GNE myopathy is a rare autosomal recessive muscle disease caused by mutations in GNE, the gene encoding the rate-limiting enzyme in sialic acid biosynthesis. GNE myopathy usually manifests in early adulthood with distal myopathy that progresses slowly and symmetrically, first involving distal muscles of the lower extremities, followed by proximal muscles with relative sparing of the quadriceps. Upper extremities are typically affected later in the disease. We report a patient with GNE myopathy who presented with asymmetric hand weakness. He had considerably decreased left grip strength, atrophy of the left anterior forearm and fibro-fatty tissue replacement of left forearm flexor muscles on T1-weighted magnetic resonance imaging. The patient was an endoscopist and thus the asymmetric hand involvement may be associated with left hand overuse in daily repetitive pinching and gripping movements, highlighting the possible impact of environmental factors on the progression of genetic muscle conditions.
OPA3-related 3-methylglutaconic aciduria, or Costeff Optic Atrophy syndrome, is a neuro-ophthalmologic syndrome of early-onset bilateral optic atrophy and later-onset spasticity, and extrapyramidal dysfunction. Urinary excretion of 3-methylglutaconic acid and of 3-methylglutaric acid is markedly increased. OPA3-related 3-methylglutaconic aciduria is due to mutations in the OPA3 gene located at 19q13.2-13.3. Here we describe two siblings with novel compound heterozygous variants in OPA3: c.1A>G (p.1M>V) in the translation initiation codon in exon 1 and a second variant, c.142+5G>C in intron 1. On cDNA sequencing the c.1A>G appeared homozygous, indicating that the allele without the c.1A>G variant is degraded. This is likely due to an intronic variant; possibly the IVS1+5 splice site variant. The older female sibling initially presented with motor developmental delay and vertical nystagmus during her first year of life and was diagnosed subsequently with optic atrophy. Her brother presented with mildly increased hip muscle tone followed by vertical nystagmus within the first 6 months of life, and was found to have elevated urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid, and optic atrophy by 1.5 years of age. Currently, ages 16 and 7, both children exhibit ataxic gaits and dysarthric speech. Immunofluorescence studies on patient's cells showed fragmented mitochondrial morphology. Thus, though the exact function of OPA3 remains unknown, our experimental results and clinical summary provide evidence for the pathogenicity of the identified OPA3 variants and provide further evidence for a mitochondrial pathology in this disease.
The GNE gene encodes the rate-limiting, bifunctional enzyme of sialic acid biosynthesis, uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Biallelic GNE mutations underlie GNE myopathy, an adult-onset progressive myopathy. GNE myopathy-associated GNE mutations are predominantly missense, resulting in reduced, but not absent, GNE enzyme activities. The exact pathomechanism of GNE myopathy remains unknown, but likely involves aberrant (muscle) sialylation. Here, we summarize 154 reported and novel GNE variants associated with GNE myopathy, including 122 missense, 11 nonsense, 14 insertion/deletions, and seven intronic variants. All variants were deposited in the online GNE variation database (http://www.dmd.nl/nmdb2/home.php?select_db=GNE). We report the predicted effects on protein function of all variants well as the predicted effects on epimerase and/or kinase enzymatic activities of selected variants. By analyzing exome sequence databases, we identified three frequently occurring, unreported GNE missense variants/polymorphisms, important for future sequence interpretations. Based on allele frequencies, we estimate the world-wide prevalence of GNE myopathy to be ?4-21/1,000,000. This previously unrecognized high prevalence confirms suspicions that many patients may escape diagnosis. Awareness among physicians for GNE myopathy is essential for the identification of new patients, which is required for better understanding of the disorder's pathomechanism and for the success of ongoing treatment trials.
