ABSTRACT
Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.
Subject(s)
GATA4 Transcription Factor/metabolism , Heart Defects, Congenital , Nuclear Proteins/metabolism , Oxidoreductases/metabolism , Transcription Factors , Animals , Heart Defects, Congenital/genetics , Mice , Mutation , Proteomics , T-Box Domain Proteins/genetics , Transcription Factors/geneticsABSTRACT
The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.
Subject(s)
DNA, Mitochondrial , Heart Defects, Congenital , DNA Copy Number Variations/genetics , DNA, Mitochondrial/genetics , Heart Defects, Congenital/genetics , Humans , Mitochondria/genetics , Mutation/geneticsABSTRACT
Congenital heart disease affects 1% of infants and is associated with impaired neurodevelopment. Right- or left-sided sulcal features correlate with executive function among people with Tetralogy of Fallot or single ventricle congenital heart disease. Studies of multiple congenital heart disease types are needed to understand regional differences. Further, sulcal pattern has not been studied in people with d-transposition of the great arteries. Therefore, we assessed the relationship between sulcal pattern and executive function, general memory, and processing speed in a meta-regression of 247 participants with three congenital heart disease types (114 single ventricle, 92 d-transposition of the great arteries, and 41 Tetralogy of Fallot) and 94 participants without congenital heart disease. Higher right hemisphere sulcal pattern similarity was associated with improved executive function (Pearson r = 0.19, false discovery rate-adjusted P = 0.005), general memory (r = 0.15, false discovery rate P = 0.02), and processing speed (r = 0.17, false discovery rate P = 0.01) scores. These positive associations remained significant in for the d-transposition of the great arteries and Tetralogy of Fallot cohorts only in multivariable linear regression (estimated change ß = 0.7, false discovery rate P = 0.004; ß = 4.1, false discovery rate P = 0.03; and ß = 5.4, false discovery rate P = 0.003, respectively). Duration of deep hypothermic circulatory arrest was also associated with outcomes in the multivariate model and regression tree analysis. This suggests that sulcal pattern may provide an early biomarker for prediction of later neurocognitive challenges among people with congenital heart disease.
Subject(s)
Heart Defects, Congenital , Child , Female , Humans , Male , Cerebral Cortex/diagnostic imaging , Cerebral Cortex/pathology , Cerebral Cortex/growth & development , Executive Function/physiology , Heart Defects, Congenital/complications , Heart Defects, Congenital/pathology , Magnetic Resonance Imaging , Neurodevelopmental Disorders/etiology , Neurodevelopmental Disorders/pathology , Adolescent , Young AdultABSTRACT
Microtia is a congenital malformation that encompasses mild hypoplasia to complete loss of the external ear, or pinna. Although the contribution of genetic variation and environmental factors to microtia remains elusive, Amerindigenous populations have the highest reported incidence. Here, using both transmission disequilibrium tests and association studies in microtia trios (parents and affected child) and microtia cohorts enrolled in Latin America, we map an â¼10-kb microtia locus (odds ratio = 4.7; P = 6.78e-18) to the intergenic region between Roundabout 1 (ROBO1) and Roundabout 2 (ROBO2) (chr3: 78546526 to 78555137). While alleles at the microtia locus significantly increase the risk of microtia, their penetrance is low (<1%). We demonstrate that the microtia locus contains a polymorphic complex repeat element that is expanded in affected individuals. The locus is located near a chromatin loop region that regulates ROBO1 and ROBO2 expression in induced pluripotent stem cellderived neural crest cells. Furthermore, we use single nuclear RNA sequencing to demonstrate ROBO1 and ROBO2 expression in both fibroblasts and chondrocytes of the mature human pinna. Because the microtia allele is enriched in Amerindigenous populations and is shared by some East Asian subjects with craniofacial malformations, we propose that both populations share a mutation that arose in a common ancestor prior to the ancient migration of Eurasian populations into the Americas and that the high incidence of microtia among Amerindigenous populations reflects the population bottleneck that occurred during the migration out of Eurasia.
Subject(s)
American Indian or Alaska Native , Congenital Microtia , Congenital Microtia/genetics , Ear, External , Founder Effect , Humans , Mutation , Nerve Tissue Proteins/genetics , Receptors, Immunologic/genetics , American Indian or Alaska Native/genetics , Roundabout ProteinsABSTRACT
Individuals with congenital heart disease (CHD) have an increased risk of neurodevelopmental impairments. Given the hypothesized complexity linking genomics, atypical brain structure, cardiac diagnoses and their management, and neurodevelopmental outcomes, unsupervised methods may provide unique insight into neurodevelopmental variability in CHD. Using data from the Pediatric Cardiac Genomics Consortium Brain and Genes study, we identified data-driven subgroups of individuals with CHD from measures of brain structure. Using structural magnetic resonance imaging (MRI; N = 93; cortical thickness, cortical volume, and subcortical volume), we identified subgroups that differed primarily on cardiac anatomic lesion and language ability. In contrast, using diffusion MRI (N = 88; white matter connectivity strength), we identified subgroups that were characterized by differences in associations with rare genetic variants and visual-motor function. This work provides insight into the differential impacts of cardiac lesions and genomic variation on brain growth and architecture in patients with CHD, with potentially distinct effects on neurodevelopmental outcomes.
Subject(s)
Brain , Heart Defects, Congenital , Magnetic Resonance Imaging , Humans , Heart Defects, Congenital/pathology , Heart Defects, Congenital/diagnostic imaging , Heart Defects, Congenital/genetics , Female , Male , Child , Brain/diagnostic imaging , Brain/pathology , Adolescent , Young Adult , White Matter/diagnostic imaging , White Matter/pathology , Adult , Child, Preschool , Diffusion Magnetic Resonance Imaging , Neurodevelopmental Disorders/diagnostic imaging , Neurodevelopmental Disorders/pathology , Neurodevelopmental Disorders/geneticsABSTRACT
BACKGROUND: Paenibacillus thiaminolyticus may be an underdiagnosed cause of neonatal sepsis. METHODS: We prospectively enrolled a cohort of 800 full-term neonates presenting with a clinical diagnosis of sepsis at 2 Ugandan hospitals. Quantitative polymerase chain reaction specific to P. thiaminolyticus and to the Paenibacillus genus were performed on the blood and cerebrospinal fluid (CSF) of 631 neonates who had both specimen types available. Neonates with Paenibacillus genus or species detected in either specimen type were considered to potentially have paenibacilliosis, (37/631, 6%). We described antenatal, perinatal, and neonatal characteristics, presenting signs, and 12-month developmental outcomes for neonates with paenibacilliosis versus clinical sepsis due to other causes. RESULTS: Median age at presentation was 3 days (interquartile range 1, 7). Fever (92%), irritability (84%), and clinical signs of seizures (51%) were common. Eleven (30%) had an adverse outcome: 5 (14%) neonates died during the first year of life; 5 of 32 (16%) survivors developed postinfectious hydrocephalus (PIH) and 1 (3%) additional survivor had neurodevelopmental impairment without hydrocephalus. CONCLUSIONS: Paenibacillus species was identified in 6% of neonates with signs of sepsis who presented to 2 Ugandan referral hospitals; 70% were P. thiaminolyticus. Improved diagnostics for neonatal sepsis are urgently needed. Optimal antibiotic treatment for this infection is unknown but ampicillin and vancomycin will be ineffective in many cases. These results highlight the need to consider local pathogen prevalence and the possibility of unusual pathogens when determining antibiotic choice for neonatal sepsis.
Subject(s)
Hydrocephalus , Neonatal Sepsis , Paenibacillus , Sepsis , Infant, Newborn , Humans , Female , Pregnancy , Uganda/epidemiology , Sepsis/complications , Sepsis/epidemiology , Sepsis/drug therapy , Anti-Bacterial Agents/therapeutic use , Disease ProgressionABSTRACT
Tethered cord syndrome (TCS) is characterized by leg pain and weakness, bladder and bowel dysfunction, orthopedic malformations such as scoliosis, and motor deficits caused by the fixation of the spinal cord to surrounding tissues. TCS is surgically treatable and often found in conjunction with other syndromic conditions. KBG syndrome is caused by variants in the ANKRD11 gene and is characterized by short stature, developmental delay, macrodontia, and a triangular face. The current study explores the prevalence of TCS in pediatric KBG patients and their associated signs and symptoms. Patients with KBG were surveyed for signs and symptoms associated with TCS and asked if they had been diagnosed with the syndrome. We found a high proportion of patients diagnosed with (11%) or being investigated for TCS (24%), emphasizing the need to further characterize the comorbid syndromes. No signs or symptoms clearly emerged as indicative of TCS in KBG patients, but some the prevalence of some signs and symptoms varied by sex. Male KBG patients with diagnosed TCS were more likely to have coordination issues and global delay/brain fog than their female counterparts. Understanding the presentation of TCS in KBG patients is critical for timely diagnosis and treatment.
Subject(s)
Abnormalities, Multiple , Bone Diseases, Developmental , Intellectual Disability , Neural Tube Defects , Tooth Abnormalities , Humans , Male , Child , Female , Abnormalities, Multiple/diagnosis , Abnormalities, Multiple/genetics , Intellectual Disability/genetics , Bone Diseases, Developmental/genetics , Tooth Abnormalities/genetics , Facies , Phenotype , Repressor Proteins/genetics , Neural Tube Defects/complications , Neural Tube Defects/diagnosis , Neural Tube Defects/epidemiology , SyndromeABSTRACT
Although DNA methylation is the best characterized epigenetic mark, the mechanism by which it is targeted to specific regions in the genome remains unclear. Recent studies have revealed that local DNA methylation profiles might be dictated by cis-regulatory DNA sequences that mainly operate via DNA-binding factors. Consistent with this finding, we have recently shown that disruption of CTCF-binding sites by rare single nucleotide variants (SNVs) can underlie cis-linked DNA methylation changes in patients with congenital anomalies. These data raise the hypothesis that rare genetic variation at transcription factor binding sites (TFBSs) might contribute to local DNA methylation patterning. In this work, by combining blood genome-wide DNA methylation profiles, whole genome sequencing-derived SNVs from 247 unrelated individuals along with 133 predicted TFBS motifs derived from ENCODE ChIP-Seq data, we observed an association between the disruption of binding sites for multiple TFs by rare SNVs and extreme DNA methylation values at both local and, to a lesser extent, distant CpGs. While the majority of these changes affected only single CpGs, 24% were associated with multiple outlier CpGs within ±1kb of the disrupted TFBS. Interestingly, disruption of functionally constrained sites within TF motifs lead to larger DNA methylation changes at nearby CpG sites. Altogether, these findings suggest that rare SNVs at TFBS negatively influence TF-DNA binding, which can lead to an altered local DNA methylation profile. Furthermore, subsequent integration of DNA methylation and RNA-Seq profiles from cardiac tissues enabled us to observe an association between rare SNV-directed DNA methylation and outlier expression of nearby genes. In conclusion, our findings not only provide insights into the effect of rare genetic variation at TFBS on shaping local DNA methylation and its consequences on genome regulation, but also provide a rationale to incorporate DNA methylation data to interpret the functional role of rare variants.
Subject(s)
CpG Islands/genetics , DNA Methylation , Epigenesis, Genetic , Genome, Human/genetics , Transcription Factors/metabolism , Adolescent , Adult , Binding Sites/genetics , Child , Child, Preschool , Chromatin Immunoprecipitation Sequencing , Cohort Studies , Female , Heart Defects, Congenital/blood , Heart Defects, Congenital/genetics , Humans , Infant , Infant, Newborn , Male , Middle Aged , Polymorphism, Single Nucleotide , Whole Genome Sequencing , Young AdultABSTRACT
We introduce mirTarRnaSeq, an R/Bioconductor package for quantitative assessment of miRNA-mRNA relationships within sample cohorts. mirTarRnaSeq is a statistical package to explore predicted or pre-hypothesized miRNA-mRNA relationships following target prediction.We present two use cases applying mirTarRnaSeq. First, to identify miRNA targets, we examined EBV miRNAs for interaction with human and virus transcriptomes of stomach adenocarcinoma. This revealed enrichment of mRNA targets highly expressed in CD105+ endothelial cells, monocytes, CD4+ T cells, NK cells, CD19+ B cells, and CD34 cells. Next, to investigate miRNA-mRNA relationships in SARS-CoV-2 (COVID-19) infection across time, we used paired miRNA and RNA sequenced datasets of SARS-CoV-2 infected lung epithelial cells across three time points (4, 12, and 24 hours post-infection). mirTarRnaSeq identified evidence for human miRNAs targeting cytokine signaling and neutrophil regulation immune pathways from 4 to 24 hours after SARS-CoV-2 infection. Confirming the clinical relevance of these predictions, three of the immune specific mRNA-miRNA relationships identified in human lung epithelial cells after SARS-CoV-2 infection were also observed to be differentially expressed in blood from patients with COVID-19. Overall, mirTarRnaSeq is a robust tool that can address a wide-range of biological questions providing improved prediction of miRNA-mRNA interactions.
Subject(s)
COVID-19 , MicroRNAs , COVID-19/genetics , Endothelial Cells , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2ABSTRACT
Neurodevelopmental disabilities are the most common noncardiac conditions in patients with congenital heart disease (CHD). Executive function skills have been frequently observed to be decreased among children and adults with CHD compared with peers, but a neuroanatomical basis for the association is yet to be identified. In this study, we quantified sulcal pattern features from brain magnetic resonance imaging data obtained during adolescence among 41 participants with tetralogy of Fallot (ToF) and 49 control participants using a graph-based pattern analysis technique. Among patients with ToF, right-hemispheric sulcal pattern similarity to the control group was decreased (0.7514 vs. 0.7553, P = 0.01) and positively correlated with neuropsychological testing values including executive function (r = 0.48, P < 0.001). Together these findings suggest that sulcal pattern analysis may be a useful marker of neurodevelopmental risk in patients with CHD. Further studies may elucidate the mechanisms leading to different alterations in sulcal patterning.
Subject(s)
Executive Function , Tetralogy of Fallot/diagnostic imaging , Tetralogy of Fallot/psychology , Adolescent , Adult , Brain/diagnostic imaging , Case-Control Studies , Cerebral Cortex/diagnostic imaging , Child , Developmental Disabilities/physiopathology , Developmental Disabilities/psychology , Female , Heart Defects, Congenital , Humans , Magnetic Resonance Imaging , Male , Neuropsychological Tests , Young AdultABSTRACT
Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.
Subject(s)
GTP-Binding Proteins/genetics , Mammals/genetics , Microfilament Proteins/genetics , Monosomy/genetics , Animals , Cell Line , Humans , Mice , Mice, Inbred C57BL , Mutation/genetics , Phenotype , Polyribosomes/genetics , Proteasome Endopeptidase Complex/genetics , RNA/genetics , RNA, Messenger/genetics , Ribosomes/genetics , TOR Serine-Threonine Kinases/genetics , Up-Regulation/geneticsABSTRACT
Quantification of proton magnetic resonance spectroscopy (1 H-MRS) data is commonly performed by referencing the ratio of the signal from one metabolite, or metabolite group, to that of another, or to the water signal. Both approaches have drawbacks: ratios of two metabolites can be difficult to interpret because study effects may be driven by either metabolite, and water-referenced data must be corrected for partial volume and relaxation effects in the water signal. Here, we introduce combined reference (CRef) analysis, which compensates for both limitations. In this approach, metabolites are referenced to the combined signal of several reference metabolites or metabolite groups. The approach does not require the corrections necessary for water scaling and produces results that are less sensitive to the variation of any single reference signal, thereby aiding the interpretation of results. We demonstrate CRef analysis using 202 1 H-MRS acquisitions from the brains of 140 infants, scanned at approximately 1 and 3 months of age. We show that the combined signal of seven reference metabolites or metabolite groups is highly correlated with the water signal, corrected for partial volume and relaxation effects associated with cerebral spinal fluid. We also show that the combined reference signal is equally or more uniform across subjects than the reference signals from single metabolites or metabolite groups. We use CRef analysis to quantify metabolite concentration changes during the first several months of life in typically developing infants.
Subject(s)
Data Analysis , Magnetic Resonance Spectroscopy , Corpus Callosum/diagnostic imaging , Female , Gray Matter/diagnostic imaging , Humans , Infant , Male , Metabolome , Reference Standards , Signal Processing, Computer-Assisted , Water , White Matter/diagnostic imagingABSTRACT
Maternal nutrition is an important factor for infant neurodevelopment. However, prior magnetic resonance imaging (MRI) studies on maternal nutrients and infant brain have focused mostly on preterm infants or on few specific nutrients and few specific brain regions. We present a first study in term-born infants, comprehensively correlating 73 maternal nutrients with infant brain morphometry at the regional (61 regions) and voxel (over 300 000 voxel) levels. Both maternal nutrition intake diaries and infant MRI were collected at 1 month of life (0.9 ± 0.5 months) for 92 term-born infants (among them, 54 infants were purely breastfed and 19 were breastfed most of the time). Intake of nutrients was assessed via standardized food frequency questionnaire. No nutrient was significantly correlated with any of the volumes of the 61 autosegmented brain regions. However, increased volumes within subregions of the frontal cortex and corpus callosum at the voxel level were positively correlated with maternal intake of omega-3 fatty acids, retinol (vitamin A) and vitamin B12, both with and without correction for postmenstrual age and sex (P < 0.05, q < 0.05 after false discovery rate correction). Omega-3 fatty acids remained significantly correlated with infant brain volumes after subsetting to the 54 infants who were exclusively breastfed, but retinol and vitamin B12 did not. This provides an impetus for future larger studies to better characterize the effect size of dietary variation and correlation with neurodevelopmental outcomes, which can lead to improved nutritional guidance during pregnancy and lactation.
Subject(s)
Brain/diagnostic imaging , Brain/growth & development , Breast Feeding/trends , Child Development/physiology , Fatty Acids, Omega-3/administration & dosage , Maternal Nutritional Physiological Phenomena/physiology , Adult , Cohort Studies , Female , Humans , Infant , Infant, Newborn , Organ Size/physiology , Pregnancy , Prospective StudiesABSTRACT
Neurodevelopmental abnormalities are the most common noncardiac complications in patients with congenital heart disease (CHD). Prenatal brain abnormalities may be due to reduced oxygenation, genetic factors, or less commonly, teratogens. Understanding the contribution of these factors is essential to improve outcomes. Because primary sulcal patterns are prenatally determined and under strong genetic control, we hypothesized that they are influenced by genetic variants in CHD. In this study, we reveal significant alterations in sulcal patterns among subjects with single ventricle CHD (n = 115, 14.7 ± 2.9 years [mean ± standard deviation]) compared with controls (n = 45, 15.5 ± 2.4 years) using a graph-based pattern-analysis technique. Among patients with CHD, the left hemisphere demonstrated decreased sulcal pattern similarity to controls in the left temporal and parietal lobes, as well as the bilateral frontal lobes. Temporal and parietal lobes demonstrated an abnormally asymmetric left-right pattern of sulcal basin area in CHD subjects. Sulcal pattern similarity to control was positively correlated with working memory, processing speed, and executive function. Exome analysis identified damaging de novo variants only in CHD subjects with more atypical sulcal patterns. Together, these findings suggest that sulcal pattern analysis may be useful in characterizing genetically influenced, atypical early brain development and neurodevelopmental risk in subjects with CHD.
Subject(s)
Cerebrum/pathology , Heart Defects, Congenital/complications , Neurodevelopmental Disorders/etiology , Adolescent , Cerebrum/diagnostic imaging , Female , Heart Defects, Congenital/genetics , Humans , Magnetic Resonance Imaging , Male , Neurodevelopmental Disorders/pathology , Neurodevelopmental Disorders/psychology , Neuropsychological TestsABSTRACT
microRNAs (miRNAs) regulate messenger RNA (mRNA) abundance and translation during key developmental processes including muscle differentiation. Assessment of miRNA targets can provide insight into muscle biology and gene expression profiles altered by disease. mRNA and miRNA libraries were generated from C2C12 myoblasts during differentiation, and predicted miRNA targets were identified based on presence of miRNA binding sites and reciprocal expression. Seventeen miRNAs were differentially expressed at all time intervals (comparing days 0, 2, and 5) of differentiation. mRNA targets of differentially expressed miRNAs were enriched for functions related to calcium signaling and sarcomere formation. To evaluate this relationship in a disease state, we evaluated the miRNAs differentially expressed in human congenital myotonic dystrophy (CMD) myoblasts and compared with normal control. Seventy-four miRNAs were differentially expressed during healthy human myocyte maturation, of which only 12 were also up- or downregulated in CMD patient cells. The 62 miRNAs that were only differentially expressed in healthy cells were compared with differentiating C2C12 cells. Eighteen of the 62 were conserved in mouse and up- or down-regulated during mouse myoblast differentiation, and their C2C12 targets were enriched for functions related to muscle differentiation and contraction.
Subject(s)
MicroRNAs/genetics , Muscle, Skeletal/cytology , Myoblasts/cytology , Myotonic Dystrophy/genetics , RNA, Messenger/genetics , Animals , Calcium Signaling/genetics , Cell Differentiation , Cell Line , Genes, X-Linked , Humans , Mice , Myoblasts/physiology , Myotonic Dystrophy/pathology , Sarcomeres/genetics , TranscriptomeABSTRACT
Eukaryotic elongation factor 1A (EEF1A), is encoded by two distinct isoforms, EEF1A1 and EEF1A2; whereas EEF1A1 is expressed almost ubiquitously, EEF1A2 expression is limited such that it is only detectable in skeletal muscle, heart, brain and spinal cord. Currently, the role of EEF1A2 in normal cardiac development and function is unclear. There have been several reports linking de novo dominant EEF1A2 mutations to neurological issues in humans. We report a pair of siblings carrying a homozygous missense mutation p.P333L in EEF1A2 who exhibited global developmental delay, failure to thrive, dilated cardiomyopathy and epilepsy, ultimately leading to death in early childhood. A third sibling also died of a similar presentation, but DNA was unavailable to confirm the mutation. Functional genomic analysis was performed in S. cerevisiae and zebrafish. In S. cerevisiae, there was no evidence for a dominant-negative effect. Previously identified putative de novo mutations failed to complement yeast strains lacking the EEF1A ortholog showing a major growth defect. In contrast, the introduction of the mutation seen in our family led to a milder growth defect. To evaluate its function in zebrafish, we knocked down eef1a2 expression using translation blocking and splice-site interfering morpholinos. EEF1A2-deficient zebrafish had skeletal muscle weakness, cardiac failure and small heads. Human EEF1A2 wild-type mRNA successfully rescued the morphant phenotype, but mutant RNA did not. Overall, EEF1A2 appears to be critical for normal heart function in humans, and its deficiency results in clinical abnormalities in neurologic function as well as in skeletal and cardiac muscle defects.
Subject(s)
Cardiomyopathy, Dilated/genetics , Peptide Elongation Factor 1/genetics , Animals , Cardiomyopathy, Dilated/metabolism , Developmental Disabilities/genetics , Epilepsy/genetics , Failure to Thrive/genetics , Genomics , Homozygote , Humans , Models, Animal , Mutation , Mutation, Missense/genetics , Peptide Elongation Factor 1/metabolism , Protein Isoforms/genetics , Saccharomyces cerevisiae/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
PURPOSE: We evaluated genome sequencing (GS) as an alternative to multigene panel sequencing (PS) for genetic testing in dilated cardiomyopathy (DCM). METHODS: Forty-two patients with familial DCM underwent PS and GS, and detection rates of rare single-nucleotide variants and small insertions/deletions in panel genes were compared. Loss-of-function variants in 406 cardiac-enriched genes were evaluated, and an assessment of structural variation was performed. RESULTS: GS provided broader and more uniform coverage than PS, with high concordance for rare variant detection in panel genes. GS identified all PS-identified pathogenic or likely pathogenic variants as well as two additional likely pathogenic variants: one was missed by PS due to low coverage, the other was a known disease-causing variant in a gene not included on the panel. No loss-of-function variants in the extended gene set met clinical criteria for pathogenicity. One BAG3 structural variant was classified as pathogenic. CONCLUSION: Our data support the use of GS for genetic testing in DCM, with high variant detection accuracy and a capacity to identify structural variants. GS provides an opportunity to go beyond suites of established disease genes, but the incremental yield of clinically actionable variants is limited by a paucity of genetic and functional evidence for DCM association.
Subject(s)
Cardiomyopathy, Dilated/genetics , Genetic Testing/methods , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Female , Genetic Predisposition to Disease/genetics , Humans , INDEL Mutation , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Sequence Analysis, DNA/methods , Whole Genome Sequencing/methodsABSTRACT
MicroRNAs (miRNAs) are regulators of myriad cellular events, but evidence for a single miRNA that can efficiently differentiate multipotent stem cells into a specific lineage or regulate direct reprogramming of cells into an alternative cell fate has been elusive. Here we show that miR-145 and miR-143 are co-transcribed in multipotent murine cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem-cell-derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2-5 (NK2 transcription factor related, locus 5) and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4 (Kruppel-like factor 4), myocardin and Elk-1 (ELK1, member of ETS oncogene family), to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells.
Subject(s)
Cell Lineage , MicroRNAs/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Animals , Cell Differentiation , Cell Proliferation , Female , Gene Expression Regulation , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/metabolism , Kruppel-Like Factor 4 , Male , Mice , Mice, Transgenic , MicroRNAs/genetics , Models, Biological , Myocardium/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Vascular Diseases/metabolism , ets-Domain Protein Elk-4/metabolismABSTRACT
Congenital heart disease (CHD) is increasingly diagnosed prenatally and the ability to screen and diagnose the genetic factors involved in CHD have greatly improved. The presence of a genetic abnormality in the setting of prenatally diagnosed CHD impacts prenatal counseling and ensures that families and providers have as much information as possible surrounding perinatal management and what to expect in the future. This review will discuss the genetic evaluation that can occur prior to birth, what different genetic testing methods are available, and what to think about in the setting of various CHD diagnoses.