Amanda M. Ackermann

Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes

Objective Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and exhibit overlapping transcriptomes and epigenomes. Notably, destruction of β-cells can stimulate repopulation via transdifferentiation of α-cells, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity. Methods We sorted human α- and β-cells and performed the “Assay for Transposase-Accessible Chromatin with high throughput sequencing” (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. Results We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The “group specific protein” (GC; or vitamin D binding protein) was restricted to α-cells, while CHODL (chondrolectin) immunoreactivity was only present in β-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. Conclusions We have determined the genetic landscape of human α- and β-cells based on chromatin accessibility and transcript levels, which allowed for detection of novel α- and β-cell signature genes not previously known to be expressed in islets. Using fine-mapping of open chromatin, we have identified thousands of potential cis-regulatory elements that operate in an endocrine cell type-specific fashion.

Oral D-galactose supplementation in PGM1-CDG

PurposePhosphoglucomutase-1 deficiency is a subtype of congenital disorders of glycosylation (PGM1-CDG). Previous casereports in PGM1-CDG patients receiving oral D-galactose (D-gal) showed clinical improvement. So far no systematic in vitro and clinical studies have assessed safety and benefits of D-gal supplementation. In a prospective pilot study, we evaluated the effects of oral D-gal in nine patients.MethodsD-gal supplementation was increased to 1.5 g/kg/day (maximum 50 g/day) in three increments over 18 weeks. Laboratory studies were performed before and during treatment to monitor safety and effect on serum transferrin-glycosylation, coagulation, and liver and endocrine function. Additionally, the effect of D-gal on cellular glycosylation was characterized in vitro.ResultsEight patients were compliant with D-gal supplementation. No adverse effects were reported. Abnormal baseline results (alanine transaminase, aspartate transaminase, activated partial thromboplastin time) improved or normalized already using 1 g/kg/day D-gal. Antithrombin-III levels and transferrin-glycosylation showed significant improvement, and increase in galactosylation and whole glycan content. In vitro studies before treatment showed N-glycan hyposialylation, altered O-linked glycans, abnormal lipid-linked oligosaccharide profile, and abnormal nucleotide sugars in patient fibroblasts. Most cellular abnormalities improved or normalized following D-gal treatment. D-gal increased both UDP-Glc and UDP-Gal levels and improved lipid-linked oligosaccharide fractions in concert with improved glycosylation in PGM1-CDG.ConclusionOral D-gal supplementation is a safe and effective treatment for PGM1-CDG in this pilot study. Transferrin glycosylation and ATIII levels were useful trial end points. Larger, longer-duration trials are ongoing.

High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting

Objective α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER “knock-in” mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. Methods We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3′ UTR of the Glucagon (Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured in Gcg-CreERT2;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Results Tamoxifen injection of Gcg-CreERT2;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal. Conclusions We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.

Current controversies in turner syndrome: Genetic testing, assisted reproduction, and cardiovascular risks

Patients with Turner syndrome (TS) require close medical follow-up and management for cardiac abnormalities, growth and reproductive issues. This review summarizes current controversies in this condition, including: 1) the optimal genetic testing for Turner syndrome patients, particularly with respect to identification of Y chromosome material that may increase the patients risk of gonadoblastoma and dysgerminoma, 2) which patients should be referred for bilateral gonadectomy and the recommended timing of such referral, 3) options for assisted reproduction in these patients and associated risks, 4) the increased risk of mortality associated with pregnancy in this population, and 5) how best to assess and monitor cardiovascular risks.

Compound heterozygous mutations in COL1A1 associated with an atypical form of type I osteogenesis imperfecta

Heterozygous mutations in the genes encoding the proα1(I) or proα2(I) chains of type I procollagen (COL1A1 and COL1A2, respectively) account for most cases of osteogenesis imperfecta (OI), a disorder characterized by reduced bone strength and increased fracture risk. COL1A1 mutations can also cause rare cases of Ehlers–Danlos syndrome (EDS), a disorder that primarily affects connective tissue and often includes reduced bone mass. Here we present a kindred of three young siblings ages 1–4 years old whose mother has a history of mild type I OI. All three children are compound heterozygotes for COL1A1 mutations, with a novel frameshift mutation (c.2522delC; p.Pro841Leufs*266) from their mother and a known missense mutation (c.3196C>T; p.R1066C) from their clinically unaffected father, which has previously been described as causing a combined type I OI/EDS phenotype. The three children exhibit features of both COL1A1 mutations: early and frequent long bone fractures, joint hyperextensibility, and blue sclerae. We describe three siblings who are the first reported surviving subjects with biallelic pathogenic COL1A1 mutations. They have a more severe form of type I OI with features of EDS that represents their compound heterozygosity for two deleterious COL1A1 mutations. Their long‐term outcomes are yet to be determined.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets

Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+]i), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+]i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

GABA and Artesunate Do Not Induce Pancreatic α-to-β Cell Transdifferentiation In Vivo

Recent reports identified activation of the GABA signaling pathway as a means to induce transdifferentiation of pancreatic α cells into β cells. These reports followed several previous studies that found that α cells were particularly well suited to conversion into β cells in mice, but only after nearly complete β cell loss or forced overexpression of key transcriptional regulators. The possibility of increasing β cell number via reprograming of α cells with a small molecule is enticing, as this could be a potential new pharmacologic therapy for diabetes. Here, we employed rigorous genetic lineage tracing of α cells, using Glucagon-CreERT2;Rosa-LSL-eYFP mice, to evaluate if activation of GABA signaling caused α-to-β cell reprogramming. In contrast to previous reports, we found that even after long-term treatment of mice with artesunate or GABA, neither α-to-β cell transdifferentiation nor insulin secretion were stimulated, putting into question whether these agents represent a viable path to a novel diabetes therapy.

Managing congenital hyperinsulinism: improving outcomes with a multidisciplinary approach

Congenital hyperinsulinism (CHI) is the most common cause of persistent hypo- glycemia in pediatric patients and is associated with significant risk of hypoglycemic seizures and developmental delays. CHI results from mutations in at least nine genes that play a role in regulating beta-cell insulin secretion. Thus, patients with CHI have dysregulated insulin secretion that is unresponsive to blood glucose level. Each different genetic etiology of CHI is associated with particular clinical characteristics that affect management decisions. Given the broad phe- notypic spectrum and relatively rare prevalence of CHI, it is important that patients with CHI be evaluated by clinicians experienced with CHI and the multiple subspecialty services that are necessary for the management of the disorder. In this review, we summarize the pathophysiology and genetic causes of CHI and then focus primarily on the most common genetic cause (muta- tions in the ATP-gated potassium (K ATP ) channel) for further discussion of diagnosis, medical and surgical management, and potential acute and chronic complications. We provide insight from relevant published studies and reports, in addition to anecdotal information from our centers clinical experience in caring for over 400 patients with CHI. Careful assessment of each patients individual pathophysiology is necessary to determine the appropriate treatment regimen, and continued close follow-up and monitoring of disease- and treatment-related complications are essential. Although significant improvements have been made in the past several years with regard to diagnosis and management, given the continued high morbidity rate in patients with CHI, improved diagnostic techniques and new therapeutic options would be welcomed.