Structural or electrophysiologic cardiac anomalies may compromise cardiac function, leading to sudden cardiac death (SCD). Genetic screening of families with severe cardiomyopathies underlines the role of genetic variations in cardiac-specific genes. The present study details the clinical and genetic characterization of a malignant dilated cardiomyopathy (DCM) case in a 1-year-old Mexican child who presented a severe left ventricular dilation and dysfunction that led to SCD. A total of 132 genes (48 structure- and 84 electrical-related genes) were examined by next generation sequencing to identify potential causative mutations in comparison to control population. In silico analysis identified only two deleterious heterozygous mutations within an evolutionarily well-conserved region of the sarcomeric genes ACTC1/cardiac actin (c.664G > A/p.Ala222Thr) and TTN/titin (c.33250G > A/p.Glu11084Lys). Further pedigree analysis revealed the father of the index case to carry with the TTN mutation. Surprisingly, the ACTC1 mutation was not harbored by any first-degree family member. Computational 3D modeling of the mutated proteins showed electrostatic and conformational shifts of cardiac actin compared to wild-type version, as well as changes in the stability of the compact/folded states of titin that normally contributes to avoid mechanic damage. In conclusion, our findings suggest a likely pathogenic de novo mutation in ACTC1 in coexpression of a TTN variant as possible causes of an early onset of a severe DCM and premature death. These results may increase the known clinical pathogenic variations that may critically alter the structure of the heart, whose fatality could be prevented when rapidly detected.

G-protein coupled receptors (GPCRs) are eukaryotic integral membrane proteins that regulate signal transduction cascade pathways implicated in a variety of human diseases and are consequently of interest as drug targets. For this reason, it is of interest to investigate the way in which specific ligands bind and trigger conformational changes in the receptor during activation and how this in turn modulates intracellular signaling. In the present study, we investigate the way in which the ligand Prostaglandin E2 interacts with three GPCRs in the E-prostanoid family: EP1, EP2, and EP3. We examine information transfer pathways based on long-time scale molecular dynamics simulations using transfer entropy and betweenness centrality to measure the physical transfer of information among residues in the system. We monitor specific residues involved in binding to the ligand and investigate how the information transfer behavior of these residues changes upon ligand binding. Our results provide key insights that enable a deeper understanding of EP activation and signal transduction functioning pathways at the molecular level, as well as enabling us to make some predictions about the activation pathway for the EP1 receptor, for which little structural information is currently available. Our results should advance ongoing efforts in the development of potential therapeutics targeting these receptors.

The ability to predict allosteric sites of protein activity is of great theoretical and practical interest in biophysics and medicinal biochemistry and plays an important role in guiding mutation analysis and drug design. Current methods emphasize the protein as a network or graph model and use probabilistic approaches to predict allosteric paths between pre-identified sites. Information theoretic measures offer a more robust framework for identifying important sites and revealing paths or relationships among them with little knowledge required or imposed on the system in advance. We have developed a method for calculating the transfer entropy in biomolecular systems from the covariance matrix of atomic fluctuations derived from molecular dynamics simulations. This approach retains full sensitivity to the amino acid composition of the protein (as opposed to coarse-grained network models) but is substantially more computationally efficient than other information theoretic approaches. We demonstrate that our approach gives good agreement with other computational methods and with experiment for ERK2 kinase, and we show that our method predicts a novel allosteric region of the kinase that helps to explain recent experimental data. Our method enables determination of information flow pathways within the system that change with the state of the system, providing insight into important residues and channels of communication. We present applications and extensions of our method, highlighting theoretical and technical challenges with convergence and timescale limitations.

ERK2 is a kinase in the MAPK/ERK pathway, which is involved in several cellular functions such as cell survival, proliferation, and differentiation. Disruption of this pathway is known to lead to the development of cancer. We performed molecular dynamics simulations of structures of unphosphorylated (inactive) ERK2 with and without the inhibitory drug FR180204, as well as simulations of phosphorylated (active) ERK2 with and without the inhibitory drug Vertex-11e. We found that both inhibitory drugs had the same effects on the driving behavior of the N-terminal region, activation lip, and part of the L16 region.

Transfer entropy is an asymmetric measure of correlation describing how much information one time series provides in the prediction of the future values of another time series. This relationship allows us to quantitatively assess three primary measures in protein-DNA systems: influential residues that drive intramolecular interactions in protein-DNA complexes, the strength of different types of interactions, and how the interactions from these significant residues impact the overall stability of the protein-DNA complex.

