Accepted Manuscript CD59 deficiency presenting as polyneuropathy and Moyamoya syndrome with endothelial abnormalities of small brain vessels Christian Klemann, Janbernd Kirschner, Sandra Ammann, Horst Urbach, Olaf MoskeEick, Barbara Zieger, Myriam Ricarda Lorenz, Klaus Schwarz, Soroush Doostkam, Stephan Ehl, Rudolf Korinthenberg PII: S1090-3798(17)30131-9 DOI: 10.1016/j.ejpn.2018.04.003 Reference: YEJPN 2409 To appear in: European Journal of Paediatric Neurology Received Date: 7 March 2017 Revised Date: 17 February 2018 Accepted Date: 4 April 2018 Please cite this article as: Klemann C, Kirschner J, Ammann S, Urbach H, Moske-Eick O, Zieger B, Lorenz MR, Schwarz K, Doostkam S, Ehl S, Korinthenberg R, CD59 deficiency presenting as polyneuropathy and Moyamoya syndrome with endothelial abnormalities of small brain vessels, European Journal of Paediatric Neurology (2018), doi: 10.1016/j.ejpn.2018.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. 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Klemann et al. 1 ACCEPTED MANUSCRIPT CD59 deficiency presenting as polyneuropathy and Moyamoya syndrome with 2 endothelial abnormalities of small brain vessels. 3 Christian Klemann1,2,3, Janbernd Kirschner2,4, Sandra Ammann1,5, Horst Urbach6, 4 Olaf Moske-Eick6,2 Barbara Zieger2, Myriam Ricarda Lorenz7, Klaus Schwarz7,8, 5 Soroush Doostkam9, Stephan Ehl1,2, Rudolf Korinthenberg2,4 6 1 7 Freilburg, Faculty of Medicine, University of Freiburg, Germany. 8 2 9 University of Freiburg, Germany RI PT 1 Center for Chronic Immunodeficiency (CCI), Medical Center – University of Center for Pediatrics, Medical Center – University of Freiburg, Faculty of Medicine, 3 11 School, Hannover, Germany. 12 4 13 Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Germany 14 5 15 Cambridge, United Kingdom. 16 6 17 Medicine, University of Freiburg, Germany 18 7 19 8 20 Cross Blood Service, Baden Württemberg – Hessen, Ulm, Germany 21 9 22 Medicine, University of Freiburg, Germany Department of Pediatric Pneumology, Allergy and Neonatology, Hannover Medical M AN U Center for Pediatrics, Department of Neuropediatrics and Muscle Disorders, Medical Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Department of Neuroradiology, Medical Center – University of Freiburg, Faculty of TE D Institute for Transfusion Medicine, University of Ulm, Ulm, Germany; Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Institut for Neuropathology, Medical Center – University of Freiburg, Faculty of EP 23 SC 10 Corresponding author: 25 Dr. med. Christian Klemann, 26 Department of Pediatric Pneumology, Allergy and Neonatology, 27 Hannover Medical School, Hannover, Germany. 28 Email: Klemann.Christian@mh-hannover.de AC C 24 29 30 Conflict of interests: none 31 Key words: MMD, MMS; high resolution vessel wall imaging; HR-VWI; CIPD; GBS; 32 pediatric stroke, early-onset stroke, hemolysis, hemolytic anemia Klemann et al. 33 34 Highlights: • Congenital isolated CD59 deficiency has previously been associated with recurrent episodes of hemolytic anemia, peripheral neuropathy, and strokes. 35 36 2 ACCEPTED MANUSCRIPT • In this 4th patient with the p.Asp49Valfs*31 mutation in CD59 reported, brain endothelium showed absent CD59 expression. Moyamoya syndrome 38 developed with contrast enhancement of the distal carotid arteries and 39 shrinkage of vascular diameter while systemic vasculitis could be excluded. 41 • Targeted therapy with eculizumab might have prevented the lethal course in our patient. SC 40 RI PT 37 42 Abstract: (word count: 214) 44 CD59 is involved in lymphocyte signal transduction and regulates complement- 45 mediated cell lysis by inhibiting the membrane attack complex. In the cases reported 46 so far, congenital isolated CD59 deficiency was associated with recurrent episodes of 47 hemolytic anemia, peripheral neuropathy, and strokes. Here, we report on a patient 48 from a consanguineous Turkish family, who had a first episode of hemolytic anemia 49 at one month of age and presented at 14 months with acute Guillain-Barré syndrome 50 (GBS). The child suffered repeated infection-triggered relapses leading to the 51 diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP). Although 52 partly steroid-responsive, the polyneuropathy failed to be stabilized by a number of 53 immunosuppressive agents. At the age of 6 years, he developed acute hemiparesis 54 and showed progressive stenosis of proximal cerebral arteries, evolving into 55 Moyamoya syndrome (MMS) with recurrent infarctions leading to death at 8 years of 56 age. Post-mortem genetic analysis revealed a pathogenic p.Asp49Valfs*31 mutation 57 in CD59. Re-analysis of brain biopsy specimens showed absent CD59 expression 58 and severe endothelial damage. Whereas strokes are a known feature of CD59 59 deficiency, MMS has not previously been described in this condition. Therefore, we 60 conclude that in MMS combined with hemolysis or neuropathy CD59 deficiency 61 should be considered. Establishing the diagnosis and targeted therapy with 62 eculizumab might have prevented the lethal course in our patient. 63 64 AC C EP TE D M AN U 43 Klemann et al. Graphical abstract: 66 TE D M AN U SC RI PT 65 3 ACCEPTED MANUSCRIPT CD59 deficiency presenting as polyneuropathy and Moyamoya syndrome with 68 endothelial abnormalities of small brain vessels. AC C EP 67 Klemann et al. 4 ACCEPTED MANUSCRIPT 69 70 71 Introduction: 72 including killing of target cells via the membrane attack complex (MAC). CD59 is the 73 only protein inhibitor of the terminal pathway of the complement cascade preventing 74 formation of the MAC [1]. Somatic mutations in the protein anchoring CD59 and 75 related proteins on the cell surface of erythrocytes result in paroxysmal nocturnal 76 hemoglobinuria presenting with recurrent hemolysis. However, deleterious autosomal 77 recessive germline mutations in CD59 have recently been described to cause severe 78 early-onset immune-mediated chronic neuropathy, chronic or relapsing Coombs- 79 negative hemolysis, and cerebrovascular events [2–7]. These observations, 80 summarized in table 1, are based on the 12 hitherto reported patients. 81 Moyamoya disease (MMD) is a rare, chronic, occlusive cerebrovasculopathy and a 82 known cause of pediatric stroke. The pathogenesis and etiology is largely unknown 83 despite devastating, permanent neurological disability if left untreated. MMD is 84 characterized by progressive stenosis of the intracranial internal carotid arteries and 85 their main branches and the development of compensatory collateral vessels at the 86 base of the brain vascularizing the hypoperfused brain distal to the occluded vessels. 87 Sometimes, similar clinical and radiologic signs are evident in children with other 88 medical conditions then termed Moyamoya syndrome (MMS). In this report, we 89 extend the clinical phenotype of CD59 deficiency to angiographic features of cerebral 90 vasculopathy progressing to MMS. RI PT SC M AN U TE D EP 91 The complement system is a complex family of proteins with a myriad of functions, Materials & methods: 93 Informed consent for retrospective collection of patient history and clinical data as 94 well as for genetic analyses was obtained from the patient and his family members in 95 accordance with local ethics committee guidelines. 96 Biopsy material from frontal left dura and cortex was investigated by histology and 97 immunohistochemistry. After formalin fixation, conventional staining for H&E, EvG 98 and PAS were performed from the paraffin-embedded biopsy material. Antibodies 99 directed towards CD59 (BioScience, 7BIO-1B-233-C100), CD34 (Dako, M 7165) and 100 CD61 (Dako, M 0753) were used for immunohistochemistry. AC C 92 Klemann et al. 5 For sequencing of CD59, 100 ng genomic DNA or cDNA corresponding to 25 ng total 102 RNA of PBMC was amplified, followed by sequencing using Big Dye Terminator v1.1 103 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany). Sequencing 104 products were separated on an Applied Biosystems 3130xl Genetic Analyzer. Primer 105 sequences are available upon request from K.S. Multiple sequence alignments were 106 performed at http://www.ncbi.nlm.nih.gov/. Databases for analyzing the frequency of 107 the variation used were SNPbase (http://www.ncbi.nlm.nih.gov/snp), 1000 Genome- 108 Database 109 Database 110 (http://db.systemsbiology.net/kaviar/), and 111 (http://exac.broadinstitute.org/). RI PT 101 SC ACCEPTED MANUSCRIPT (http://browser.1000genomes.org/Homo_sapiens/Info/Index), (http://evs.gs.washington.edu), Kaviar-Database ExAC-Database M AN U 112 EVS- Results: 114 Our patient was born as the first son of healthy consanguineous Turkish parents (fig. 115 1A). No relationship to previously reported Turkish patients could be established. 