Brendan Floyd

All Publications

• Multisite Validation of a Functional Assay to AdjudicateSCN5ABrugada Syndrome-Associated Variants. Circulation. Genomic and precision medicine Ma, J. G., O'Neill, M. J., Richardson, E., Thomson, K. L., Ingles, J., Muhammad, A., Solus, J. F., Davogustto, G., Anderson, K. C., Shoemaker, M. B., Stergachis, A. B., Floyd, B. J., Dunn, K., Parikh, V. N., ChubbW, H., Perrin, M. J., Roden, D. M., Vandenberg, J. I., Ng, C., Glazer, A. M. 2024: e004569

  Abstract

  BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and 79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification.METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was generated and independently studied at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function.RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values yielded 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic.CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

  View details for DOI 10.1161/CIRCGEN.124.004569

  View details for PubMedID 38953211

• Proactive Variant Effect Mapping Aids Diagnosis in Pediatric Cardiac Arrest. Circulation. Genomic and precision medicine Floyd, B. J., Weile, J., Kannankeril, P. J., Glazer, A. M., Reuter, C. M., MacRae, C. A., Ashley, E. A., Roden, D. M., Roth, F. P., Parikh, V. N. 2023

  View details for DOI 10.1161/CIRCGEN.122.003792

  View details for PubMedID 36716194

• Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function MOLECULAR CELL Floyd, B. J., Wilkerson, E. M., Veling, M. T., Minogue, C. E., Xia, C., Beebe, E. T., Wrobel, R. L., Cho, H., Kremer, L. S., Alston, C. L., Gromek, K. A., Dolan, B. K., Ulbrich, A., Stefely, J. A., Bohl, S. L., Werner, K. M., Jochem, A., Westphall, M. S., Rensvold, J. W., Taylor, R. W., Prokisch, H., Kim, J. P., Coon, J. J., Pagliarini, D. J. 2016; 63 (4): 621–32

  Abstract

  Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

  View details for DOI 10.1016/j.molcel.2016.06.033

  View details for Web of Science ID 000381620300010

  View details for PubMedID 27499296

  View details for PubMedCentralID PMC4992456

• Quantification of Mitochondrial Acetylation Dynamics Highlights Prominent Sites of Metabolic Regulation JOURNAL OF BIOLOGICAL CHEMISTRY Still, A. J., Floyd, B. J., Hebert, A. S., Bingman, C. A., Carson, J. J., Gunderson, D. R., Dolan, B. K., Grimsrud, P. A., Dittenhafer-Reed, K. E., Stapleton, D. S., Keller, M. P., Westphall, M. S., Denu, J. M., Attie, A. D., Coon, J. J., Pagliarini, D. J. 2013; 288 (36): 26209–19

  Abstract

  Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism. Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions.

  View details for DOI 10.1074/jbc.M113.483396

  View details for Web of Science ID 000330623300051

  View details for PubMedID 23864654

  View details for PubMedCentralID PMC3764825

• Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants. medRxiv : the preprint server for health sciences Ma, J. G., O'Neill, M. J., Richardson, E., Thomson, K. L., Ingles, J., Muhammad, A., Solus, J. F., Davogustto, G., Anderson, K. C., Benjamin Shoemaker, M., Stergachis, A. B., Floyd, B. J., Dunn, K., Parikh, V. N., Chubb, H., Perrin, M. J., Roden, D. M., Vandenberg, J. I., Ng, C. A., Glazer, A. M. 2023

  Abstract

  Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.

  View details for DOI 10.1101/2023.12.19.23299592

  View details for PubMedID 38196587

  View details for PubMedCentralID PMC10775332

• Exploring the feasibility of using long-term stored newborn dried blood spots to identify metabolic features for congenital heart disease screening. Biomarker research Ceresnak, S. R., Zhang, Y., Ling, X. B., Su, K. J., Tang, Q., Jin, B., Schilling, J., Chou, C. J., Han, Z., Floyd, B. J., Whitin, J. C., Hwa, K. Y., Sylvester, K. G., Chubb, H., Luo, R. Y., Tian, L., Cohen, H. J., McElhinney, D. B. 2023; 11 (1): 97

