first feature of their multisystem autoimmune disorder and for one proband is currently the only feature at the age of two years.…”

Section: Discussionmentioning

confidence: 99%

Recessively InheritedLRBAMutations Cause Autoimmunity Presenting as Neonatal Diabetes

et al. 2017

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Young-onset autoimmune diabetes associated with additional autoimmunity usually reflects a polygenic predisposition but rare cases result from monogenic autoimmunity. Diagnosing monogenic autoimmunity is crucial for patients' prognosis and clinical management. We sought to identify novel genetic causes of autoimmunity presenting with neonatal diabetes (NDM; diagnosis <6 months).We performed exome sequencing in a patient with NDM and autoimmune lymphoproliferative syndrome and his unrelated, unaffected parents and identified compound heterozygous null mutations in LRBA. Biallelic LRBA mutations cause Common Variable Immunodeficiency-8, however NDM has not been confirmed in this disorder.We sequenced LRBA in 169 additional

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Evaluation of a target region capture sequencing platform using monogenic diabetes as a study-model

Cited by 56 publications

References 29 publications

HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay

HIV Integration Site Analysis of Cellular Models of HIV Latency with a Probe-Enriched Next-Generation Sequencing Assay

Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

Recessively InheritedLRBAMutations Cause Autoimmunity Presenting as Neonatal Diabetes

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