Genetics of Type 2 Diabetes - Pitfalls and Possibilities

Type 2 diabetes (T2D) is a complex disease that is caused by a complex interplay between genetic, epigenetic and environmental factors. While the major environmental factors, diet and activity level, are well known, identification of the genetic factors has been a challenge. However, recent years have seen an explosion of genetic variants in risk and protection of T2D due to the technical development that has allowed genome-wide association studies and next-generation sequencing. Today, more than 120 variants have been convincingly replicated for association with T2D and many more with diabetes-related traits. Still, these variants only explain a small proportion of the total heritability of T2D. In this review, we address the possibilities to elucidate the genetic landscape of T2D as well as discuss pitfalls with current strategies to identify the elusive unknown heritability including the possibility that our definition of diabetes and its subgroups is imprecise and thereby makes the identification of genetic causes difficult.Peer reviewe

Genome editing of human pancreatic beta cell models : problems, possibilities and outlook

Understanding the molecular mechanisms behind beta cell dysfunction is essential for the development of effective and specific approaches for diabetes care and prevention. Physiological human beta cell models are needed for this work. We review the possibilities and limitations of currently available human beta cell models and how they can be dramatically enhanced using genome-editing technologies. In addition to the gold standard, primary isolated islets, other models now include immortalised human beta cell lines and pluripotent stem cell-derived islet-like cells. The scarcity of human primary islet samples limits their use, but valuable gene expression and functional data from large collections of human islets have been made available to the scientific community. The possibilities for studying beta cell physiology using immortalised human beta cell lines and stem cell-derived islets are rapidly evolving. However, the functional immaturity of these cells is still a significant limitation. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) has enabled precise engineering of specific genetic variants, targeted transcriptional modulation and genome-wide genetic screening. These approaches can now be exploited to gain understanding of the mechanisms behind coding and non-coding diabetes-associated genetic variants, allowing more precise evaluation of their contribution to diabetes pathogenesis. Despite all the progress, genome editing in primary pancreatic islets remains difficult to achieve, an important limitation requiring further technological development.Peer reviewe

Turning Vice into Virtue : Using Batch-Effects to Detect Errors in Large Genomic Data Sets

It is often unavoidable to combine data from different sequencing centers or sequencing platforms when compiling data sets with a large number of individuals. However, the different data are likely to contain specific systematic errors that will appear as SNPs. Here, we devise a method to detect systematic errors in combined data sets. To measure quality differences between individual genomes, we study pairs of variants that reside on different chromosomes and co-occur in individuals. The abundance of these pairs of variants in different genomes is then used to detect systematic errors due to batch effects. Applying our method to the 1000 Genomes data set, we find that coding regions are enriched for errors, where similar to 1% of the higher frequency variants are predicted to be erroneous, whereas errors outside of coding regions are much rarer (Peer reviewe

Optimization of Regeneration Protocol and Agrobacterium Mediated Transformation in Carnation (Dianthus caryophyllus L.)

An efficient and reproducible regeneration protocol for carnation genotypes Arka Flame and IIHRS-1 has been developed from leaf and stem explants. Although IIHRS-1 showed slightly higher regeneration (55%) compared to Arka Flame (49.2%), there was no significant difference in their regeneration response. However, significant difference in regeneration potential was observed with leaf explant exhibiting higher regeneration potential (5.5 shoots/explant) as compared to (4.9) stem explant. Among various plant growth regulator combinations tested for regeneration, the best regeneration response and maximum regeneration potential was obtained in MS medium supplemented with NAA (0.1 mg/l) and TDZ (1.0mg /l) for both the explants and genotypes used. The medium also proved suitable for inducing elongation of regenerated shoots. Rooting of in vitro formed shootlets could be induced at greater frequency in MS medium supplemented with IAA (1.0 mg/l). Based on this protocol, transformation was carried out in genotype IIHRS-1 using leaf explants with Agrobacterium tumefaciens LBA 4404 with binary vector pROK2 containing baculovirus chitinase gene under the control of 35S promoter with npt II serving as selectable marker. There was regeneration of putative transformants at a frequency of 28.9%. However, great difficulty was encountered in rooting of shoots. Hence a few shoots regenerated on selection medium at random were tested for transgene integration. Out of the three shoots tested for npt II amplification, two shoots tested positive. The presence of transgene was confirmed through PCR amplification of npt II gene and dot blot analysis of chitinase gene