  Abstract

  Congenital heart disease (CHD) represents a significant contributor to both morbidity and mortality in neonates and children. There's currently no analogous dried blood spot (DBS) screening for CHD immediately after birth. This study was set to assess the feasibility of using DBS to identify reliable metabolite biomarkers with clinical relevance, with the aim to screen and classify CHD utilizing the DBS. We assembled a cohort of DBS datasets from the California Department of Public Health (CDPH) Biobank, encompassing both normal controls and three pre-defined CHD categories. A DBS-based quantitative metabolomics method was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We conducted a correlation analysis comparing the absolute quantitated metabolite concentration in DBS against the CDPH NBS records to verify the reliability of metabolic profiling. For hydrophilic and hydrophobic metabolites, we executed significant pathway and metabolite analyses respectively. Logistic and LightGBM models were established to aid in CHD discrimination and classification. Consistent and reliable quantification of metabolites were demonstrated in DBS samples stored for up to 15 years. We discerned dysregulated metabolic pathways in CHD patients, including deviations in lipid and energy metabolism, as well as oxidative stress pathways. Furthermore, we identified three metabolites and twelve metabolites as potential biomarkers for CHD assessment and subtypes classifying. This study is the first to confirm the feasibility of validating metabolite profiling results using long-term stored DBS samples. Our findings highlight the potential clinical applications of our DBS-based methods for CHD screening and subtype classification.

  View details for DOI 10.1186/s40364-023-00536-y

  View details for PubMedID 37957758

  View details for PubMedCentralID PMC10644604

• SUCCESSFUL CARDIAC TRANSPLANTATION AND LONG-TERM FOLLOW-UP IN DNAJC19-ASSOCIATED DILATED CARDIOMYOPATHY WITH ATAXIA Tahata, S., Tise, C., Floyd, B., Cusmano-Ozog, K., Ruzhnikov, M., Enns, G. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2022: 302

  View details for Web of Science ID 000772339900104

• Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function. iScience Rosier, K., McDevitt, M. T., Smet, J., Floyd, B. J., Verschoore, M., Marcaida, M. J., Bingman, C. A., Lemmens, I., Dal Peraro, M., Tavernier, J., Cravatt, B. F., Gounko, N. V., Vints, K., Monnens, Y., Bhalla, K., Aerts, L., Rashan, E. H., Vanlander, A. V., Van Coster, R., Regal, L., Pagliarini, D. J., Creemers, J. W. 2021; 24 (12): 103460

  Abstract

  Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPLL isoform localizes to mitochondria, whereas PREPLS remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPLL is involved in mitochondrial homeostasis.

  View details for DOI 10.1016/j.isci.2021.103460

  View details for PubMedID 34888501

• Aicardi-Goutières syndrome may present with positive newborn screen for X-linked adrenoleukodystrophy. American journal of medical genetics. Part A Tise, C. G., Morales, J. A., Lee, A. S., Velez-Bartolomei, F. n., Floyd, B. J., Levy, R. J., Cusmano-Ozog, K. P., Feigenbaum, A. S., Ruzhnikov, M. R., Lee, C. U., Enns, G. M. 2021

  Abstract

  We report three unrelated probands, two male and one female, diagnosed with Aicardi-Goutières syndrome (AGS) after screening positive on California newborn screening (CA NBS) for X-linked adrenoleukodystrophy (X-ALD) due to elevated C26:0 lysophosphatidylcholine (C26:0-LPC). Follow-up evaluation was notable for elevated C26:0, C26:1, and C26:0/C22:0 ratio, and normal red blood cell plasmalogens levels in all three probands. Diagnoses were confirmed by molecular sequencing prior to 12 months of age after clinical evaluation was inconsistent with X-ALD or suggestive of AGS. For at least one proband, the early diagnosis of AGS enabled candidacy for enrollment into a therapeutic clinical trial. This report demonstrates the importance of including AGS on the differential diagnosis for individuals who screen positive for X-ALD, particularly infants with abnormal neurological features, as this age of onset would be highly unusual for X-ALD. While AGS is not included on the Recommended Universal Screening Panel, affected individuals can be identified early through state NBS programs so long as providers are aware of a broader differential that includes AGS. This report is timely, as state NBS algorithms for X-ALD are actively being established, implemented, and refined.

