Discovering mechanisms of human genetic variation and controlling cell states at scale.

Population-scale sequencing efforts have catalogued substantial genetic variation in humans such that variant discovery dramatically outpaces interpretation. We discuss how single-cell sequencing is poised to reveal genetic mechanisms at a rate that may soon approach that of variant discovery. The functional genomics toolkit is sufficiently modular to systematically profile almost any type of variation within increasingly diverse contexts and with molecularly comprehensive and unbiased readouts. As a result, we can construct deep phenotypic atlases of variant effects that span the entire regulatory cascade. The same conceptual approach to interpreting genetic variation should be applied to engineering therapeutic cell states. In this way, variant mechanism discovery and cell state engineering will become reciprocating and iterative processes towards genomic medicine.

Highly multiplexed design of an allosteric transcription factor to sense novel ligands.

Allosteric transcription factors (aTF), widely used as biosensors, have proven challenging to design for detecting novel molecules because mutation of ligand-binding residues often disrupts allostery. We developed Sensor-seq, a high-throughput platform to design and identify aTF biosensors that bind to non-native ligands. We screened a library of 17,737 variants of the aTF TtgR, a regulator of a multidrug exporter, against six non-native ligands of diverse chemical structures - four derivatives of the cancer therapeutic tamoxifen, the antimalarial drug quinine, and the opiate analog naltrexone - as well as two native flavonoid ligands, naringenin and phloretin. Sensor-seq identified novel biosensors for each of these ligands with high dynamic range and diverse specificity profiles. The structure of a naltrexone-bound design showed shape-complementary methionine-aromatic interactions driving ligand specificity. To demonstrate practical utility, we developed cell-free detection systems for naltrexone and quinine. Sensor-seq enables rapid, scalable design of new biosensors, overcoming constraints of natural biosensors.

Discovering chromatin dysregulation induced by protein-coding perturbations at scale.

Although population-scale databases have expanded to millions of protein-coding variants, insight into variant mechanisms has not kept pace. We present PROD-ATAC, a high-throughput method for discovering the effects of protein-coding variants on chromatin. A pooled library of variants is expressed in a disease-agnostic cell line, and single-cell ATAC resolves each variant's effect on chromatin. Using PROD-ATAC, we characterized the effects of >100 oncofusions (a class of cancer-causing chimeric proteins) and controls and revealed that pioneer activity is a common feature of fusions spanning an enormous range of fusion frequencies. Further, fusion-induced dysregulation can be context-agnostic as observed mechanisms often overlapped with cancer and cell-type specific prior knowledge. We also showed that gain-of-function pioneering is common among oncofusions. This work provides a global view of fusion-induced chromatin. We uncovered convergent mechanisms among disparate oncofusions and shared modes of dysregulation across different cancers. PROD-ATAC is generalizable to any set of protein-coding variants.

An oligogenic case of severe neonatal thrombocytopenia and a purportedly benign variant in GFI1B requiring reinterpretation.

Although thrombocytopenia in neonatal intensive care patients is rarely due to inherited disorders, the number of genetic variants implicated in platelet defects has grown dramatically with increasing genome-wide sequencing. Here we describe a case of severe, oligogenic neonatal thrombocytopenia and reinterpret a reportedly benign mutation that is likely pathogenic. Despite this patient's synonymous mutation (GFI1B 576 C>T, Phe192=) being annotated as benign, GFI1B is a well-known regulator of megakaryopoiesis, this variant alters splicing and megakaryocyte maturation, and our analysis of existing genome-wide associated studies demonstrates that it likely causes gray platelet syndrome. This variant has not been reported in a case of life-threatening thrombocytopenia. We propose that the severity of this patient's phenotype is due to synergistic epistasis between the intrinsic platelet defect caused by this mutation and her concomitant inherited PMM2 congenital glycosylation disorder neither of which have been associated with such a severe phenotype. This case highlights the importance of whole-exome/genome sequencing for critically ill patients, reexamining variant interpretation when clinically indicated, and the need to study diverse genetic variation in hematopoiesis.

