MYH7 Research Articles

Abstract 516: Endothelial Foxp1 Regulates Cardiac Remodeling via Tgfbeta and Et-1 Signal Pathway

Rationale: Cardiac fibrosis and hypertrophy is common features in left ventricular remodeling that leads to heart failure. Forkhead box protein P-1 (Foxp1), a large modular transcriptional repressor that binds to DNA via the highly conserved forkhead DNA-binding domain, is crucial in coordinating the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation during cardiac development. However, its role in adult pathological cardiac remodel remains to be clarified. Objective: We seek to determine the role and the underlying mechanisms of Foxp1 in cardiac fibrosis and hypertrophy. Methods and Results: Foxp1 was highly expressed in endocardium and cardiac microvascular endothelial cells, significantly reduced following AngII stimulation. Loss of endothelial Foxp1 increased AngII-mediated cardiac fibrosis, which was accompanied by myofibroblast proliferation and endothelial-mesenchymal transition (endMT) with elevated expression of fibrotic matrix genes. TGF-β signal pathway was identified as Foxp1 direct downstream target gene in endothelial cells to regulate the fibrosis. Increased AngII-mediated cardiac hypertrophy was observed when loss of endothelial Foxp1, with elevated cardiac hypertrophic genes and fetal myosin gene (myh7) expression but reduced adult mature myosin gene (myh6) expression via endothelial ET-1 signal. The pathological remodel in endothelial Foxp1 deficient mutant mice finally led to worsen cardiac diastolic dysfunction as shown by elevated E/e’ ratio in echocardiogram. Moreover, the similar pathological remodel and worsen cardiac diastolic and systolic dysfunction as shown by reduction of LV fractional shortening (FS) and ejection fraction (EF) were observed in pressure overload transverse aortic constriction (TAC) model when loss of endothelial Foxp1. Conclusions: This study uncovered a previously unrecognized anti-fibrotic and anti-hypertrophic role of Foxp1 in pathological cardiac remodel, which is achieved through the regulation of endothelial TGF-β/ET-1 signals, implying the promising therapeutic targets in pathological cardiac remodeling diseases and heart failure.

A rare missense mutation in MYH6 associates with non-syndromic coarctation of the aorta.

AimsCoarctation of the aorta (CoA) accounts for 4–8% of congenital heart defects (CHDs) and confers substantial morbidity despite treatment. It is increasingly recognized as a highly heritable condition. The aim of the study was to search for sequence variants that affect the risk of CoA.Methods and resultsWe performed a genome-wide association study of CoA among Icelanders (120 cases and 355 166 controls) based on imputed variants identified through whole-genome sequencing. We found association with a rare (frequency = 0.34%) missense mutation p.Arg721Trp in MYH6 (odds ratio = 44.2, P = 5.0 × 10−22), encoding the alpha-heavy chain subunit of cardiac myosin, an essential sarcomere protein. Approximately 20% of individuals with CoA in Iceland carry this mutation. We show that p.Arg721Trp also associates with other CHDs, in particular bicuspid aortic valve. We have previously reported broad effects of p.Arg721Trp on cardiac electrical function and strong association with sick sinus syndrome and atrial fibrillation.ConclusionThrough a population approach, we found that a rare missense mutation p.Arg721Trp in the sarcomere gene MYH6 has a strong effect on the risk of CoA and explains a substantial fraction of the Icelanders with CoA. This is the first mutation associated with non-familial or sporadic form of CoA at a population level. The p.Arg721Trp in MYH6 causes a cardiac syndrome with highly variable expressivity and emphasizes the importance of sarcomere integrity for cardiac development and function.

