Publications by authors named "Elvir Becirovic"

39 Publications

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

Nat Commun 2020 11 3;11(1):5555. Epub 2020 Nov 3.

Hannover Medical School, Institute for Neurophysiology, 30625, Hannover, Germany.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.
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http://dx.doi.org/10.1038/s41467-020-19304-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641277PMC
November 2020

Bihavioral Addictions in Childhood and Adolescence - Pandemic Knocking Door.

Psychiatr Danub 2020 Oct;32(Suppl 3):382-385

Department of Psychiatry, University Clinical Center Tuzla, Ul. Rate Dugonjića bb, 75000 Tuzla, Bosnia and Herzegovina,

Introduction: Addiction is not solely "substance dependence". Diminished control is a core defining concept of psychoactive substance addiction. Several behaviors, besides psychoactive substance ingestion, produce short-term reward that may engender diminished control over the behavior. Growing evidence suggests that behavioral addictions resemble substance addictions in many domains, including phenomenology, tolerance, comorbidity, overlapping genetic contribution, neurobiological mechanisms, and response to treatment. This similarity has given rise to the concept of non-substance or behavioral addictions, i.e., syndromes analogous to substance addiction, but with a behavioral focus. The type of excessive behaviors identified as being addictive include gambling, use of computers, playing video games, use of the internet, exercise, and shopping. Behavioral addictions have been proposed as a new class in DSM-5, but the only category included is gambling disorder. Internet gaming disorder is included in the appendix as a condition for further study. The ICD-11 included also the definition of a new disorder, gaming disorder. To present actual knowledge about behavioral addictions in childhood and adolescence.

Methods: Analysis of data in available literature in data basis and textbooks.

Results: Some behavioral addictions are becoming more common in children and adolescents. Dominant are gaming and gambling addiction that are also best researched.

Conclusions: Behavioral addiction becomes an epidemic in children and adolescents.
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October 2020

Psychological Disorders in Childhood and Adolescent Age - New Classifications.

Psychiatr Danub 2020 Oct;32(Suppl 3):311-315

Department of Psychiatry, University Clinical Center Tuzla, Ul. Rate Dugonjića bb, 75000 Tuzla, Bosnia and Herzegovina,

Introduction: The eleventh revision of the International Classification of Diseases (ICD-11) is planned to be published in 2018. So called, "beta version" of the chapter of mental and behavioral disorders (ICD-11) is already available and it is considered that there will be no significant deviations in the final version. The DSM-5 was released in 2013. Changes related to mental disorders in child and adolescent psychiatry have been made in both of these classifications. To identify changes in the classifications of mental disorders in childhood and adolescent age in beta version of ICD-11 and DSM-5.

Methods: Review of mental disorders in childhood and adolescent age and their classification in ICD-11 and DSM-5.

Results: For disorders that are classified as "mental retardation" in ICD-10, a new term "intellectual development disorders" has been introduced in ICD-11, ie "intellectual disabilities" in DSM-5. Hyperactivity disorders and attention deficit is a separate entity in relation to ICD-10, in which it is classified as a hyperkinetic disorder. Asperger's syndrome, which is isolated from autism spectrum disorders in DSM-5, does not appear under that name in ICD-11 either. Elimination disorders are in a separate block MKB-11 and DSM-5. Speech and language disorders are classified as communication disorders in the DSM-5 classification. Selective mutism and anxiety separation disorder in childhood are in the block of anxiety and fear-related disorders in ICD-11, and among anxiety disorders in DSM-5, respectively. Reactive emotional disorder and disinhibited attachment disorder of childhood are classified as stress-related disorders in ICD-11 and DSM-5.

Conclusions: The new classifications (ICD-11 and DSM-5) classify mental disorders in child and adolescent psychiatry somewhat differently from their antecedents. New entities have also been formed.
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October 2020

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Sci Adv 2020 Aug 19;6(34):eaba5614. Epub 2020 Aug 19.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin () in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.
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http://dx.doi.org/10.1126/sciadv.aba5614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438099PMC
August 2020

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation.

Mol Ther Nucleic Acids 2020 Sep 31;21:1050-1061. Epub 2020 Jul 31.

Center for Integrated Protein Science Munich CIPSM, Munich, Germany; Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.
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http://dx.doi.org/10.1016/j.omtn.2020.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452116PMC
September 2020

Memory efficiency in patients with drug-resistant epilepsy.

Acta Clin Belg 2020 Jun 12:1-5. Epub 2020 Jun 12.

Department of Neurology, Primary Health Center University of Tuzla, Tuzla, Bosnia and Herzegovina.

Objective: To evaluate memory in patients with drug-resistant epilepsy.

Methods: Following an examination, 50 patients were diagnosed in accordance with the 2005 proposal of the International League Against Epilepsy and the definition of drug-resistant epilepsy from 2010. The neuropsychological examination used the Wechsler Memory Scale. It assessed seven structural types of memory: general knowledge, orientation, mental control, logical memory, number memory, associative memory, and visual reproduction. The values were compared with 50 subjects without epilepsy.

Results: Patients with epilepsy had statistically significantly lower values in five of seven structural units of memory. The average value of overall memory efficacy in subjects with epilepsy was 96.5 ± 19.6, while in subjects without epilepsy it was 118 ± 15.6 (p = 0.0002). Memory impairments are greater in those taking polytherapy (p = 0.0429). The overall memory efficiency correlated significantly negatively with seizure frequency (p = 0.0015) and insignificantly negative with the duration of epilepsy (p = 0.1935).

Conclusion: Patients with drug-resistant epilepsy have lower memory efficiency. Memory impairments are greater in those taking polytherapy, as with those with more frequent seizures. The duration of epilepsy has no significant effect on overall memory performance.
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http://dx.doi.org/10.1080/17843286.2020.1778347DOI Listing
June 2020

Neuropathic and cAMP-induced pain behavior is ameliorated in mice lacking CNGB1.

Neuropharmacology 2020 07 6;171:108087. Epub 2020 Apr 6.

Institute of Pharmacology and Clinical Pharmacy, Goethe University, 60438, Frankfurt am Main, Germany.

Cyclic nucleotide-gated (CNG) channels, which are directly activated by cAMP and cGMP, have long been known to play a key role in retinal and olfactory signal transduction. Emerging evidence indicates that CNG channels are also involved in signaling pathways important for pain processing. Here, we found that the expression of the channel subunits CNGA2, CNGA3, CNGA4 and CNGB1 in dorsal root ganglia, and of CNGA2 in the spinal cord, is transiently altered after peripheral nerve injury in mice. Specifically, we show using in situ hybridization and quantitative real-time RT-PCR that CNG channels containing the CNGB1b subunit are localized to populations of sensory neurons and predominantly excitatory interneurons in the spinal dorsal horn. In CNGB1 knockout (CNGB1) mice, neuropathic pain behavior is considerably attenuated whereas inflammatory pain behavior is normal. Finally, we provide evidence to support CNGB1 as a downstream mediator of cAMP signaling in pain pathways. Altogether, our data suggest that CNGB1-positive CNG channels specifically contribute to neuropathic pain processing after peripheral nerve injury.
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http://dx.doi.org/10.1016/j.neuropharm.2020.108087DOI Listing
July 2020

Sexual Dysfunctions in Bosnian War Veterans.

Psychiatr Danub 2019 Dec;31(Suppl 5):839-846

Department of Psychiatry, University Clinical Center Tuzla, 75000 Tuzla, Bosnia and Herzegovina,

Sexual functioning of war veterans is significantly under-explored. During devastating aggression on Bosnia-Herzegovina (BiH) around 400 thousand soldiers were included in combats. It is estimated that more than 100 000 persons were killed, and more than 60 000 them were soldiers. Vast majority of them were deployed since war is ended. We found high prevalence of sexual dysfunctions in war veterans. Also significant difference in several areas of sexual functioning between war veterans with and without symptoms of posttraumatic stress disorder was found.
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December 2019

Post-traumatic Stress Disorder, Metacognitions, Cognitive and Global Functioning in Bosnian War Veterans.

Mater Sociomed 2019 Dec;31(4):258-261

Faculty of Pharmacy, University of Tuzla, Univerzitetska br. 8, 75.000 Tuzla.

Introduction: Cognitive impairment is common finding in individuals with PTSD. Dysfunctional metacognitions in variety of anxiety disorders can represent generic vulnerability for anxiety disorders, as well as elements that contribute to maintaining the disorder. There is little empirical information about metacognition in war veterans with PTSD, and its relation to cognitive and/or social, occupational and psychological functioning.