Neuronal Dysfunction Is Linked to the Famine-Associated Risk of Proliferative Retinopathy in Patients With Type 2 Diabetes

Diabetic retinopathy; Famine; Neuronal functionRetinopatía diabética; Hambruna; Función neuronalRetinopatia diabètica; Fam; Funció neuronalPersons with type 2 diabetes born in the regions of famine exposures have disproportionally elevated risk of vision-threatening proliferative diabetic retinopathy (PDR) in adulthood. However, the underlying mechanisms are not known. In the present study, we aimed to investigate the plausible molecular factors underlying progression to PDR. To study the association of genetic variants with PDR under the intrauterine famine exposure, we analyzed single nucleotide polymorphisms (SNPs) that were previously reported to be associated with type 2 diabetes, glucose, and pharmacogenetics. Analyses were performed in the population from northern Ukraine with a history of exposure to the Great Ukrainian Holodomor famine [the Diagnostic Optimization and Treatment of Diabetes and its Complications in the Chernihiv Region (DOLCE study), n = 3,583]. A validation of the top genetic findings was performed in the Hong Kong diabetes registry (HKDR, n = 730) with a history of famine as a consequence of the Japanese invasion during WWII. In DOLCE, the genetic risk for PDR was elevated for the variants in ADRA2A, PCSK9, and CYP2C19*2 loci, but reduced at PROX1 locus. The association of ADRA2A loci with the risk of advanced diabetic retinopathy in famine-exposed group was further replicated in HKDR. The exposure of embryonic retinal cells to starvation for glucose, mimicking the perinatal exposure to famine, resulted in sustained increased expression of Adra2a and Pcsk9, but decreased Prox1. The exposure to starvation exhibited a lasting inhibitory effects on neurite outgrowth, as determined by neurite length. In conclusion, a consistent genetic findings on the famine-linked risk of ADRA2A with PDR indicate that the nerves may likely to be responsible for communicating the effects of perinatal exposure to famine on the elevated risk of advanced stages of diabetic retinopathy in adults. These results suggest the possibility of utilizing neuroprotective drugs for the prevention and treatment of PDR.This work was supported by the Swedish Research Council (Dnr2015-03574 and Dnr349-2006-237), Strategic Research Area Exodiab (Dnr2009-1039), the Novonordisk Foundation (NNF12OC1016467), Swedish Foundation for Strategic Research (DnrIRC15-0067), the Steno Diabetes Center Copenhagen, Bergen Research Foundation and Trond Mohn Foundation (BFS811294), and the University of Bergen

Lipid-Associated Variants near ANGPTL3 and LPL Show Parent-of-Origin Specific Effects on Blood Lipid Levels and Obesity

Parent-of-origin effects (POE) and sex-specific parental effects have been reported for plasma lipid levels, and a strong relationship exists between dyslipidemia and obesity. We aim to explore whether genetic variants previously reported to have an association to lipid traits also show POE on blood lipid levels and obesity. Families from the Botnia cohort and the Hungarian Transdanubian Biobank (HTB) were genotyped for 12 SNPs, parental origin of alleles were inferred, and generalized estimating equations were modeled to assess parental-specific associations with lipid traits and obesity. POE were observed for the variants at the TMEM57, DOCK7/ANGPTL3, LPL, and APOA on lipid traits, the latter replicated in HTB. Sex-specific parental effects were also observed; variants at ANGPTL3/DOCK7 showed POE on lipid traits and obesity in daughters only, while those at LPL and TMEM57 showed POE on lipid traits in sons. Variants at LPL and DOCK7/ANGPTL3 showed POE on obesity-related traits in Botnia and HTB, and POE effects on obesity were seen to a higher degree in daughters. This highlights the need to include analysis of POEs in genetic studies of complex traits.Peer reviewe

Silencing of the FTO gene inhibits insulin secretion : An in vitro study using GRINCH cells