  View details for DOI 10.1002/ajmg.a.62160

  View details for PubMedID 33683010

• Systems Biochemistry Approaches to Defining Mitochondrial Protein Function. Cell metabolism Sung, A. Y., Floyd, B. J., Pagliarini, D. J. 2020; 31 (4): 669–78

  Abstract

  Defining functions for the full complement of proteins is a grand challenge in the post-genomic era and is essential for our understanding of basic biology and disease pathogenesis. In recent times, this endeavor has benefitted from a combination of modern large-scale and classical reductionist approaches-a process we refer to as "systems biochemistry"-that helps surmount traditional barriers to the characterization of poorly understood proteins. This strategy is proving to be particularly effective for mitochondria, whose well-defined proteome has enabled comprehensive analyses of the full mitochondrial system that can position understudied proteins for fruitful mechanistic investigations. Recent systems biochemistry approaches have accelerated the identification of new disease-related mitochondrial proteins and of long-sought "missing" proteins that fulfill key functions. Collectively, these studies are moving us toward a more complete understanding of mitochondrial activities and providing a molecular framework for the investigation of mitochondrial pathogenesis.

  View details for DOI 10.1016/j.cmet.2020.03.011

  View details for PubMedID 32268114

• Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity MOLECULAR CELL Stefely, J. A., Licitra, F., Laredj, L., Reidenbach, A. G., Kemmerer, Z. A., Grangeray, A., Jaeg-Ehret, T., Minogue, C. E., Ulbrich, A., Hutchins, P. D., Wilkerson, E. M., Ruan, Z., Aydin, D., Hebert, A. S., Guo, X., Freiberger, E. C., Reutenauer, L., Jochem, A., Chergova, M., Johnson, I. E., Lohman, D. C., Rush, M. P., Kwiecien, N. W., Singh, P. K., Schlagowski, A. I., Floyd, B. J., Forsman, U., Sindelar, P. J., Westphall, M. S., Pierrel, F., Zoll, J., Dal Peraro, M., Kannan, N., Bingman, C. A., Coon, J. J., Isope, P., Puccio, H., Pagliarini, D. J. 2016; 63 (4): 608–20

  Abstract

  The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

  View details for DOI 10.1016/j.molcel.2016.06.030

  View details for Web of Science ID 000381620300009

  View details for PubMedID 27499294

  View details for PubMedCentralID PMC5012427

• Mitochondrial ADCK3 Employs an Atypical Protein Kinase-like Fold to Enable Coenzyme Q Biosynthesis MOLECULAR CELL Stefely, J. A., Reidenbach, A. G., Ulbrich, A., Oruganty, K., Floyd, B. J., Jochem, A., Saunders, J. M., Johnson, I. E., Minogue, C. E., Wrobel, R. L., Barber, G. E., Lee, D., Li, S., Kannan, N., Coon, J. J., Bingman, C. A., Pagliarini, D. J. 2015; 57 (1): 83–94

  Abstract

  The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

  View details for DOI 10.1016/j.molcel.2014.11.002

  View details for Web of Science ID 000347711100008

  View details for PubMedID 25498144

  View details for PubMedCentralID PMC4289473

• SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed beta cells JOURNAL OF CLINICAL INVESTIGATION Kebede, M. A., Oler, A. T., Gregg, T., Balloon, A. J., Johnson, A., Mitok, K., Rabaglia, M., Schueler, K., Stapleton, D., Thorstenson, C., Wrighton, L., Floyd, B. J., Richards, O., Raines, S., Eliceiri, K., Seidah, N. G., Rhodes, C., Keller, M. P., Coon, J. L., Audhya, A., Attie, A. D. 2014; 124 (10): 4240–56

  Abstract

  We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin(ob) mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.

  View details for DOI 10.1172/JCI74072

  View details for Web of Science ID 000342649900022

  View details for PubMedID 25157818

  View details for PubMedCentralID PMC4191024