The current landscape of genetic test stewardship: A multi-center prospective study.

Genetic counselors (GCs) play a pivotal role in selecting clinically appropriate and cost-effective genetic testing. Several single-institution reports over the past decade provide evidence of the value GCs bring to this stewardship role across diverse settings in healthcare, including hospital laboratories, commercial laboratories, and insurance companies. This multi-center, prospective, and quantitative study describes the outcomes of GC review of genetic test requests over a four-week period at six hospital laboratories and three commercial laboratories, thus expanding our understanding of this emerging specialty in the genetic counseling field. This study also highlights the added value of utilizing GC expertise in stewardship efforts, namely selecting the most appropriate genetic testing and realizing significant cost savings. GC review of genetic test requests led to an average order modification rate of 22%-25%. It also resulted in significant cost savings to institutions. The projected average annual savings after GC review of genetic test requests approximated $665,600 for hospital laboratories and $1,651,000 for commercial laboratories. These study findings demonstrate the significant value of GC-led genetic test stewardship programs, allow for comparisons across institutions currently performing genetic test stewardship, and support the implementation of a GC-led stewardship program at institutions who currently do not have one.

Characterization of a novel pathogenic variant in the FECH gene associated with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an autosomal recessive deficiency in heme biosynthesis due to pathogenic variants in the ferrochelatase gene (FECH). Patients present with lifelong photosensitivity and potential liver disease. Here we report a novel FECH variant designated c.904_912+1del found in trans with the c.315-48T>C hypomorphic variant, in one family with three affected individuals. These patients presented with immediate painful cutaneous photosensitivity but no hepatic manifestations. All have elevated protoporphyrin levels consistent with a diagnosis of EPP. Genetic, biochemical, and functional assay results obtained for this family suggest that the unique variant c.904_912+1del is likely pathogenic and thus causative of EPP.

Assessing energetic contributions to binding from a disordered region in a protein-protein interaction .

Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction.

Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.

Dissecting cooperative and additive binding energetics in the affinity maturation pathway of a protein-protein interface.

When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor Vbeta domain that exhibit an overall affinity range of approximately 1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the Vbeta domain, exhibit negative cooperativity.