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1.
Front Genet ; 14: 1095600, 2023.
Article in English | MEDLINE | ID: mdl-36713073

ABSTRACT

Newborn screening (NBS) assays for spinal muscular atrophy (SMA) typically use a polymerase chain reaction (PCR) based assay to identify individuals with homozygous deletion in exon 7 of the SMN1 gene. Due to high DNA sequence homology between SMN1 and SMN2, it has previously been difficult to accurately bioinformatically map short reads from next-generation DNA sequencing (NGS) to SMN1, resulting in low analytical performance and preventing NGS being used for SMA screening. Advances in bioinformatics have allowed NGS to be used in diagnostic settings, but to date these assays have not reached the scale required for high volume population newborn screening and have not been performed on the dried blood spot samples that NBS programs currently use. Here we integrate an NGS assay using hybridisation-based capture with a customised bioinformatics algorithm and purpose designed high throughput reporting software into an existing NBS program to achieve a laboratory workflow for population SMA screening. We tested the NGS assay on over 2500 newborns born over 2 weeks in a NBS program in a technical feasibility study and show high sensitivity and specificity. Our results suggest NGS may be an alternate method for SMA screening by NBS programs, providing a multiplex testing platform on which potentially hundreds of inherited conditions could be simultaneously tested.

2.
Prenat Diagn ; 43(1): 109-116, 2023 01.
Article in English | MEDLINE | ID: mdl-36484552

ABSTRACT

OBJECTIVE: European and Australian guidelines for cystic fibrosis (CF) reproductive carrier screening recommend testing a small number of high frequency CF causing variants, rather than comprehensive CFTR sequencing. The study objective was to determine variant detection rates of commercially available targeted reproductive carrier screening tests in Australia. METHODS: Next-generation DNA sequencing of the CFTR gene was performed on 2552 individuals from a whole population sample to identify CF causing variants. The variant detection rates of two commercially available Australian reproductive carrier screening tests, which target 50 or 175 CF causing variants, in this population were calculated. The ethnicity of individuals was determined using principal component analysis. RESULTS: Variant detection rates of the tests for 50 and 175 CF causing variants were 88.2% and 90.8%, respectively. No CF causing variants in individuals of East Asian ethnicity (n = 3) were detected by either test, while >86.6% (n = 69) of CF causing variants in Europeans would be identified by either test. CONCLUSIONS: Reproductive carrier screening tests for a targeted set of high frequency CF variants are unable to detect approximately 10% of CF variants in a multiethnic Australian population, and individuals of East Asian ethnicity are disproportionally affected by this test limitation.


Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/diagnosis , Cystic Fibrosis/epidemiology , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Australia/epidemiology , Genetic Testing , Ethnicity , Mutation
3.
BMC Pediatr ; 21(1): 154, 2021 03 31.
Article in English | MEDLINE | ID: mdl-33789612

ABSTRACT

BACKGROUND: Cystic Fibrosis (CF) is one of the most prevalent autosomal recessive inherited disease in Caucasians. Rates of CF were thought to be negligible in non-Caucasians but growing epidemiological evidence shows CF is more common in Indian, African, Hispanic, Asian, and other ethnic groups than previously thought. Almost all second-tier molecular diagnostic tools currently used to confirm the diagnosis of CF consist of panels of the most common CF-causing DNA variants in Caucasians. However non-Caucasian individuals with CF often have a different spectrum of pathogenic variants than Caucasians, limiting the clinical utility of existing molecular diagnostic panels in this group. As a consequence of racially inequitable CF testing frameworks, non-Caucasians with CF encounter greater delays in diagnosis and associated harms than Caucasians. An unbiased approach of detecting CF-causing DNA variants using full gene sequencing could potentially address racial inequality in current CF testing. CASE PRESENTATION: We present the case of a female baby from rural India who had a borderline first-tier newborn screening result for CF. Instead of choosing a targeted CF panel for second-tier testing, we used next-generation DNA sequencing to comprehensively analyze the cystic fibrosis transmembrane conductance regulator gene as an unbiased approach for molecular confirmation of CF. Sequencing identified two pathogenic variants that cause CF. One variant (c.1521_1523delCTT) is the most common cause of CF, while the other variant (c.870-1G > C) is absent from all population allele databases and has not been found in the Indian population previously. The rare variant would not have been detected by all currently available targeted CF panels used for second- or third-tier molecular CF testing. CONCLUSIONS: Our use of full gene sequencing as a second-tier CF test in a non-Caucasian patient avoided the problems of missed diagnosis from using Caucasian-biased targeted CF panels currently recommended for second-tier testing. Full gene sequencing should be considered as the standard methodology of second-tier CF testing to enable equal opportunity for CF diagnosis in non-Caucasians.