Aim: to determine the values and reciprocal correlations of different aspects of metacognition, with cognitive and global functioning in outpatient war veterans with PTSD.

Methods: The study was conducted on 25 war veterans (24 male), with confirmed diagnosis of PTSD by a trained psychiatrist, average age 48,5±6,2 (38-63) years, with average duration of symptoms of 9,9±4,7 (0,5-16) years. We used the Metacognitions questionnaire, Mini Mental Status Examination, and Global Assessment of Functioning Scale to assess metacognition, cognitive impairment, and global functioning. Median values of Metacognitions questionnaire subcomponents, Global Assessment of Functioning Scale and Mini Mental Status Examination were determined, and also reciprocal correlations of all parameters expressed with Spearman Rank Correlation.

Results: 12 patients (48%) had impaired cognitive function. Significant negative correlation of score on Mini Mental Status Examination, and negative beliefs about worry is observed (r=-0,4278, p=0,034), as well as non significant correlations between rest of metacognition subscales and score on Mini Mental Status Examination. Cognitive self-consciousness showed high positive correlation with Global Assessment of Functioning Scale (r =0,7436, p<0,0001).

Conclusion: Follow up of metacognitions, cognitive and global functioning, and its relations, may have an important role in assessment of war veterans with posttraumatic stress disorder.
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http://dx.doi.org/10.5455/msm.2019.31.258-261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007607PMC
December 2019

Enigmatic rhodopsin mutation creates an exceptionally strong splice acceptor site.

Hum Mol Genet 2020 01;29(2):295-304

Center for Integrated Protein Science Munich (CIPSM), 81377 Munich, Germany.

The c.620 T > G mutation in rhodopsin found in the first mapped autosomal dominant retinitis pigmentosa (adRP) locus is associated with severe, early-onset RP. Intriguingly, another mutation affecting the same nucleotide (c.620 T > A) is related to a mild, late-onset RP. Assuming that both mutations are missense mutations (Met207Arg and Met207Lys) hampering the ligand-binding pocket, previous work addressed how they might differentially impair rhodopsin function. Here, we investigated the impact of both mutations at the mRNA and protein level in HEK293 cells and in the mouse retina. We show that, in contrast to c.620 T > A, c.620 T > G is a splicing mutation, which generates an exceptionally strong splice acceptor site (SAS) resulting in a 90 bp in-frame deletion and protein mislocalization in vitro and in vivo. Moreover, we identified the core element underlying the c.620 T > G SAS strength. Finally, we demonstrate that the c.620 T > G SAS is very flexible in branch point choice, which might explain its remarkable performance. Based on these results, we suggest that (i) point mutations should be routinely tested for mRNA splicing to avoid dispensable analysis of mutations on protein level, which do not naturally exist. (ii) Puzzling disease courses of mutations in other genes might also correlate with their effects on mRNA splicing. (iii) Flexibility in branch point choice might be another factor influencing the SAS strength. (iv) The core splice element identified in this study could be useful for biotechnological applications requiring effective SAS.
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http://dx.doi.org/10.1093/hmg/ddz291DOI Listing
January 2020

Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy.

J Clin Invest 2018 12 12;128(12):5663-5675. Epub 2018 Nov 12.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
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http://dx.doi.org/10.1172/JCI96098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264655PMC
December 2018

In Vitro Evaluation of AAV Vectors for Retinal Gene Therapy.

Methods Mol Biol 2019 ;1834:383-390

Department of Pharmacy-Center for Drug Research, Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

Gene therapy holds promise for treating previously untreatable retinal disorders. The most promising approaches use gene transfer vectors derived from adeno-associated virus (AAV) to supplement a gene function in the affected cell type. One example is gene therapy for achromatopsia which affects daylight vision. In this case, recombinant AAV (rAAV) vectors are being developed to specifically target cone photoreceptors. Development of rAAV vectors could be facilitated by the use of in vitro models. In this chapter we provide a protocol which utilizes mouse 661W cells, an in vitro model of cone photoreceptors for evaluation of the transduction efficacy of rAAV vectors.
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http://dx.doi.org/10.1007/978-1-4939-8669-9_24DOI Listing
April 2019

Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis.

J Cell Sci 2018 08 17;131(16). Epub 2018 Aug 17.

Animal Physiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina and via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.
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http://dx.doi.org/10.1242/jcs.218487DOI Listing
August 2018

Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy.

Int J Mol Sci 2018 Mar 7;19(3). Epub 2018 Mar 7.

Center for Integrated Protein Science Munich (CIPSM), Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr, 5-13, 81377 Munich, Germany.

The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG) channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP). Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and voltage-gated potassium channels (KCN). In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.
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http://dx.doi.org/10.3390/ijms19030749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877610PMC
March 2018

Construction and Cloning of Minigenes for Analysis of Potential Splice Mutations.

Bio Protoc 2018 Mar 5;8(5):e2760. Epub 2018 Mar 5.

Center for Integrated Protein Science Munich CIPSM, München, Germany.

Disease-associated mutations influencing mRNA splicing are referred to as splice mutations. The majority of splice mutations are found on exon-intron boundaries defining canonical donor and acceptor splice sites. However, mutations in the coding region (exonic mutations) can also affect mRNA splicing. Exact knowledge of the disease mechanism of splice mutations is essential for developing optimal treatment strategies. Given the large number of disease-associated mutations thus far identified, there is an unmet need for methods to systematically analyze the effects of pathogenic mutations on mRNA splicing. As splicing can vary between cell types, splice mutations need to be tested under native conditions if possible. A commonly used tool for the analysis of mRNA splicing is the construction of minigenes carrying exonic and intronic sequences. Here, we describe a protocol for the design and cloning of minigenes into recombinant adeno-associated virus (rAAV) vectors for gene delivery and investigation of mRNA splicing in a native context. This protocol was developed for minigene-based analysis of mRNA splicing in retinal cells, however, in principle it is applicable to any cell type, which can be transduced with rAAV vectors.
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http://dx.doi.org/10.21769/BioProtoc.2760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203986PMC
March 2018

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice.

Front Mol Neurosci 2017 9;10:436. Epub 2018 Jan 9.

Center for Integrated Protein Science and Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians University, Munich, Germany.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3 mice show normal visual, photic, and non-photic circadian function. In addition, HCN3 mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.
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http://dx.doi.org/10.3389/fnmol.2017.00436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767300PMC
January 2018

Patients and animal models of CNGβ1-deficient retinitis pigmentosa support gene augmentation approach.

J Clin Invest 2018 01 20;128(1):190-206. Epub 2017 Nov 20.

Department of Ophthalmology Pathology & Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA.

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.
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http://dx.doi.org/10.1172/JCI95161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749539PMC
January 2018

Design and Development of AAV-based Gene Supplementation Therapies for Achromatopsia and Retinitis Pigmentosa.

Methods Mol Biol 2018 ;1715:33-46

Department of Pharmacy, Center for Drug Research, Center for Integrated Protein Science Munich CiPSM, Ludwig-Maximilian-University, Munich, Germany.

Achromatopsia (ACHM) and retinitis pigmentosa (RP) are inherited disorders caused by mutations in cone and rod photoreceptor-specific genes, respectively. ACHM strongly impairs daylight vision, whereas RP initially affects night vision and daylight vision at later stages. Currently, gene supplementation therapies utilizing recombinant adeno-associated virus (rAAV) vectors are being developed for various forms of ACHM and RP. In this chapter, we describe the procedure of designing and developing specific and efficient rAAV vectors for cone- and rod-specific gene supplementation.
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http://dx.doi.org/10.1007/978-1-4939-7522-8_3DOI Listing
July 2018

Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants.

Sci Rep 2017 05 24;7(1):2321. Epub 2017 May 24.

Center for Integrated Protein Science Munich CiPSM, Ludwig-Maximilians-Universität München, Munich, Germany.

Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (per), mutant peripherin-2 (per), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to per and Rom-1. Furthermore, both mutants are preferentially forming non-covalent per-per, per-per, and Rom-1-per dimers. However, only per-per, but not per-per or Rom-1-per complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent per-per dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of per and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals.
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http://dx.doi.org/10.1038/s41598-017-02514-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443838PMC
May 2017

Family Atmosphere and Relationships as Predictors of Heroin Addiction.

Psychiatr Danub 2017 May;29(Suppl 2):129-133

Psychiatric Clinic, University Clinical Center Tuzla, Rate Dugonjica bb, 75000 Tuzla, Bosnia and Herzegovina,

Introduction: Studies show that dysfunctional family relationships are important predictors of addictions to all psychoactive substances.

Objective: To establish if there is a connection between family relations and heroin addiction and if found to exist, what is the quality of this connection.

Subjects And Methods: This research was conducted on the sample comprised of 160 subjects divided into two groups. The first group consisted of 61 heroin addicts treated at the Tuzla University Clinical Centre Psychiatric Hospital. The second group consisted of 99 subjects who were students at the Tuzla University Faculties of Philosophy and Electrical Engineering and who were not using any psychoactive substances. The subjects were tested with the Quality of Family Interactions Scale (KOBI) which measures the interactions between children and parents in two dimensions, described in literature as 'acceptance' and 'rejection'.

Results: The research team established statistically significant differences between the heroin addicts and the students, the non-users, in terms of their family relationships. The results show that the addicts families were characterized by lack of understanding, by conflicts, rejection, non-acceptance by parents, while the non-users families were characterized by understanding, acceptance by parents and good communication.

Conclusions: There is a connection between inter-family relationships and addiction. Namely, rejection and non-acceptance of children/persons by their families and parents, bad communication and dysfunctional family relationships are significant predictors of heroin addiction.
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May 2017

A deep intronic CLRN1 (USH3A) founder mutation generates an aberrant exon and underlies severe Usher syndrome on the Arabian Peninsula.

Sci Rep 2017 05 3;7(1):1411. Epub 2017 May 3.

Bioscientia Center for Human Genetics, Ingelheim, Germany.

Deafblindness is mostly due to Usher syndrome caused by recessive mutations in the known genes. Mutation-negative patients therefore either have distinct diseases, mutations in yet unknown Usher genes or in extra-exonic parts of the known genes - to date a largely unexplored possibility. In a consanguineous Saudi family segregating Usher syndrome type 1 (USH1), NGS of genes for Usher syndrome, deafness and retinal dystrophy and subsequent whole-exome sequencing each failed to identify a mutation. Genome-wide linkage analysis revealed two small candidate regions on chromosome 3, one containing the USH3A gene CLRN1, which has never been associated with Usher syndrome in Saudi Arabia. Whole-genome sequencing (WGS) identified a homozygous deep intronic mutation, c.254-649T > G, predicted to generate a novel donor splice site. CLRN1 minigene-based analysis confirmed the splicing of an aberrant exon due to usage of this novel motif, resulting in a frameshift and a premature termination codon. We identified this mutation in an additional two of seven unrelated mutation-negative Saudi USH1 patients. Locus-specific markers indicated that c.254-649T > G represents a founder allele that may significantly contribute to deafblindness in this population. Our finding underlines the potential of WGS to uncover atypically localized, hidden mutations in patients who lack exonic mutations in the known disease genes.
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http://dx.doi.org/10.1038/s41598-017-01577-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431179PMC
May 2017

Gene therapy for achromatopsia.

J Gene Med 2017 Mar;19(3)

Center for Integrated Protein Science Munich CiPSM and Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany.

The present review summarizes the current status of achromatopsia (ACHM) gene therapy-related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide-gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno-associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy.
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http://dx.doi.org/10.1002/jgm.2944DOI Listing
March 2017

PREDICTORS FOR POST- STROKE DELIRIUM OUTCOME.

Mater Sociomed 2016 Oct 17;28(5):382-386. Epub 2016 Oct 17.

Department of Psichiatry, University Clinical Centre Tuzla, Bosnia and Herzegovina.

Background: There have been only a small number of studies that have evaluated the outcome of post-stroke delirium.

Objectives: To evaluate the effects of gender, age, stroke localization, delirium severity, previous illnesses, associated medical complications on delirium outcome as well as, to determine effects of delirium on cognitive functioning one year after stroke.

Patients And Methods: Comprehensive neuropsychological assessments were performed within the first week of stroke onset, at hospital discharge, and followed-up for 3, 6 and 12 months after stroke. We used diagnostic tools such as Glazgow Coma Scale, Delirium Rating Scale, National Institutes of Health Stroke Scale and Mini-Mental State.

Results: Patients who developed post-stroke delirium had significantly more complications (p = 0.0005). Direct logistic regression was performed to assess the impact of several factors on the likelihood that patients will die. The strongest predictor of outcome was age, mean age ≥ 65 years with a odds ratio (OR) 4.9. Cox's regression survival was conducted to assess the impact of multiple factors on survival. The accompanying medical complications were the strongest predictor of respondents poore outcome with Hazard-risk 3.3. Cognitive assessments including Mini Mental State score have showen that post-stroke delirium patients had significant cognitive impairment, three (p = 0.0005), six months (p = 0.0005) and one year (p = 0.0005) after stroke, compared to patients without delirium.

Conclusion: Patient gender, age, localization of stroke, severity of delirium, chronic diseases and emerging complications significantly affect the outcome of post- stroke delirium. Delirium significantly reduced cognitive functioning of after stroke patients.
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http://dx.doi.org/10.5455/msm.2016.28.382-386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5149427PMC
October 2016

AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.

Front Neurosci 2016 28;10:356. Epub 2016 Jul 28.

Department of Pharmacy - Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität MünchenMunich, Germany; Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität MünchenMunich, Germany.

Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types.
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http://dx.doi.org/10.3389/fnins.2016.00356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963399PMC
August 2016

Peripherin-2 differentially interacts with cone opsins in outer segments of cone photoreceptors.

Hum Mol Genet 2016 06 30;25(12):2367-2377. Epub 2016 Mar 30.

Munich Center for Integrated Protein Science CIPS , 81377 München, Germany,

Peripherin-2 is a glycomembrane protein exclusively expressed in the light-sensing compartments of rod and cone photoreceptors designated as outer segments (OS). Mutations in peripherin-2 are associated with degenerative retinal diseases either affecting rod or cone photoreceptors. While peripherin-2 has been extensively studied in rods, there is only little information on its supramolecular organization and function in cones. Recently, we have demonstrated that peripherin-2 interacts with the light detector rhodopsin in OS of rods. It remains unclear, however, if peripherin-2 also binds to cone opsins. Here, using a combination of co-immunoprecipitation analyses, transmission electron microscopy (TEM)-based immunolabeling experiments, and quantitative fluorescence resonance energy transfer (FRET) measurements in cone OS of wild type mice, we demonstrate that peripherin-2 binds to both, S-opsin and M-opsin. However, FRET-based quantification of the respective interactions indicated significantly less stringent binding of peripherin-2 to S-opsin compared to its interaction with M-opsin. Subsequent TEM-studies also showed less co-localization of peripherin-2 and S-opsin in cone OS compared to peripherin-2 and M-opsin. Furthermore, quantitative FRET analysis in acutely isolated cone OS revealed that the cone degeneration-causing V268I mutation in peripherin-2 selectively reduced binding to M-opsin without affecting the peripherin-2 interaction to S-opsin or rhodopsin. The differential binding of peripherin-2 to cone opsins and the mutant-specific interference with the peripherin-2/M-opsin binding points to a novel role of peripherin-2 in cones and might contribute to understanding the differential penetrance of certain peripherin-2 mutations in rods and cones. Finally, our results provide a proof-of-principle for quantitative FRET measurements of protein-protein interactions in cone OS.
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http://dx.doi.org/10.1093/hmg/ddw103DOI Listing
June 2016

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors.

PLoS Genet 2016 Jan 21;12(1):e1005811. Epub 2016 Jan 21.

Munich Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, München, Germany.

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization.
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http://dx.doi.org/10.1371/journal.pgen.1005811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722987PMC
January 2016

Metabolic syndrome in schizophrenia - who is more to blame: FGA polypharmacy or clozapine monotherapy?

Psychiatr Danub 2015 Dec;27(4):378-84

Department of Psychiatry, University Clinical Center Tuzla, Solina b.b., 75000 Tuzla, Bosnia and Hercegovina,

Background: To establish the prevalence of metabolic syndrome and its parameters in group of patients with schizophrenia in polypharmacy - receiving first generation antipsychotics versus clozapine alone treated group.

Subjects And Methods: 48 outpatients with schizophrenia divided into two groups: the first group of 21 patients in polypharmacy with first generation antipsychotics, and the second group of 27 patients treated with clozapine alone were assessed for the presence of metabolic syndrome. We used logistic regression models to assess the relationship between metabolic syndrome and antipsychotic therapy, gender and age.