This study describes the organization of mature hyaline xiphoid cartilage during repair in animals submitted to electrical current stimulation. Twenty male Wistar rats, 90 days old, were divided into a control group (CG) and a treated group (TG). A cylindrical full-thickness cartilage defects were created with a 3-mm punch in anesthetized animals. After 24h, TG received daily applications of a continuous electrical current (1Hz/20?A) for 5min. The animals were sacrificed after 7, 21 and 35 days for structural analysis. In CG, the repair tissue presented fibrous characteristics, with fibroblastic cells being infiltrated and permeated by blood vessels. Basophilic foci of cartilage tissue were observed on day 35. In TG, the repair tissue also presented fibrous characteristics, but a larger number of thick collagen fibers were seen on day 21. A large number of cartilaginous nests were observed on day 35. Cell numbers were significantly higher in TG. Calcification points were detected in TG on day 35. There was no difference in elastic fibers between groups. Ultrastructural analysis revealed the presence of chondrocyte-like cells in CG at all time points, but only on days 21 and 35 in TG. The amount of cuprolinic blue-stained proteoglycans was higher in TG on day 35. Microcurrent stimulation accelerates the repair process in non-articular hyaline cartilage.
Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1?,25(OH)2D3) in the development of those disorders in two individuals.
UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the three previously described human GNE isoforms (hGNE1-hGNE3), our database and polymerase chain reaction analysis yielded five additional human isoforms (hGNE4-hGNE8). hGNE1 is the ubiquitously expressed major isoform, while the hGNE2-hGNE8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. hGNE2 and hGNE7 display a 31-residue N-terminal extension compared to hGNE1. On the basis of similarities to kinases and helicases, this extension does not seem to hinder the epimerase enzymatic active site. hGNE3 and hGNE8 contain a 55-residue N-terminal deletion and a 50-residue N-terminal extension compared to hGNE1. The size and secondary structures of these fragments are similar, and modeling predicted that these modifications do not affect the overall fold compared to that of hGNE1. However, the epimerase enzymatic activity of GNE3 and GNE8 is likely absent, because the deleted fragment contains important substrate binding residues in homologous bacterial epimerases. hGNE5-hGNE8 have a 53-residue deletion, which was assigned a role in substrate (UDP-GlcNAc) binding. Deletion of this fragment likely eliminates epimerase enzymatic activity. Our findings imply that GNE is subject to evolutionary mechanisms to improve cellular functions, without increasing the number of genes. Our expression and modeling data contribute to elucidation of the complex functional and regulatory mechanisms of human GNE and may contribute to further elucidating the pathology and treatment strategies of the human GNE-opathies sialuria and hereditary inclusion body myopathy.
We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2. This was ruled out because of the presence of platelet ?-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patients melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patients G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual.
Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder characterized by multiple congenital anomalies. The syndrome is primarily ascribed to a ?3.7 Mb de novo deletion on chromosome 17p11.2. Haploinsufficiency of multiple genes likely underlies the complex clinical phenotype. RAI1 (Retinoic Acid Induced 1) is recognized as a major gene involved in the SMS phenotype. Extensive genetic and clinical analyses of 36 patients with SMS-like features, but without the 17p11.2 microdeletion, yielded 10 patients with RAI1 variants, including 4 with de novo deleterious mutations, and 6 with novel missense variants, 5 of which were familial. Haplotype analysis showed two major RAI1 haplotypes in our primarily Caucasian cohort; the novel RAI1 variants did not occur in a preferred haplotype. RNA analysis revealed that RAI1 mRNA expression was significantly decreased in cells of patients with the common 17p11.2 deletion, as well as in those with de novo RAI1 variants. Expression levels varied in patients with familial RAI1 variants and in non-17p11.2 deleted patients without identified RAI1 defects. No correlation between SNP haplotype and RAI1 expression was found. Two clinical features, ocular abnormalities and polyembolokoilomania (object insertion), were significantly correlated with decreased RAI1 expression. While not significantly correlated, the presence of hearing loss, seizures, hoarse voice, childhood onset of obesity and specific behavioral aspects and the absence of immunologic abnormalities and cardiovascular or renal structural anomalies, appeared to be specific for the de novo RAI1 subgroup. Recognition of the combination of these features will assist in referral for RAI1 analysis of patients with SMS-like features without detectable microdeletion of 17p11.2. Moreover, RAI1 expression emerged as a genetic target for development of therapeutic interventions for SMS.
Hermansky-Pudlak Syndrome (HPS) is an autosomal-recessive condition characterized by oculocutaneous albinism and a bleeding diathesis due to absent platelet delta granules. HPS is a genetically heterogeneous disorder of intracellular vesicle biogenesis. We first screened all our patients with HPS-like symptoms for mutations in the genes responsible for HPS-1 through HPS-6 and found no functional mutations in 38 individuals. We then examined all eight genes encoding the biogenesis of lysosome-related organelles complex-1, or BLOC-1, proteins in these individuals. This identified a homozygous nonsense mutation in PLDN in a boy with characteristic features of HPS. PLDN is mutated in the HPS mouse model pallid and encodes the protein pallidin, which interacts with the early endosomal t-SNARE syntaxin-13. We could not detect any full-length pallidin in our patients cells despite normal mRNA expression of the mutant transcript. We could detect an alternative transcript that would skip the exon that harbored the mutation, but we demonstrate that if this transcript is translated into protein, although it correctly localizes to early endosomes, it does not interact with syntaxin-13. In our patients melanocytes, the melanogenic protein TYRP1 showed aberrant localization, an increase in plasma-membrane trafficking, and a failure to reach melanosomes, explaining the boys severe albinism and establishing his diagnosis as HPS-9.
Costeff Syndrome, which is caused by mutations in the OPTIC ATROPHY 3 (OPA3) gene, is an early-onset syndrome characterized by urinary excretion of 3-methylglutaconic acid (MGC), optic atrophy and movement disorders, including ataxia and extrapyramidal dysfunction. The OPA3 protein is enriched in the inner mitochondrial membrane and has mitochondrial targeting signals, but a requirement for mitochondrial localization has not been demonstrated. We find zebrafish opa3 mRNA to be expressed in the optic nerve and retinal layers, the counterparts of which in humans have high mitochondrial activity. Transcripts of zebrafish opa3 are also expressed in the embryonic brain, inner ear, heart, liver, intestine and swim bladder. We isolated a zebrafish opa3 null allele for which homozygous mutants display increased MGC levels, optic nerve deficits, ataxia and an extrapyramidal movement disorder. This correspondence of metabolic, ophthalmologic and movement abnormalities between humans and zebrafish demonstrates a phylogenetic conservation of OPA3 function. We also find that delivery of exogenous Opa3 can reduce increased MGC levels in opa3 mutants, and this reduction requires the mitochondrial localization signals of Opa3. By manipulating MGC precursor availability, we infer that elevated MGC in opa3 mutants derives from extra-mitochondrial HMG-CoA through a non-canonical pathway. The opa3 mutants have normal mitochondrial oxidative phosphorylation profiles, but are nonetheless sensitive to inhibitors of the electron transport chain, which supports clinical recommendations that individuals with Costeff Syndrome avoid mitochondria-damaging agents. In summary, this paper introduces a faithful Costeff Syndrome model and demonstrates a requirement for mitochondrial OPA3 to limit HMG-CoA-derived MGC and protect the electron transport chain against inhibitory compounds.
Griscelli syndrome (GS), a rare autosomal recessive disorder characterized by partial albinism and immunological impairment and/or severe neurological impairment, results from mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes. We identified a Hispanic patient born of a consanguineous union who presented with immunodeficiency, partial albinism, hepatic dysfunction, hemophagocytosis, neurological impairment, nystagmus, and silvery hair indicative of Griscelli Syndrome Type 2 (GS2). We screened for point mutations, but only exons 2-6 of the patients DNA could be PCR-amplified. Whole genome analysis using the Illumina 1M-Duo DNA Analysis BeadChip identified a homozygous deletion in the patients DNA. The exact breakpoints of the 47.5-kb deletion were identified as chr15q15-q21.1: g.53332432_53379990del (NCBI Build 37.1); the patient lacks the promoter and 5UTR regions of RAB27A, thus confirming the diagnosis of GS2.