Subject(s)
Cystic Fibrosis , Cystic Fibrosis/diagnosis , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA , Female , Humans , India , Infant, Newborn , Mutation , Sequence Analysis, DNA
4.
J Mol Diagn ; 19(4): 602-612, 2017 07.
Article in English | MEDLINE | ID: mdl-28502726

ABSTRACT

The sensitivity and specificity of next-generation sequencing laboratory developed tests (LDTs) are typically determined by an analyte-specific approach. Analyte-specific validations use disease-specific controls to assess an LDT's ability to detect known pathogenic variants. Alternatively, a methods-based approach can be used for LDT technical validations. Methods-focused validations do not use disease-specific controls but use benchmark reference DNA that contains known variants (benign, variants of unknown significance, and pathogenic) to assess variant calling accuracy of a next-generation sequencing workflow. Recently, four whole-genome reference materials (RMs) from the National Institute of Standards and Technology (NIST) were released to standardize methods-based validations of next-generation sequencing panels across laboratories. We provide a practical method for using NIST RMs to validate multigene panels. We analyzed the utility of RMs in validating a novel newborn screening test that targets 70 genes, called NEO1. Despite the NIST RM variant truth set originating from multiple sequencing platforms, replicates, and library types, we discovered a 5.2% false-negative variant detection rate in the RM truth set genes that were assessed in our validation. We developed a strategy using complementary non-RM controls to demonstrate 99.6% sensitivity of the NEO1 test in detecting variants. Our findings have implications for laboratories or proficiency testing organizations using whole-genome NIST RMs for testing.


Subject(s)
Genome, Human , High-Throughput Nucleotide Sequencing/methods , Multigene Family , DNA Copy Number Variations , Gene Deletion , Genetic Variation , High-Throughput Nucleotide Sequencing/standards , Humans , Infant, Newborn , Infant, Newborn, Diseases/genetics , Mutagenesis, Insertional , Polymorphism, Single Nucleotide , Reference Standards , Validation Studies as Topic
5.
J Clin Invest ; 121(11): 4303-10, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22019586

ABSTRACT

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.


Subject(s)
Antigen-Presenting Cells/immunology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Interleukins/metabolism , Th17 Cells/immunology , Animals , CD11b Antigen/metabolism , Cell Differentiation/immunology , Diabetes Mellitus, Type 1/metabolism , Female , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Interleukin-2/genetics , Interleukin-2/metabolism , Interleukin-21 Receptor alpha Subunit/deficiency , Interleukin-21 Receptor alpha Subunit/genetics , Interleukin-21 Receptor alpha Subunit/metabolism , Interleukins/genetics , Mice , Mice, Congenic , Mice, Inbred NOD , Mice, Knockout , Signal Transduction/immunology , Tretinoin/metabolism
6.
Cell ; 139(7): 1255-67, 2009 Dec 24.
Article in English | MEDLINE | ID: mdl-20064372

ABSTRACT

During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.


Subject(s)
Host-Pathogen Interactions , Influenza A Virus, H1N1 Subtype/metabolism , Viral Proteins/metabolism , Apoptosis , Epithelial Cells/virology , Gene Expression Profiling , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H1N1 Subtype/pathogenicity , Interferons/metabolism , Lung/cytology , Lung/virology , Proteomics , RNA, Small Interfering/metabolism , RNA, Viral/metabolism , Two-Hybrid System Techniques , Viral Nonstructural Proteins/metabolism , Wnt Proteins/metabolism
7.
Expert Rev Mol Med ; 10: e15, 2008 May 27.
Article in English | MEDLINE | ID: mdl-18503727

ABSTRACT

Asthma is a chronic inflammatory disease of the airways, involving recurrent episodes of airway obstruction and wheezing. A common pathological feature in asthma is the presence of a characteristic allergic airway inflammatory response involving extensive leukocyte infiltration, mucus overproduction and airway hyper-reactivity. The pathogenesis of allergic airway inflammation is complex, involving multiple cell types such as T helper 2 cells, regulatory T cells, eosinophils, dendritic cells, mast cells, and parenchymal cells of the lung. The cellular response in allergic airway inflammation is controlled by a broad range of bioactive mediators, including IgE, cytokines and chemokines. The asthmatic allergic inflammatory response has been a particular focus of efforts to develop novel therapeutic agents. Animal models are widely used to investigate inflammatory mechanisms. Although these models are not perfect replicas of clinical asthma, such studies have led to the development of numerous novel therapeutic agents, of which some have already been successful in clinical trials.