Prognostic factors in paediatric restrictive cardiomyopathy

Restrictive cardiomyopathy (RCM) is the rarest and most serious form of heart muscle disease, accounting for 2–5% of pediatric cardiomyopathies. Identification of prognostic factors to identify high-risk patients that need to be addressed for early heart transplantation. We enrolled 47 patients that had a diagnosis of RCM between 1996 and 2017 in our center. The clinical, ultrasonography, and hemodynamic data were collected at the time of diagnosis, as well as genetic analysis in order to identify early predictors of poor prognosis (transplantation or death). The mean age at diagnosis was 5.9 years with a gender ratio of 1.76 (F/M). 57.5% had a pure RCM and 42.5% an hypertrophic cardiomyopathy with restrictive phenotype (HRCM). The median follow-up was 10.2 months. Overall survival at 1, 2 and 5 years was 43% 35% and 15% respectively. There was no statistically significant difference in terms of survival between RCM and HRCM (P = 0.651) and between age groups (P = 0.582). Clinical findings at diagnosis were dominated by congestive signs (63.9%), which were related to a poor outcome (P = 0/041). The relationship between a mildly altered left ventricular ejection fraction (LVEF 45-55%) and mortality was statistically significant (P = 0.01). Imaging findings didn’t show a late gadolinium enhancement. Regarding catheterism data, neither cardiac output nor pulmonary vascular resistance nor pulmonary capillary pressure were related to a poor outcome. The rentability of family screening was 60%: we identified 1 homozygous and 4 heterozygous mutations on TNNI3 gene including a neomutation, and 1 on MYH7 gene. Adverse prognostic factors were dominated by congestive signs at diagnosis and left ventricular dysfunction. Patient with these factors should be addressed early for Heart transplantation. Autosomic dominant mutation in TNNI3 was the most frequent genetic finding. Family screening and genetic counseling is thus important.

Molecular and histomorphological evaluation of female rats' urethral tissues after an innovative trauma model of prolonged vaginal distention: immediate, short-term and long-term effects.

An animal model of vaginal distention (VD) was developed to reproduce the acute urethral injury and deficiency underlying stress urinary incontinence (SUI). Data on the chronic effects of urethral trauma and the recovery process are still scarce. We investigated acute, short- and long-term histomorphological and molecular changes in the urethra of rats post 12-h intermittent VD. We evaluated the urethra of four groups of female rats (n = 72): control without trauma, 1h, 7days and 30days post VD. We compared the gene and protein expression of the VEGF and NGF growth factors, collagens (COL1a1 and COL3a1), desmin, smooth muscle myosin (MYH11), skeletal muscle myosins (MYH1, MYH2 and MYH3) and cell proliferation marker MKi67. We used real-time RT-qPCR, and immunohistochemistry. Histology showed urethral damage after VD mainly involving the muscular layers. VEGF, NGF, desmin and MKi67 mRNA were significantly upregulated in the urethras of rats 1-h post VD compared with controls (P < 0.05 for all). By 7 days post trauma, COL1a1, MYH11 and MYH3 genes were overexpressed compared with controls (p < 0.05 for all). The COL3a1 protein level was increased by 2.6 times by day 7, while MYH2 protein was significantly decreased (around two times) from 7 to 30 days post VD compared with controls (p < 0.05 for both). The 12-h intermittent VD causes chronic alterations in the urethra represented by increased COL3a1 and decreased MYH2 protein levels in the long term. The model can potentially be used to study the mechanisms of urethral injury and recovery as well as the physiopathology of SUI.

Successful knock-in of Hypertrophic Cardiomyopathy-mutation R723G into the MYH7 gene mimics HCM pathology in pigs

Familial Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiac disease. About 30% of the patients are heterozygous for mutations in the MYH7 gene encoding the ß-myosin heavy chain (MyHC). Hallmarks of HCM are cardiomyocyte disarray and hypertrophy of the left ventricle, the symptoms range from slight arrhythmias to sudden cardiac death or heart failure. To gain insight into the underlying mechanisms of the diseases’ etiology we aimed to generate genome edited pigs with an HCM-mutation. We used TALEN-mediated genome editing and successfully introduced the HCM-point mutation R723G into the MYH7 gene of porcine fibroblasts and subsequently cloned pigs that were heterozygous for the HCM-mutation R723G. No off-target effects were determined in the R723G-pigs. Surprisingly, the animals died within 24 h post partem, probably due to heart failure as indicated by a shift in the a/ß-MyHC ratio in the left ventricle. Most interestingly, the neonatal pigs displayed features of HCM, including mild myocyte disarray, malformed nuclei, and MYH7-overexpression. The finding of HCM-specific pathology in neonatal R723G-piglets suggests a very early onset of the disease and highlights the importance of novel large animal models for studying causative mechanisms and long-term progression of human cardiac diseases.

MYOCARDITIS AS A LEGITIMATE PHENOMENON IN NON-COMPACTION MYOCARDIUM: DIAGNOSTICS, MANAGEMENT AND INFLUENCE ON OUTCOMES

Aim. To evaluate the prevalence of myocarditis in adult patients with non-compaction myocardium (NCM) of the left ventricle (LV), and its influence on the disease course, results of treatment and outcomes. Material and methods. To the study, 103 adult patients included, with NCM, 61 males, mean age 45,6±14,9 y. o. (from 18 to 78). Mean end diastolic LV size was 6,0±0,8 сm, EF LV 38,8±14,5%. Diagnosis of NCM had been done by echocardiography, multispiral computed tomography (n=81) and magnetic resonance tomography (n=39). DNA-diagnostics was performed by NGS method with further Senger sequencing. Pathogenic mutations were found in 9% of patients in the genes MYH7, MyBPC3, LAMP2, DES, DSP, TTN. The investigation also included anticardiac antibodies, genome of cardiotropic viruses by PCR, coronary arteriography (n=26), scintigraphy (n=25). Morphological assessment of the myocardium was done in 19 patients (12 endomyocardial biopsies), 1 intraoperation biopsy, 3 explanted hearts, incl. 2 after biopsy, and 5 autopsies. Mean follow-up 12 [2; 32] months. Results. Myocarditis was found in 53,4%, incl. virus-positive in 32,7% of those, with morphology done for 19 patients (active myocarditis in 10, borderline in 6; with minimal signs of activeness in 3). Viral genome in myocardium found in 8 patients (42,1%). The prevalence of myocarditis 44,4% in an arrhythmic variant of NCM, 12,5% in chronic ischemic, 57,5% in dilation cardiomyopathy, 50,0% in NCM patients with other cardiomyopathies. Special cases were acute/subacute myocarditis in NCM (10,7% of all), acute necrosis (infarction) in 4,9%. Comorbidity with myocarditis in NCM led to significantly more severe LV dysfunction (CHF FC 2 [1; 3] v 1,75 [0; 2], p 0,05). Only in myocarditis patients, as a result of basis (antiviral, immune suppression) and cardiotropic therapy there was significant increase of EF (in acute myocarditis from 25,4±7,9 to 38,6±9,5%, p<0,01), decrease of LV size and pulmonary systolic pressure. Conclusion. Myocarditis is typical phenomenon developing in patients with the primary, i. e. genetically verified NCM. The nature of myocarditis in NCM varies (primary infectious-autoimmune, secondary as a response on genetic/ischemic damage of cardiomyocytes), however regardless of this, it leads to significant worsening of structural and functional parameters, increase of the life-threatening arrhythmias rate, and outcomes (death + transplantation, proper shocks), demanding for active basic therapy.