A critical role of the FOX transcription factors in the development of different tissues has been shown. Among these genes, FOXN1 encodes a protein whose alteration is responsible for the Nude/SCID phenotype. Recently, our group reported on a human Nude/SCID fetus, which also had severe neural tube defects, namely anencephaly and spina bifida. This led to hypothesize that FOXN1 could have a role in the early stages of central nervous system development. Here we report on a second fetus that carried the R255X homozygous mutation in FOXN1 that has been examined for the presence of CNS developmental anomalies. At 16 postmenstrual weeks of gestation, the abdominal ultrasonography of the Nude/SCID fetus revealed a morphologically normal brain, but with absence of cavum septi pellucidi (CSP). Moreover, after confirmation of the diagnosis of severe Nude/SCID, the fetus was further examined postmortem and a first gross examination revealed an enlargement of the interhemispheric fissure. Subsequently, a magnetic resonance imaging failed to identify the corpus callosum in any section. In conclusion, our observations did not reveal any gross abnormalities in the CNS anatomy of the Nude/SCID fetus, but alteration of the corpus callosum, suggesting that FOXN1 alterations could play a role as a cofactor in CNS development in a similar fashion to other FOX family members.
The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. Non-allosteric GNE gene mutations cause the muscular disorder GNE myopathy (also known as hereditary inclusion body myopathy), whose exact pathology remains unknown. Increased knowledge of GNE regulation, including isoform regulation, may help elucidate the pathology of GNE myopathy. While eight mRNA transcripts encoding human GNE isoforms are described, we only identified two mouse Gne mRNA transcripts, encoding mGne1 and mGne2, homologous to human hGNE1 and hGNE2. Orthologs of the other human isoforms were not identified in mice. mGne1 appeared as the ubiquitously expressed, major mouse isoform. The mGne2 encoding transcript is differentially expressed and may act as a tissue-specific regulator of sialylation. mGne2 expression appeared significantly increased the first 2 days of life, possibly reflecting the high sialic acid demand during this period. Tissues of the knock-in Gne p.M712T mouse model had similar mGne transcript expression levels among genotypes, indicating no effect of the mutation on mRNA expression. However, upon treatment of these mice with N-acetylmannosamine (ManNAc, a Gne substrate, sialic acid precursor, and proposed therapy for GNE myopathy), Gne transcript expression, in particular mGne2, increased significantly, likely resulting in increased Gne enzymatic activities. This dual effect of ManNAc supplementation (increased flux through the sialic acid pathway and increased Gne activity) needs to be considered when treating GNE myopathy patients with ManNAc. In addition, the existence and expression of GNE isoforms needs consideration when designing other therapeutic strategies for GNE myopathy.
GNE myopathy, previously termed hereditary inclusion body myopathy (HIBM), is an adult-onset neuromuscular disorder characterized by progressive muscle weakness. The disorder results from biallelic mutations in GNE, encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid synthesis. GNE myopathy, associated with impaired glycan sialylation, has no approved therapy. Here we test potential sialylation-increasing monosaccharides for their effectiveness in prophylaxis (at the embryonic and neonatal stages) and therapy (after the onset of symptoms) by evaluating renal and muscle hyposialylation in a knock-in mouse model (Gne p.M712T) of GNE myopathy. We demonstrate that oral mannosamine (ManN), but not sialic acid (Neu5Ac), mannose (Man), galactose (Gal), or glucosamine (GlcN), administered to pregnant female mice has a similar prophylactic effect on renal hyposialylation, pathology and neonatal survival of mutant offspring, as previously shown for N-acetylmannosamine (ManNAc) therapy. ManN may be converted to ManNAc by a direct, yet unknown, pathway, or may act through another mode of action. The other sugars (Man, Gal, GlcN) may either not cross the placental barrier (Neu5Ac) and/or may not be able to directly increase sialylation. Because GNE myopathy patients will likely require treatment in adulthood after onset of symptoms, we also administered ManNAc (1 or 2g/kg/day for 12 weeks), Neu5Ac (2 g/kg/day for 12 weeks), or ManN (2 g/kg/day for 6 weeks) in drinking water to 6 month old mutant Gne p.M712T mice. All three therapies markedly improved the muscle and renal hyposialylation, as evidenced by lectin histochemistry for overall sialylation status and immunoblotting of specific sialoproteins. These preclinical data strongly support further evaluation of oral ManNAc, Neu5Ac and ManN as therapy for GNE myopathy and conceivably for certain glomerular diseases with hyposialylation.
Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.
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