Given recent emphasis on non-local or large-scale effects (e.g. DNA flexibility, the role of water and ions, the biophysics of the major and minor grooves, etc), it is of interest to ask to what extent the (relatively small) local interactions (e.g. individual hydrogen bonds) contribute to the overall stability of the complex. These specific interactions were visualized and analyzed through molecular surfaces using protein-DNA interactions of the lac repressor headpiece assessed from PDB codes 1L1M, 2BJC, and 1CJG in the program Flare.

Transfer entropy methods provide an approach to understanding asymmetric information flow in coupled systems. In biomolecular systems, transfer entropy can be useful for identification of “driving” and “responding” residues as well as pathways or networks of residues that are coupled in their information flow. Unfortunately, most methods for calculating transfer entropy require very long simulations and almost equally long calculations of joint probability histograms to compute the information transfer. Available approximate methods based on graph/network theory approaches are rapid but lose sensitivity to the chemical nature of the biomolecules and thus are not applicable in mutation studies. We show that reliable estimates of the transfer entropy can be obtained from the variance-covariance matrix of atomic fluctuations, which converges quickly and retains sensitivity to the full chemical profile of the biomolecular system. We validate our method on ERK2, a well-studied kinase involved in the MAPK signaling cascade for which considerable computational, experimental, and mutation data are available. We present the results of transfer entropy analysis on data obtained from molecular dynamics simulations of wild type active and inactive ERK2, along with mutants Q103A, I84A, L73P, and G83A. We show that our method is consistent with the results of computational and experimental studies on ERK2, and we present a method for interpreting networks of interconnected residues in the protein from a perspective of allosteric coupling. Of particular interest are new insights gained about possible allosteric activity of the extreme N-terminal region of the kinase, which to date has been under-explored in the literature. Our results highlight the advantages and disadvantages of various methods for calculating transfer entropy and show the important role of transfer entropy analysis for understanding allosteric behavior in biomolecular systems.

Transfer entropy methods provide an approach to understanding asymmetric information flow in coupled systems, with particular application to understanding allosteric interactions in biomolecular systems. Transfer entropy analysis holds the potential to reveal pathways or networks of residues that are coupled in their information flow and thus give new insights into folding and binding dynamics. Most current methods for calculating transfer entropy require very long simulations and almost equally long calculations of joint probability histograms to compute the information transfer that make these methods either functionally intractable or statistically unreliable. Available approximate methods based on graph and network theory approaches are rapid but lose sensitivity to the chemical nature of the biomolecules and thus are not applicable in mutation studies. We show that reliable estimates of the transfer entropy can be obtained from the variance-covariance matrix of atomic fluctuations, which converges quickly and retains sensitivity to the full chemical profile of the biomolecular system. We validate our method on ERK2, a well-studied kinase involved in the MAPK signaling cascade for which considerable computational, experimental, and mutation data are available. We present the results of transfer entropy analysis on data obtained from molecular dynamics simulations of wild type active and inactive ERK2, along with mutants Q103A, I84A, L73P, and G83A. We show that our method is consistent with the results of computational and experimental studies on ERK2, and we provide a method for interpreting networks of interconnected residues in the protein from a perspective of allosteric coupling. We introduce new insights about possible allosteric activity of the extreme N-terminal region of the kinase, which to date has been under-explored in the literature and may provide an important new direction for kinase studies. We also describe evidence that suggests activation may occur by different paths or routes in different mutants. Our results highlight systematic advantages and disadvantages of each method for calculating transfer entropy and show the important role of transfer entropy analysis for understanding allosteric behavior in biomolecular systems.

HomeCirculation: Genomic and Precision MedicineVol. 14, No. 5MYH7 p.Glu903Gln Is a Pathogenic Variant Associated With Hypertrophic Cardiomyopathy Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBMYH7 p.Glu903Gln Is a Pathogenic Variant Associated With Hypertrophic Cardiomyopathy Glauber M. Dias, PhD, Arsonval Lamounier Júnior, MD, MSc, Maila Seifert, MD, MSc, Hector Barájas-Martinez, PhD, Daniel Barr, PhD, Eduardo B. Sternick, MD, PhD, Enrique Medina-Acosta, PhD, Antônio C. Campos de Carvalho, MD, PhD and Fernando E.S. Cruz Filho, MD, PhD Glauber M. DiasGlauber M. Dias Correspondence to: Glauber M. Dias, PhD, Laboratório de Biologia Celular e Tecidual - LBCT, Centro de Biociências e Biotecnologia - CBB, Avenida Alberto Lamego, 2000 - Parque Califórnia, Campos dos Goytacazes, RJ – Brazil. Email E-mail Address: [email protected] https://orcid.org/0000-0002-7221-1515 Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil (G.M.D., M.S., A.C.C.d.C., F.E.S.C.F.). Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brazil (G.M.D., E.M.-A.). , Arsonval Lamounier JúniorArsonval Lamounier Júnior Health in Code SL, A Coruña, Spain (A.L.J.). Universidade da Coruña, A Coruña, Spain (A.L.J.). , Maila SeifertMaila Seifert https://orcid.org/0000-0003-3799-5315 Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil (G.M.D., M.S., A.C.C.d.C., F.E.S.C.F.). , Hector Barájas-MartinezHector Barájas-Martinez https://orcid.org/0000-0002-2704-3807 Lankenau Institute for Medical Research, Philadelphia (H.B.-M.). , Daniel BarrDaniel Barr https://orcid.org/0000-0003-4660-5649 Department of Chemistry, University of Mary, Bismarck (D.B.). , Eduardo B. SternickEduardo B. Sternick https://orcid.org/0000-0003-3249-3711 Biocor Instituto, Belo Horizonte, Brazil (E.B.S.). , Enrique Medina-AcostaEnrique Medina-Acosta https://orcid.org/0000-0002-2529-0548 Universidade Estadual do Norte Fluminense Darcy Ribeiro, Brazil (G.M.D., E.M.-A.). , Antônio C. Campos de CarvalhoAntônio C. Campos de Carvalho https://orcid.org/0000-0002-0062-3043 Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil (G.M.D., M.S., A.C.C.d.C., F.E.S.C.F.). Universidade Federal do Rio de Janeiro, Brazil (A.C.C.d.C.). Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa, Brazil (A.C.C.d.C.). and Fernando E.S. Cruz FilhoFernando E.S. Cruz Filho Instituto Nacional de Cardiologia, Rio de Janeiro, Brazil (G.M.D., M.S., A.C.C.d.C., F.E.S.C.F.). Originally published24 Sep 2021https://doi.org/10.1161/CIRCGEN.121.003476Circulation: Genomic and Precision Medicine. 2021;14:e003476Pathogenic variants in the MYH7 gene, encoding the beta-myosin heavy chain protein, constitute the leading cause among genotype-positive hypertrophic cardiomyopathy (HCM) patients.1 Clinical information about most of these variants is scarce, creating barriers in the clinical interpretation of the genetic findings. Around 1560 missense-type variants in MYH7 are listed in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar), with 260 disease-causing variants and 1300 as of uncertain significance or with conflicting interpretations on their pathogenicity, despite the specific pathogenicity criteria for this gene.2Here, we screened an HCM cohort with 134 consecutive unrelated probands from a quaternary health care center in Rio de Janeiro, Brazil, with a target-next-generation sequencing panel of 53 cardiomyopathies genes. All patients gave written informed consent, and the study was approved by the regional research ethics committee (CAAE 44740415.7.0000.5272). We identified 6 probands carrying the MYH7 p.Glu903Gln missense variant (NM_000257.4:c.2707G>C) and analyzed the genotype-phenotype correlation and the variant’s pathogenicity. Clinical records were reviewed and pedigrees constructed upon family screening by the Sanger method. This change and 3 other missense variants affecting the same amino acid (p.Glu903Lys/Gly/Asp) are described as variant of uncertain significance in ClinVar.Thirty-two affected individuals (those with left ventricle hypertrophy, according to the guidelines criteria,1,3 or with major cardiovascular outcomes: heart failure, heart failure-related death, sudden cardiac death, and unspecified cardiac death) were identified. From those 24 were MYH7 p.Glu903Gln variant carriers, and 8 cardiac-related deaths could not be tested. The mean follow-up period was 6.5±9.1 years, and the mean diagnosis age was 34.6±17 years. Clinical features of the subjects are summarized in Figure [A]. No MYH7 p.Glu903Gln genotype negative individuals presented disease signs. Familial co-segregation of this variant with the phenotype was documented (combined logarithm of the odds score=5.7)4 (Figure [B]).Download figureDownload PowerPointFigure. MYH7 p.Glu903Gln: Clinical features, pedigrees and 3D-modeling.A, MYH7 p.Glu903Gln clinical features. [*] Affected individuals include those with left ventricle hypertrophy, heart failure (HF), HF-related death, sudden cardiac death (SCD), and unspecified cardiac death (UCD). [†] 46 individuals: 32 affected, 8 unaffected carriers, and 6 unknown phenotype carriers. B, MYH7 p.Glu903Gln Pedigrees. a-f: The 6 pedigrees reported in this study. Square: male individual; circle: female individual; arrow: proband; white symbols: unknown phenotype (not clinically evaluated); N: Normal individuals (unaffected); black symbols: affected individuals; (−/+) MYH7 p.Glu903Gln simple heterozygous carrier; (−/−) MYH7 p.Glu903Gln noncarrier; +(o) MYH7 p.Glu903Gln obligate carrier; (+) presumed carrier; number inside symbols: number of individuals by gender. A minus sign (−) after the cause of death in affected individuals identifies subjects without echocardiography evaluation. All other affected individuals presented echo signs of cardiomyopathy. [**] Unspecified cardiac deaths (UCD) were considered those without previous established diagnosed or postmortem autopsy, reported by the families as heart attack or cardiac disease at any age, which the reports did not suggest a sudden presentation. C, Three-dimensional Modeling of the human beta-myosin S2 fragment. Structures A and B show the electrostatic surfaces of the S2 protein chain visualized by APBS for the wild type and p.Glu903Gln mutant. C and D show the protein electric fields visualized in Flare Version 4.0.0 (Cresset, Litlington, Cambridgeshire, United Kingdom; http://www.cresset-group.com/flare/) for the 3 negatively charged rings at the end of the S2 chain. In all cases, black circles indicate regions where significant electron density has been lost because of the p.Glu903Gln variant. HCM indicates hypertrophic cardiomyopathy; ICD, implantable cardioverter-defibrillator; LGE, late gadolinium enhancement; LVOTO, left ventricular outflow tract obstruction; MLVW, maximal left ventricular wall thickness; and VT/VF, ventricular tachycardia/ventricular fibrillation.Earlier disease onset was observed in male carriers (mean age 16±11.9 