Subject(s)
Anti-Asthmatic Agents/therapeutic use , Asthma/immunology , Disease Models, Animal , Inflammation/immunology , Mice , Animals , Anti-Asthmatic Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antigen Presentation , Asthma/drug therapy , Asthma/pathology , Asthma/physiopathology , Bronchial Hyperreactivity/physiopathology , Chemotaxis, Leukocyte/physiology , Cytokines/physiology , Dendritic Cells/immunology , Eosinophils/immunology , Epithelial Cells/immunology , Epithelial Cells/pathology , Humans , Immunoglobulin E/immunology , Inflammation/drug therapy , Inflammation/pathology , Inflammation/physiopathology , Mast Cells/immunology , Mice/genetics , Mice/immunology , Mice/physiology , Mice, Knockout , Mice, Transgenic , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology
8.
J Immunol ; 177(11): 7794-801, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17114450

ABSTRACT

The fatty acid-binding protein (FABP) family consists of a number of conserved cytoplasmic proteins with roles in intracellular lipid transport, storage, and metabolism. Examination of a comprehensive leukocyte gene expression database revealed strong expression of the adipocyte FABP aP2 in human monocyte-derived dendritic cells (DCs). We isolated bone marrow-derived DC from aP2-deficient mice, and showed that expression of DC cytokines including IL-12 and TNF was significantly impaired in these cells. Degradation of IkappaBalpha was also impaired in aP2-deficient DCs, indicative of reduced signaling through the IkappaB kinase-NF-kappaB pathway. The cytokine defect was selective because there was no effect on Ag uptake or expression of MHC class II, CD40, CD80, or CD86. In an MLR, aP2-deficient DCs stimulated markedly lower T cell proliferation and cytokine production than did wild-type DCs. Moreover, aP2-deficient mice immunized with keyhole limpet hemocyanin/CFA showed reduced production of IFN-gamma by restimulated draining lymph node cells, suggesting a similar defect in DC function in vivo. Similarly, infection of aP2-deficient mice with the natural mouse pathogen ectromelia virus resulted in substantially lower production of IFN-gamma by CD8+ T cells. Thus, FABP aP2 plays an important role in DC function and T cell priming, and provides an additional link between metabolic processes and the regulation of immune responses.


Subject(s)
Dendritic Cells/immunology , Fatty Acid-Binding Proteins/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Animals , Blotting, Western , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Dendritic Cells/metabolism , Fatty Acid-Binding Proteins/metabolism , Gene Expression , I-kappa B Proteins/metabolism , Interleukin-12/metabolism , Lymphocyte Culture Test, Mixed , Mice , NF-KappaB Inhibitor alpha , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/metabolism
9.
J Clin Invest ; 116(8): 2183-2192, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16841093

ABSTRACT

The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. We report that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by human airway epithelial cells and shows a striking upregulation following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13. Regulation of aP2 mRNA expression by Th2 cytokines was highly dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2-deficient mice in a model of allergic airway inflammation and found that infiltration of leukocytes, especially eosinophils, into the airways was highly dependent on aP2 function. T cell priming was unaffected by aP2 deficiency, suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-hematopoietic cells, most likely bronchial epithelial cells, as the site of action of aP2 in allergic airway inflammation. Thus, aP2 regulates allergic airway inflammation and may provide a link between fatty acid metabolism and asthma.


Subject(s)
Adipocytes/physiology , Asthma/immunology , Fatty Acid-Binding Proteins/metabolism , Hypersensitivity/immunology , Inflammation/physiopathology , 3T3 Cells , Adipocytes/immunology , Animals , Bronchi/physiology , Cell Culture Techniques , Disease Models, Animal , Fatty Acid-Binding Proteins/deficiency , Fatty Acid-Binding Proteins/genetics , Fatty Acids/metabolism , Interleukin-12/immunology , Interleukin-4/immunology , Mice , Mice, Knockout , RNA, Messenger/genetics , Respiratory Mucosa/physiology , Th2 Cells/immunology , Transcription, Genetic
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