A MYH3 mutation identified for the first time in a Chinese family with Sheldon-Hall syndrome (DA2B)

Sheldon-Hall syndrome is the most common type of distal arthrogryposis syndromes, also known as distal arthrogryposis 2B (DA2B). Sheldon-Hall syndrome is caused by mutations in the TPM2, TNNI2, TNNT3 or MYH3 gene and characterized by ulnar deviation, camptodactyly, overlapping fingers and scoliosis from birth. We investigated a Chinese family with multiple members who clinically presented with distal arthrogryposis of the hands. In total, 261 subjects including one proband and ten family members from the non-consanguineous Chinese family and 250 healthy volunteers were included and had their genomic DNA extracted. A novel missense mutation in exon 13 of the MYH3 gene, c.1160A > G (p.Tyr387Cys), was identified in the proband and his father through whole-exome sequencing. The proband and six affected family members were confirmed to carry this mutation by Sanger sequencing, although the mutation was not detected in the four unaffected individuals or 250 volunteers. This is the first report of a novel MYH3 mutation being identified as the cause of DA2B in a Chinese family. Our findings confirm that MYH3 gene mutations can be a pathogenic cause of DA2B in Asian patients. This study increases the mutational spectrum in MYH3 and aids genetic counseling and prenatal diagnosis.

Novel Genetic Variants of Sporadic Atrial Septal Defect (ASD) in a Chinese Population Identified by Whole-Exome Sequencing (WES).

BackgroundRecently, mutations in several genes have been described to be associated with sporadic ASD, but some genetic variants remain to be identified. The aim of this study was to use whole-exome sequencing (WES) combined with bioinformatics analysis to identify novel genetic variants in cases of sporadic congenital ASD, followed by validation by Sanger sequencing.Material/MethodsFive Han patients with secundum ASD were recruited, and their tissue samples were analyzed by WES, followed by verification by Sanger sequencing of tissue and blood samples. Further evaluation using blood samples included 452 additional patients with sporadic secundum ASD (212 male and 240 female patients) and 519 healthy subjects (252 male and 267 female subjects) for further verification by a multiplexed MassARRAY system. Bioinformatic analyses were performed to identify novel genetic variants associated with sporadic ASD.ResultsFrom five patients with sporadic ASD, a total of 181,762 genomic variants in 33 exon loci, validated by Sanger sequencing, were selected and underwent MassARRAY analysis in 452 patients with ASD and 519 healthy subjects. Three loci with high mutation frequencies, the 138665410 FOXL2 gene variant, the 23862952 MYH6 gene variant, and the 71098693 HYDIN gene variant were found to be significantly associated with sporadic ASD (P<0.05); variants in FOXL2 and MYH6 were found in patients with isolated, sporadic ASD (P<5×10−4).ConclusionsThis was the first study that demonstrated variants in FOXL2 and HYDIN associated with sporadic ASD, and supported the use of WES and bioinformatics analysis to identify disease-associated mutations.

Testing patterns for genetically triggered aortic and arterial aneurysms and dissections at an academic center

ObjectiveThe contemporary practice of testing for genetically triggered aortic and arterial aneurysms and dissections is not well described. This study aimed to describe this practice at a tertiary care academic center and to ascertain the yield of testing in establishing the diagnosis in patients referred on the basis of clinical suspicion. MethodsThis is a retrospective cohort study of patients referred for vascular genetic testing at an academic medical center between 2010 and 2015. Patients were identified by Current Procedural Terminology diagnostic codes 81405, 81408, and 81479 for genetic testing (Marfan syndrome, Loeys-Dietz syndrome, aneurysms-osteoarthritis syndrome, COL3A1, and familial thoracic aortic aneurysm panel [ACTA2, COL3A1, TGFBR1, TGFBR2, SMAD3, TGFB2, MYLK, MYH11, and PRKG1 genes]) and by review of the collagen vascular laboratory database for genetic testing results. Data abstracted included demographics, clinical history, reason for referral, family history, referring provider type, and outcomes of genetic testing. ResultsNinety-six patients (44.3% male; median age, 40.8 years) were referred for suspected genetic vascular disease. Genetic testing was performed in 75 cases thought to have heritable mutations related to aortic or arterial aneurysms and dissections. The most common reason for genetic testing was a personal history of aortic or arterial aneurysms and dissections (62.3%; mean age, 45.8 ± 11.1 years), followed by a family history of aortic or arterial aneurysms and dissections without a personal history (26.6%; age, 28.8 ± 17.9 years). The most common genetic testing performed was a familial thoracic aortic aneurysm gene panel (44%), followed by single gene testing for vascular Ehlers-Danlos syndrome (33.3%). Genetic testing identified a pathogenic mutation in 36% of the cases. The highest likelihood of identifying a pathogenic mutation was in those who had a family history with an already diagnosed mutation (57.1%), followed by patients with aortic root and ascending aortic aneurysm or dissection (42.3%). ConclusionsIn patients with suspected genetically triggered vascular disease, the yield of clinical vascular genetic testing is reasonable when selective genetic testing is performed on the basis of personal or family history. These tests should be obtained with appropriate expertise in genetic counseling and interpretation of genetic testing results. Negative genetic test results in the setting of a positive family history demonstrate the limits of testing and known mutations leading to genetically triggered aortic and arterial aneurysms and dissections and support the need for novel gene discovery.

THE FREQUENCY OF MUTATIONS IN KNOWN CARDIOMYOPATHY GENES AMONG CHILDREN WITH HYPERTROPHIC CARDIOMYOPATHY: THE NATIONAL HEART, LUNG AND BLOOD INSTITUTE-FUNDED MULTICENTER PEDIATRIC CARDIOMYOPATHY REGISTRY STUDY

Hypertrophic cardiomyopathy (HCM) is a rare disease in children often caused by pathogenic variants in MYH7 or MYBPC3 genes. Exome sequencing was completed in 160 children with HCM. Genetic variants in 36 HCM genes were interpreted using American College of Medical Genetics guidelines. Individuals

Whole‐exome sequencing and microRNA profiling reveal PI3K/AKT pathway’s involvement in juvenile myelomonocytic leukemia

BackgroundClinical studies and genetic analyses have revealed that juvenile myelomonocytic leukemia (JMML) is caused by somatic and/or germline mutations of genes involved in the RAS/MAPK signalling pathway. Given the vastly different clinical prognosis among individual patients that have had this disease, mutations in genes of other pathways may be involved.MethodsIn this study, we conducted whole‐exome and cancer‐panel sequencing analyses on a bone marrow sample from a 2‐year old juvenile myelomonocytic leukemia patient. We also measured the microRNA profile of the same patient’s bone marrow sample and the results were compared with the normal mature monocytic cells from the pooled peripheral blood.ResultsWe identified additional novel mutations in the PI3K/AKT pathway and verified with a cancer panel targeted sequencing. We have confirmed the previously tested PTPN11 gene mutation (exon 3 181G&gt;T) in the same sample and identified new nonsynonymous mutations in NTRK1, HMGA2, MLH3, MYH9 and AKT1 genes. Many of the microRNAs found to be differentially expressed are known to act as oncogenic MicroRNAs (onco‐MicroRNAs or oncomiRs), whose target genes are enriched in the PI3K/AKT signalling pathway.ConclusionsOur study suggests an alternative mechanism for JMML pathogenesis in addition to RAS/MAPK pathway. This discovery may provide new genetic markers for diagnosis and new therapeutic targets for JMML patients in the future.

Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells

Targeted integration of exogenous genes into so-called safe harbors/friend sites, offers the advantages of expressing normal levels of target genes and preventing potentially adverse effects on endogenous genes. However, the ideal genomic loci for this purpose remain limited. Additionally, due to the inherent and unresolved issues with the current genome editing tools, traditional embryonic stem (ES) cell-based targeted transgenesis technology is still preferred in practical applications. Here, we report that a high and repeatable homologous recombination (HR) frequency (>95%) is achieved when an approximate 6kb DNA sequence flanking the MYH9 gene exon 2 site is used to create the homology arms for the knockout/knock-in of diverse nonmuscle myosin II (NM II) isoforms in mouse ES cells. The easily obtained ES clones greatly facilitated the generation of multiple NM II genetic replacement mouse models, as characterized previously. Further investigation demonstrated that though the targeted integration site for exogenous genes is shifted to MYH9 intron 2 (about 500bp downstream exon 2), the high HR efficiency and the endogenous MYH9 gene integrity are not only preserved, but the expected expression of the inserted gene(s) is observed in a pre-designed set of experiments conducted in mouse ES cells. Importantly, we confirmed that the expression and normal function of the endogenous MYH9 gene is not affected by the insertion of the exogenous gene in these cases. Therefore, these findings suggest that like the commonly used ROSA26 site, the MYH9 gene locus may be considered a new safe harbor for high-efficiency targeted transgenesis and for biomedical applications.

Abstract 166: The Brain and Aortic Aneurysm Screening (BAAS) Study - Screening for Occult Intracranial Aneurysms in Those With Abdominal Aortic Aneurysms

Introduction: Abdominal aortic aneurysm (AAA) rupture has a mortality rate of 64% and intracranial aneurysm (IA) rupture has a mortality rate of 50%. Despite the high mortality rates, early detection and treatment of these vascular lesions prior to rupture can prevent devastating clinical outcomes, underscoring the need to identify high-risk populations for screening. Current evidence demonstrates shared genetic and environmental risk factors for both AAA and IA. Methods: The BAAS study sought to establish the co-prevalence of AAA and IA. In one arm of BAAS, we recruited patients presenting with AAA and screened them for IA. All participants underwent a detailed interview to collect demographic, radiographic, and clinical data and also had genetic counseling. Results: Of the 126 individuals with an AAA enrolled, 65% (82/126) completed the MRA. Difficulty with logistics of obtaining the research MRA with long distance travel to clinic was the principal reason for failure to obtain MRA. We identified 20.7% (17/82) with one or more IA, including 21 aneurysms found during systematic screening. IA ranged in size from 1.5-14 mm (height) x 1.4-7 mm (width) and varied in location. 9.5% (2/21) had a maximum diameter of 7 mm or more. One participant with a positive MRA underwent early coil embolization with the remainder undergoing continued clinical surveillance. Of the 15 individuals completing the genetic counseling screening, two underwent recommended clinical genetic testing, revealing one negative screen and one variant of unknown significance in the MYH11 gene. Conclusion: Consistent with our prior cost effectiveness models, these prospective data support the clinical utility of routine screening for IA in patients presenting with AAA.

Phenotypic diversity identified by cardiac magnetic resonance in a large hypertrophic cardiomyopathy family with a single MYH7 mutation

Limited data is available on phenotypic variations with the same genotype in hypertrophic cardiomyopathy (HCM). The present study aims to explore the relationship between genotype and phenotype characterized by cardiovascular magnetic resonance (CMR) in a large Chinese family. A proband diagnosed with HCM from a multigenerational family underwent next-generation sequencing based on a custom sureSelect panel, including 117 candidate pathogenic genes associated with cardiomyopathies. All genetic results were confirmed by the Sanger sequencing method. All confirmed mutation carriers underwent CMR exam and myocardial tissue characterization using T1 mapping and late gadolinium enhancement (LGE) on a 3T scanner (Siemens Trio, Gemany). After clinical and genetic screening of 36 (including the proband) members of a large Chinese family, nineteen family members are determined to carry the single p.T1377M (c.4130C>T) mutation in the MYH7 gene. Of these 19 mutation carriers, eight are diagnosed with HCM, one was considered as borderline affected and ten are not clinically or phenotypically affected. Different HCM phenotypes are present in the nine affected individuals in this family. In addition, we have found different tissue characteristics assessed by T1 mapping and LGE in these individuals. We describe a family that demonstrates the diverse HCM phenotypes associated with a single MYH7 mutation.