years) than in females (41±13.2 years; P<0.001—Mann-Whitney test). The Kaplan-Meier survival analysis including carriers and first-degree relatives without genetic testing, but reported with major cardiovascular outcomes, showed a worse prognosis in male than female carriers (30% versus 10% of mortality at 40 years, respectively; P=0.05). Gender differences in the age of diagnosis and survival function were described in previous unselect HCM cohorts5; however, it seems to be more marked here. All probands and one relative had high sudden cardiac death risk scores, considering the European and North American HCM guidelines.1,3Three families carrying additional genetic variants in SCN5A (p.Arg800Cys) and MYBPC3 (p.Leu455Phe and p.Arg17Gln) were also identified (Figure [B]); all classified as variant of uncertain significance. Familial co-segregation analysis of these variants supports the view that they were not the cause of HCM. In the pedigree with the SCN5A variant, no electrocardiographic abnormalities (eg, QTc prolongation or Brugada syndrome patterns) suggesting sodium channel involvement were observed. In family (d), a more pronounced phenotype was described in the proband carrying the additional genetic variant, MYBPC3 p.Leu455Phe; however, his 37-year-old mother, also a carrier of both variants, had a mild phenotype.MYH7 p.Glu903Gln is absent in individuals from a large international sequence database (gnomAD database; https://gnomad.broadinstitute.org), and all in silico predictors are concordant with having a deleterious effect. The p.Glu903Gln variant is located within a known functional domain (amino acids 181-937) that shows a statistically significant clustering of pathogenic variants in HCM.4 Three-dimensional modeling of the MYH7 protein structure with S2 fragment (amino acids 838-1371) mutated by p.Glu903Gln variant and the wild-type and protein fields were evaluated in Flare (http://www.cresset-group.com/flare), PDB2PQR, and APBS software (https://www.poissonboltzmann.org). The Glu903 provides a significant component of the negative electrostatic field in the Ring 1 region of the S2 domain, and upon the variant, this region shows a loss of negative charge, leading to a significant shrinking of Ring 1, compared with the wild type (Figure [C]). This result suggests a deleterious effect of the variant over the protein structure.The high prevalence of the MYH7 p.Glu903Gln in our HCM cohort prompted us to reconstruct the most likely common extended haplotype (CTCTGCTGGTTGGGTAGA [upper strand], where G is the rs730880756 pathogenic allele) across the MYH7 gene using the genotypes of 18 single nucleotide variant sites spanning the pathogenic site and the 1000 Genomes Project Phase 3 data (https://www.internationalgenome.org/category/phase-3). The spanned chromosomal region has the characteristics of a robust haplotype block (linkage disequilibrium r2 >0.8), suggesting the hypothesis of a founder variant in the study cohort.The described allele variant meets the PS4, PP1_Strong, PM1, PM2, and PP3 adapted pathogenicity criteria of the American College of Medical Genetics and Genomics/Association for Molecular Pathology4 that support its association with HCM phenotype. Accordingly, the classification of other missense variants in the same amino acid (p.Glu903Lys/Gly/Asp) was updated to likely pathogenic. Additional investigations could be helpful to explore the hypothesis of a founder variant and a better clinical characterization. This description can contribute to the clinical interpretation of the genetic findings and precision medicine in HCM.Study LimitationsA uniform clinical evaluation was not performed across all the subjects of this study, and there were individuals not clinically evaluated in the pedigrees. There was no molecular or clinical diagnosis of the individuals who suddenly died.AcknowledgmentsWe are grateful to all participants in this study.Sources of FundingThis study was supported by grants from Brazilian research agencies Fundação Pró-Coração - FUNDACOR, FAPERJ, DECIT/MS, and Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq.Disclosures None.FootnotesFor Sources of Funding and Disclosures, see page 664.Correspondence to: Glauber M. Dias, PhD, Laboratório de Biologia Celular e Tecidual - LBCT, Centro de Biociências e Biotecnologia - CBB, Avenida Alberto Lamego, 2000 - Parque Califórnia, Campos dos Goytacazes, RJ – Brazil. Email [email protected]brReferences1. Elliott PM, Anastasakis A, Borger MA, Borggrefe M, Cecchi F, Charron P, Hagege AA, Lafont A, Limongelli G, Mahrholdt H, et al.; Authors/Task Force Members.2014 ESC Guidelines on diagnosis and management of hypertrophic cardiomyopathy: the Task Force for the Diagnosis And Management Of Hypertrophic Cardiomyopathy of the European Society of Cardiology (ESC).Eur Heart J. 2014; 35:2733–2779. doi: 10.1093/eurheartj/ehu284CrossrefMedlineGoogle Scholar2. Mattivi CL, Bos JM, Bagnall RD, Nowak N, Giudicessi JR, Ommen SR, Semsarian C, Ackerman MJ. Clinical utility of a phenotype-enhanced MYH7-specific variant classification framework in hypertrophic cardiomyopathy genetic testing.Circ Genom Precis Med. 2020; 13:453–459. doi: 10.1161/CIRCGEN.120.003039LinkGoogle Scholar3. Gersh BJ, Maron BJ, Bonow RO, Dearani JA, Fifer MA, Link MS, Naidu SS, Nishimura RA, Ommen SR, Rakowski H, et al.; American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines; American Association for Thoracic Surgery; American Society of Echocardiography; American Society of Nuclear Cardiology; Heart Failure Society of America; Heart Rhythm Society; Society for Cardiovascular Angiography and Interventions; Society of Thoracic Surgeons.2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines.Circulation. 2011; 124:2761–2796. doi: 10.1161/CIR.0b013e318223e230LinkGoogle Scholar4. Kelly MA, Caleshu C, Morales A, Buchan J, Wolf Z, Harrison SM, Cook S, Dillon MW, Garcia J, Haverfield E, et al.. Adaptation and validation of the ACMG/AMP variant classification framework for MYH7-associated inherited cardiomyopathies: recommendations by ClinGen’s Inherited Cardiomyopathy Expert Panel.Genet Med. 2018; 20:351–359. doi: 10.1038/gim.2017.218CrossrefMedlineGoogle Scholar5. Lakdawala NK, Olivotto I, Day SM, Han L, Ashley EA, Michels M, Ingles J, Semsarian C, Jacoby D, Jefferies JL, et al.. Associations between female sex, sarcomere variants, and clinical outcomes in hypertrophic cardiomyopathy.Circ Genom Precis Med. 2021; 14:e003062. doi: 10.1161/CIRCGEN.120.003062LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails October 2021Vol 14, Issue 5Article InformationMetrics Download: 99 © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCGEN.121.003476PMID: 34555931 Originally publishedSeptember 24, 2021 Keywordsheart failurehypertrophic cardiomyopathygenetic testingMYH7genotypephenotypePDF download SubjectsGenetic, Association StudiesGeneticsPrecision MedicineTranslational Studies

