The majority of clinical genetic testing focuses almost exclusively on regions of the genome that directly encode proteins. The important role of variants in non-coding regions in penetrant disease ...is, however, increasingly being demonstrated, and the use of whole genome sequencing in clinical diagnostic settings is rising across a large range of genetic disorders. Despite this, there is no existing guidance on how current guidelines designed primarily for variants in protein-coding regions should be adapted for variants identified in other genomic contexts.
We convened a panel of nine clinical and research scientists with wide-ranging expertise in clinical variant interpretation, with specific experience in variants within non-coding regions. This panel discussed and refined an initial draft of the guidelines which were then extensively tested and reviewed by external groups.
We discuss considerations specifically for variants in non-coding regions of the genome. We outline how to define candidate regulatory elements, highlight examples of mechanisms through which non-coding region variants can lead to penetrant monogenic disease, and outline how existing guidelines can be adapted for the interpretation of these variants.
These recommendations aim to increase the number and range of non-coding region variants that can be clinically interpreted, which, together with a compatible phenotype, can lead to new diagnoses and catalyse the discovery of novel disease mechanisms.
On whole-exome sequencing, a child with clinical hypothyroidism but borderline-abnormal thyroid hormone levels was found to have a heterozygous nonsense mutation in THRα, encoding a mutant protein ...inhibiting wild-type receptor action in a dominant negative manner.
Thyroid hormones have diverse actions, which include regulation of skeletal growth, maturation of the central nervous system, cardiac and gastrointestinal function, and energy homeostasis. In addition, thyroid hormones control their own production by feedback inhibition of hypothalamic thyrotropin-releasing hormone and pituitary thyroid-stimulating hormone, which direct their synthesis or release. These physiological effects are principally mediated by hormone action through nuclear receptor proteins that act as ligand-inducible transcription factors and either positively or negatively regulate the expression of target genes in different tissues in a hormone-dependent manner.
The receptors are encoded by two genes (
THRA
and
THRB
), each of . . .
We carried out a meta-analysis of data from three genome-wide association (GWA) studies of type 1 diabetes (T1D), testing 305,090 SNPs in 3,561 T1D cases and 4,646 controls of European ancestry. We ...obtained further support for 4q27 (IL2-IL21, P = 1.9 x 10(-8)) and, after genotyping an additional 6,225 cases, 6,946 controls and 2,828 families, convincing evidence for four previously unknown and distinct risk loci in chromosome regions 6q15 (BACH2, P = 4.7 x 10(-12)), 10p15 (PRKCQ, P = 3.7 x 10(-9)), 15q24 (CTSH, P = 3.2 x 10(-15)) and 22q13 (C1QTNF6, P = 2.0 x 10(-8)).
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Type 1 diabetes and celiac disease, both of which are associated with HLA class II genes, cosegregate in populations, suggesting a common genetic origin. In this article, the authors tested whether ...any non-HLA loci are shared. They report susceptibility alleles shared by both diseases, indicating that common biologic mechanisms underlie these immune-mediated disorders.
Type 1 diabetes and celiac disease cosegregate in populations, suggesting a common genetic origin. These authors report susceptibility alleles shared by both diseases, indicating that common biologic mechanisms underlie these immune-mediated disorders.
Type 1 diabetes is caused by autoimmune destruction of the insulin-producing beta cells in the pancreatic islets. The disease affects approximately 0.4% of persons of European origin and is strongly clustered in families. The major susceptibility genes — the HLA class II loci, HLA-DQB1 and HLA-DRB1 on chromosome 6p21 — act in combination with many other non-HLA loci across the genome,
1
,
2
with unknown environmental factors playing a major role.
3
–
6
Celiac disease, which results from an immune, inflammatory reaction in the small intestine to proteins in ingested barley, wheat, and rye gluten, occurs in approximately 0.1% of persons of . . .
In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and ...intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced α
β
activation and secretion. Common sequence variation of
and
, associated with GPVI-induced α
β
activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.
Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and ...fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure.
129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301).
The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation r2 = 0.88, P<0.0001 in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001).
Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mammalian mitochondria can be transferred between cells both in culture and in vivo. There is evidence that isolated mitochondria enter cells by endocytosis, but the mechanism has not been fully ...characterised. We investigated the entry mechanism of isolated mitochondria into human osteosarcoma (HOS) cells. Initially we confirmed that respiratory-competent cells can be produced following incubation of HOS cells lacking mitochondrial DNA (mtDNA) with functional exogenous mitochondria and selection in a restrictive medium. Treatment of HOS cells with inhibitors of different endocytic pathways suggest that uptake of EGFP-labelled mitochondria occurs via an actin-dependent endocytic pathway which is consistent with macropinocytosis. We later utilised time-lapse microscopy to show that internalised mitochondria were found in large, motile cellular vesicles. Finally, we used confocal imaging to show that EGFP-labelled mitochondria colocalise with a macropinocytic cargo molecule during internalisation, HOS cells produce membrane ruffles interacting with external mitochondria during uptake and EGFP-labelled mitochondria are found within early macropinosomes inside cells. In conclusion our results are consistent with isolated mitochondria being internalised by macropinocytosis in HOS cells.
Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. ...Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.
Autoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4
T cells. Linking such regulatory regions to gene promoters in disease-relevant ...cell contexts facilitates identification of candidate disease genes.
Within 4 h, activation of CD4
T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases, we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach.
Our systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes.
Blood cells contain functionally important intracellular structures, such as granules, critical to immunity and thrombosis. Quantitative variation in these structures has not been subjected ...previously to large-scale genetic analysis. We perform genome-wide association studies of 63 flow-cytometry derived cellular phenotypes-including cell-type specific measures of granularity, nucleic acid content and reactivity-in 41,515 participants in the INTERVAL study. We identify 2172 distinct variant-trait associations, including associations near genes coding for proteins in organelles implicated in inflammatory and thrombotic diseases. By integrating with epigenetic data we show that many intracellular structures are likely to be determined in immature precursor cells. By integrating with proteomic data we identify the transcription factor FOG2 as an early regulator of platelet formation and α-granularity. Finally, we show that colocalisation of our associations with disease risk signals can suggest aetiological cell-types-variants in IL2RA and ITGA4 respectively mirror the known effects of daclizumab in multiple sclerosis and vedolizumab in inflammatory bowel disease.
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