Epilepsy panels in clinical practice: Yield, variants of uncertain significance, and treatment implications.

OBJECTIVE: There has been increasing utilization of genetic testing for pediatric epilepsy in recent years. Little systematic data is available examining how practice changes have impacted testing yields, diagnostic pace, incidence of variants of uncertain significance (VUSs), or therapeutic management. METHODS: A retrospective chart review was performed at Children's Hospital Colorado from February 2016 through February 2020. All patients under 18 years for whom an epilepsy gene panel was sent were included. RESULTS: A total of 761 epilepsy gene panels were sent over the study period. During the study period, there was a 292% increase in the average number of panels sent per month. The time from seizure onset to panel result decreased over the study period from a median of 2.9 years to 0.7 years. Despite the increase in testing, the percentage of panels yielding a disease-causing result remained stable at 11-13%. A total of 90 disease-causing results were identified, > 75% of which provided guidance in management. Children were more likely to have a disease-causing result if they were < 3 years old at seizure onset (OR 4.4, p < 0.001), had neurodevelopmental concerns (OR 2.2, p = 0.002), or had a developmentally abnormal MRI (OR 3.8, p < 0.001). A total of 1417 VUSs were identified, equating to 15.7 VUSs per disease-causing result. Non-Hispanic white patients had a lower average number of VUSs than patients of all other races/ethnicities (1.7 vs 2.1, p < 0.001). SIGNIFICANCE: Expansion in the volume of genetic testing corresponded to a decrease in the time from seizure onset to testing result. Diagnostic yield remained stable, resulting in an increase in the absolute number of disease-causing results annually-most of which have implications for management. However, there has also been an increase in total VUSs, which likely resulted in additional clinical time spent on VUS resolution.

N-terminus DUX4-immunohistochemistry is a reliable methodology for the diagnosis of DUX4-fused B-lymphoblastic leukemia/lymphoma (N-terminus DUX4 IHC for DUX4-fused B-ALL).

B-lymphoblastic leukemia/lymphoma (B-ALL) is the most common pediatric malignancy and the most commonly diagnosed adult lymphoblastic leukemia. Recent advances have broadened the spectrum of B-ALL, with DUX4 gene fusions implicated in a subclass occurring in adolescents and young adults and harboring a favorable prognosis. DUX4 fusions have been challenging to identify. We aimed to determine whether expression of the DUX4 oncoprotein, as detected by targeted immunohistochemistry, might serve as a surrogate for molecular detection of DUX4 fusions in B-ALL. A cohort of investigational B-ALLs was generated with enrichment for DUX4 fusions by the inclusion of cases with characteristic demographic features and immunophenotypic properties. B-ALLs with mutually exclusive cytogenetics were collected. Immunohistochemical staining by a monoclonal antibody raised against the N-terminus of the DUX4 protein was performed. N-DUX4 immunohistochemistry demonstrated strong, crisp nuclear staining in blasts of seven investigational cases, six of which had nucleic acid material available for molecular evaluation. Five of these cases demonstrated RNA-seq DUX4-fusion positivity. One N-DUX4 immunohistochemistry positive case lacked a definitive DUX4-fusion by RNA-seq, though demonstrated a gene expression profile characteristic of DUX4-rearranged B-ALLs, a CD2+ immunophenotype, and a lack of staining by C-terminus DUX4 antibody immunohistochemistry. At least 83.3% [5/6] positive predictive value. N-DUX4 immunohistochemistry was negative in blasts of three RNA-seq DUX4-fusion-negative cases (3/3; 100% negative predictive value). B-ALLs with mutually exclusive cytogenetic profiles were all N-DUX4 negative (0/10, specificity 100%). N-DUX4 immunohistochemistry is reliable for the distinction of DUX4-rearranged B-ALLs from other B-ALLs. We recommend its use for subclassification of B-ALLs in adolescents and young adults and in B-ALLs that remain "not otherwise specified."

Efficacy of intermittent combined RAF and MEK inhibition in a patient with concurrent BRAF- and NRAS-mutant malignancies.

Vemurafenib, a RAF inhibitor, extends survival in patients with BRAF(V600)-mutant melanoma but activates extracellular signal-regulated kinase (ERK) signaling in RAS-mutant cells. In a patient with a BRAF(V600K)-mutant melanoma responding to vemurafenib, we observed accelerated progression of a previously unrecognized NRAS-mutant leukemia. We hypothesized that combining vemurafenib with a MAP-ERK kinase (MEK) inhibitor would inhibit ERK activation in the melanoma and prevent ERK activation by vemurafenib in the leukemia, and thus suppress both malignancies. We demonstrate that intermittent administration of vemurafenib led to a near-complete remission of the melanoma, and the addition of the MEK inhibitor cobimetinib (GDC-0973) caused suppression of vemurafenib-induced leukemic proliferation and ERK activation. Antimelanoma and antileukemia responses have been maintained for nearly 20 months, as documented by serial measurements of tumor-derived DNA in plasma in addition to conventional radiographic and clinical assessments of response. These data support testing of intermittent ERK pathway inhibition in the therapy for both RAS-mutant leukemia and BRAF-mutant melanoma.

Multi-tier regulation of the streptomycete morphogenetic peptide SapB.

Streptomyces coelicolor is a morphologically complex bacterium requiring the secretion of surface-active proteins to progress through its life cycle. SapB represents an important class of these biosurfactants, as illustrated by its ability to restore aerial hyphae formation when applied exogenously to developmental mutants. However, such aerial hyphae fail to sporulate, exemplifying the need to co-ordinate the timing of SapB production with other developmental events. SapB has an unusual lantibiotic structure. Its structural gene, ramS, is only 38 nucleotides downstream of the gene encoding its putative modification enzyme, RamC. Transient, co-ordinated expression of the operon was thought to be controlled by the response regulator RamR. However, we show that ramS is transcribed throughout the cell cycle with a dual expression profile dissimilar to the tightly controlled ramC expression. Surprisingly, post-translational modification relies on prior membrane localization of the precursor peptide, RamS, as demonstrated by the absence of RamS modification in S. coelicolor hyphae treated with the Bacillus subtilis lipoprotein surfactin. Our results demonstrate that interspecies interaction can also be mediated by interference of post-translational events. Further, temporal and spatial regulation of irreversible post-translational modification of a surface-active morphogenetic peptide suggests a new model for the control of key developmental events.

Signal perception by FNR: the role of the iron-sulfur cluster.

The metabolic flexibility of bacteria is key to their ability to survive and thrive in a wide range of environments. Optimal switching from one metabolic pathway to another is a key requirement for this flexibility. Respiration is a good example: many bacteria utilize O(2) as the terminal electron acceptor, but can switch to a range of other acceptors, such as nitrate, when O(2) becomes limiting. Sensing environmental levels of O(2) is the key step in switching from aerobic to anaerobic respiration. In Escherichia coli, the fumarate and nitrate reduction transcriptional regulator (FNR) controls this switch. Under O(2)-limiting conditions, FNR binds a [4Fe-4S](2+) cluster, generating a transcriptionally active dimeric form. Exposure to O(2) results in conversion of the cluster into a [2Fe-2S](2+) form, leading to dissociation of the protein into inactive monomers. The mechanism of cluster conversion, together with the nature of the reaction products, is of considerable current interest, and a near-complete description of the process has now emerged. The [4Fe-4S](2+) into [2Fe-2S](2+) cluster conversion proceeds via a two-step mechanism. In step 1, a one-electron oxidation of the cluster takes place, resulting in the release of a Fe(2+) ion, the formation of an intermediate [3Fe-4S](1+) cluster, together with the generation of a superoxide anion. In step 2, the intermediate [3Fe-4S](1+) cluster rearranges spontaneously to form the [2Fe-2S](2+) cluster, releasing two sulfide ions and an Fe(3+) ion in the process. The one-electron activation of the cluster, coupled to catalytic recycling of the superoxide anion back to oxygen via superoxide dismutase and catalase, provides a novel means of amplifying the sensitivity of [4Fe-4S](2+) FNR to its signal molecule.

Influence of the environment on the [4Fe-4S]2+ to [2Fe-2S]2+ cluster switch in the transcriptional regulator FNR.

In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by the fumarate and nitrate reduction transcriptional regulator FNR. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in the conversion of the cluster to a [2Fe-2S]2+ form, leading to dissociation of the protein into transcriptionally inactive monomers. The [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion proceeds in two steps. Step 1 involves the one-electron oxidation of the cluster, resulting in the release of Fe2+, generating a [3Fe-4S]1+ cluster intermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, in both native and reconstituted [4Fe-4S] FNR, the reaction environment and, in particular, the presence of Fe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstrate that while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced by both Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 is enhanced to the extent that step 1 becomes the rate determining step and the [3Fe-4S]1+ intermediate is no longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presence of Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reaction in the cell cytoplasm, provides an explanation for apparently contradictory results reported from different laboratories.

RsmA is an anti-sigma factor that modulates its activity through a [2Fe-2S] cluster cofactor.

The rsmA gene of Streptomyces coelicolor lies directly upstream of the gene encoding the group 3 sigma factor sigma(M). The RsmA protein is a putative member of the HATPase_c family of anti-sigma factors but is unusual in that it contains seven cysteine residues. Bacterial two-hybrid studies demonstrate that it interacts specifically with sigma(M), and in vitro studies of the purified proteins by native PAGE and transcription assays confirmed that they form a complex. Characterization of RsmA revealed that it binds ATP and that, as isolated, it contains significant quantities of iron and inorganic sulfide, in equal proportion, with spectroscopic properties characteristic of a [2Fe-2S] cluster-containing protein. Importantly, the interaction between RsmA and sigma(M) is dependent on the presence of the iron-sulfur cluster. We propose a model in which RsmA regulates the activity of sigma(M). Loss of the cluster, in response to an as yet unidentified signal, activates sigma(M) by abolishing its interaction with the anti-sigma factor. This represents a major extension of the functional diversity of iron-sulfur cluster proteins.