Inborn errors of metabolism: Lessons from iPSC models

The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs

Trabeculated Myocardium in Hypertrophic Cardiomyopathy: Clinical Consequences

Aims: Hypertrophic cardiomyopathy (HCM) is often accompanied by increased trabeculated myocardium (TM)-which clinical relevance is unknown. We aim to measure the left ventricular (LV) mass and proportion of trabeculation in an HCM population and to analyze its clinical implication. Methods and Results: We evaluated 211 patients with HCM (mean age 47.8 +/- 16.3 years, 73.0% males) with cardiac magnetic resonance (CMR) studies. LV trabecular and compacted mass were measured using dedicated software for automatic delineation of borders. Mean compacted myocardium (CM) was 160.0 +/- 62.0 g and trabecular myocardium (TM) 55.5 +/- 18.7 g. The percentage of trabeculated myocardium (TM%) was 26.7% +/- 6.4%. Females had significantly increased TM% compared to males (29.7 +/- 7.2 vs. 25.6 +/- 5.8, p < 0.0001). Patients with LVEF < 50% had significantly higher values of TM% (30.2% +/- 6.0% vs. 26.6% +/- 6.4%, p = 0.02). Multivariable analysis showed that female gender and neutral pattern of hypertrophy were directly associated with TM%, while dynamic obstruction, maximal wall thickness and LVEF% were inversely associated with TM%. There was no association between TM% with arterial hypertension, physical activity, or symptoms. Atrial fibrillation and severity of hypertrophy were the only variables associated with cardiovascular death. Multivariable analysis failed to demonstrate any correlation between TM% and arrhythmias. Conclusions: Approximately 25% of myocardium appears non-compacted and can automatically be measured in HCM series. Proportion of non-compacted myocardium is increased in female, non-obstructives, and in those with lower contractility. The amount of trabeculation might help to identify HCM patients prone to systolic heart failure

iPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

BACKGROUND Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. METHODS We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. RESULTS Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. CONCLUSIONS Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.The funding for this research was provided by the Spanish Ministry of Science and Innovation-MCIN (grants PID2021-123925OB-I00, PID2019-104776RB-I00, CB06/01/1056, and CB16/11/00399 financed by MCIN/AEI/10.13039/501100011033), AGAUR (2021-SGR-974), Fundació La Marató de TV3 (201534-30), Fundación BBVA (BIO14_298), Fundació Obra Social la Caixa, and CERCA Program/ Generalitat de Catalunya. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN, and the Pro CNIC Foundation. I. Lazis was partially supported by a predoctoral fellowship from MCIN (PRE2019-087901).S

Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.This study was supported by grants PID2019-104776RB-I00 and PID2020-120326RB-I00, CB16/11/00399 (CIBER CV) financed by MCIN/AEI/10.13039/501100011033, a grant from the Fundación BBVA (Ref. BIO14_298), and a grant from Fundació La Marató de TV3 (Ref. 20153431) to J.L.d.l.P. M.S.-A. was supported by a PhD contract from the Severo Ochoa Predoctor-al Program (SVP-2014-068723) of the MCIN/AEI/10.13039/501100011033. J.R.G.-B. was supported by SEC/FEC-INV-BAS 21/021. A.R. was funded by grants from MCIN (PID2021123925OB-I00), TerCel (RD16/0011/0024), AGAUR (2017-SGR-899), and Fundació La Marató de TV3 (201534-30). J.M.P.-P. was supported by RTI2018-095410-B-I00 (MCIN) and PY2000443 (Junta de Andalucía). B.I. was supported by the European Commission (H2020-HEALTH grant No. 945118) and by MCIN (PID2019-107332RB-I00). DO’R was sup-ported by the Medical Research Council (MC-A658-5QEB0) and KAMcG by the British Heart Foundation (RG/19/6/34387, RE/18/4/34215). The cost of this publication was supported in part with funds from the European Regional Devel-opment Fund. The Centro Nacional de Investigaciones Cardiovasculares is sup-ported by the ISCIII, the MCIN, and the Pro Centro Nacional de Investigaciones Cardiovasculares Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020001041-S) financed by MCIN/AEI/10.13039/501100011033. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising.S

Análisis proteómico de la infección de plantas de tomate por Pseudomonas syringæ. Validación en distintos sistemas planta-patógeno

[ES] Las plantas se encuentran sometidas continuamente a estreses bióticos y abióticos de diversa naturaleza. A lo largo de la evolución, han ido desarrollando mecanismos de resistencia frente a cada estrés, llegando a originar un sistema defensivo altamente eficaz, formado tanto por barreras físico-químicas constitutivas como por defensas inducibles. Las principales defensas que una planta emplea en su lucha contra las agresiones medioambientales son la síntesis de proteínas defensivas y de compuestos químicos con propiedades antimicrobianas. Todo ello tiene el objetivo de mantener la homeostasis de la planta frente a las agresiones del entorno. En el estudio de las proteínas que la planta emplea para combatir las diferentes agresiones patogénicas, la Proteómica desempeña un papel esencial para descifrar los mecanismos que tienen lugar durante la infección y poder así desarrollar variedades vegetales más resistentes. El objetivo del presente estudio es analizar el comportamiento diferencial de plantas de tomate de la variedad ‘Rio Grande’ frente a dos infecciones bacterianas de diferente naturaleza, una de ellas avirulenta, que no da lugar al desarrollo de la enfermedad y otra virulenta, en la cual el patógeno invade la planta y tiene lugar la aparición de síntomas. Asimismo, se pretende comparar los resultados obtenidos de nuestro estudio con bacterias y los publicados para otras infecciones causadas por viroides o por virus en tomate y llevar a cabo su validación en varios sistemas planta-patógeno (bacteria, virus y viroide). Para ello, se realizó en primer lugar un análisis proteómico comparativo mediante 2D-DIGE de plantas de tomate ‘Rio Grande’, portadoras del gen de resistencia Pto, infectadas con la cepa virulenta (ΔavrPto) y con la cepa avirulenta (AvrPto) de la bacteria Pseudomonas syringæ pv. tomato DC3000, a diferentes tiempos. Se identificaron proteínas que se acumularon de manera diferencial en plantas infectadas con respecto a las plantas control y proteínas con acumulación diferencial entre ambos tipos de infección. Asimismo, se realizó un estudio comparativo entre nuestros datos y los estudios proteómicos realizados en plantas de tomate infectadas por el Viroide de la Exocortis de los Cítricos (CEVd) y el Virus del Rizado Amarillo del Tomate (TYLCV). Por último se validaron nuestros datos proteómicos obtenidos, así como las comparaciones realizadas para las tres infecciones, mediante RT-PCR y Western Blot de distintas proteínas comunes a la infección bacteriana y al menos un tipo diferente de infección. El establecimiento de las defensas de las plantas requiere la regulación de un amplio número de proteínas que están implicadas a diferentes niveles. Nuestros resultados se unen a los realizados en los últimos años para analizar las interacciones planta-patógeno mediante estrategias proteómicas, constituyendo en su conjunto una información muy valiosa sobre la regulación de estas proteínas y su función en los diferentes mecanismos defensivos.[EN] Plants are constantly exposed to diverse biotic and abiotic environmental stresses. Throughout evolution, plants have developed resistance mechanisms to cope with each stress, thus originating a highly efficient defence system, composed by both constitutive physical and chemical barriers as well as inducible defences. The main strategies used by plants against environmental aggressions are the synthesis of defence proteins and chemical compounds with antimicrobial properties. This has the purpose of preserving plant homeostasis against environmental challenges. Studying the proteins used by plants to face different pathogenic aggressions, Proteomics plays an essential role to understand the mechanisms that take place upon infection, thus allowing to develop more resistant plant varieties. The aim of this project is to analyze the differential behavior of the tomato cultivar ‘Rio Grande’ under two different plant-pathogen scenarios. One of them involves the use of an avirulent bacteria, in which disease development does not take place, and a virulent one, which does produce disease and symptom development. Furthermore, it is intended to compare the results obtained in our bacterial study with other published results dealing with infections caused by viroids and viruses in tomato in order to validate different plant-pathogen systems (bacteria, virus and viroid). For this purpose, we made a comparative proteomic analysis by 2D-DIGE in ‘Rio Grande’ tomato plants, which contain the resistant gene Pto, infected with the avirulent strain (ΔavrPto) and with the virulent strain (AvrPto) of the bacteria Pseudomonas syringæ pv. tomato DC3000, in different times. We identified differentially accumulated proteins in infected plants with respect control plants and between different types of infection. Furthermore, we made a comparative study between our data and other proteomic approaches described in tomato plants infected with Citrus Exocortis Viroid (CEVd) and with Tomato Yellow Leaf Curl Virus (TYLCV). Finally, we validated our proteomic results, as well as the comparisons made for the three infections, by RT-PCR and Western Blots of several common proteins to the bacterial infection and at least another type of infection. The establishment of the plant defences requires the regulation of a large number of proteins involved in different levels. Our findings join to the most recent proteomic studies on plant-pathogen interactions, making together a valuable body of information about the regulation of these proteins and their function in different plant defence mechanisms.Escribá Piera, R. (2014). Análisis proteómico de la infección de plantas de tomate por Pseudomonas syringæ. Validación en distintos sistemas planta-patógeno. http://hdl.handle.net/10251/47715.Archivo delegad

Investigating genetic and mechanistic interactors in familial cardoimyopathy through advanced disease modeling

[eng] Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. HCM is a highly complex condition defined by clinical and genetic heterogeneity. During last decades, our understanding of the diverse genetic landscape and the pathological molecular mechanisms underlying HCM has increased significantly. However, studying the effect of genetic modifiers of cardiomyopathies is limited by their complex genetic aetiology. A better understanding of the complex genetic mechanisms underlying cardiac diseases is an imperative hallmark for precision medicine. With this aim, we sought to investigate the differing molecular and genetic mechanisms of two siblings with an extensive family history of HCM but divergent clinical manifestations using patient-specific induced pluripotent stem cells (hiPSCs). For this purpose, we generated patient-specific iPSC from the male, diagnosed with a severe hypertrophic phenotype, and from the female, with mild hypertrophy, whose genetic testing revealed a common pathogenic mutation in the MYBPC3 gene (K600Nfs*2). Morphological characterization of iPSC-derived cardiomyocytes from mutant carriers revealed that sarcomeric alignment and structure was not compromised. However, MYBPC3 deficient iPSC-CMs showed reduced contractile force generation without cell shape remodelling. We then took advantage of the CRISPR/Cas9 gene-editing technology to generate MYBPC3-corrected isogenic controls in order to better ascribe genotype-phenotype correlations. Functional evaluation of mutant and isogenic iPSC-CMs revealed that cardiomyocytes from the symptomatic patient presented a hypercontractile phenotype as well as faster calcium transients. Further analysis on the mitochondrial bioenergetics indicated an inefficient ATP consumption in sarcomeres from both mutant carriers. In order to explore whether additional genetic variants were modifying the pathological outcomes in the symptomatic carrier, we performed a whole- exome sequencing of the mutant carriers. We identified a variant of unknown significance (VUS) in the MYH7 gene (I1927F), the second most common mutated gene in HCM, uniquely present in the severe HCM individual. Although the identified VUS has been previously described in HCM patients, there is not sufficient clinical and functional evidence to ascertain pathogenicity. To precisely evaluate the effect of the VUS, we generated a MYH7 I1927F corrected isogenic iPSC line using CRISPR/Cas9. Functional evaluation of double and single mutant iPSC-CMs revealed that the additional presence of the MYH7 variant was responsible for the faster cardiac contraction, strongly supporting a severe pathogenic contribution. Our study provides a unique platform to functionally assess the effect of genetic modifiers

Trabeculated Myocardium in Hypertrophic Cardiomyopathy: Clinical Consequences.

Hypertrophic cardiomyopathy (HCM) is often accompanied by increased trabeculated myocardium (TM)-which clinical relevance is unknown. We aim to measure the left ventricular (LV) mass and proportion of trabeculation in an HCM population and to analyze its clinical implication. We evaluated 211 patients with HCM (mean age 47.8 ± 16.3 years, 73.0% males) with cardiac magnetic resonance (CMR) studies. LV trabecular and compacted mass were measured using dedicated software for automatic delineation of borders. Mean compacted myocardium (CM) was 160.0 ± 62.0 g and trabecular myocardium (TM) 55.5 ± 18.7 g. The percentage of trabeculated myocardium (TM%) was 26.7% ± 6.4%. Females had significantly increased TM% compared to males (29.7 ± 7.2 vs. 25.6 ± 5.8, p < 0.0001). Patients with LVEF < 50% had significantly higher values of TM% (30.2% ± 6.0% vs. 26.6% ± 6.4%, p = 0.02). Multivariable analysis showed that female gender and neutral pattern of hypertrophy were directly associated with TM%, while dynamic obstruction, maximal wall thickness and LVEF% were inversely associated with TM%. There was no association between TM% with arterial hypertension, physical activity, or symptoms. Atrial fibrillation and severity of hypertrophy were the only variables associated with cardiovascular death. Multivariable analysis failed to demonstrate any correlation between TM% and arrhythmias. Approximately 25% of myocardium appears non-compacted and can automatically be measured in HCM series. Proportion of non-compacted myocardium is increased in female, non-obstructives, and in those with lower contractility. The amount of trabeculation might help to identify HCM patients prone to systolic heart failure.This work was supported by a grant from the Foundation Marató TV3 2018/C/2015 and by the Spanish MICINN and AEI, as well as European Commission FEDER funds, under grant RTI2018-098156-B-C53. Investigators are part of the cardiovascular research network and Cell Therapy network (TerCel) of the Carlos III Health Institute (SAF2015-71863-REDT, RTI2018-095377-B-I00, CIBERCV CB16/11/00385, RD16/0011/0021, RD16/0011/0024, CB16/11/00399, RIC; RD12/0042/0049), MCIU (SAF2016-78370-R) and IMIB (Instituto Murciano de Investigación Biosanitaria). María Sabater was supported by a grant from FFIS. Investigators are part of clinical group of ERN Guard-Heart, CIBERCV, CIBERER and University of Murcia.S

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve

Background:The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. Methods:We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Results:Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. Conclusions:These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers