Developmental stage-specific differentiation of stem or progenitor cells into safe and functional cells is of fundamental importance in regenerative medicine, including β-cell replacement. However, the differentiation of islet progenitor cells (IPCs) into insulin-secreting β cells remains elusive. Here, we report that the multifunctional molecule nicotinamide (NIC) is a specific differentiation regulator of mouse IPCs. The differentiated cells regulated by NIC exhibited many characteristics of adult β cells, including ameliorating preclinical diabetes and a highly comparable transcriptome profile. Gene set enrichment analysis showed that during differentiation, numerous IPC transcription factor genes, including Ngn3, Pax4, Fev, and Mycl1, were all down regulated. Pharmacological, biochemical, and gene knockdown analyses collectively demonstrated that NIC regulated the differentiation via inhibiting Sirt1 (silent information regulator transcript 1). Finally, NIC also regulates human IPC differentiation. Thus, our study advances islet developmental biology and impacts on translational research and regenerative therapies to diabetes and other diseases. Stem Cells 2017;35:1341-1354.

Different genes encode the α-actin isoforms that are predominantly expressed in heart and skeletal muscle. Mutations in the skeletal muscle α-actin gene (ACTA1) cause muscle diseases that are mostly lethal in the early postnatal period. We previously demonstrated that the disease phenotype of ACTA1 mouse models could be rescued by transgenic over-expression of cardiac α-actin (ACTC1). ACTC1 is the predominant striated α-actin isoform in the heart but is also expressed in developing skeletal muscle. To develop a translatable therapy, we investigated the genetic regulation of Actc1 expression. Using strains from The Collaborative Cross (CC) genetic resource, we found that Actc1 varies in expression by up to 24-fold in skeletal muscle. We defined significant expression quantitative trait loci (eQTL) associated with early adult Actc1 expression in soleus and heart. eQTL in both heart and soleus mapped to the Actc1 locus and replicate an eQTL mapped for Actc1 in BXD heart and quadriceps. We built on this previous work by analysing genes within the eQTL peak regions to prioritise likely candidates for modifying Actc1 expression. Additionally we interrogated the CC founder haplotype contributions to enable prioritisation of genetic variants for functional analyses. Methylation around the Actc1 transcriptional start site in early adult skeletal muscle negatively correlated with Actc1 expression in a strain-dependent manner, while other marks of regulatory potential (histone modification and chromatin accessibility) were unaltered. This study provides novel insights into the complex genetic regulation of Actc1 expression in early adult skeletal muscles.

Genome-wide association studies have found >60 loci that confer genetic susceptibility to type 1 diabetes (T1D). Many of these are defined only by anonymous single nucleotide polymorphisms: the underlying causative genes, as well as the molecular bases by which they mediate susceptibility, are not known. Identification of how these variants affect the complex mechanisms contributing to the loss of tolerance is a challenge. In this study, we performed systematic analyses to characterize these variants. First, all known genes in strong linkage disequilibrium (r(2) > 0.8) with the reported single nucleotide polymorphisms for each locus were tested for commonly occurring nonsynonymous variations. We found only a total of 22 candidate genes at 16 T1D loci with common nonsynonymous alleles. Next, we performed functional studies to examine the effect of non-HLA T1D risk alleles on regulating expression levels of genes in four different cell types: EBV-transformed B cell lines (resting and 6 h PMA stimulated) and purified CD4(+) and CD8(+) T cells. We mapped cis-acting expression quantitative trait loci and found 24 non-HLA loci that affected the expression of 31 transcripts significantly in at least one cell type. Additionally, we observed 25 loci that affected 38 transcripts in trans. In summary, our systems genetics analyses defined the effect of T1D risk alleles on levels of gene expression and provide novel insights into the complex genetics of T1D, suggesting that most of the T1D risk alleles mediate their effect by influencing expression of multiple nearby genes.

The data shows results acquired in a large cohort of 5668 ethnic Arabs involved in a common variants association study for coronary artery disease (CAD) and myocardial infarction (MI) using the Affymetrix Axiom Genotyping platform ("A genome-wide association study reveals susceptibility loci for myocardial infarction/coronary artery disease in Saudi Arabs" Wakil et al. (2015) [1] ). Several loci were described that conferred risk for CAD or MI, some of which were validated in an independent set of samples. Principal Component (PCA) analysis suggested that the Saudi Cohort was close to the CEU and TSI populations, thus pointing to similarity with European populations.