Results: Metabolic syndrome was found in 52.1% of all subjects. Compared to first generation antipsychotics polypharmacy, the monopharmacy with clozapine was associated with elevated rates of metabolic syndrome (28.6% vs. 70.4%, p=0.004). With regard to particular parameters of metabolic syndrome, the elevated plasma triglycerides were significantly more present in subjects within Clozapine group (p=0.03). Logistic regression analysis showed that female gender (p=0.004), and clozapine treatment (p=0.005) were significantly associated with metabolic syndrome.

Conclusion: Compared to polypharmacy with first generation antipsychotics, the higher prevalence of metabolic syndrome is found in patients treated with Clozapine alone. The most prevalent metabolic disorder is dyslipidemia.
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December 2015

Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod photoreceptors.

Hum Mol Genet 2014 Nov 24;23(22):5989-97. Epub 2014 Jun 24.

Munich Center for Integrated Protein Science CIPSM and Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany,

Outer segments (OSs) of rod photoreceptors are cellular compartments specialized in the conversion of light into electrical signals. This process relies on the light-triggered change in the intracellular levels of cyclic guanosine monophosphate, which in turn controls the activity of cyclic nucleotide-gated (CNG) channels in the rod OS plasma membrane. The rod CNG channel is a macromolecular complex that in its core harbors the ion-conducting CNGA1 and CNGB1a subunits. To identify additional proteins of the complex that interact with the CNGB1a core subunit, we applied affinity purification of mouse retinal proteins followed by mass spectrometry. In combination with in vitro and in vivo co-immunoprecipitation and fluorescence resonance energy transfer (FRET), we found that the tetraspanin peripherin-2 links CNGB1a to the light-detector rhodopsin. Using immunoelectron microscopy, we found that this peripherin-2/rhodopsin/CNG channel complex localizes to the contact region between the disk rims and the plasma membrane. FRET measurements revealed that the fourth transmembrane domain (TM4) of peripherin-2 is required for the interaction with rhodopsin. Quantitatively, the binding affinity of the peripherin-2/rhodopsin interaction was in a similar range as that observed for rhodopsin dimers. Finally, we demonstrate that the p.G266D retinitis pigmentosa mutation found within TM4 selectively abolishes the binding of peripherin-2 to rhodopsin. This finding suggests that the specific disruption of the rhodopsin/peripherin-2 interaction in the p.G266D mutant might contribute to the pathophysiology in affected persons.
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http://dx.doi.org/10.1093/hmg/ddu323DOI Listing
November 2014

Gene therapy restores vision and delays degeneration in the CNGB1(-/-) mouse model of retinitis pigmentosa.

Adv Exp Med Biol 2014 ;801:733-9

Center for Integrated Protein Science Munich (CIPSM), Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany,

Retinitis pigmentosa (RP) is a severe retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. Here, we used CNGB1-deficient (CNGB1(-/-)) mice, a mouse model for autosomal recessive RP, to evaluate the efficacy of adeno-associated virus (AAV) vector-mediated gene therapy for the treatment of RP. The treatment restored normal expression of rod CNG channels and rod-driven light responses in the CNGB1(-/-) retina. This led to a substantial delay of retinal degeneration and long-term preservation of retinal morphology. Finally, treated CNGB1(-/-) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this study holds promise for the treatment of rod channelopathy-associated retinitis pigmentosa by AAV-mediated gene replacement.
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http://dx.doi.org/10.1007/978-1-4614-3209-8_92DOI Listing
July 2014

Sick sinus syndrome in HCN1-deficient mice.

Circulation 2013 Dec 11;128(24):2585-94. Epub 2013 Nov 11.

Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie (S.F., S.C.K., S.I.H.H., E.B., F.A., R.B., V.H., C.P., M.B., C.A.W.-S.) and Medizinische Klinik und Poliklinik I, Klinikum Großhadern (C.K., P.L., T.Z.), Ludwig Maximilians University, Munich, Germany; DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany (C.K., P.L., T.Z., M.B., C.A.W.-S.); Max-Planck-Institut für Psychiatrie München, Germany (C.T.W.); and School of Physics & Astronomy, University of Manchester, UK (H.Z.).

Background: Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND.

Methods And Results: Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output.

Conclusions: We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003712DOI Listing
December 2013
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