Insertional Mutagenesis Confounds the Mechanism of the Morbid Phenotype of a PLNR9C Transgenic Mouse Line

BackgroundA mouse line with heterozygous transgenic expression of phospholamban carrying a substitution of cysteine for arginine 9 (TgPLNR9C) under the control of α-myosin heavy chain (αMHC) promoter features dilated cardiomyopathy, heart failure, and premature death. Methods and ResultsDetermination of transgene chromosomal localization by conventional methods shows that in this line the transgenic array of 13 PLNR9C expression cassettes, arranged in a head-to-tail tandem orientation, have integrated into the bidirectional promoter of the αMHC (Myh6) gene and the gene for the regulatory noncoding RNA Myheart (Mhrt), both of which are known to be involved in cardiac development and pathology. Expression of the noncoding RNA Mhrt in TgPLNR9C mice exhibits profound deregulation, despite the presence of the second, intact allele. ConclusionsThe TgPLNR9C mouse strain is, in the best case, a functionally ambiguous phenocopy of the human PLNR9C heterozygote, because a similar constellation of genetically programmed events can not occur in a patient. Publications featuring “cardiac-specific overexpression” are focused on the phenotype and typically forgo the definition of the transgene integration site or transgene temporal expression profile, so caution should be exercised in attributing clinical relevance to pathologic phenomena observed in αMHC-driven transgenes.

High proportion of genetic cases in patients with advanced cardiomyopathy including a novel homozygous Plakophilin 2-gene mutation.

Cardiomyopathies might lead to end-stage heart disease with the requirement of drastic treatments like bridging up to transplant or heart transplantation. A not precisely known proportion of these diseases are genetically determined. We genotyped 43 index-patients (30 DCM, 10 ARVC, 3 RCM) with advanced or end stage cardiomyopathy using a gene panel which covered 46 known cardiomyopathy disease genes. Fifty-three variants with possible impact on disease in 33 patients were identified. Of these 27 (51%) were classified as likely pathogenic or pathogenic in the MYH7, MYL2, MYL3, NEXN, TNNC1, TNNI3, DES, LMNA, PKP2, PLN, RBM20, TTN, and CRYAB genes. Fifty-six percent (n = 24) of index-patients carried a likely pathogenic or pathogenic mutation. Of these 75% (n = 18) were familial and 25% (n = 6) sporadic cases. However, severe cardiomyopathy seemed to be not characterized by a specific mutation profile. Remarkably, we identified a novel homozygous PKP2-missense variant in a large consanguineous family with sudden death in early childhood and several members with heart transplantation in adolescent age.

Diagnosis and treatment of MYH9-RD in an Australasian cohort with thrombocytopenia

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (döhle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if döhle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.

Non-muscle myosin IIB (Myh10) is required for epicardial function and coronary vessel formation during mammalian development.

The coronary vasculature is an essential vessel network providing the blood supply to the heart. Disruptions in coronary blood flow contribute to cardiac disease, a major cause of premature death worldwide. The generation of treatments for cardiovascular disease will be aided by a deeper understanding of the developmental processes that underpin coronary vessel formation. From an ENU mutagenesis screen, we have isolated a mouse mutant displaying embryonic hydrocephalus and cardiac defects (EHC). Positional cloning and candidate gene analysis revealed that the EHC phenotype results from a point mutation in a splice donor site of the Myh10 gene, which encodes NMHC IIB. Complementation testing confirmed that the Myh10 mutation causes the EHC phenotype. Characterisation of the EHC cardiac defects revealed abnormalities in myocardial development, consistent with observations from previously generated NMHC IIB null mouse lines. Analysis of the EHC mutant hearts also identified defects in the formation of the coronary vasculature. We attribute the coronary vessel abnormalities to defective epicardial cell function, as the EHC epicardium displays an abnormal cell morphology, reduced capacity to undergo epithelial-mesenchymal transition (EMT), and impaired migration of epicardial-derived cells (EPDCs) into the myocardium. Our studies on the EHC mutant demonstrate a requirement for NMHC IIB in epicardial function and coronary vessel formation, highlighting the importance of this protein in cardiac development and ultimately, embryonic survival.

MicroRNA 499 gene expression in patients on hemodialysis with cardiovascular complications.

