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Instituto de Salud Carlos III (ISCIII), European Regional Development Fund/European Social Fund "A way to make Europe"/" Investing in your future" [PI18/0004, PI20/0320, PT17/0015/0043]; ISCIII; MCIN; Pro-CNIC Foundation; Severo Ochoa Centers of Excellence program [CEX2020-001041-S]; ISCIII [CM20/00101], Frutos F. de, Ochoa JP, Navarro-Peñalver M, Baas A, Bjerre JV, Zorio E, Méndez I, Lorca R, Verdonschot JAJ, García-Granja PE, Bilinska Z, Fatkin D, Fuentes-Cañamero ME, García-Pinilla JM, García-Álvarez MI, Girolami F, Barriales-Villa R, Díez-López C, Lopes LR, Wahbi K, García-Álvarez A, Rodríguez-Sánchez I, Rekondo-Olaetxea J, Rodríguez-Palomares JF, Gallego-Delgado M, Meder B, Kubanek M, Hansen FG, Restrepo-Córdoba MA, Palomino-Doza J, Ruiz-Guerrero L, Sarquella-Brugada G, Perez-Perez AJ, Bermúdez-Jiménez FJ, Ripoll-Vera T, Rasmussen TB, Jansen M, Sabater-Molina M, Elliot PM, Garcia-Pavia P; European Genetic Cardiomyopathies Initiative Investigators

International audience; Bicuspid aortic valve (BAV) is the most common congenital heart defect (CHD), affecting 1-2% of the population. BAV is associated with thoracic aortic aneurysms (TAAs). Deleterious copy number variations (CNVs) were found previously in up to 10% of CHD cases. This study aimed at unravelling the contribution of deleterious deletions or duplications in 95 unrelated BAV/TAA patients. Seven unique or rare CNVs were validated, harbouring protein-coding genes with a role in the cardiovascular system. Based on the presence of overlapping CNVs in patients with cardiovascular phenotypes in the DECIPHER database, the identification of similar CNVs in whole-exome sequencing data of 67 BAV/TAA patients and suggested topological domain involvement from Hi-C data, supportive evidence was obtained for two genes (DGCR6 and TBX20) of the seven initially validated CNVs. A rare variant burden analysis using next-generation sequencing data from 637 BAV/TAA patients was performed for these two candidate genes. This revealed a suggestive genetic role for TBX20 in BAV/TAA aetiology, further reinforced by segregation of a rare TBX20 variant with the phenotype within a BAV/TAA family. To conclude, our results do not confirm a significant contribution for deleterious CNVs in BAV/TAA as only one potentially pathogenic CNV (1.05%) was identified. We cannot exclude the possibility that BAV/TAA is occasionally attributed to causal CNVs though, or that certain CNVs act as genetic risk factors by creating a sensitised background for BAV/TAA. Finally, accumulative evidence for TBX20 involvement in BAV/TAA aetiology underlines the importance of this transcription factor in cardiovascular disease.

Background Nonischaemic cardiomyopathy (NICM) represents a heterogenic disorder with a variable arrhythmogenic substrate. Its location is often epicardial and catheter ablation in this location proved to be an effective therapeutic modality in NICM patients with recurrent ventricular tachycardias (VTs). Purpose To determine the impact of the type of genetic mutation on the long-term outcome of endo-epicardial ablation in patients with NICM. Methods We investigated 82 patients (age 47±15 years, 10 women) with NICM who underwent endo-epicardial ablation for frequent VTs. Of them, 59% had a history of failed endocardial ablation. Patients had a left ventricular ejection fraction of 44±14% and all were implanted with cardioverter-defibrillator. One hundred candidate genes were examined using the new generation sequencing technique. Results Mutation in genes coding desmosomal complex (genes: PKP2, DSC, DSP, and DSG) was found in 30% of patients (“desmosomal” group). In 23% of patients, other gene mutations (genes: LMNA/C, MYH7, DES, TTN, RYR2, TPM1, MYPN, FLNC, and SCN5A) were detected (“non-desmosomal” group). In 46% of subjects no pathogenic mutation could be identified (“none” group). During a mean follow up of 34±33 months, patients in the “non-desmosomal” group were at significantly higher risk of VT recurrence and death/heart transplant compared to patients in the “desmosomal” group (Figure 1). Conclusion Potentially pathogenic mutation can be detected in about half of patients with NICM undergoing endo-epicardial VT ablation. Most commonly, mutations can be found in genes coding desmosomal complex and the endo-epicardial ablation is then associated with a satisfactory low VT recurrence rate and excellent survival in the long-term. On the other hand, patients with a mutation in non-desmosomal genes have poor outcomes despite endo-epicardial ablation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Supported by Ministry of Health of the Czech Republic, grant nr. NV18-02-00237 Figure 1

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Health of the Czech Republic Introduction The complex diagnostic work up in SCA survivors often does not yield a concrete cardiological diagnosis. Moreover, there is conflicting evidence whether genetic testing could support or guide clinical diagnostics. Purpose To assess the molecular architecture of idiopathic ventricular fibrillation in cases without apparent evidence of specific structural or arrhythmic cardiac disease at initial diagnostic work up in a representative Czech cohort. Patients and Methods Between 2013 - 2020 we have ascertained 100 SCA survivors (54 M / 46 F; age range at cardiac arrest 5-69 years). Genetic counselling was followed by massively parallel DNA sequencing using custom-made panels comprising 100 cardiac conditions-related genes. Subsequently, thorough cardiological screening examinations in first degree relatives were carried out. Presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results Highly likely or certain molecular aetiology (i.e. based on the presence of Class 4 or 5 variants) was disclosed in 20/100 (20%) in PKP2 (3x), SCN5A (4x), RYR2 (3x), TTN (2x), PLN, FLNC, PRKAG2, KCNH2, KCNQ1, SLC4A, TNNT2, and DSP. Interestingly, the KCNE1 p.Asp85Asn (LQT 5 lite) variant, was detected in further 3/100 cases, representing a recognized risk factor for ventricular arrhythmias. Conclusions Genetic testing facilitates stratification of the cause of arrhythmia in a substantial portion of SCA survivors. The utility of positive outcomes of genetic testing was substantiated in 10/20 gene positive patients, where the genetic stratification led to diagnosis of concealed arrhythmogenic cardiomyopathy, whose extent of morphological changes was under the diagnostic sensibility of imaging modalities or ECG. Our results enable individualized care in SCA survivors and assure targeted preventive approaches in their relatives.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Supported by Ministry of Health of the Czech Republic Introduction Post mortem genetic analysis in sudden cardiac death (SCD) represents an important diagnostic tool for the primary prevention of cardiac arrest in victim´s relatives. Purpose To assess the underlying molecular pathogenesis of SCD in a representative Czech cohort and to evaluate the effects of primary prevention of SCD in genetic relatives. Patients and Methods Between 2016 and 2020 we have ascertained 100 SCD cases (29 females/71 males; age range 0-52 years). According to autopsy protocols, cases with SCD were divided into categories of sudden arrhythmic death (SADS), sudden unexplained death (in infants; SUD/SUDI), thoracic aortic aneurysm/dissection and cardiomyopathy hypertrophic, arrhythmic, dilated (HCM, ACM, DCM) and sudden infant death syndrome (SIDS). DNA was isolated from post mortem biopsies / relatives blood and subjected to massively parallel sequencing (Illumina, USA) comprising custom-made candidate gene panel (100 genes). Genetic counselling and cardiological examinations were carried out in 245 family members. Results According to post mortem-established diagnosis, we identified 20 victims with SADS and SUD/SUDI, 11 with HCM and DCM, 19 with ACMG, 8 SIDS cases and 9 acute dissection cases. Most of victims died at sleep or at rest, while only 10/100 victims died during strenuous sport activities. About 50% of SCD victims did not report any apparent cardiac complaints. Highly likely or certain molecular etiology (i.e. based on presence of ACMG.net Class 4 to 5 variants) was disclosed in 19/100 (19%) in RYR2, KCNH2, SCN5A, FLNC (stop), TTN, RBM 20, LMNA/C, PRKAG2, MYBPC3, DSC2, FHL1, TGFBR1 and Col3A1 genes (see Tab). Finally, we identified 52/241 phenotype/genotype positive family members who are at risk of cardiac arrest and were offered corresponding cardiological care. Conclusion Multidisciplinary cooperation, together with centralized and standardized molecular genetic testing, enables the primary prevention of cardiac arrest in relatives of SCD victims. Results of post mortem genetic analysis Post mortem diagnosis Nr. Gender Age (years) Nr. of positive cases (DNA variant class IV or V) Gene Nr. examined relatives/phenotype or genotype positive cases SADS 20 8 females12 males 3-52 5/20 (25%) KCNH2 3x RYR2RANGFR 56/11 SUD/SUDI 20 5 females, 15 males 0-50 1/18 (5%, 2 non informative cases) RYR2 45/9 HCM 11 0 females11 males 14-52 3/11 (27%) MYBPC3FHL1PRKAG2 26/9 DCM 11 3 females8 males 8-48 4/11 (36%) TTN (3x)RBM 20FLNC (stop) 24/7 ACM 19 9 females10 males 17 - 49 4/19 (21%) SCN5AFLNC (stop)DSC2LMNA/C 58/9 SIDS 8 3 females5 males < 1 0/8 - 12/0 Acute dissection 9 1 female8 males 16-49 2/9 (22%) TGFBR1Col3A1 24/7