Expression of fat mass and obesity-associated gene (FTO) and ADP-ribosylation factor-like 15 (ARL15) in human islets is inversely correlated with HbA(1c). However, their impact on insulin secretion is still ambiguous. Here in, we investigated the role of FTO and ARL15 using GRINCH (Glucose-Responsive Insulin-secreting C-peptide-modified Human proinsulin) clonal rat beta-cells. GRINCH cells have inserted GFP into the human C-peptide insulin gene. Hence, secreted CpepGFP served to monitor insulin secretion. mRNA silencing of FTO in GRINCH cells showed a significant reduction in glucose but not depolarization-stimulated insulin secretion, whereas ARL15 silencing had no effect. A significant down-regulation of insulin mRNA was observed in FTO knockdown cells. Type-2 Diabetic islets revealed a reduced expression of FTO mRNA. In conclusion, our data suggest that fluorescent CpepGFP released from GRINCH cells may serve as a convenient marker for insulin secretion. Silencing of FTO expression, but not ARL15, inhibits insulin secretion by affecting metabolic signaling.Peer reviewe

High Prevalence of Gestational Diabetes Mellitus in Rural Tanzania-Diagnosis Mainly Based on Fasting Blood Glucose from Oral Glucose Tolerance Test

Gestational diabetes mellitus (GDM) is associated with poor pregnancy outcomes and increased long-term risk of metabolic diseases for both mother and child. In Tanzania, GDM prevalence increased from 0% in 1991 to 19.5% in 2016. Anaemia has been proposed to precipitate the pathogenesis of GDM. We aimed to examine the prevalence of GDM in a rural area of Tanzania with a high prevalence of anaemia and to examine a potential association between haemoglobin concentration and blood glucose during pregnancy. The participants were included in a population-based preconception, pregnancy and birth cohort study. In total, 538 women were followed during pregnancy and scheduled for an oral glucose tolerance test (OGTT) at week 32-34 of gestation. Gestational diabetes mellitus was diagnosed according to the WHO 2013 guidelines. Out of 392 women screened, 39% (95% CI: 34.2-44.1) had GDM, the majority of whom (94.1%) were diagnosed based solely on the fasting blood sample from the OGTT. No associations were observed between haemoglobin or ferritin and glucose measurements during pregnancy. A very high prevalence of GDM was found in rural Tanzania. In view of the laborious, costly and inconvenient OGTT, alternative methods such as fasting blood glucose should be considered when screening for GDM in low- and middle-income countries.Peer reviewe

Fabrication of α‑Fe2O3 Nanostructures: synthesis, characterization, and their promising application in the treatment of Carcinoma A549 Lung Cancer Cells

In the present work, iron nanoparticles were synthesized in the α-Fe2O3 phase with the reduction of potassium hexachloroferrate(III) by using l-ascorbic acid as a reducing agent in the presence of an amphiphilic non-ionic polyethylene glycol surfactant in an aqueous solution. The synthesized α-Fe2O3 NPs were characterized by powder X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, atomic force microscopy, dynamic light scattering, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and ultraviolet–visible spectrophotometry. The powder X-ray diffraction analysis result confirmed the formation of α-Fe2O3 NPs, and the average crystallite size was found to be 45 nm. The other morphological studies suggested that α-Fe2O3 NPs were predominantly spherical in shape with a diameter ranges from 40 to 60 nm. The dynamic light scattering analysis revealed the zeta potential of α-Fe2O3 NPs as −28 ± 18 mV at maximum stability. The ultraviolet–visible spectrophotometry analysis shows an absorption peak at 394 nm, which is attributed to their surface plasmon vibration. The cytotoxicity test of synthesized α-Fe2O3 NPs was investigated against human carcinoma A549 lung cancer cells, and the biological adaptability exhibited by α-Fe2O3 NPs has opened a pathway to biomedical applications in the drug delivery system. Our investigation confirmed that l-ascorbic acid-coated α-Fe2O3 NPs with calculated IC50 ≤ 30 μg/mL are the best suited as an anticancer agent, showing the promising application in the treatment of carcinoma A549 lung cancer cells

The type 1 diabetes gene TYK2 regulates beta-cell development and its responses to interferon-alpha

The TYK2 gene is associated with development of type 1 diabetes. Here the authors show that TYK2 regulates beta-cell development, but at the same time TYK2 inhibition in the islets prevents IFN alpha responses and enhances their survival against CD8(+) T-cell cytotoxicity; representing a potent therapeutic target to halt T1D progression. Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic beta-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Iota interferon (IFN-Iota) mediated intracellular signalling. To study the role of TYK2 in beta-cell development and response to IFN alpha, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFN alpha-induced antigen processing and presentation, including MHC Class Iota and Class Iota Iota expression, enhancing their survival against CD8(+) T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in beta-cell development and support TYK2 inhibition in adult beta-cells as a potent therapeutic target to halt T1D progression.Peer reviewe