Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans. METHODS: We examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. RESULTS: We demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack. CONCLUSION: The Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Biallelic truncating variants in the muscular A-type lamin-interacting protein (MLIP) gene cause myopathy with hyperCKemia.

BACKGROUND AND PURPOSE: Muscular A-type lamin-interacting protein (MLIP) is most abundantly expressed in cardiac and skeletal muscle. In vitro and animal studies have shown its regulatory role in myoblast differentiation and in organization of myonuclear positioning in skeletal muscle, as well as in cardiomyocyte adaptation and cardiomyopathy. We report the association of biallelic truncating variation in the MLIP gene with human disease in five individuals from two unrelated pedigrees. METHODS: Clinical evaluation and exome sequencing were performed in two unrelated families with elevated creatine kinase level. RESULTS: Family 1. A 6-year-old girl born to consanguineous parents of Arab-Muslim origin presented with myalgia, early fatigue after mild-to-moderate physical exertion, and elevated creatine kinase levels up to 16,000 U/L. Exome sequencing revealed a novel homozygous nonsense variant, c.2530C>T; p.Arg844Ter, in the MLIP gene. Family 2. Three individuals from two distantly related families of Old Order Amish ancestry presented with elevated creatine kinase levels, one of whom also presented with abnormal electrocardiography results. On exome sequencing, all showed homozygosity for a novel nonsense MLIP variant c.1825A>T; p.Lys609Ter. Another individual from this pedigree, who had sinus arrhythmia and for whom creatine kinase level was not available, was also homozygous for this variant. CONCLUSIONS: Our findings suggest that biallelic truncating variants in MLIP result in myopathy characterized by hyperCKemia. Moreover, these cases of MLIP-related disease may indicate that at least in some instances this condition is associated with muscle decompensation and fatigability during low-to-moderate intensity muscle exertion as well as possible cardiac involvement.

Multi-system neurological disorder associated with a CRYAB variant.

We report a multiplex family with extended multisystem neurological phenotype associated with a CRYAB variant. Two affected siblings were evaluated with whole exome sequencing, muscle biopsy, laser microdissection, and mass spectrometry-based proteomic analysis. Both patients and their mother manifested a combination of early-onset cataracts, cardiomyopathy, cerebellar ataxia, optic atrophy, cognitive impairment, and myopathy. Whole exome sequencing identified a heterozygous c.458C>T variant mapped to the C-terminal extension domain of the Alpha-crystallin B chain, disrupting its function as a molecular chaperone and its ability to suppress protein aggregation. In accordance with the molecular findings, muscle biopsies revealed subsarcolemmal deposits that appeared dark with H&E and trichrome staining were negative for the other routine histochemical staining and for amyloid with the Congo-red stain. Electron microscopy demonstrated that the deposits were composed of numerous parallel fibrils. Laser microdissection and mass spectrometry-based proteomic analysis revealed that the inclusions are almost exclusively composed of crystallized chaperones/heat shock proteins. Moreover,  a structural model suggests that Ser153 could be involved in monomer stabilization, dimer association, and possible binding of partner proteins. We propose that our report potentially expands the complex phenotypic spectrum of alpha B-crystallinopathies with possible effect of a CRYAB variant on the central nervous system.

Pax7, Pax3 and Mamstr genes are involved in skeletal muscle impaired regeneration of dy2J/dy2J mouse model of Lama2-CMD.

Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively. Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and wild-type (WT) mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEGs) was found in dy2J/dy2J and mdx models (1834 common DEGs, false discovery rate [FDR] < 0.05). Enrichment pathway analysis using ingenuity pathway analysis showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (P-values 3E-10-9E-37). Via canonical pathway analysis, actin cytoskeleton, integrin, integrin-linked kinase, NF-kB, renin-angiotensin, epithelial-mesenchymal transition, and calcium signaling were also enriched and upregulated in both models (FDR < 0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

A novel homozygous MSTO1 mutation in Ashkenazi Jewish siblings with ataxia and myopathy.

MSTO1 is a cytoplasmic protein that modulates mitochondrial dynamics by promoting mitochondrial fusion. Mutations in the MSTO1 gene are responsible for an extremely rare condition characterized by early-onset myopathy and cerebellar ataxia. We report here two siblings from a large Ashkenazi Jewish family, presenting with a progressive neuromuscular disease characterized by ataxia and myopathy. By whole exome sequencing, we found a novel homozygous missense mutation (c.1403T>A, p.Leu468Gln) in MSTO1. Studies performed on fibroblasts from the index patient demonstrated the pathogenic role of the identified variant; we found that MSTO1 protein level was reduced and that mitochondrial network was fragmented or formed enlarged structures. Moreover, patient's cells showed reduced mitochondrial DNA amount. Our report confirms that MSTO1 mutations are typically recessive, and associated with clinical phenotypes characterized by early-onset muscle impairment and ataxia, often with upper motor neuron signs and varied cognitive impairment.

Spatial Intratumoral Heterogeneity Expression of PD-L1 Antigen in Head and Neck Squamous Cell Carcinoma.

INTRODUCTION: Immune-checkpoint inhibitors have demonstrated a significant survival benefit in metastatic and non-resectable head and neck squamous cell carcinoma (HNSCC). Patients with a combined positivity score (CPS) of 20 and higher benefit the most from therapy. Inaccurate definition of the CPS category might lead to the incorrect stratification of patients to immunotherapy. This study's main aim was to investigate programmed death-ligand 1 (PD-L1) antigen expression in HNSCC in diverse clinical situations and histological settings. MATERIALS AND METHODS: This is a prospective cohort study conducted in a tertiary referral medical center. Tissues were investigated for PD-L1 expression using the FDA-approved 22C3 immunohistochemistry assay (Dako). We analyzed potential associations between the CPS category and meaningful demographic, clinical, and outcome metrics. Furthermore, we investigated morphologically separate sites for CPS scores in whole surgical tissue specimens and matched preoperative biopsies. RESULTS: We analyzed 36 patients, of whom 26 had oral cavity SCC and 10 had laryngeal SCC. The overall, disease-specific, and progression-free survival of the HNSCC group of patients were not associated with the CPS category (p = 0.45, p = 0.31, and p = 0.88, respectively). There was a significant (18%, 95% CI 0.65-0.9) inconsistency between the CPS category determined in biopsies versus whole carcinoma analyses. We also found an uneven distribution of whole-tumor CPS attributed to spatial carcinoma invasiveness, tumor differentiation, and inflammatory cell infiltration heterogeneity. DISCUSSION AND CONCLUSIONS: Our data suggest that careful selection of tumor area for CPS analysis is important. PD-L1 antigen expression, clinically represented by CPS, may be up- or down-categorized in different clinical and pathological circumstances. The high whole-tissue CPS category scatter may clinically result in potential treatment modifications. We argue that CPS analysis requires not only adequacy (at least 100 viable tumor cells), but also correct representation of the tumor microenvironment.

Abundance of P-glycoprotein and Breast Cancer Resistance Protein Measured by Targeted Proteomics in Human Epileptogenic Brain Tissue.

Our goal was to measure the absolute differential abundance of key drug transporters in human epileptogenic brain tissue and to compare them between patients and at various distances from the epileptogenic zone within the same patient. Transporter protein abundance was quantified in brain tissue homogenates from patients who underwent epilepsy surgery, using targeted proteomics, and correlations with clinical and tissue characteristics were assessed. Fourteen brain samples (including four epileptogenic hippocampal samples) were collected from nine patients. Among the quantifiable drug transporters, the abundance (median, range) ranked: breast cancer resistance protein (ABCG2/BCRP; 0.55, 0.01-3.26 pmol/g tissue) > P-glycoprotein (ABCB1/MDR1; 0.30, 0.02-1.15 pmol/g tissue) > equilibrative nucleoside transporter 1 (SLC29A1/ENT1; 0.06, 0.001-0.35 pmol/g tissue). The ABCB1/ABCG2 ratio (mean 0.27, range 0.08-0.47) was comparable with literature values from nonepileptogenic brain tissue (mean 0.5-0.8). Transporter abundance was lower in the hippocampi than in the less epileptogenic neocortex of the same patients. ABCG2/BCRP and ABCB1/MDR1 expression strongly correlated with that of glucose transporter 1 (SLC2A1/GLUT1) (r = 0.97, p < 0.001; r = 0.90, p < 0.01, respectively). Low transporter abundance was found in patients with overt vascular pathology, whereas the highest abundance was seen in a sample with normally appearing blood vessels. In conclusion, drug transporter abundance highly varies across patients and between epileptogenic and less epileptogenic brain tissue of the same patient. The strong correlation in abundance of ABCB1/MDR1, ABCG2/BCRP, and SLC2A1/GLUT1 suggests variation in the content of the functional vasculature within the tissue samples. The epileptogenic tissue can be depleted of key drug transport mechanisms, warranting consideration when selecting treatments for patients with drug-resistant epilepsy.

Single Exon Skipping Can Address a Multi-Exon Duplication in the Dystrophin Gene.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10-15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14-17 duplication. Two strategies were employed; the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.

Nemaline body myopathy caused by a novel mutation in troponin T1 (TNNT1).

INTRODUCTION: Nemaline myopathy is a rare disorder characterized by skeletal muscle weakness of varying severity and onset, with the presence of nemaline rods on muscle biopsy. Congenital nemaline body myopathy due to mutations in TNNT1 has hitherto only been described as a result of a single founder mutation in patients of Amish origin and in 2 other individuals with different recessive mutations. METHODS: Autozygosity mapping and whole exome sequencing were applied after we identified 9 Palestinian patients from 7 unrelated families who have nemaline myopathy. RESULTS: All patients were homozygous for a novel complex rearrangement of the TNNT1 gene (c.574_577delinsTAGTGCTGT | NM_003283) leading to C-terminal truncation of the protein (p.L203* | NP_003274.3). Their clinical course was remarkable for early respiratory failure and striking stiffness of the cervical spine. CONCLUSIONS: This report exemplifies the utility of combining autozygosity mapping and whole exome sequencing and expands the phenotype associated with TNNT1 mutations.

Myelin-associated glycoprotein gene mutation causes Pelizaeus-Merzbacher disease-like disorder.

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the proteasome. Our findings identify involvement of myelin-associated glycoprotein in this family with a disorder affecting the central and peripheral nervous system, and suggest that loss of the protein function is responsible for the unique clinical phenotype.

Minocycline Effectively Protects the Rabbit's Spinal Cord From Aortic Occlusion-Related Ischemia.

OBJECTIVES: To identify the minocycline anti-inflammatory and antiapoptotic mechanisms through which it is believed to exert spinal cord protection during aortic occlusion in the rabbit model. DESIGN: An animal model of aortic occlusion-related spinal cord ischemia. Randomized study with a control group and pre-ischemia and post-ischemia escalating doses of minocycline to high-dose minocycline in the presence of either hyperglycemia, a pro-apoptotic maneuver, or wortmannin, a specific phosphatidylinositol 3-kinase antagonist. SETTING: Tertiary medical center and school of medicine laboratory. PARTICIPANTS: Laboratory animals-rabbits. INTERVENTIONS: Balloon obstruction of infrarenal aorta introduced via femoral artery incision. RESULTS: Severe hindlimb paralysis (mean Tarlov score 0.36±0.81 out of 3) was observed in all the control group animals (9 of 11 with paraplegia and 2 of 11 with paraparesis) compared with 11 of 12 neurologically intact animals (mean Tarlov score 2.58±0.90 [p = 0.001 compared with control]) in the high-dose minocycline group. This protective effect was observed partially during a state of hyperglycemia and was completely abrogated by wortmannin. Minocycline administration resulted in higher neurologic scores (p = 0.003) and a shift to viable neurons and more apoptotic-stained nuclei resulting from reduced necrosis (p = 0.001). CONCLUSIONS: In a rabbit model of infrarenal aortic occlusion, minocycline effectively reduced paraplegia by increasing the number of viable neurons in a dose-dependent manner. Its action was completely abrogated by inhibiting the phosphatidylinositol 3-kinase pathway and was inhibited partially by the pro-apoptotic hyperglycemia maneuver, indicating that the activation of cell salvage pathways and mitochondrial sites are possible targets of minocycline action in an ischemic spinal cord.

Nonsyndromic Early-Onset Cone-Rod Dystrophy and Limb-Girdle Muscular Dystrophy in a Consanguineous Israeli Family are Caused by Two Independent yet Linked Mutations in ALMS1 and DYSF.

Genetic analysis of clinical phenotypes in consanguineous families is complicated by coinheritance of large DNA regions carrying independent variants. Here, we characterized a family with early onset cone-rod dystrophy (CRD) and muscular dystrophy. Homozygosity mapping (HM) followed by whole exome sequencing revealed a nonsense mutation, p.R270*, in ALMS1 and two novel potentially disease-causing missense variants, p.R1581C and p.Y2070C, in DYSF. ALMS1 and DYSF are genetically and physically linked on chromosome 2 in a genomic region suggested by HM and associated with Alström syndrome, which includes CRD, and with limb girdle muscular dystrophy, respectively. Affected family members lack additional systemic manifestations of Alström syndrome but exhibit mild muscular dystrophy. RNA-seq data did not reveal any significant variations in ALMS1 transcripts in the human retina. Our study thus implicates ALMS1 as a nonsyndromic retinal disease gene and suggests a potential role of variants in interacting cilia genes in modifying clinical phenotypes.