BACKGROUND:Although 21 causative mutations have been associated with PRKAG2 syndrome, our understanding of the syndrome remains incomplete. The aim of this project is to further investigate its unique genetic background, clinical manifestations, and underlying structural changes. METHODS:We recruited 885 hypertrophic cardiomyopathy (HCM) probands and their families internationally. Targeted next-generation sequencing of sudden cardiac death (SCD) genes was performed. The role of the identified variants was assessed using histological techniques and computational modeling. FINDINGS:Twelve PRKAG2 syndrome kindreds harboring 5 distinct variants were identified. The clinical penetrance of 25 carriers was 100.0%. Twenty-two family members died of SCD or heart failure (HF). All probands developed bradycardia (HRmin, 36.3 ± 9.8 bpm) and cardiac conduction defects, and 33% had evidence of atrial fibrillation/paroxysmal supraventricular tachycardia (PSVT) and 67% had ventricular preexcitation, respectively. Some carriers presented with apical hypertrophy, hypertension, hyperlipidemia, and renal insufficiency. Histological study revealed reduced AMPK activity and major cardiac channels in the heart tissue with K485E mutation. Computational modelling suggests that K485E disrupts the salt bridge connecting the β and γ subunits of AMPK, R302Q/P decreases the binding affinity for ATP, T400N and H401D alter the orientation of H383 and R531 residues, thus altering nucleotide binding, and N488I and L341S lead to structural instability in the Bateman domain, which disrupts the intramolecular regulation. INTERPRETATION:Including 4 families with 3 new mutations, we describe a cohort of 12 kindreds with PRKAG2 syndrome with novel pathogenic mechanisms by computational modelling. Severe clinical cardiac phenotypes may be developed, including HF, requiring close follow-up.

The yeast protein GCN4 is a transcriptional activator in the basic leucine zipper (bZip) family, whose distinguishing feature is the "chopstick-like" homodimer of alpha helices formed at the DNA-binding interface. While experiments have shown that truncated versions of the protein retain biologically relevant DNA-binding affinity, we present the results of a computational study revealing that these variants show a wide variety of dynamical modes in their interaction with the target DNA sequence. We have performed all-atom molecular dynamics simulations of the full-length GCN4 protein as well as three truncated variants; our data indicate that the truncated mutants show dramatically different correlation patterns. We conclude that although the truncated mutants still retain DNA-binding ability, the bZip interface present in the full-length protein provides important stability for the protein-DNA complex.

Introduction The major structure elements of the AMP-activated protein kinase (AMPK) are α, β, and γ sunbunits. Mutations in γ2 subunit (PRKAG2) have been associated with a cardiac syndrome including inherited ventricular preexcitation, conduction disorder and hypertrophy mimicking hypertrophic cardiomyopathy. The aim of the present study was to identify PRKAG2 syndrome among patients presenting with left ventricular hypertrophy (LVH). Methods and Results Nineteen unrelated subjects with unexplained LVH were clinically and genetically evaluated. Among 4 patients with bradycardia, manifestations of preexcitation were only found in a 19 year old male who also developed congestive heart failure 3 years later. Electrophysiological study of this case identified the coexistence of an AV accessory pathway and AV conduction defect. Histological analysis of his ventricular tissue isolated by biopsy confirmed excessive glycogen accumulation, prominent myofibrillar disarray and interstitial fibrosis. Direct sequencing of his DNA revealed a heterozygous mutation in PRKAG2 consisting of an A-to-G transition at nucleotide 1453 (c.1453A>G), predicting a substitution of a glutamic acid for lysine at highly-conserved residue 485 (p.Lys485Glu, K485E), which was absent in his unaffected family members and in 215 healthy controls. To assess the role of K485 in the structure and function of the protein, computational modeling calculations and conservation analyses were performed. Electrostatic calculations indicate that K485 forms a salt bridge with the conserved D248 residue in the AMPK β subunit, which is critical for proper regulation of the enzyme, and the K485E mutant disrupts the connection. Conclusions Our study identifies a novel de novo PRKAG2 mutation in a young, in which progression of the disease warrants close medical attention. It also underlines the importance of molecular screening of PRKAG2 gene in patients with unexplained LVH, ventricular preexcitation, conduction defect, and/or early onset of heart failure.