Introduction Hereditary cardiomyopathy is associated with an increased risk of ventricular arrhythmia and sudden cardiac death (SCD). Genetic stratification substantiates risk assessment and enables the primary prevention of SCD in relatives at risk. We have analyzed the genetic aetiology of SCD in a representative Czech cohort with post mortem diagnosis of various forms of cardiomyopathy and compared it to living cases with these cardiac disorders. Patients and methods Between 2018 and 2019, altogether 47 victims of SCD with post mortem diagnosis of hypertrophic- (HCM; 18/47), arrhythmogenic- (ACM; 19/47) and dilated cardiomyopathy (DCM; 10/47) were identified. Concurrently, genetic testing was performed in 114 living patients (HCM 54/114, ACM 22/114, DCM 38/114). Genetic counselling and cardiologic examination had been carried out in first-degree relatives in all patients/SCD victims. Massively parallel sequencing (MiSeq platform; Illumina.com) was utilized for a custom-made panel comprising 100 candidate genes (Sophia Genetics, Switzerland). The presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results The causative detection rate (according to ACMG.net classes 4 or 5) in SCD victims with DCM was 60% (6/10) and in living patients with DCM 47.4% (18/38). Variants in TTN, RBM20, DES and FLNC (mainly truncating variants) prevailed in both groups. The detection rate in ACM was 5% (1/19 in SCN5A gene) in SCD victims and 31.8% (7/22) in living patients. Interestingly, the most prevalent mutated gene PKP2 in living patients was not detected in SCD victims. The detection rate in SCD victims with post mortem diagnosis of HCM was 16% (3/18) and in living patients 35% (19/54). The most prevalent gene was MYBPC3 in both groups, while PRKAG2 was detected in one SCD victim and in one living case who survived cardiac arrest. Conclusion Post-mortem genetic analysis in DCM yields a high detection rate and allows potentially effective primary prevention of SCD in relatives at risk. In contrast, the molecular autopsy of HCM and ACM renders a much lower yield which is below the mutation detection rate in living phenotype positive individuals. The results help to improve the genetic counselling in affected families in Czech Republic. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Ministry of Health of the Czech Republic

Funding Acknowledgements Supported by Ministry of Health of the Czech Republic, grant nr. NV18-02-00237. All rights reserved." Introduction "Molecular autopsy" in sudden cardiac death (SCD) is an important diagnostic tool for primary prevention of cardiac arrest in victim´s relatives and requires multicentric and multidisciplinary collaboration. Purpose To establish a national network for SCD diagnostics, to assess the acceptance of genetic testing in at risk relatives and to elucidate the genetic etiology of SCD in a representative Czech cohort aged below 45 years. Patients and Methods Between 2016 and 2019 we ascertained 70 SCD (50 M / 20 F), with positive family history for cardiac arrest in 8/70 cases. Only one family did not agree with molecular autopsy. Genetic counselling and cardiological screening examination was carried out in first degree relatives of SCD survivors accompanied by custom-made targeted panel massively parallel DNA sequencing comprising 100 cardiac conditions-related genes. Presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results According to post-mortem diagnosis most victims died of HCM (17/70), ACM (16/70), no structural heart disease was found in 15/70 cases, DCM/LVNC in 9/70, 6/70 were SIDS cases, other rare diagnoses comprised aortic dissection, or myocarditis. Most of victims died at sleep, only 9/70 victims died during strenuous activities. About 50% of SCD victims did not have cardiac complaints before death. Very likely or certain molecular etiology (i.e. based on presence of ACMG.net Class 4 to 5 variants) was disclosed in 12/70 (17%) in RYR2, FLNC, TTN, KCNH2 and KCNQ1 genes, whilst potentially causative DNA variants (Classes 3-4) were observed in 13/70 (18%) cases. In SIDS we did not find any pathogenic variants. Interestingly, the KCNE1 p.Asp85Asn (LQT 5 lite) variant, was detected in 3/70 cases as a recognized risk factor for ventricular arrhythmias. Conclusion The multidisciplinary cooperation in Czech Republic has been established, family survivors are willing to receive genetic and cardiological care, while centralised molecular genetic analysis enables reliable results which are in accordance with other multicentric studies. The molecular autopsy should, especially in SIDS, be expanded to whole exome sequencing.

Aims The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. This disease of intermediate filaments causes not only a contractile dysfunction in cardiomyopathy and skeletal myopathy, but also a secondary mitochondrial dysfunction. We aimed to describe prevalence, phenotypic expression and mitochondrial function in desmin mutation carriers identified in a large cohort of patients with unexplained cardiomyopathy. Methods and results A representative cohort of 327 Czech patients with unexplained cardiomyopathy underwent whole exome sequencing. The cohort consisted of cases with familial and sporadic dilated cardiomyopathy (81%), left ventricular noncompaction cardiomyopathy (LVNC) (13%), and less frequently of restrictive (3%) or arrhythmogenic cardiomyopathy (3%). Clinical and laboratory data of desmin mutation carriers were collected. Morphology, desmin expression and mitochondrial function were studied in available myocardial and skeletal muscle specimens. Rare, conserved and possibly pathogenic desmin variants were identified in 6 (1.8%) probands. Two desmin mutations previously classified as variants of uncertain significance (p.K43E, p.S57L), one novel desmin variant (p.A210D) and two known pathogenic desmin mutations (p.R406W, p.R454W) were in affected individuals associated with characteristic pathological desmin aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel desmin variant p.Q364H had decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal biopsy and presented with familial left ventricular non-compaction cardiomyopathy, a novel phenotype of desminopathy. Assessment of mitochondrial function in four carriers of desmin mutations with fresh-frozen skeletal and/or myocardial muscle specimens confirmed decreased metabolic capacity of mitochondrial respiratory chain complexes, which was in case of myocardial succinate respiration more profound than in end-stage heart failure of other etiologies. Genetic testing corrected an inappropriate clinical diagnosis in two probands previously diagnosed with mitochondrial disease and inflammatory cardiomyopathy. During a median follow-up of 56 months, 5 (83%) probands developed end-stage heart failure. Conclusions Desminopathy is a rare cause of cardiomyopathy and/or skeletal muscle myopathy with a pleomorphic clinical presentation and poor prognosis. The presence of desminopathy should be considered also in individuals with left ventricular non-compaction cardiomyopathy, and in the differential diagnosis of mitochondrial diseases and inflammatory cardiomyopathy. Acknowledgement/Funding Research grant of the Ministry of Health, Czech Republic [MZ 15-27682A], No. 00023001 (IKEM, Prague, CZ), the Czech Science Foundation [14-36804G].

Background External aortic root support (PEARS) is a novel prophylactic aortic root surgery. Purpose The study aimed to determine the severity of inflammatory response after the personalized external aortic root support (PEARS) procedure in comparison to after the standard prophylactic aortic root surgery (SPARS). Materials and methods The study was a single-centre, retrospective, based on hospital record analysis of patients who underwent the PEARS procedure (PEARS group) or SPARS (SPARS group) during 1998–2017. C-reactive protein (CRP), white blood count (WBC), and echocardiography were routinely obtained. Fever was defined as body temperature ≥38°C. Diagnosis of pericarditis included a minimum of three signs from chest pain, pericardial effusion, ST elevation, elevated CRP, and body temperature. Results PEARS and SPARS groups consisted of 13 and 14 patients, respectively, scheduled for prophylactic aortic root surgery. A majority of patients in both groups had Marfan syndrome with causal mutation in the fibrillin 1 (FBN1) gene (62% vs 79%). Patient baseline characteristics were similar in the two groups, except aortic root was significantly larger in the SPARS group than in the PEARS group (60±12 mm vs 48±5 mm; P=0.003). All surgical procedures were successful and without major complications. The peak values of CRP and WBC were significantly higher in the PEARS group (264.5±84.4 mg/L vs 184.6±89.6 mg/L; P=0.034 and 15.2±3.8 109/L vs 11.9±3.3 109/L; P=0.029). Early and recurrent fever requiring hospital readmission was significantly more frequent in the PEARS group (77% vs 36%; P=0.032 and 46% vs 7%; P=0.020). Early and recurrent pericarditis requiring hospital readmission was also more frequent in the PEARS group (31% vs 0%; P=0.024 and 31% vs 0%; P=0.024). Inflammatory characteristics Postprocedural inflammatory characteristics PEARS group SPARS group P value (N=13) (N=14) Peak level of CRP (mg/L) 264.5±84.4 184.6±89.6 0.034 Peak WBC (109/L) 15.2±3.8 11.9±3.3 0.029 ST elevation (N) 11 (85) 6 (43) 0.024 Early fever (N) 10 (77) 5 (36) 0.032 Recurrent fever (N) 6 (46) 1 (7) 0.020 Early pericarditis (N) 4 (31) 0 (0) 0.024 Recurrent pericarditis (N) 4 (31) 0 (0) 0.024 CRP, C-reactive protein; WBC, white blood count. Echocardiography-signs of inflammation Conclusions The PEARS procedure is an extremely promising surgical technique, but the postoperative inflammatory response occurs frequently and more severely in comparison to SPARS. Clearly, these findings warrant further investigation.

Cullin 4B (CUL4B), lysosomal-associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.

Contains fulltext : 176973.pdf (Publisher’s version ) (Open Access) Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter >/= 4.0 cm in adults, or a Z-score >/= 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

Familial multiple coagulation factor deficiency (FMFD) of factors II, VII, IX, X, protein C, and protein S is a very rare bleeding disorder with autosomal recessive inheritance. The phenotypic presentation is variable with respect to the residual activities of the affected proteins, its response to oral administration of vitamin K, and to the involvement of skeletal abnormalities. The disease may result either from a defective resorption/transport of vitamin K to the liver, or from a mutation in one of the genes encoding γ-carboxylase or other proteins of the vitamin K cycle. We have recently presented clinical details of a Lebanese family and a German family with 10 and 4 individuals, respectively, where we proposed autosomal recessive inheritance of the FMFD phenotype. Biochemical investigations of vitamin K components in patients' serum showed a significantly increased level of vitamin K epoxide, thus suggesting a defect in one of the subunits of the vitamin K 2,3-epoxide reductase (VKOR) complex. We now have performed a genome-wide linkage analysis and found significant linkage of FMFD to chromosome 16. A total maximum 2-point LOD score of 3.4 at θ = 0 was obtained in the interval between markers D16S3131 on 16p12 and D16S419 on 16q21. In both families, patients were autozygous for 26 and 28 markers, respectively, in an interval of 3 centimorgans (cM). Assuming that FMFD and warfarin resistance are allelic, conserved synteny between human and mouse linkage groups would restrict the candidate gene interval to the centromeric region of the short arm of chromosome 16.

Contains fulltext : 47856.pdf (Publisher’s version ) (Closed access) Previously, the DFNA21 locus was positioned telomeric to the DFNA13 locus based on testing of candidate loci. One family member in this region did not carry the at risk haplotype, although he had the same nonspecific midfrequency hearing impairment as other affected family members. Hence, we performed a whole genome linkage scan excluding other regions of the genome and confirming the localization of DFNA21 to 6p22.3-24.1. The DFNA21 interval was determined to span 12.4 Mb (approximately 22 cM) and is delimited on the telomeric side by BV097155 and on the centromeric side by D6S1691. A maximum lod score of 3.51 (theta = 0.066), was calculated for marker D6S1721. The DFNA21 region does not overlap the adjacent DFNA31 and DFNA13 loci and contains 31 known genes. The coding regions and exon-intron boundaries of four candidate genes, SOX4, MYLIP, CAP2, and RPEL1, were sequenced, but no mutations were identified.

Familial multiple coagulation factor deficiency (FMFD) of factors II, VII, IX, X, protein C and protein S is a very rare bleeding disorder with autosomal recessive inheritance [1]. The disease may result either from a defective resorption/transport of vitamin K to the liver, or from a mutation in one of the genes encoding gamma-carboxylase (GGCX) or other proteins of the Vitamin K 2,3-epoxide reductase (VKOR, Fig. 1) [2, 3].