MicroRNA (miRNA) 499 is an evolutionarily conserved muscle-specific miRNA that is encoded by an intron of the myh7 gene and is likely to play a role in myosin gene regulation. It has been shown to be involved in inhibiting apoptosis and myocardial infarction induced by ischemia and anoxia. It is unknown whether levels of miRNAs are affected in patients undergoing hemodialysis. The aim of this study was to assess circulating levels of miRNA 499 in hemodialysis patients and whether the levels are affected by dialyzer membranes (high flux vs. low flux). The studied population consisted of 32 end stage renal disease (ESRD) patients (22 males and 10 females) with age ranged from 38% to 75% years on regular hemodialysis (4 hours, 3 times weekly) for at least 1 year duration with cardiovascular events in the last 6 months and 32 healthy controls (20 males and 12 females) with an age range from 54 to 60 years. Patients were involved into a two-stage sequential study; high-flux hemodialysis stage (stage I), then low-flux hemodialysis stage (stage II). Expressed levels of plasma miRNA 499 have been measured by Real Time-PCR. Lipid profile, serum phosphorus, serum calcium, serum creatinine, and blood urea were measured in all patients. In this study, 2 patients with an open-heart surgery showed highly elevation in the miRNA 499, while the other patients, showing different degrees of ischemia, had different levels of elevated miRNA 499. Statistically significant higher levels of miRNA 499 in plasma were observed in all the studied patients with cardiovascular diseases compared to the levels of miRNA 499 found in healthy controls (P < 0.0001). MicroRNA 499 was found to be a dialyzable marker. A significant decrease in plasma levels of miRNA 499 was obtained after either high-flux or low-flux dialysis compared to plasma levels of miRNA 499 found before dialysis (P < 0.0001). On comparing both types of hemodialysis membranes with respect to miRNA 499 clearance, we found that low-flux membrane showed better clearance for miRNA 499 than high-flux membrane with a statistically significant difference between them (P < 0.001). In conclusion, miRNA 499 levels are elevated in patients with ESRD with cardiovascular complications. High-flux membrane seems to be less efficient in miRNA 499 clearance in cardiac patients on hemodialysis.

A Missense Variant in PLEC Increases Risk of Atrial Fibrillation

BackgroundGenome-wide association studies (GWAS) have yielded variants at >30 loci that associate with atrial fibrillation (AF), including rare coding mutations in the sarcomere genes MYH6 and MYL4. ObjectivesThe aim of this study was to search for novel AF associations and in doing so gain insights into the mechanisms whereby variants affect AF risk, using electrocardiogram (ECG) measurements. MethodsThe authors performed a GWAS of 14,255 AF cases and 374,939 controls, using whole-genome sequence data from the Icelandic population, and tested novel signals in 2,002 non-Icelandic cases and 12,324 controls. They then tested the AF variants for effect on cardiac electrical function by using measurements in 289,297 ECGs from 62,974 individuals. ResultsThe authors discovered 2 novel AF variants, the intergenic variant rs72700114, between the genes LINC01142 and METTL11B (risk allele frequency = 8.1%; odds ratio [OR]: 1.26; p = 3.1 × 10−18), and the missense variant p.Gly4098Ser in PLEC (frequency = 1.2%; OR: 1.55; p = 8.0 × 10−10), encoding plectin, a cytoskeletal cross-linking protein that contributes to integrity of cardiac tissue. The authors also confirmed 29 reported variants. p.Gly4098Ser in PLEC significantly affects various ECG measurements in the absence of AF. Other AF variants have diverse effects on the conduction system, ranging from none to extensive. ConclusionsThe discovery of a missense variant in PLEC affecting AF combined with recent discoveries of variants in the sarcomere genes MYH6 and MYL4 points to an important role of myocardial structure in the pathogenesis of the disease. The diverse associations between AF variants and ECG measurements suggest fundamentally different categories of mechanisms contributing to the development of AF.