Patients with the same genetic mutation who differ in disease severity may possess genetic modifying factors. Such genetic modifiers have been identified for neuromuscular disorders; many of these have been found from mouse studies. The Collaborative Cross (CC) is a resource with the potential to effectively map genetic modifiers more effectively than previously available methods. Derived from eight genetically diverse founder mouse strains, the genome of each CC offspring strain is a mosaic of chromosomal segments inherited randomly from the founders. As a proxy measure of skeletal muscle function, we analysed voluntary running wheel activity of over 50 CC mouse strains at two different ages (6 weeks and 6 months). Marked phenotypic variation was seen across strains for all related traits measured (distance run per day, maximum speed, average speed, and time spent running per day). A select number of strains were chosen to breed with dystrophin-deficient muscular dystrophy (mdx) mice, and we evaluated voluntary running wheel activity of the resulting progeny. Progeny of some strains performed equal to, or worse than, mdx mice on the standard C57BL/10 background. However, the dystrophin-deficient progeny of one of the highest performing CC strains had significantly improved voluntary exercise performance. Comparison of the CC strains' genomes allowed mapping of a key modifier gene to a small chromosome region that is currently being scrutinised. Microarray analysis of skeletal muscles from “rescued” and “standard” mdx mice indicates a relatively small number of differentially expressed genes between the two cohorts. Pathway analysis of differentially expressed genes highlights the Parkinson's pathway. The genetic modifier/s for improved voluntary exercise ability by mdx mice could be targeted for modulation as a potential therapeutic avenue for muscular dystrophy.

BACKGROUND:Multiple loci have been identified for coronary artery disease (CAD) by genome-wide association studies (GWAS), but no such studies on CAD incidence has been reported yet for any Middle Eastern population. METHODS:In this study, we performed a GWAS for CAD and myocardial infarction (MI) incidence in 5668 Saudis of Arab descent using the Affymetrix Axiom Genotyping platform. RESULTS:We describe SNPs at 16 loci that showed significant (P < 5 × 10(-8)) or suggestive GWAS association (P < 1 × 10(-5)) with CAD or MI, in the ethnic Saudi Arab population. Among the four variants reaching GWAS significance in the present study, the rs10738607_G [0.78(0.71-0.85); p = 2.17E-08] in CDNK2A/B gene was associated with CAD. Two other SNPs on the same gene, rs10757274_G [0.79(0.73-0.86); p = 2.98E-08] and rs1333045_C [0.79(0.73-0.86); p = 1.15E-08] as well as the rs9982601_T [1.38(1.23-1.55); p = 3.49E-08] on KCNE2 were associated with MI. These variants have been previously described in other populations. Several SNPs, including the rs7421388 (PLCL1) and rs12541758 (TRPA1) displaying a suggestive GWAS association (P < 1 × 10(-5)) with CAD as well as rs41411047 (RNF13), rs32793 (PDZD2) and rs4739066 (YTHDF3), similarly showing weak association with MI, were confirmed in an independent dataset. Furthermore, our estimation of heritability of CAD and MI based on observed genome-wide sharing in unrelated Saudi Arabs was approximately 33% and 44%, respectively. CONCLUSIONS:Our study has identified susceptibility variants for CAD/MI in ethnic Arabs. These findings provide further insights into pathways contributing to the susceptibility for CAD and will enable more comprehensive genetic studies of these diseases in Middle East populations.

Analysis of voluntary running wheel activity in mice is a useful phenotypic measure of skeletal muscle function. We have collected voluntary running wheel activity and total body weight data from >50 strains of mice belonging to the ‘Collaborative Cross’ (aka The Gene Mine), and compared these to known models of neuromuscular disease. The Gene Mine is a mouse reference population derived from eight genetically diverse founder strains, with the genome of each offspring strain being a mosaic of chromosomal segments inherited randomly from the founders. The Gene Mine is designed specifically for complex trait analysis and the identification of quantitative trait loci (QTL). Our analyses of voluntary running wheel activity has involved measuring distance, maximum speed, average speed and time spent on the wheel over a six day consecutive period in male and female Gene Mine mice at two different ages (6 weeks and 6–9 months). We also recorded real-time activity levels and are imaging selected mouse strains by magnetic resonance imaging and computed tomography. Our results indicate marked phenotypic variation across all traits measured, including comparisons of young and older Gene Mine mice. We are now mapping QTL for the various phenotypes and comparing these with skeletal muscle gene expression. We previously crossed skeletal muscle actin knockout mice with Gene Mine strains to identify strains capable of extending the usual early lethal phenotype of this model. The phenotype resource we and others are building can now be used to search for strains, and from there QTL, that modify voluntary running wheel activity, weight and muscle bulk in various models of neuromuscular diseases. Analysis of voluntary running wheel activity in mice is a useful phenotypic measure of skeletal muscle function. We have collected voluntary running wheel activity and total body weight data from >50 strains of mice belonging to the ‘Collaborative Cross’ (aka The Gene Mine), and compared these to known models of neuromuscular disease. The Gene Mine is a mouse reference population derived from eight genetically diverse founder strains, with the genome of each offspring strain being a mosaic of chromosomal segments inherited randomly from the founders. The Gene Mine is designed specifically for complex trait analysis and the identification of quantitative trait loci (QTL). Our analyses of voluntary running wheel activity has involved measuring distance, maximum speed, average speed and time spent on the wheel over a six day consecutive period in male and female Gene Mine mice at two different ages (6 weeks and 6–9 months). We also recorded real-time activity levels and are imaging selected mouse strains by magnetic resonance imaging and computed tomography. Our results indicate marked phenotypic variation across all traits measured, including comparisons of young and older Gene Mine mice. We are now mapping QTL for the various phenotypes and comparing these with skeletal muscle gene expression. We previously crossed skeletal muscle actin knockout mice with Gene Mine strains to identify strains capable of extending the usual early lethal phenotype of this model. The phenotype resource we and others are building can now be used to search for strains, and from there QTL, that modify voluntary running wheel activity, weight and muscle bulk in various models of neuromuscular diseases.

Significance In type 1 diabetes (T1D), the insulin-producing pancreatic β-cells are destroyed by the immune system. Both genetic and environmental factors contribute to T1D risk. Candidate genes for T1D identified by genome-wide association studies have been proposed to act at both the immune system and the β-cell levels. This study shows that the risk variant rs3825932 in the candidate gene cathepsin H ( CTSH ) predicts β-cell function in both model systems and human T1D. Collectively, our data indicate that higher CTSH expression in β-cells may protect against immune-mediated damage and preserve β-cell function, thereby representing a possible therapeutic target. Our study reinforces the concept that candidate genes for T1D may affect disease progression by modulating survival and function of the β-cells.

The Collaborative Cross (CC) was designed to facilitate rapid gene mapping and consists of hundreds of recombinant inbred lines descended from eight diverse inbred founder strains. A decade in production, it can now be applied to mapping projects. Here, we provide a proof of principle for rapid identification of major-effect genes using the CC. To do so, we chose coat color traits since the location and identity of many relevant genes are known. We ascertained in 110 CC lines six different coat phenotypes: albino, agouti, black, cinnamon, and chocolate coat colors and the white-belly trait. We developed a pipeline employing modifications of existing mapping tools suitable for analyzing the complex genetic architecture of the CC. Together with analysis of the founders' genome sequences, mapping was successfully achieved with sufficient resolution to identify the causative genes for five traits. Anticipating the application of the CC to complex traits, we also developed strategies to detect interacting genes, testing joint effects of three loci. Our results illustrate the power of the CC and provide confidence that this resource can be applied to complex traits for detection of both qualitative and quantitative trait loci.

Cardiac actin (ACTC) is the fetal homologue of skeletal muscle actin (ACTA1), and is switched off at birth in skeletal muscle but remains highly expressed in adult heart and in regenerating muscle. ACTC is 99% identical to ACTA1 protein, differing at only 4 amino acids. We previously showed an ACTC transgene could functionally replace ACTA1 in postnatal skeletal muscle of Acta1 knockout mice that normally die within a few days of birth, indicating that ACTC could have a therapeutic role in skeletal muscle actin diseases. We harnessed the power of two different recombinant inbred (RI) mouse models including BXD and the more recently developed Collaborative Cross (CC), to identify genetic elements controlling Actc expression. RI mice are genetic reference populations consisting of large numbers of inbred mouse strains descended from either 2 (BXD) or 8 (CC) founder strains. Characterization of these strains for a trait of interest allows for the mapping of gene(s) mediating that trait to genomic intervals called quantitative trait loci (QTL). We have identified in both RI sets a highly significant QTL regulating Actc expression in skeletal muscle. This QTL also regulates Actc expression in particular non-skeletal muscle tissues (eye and lung). Identification of the regulatory genetic element may provide a target for modifying expression of cardiac actin by therapeutic intervention. Cardiac actin (ACTC) is the fetal homologue of skeletal muscle actin (ACTA1), and is switched off at birth in skeletal muscle but remains highly expressed in adult heart and in regenerating muscle. ACTC is 99% identical to ACTA1 protein, differing at only 4 amino acids. We previously showed an ACTC transgene could functionally replace ACTA1 in postnatal skeletal muscle of Acta1 knockout mice that normally die within a few days of birth, indicating that ACTC could have a therapeutic role in skeletal muscle actin diseases. We harnessed the power of two different recombinant inbred (RI) mouse models including BXD and the more recently developed Collaborative Cross (CC), to identify genetic elements controlling Actc expression. RI mice are genetic reference populations consisting of large numbers of inbred mouse strains descended from either 2 (BXD) or 8 (CC) founder strains. Characterization of these strains for a trait of interest allows for the mapping of gene(s) mediating that trait to genomic intervals called quantitative trait loci (QTL). We have identified in both RI sets a highly significant QTL regulating Actc expression in skeletal muscle. This QTL also regulates Actc expression in particular non-skeletal muscle tissues (eye and lung). Identification of the regulatory genetic element may provide a target for modifying expression of cardiac actin by therapeutic intervention.

Embryonic stem cells (ESCs) have been promised as a renewable source for regenerative medicine, including providing a replacement therapy in type 1 diabetes. However, they have not yet been differentiated into functional insulin-secreting β cells. This is due partially to the knowledge gap regarding the transcription factors (TFs) required for pancreas development. We hypothesize that, if directed differentiation in vitro recapitulates the developmental process in vivo, ESCs provide a powerful model to discover novel pancreatic TF genes. Guided by knowledge of their normal development and using RT-PCR and immunochemical analyses, we have established protocols for directed differentiation of mouse ESCs into pancreatic progenitors. Microarray analyses of these differentiating ESC cells at days 0, 4, 8 and 15 confirmed their sequential differentiation. By day 15, we found up-regulation of a group of pancreatic progenitor marker genes including Pdx1, Ptf1a, Nkx6.1, Pax4 and Pax6. Consistently, Pdx1-immunoreactive cells were detected on day 15. Most of these Pdx1(+) cells also expressed Nkx6.1. Bioinformatic analyses of sequential datasets allowed identification of over 20 novel TF genes potentially important for pancreas development. The dynamic expression of representative known and novel genes was confirmed by quantitative real time RT-PCR analysis. This strategy may be modified to study novel regulatory molecules for development of other tissue and organ systems.

OBJECTIVE Interactions between genetic and environmental factors lead to immune dysregulation causing type 1 diabetes and other autoimmune disorders. Recently, many common genetic variants have been associated with type 1 diabetes risk, but each has modest individual effects. Familial clustering of type 1 diabetes has not been explained fully and could arise from many factors, including undetected genetic variation and gene interactions. RESEARCH DESIGN AND METHODS To address this issue, the Type 1 Diabetes Genetics Consortium recruited 3,892 families, including 4,422 affected sib-pairs. After genotyping 6,090 markers, linkage analyses of these families were performed, using a novel method and taking into account factors such as genotype at known susceptibility loci. RESULTS Evidence for linkage was robust at the HLA and INS loci, with logarithm of odds (LOD) scores of 398.6 and 5.5, respectively. There was suggestive support for five other loci. Stratification by other risk factors (including HLA and age at diagnosis) identified one convincing region on chromosome 6q14 showing linkage in male subjects (corrected LOD = 4.49; replication P = 0.0002), a locus on chromosome 19q in HLA identical siblings (replication P = 0.006), and four other suggestive loci. CONCLUSIONS This is the largest linkage study reported for any disease. Our data indicate there are no major type 1 diabetes subtypes definable by linkage analyses; susceptibility is caused by actions of HLA and an apparently random selection from a large number of modest-effect loci; and apart from HLA and INS, there is no important susceptibility factor discoverable by linkage methods.

Iron homeostasis is crucial to many biological functions in nearly all organisms, with roles ranging from oxygen transport to immune function. Disruption of iron homeostasis may result in iron overload or iron deficiency. Iron deficiency may have severe consequences, including anemia or changes in immune or neurotransmitter systems. Here we report on the variability of phenotypic iron tissue loss and splenomegaly and the associated quantitative trait loci (QTLs), polymorphic areas in the mouse genome that may contain one or more genes that play a role in spleen iron concentration or spleen weight under each dietary treatment. Mice from 26 BXD/Ty recombinant inbred strains, including the parent C57BL/6 and DBA/2 strains, were randomly assigned to adequate iron or iron-deficient diets at weaning. After 120 days, splenomegaly was measured by spleen weight, and spleen iron was assessed using a modified spectrophotometry technique. QTL analyses and gene expression comparisons were then conducted using the WebQTL GeneNetwork. We observed wide, genetic-based variability in splenomegaly and spleen iron loss in BXD/Ty recombinant inbred strains fed an iron-deficient diet. Moreover, we identified several suggestive QTLs. Matching our QTLs with gene expression data from the spleen revealed candidate genes. Our work shows that individual differences in splenomegaly response to iron deficiency are influenced at least partly by genetic constitution. We propose mechanistic hypotheses by which splenomegaly may result from iron deficiency.

Objective: Despite great progress in understanding the transcriptional regulation of the development of insulin-secreting A cells, the quantitative temporal expression of insulin gene(s) remains largely unknown. We here aimed to quantify insulin gene transcripts during development.Methods: We described bioinformatics algorithms to quantify (insulin) gene transcript abundance in sequential microarray data sets at the global level. Several molecular techniques were used to confirm our analyses.Results: We demonstrated that the expression of insulin genes was up-regulated at approximately 14-fold, 700- to 2000-fold, and 5000- to 6000-fold in Pdx1- and Ngn3-expressing cells and adult islets compared with definitive endodermal or embryonic stem cells, respectively. The expression of multiple genes encoding molecules involved in posttranslational modi. cations of insulin and glucose sensing was also elevated in the same period. All islet and associated genes determined with microarray data were confirmed not only to be up-regulated by real-time quantitative reverse transcriptase polymerase chain reaction but also that the magnitude of their increase quantified with these 2 methods was statistically highly correlated. Consistent with the above, green fluorescence protein expression under the control of the mouse insulin 1 promoter could be visualized in the pancreas from embryonic day (E) 11.5, increasing progressively through E13.5 to E15.5.Conclusion: Our study provides a novel insight into islet developmental biology.

Aim: Type 1 diabetes susceptibility is influenced by a number of genes, of which those with the strongest effects map to the human leucocyte antigen (HLA) complex. Evidence for linkage of non‐HLA genes in several affected sib‐pair analyses was increased if the HLA or gender status of the sibs was considered independently. We investigated whether linkage status at the HLA complex differentially affected transmission of alleles to sibs depending on their HLA or gender linkage status. Methods: Genotypes of 2437 markers typed on 11 279 individuals from 2363 families, stratified according to HLA and gender, were examined using the transmission disequilibrium test. Results: Several significant single nucleotide polymorphisms (SNPs) loci were found near the class II genes; no significant SNPs were found by these analyses near the class I or class II genes. Other significant effects were found when the gender of the sibs or the parents was considered. There was not a significant difference in HLA‐DRB genotypes between the stratified sets. Conclusions: These results suggest the presence of novel recessive susceptibility gene(s) within the HLA complex.

"Systems Genetics" detects variation in phenotypic traits and integrates this with underlying genetic variation. A powerful application of systems genetics is analyzing effects of genome-wide genetic variants on transcriptome-wide variation in gene expression. We see systems genetics as a new powerful technology which will empower research in genetics and in other disciplines. Here, we present preliminary analyses of systems genetics approaches to defining genetic interactions within the thymus, which is the key site for T lymphocyte development and imposition of immunological tolerance.