What is the context? Low platelets (thrombocytopenia) in the neonatal population is not frequently inherited. As we perform unbiased DNA sequencing in more patients, the number of inherited platelet disorders and implicated variants is growing.The gene GFI1B encodes for a transcription factor that regulates megakaryocytes, the cell type that produces platelets. A synonymous substitution in GFI1B (576 C>T, Phe192=) is annotated as benign; however, experimental studies have shown that it inhibits megakaryocyte production.There is growing appreciation for oligogenic inheritance, where multiple causal variants contribute to clinical phenotypes.What is new? We present a case of life-threatening neonatal macrothrombocytopenia (large, hypogranulated sparse platelets) that has an oligogenic cause. We reinterpret the synonymous substitution GFI1B 576 C>T as pathogenic.This patient's severe phenotype was likely due to the combined effect of GFI1B 576 C>T and her inherited glycosylation disorder (PMM2-CDG). Neither variant alone causes severe thrombocytopenia, but the combined intrinsic platelet defect (GFI1B mutation) and consumption (PMM2-CDG) likely produced her life-threatening phenotype.What is the impact? GFI1B is a critical regulator of megakaryocyte production. The purportedly benign mutation 576 C>T is likely pathogenic causing thrombocytopenia by impairing megakaryocyte maturation.As more patients have unbiased genome sequencing, oligogenic and polygenic inheritance will become increasingly appreciated as causes of platelet disorders.NICU providers should consider whole genome or exome sequencing of neonates with severe thrombocytopenia after reversible causes are ruled out.

Intrathecal Baclofen for Hypertonia Secondary to Glutaric Aciduria Type I.

Glutaric aciduria type I (GA1) is a rare organic aciduria characterized by basal ganglia dysfunction and severe dystonia and spasticity for which enteral baclofen is currently first-line therapy. Intrathecal baclofen (ITB) is a promising alternative, given the dose titratability and concentrated delivery of medication to therapeutic targets within the central nervous system. However, the response to ITB in patients with this rare condition has not been previously reported. We present a 15-year-old girl with GA1 and associated hypertonia refractory to extensive, multimodal adjuvant medical therapy including enteral baclofen. An ITB pump was implanted, and after an appropriate baclofen titration, her hypertonia and enteral pharmacologic regimen were both reduced. We demonstrate that ITB is a viable modality for treating refractory dystonia and spasticity secondary to GA1; it can objectively reduce hypertonia, subjectively improve quality of life, and minimize the side effect profile of otherwise extensive pharmacologic therapies.

A protocol for the retina surgeon's safe initial intravitreal injections.

PURPOSE: To determine the safety of a surgeon's initial consecutive intravitreal injections using a specific protocol and to review the complications that may be attributed to the injection procedure. DESIGN: A retrospective chart review. PARTICIPANTS: Fifty-nine patients (30 females, 29 males) received intravitreal injections of pegaptanib, bevacizumab, or ranibizumab as part of their treatment for neovascular age-related macular degeneration. The average patient age was 80 years. Twenty-two patients were diagnosed with or suspected of having glaucoma. Each patient received an average of 5.8 injections. METHODS: The charts of 59 patients who received a total of 345 intravitreal injections (104 pegaptanib, 74 bevacizumab, 167 ranibizumab) were reviewed. All injections were performed in an office-based setting. Povidone-iodine, topical antibiotics, and eye speculum were used as part of the pre injection procedure. Vision and intraocular pressure were evaluated immediately following each injection. MAIN OUTCOME MEASURES: Incidence of post injection complications, including but not limited to endophthalmitis, retinal detachment, traumatic cataract, and vitreous hemorrhage. RESULTS: There were no cases of endophthalmitis, toxic reactions, traumatic cataracts, retinal detachment, or vitreous hemorrhage. There was one case each of lid swelling, transient floaters, retinal pigment epithelial tear, corneal edema, and corneal abrasion. There were five cases of transient no light perception following pegaptanib injections. CONCLUSION: The incidence of serious complications was very low for the intravitreal injections given. A surgeon's initial intravitreal injections may be performed with a very high degree of safety using this protocol.

Effect of prophylactic intraocular pressure-lowering medication on intraocular pressure spikes after intravitreal injections.

OBJECTIVE: To determine if prophylactic use of intraocular pressure (IOP)-lowering medication is effective in reducing the IOP spikes after intravitreal injections of pegaptanib, bevacizumab, and ranibizumab. METHODS: Seventy-one patients with exudative age-related macular degeneration received intravitreal injections of 1 of 3 anti-vascular endothelial growth factor medications: 30 patients received pegaptanib (0.09 mL), 47 patients received bevacizumab (0.05 mL), and 42 patients received ranibizumab (0.05 mL). Intraocular pressure-lowering medication, 1 hour prior to the injection, was used 63%, 74%, and 66% of the time in eyes that received pegaptanib, ranibizumab, and bevacizumab, respectively. Intraocular pressure was measured prior to injection, within 1 minute after injection, and every 5 to 10 minutes until the pressure was reduced to a safe level. RESULTS: All 3 intravitreal injections caused significant initial IOP spikes (mean [SD] IOP of 38.5 [11.56] mm Hg in the pegaptanib group, 37.75 [8.36] mm Hg in the ranibizumab group, and 34.88 [10.45] mm Hg in the bevacizumab group). The IOP reduced to less than 30 mm Hg in all 3 groups within 20 minutes. Prophylactic medication did not prevent postinjection IOP spikes. Patients with and without glaucoma showed a similar rate of IOP normalization over time in all 3 groups. CONCLUSION: Intraocular pressure spikes after intravitreal injection of pegaptanib, ranibizumab, and bevacizumab are common and in most cases transient. Routine prophylactic use of IOP-lowering medications is essentially ineffective in preventing IOP spikes after intravitreal injection of pegaptanib, ranibizumab, and bevacizumab and therefore not necessary before the injection.

Pharmacoeconomic analysis of prostaglandin and prostamide therapy for patients with glaucoma or ocular hypertension.

BACKGROUND: To determine monthly cost and cost effectiveness of bilateral prostaglandin/prostamide therapy for lowering intraocular pressure (IOP) in patients taking bimatoprost 0.03% (Lumigan, Allergan, Inc.), latanoprost 0.005% (Xalatan, Pfizer, Inc.), or travoprost 0.004% (Travatan, Alcon Laboratories, Inc.). METHODS: Drops in five new 2.5-mL bottles were counted and then averaged for each drug. Average retail price was determined by surveys of pharmacies. Drop count, average retail price, average wholesale price, and IOP reduction data were used to compute annual cost, and cost effectiveness (annual cost-per-mm Hg of IOP reduction) of the three drugs. RESULTS: Drops per 2.5-mL bottle averaged 113 for bimatoprost 0.03%, 84 for latanoprost 0.005%, and 83 for travoprost 0.004%. Average retail cost (2005) per bottle was $69.99 for bimatoprost 0.03%, $61.69 for latanoprost 0.005%, and $66.37 for travoprost 0.004%. The monthly retail cost of bilateral therapy was $37.92 for bimatoprost 0.03%, $44.75 for latanoprost 0.005%, and $49.25 for travoprost 0.004%. Cost effectiveness ranges were $57 to $65 per mm Hg reduction in IOP per year for bimatoprost, 0.03%, $67 to $90 per mm Hg for latanoprost 0.005%, and $74 to $84 per mm Hg for travoprost 0.004%. CONCLUSION: Bimatoprost 0.03% had the lowest monthly and annual costs and the greatest cost effectiveness for lowering IOP compared with latanoprost 0.005% and travoprost 0.004%.

Intraocular pressure effects of pegaptanib (Macugen) injections in patients with and without glaucoma.

PURPOSE: To determine if pegaptanib (Macugen) [Eyetech Pharmaceuticals; Pfizer Inc, New York, New York, USA] injections cause adverse intraocular pressure (IOP) effects in patients with and without glaucoma. DESIGN: Retrospective chart review. METHODS: Review of 75 eyes treated with intravitreal injections of pegaptanib (0.09 ml). The effect of these injections on IOP was analyzed. Most patients received prophylactic IOP-lowering medications. RESULTS: The mean preinjection IOP based on the first injection of each patient was 14 +/- 3 mm Hg; at one minute postinjection, 38 +/- 14 mm Hg, at three to 10 minutes was 34 +/- 9 mm Hg, and at 11 to 20 minutes was 26 +/-10 mm Hg, respectively. Most patients' IOPs had significantly diminished by 30 minutes postinjection. CONCLUSION: Pegaptanib injections result in a significant transient rise in IOP that considerably diminishes by 30 minutes postinjection but can take up to one hour. This may be damaging to the optic nerve, particularly in patients with advanced glaucoma.