We demonstrate the successful application of exome sequencing to discover a gene for an autosomal dominant disorder, Kabuki syndrome (OMIM%147920). We subjected the exomes of ten unrelated probands ...to massively parallel sequencing. After filtering against existing SNP databases, there was no compelling candidate gene containing previously unknown variants in all affected individuals. Less stringent filtering criteria allowed for the presence of modest genetic heterogeneity or missing data but also identified multiple candidate genes. However, genotypic and phenotypic stratification highlighted MLL2, which encodes a Trithorax-group histone methyltransferase: seven probands had newly identified nonsense or frameshift mutations in this gene. Follow-up Sanger sequencing detected MLL2 mutations in two of the three remaining individuals with Kabuki syndrome (cases) and in 26 of 43 additional cases. In families where parental DNA was available, the mutation was confirmed to be de novo (n = 12) or transmitted (n = 2) in concordance with phenotype. Our results strongly suggest that mutations in MLL2 are a major cause of Kabuki syndrome.
Key points
The contractile properties of human fetal cardiac muscle have not been previously studied.
Small‐scale approaches such as isolated myofibril and isolated contractile protein biomechanical ...assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well‐controlled conditions.
We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59–134 days.
Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression.
Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell‐derived cardiomyocytes.
Little is known about the contractile properties of human fetal cardiac muscle during development. Understanding these contractile properties, and how they change throughout development, can provide valuable insight into human heart development, and provide a framework to study the early stages of cardiac diseases that develop in utero. We characterized the contractile properties of isolated human fetal cardiac myofibrils across 8–19 weeks of gestation. Mechanical measurements revealed that in early stages of gestation there is low specific force and slow rates of force development and relaxation, with increases in force and the rates of activation and relaxation as gestation progresses. The duration and slope of the initial, slow phase of relaxation, related to myosin detachment and thin filament deactivation rates, decreased with gestation age. F‐actin sliding on human fetal cardiac myosin‐coated surfaces slowed significantly from 108 to 130 days of gestation. Electron micrographs showed human fetal muscle myofibrils elongate and widen with age, but features such as the M‐line and Z‐band are apparent even as early as day 52. Protein isoform analysis revealed that β‐myosin is predominantly expressed even at the earliest time point studied, but there is a progressive increase in expression of cardiac troponin I (TnI), with a concurrent decrease in slow skeletal TnI. Together, our results suggest that cardiac myofibril force production and kinetics of activation and relaxation change significantly with gestation age and are influenced by the structural maturation of the sarcomere and changes in contractile filament protein isoforms.
Key points
The contractile properties of human fetal cardiac muscle have not been previously studied.
Small‐scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well‐controlled conditions.
We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59–134 days.
Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression.
Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell‐derived cardiomyocytes.
Beckwith-Wiedemann syndrome (BWS) is a human genomic imprinting disorder characterized by lateralized overgrowth, macroglossia, abdominal wall defects, congenital hyperinsulinism, and predisposition ...to embryonal tumors. One of the molecular etiologies underlying BWS is paternal uniparental isodisomy of chromosome 11p15.5 (pUPD11). About 8% of pUPD11 cases are due to genome-wide paternal uniparental isodisomy (GWpUPD). About 30 cases of live-born patients with GWpUPD have been described, most of whom were mosaic and female. We present male patients with BWS due to GWpUPD, elucidate the underlying mechanism, and make recommendations for management.
Three male patients with GWpUPD underwent clinical and molecular evaluation by single-nucleotide polymorphism (SNP) microarrays in different tissues. Previously published cases of GWpUPD were reviewed.
SNP microarray demonstrated a GWpUPD cell population with sex chromosomes XX and biparental cell population with sex chromosomes XY, consistent with dispermic androgenetic chimerism.
SNP microarray is necessary to distinguish GWpUPD cases and the underlying mechanisms. The percentage of GWpUPD cell population within a specific tissue type correlated with the amount of tissue dysplasia. Males with BWS due to GWpUPD are important to distinguish from other molecular etiologies because the mechanism indicates risk for germ cell tumors and autosomal recessive diseases in addition to other BWS features.
Distal arthrogryposis (DA) syndromes are the most common of the heritable congenital-contracture disorders, and ∼50% of cases are caused by mutations in genes that encode contractile proteins of ...skeletal myofibers. DA type 5D (DA5D) is a rare, autosomal-recessive DA previously defined by us and is characterized by congenital contractures of the hands and feet, along with distinctive facial features, including ptosis. We used linkage analysis and whole-genome sequencing of a multiplex consanguineous family to identify in endothelin-converting enzyme-like 1 (ECEL1) mutations that result in DA5D. Evaluation of a total of seven families affected by DA5D revealed in five families ECEL1 mutations that explain ∼70% of cases overall. ECEL1 encodes a neuronal endopeptidase and is expressed in the brain and peripheral nerves. Mice deficient in Ecel1 exhibit perturbed terminal branching of motor neurons to the endplate of skeletal muscles, resulting in poor formation of the neuromuscular junction. Our results distinguish a second developmental pathway that causes congenital-contracture syndromes.
Distal arthrogryposis is the most common known heritable cause of congenital contractures (e.g. clubfoot) and results from mutations in genes that encode proteins of the contractile complex of ...skeletal muscle cells. Mutations are most frequently found in MYH3 and are predicted to impair the function of embryonic myosin. We measured the contractile properties of individual skeletal muscle cells and the activation and relaxation kinetics of isolated myofibrils from two adult individuals with an R672C substitution in embryonic myosin and distal arthrogryposis syndrome 2A (DA2A) or Freeman-Sheldon syndrome. In R672C-containing muscle cells, we observed reduced specific force, a prolonged time to relaxation and incomplete relaxation (elevated residual force). In R672C-containing muscle myofibrils, the initial, slower phase of relaxation had a longer duration and slower rate, and time to complete relaxation was greatly prolonged. These observations can be collectively explained by a small subpopulation of myosin cross-bridges with greatly reduced detachment kinetics, resulting in a slower and less complete deactivation of thin filaments at the end of contractions. These findings have important implications for selecting and testing directed therapeutic options for persons with DA2A and perhaps congenital contractures in general.
The objective of this study was to determine the effect of offering animals a multiforage choice (MF) of fresh herbages on dry matter intake (DMI), live weight gain, and animal welfare, in comparison ...with a monotonous diet of ryegrass (Lolium perenne L.). Twenty ram lambs (30.5 ± 0.9 kg initial live weight; mean ± SEM), were randomly allocated to either a diet consisting of diverse MF choice or a single forage ryegrass (SF) diet (n = 10 per treatment) for 35 d. Both diets were fed ad libitum; however, the MF diet was composed of set dry matter ratios of 24% chicory (Cichorium intybus L.), 30% lucerne (Medicago sativa L.), 25% plantain (Plantago lanceolata L.), and 21% ryegrass. The DMI of the MF lambs was 48% greater (P < 0.01) and the within animal day-to-day coefficient of variation (CV) of intake was 26% lower (P < 0.01) than the SF lambs. The average daily gain (ADG) of lambs offered the MF diet was 92% greater (P < 0.01) than the lambs offered the SF diet. The within-animal day-to-day CV of intake was negatively related to ADG (r = −0.59; P < 0.01). The MF lamb’s urinary N concentration was 30% lower (P < 0.01) than that of the SF lambs. The SF lambs spent more time (P < 0.05) exhibiting stereotypic behaviors in the afternoon and spent more time observing other animals than the MF. Overall, allocating an MF choice of fresh herbages as opposed to a single forage diet of ryegrass increases DMI and thereby animal performance, while potentially reducing urinary N excretion.
Glycosylation is a critical post/peri-translational modification required for the appropriate development and function of the immune system. As an example, abnormalities in glycosylation can cause ...antibody deficiency and reduced lymphocyte signaling, although the phenotype can be complex given the diverse roles of glycosylation. Human MGAT2 encodes N-acetylglucosaminyltransferase II, which is a critical enzyme in the processing of oligomannose to complex N-glycans. Complex N-glycans are essential for immune system functionality, but only one individual with MGAT2-CDG has been described to have an abnormal immunologic evaluation. MGAT2-CDG (CDG-IIa) is a congenital disorder of glycosylation (CDG) associated with profound global developmental disability, hypotonia, early onset epilepsy, and other multisystem manifestations. Here, we report a 4-year old female with MGAT2-CDG due to a novel homozygous pathogenic variant in MGAT2, a 4-base pair deletion, c.1006_1009delGACA. In addition to clinical features previously described in MGAT2-CDG, she experienced episodic asystole, persistent hypogammaglobulinemia, and defective ex vivo mitogen and antigen proliferative responses, but intact specific vaccine antibody titers. Her infection history has been mild despite the testing abnormalities. We compare this patient to the 15 previously reported patients in the literature, thus expanding both the genotypic and phenotypic spectrum for MGAT2-CDG.
Key points
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The contractile properties of human fetal skeletal muscle are unknown.
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Reductionist approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow ...study of activation and relaxation properties of skeletal muscle from different sources.
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We have tested the contractile properties of human fetal skeletal myofibrils and myosin in comparison with myosin and myofibrils from human adult skeletal muscle and rabbit psoas muscle.
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Human fetal skeletal myofibrils have much slower kinetics of activation and relaxation compared to myofibrils from adult human or rabbit psoas skeletal muscle.
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Investigations using altered substrate and product conditions for both the in vitro motility assay and myofibril mechanics/kinetics indicate that fetal muscle acto‐myosin crossbridges cycle more slowly than, but with similar rate‐limiting steps to, the adult myosin isoforms.
Little is known about the contraction and relaxation properties of fetal skeletal muscle, and measurements thus far have been made with non‐human mammalian muscle. Data on human fetal skeletal muscle contraction are lacking, and there are no published reports on the kinetics of either fetal or adult human skeletal muscle myofibrils. Understanding the contractile properties of human fetal muscle would be valuable in understanding muscle development and a variety of muscle diseases that are associated with mutations in fetal muscle sarcomere proteins. Therefore, we characterised the contractile properties of developing human fetal skeletal muscle and compared them to adult human skeletal muscle and rabbit psoas muscle. Electron micrographs showed human fetal muscle sarcomeres are not fully formed but myofibril formation is visible. Isolated myofibril mechanical measurements revealed much lower specific force, and slower rates of isometric force development, slow phase relaxation, and fast phase relaxation in human fetal when compared to human adult skeletal muscle. The duration of slow phase relaxation was also significantly longer compared to both adult groups, but was similarly affected by elevated ADP. F‐actin sliding on human fetal skeletal myosin coated surfaces in in vitro motility (IVM) assays was much slower compared with adult rabbit skeletal myosin, though the Km(app) (apparent (fitted) Michaelis–Menten constant) of F‐actin speed with ATP titration suggests a greater affinity of human fetal myosin for nucleotide binding. Replacing ATP with 2 deoxy‐ATP (dATP) increased F‐actin speed for both groups by a similar amount. Titrations of ADP into IVM assays produced a similar inhibitory affect for both groups, suggesting ADP binding may be similar, at least under low load. Together, our results suggest slower but similar mechanisms of myosin chemomechanical transduction for human fetal muscle that may also be limited by immature myofilament structure.
Feeding intolerance in Prader-Willi syndrome (PWS) infants is well-recognized, but their swallow physiology is not well understood. Swallow dysfunction increases risks of respiratory compromise and ...choking, which have a high incidence in PWS. To investigate swallow pathology in PWS infants we undertook a retrospective review of videofluoroscopic swallow studies (VFSS) in infants with PWS seen at our institution. We hypothesize that VFSS will characterize swallow pathology suspected by clinical observation during a feeding evaluation and may help determine feeding safety in these infants.Retrospective review of 23 VFSS on 10 PWS infants (average age 9.7 ± 8.4 months; range 3 weeks-29 months). Logistic regression models evaluated associations between gender, genetic subtype, and growth hormone (GH) use on aspiration incidence. Polysomnographic (PSG) studies conducted on the same participant ±1 year from VFSS were examined to characterize respiratory abnormalities.There was a high rate of swallowing dysfunction (pharyngeal residue 71%, aspiration events 87%) and disordered sleep. All aspiration events were silent. There were no differences in rates of aspiration for gender, genetic subtype, or GH use.A high incidence of aspiration was identified indicating swallow dysfunction may frequently be present in infants with PWS. Comprehensive evaluation of feeding and swallowing is essential and requires a multidisciplinary approach. Providers should recognize risk factors for swallow dysfunction and consider a multidisciplinary approach to guide decision making and optimize feeding safety in PWS.
The non‐POU domain‐containing octamer‐binding (NONO) protein is involved in multiple steps of gene regulation such as RNA metabolism and DNA repair. Hemizygous pathogenic variants in the NONO gene ...were confirmed to cause a rare X‐linked syndromic disorder. Through our in‐house diagnostics and subsequent matchmaking, we identified six unrelated male individuals with pathogenic or likely pathogenic NONO variants. For a detailed comparison, we reviewed all published characterizations of the NONO‐associated disorder. The combined cohort consists of 16 live‐born males showing developmental delay, corpus callosum anomalies, non‐compaction cardiomyopathy and relative macrocephaly as leading symptoms. Seven prenatal literature cases were characterized by cardiac malformations. In this study, we extend the phenotypic spectrum through two more cases with epilepsy as well as two more cases with hematologic anomalies. By RNA expression analysis and structural modeling of a new in‐frame splice deletion, we reinforce loss‐of‐function as the pathomechanism for the NONO‐associated syndromic disorder.
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