Transcriptional up-regulation of BAG3, a Chaperone Assisted Selective Autophagy factor, in animal models of KY-deficient hereditary myopathy

The importance of kyphoscoliosis peptidase (KY) in skeletal muscle physiology has recently been emphasised by the identification of novel human myopathies associated with KY deficiency. Neither the pathogenic mechanism of KY deficiency nor a specific role for KY in muscle function have been established. However, aberrant localisation of FLNC in muscle fibers has been shown in humans and mice with loss of function mutations in the KY gene. FLNC turnover has been proposed to be controlled by Chaperone Assisted Selective Autophagy (CASA), a client-specific and tension-induced pathway that is required for muscle maintenance. Here, we have generated new C2C12 myoblast and zebrafish models of KY-deficiency by CRISPR/Cas9 mutagenesis. To obtain insights into the pathogenic mechanism caused by KY deficiency, expression of the co-chaperone BAG3 and other CASA factors was analyzed in the cellular, zebrafish and ky/ky mouse models. Ky-deficient C2C12 derived clones show trends of higher transcription of CASA factors in differentiated myotubes. The ky-deficient zebrafish model (kyyo1/kyyo1) lacks overt signs of pathology but shows significantly increased bag3 and flnca/b expression in embryos and adult muscle. Additionally, kyyo1/kyyo1 embryos challenged by swimming in viscous media show an inability to further increase expression of these factors in contrast to WT controls. The ky/ky mouse shows elevated expression of Bag3 in the non-pathological EDL and evidence of impaired BAG3 turnover in the pathological soleus. Thus, upregulation of CASA factors appears to be an early and primary molecular hallmark of KY deficiency

The adhesion G protein-coupled receptor GPR56/ADGRG1 is an inhibitory receptor on human NK cells

Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. We here explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit, a homolog of Blimp-1, and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in the ADGRG1 gene, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance

West Nile Virus–associated Flaccid Paralysis

The causes and frequency of acute paralysis and respiratory failure with West Nile virus (WNV) infection are incompletely understood. During the summer and fall of 2003, we conducted a prospective, population-based study among residents of a 3-county area in Colorado, United States, with developing WNV-associated paralysis. Thirty-two patients with developing paralysis and acute WNV infection were identified. Causes included a poliomyelitislike syndrome in 27 (84%) patients and a Guillain-Barré–like syndrome in 4 (13%); 1 had brachial plexus involvement alone. The incidence of poliomyelitislike syndrome was 3.7/100,000. Twelve patients (38%), including 1 with Guillain-Barré–like syndrome, had acute respiratory failure that required endotracheal intubation. At 4 months, 3 patients with respiratory failure died, 2 remained intubated, 25 showed various degrees of improvement, and 2 were lost to followup. A poliomyelitislike syndrome likely involving spinal anterior horn cells is the most common mechanism of WNV-associated paralysis and is associated with significant short- and long-term illness and death

Novel homozygous missense mutation in GAN associated with Charcot-Marie-Tooth disease type 2 in a large consanguineous family from Israel.

BACKGROUND: CMT-2 is a clinically and genetically heterogeneous group of peripheral axonal neuropathies characterized by slowly progressive weakness and atrophy of distal limb muscles resulting from length-dependent motor and sensory neurodegeneration. Classical giant axonal neuropathy (GAN) is an autosomal recessively inherited progressive neurodegenerative disorder of the peripheral and central nervous systems, typically diagnosed in early childhood and resulting in death by the end of the third decade. Distinctive phenotypic features are the presence of "kinky" hair and long eyelashes. The genetic basis of the disease has been well established, with over 40 associated mutations identified in the gene GAN, encoding the BTB-KELCH protein gigaxonin, involved in intermediate filament regulation. METHODS: An Illumina Human CytoSNP-12 array followed by whole exome sequence analysis was used to identify the disease associated gene mutation in a large consanguineous family diagnosed with Charcot-Marie-Tooth disease type 2 (CMT-2) from which all but one affected member had straight hair. RESULTS: Here we report the identification of a novel GAN missense mutation underlying the CMT-2 phenotype observed in this family. Although milder forms of GAN, with and without the presence of kinky hair have been reported previously, a phenotype distinct from that was investigated in this study. All family members lacked common features of GAN, including ataxia, nystagmus, intellectual disability, seizures, and central nervous system involvement. CONCLUSIONS: Our findings broaden the spectrum of phenotypes associated with GAN mutations and emphasize a need to proceed with caution when providing families with diagnostic or prognostic information based on either clinical or genetic findings alone

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targetedpanel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations

Fatal cerebral edema associated with serine deficiency in CSF

Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and enzyme measurement (in one patient) excluded 3-PGDH deficiency. Deficiencies in other serine biosynthesis enzymes were highly unlikely on clinical grounds. On basis of the fasting state, ketone bodies and lactate in plasma, urine and CSF, we speculate that reduced serine levels were due to its use as gluconeogenic substrate, conversion to pyruvate by brain serine racemase or decreased L-serine production because of a lack of glucose. These are the first strikingly similar cases of patients with a clear secondary serine deficiency associated with a toxic encephalopathy

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution.

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations