Combined analysis of 19 common validated type 2 diabetes susceptibility gene variants shows moderate discriminative value and no evidence of gene–gene interaction

Aims/hypothesis  The list of validated type 2 diabetes susceptibility variants has recently been expanded from three to 19. The variants identified are common and have low penetrance in the general population. The aim of the study is to investigate the combined effect of the 19 variants by applying receiver operating characteristics (ROC) to demonstrate the discriminatory value between glucose-tolerant individuals and type 2 diabetes patients in a cross-sectional population of Danes. Methods  The 19 variants were genotyped in three study populations: the population-based Inter99 study; the ADDITION study; and additional type 2 diabetic patients and glucose-tolerant individuals. The case–control studies involved 4,093 type 2 diabetic patients and 5,302 glucose-tolerant individuals. Results  Single-variant analyses demonstrated allelic odds ratios ranging from 1.04 (95% CI 0.98–1.11) to 1.33 (95% CI 1.22–1.45). When combining the 19 variants, subgroups with extreme risk profiles showed a threefold difference in the risk of type 2 diabetes (lower 10% carriers with ≤15 risk alleles vs upper 10% carriers with ≥22 risk alleles, OR 2.93 (95% CI 2.38–3.62, p = 1.6 × 10⁻²⁵). We calculated the area under a ROC curve to estimate the discrimination rate between glucose-tolerant individuals and type 2 diabetes patients based on the 19 variants. We found an area under the ROC curve of 0.60. Two-way gene–gene interaction showed few nominal interaction effects. Conclusions/interpretation  Combined analysis of the 19 validated variants enables detection of subgroups at substantially increased risk of type 2 diabetes; however, the discrimination between glucose-tolerant and type 2 diabetes individuals is still too inaccurate to achieve clinical value. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

The heritable basis of gene–environment interactions in cardiometabolic traits

Aims/hypothesis

Little is known about the heritable basis of gene–environment interactions in humans. We therefore screened multiple cardiometabolic traits to assess the probability that they are influenced by genotype–environment interactions.

Methods

Fourteen established environmental risk exposures and 11 cardiometabolic traits were analysed in the VIKING study, a cohort of 16,430 Swedish adults from 1682 extended pedigrees with available detailed genealogical, phenotypic and demographic information, using a maximum likelihood variance decomposition method in Sequential Oligogenic Linkage Analysis Routines software.

Results

All cardiometabolic traits had statistically significant heritability estimates, with narrow-sense heritabilities (h ²) ranging from 24% to 47%. Genotype–environment interactions were detected for age and sex (for the majority of traits), physical activity (for triacylglycerols, 2 h glucose and diastolic BP), smoking (for weight), alcohol intake (for weight, BMI and 2 h glucose) and diet pattern (for weight, BMI, glycaemic traits and systolic BP). Genotype–age interactions for weight and systolic BP, genotype–sex interactions for BMI and triacylglycerols and genotype–alcohol intake interactions for weight remained significant after multiple test correction.

Conclusions/interpretation

Age, sex and alcohol intake are likely to be major modifiers of genetic effects for a range of cardiometabolic traits. This information may prove valuable for studies that seek to identify specific loci that modify the effects of lifestyle in cardiometabolic disease.

     

Gene–gene interaction between CD40 and CD226 gene on systemic lupus erythematosus in the Chinese Han population

The aim of the study is to investigate the impact of CD40 and CD226 gene single-nucleotide polymorphism (SNP) and additional gene–gene interaction on systemic lupus erythematosus (SLE) risk in Chinese Han populations. Three SNPs were selected for genotyping in the case–control study: rs4810485, rs763361, and rs3765456. Logistic regression was performed to investigate association between SNP within CD40 and CD226 and SLE. Generalized multifactor dimensionality reduction (GMDR) was used to analyze the interaction among three SNPs. Logistic regression analysis showed that SLE risk was significantly higher in carriers of T allele of rs4810485 in CD40 gene than those with GG genotype (GT+ TT vs GG), adjusted OR (95 % CI) 1.84 (1.40–2.29). In addition, we also found SLE risk was also significantly higher in carriers of rs763361 T allele within CD226 gene than those with CC genotype (CT+ TT vs CC), adjusted OR (95 % CI) 1.89 (1.38–2.13). GMDR analysis suggested a potential gene–gene interaction between rs4810485 and rs763361. Overall, cross-validation consistency of the two-locus model was 10/10, and the testing accuracy was 62.17 %. We also found that subjects with GT or TT of rs4810485 and CT or TT of rs763361 genotype have the highest SLE risk, compared with subjects with GG of rs4810485 and CC of rs763361 genotype, and OR (95 % CI) was 2.14 (1.67–3.08), after covariates adjustment. Our results support an important association of rs4810485 in CD40 gene and rs763361 in CD226 gene polymorphism, combined effect of rs4810485 and rs763361 with increased risk of SLE.     

Bernard–Soulier syndrome: novel nonsense mutation in GPIbβ gene affecting GPIb–IX complex expression

Bernard–Soulier syndrome (BSS) is a rare autosomal recessive genetic disorder characterized by thrombocytopenia, circulating giant platelets, and prolonged bleeding time. BSS is explained by a defect in primary hemostasis owing to quantitative or qualitative defect in the GPIb–IX–V complex, composed of four subunits: GPIbα, GPIbβ, GPIX, and GPV. In this study, we report a novel GPIbβ defect in a Tunisian family, in which Serine 23 is substituted by a Stop codon causing a premature termination of translation. This defect was homozygous in the BSS patient and heterozygote in both the parents and sisters of the patient. We studied the effect of this mutation on the expression of the GPIb–IX complex by western blot, flow cytometry, and confocal microscopy: GPIbα and GPIX were absent on the surface of platelets, whereas they were present in the cytoplasm. These results led to conclude that the novel Ser 23 Stop mutation in GPIbβ is responsible of BSS in the studied family and hampers the complex to form on the platelets surface.     

Renin–angiotensin system gene polymorphisms: association with susceptibility to Henoch–Schonlein purpura and renal involvement

The clinical course of Henoch–Schönlein Purpura (HSP) in children is variable, with some patients having a much more rapidly progressing course than others. We investigated whether polymorphisms of the renin–angiotensin system (RAS) genes are involved in HSP. Three RAS genotypes were examined in 114 children with HSP and in 164 healthy children: the angiotensin I converting enzyme (ACE) insertion/deletion polymorphism, the M235T mutation in the angiotensinogen gene (Agt), and the A1166C in the angiotensin II type I receptor (AT1R) gene. Significant differences were observed between HSP patients and control group in the frequency of ACE and Agt genotypes (p=0.004 and p=0.003, respectively). The TT genotype of Agt gene was associated with a 3.5-fold increased risk for Henoch–Schönlein nephritis (HSN) compared with the MM/MT genotype (odds ratio, 3.5; 95% confidence interval, 1.2–10.4). There was a trend to a higher prevalence of the TT genotype of the Agt gene among patients with nephrotic range proteinuria when compared to the patients with mild proteinuria, although the difference did not reach a statistical significance. The results of this study suggest that polymorphisms of ACE gene and Agt gene likely influence the risk of developing HSP. However, among the three genes of the RAS studies, only Agt gene was associated with the susceptibility to HSN. RAS gene polymorphisms studied are not associated with the presence of nephrotic range proteinuria. Additional studies are warranted to verify the correlation between RAS gene polymorphisms and susceptibility to HSP.     

Age-related alterations of articular cartilage in pituitary adenylate cyclase–activating polypeptide (PACAP) gene–deficient mice

GeroScience - Pituitary adenylate cyclase activating polypeptide (PACAP) is an evolutionarly conserved neuropeptide which is produced by various neuronal and non-neuronal cells, including cartilage...     

Establishing a low-expression renalase gene model in cardiac tissue of Sprague–Dawley rats

Background

Renalase is a novel secretory amino oxidase expressed in the kidney and heart. To study the protective mechanism of renalase in local heart tissue, we established a low-expression renalase model with lentivirus (LV)-mediated RNA interference technology.

Materials and methods

Three renalase-targeting oligonucleotides were designed after analyzing the mRNA of renalase. LV particles were prepared with LV expression systems (using the Trono 3 plasmid component system), after which LV-RNLS-shRNAs and LV-NC-shRNA were transfected into H9C2 cells in different cell culture plates. The optimal oligonucleotide was screened by real-time PCR and Western blot. These techniques were also used to detect renalase gene expression in the heart tissue.

Results

In the cell screening experiment, the efficacy of the inhibition of renalase mRNA expression was 93.7?% and that of renalase protein expression was 83.1?% in H9C2 cells. When the oligonucleotide was injected into the pericardial cavities of the SD rats on the 10th day, it inhibited 63.9?% of the expression of renalase protein in the heart tissue.

Conclusion

LV-RNLS-RNAi (19813-1) can be used to establish an optimal renalase low-expression model for further research on the renalase system.

     

Influences of XDH genotype by gene–gene interactions with SUCLA2 for thiopurine-induced leukopenia in Korean patients with Crohn’s disease

Background The impact of genetic variation in the thiopurine S-methyltransferase (TPMT) gene on thiopurine-induced leukopenia has been well demonstrated. Although xanthine dehydrogenase (XDH) is the second major contributor to azathioprine breakdown, polymorphisms in XDH have rarely been studied in IBD patients. We aim to access association between XDH variants and thiopurine-induced leukopenia by gene–gene interaction in a Crohn’s disease (CD) population. Study A total of 964 CD patients treated with thiopurines were recruited from a tertiary referral center. The association between four XDH variants (p.Gly172Arg, p.Asn1109Thr, p.Arg149Cys, and p.Thr910Lys) and thiopurine-induced leukopenia was analyzed in cases with early leukopenia (n?=?66), late leukopenia (n?=?264), and in controls without leukopenia (n?=?632). Three non-synonymous SNPs, which we previously reported association with thiopurine-induced leukopenia, NUDT15 (p.Arg139Cys), SUCLA2 (p.Ser199Thr), and TPMT *3C were selected for epistasis analysis with the XDH variants. Results There was no significant association for two variants of XDH and thiopurine-induced leukopenia. In the epistasis analysis, only XDH (p.Asn1109Thr) * SUCLA2 (p.Ser199Thr) showed a statistically significant association with early leukopenia [odds ratio (OR)?=?0.16; p?=?0.03]. After genotype stratification, a positive association on the background of SUCLA2 wild-type (199Ser) between the XDH (p.Asn1109Thr) and early leukopenia (OR?=?4.39; p?=?0.01) was detected. Conclusion Genes associated with thiopurine-induced leukopenia can act in a complex interactive manner. Further studies are warranted to explore the mechanisms underlying the effects of the combination of XDH (p.Asn1109Thr) and SUCLA2 (199Ser) on thiopurine-induced leukopenia.     

DNA bending by an adenine–thymine tract and its role in gene regulation

To gain insight into the structural basis of DNA bending by adenine-thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-A resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2-DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein-DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.     

α2-Heremans–Schmid glycoprotein gene polymorphisms are associated with adipocyte insulin action

Aims/hypothesis The aim of this study was to investigate the effect of single-nucleotide polymorphisms (SNPs) in the gene encoding the human ₂-Heremans–Schmid glycoprotein (AHSG) on obesity and insulin action in adipocytes.Methods We screened 24 individuals for SNPs in AHSG. Six haplotype-tagging SNPs were genotyped in 188 lean and 176 obese otherwise healthy women for whom common blood chemistry phenotypes were also available. Adipocyte lipolysis and lipogenesis phenotypes were quantified in a subset of 117 lean and 174 obese women.Results The –469T>G SNP, which is located in the 5 region of AHSG, was associated with insulin-mediated inhibition of lipolysis and stimulation of lipogenesis, as well as basal and 8-bromocyclic AMP-stimulated lipolysis. Three AHSG SNPs were associated with circulating levels of cholesterol. None of the six genotyped SNPs or inferred haplotypes were associated with BMI, calculated percent body fat, waist circumference, circulating levels of glucose or insulin, or homeostasis model assessment of insulin resistance, which was used as an estimate of in vivo insulin sensitivity.Conclusions/interpretation Our results are in agreement with a threshold model of susceptibility for insulin resistance and type 2 diabetes, in which specific genetic loci regulate intermediate molecular phenotypes. When an individuals set of susceptibility alleles at such loci exceeds a threshold, clinical disease occurs. Lipolysis in adipocytes appears to be a phenotype that is particularly sensitive to variation in AHSG.     

Pharmacotherapy to gene editing: potential therapeutic approaches for Hutchinson–Gilford progeria syndrome

GeroScience - Hutchinson–Gilford progeria syndrome (HGPS), commonly called progeria, is an extremely rare disorder that affects only one child per four million births. It is characterized by...     

Synergistic effects of gene polymorphisms of the renin–angiotensin–aldosterone system on essential hypertension in Kazakhs in Xinjiang

Objective: To assess the synergistic effects of gene polymorphisms of the renin–angiotensin–aldosterone system (RAAS) on essential hypertension (EH) in Kazakhs in Xinjiang. Methods: A cross-sectional case-control association study was conducted in 52 1 hypertensive and 623 normotensive subjects of Kazakh ethnicity on eight common single nucleotide polymorphisms (SNPs) interspersed over five genes of the RAAS. SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Interactions among the SNPs were analyzed by the multifactor dimensionality reduction method (MDR). Results: In single-locus analysis, subjects with AGT -6G, ACE D, and CYP11B2 -344C had increased susceptibility to EH (OR: 1.249; 1.425; 1.201). When subgrouped by sex, males with the t allele of REN Taq I had decreased risk for EH (OR: 0.529), and those with AGT -6G and CYP11B2 -344 C had increased risk for EH (OR: 1.498; 1.449). In females, carrying ACE D increased the risk for EH. (OR: 1.327). In six AGT haplotypes, H1 was protective, while H3 increased susceptibility to EH (OR: 0.683; 2.025). Interaction analysis by MDR showed that there was a strong synergistic effect between ACE I/D and CY11B2 (T-344C) and a moderate interaction between both ACE I/D and CY11B2 T-344C and AGT A-6G. Conclusions: There was a strong synergistic effect between ACE I/D and CY11B2 T-344C and a moderate effect between both ACE I/D and CY11B2 T-344C and AGT A-6G. AGT -6G, ACE D, and CY11B2 -344C increased susceptibility to EH. REN Taq I, AGT -6G, CY11B2 -344 C and ACE D were associated with male and female EH, respectively. H1 and H3 of AGT were protective and risk haplotypes, respectively.     

Association of peroxisome proliferator-activated receptor delta and additional gene–smoking interaction on cardiovascular disease

Aims: To investigate the impact of peroxisome proliferator–activator receptor delta (PPARD) gene polymorphism and additional gene–smoking interaction on cardiovascular disease (CVD) risk based on this Chinese population. Methods: A total of 1048 subjects (617 males, 431 females) with a mean age of 52.9 ± 14.1 years old were selected, including 520 CVD patients and 528 normal control subjects. The logistic regression model was used to examine the association between three SNPs and CVD risk, odds ratio (OR), and 95% confident interval (95%CI) were calculated. Generalized multifactor dimensionality reduction (GMDR) was employed to investigate the gene–smoking interaction. Results: Genotypes of variants in rs2016520 and rs9794 were associated with decreased CVD risk, and CVD risk was significantly lower in carriers of C allele of the rs2016520 polymorphism than those with the TT genotype (TC+CC versus TT), adjusted OR (95%CI) = 0.71 (0.56–0.86). In addition, we also found that CVD risk was also significantly lower in carriers of the G allele of the rs9794 polymorphism than those with the CC genotype (CG+ GG versus CC), adjusted OR (95%CI) = 0.69 (0.53–0.86). GMDR analysis suggested a potential gene–environment interaction between rs2016520 and smoking. Overall, the two-locus models had a cross-validation consistency of 10 of 10, and had the testing accuracy of 62.17%, and never smokers with TC or CC of the rs2016520 genotype have the lowest CVD risk, compared to smokers with TT of rs2016520, OR (95%CI) was 0.42 (0.23–0.66). Conclusions: The minor allele of rs2016520 and rs9794 in PPAR-δ and interaction between rs2016520 and non-smoking were associated with decreased risk of CVD.     

Identification of gene ontologies linked to prefrontal–hippocampal functional coupling in the human brain

Functional interactions between the dorsolateral prefrontal cortex and hippocampus during working memory have been studied extensively as an intermediate phenotype for schizophrenia. Coupling abnormalities have been found in patients, their unaffected siblings, and carriers of common genetic variants associated with schizophrenia, but the global genetic architecture of this imaging phenotype is unclear. To achieve genome-wide hypothesis-free identification of genes and pathways associated with prefrontal–hippocampal interactions, we combined gene set enrichment analysis with whole-genome genotyping and functional magnetic resonance imaging data from 269 healthy German volunteers. We found significant enrichment of the synapse organization and biogenesis gene set. This gene set included known schizophrenia risk genes, such as neural cell adhesion molecule (NRCAM) and calcium channel, voltage-dependent, beta 2 subunit (CACNB2), as well as genes with well-defined roles in neurodevelopmental and plasticity processes that are dysfunctional in schizophrenia and have mechanistic links to prefrontal–hippocampal functional interactions. Our results demonstrate a readily generalizable approach that can be used to identify the neurogenetic basis of systems-level phenotypes. Moreover, our findings identify gene sets in which genetic variation may contribute to disease risk through altered prefrontal–hippocampal functional interactions and suggest a link to both ongoing and developmental synaptic plasticity.Imaging genetics is widely used to identify neural circuits linked to genetic risk for heritable neuropsychiatric disorders, such as schizophrenia, autism, or bipolar disorder (1). A well-established imaging genetics phenotype is functional connectivity between the right dorsolateral prefrontal cortex (DLPFC) and the left hippocampus (HC) during working memory (WM) performance (2–4). Specifically, impaired interaction of the HC and prefrontal cortex (PFC) has been proposed as a core abnormality during neurodevelopment in schizophrenia. The hippocampus provides input to the DLPFC through long-range glutamatergic connections, which have been linked to the glutamate hypothesis of the illness. Moreover, selective lesions of the hippocampus in primates and rodents have been shown to result in postpubescent changes in prefrontal regions that are consistent with neuropathological findings in schizophrenic patients (5, 6). Brain physiology during WM performance is highly heritable (7), and anomalies of prefrontal–hippocampal functional coupling during WM have been identified in schizophrenia patients (1, 2, 4, 8), their unaffected first-grade relatives (4), healthy carriers of genome-wide supported schizophrenia risk variants and subjects at risk (4, 9–12), and in genetic animal models of the disorder (13). These studies provide strong support for a role of this neural systems-level phenotype in schizophrenia pathophysiology and correspond well to current theories that conceptualize the illness as a “brain disconnection syndrome” rooted in disturbed synaptic plasticity processes (14, 15).Previous studies have characterized abnormal prefrontal–hippocampal interactions in subjects with genetic risk factors for schizophrenia (4, 9, 10, 16). In particular, genome-wide association studies (GWAS) have become a standard approach for identifying common variants that may contribute to risk phenotypes in structural and functional neuroimaging data (10, 16, 17). However, although this approach has been effective in identifying genetic risk variants for imaging phenotypes, post hoc interpretation of results is challenging. Detected risk variants often fall within intronic sequences, where a lack of prior knowledge on functionality hinders a mechanistic explanation of how they impact brain function (18).Increasing evidence suggests that common genetic risk variants for psychiatric disorders are not distributed randomly but rather lie among sets of genes with overlapping functions (19–22). Gene set enrichment analysis (GSEA) is a data analytical approach that leverages a priori knowledge to gain insight into the biological functions of genes and pathways in the analysis of genetic data (23, 24). This approach relies on analysis of sets of genes grouped by common biological characteristics, such as a shared role in particular molecular functions or metabolic pathways. GSEA can then be used to test whether genes that are more strongly associated with a phenotype of interest tend to significantly aggregate within specific biologically based “gene sets.” As an adjunct to established GWA studies and candidate gene approaches, GSEA has successfully identified genes sets with established risk genes for complex diseases such as lung cancer, Parkinson’s disease, and psychiatric disorders, yielding insight into plausible biological processes and molecular mechanisms warranting further investigation (24–26).Although in principle the same strategy can be applied to other quantitative risk-associated phenotypes (27), no prior study has attempted to identify shared biological pathways linked to individual variation in DLPFC–HC functional coupling through a combination of GSEA, whole-genome genotype data, and neuroimaging. Here we used GSEA to test the association of ontology-based gene sets derived from common genetic variants with prefrontal–hippocampal interactions in 269 healthy volunteers who performed the n-back WM task during functional magnetic resonance imaging (fMRI), a well-established paradigm to challenge DLPFC–HC interactions. Given the reviewed evidence (14, 15), we hypothesized that we would identify gene sets linked to developmental plasticity and synaptic neurotransmission, including previously identified risk genes for schizophrenia.     

Synthetic gene network restoring endogenous pituitary–thyroid feedback control in experimental Graves’ disease

Graves’ disease is an autoimmune disorder that causes hyperthyroidism because of autoantibodies that bind to the thyroid-stimulating hormone receptor (TSHR) on the thyroid gland, triggering thyroid hormone release. The physiological control of thyroid hormone homeostasis by the feedback loops involving the hypothalamus–pituitary–thyroid axis is disrupted by these stimulating autoantibodies. To reset the endogenous thyrotrophic feedback control, we designed a synthetic mammalian gene circuit that maintains thyroid hormone homeostasis by monitoring thyroid hormone levels and coordinating the expression of a thyroid-stimulating hormone receptor antagonist (TSH_Antag), which competitively inhibits the binding of thyroid-stimulating hormone or the human autoantibody to TSHR. This synthetic control device consists of a synthetic thyroid-sensing receptor (TSR), a yeast Gal4 protein/human thyroid receptor-α fusion, which reversibly triggers expression of the TSH_Antag gene from TSR-dependent promoters. In hyperthyroid mice, this synthetic circuit sensed pathological thyroid hormone levels and restored the thyrotrophic feedback control of the hypothalamus–pituitary–thyroid axis to euthyroid hormone levels. Therapeutic plug and play gene circuits that restore physiological feedback control in metabolic disorders foster advanced gene- and cell-based therapies.Thyroid hormones impact the function of all tissues and affect essentially every major pathway, including thermogenesis, carbohydrate metabolism, and lipid homeostasis (1). The thyroid gland releases a mixture of the thyroid hormones triiodothyronine (T3; 20%) and thyroxine (T4; 80%), which is converted to the more potent agonist T3 by type II deiodinase (DIO2) in the CNS (hypothalamus and the pituitary gland) or DIO1/2 in the peripheral tissues (e.g., liver, muscle, and heart) (2, 3). DIO1 and DIO2 sensitize target cells to T4 by triggering its deiodination to the more potent hormone T3 (3). The thyroid hormones, particularly T3, exert their action by nuclear thyroid hormone receptors, TRα and TRβ, which are differentially expressed in tissues and have distinct roles in thyroid hormone control of various target genes, such as uncoupling protein 1, HMG-CoA reductase, and phosphoenolpyruvate carboxy kinase (1). The TRα and TRβ can activate or repress gene expression depending on the target–promoter context in the target cells (4). The concentration of thyroid hormones in the body is tightly regulated by the combination of classical activation loops initiated at low thyroid hormone levels and negative feedback loops initiated by high thyroid hormone levels operating along the hypothalamus–pituitary–thyroid axis (2, 3) (Fig. 1A). At low thyroid hormone (T3 and T4) levels, the hypothalamus releases the thyroid-stimulating hormone (TSH) -releasing hormone, which binds and activates the TSH-releasing hormone receptor to stimulate the release of the TSH by the pituitary gland (activation loop) (5). TSH binding to the thyroid-stimulating hormone receptor (TSHR) in the thyroid gland stimulates the production and release of the T3/T4 mixture, which completes the activation loops along the hypothalamus–pituitary–thyroid axis (2, 3). To maintain the thyroid hormones at a homeostasis level and prevent hyperthyroidism by ill-controlled activation of the hypothalamus–pituitary–thyroid axis, the circulating thyroid hormones trigger a negative feedback loop by binding to the TRs in the hypothalamus and the pituitary gland and repress TSH-releasing hormone and TSH release, respectively (6) (Fig. 1A).Open in a separate windowFig. 1.A synthetic thyroid hormone-responsive mammalian gene switch interfacing with the hypothalamus–pituitary–thyroid axis. (A) At low thyroid hormone (T3 and T4), the hypothalamus stimulates thyrotrophic cells in the pituitary gland to release the TSH (activation loop). Binding of TSH to the TSHR in the thyroid gland (activation loop) triggers production and release of a mixture of the thyroid hormones T3 (20%) and T4 (80%), which is converted by DIO2 in the CNS (hypothalamus and the pituitary gland) and DIO1/2 in the peripheral tissues, such as the liver, muscle, and heart, to the more potent agonist T3. On reaching a specific threshold, the homeostasis of circulating thyroid hormone levels is maintained by negative feedback loops repressing thyroid-stimulating hormone-releasing hormone (TRH) and TSH production and release by the hypothalamus and the pituitary gland, respectively. In Graves’ disease, autoantibodies, such as M22, constitutively activate T3 and T4 release by the thyroid gland, thereby disrupting the physiological negative feedback loops maintaining TRH and TSH homeostasis and resulting in chronically increased thyroid hormone levels. The synthetic sensor–effector TSR-TSH_Antag circuit, which constantly monitors systemic T3 levels and coordinates corresponding production of the TSH_Antag to neutralize excessive activation of TSHR by the autoantibody, overrules antibody-triggered hyperthyroidism and restores thyroid hormone homeostasis by a synthetic negative feedback loop. TRHR, thyroid-stimulating hormone-releasing hormone receptor. (B) The synthetic thyroid hormone-responsive gene switch (TSR) consists of a fusion protein, combining the ligand-binding domain of the TR with the DNA-binding domain of Gal4 (yeast Gal4 protein), which binds to a Gal4-specific operator sequence (upstream activating sequence) linked to a minimal promoter (P_hCMVmin) to control transgene expression. In the absence of the thyroid hormones T3 and T4, TSR a priori associates with corepressors, such as silencing mediator for retinoid or thyroid hormone receptors (SMRT)/nuclear receptor corepressor 2 (NcoR2), triggers histone deacetylation, and inhibits gene repression. In the presence of T3 and T4 (converted to T3 by DIO2), TSR is known to interact with coactivators, such as steroid receptor coactivator-1 (SRC-1) and thyroid hormone receptor-associated protein complex 220-kDa component (TRAP 220), that trigger histone acetylation and mediate gene expression. (C) T3- and T4-induced SEAP expression in different cell lines. Mammalian cells were cotransfected with TSR-encoding expression vector (pSP27; P_hCMV-TSR-pA) and P_UAS5-driven SEAP expression vector (pSP30; P_UAS5-SEAP-pA) grown in presence of 0 and 100 nM T3 or T4, and SEAP expression in the supernatant was profiled after 48 h. Data are the means ± SD of three independent experiments done in triplicate.In humans, hyperthyroidism is mostly the result of an overactive thyroid gland caused by thyroid-stimulating autoantibodies (Graves’ disease) or autonomous TSH-secreting pituitary and thyroid hormone-secreting thyroid adenomas, resulting in elevated blood thyroid hormone levels (7). Hyperthyroid symptoms include nervousness, increased sweating, weight loss, tachycardia, palpitations, hyperactivity, and tremor and are associated with serious complications, including life-threatening cardiac arrhythmia or psychosis if left untreated (8). Graves’ disease, the most common cause of hyperthyroidism, is an autoimmune disorder characterized by the production of thyroid-stimulating hormone receptor-stimulating antibodies (TSAbs) that trigger constitutive thyroid hormone production and release from the thyroid gland (9–11). The TSHR is also expressed in ocular connective tissue and represents a candidate autoantigen for development of Graves’ orbitopathy, an ocular manifestation of the disease that, in rare cases, may result in sight-impeding expansion of orbital tissue with inflammation, exophthalmos, and optic nerve compression (9). Current treatments for Graves’ disease are based on the suppression of thyroid hormone production with antithyroid drugs (thionamides) and the destruction of the thyroid gland with ¹³¹I-radiotherapy or surgical removal of the thyroid gland (10, 12, 13). The relapse rate of antithyroid drugs is very high (50–60%); therefore, most patients in the United States are treated by radioiodine or less frequently, surgical removal of the thyroid gland, with the necessity for lifelong thyroid hormone replacement in both cases (10, 11). An ideal treatment for hyperthyroidism would restore the highly effective control of the thyroid gland’s function by the pituitary gland and thus, reestablish the physiological feedback control mechanism of the hypothalamus–pituitary–thyroid axis without destruction of the thyroid gland (14–17). One such treatment would involve blocking TSAb by a soluble TSH variant; however, determining the precise dosage and optimal administration time point of these modified TSH variants remains challenging (18). Here, we present a synthetic biology-inspired gene circuit that can dynamically coordinate the therapeutic expression of a thyroid-stimulating hormone receptor antagonist (TSH_Antag) that competes with endogenous TSH or TSAb in the case of increased thyroid hormone levels, restore the feedback control mechanism along the hypothalamus–pituitary axis, and reset the homeostasis levels of the thyroid hormones.     

Mitochondrial DNA gene therapy: a gene therapy for aging?

Mutations in mitochondrial DNA cause a group of diverse diseases that affect an estimated half a million people worldwide. These disorders are remarkably resistant to conventional treatments, and thus several gene therapy approaches are being explored. As some of these approaches develop towards maturity, one can't help thinking that some day they may be used against a much more common health problem currently affecting about 6 billion people- aging, which also has been quite resistant to treatment. Unfortunately, we still do not know whether mtDNA mutations significantly contribute to the aging process or not. The prospect of success in mtDNA gene therapy makes getting the answer a high priority.     

Cardiometabolic assessment of lamin A/C gene mutation carriers: a phenotype–genotype correlation

AimsMutations of the LMNA gene encoding lamin A/C induce heterogeneous phenotypes ranging from cardiopathies and myopathies to lipodystrophies. The aim of this study was to compare cardiometabolic complications in patients with heterozygous LMNA mutations at the 482nd codon, the ‘hotspot’ for partial lipodystrophy, with carriers of other, non-R482 LMNA mutations.Methods and resultsThis study included 29 patients with R482 LMNA mutations, 29 carriers of non-R482 LMNA mutation and 19 control subjects. Cardiac and metabolic phenotypes were compared between groups. A family history of either cardiac implantable electronic devices (CIEDs; P < 0.001) or sudden death (P < 0.01) was more frequent in non-R482 than R482 carriers. The non-R482 carriers also had more abnormalities on electrocardiography and received CIEDs more often than R482 carriers (P < 0.001). On cardiac ultrasound, non-R482 patients had greater frequencies of left atrial enlargement (P < 0.05) and lower left ventricular ejection fractions (P < 0.01) than R482 carriers. In contrast, R482 carriers had lower BMI (P < 0.05), leptin (P < 0.01) and fat mass (P < 0.001), but higher intra-/total abdominal fat-mass ratios (P < 0.001) and prevalences of diabetes (P < 0.01) and hypertriglyceridaemia (P < 0.05) than non-R482 carriers, with a trend towards more coronary artery disease. However, non-R482 carriers had higher intra-/total abdominal fat-mass ratios (P < 0.02) and prevalences of diabetes (P < 0.001) and hypertriglyceridaemia (P < 0.05) than the controls.ConclusionNon-R482 carriers present more frequently with arrhythmias than R482 carriers, who twice as often have diabetes, suggesting that follow-up for laminopathies could be adjusted for genotype. Non-R482 mutations require ultra-specialized cardiac follow-up, and coronary artery disease should not be overlooked. Although overlapping phenotypes are found, LMNA mutations essentially lead to tissue-specific diseases, favouring genotype-specific pathophysiological mechanisms.