Erythropoietin Receptor Signaling Is Membrane Raft Dependent

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins

Osteoporotic Vertebral Fractures During Pregnancy: Be Aware of a Potential Underlying Genetic Cause

Context: Although the baby growing in its mother's womb needs calcium for skeletal development, osteoporosis and fractures very rarely occur during pregnancy. Case Presentation: A 27-year-old woman in the seventh month of her first pregnancy contracted midthoracic back pain after lifting an object. The pain was attributed to her pregnancy, but it remained postpartum. Her past medical history was uneventful, except for severely reduced vision of her left eye since birth. Family history revealed that her maternal grandmother had postmenopausal osteoporosis and her half-brother had three fractures during childhood after minor trauma. Her height was 1.58 m; she had no blue sclerae or joint hyperlaxity. Laboratory examination including serum calcium, phosphate, alkaline phosphatase, creatinine, beta-carboxyterminal cross-linking telopeptide of type I collagen, 25-hydroxyvitamin D, and TSH was normal. Multiple thoracic vertebral fractures were diagnosed on x-ray examination, and dual-energy x-ray absorptiometry scanning showed severe osteoporosis (Z-scores: L2-L4, -5.6 SD; femur neck, -3.9 SD). DNA analyses revealed two compound heterozygous missense mutations in LRP5. The patient's mother carried one of the LRP5 mutations and was diagnosed with osteoporosis. Her half-brother, treated with cabergoline for a microprolactinoma, also had osteoporosis of the lumbar spine on dual-energy x-ray absorptiometry and carried the same LRP5 mutation. The patient was treated with risedronate for 2.5 years. Bone mineral density and back pain improved. She stopped bisphosphonate use 6 months before planning a second pregnancy. Conclusion: Our patient was diagnosed with osteoporosis pseudoglioma syndrome/familial exudative vitreoretinopathy. Potential underlying genetic causes should be considered in pregnancy-associated osteoporosis with implications for patients and relatives. More studies regarding osteoporosis treatment preceding conception are desirable

Mutation update on the CHD7 gene involved in CHARGE syndrome

CHD7 is a member of the chromodomain helicase DNA-binding (CHD) protein family that plays a role in transcription regulation by chromatin remodeling. Loss-of-function mutations in CHD7 are known to cause CHARGE syndrome, an autosomal-dominant malformation syndrome in which several organ systems, for example, the central nervous system, eye, ear, nose, and mediastinal organs, are variably involved. In this article, we review all the currently described CHD7 variants, including 183 new pathogenic mutations found by our laboratories. In total, we compiled 528 different pathogenic CHD7 alterations from 508 previously published patients with CHARGE syndrome and 294 unpublished patients analyzed by our laboratories. The mutations are equally distributed along the coding region of CHD7 and most are nonsense or frameshift mutations. Most mutations are unique, but we identified 94 recurrent mutations, predominantly arginine to stop codon mutations. We built a locus-specific database listing all the variants that is easily accessible at . In addition, we summarize the latest data on CHD7 expression studies, animal models, and functional studies, and we discuss the latest clinical insights into CHARGE syndrome. Hum Mutat 33:11491160, 2012. (c) 2012 Wiley Periodicals, Inc

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

Purpose: To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods: We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon-intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results: The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions: The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis