NHGRI/DIR Cytogenetics and Microscopy Core

National Human Genome Research Institute

Investigators

Linked publications & trials

Abstract

Summary: In total, 12,016 hours of cytogenetic experiments including analyzes and the use of microscope systems and processing workstations were used by the Cytogenetic and Microscopy Core in the reporting period to serve the NHGRI scientific community. The Cytogenetic Section experiments include cell culture, harvest, slide making and processing of all these techniques and microscope analyses related with cytogenetic, molecular cytogenetic and RNAscope research projects. The Microscopy Section work included the training of investigators and institute trainees in how to use Confocal Laser Scanning Microscopy in studies that comprise of Fluorescence Recovery After Photo-bleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET), Photo-activation of Green Fluorescent Protein (PA-GFP), nuclear/organelle/cytoplasmic colocalization studies, Two-Dimensional (2D), Three-Dimensional (3D) and Four-Dimensional (4D) cell morphology and volumetric studies, response to stimuli (drug), quantitative analysis (fluorescence, area, counts, etc.), along with live-cell, digitizing slides and super resolution microscopy. Microscopy usage is described by the metric of hours logged by Principal Investigators or their trainees. The Core maintains two confocal systems (Zeiss LSM 880 + Airyscan and Spinning disc), one long-term live-cell system, one automated slide scanner, two epi-fluorescence microscopes all fitted with CCD cameras and five computer workstations. Below is an abbreviated list of projects that the Core collaborated in the past year: The laboratory of Dr. Gahl (MGB) is studying Smith-Magenis syndrome, a complex disorder. The syndrome is primarily caused by de novo interstitial deletions of chromosome 17p11.2. However, atypical deletions that can range from 1.5 to 9 Mb in size and heterozygous point mutations of the RAI1 gene are also associated with the phenotype. The deletions are detectable by cytogenetic G-banding and/or by fluorescence in situ hybridization (FISH) analyses. The Core is working on experiments to define the exact deleted region in several patients. The laboratory of Dr. Liu (TFGB) since 2019, launched a longitudinal natural history study of patients with germline RUNX1 variants to increase our understanding of familial platelet disorder (FPD), which will hopefully lead to better clinical management and potentially new therapies. They have since been generating induced pluripotent stem cells (iPSC). To guarantee the quality of our results the NHGRI Cytogenetics section of the Core has been doing karyotyping studies, as our iPSC bank continues to grow with the enrollment of more patients and the establishment of new collaborations. Dr. Kastner lab (MGB) is studying the mechanism of inflammation in patients with trisomy 8 mosaicism. In some of their patients we found constitutional trisomy 8 mosaicism (diagnosed by blood karyotype) and is fresh tissue from the esophagus, stomach, and buccal cells. They were originally planning to look at the copy number of chromosome 8 genes in the bulk tissue, but also, they would like to know if non-hematopoietic cells in the GI tract also have trisomy 8. Performing FISH may be a way to clarify which type of cells carry trisomy 8. Dr. Biesecker (CPHR) lab is studying Proteus syndrome that is a rare, progressive overgrowth disorder caused by a somatic activating variant c.49G>A, p.(E17K) in AKT1. They created mouse model with endogenously regulated expression of a conditional allele containing the Proteus syndrome variant. To test the Happle hypothesis, which states that the Proteus syndrome variant only survives by mosaicism, we activated the variant allele by crossing Akt1 conditional and ACTB-Cre mice which express CRE ubiquitously. Embryos from this cross were not viable and died between E11.5 and E17.5, supporting the Happle hypothesis. To determine the extent of the vascular defects in these embryos, they used the iDISCO clearing method to perform whole mount immunolabeling and volume imaging on mutant and wild type embryos. Endothelial and smooth muscle cells were labeled with antibodies to PECAM-1 and smooth muscle actin (SMA), respectively, to visualize the vasculature in embryos collected from E11.5 to E14.5. Cleared and stained embryos were imaged in the NHGRI Cytogenetic and Microscopy Core. Dr. Giniger lab (Adjunct MGB) is working with the Axon Guidance and Neural Connectivity Section of NINDS has two general groups of projects we perform in the NHGRI Microscopy Core. One portion of the lab studies how nerves grow during early nervous system development. The focus of these experiments is to image individual axons as they grow in living tissue, tracking their structure, dynamics, and the activity of the molecules that promote and guide their growth. The other half of the lab studies the mechanisms of neurodegenerative disease and their association with aging. A major focus in this project is to use imaging of fluorescent reporters to quantify how the internal state of a neuron changes as it progresses over the course of disease. Dr. Sidranskys lab (MGB) is investigating heterozygote mutations in the gene GBA1 is the most common genetic risk factor for Parkinson disease. Homozygous mutations in GBA1 cause the lysosomal storage disorder Gaucher disease, but despite carrying biallelic mutations, only a minority of patients with Gaucher disease develop Parkinson disease. To understand the molecular differences between individuals who develops the neurodegenerative disorder from those who are spared, we use iPSCs. Using CRISPR we genetically engineered the iPSCs to optimize differentiation into glutamatergic and dopaminergic neurons. Karyotyping of the clones is a part of our quality controls to ensure that the CRISPR editing did not induce any large chromosomal shifts. After unilateral injection of AAV-Atp10b RNAi, knock down efficiency was validated using RNAscope in-situ hybridization technology. Reduction of Atp10b mRNA in DA-neuron was confirmed by multiplex RNAscope assay using Atp10b probe and TH probe. Dr. Collins lab (CPHR) is investigating children with Hutchinson-Gilford Progeria Syndrome (HGPS or progeria) age at seven times the normal rate and die of heart attacks and strokes in their teen years. To understand the potential heart contribution to the death of progeria patients, they have been studying cardiac manifestations in the LMNAG608G/G608G homozygous (G608G DC) mouse model. Microscopy studies showed cardiomyocyte nuclear blebbing, mitotic defects, disorganized sarcomeres, and increased fibrosis in progeria G608G DC mice heart compared to WT control. She further identified significant upregulation of Nppa and Nppb, two known heart failure genes, in middle- and late-stage G608G DC mice by snRNA-seq and validated the overexpression of these genes by RNAscope in situ hybridization. Also, the lab uses the slide scanner that has enabled detailed histologic studies on multiple tissues from HGPS mouse models including demonstration of decreased trabecular volume and abnormal growth plate structure in long bones, as well as improved bone, vascular and skin structural parameters in HGPS mice treated with a DNA base editor to correct the mutation.