Oncofetal H19 RNA promotes tumor metastasis.

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-ß and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-ß treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

Congenital myopathy is caused by mutation of HACD1.

Congenital myopathies are heterogeneous inherited diseases of muscle characterized by a range of distinctive histologic abnormalities. We have studied a consanguineous family with congenital myopathy. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous non-sense mutation in 3-hydroxyacyl-CoA dehydratase 1 (HACD1) in affected individuals. The mutation results in non-sense mediated decay of the HACD1 mRNA to 31% of control levels in patient muscle and completely abrogates the enzymatic activity of dehydration of 3-hydroxyacyl-CoA, the third step in the elongation of very long-chain fatty acids (VLCFAs). We describe clinical findings correlated with a deleterious mutation in a gene not previously known to be associated with congenital myopathy in humans. We suggest that the mutation in the HACD1 gene causes a reduction in the synthesis of VLCFAs, which are components of membrane lipids and participants in physiological processes, leading to congenital myopathy. These data indicate that HACD1 is necessary for muscle function.

Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with a loss-of-function mutation in CDK5.

Lissencephaly comprises a heterogeneous group of developmental brain disorders of varying severity, involving abnormal cortical gyration. We studied a highly consanguineous Israeli Moslem family with a lethal form of autosomal recessive lissencephaly with cerebellar hypoplasia (LCH). Using microarray-based homozygosity mapping in the reported family, combined with whole exome sequencing in one affected infant, we identified a homozygous splice site mutation g.IVS8+1G>A in cyclin-dependent kinase 5 (CDK5), causing complete skipping of exon 8, and leading to a frame shift and premature stop codon (p.V162SfsX19). The mutation co-segregated with the disease phenotype in all 29 study participants (4 patients and 25 healthy relatives), and was not identified in 200 ethnically matched control chromosomes. The p.V162SfsX19 mutation causes lack of endogenous CDK5 expression in affected dermal fibroblasts and brain tissue at the mRNA and protein levels, consistent with nonsense-mediated mRNA decay. Functional analysis of the p.V162SfsX19 mutation, using a yeast complementation assay, showed loss-of-function of the mutant CDK5 gene product, thereby implicating its role in the pathogenesis of autosomal recessive LCH in the studied family.

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization.

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.

CD59 deficiency is associated with chronic hemolysis and childhood relapsing immune-mediated polyneuropathy.

CD59 deficiency is a common finding in RBCs and WBCs in patients with chronic hemolysis suffering from paroxysmal nocturnal hemoglobinuria in which the acquired mutation in the PIGA gene leads to membrane loss of glycosylphosphatidylinositol-anchored membrane proteins, including CD59. The objective of the present study was to elucidate the molecular basis of childhood familial chronic Coombs-negative hemolysis and relapsing polyneuropathy presenting as chronic inflammatory demyelinating polyradiculoneuropathy in infants of North-African Jewish origin from 4 unrelated families. A founder mutation was searched for using homozygosity mapping followed by exome sequencing. The expression of CD59, CD55, and CD14 was examined in blood cells by flow cytometry followed by Western blot of the CD59 protein. A homozygous missense mutation, p.Cys89Tyr in CD59, was identified in all patients. The mutation segregated with the disease in the families and had a carrier rate of 1:66 among Jewish subjects of North-African origin. The mutated protein was present in the patients' cells in reduced amounts and was undetectable on the membrane surface. Based on the results of the present study, we conclude that the Cys89Tyr mutation in CD59 is associated with a failure of proper localization of the CD59 protein in the cell surface. This mutation is manifested clinically in infancy by chronic hemolysis and relapsing peripheral demyelinating disease.

Rare Median Nerve and Digital Nerve Tumor Resection and Distal Nerve Transfers: A Report of 2 Cases.

CASES: We present 2 cases of median nerve reconstruction using distal nerve transfers after resection of unusual benign median nerve tumors. Critical sensation was restored in case 1 by transferring the fourth common digital nerve to first web digital nerves. Thumb opposition was regained by transferring the abductor digiti minimi ulnar motor nerve branch to the recurrent median motor nerve branch. Critical sensation was restored in case 2 by transferring the long finger ulnar digital nerve to the index finger radial digital nerve. CONCLUSION: Distal nerve transfers, even with short grafts, are reliable median nerve deficit treatments, sparing the need for larger autologous nerve grafts and late tendon opponensplasties.

Induced muscle and liver absence of Gne in postnatal mice does not result in structural or functional muscle impairment.

Background: GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice. Objective: To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle. Methods: A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene. Results: Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver. Conclusions: We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.