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. 2013 Jun;21(6):637-42.
doi: 10.1038/ejhg.2012.226. Epub 2012 Oct 24.

Dynamin 2 Homozygous Mutation in Humans With a Lethal Congenital Syndrome

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Free PMC article

Dynamin 2 Homozygous Mutation in Humans With a Lethal Congenital Syndrome

Olga S Koutsopoulos et al. Eur J Hum Genet. .
Free PMC article

Abstract

Heterozygous mutations in dynamin 2 (DNM2) have been linked to dominant Charcot-Marie-Tooth neuropathy and centronuclear myopathy. We report the first homozygous mutation in the DNM2 protein p.Phe379Val, in three consanguineous patients with a lethal congenital syndrome associating akinesia, joint contractures, hypotonia, skeletal abnormalities, and brain and retinal hemorrhages. In vitro membrane tubulation, trafficking and GTPase assays are consistent with an impact of the DNM2p.Phe379Val mutation on endocytosis. Although DNM2 has been previously implicated in axonal and muscle maintenance, the clinical manifestation in our patients taken together with our expression analysis profile during mouse embryogenesis and knockdown approaches in zebrafish resulting in defects in muscle organization and angiogenesis support a pleiotropic role for DNM2 during fetal development in vertebrates and humans.

Figures

Figure 1
Figure 1
Identification of a homozygous DNM2 mutation. (a) Pedigree of the consanguineous Pakistani family investigated. Heterozygous carriers are indicated by dots. (b) Direct sequencing analysis. A single base substitution (c.1135 T>G; Genbank "file:///D:\CopyEditJobs\NPG\EJHG\ejhg2012226\genbank" >NM_001005360) in exon 9 of DNM2 was identified (indicated by black box) and was found homozygous in all affected and heterozygous in unaffected family members. (c) Hands of AFB3 and AFB2 display contractures. (d) Quadriceps biopsies depicting the presence of centralized nuclei in AFB2. Some fibers with centralized nuclei are indicated by arrows. Scale bars represent 50 μm. (e) Anteroposterior radiograph of the thorax and abdomen of AFB3 depicting thin ribs with normal length and bone density. Note the deep position of the endotracheal tube. (f) MRI analysis of patient AFB3. From left to right: axial T1-weighted, susceptibility, T2-weighted images of the cerebrum at the level of the basal ganglia, and axial T2-weighted image at the level of the brainstem. Bilateral hyperintense signal in the occipital subdural space (arrows) and posterior horns of the ventricles (arrowheads) with low signal intensity on the susceptibility image and hypointense signal on T2-weighted image are consistent with bilateral subdural hematomas and intraventricular hemorrhage. Symmetric areas of low signal intensity within the thalamus and globus pallidus (asterisk) are suggestive of early subacute 3–7 day-old hemorrhage. The axial T2-weighted image at the level of the brainstem indicates hemorrhagic changes in the brainstem.
Figure 2
Figure 2
Effect of the p.Phe379Val mutation on DNM2 function. (a) DNM2 protein domains with the p.Phe379Val point mutation indicated (top); GED, GTPase effector domain; MID, middle domain; PH, Pleckstrin homology domain; PRD, proline-rich domain. Protein alignment of the dynamin family members showing conservation of amino-acid F379 in vertebrates (bottom); ce, Caenorhabditis elegans; dm, Drosophila melanogaster; dr, Danio rerio; hs, Homo sapiens; mm, Mus musculus. (b) DNM2 protein levels are comparable between AFB2 (p.Phe379Val) and control fibroblasts as compared by western blot analysis using the in-house polyclonal antibody R2680 against DNM2. (c) p.Phe379Val patient fibroblasts exhibit reduced transferrin uptake compared with untreated controls. Arbitary units describe mean fluorescence of transferrin uptake. Error bars represent SD (P-values *0.00616 and **0.0003522). (d) The p.Phe379Val protein exhibits ∼20% lower GTPase activity using a malachite green-based assay (P-value *0.05 for three independent experiments using the Student's t-test). Error bars represent SD. (e) Electron micrographs depicting wild-type (WT) and p.Phe379Val DNM2 incubated in the presence of small unilammellar vesicles. The p.Phe379Val protein does not tubulate liposomes and typical striated polymers of DNM2 observed in the case of WT DNM2 are absent from vesicles incubated with the DNM2p.Phe379Val protein.
Figure 3
Figure 3
DNM2 expression and function during embryonic development. (a) Dnm2 mRNA can be detected in several tissues during murine embryonic development. In situ hybridization experiments were performed with a Dnm2 mRNA probe (Dnm2) on sagital sections of 14.5-day (top) and 16.5-day (bottom) mouse embryos. Controls represent embryo sections incubated with all reagents but lacking mRNA probe to measure levels of nonspecific reaction (left panels). No Dnm2 mRNA was evident in muscle at this stage. (b) Morphology of muscle fibers and vessels in the tail of control (left panels) and dnm2 MO-injected (right panels) zebrafish embryos at 72 hpf. SHG revealing the myosin fibers organization and the periodic structure of sarcomeric thick filaments (top panels). Note the mild fiber misalignment in the MO-injected embryos (arrow). Confocal reconstructions of the GFP-positive endothelial cells revealing the vascular system of the transgenic Tg(flk1:eGFP) zebrafish (middle panels). In dnm2 MO-injected embryos, the inter-segmental vessels display deficient or abnormal branching (asterisk and white arrows, respectively).

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