116 Pregnancy was uneventful; birth was complicated by meconium aspiration and 117 sepsis requiring antibiotic treatment. The patient presented at the age of 1 month 118 with an episode of hemolysis (minimal hemoglobin 7.0 g/dl) prompting an erythrocyte 119 transfusion. LDH was increased while direct and indirect Coombs tests were 120 negative. Osmotic testing of erythrocytes, Hb-electrophoresis and G6PDH were 121 normal. In the year thereafter no new episodes of hemolysis occurred and neuro- 122 cognitive and motor development were normal. 123 After the development of the independent walking capability, at the age of 14 months 124 the boy fell ill with apathy, areflexia and progressive tetraparesis including loss of 125 control of head position and dysphagia after a mild gastroenteritis. PCR for 126 Coxsackie A2 virus in stool was positive and again Coombs-negative hemolytic 127 anemia became apparent. Guillain-Barré syndrome (GBS) was diagnosed and the 128 condition improved markedly under a high dose of immunoglobins i.v. (IVIG). No 129 nerve conduction studies were performed during this episode. A broad metabolic 130 workup was negative. At the age of 17 months a relapse of the neurological 131 symptoms was again treated successfully with IVIG. AC C EP TE D 113 Klemann et al. 6 ACCEPTED MANUSCRIPT At the age of 27 months a severe relapse of GBS unresponsive to IVIG prompted 133 referral to our department of child neurology. CSF analysis was acellular with an 134 increased protein level (164 mg/dl, normal value 15 - 40 mg/dl) without signs of local 135 IgG synthesis. Similar results were found in repeated CSF investigations in the 136 following years. Nerve conduction studies in three nerves showed severe slowing of 137 conduction velocity (15-19 m/sec), dispersed motor action potentials with diminished 138 amplitude (0.4-0.5 mV), and a conduction block proving primary demyelination with 139 secondary axonal injury. This gave rise to a diagnosis of chronic inflammatory 140 demyelinating polyneuropathy (CIDP) and intravenous treatment with steroids was 141 initiated rapidly improving the clinical condition. Under continued treatment with oral 142 steroids the clinical course was initially satisfying without any signs of hemolysis and 143 positive neurocognitive and motor development (standing and walking few steps with 144 aids). Tendon reflexes remained absent throughout the further clinical course. Nine 145 months later, again following a febrile infection CIDP and hemolysis relapsed 146 prompting increasing the steroid dose and additional treatment with azathioprine. In 147 the following years the clinical course was characterized by numerous infection- 148 associated relapses of CIDP which responded well to pulsed treatment with 149 corticosteroids, but immunoglobulins were without lasting effect. Only after 150 introduction of mycophenolate-mofetil the neuropathy stabilized with a gradual 151 increase of the unaided walking distance from 50 to 500 meters. 152 Unexpectedly, at the age of 6 years the boy suffered an acute stroke with right-sided 153 dysesthesia and paresis. MRI showed multiple left-sided thromboembolic and/or 154 hemodynamic infarctions. With TOF-MRA on the left side severe stenosis involving 155 the carotid-T and the proximal M1 and A1-segments and signs of a fine collateral 156 network, and on the right side a distinct but less pronounced stenosis of the M1 origin 157 was found (fig. 2A-C). At follow-up 18 months later, progression of both left- and 158 right-sided stenoses, and on high-resolutionVessel-Wall Imaging (HR-VWI) diffuse 159 and circular enhancement of arterial walls affecting the distal ICA and proximal M1- 160 and A1-segments with shrinkage of the vessel diameter were shown (fig. 2D-E). 161 Systemic vasculitis diseases were excluded by whole body MRI, PET scan, and 162 repeated blood works including repeatedly normal von Willebrand factor antigen and 163 activity, factor VIII activity and normal D-Dimers (in episodes of absent hemolysis). A 164 combined meningeal and brain biopsy did not confirm an inflammatory process but 165 showed a severely damaged intima of blood vessels, as indicated by a loss of the AC C EP TE D M AN U SC RI PT 132 Klemann et al. 7 ACCEPTED MANUSCRIPT endothelial marker CD34, (fig. 3A) and an aggregation of platelets shown by the 167 immunoreactivity for the platelet marker CD61 (fig. 3B). Rituximab along with 168 steroids, antithrombotic treatment and MMF initially seemed to stabilize the 169 vasculopathy, but during the following year the arterial stenoses showed further 170 progression despite additional aggressive treatment with cyclophosphamide. 171 Eventually this progression resulted in the occlusion of most large cerebral arteries 172 but only a faint basal collateral network. Under the diagnosis of MMS and due to 173 repeated TIAs and minor strokes burr-hole trepanations and synangioses were 174 performed resulting in a clear cortical collateral network (fig. 2F). However, despite 175 these measures and maximal care the boy died at the age of 8 years due to multiple 176 malignant cerebral infractions (fig. 2G). 177 Genetic analyses investigating known genes predisposing for Moya-Moya disease 178 proofed normal. Years later, following recently published reports of chronic 179 neuropathy, hemolysis and recurrent brain ischemic infarctions in pediatric patients 180 due to a germline CD59 deficiency, a post-mortem genetic investigation revealed a 181 previously reported homozygous point mutation at c.146delA (hg19, NM_203330) on 182 Exon 5 of CD59 (fig. 1B). This deletion leads to a frameshift at position 49 183 (p.Asp49Valfs*31) with a premature stop. The variation was not listed in SNPbase, 184 1000 Genome-Database, EVS-Database, Kaviar-Database, and ExAC-Database. 185 Both parents and the sister were heterozygous for the mutation. 186 Educated by the genetic analysis, a reanalysis of the brain and dural biopsies 187 showed subacute cerebral infarction with macrophages and capillary proliferation. 188 Several cerebral and meningeal blood vessels showed severe endothelial damage 189 with 190 Immunohistochemically analysis of brain tissue for CD59 expression showed a 191 complete absence of CD59 staining when compared to a healthy control (fig. 3 192 C&D). EP TE D M AN U SC RI PT 166 aggregation AC C platelet both inside and outside the infarcted areas. Klemann et al. 8 ACCEPTED MANUSCRIPT Discussion 194 CD59 (also known as MAC-inhibitory protein, membrane inhibitor of reactive lysis, 195 or protectin) is a complement regulatory protein linked to the cell surface by a GPI- 196 anchor protein [1]. Somatic mutations in hematopoetic stem cells affecting the GPI 197 anchor of several proteins reduce, amongst other proteins, the expression of CD59 198 leading to paroxysmal nocturnal hemoglobinuria (PNH) [8]. PNH is characterized by 199 Coombs-negative hemolytic anemia due to the lack of inhibition of the complement 200 cascade [8]. About half of all PNH patients additionally suffer of thrombosis, including 201 cerebral venous thrombosis in about 4 % of patients [8]. Anecdotal reports have 202 described that besides acquired mutations in GPI-anchors - and thus secondary lack 203 of CD59 – germline mutations in CD59 can cause PNH [2,3]. In contrast to PNH, 204 germline CD59 mutations affect all cells of the body and thus the more severe 205 phenotype is comprehensible. Recent reports of inherited CD59 deficiency leading to 206 early-onset CIDP, coombs negative hemolytic anemia, and strokes led us to 207 reanalyze post mortally the case of a boy of Turkish descent with similar 208 presentation. 209 p.Asp49Valfs*31 mutation (fig. 1) in addition to the 8 reported patients with other 210 mutations in CD59 (tbl. 1). Of note, no relationship to the other affected Turkish 211 patients could be established over the past 4 generations, albeit this does not 212 contradict the presence of a founder mutation in the Turkish population. 213 Although at first sight the combination of hemolytic anemia, recurrent polyneuropathy 214 and cerebrovascular disease sounds quite uniform in all reported patients with 215 congenital CD59 deficiency, at a closer look there are some remarkable differences 216 even between patients with the same mutation that are probably due to variable 217 pathophysiological pathways depending on the genetic background or on exogenous 218 influences. With the exception of the first reported Japanese case all patients 219 suffered from usually very early onset, severe, chronic, relapsing-remitting 220 polyneuropathy that in some of them lead to severe disability and even death (tbl.1). 221 In our patient as well as in the cases reported by Nevo et al., Ben-Zeev et al., and 222 Höchsmann et al. electrophysiological criteria showed that the neuropathy was 223 clearly of the classical demyelinating CIDP type with secondary axonal damage, 224 whereas the cases of Haliloglu et al. suffered from primary axonal neuropathy (in one 225 case with secondary demyelination) [4–7]. We conclude that CD59 deficiency can identified the 4th patient reported with a homozygous AC C EP TE D We M AN U SC RI PT 193 Klemann et al. 9 ACCEPTED MANUSCRIPT lead to a primary insult either at the Schwann cell or to the axon itself. 227 Pathophysiologically, CD59 deficiency might increase the vulnerability of spinal roots 228 and peripheral nerves due to diminished protection from the MAC; a previous in vitro 229 study demonstrated protection from complement-mediated lysis of neurons when 230 CD59 expression was increased [9]. Alternatively, autoimmunity could be causative, 231 as experimental investigations in mice lacking CD59 have shown an increased 232 susceptibility for autoimmune-phenomena [10]. Clinically, the occurrence of CIDP or 233 chronic axonal neuropathy already in infancy is extremely rare so in such cases 234 CD59 deficiency should be considered as being disease causing. 235 Not all cases with CD59 deficiency reported in the literature have suffered strokes 236 (tbl. 1). The affected children might have been too young at the age of reporting, but 237 this seems improbable for the 13 to 55 years old cases in the North African Jewish 238 families of Nevo et al [4]. If reported the strokes have usually been diagnosed by MRI 239 showing supra- or infratentorial infarctions due to assumed thrombo-embolic events 240 in the anterior or posterior cerebral circulation (tbl. 1). In one case hemorrhagic 241 stroke due to an occlusion of the PICA (posterior inferior cerebellar artery) has been 242 demonstrated by MR-angiography [7]. In contrast, serial MRI, TOF-MRA, HR-VWI 243 and DSA show relentless progression of stenoses of the proximal cerebral arteries at 244 the Circulus of Willisii in our patient and thus MMS was diagnosed (fig. 2). 245 Moyamoya (meaning a "hazy puff of smoke" in Japanese) is the term for a radiologic 246 finding of progressive intracranial vascular stenoses of the distal internal carotid 247 arteries and the proximal medial, anterior and less frequently posterior cerebral 248 arteries associated with enlarged collateral supply via the lenticulostriate and 249 thalamoperforating arteries and in later stages leptomeningeal and transdural arteries 250 [11–13]. Strokes and epileptic seizures constitute the prevailing presentation in 251 childhood [12]. The etiology of Moyamoya is multifactorial and largely unknown. 252 ‘Idiopathic Moyamoya disease’ (MMD) is considered to be an entity to be separated 253 from secondary occurrences termed ‘Moyamoya syndrome’ (MMS). In far-eastern 254 countries the largest part of idiopathic cases is caused by a single mutation in the 255 RNF213 gene. 256 GUCY A3, COL4A1 or MOPD2. MMS is associated 257 Neurofibromatosis 258 atherosclerosis, cerebral vasculitis and others [12, 13]. A single case of MMS has AC C EP TE D M AN U SC RI PT 226 Other hereditary factors comprise mutations in ACTA2, TIMP2, type 1, Alagille syndrome, with Trisomy 21, radiotherapy, cerebrovascular Klemann et al. 10 ACCEPTED MANUSCRIPT been reported in the context of PNH, but whether pro-inflammatory or pro-thrombotic 260 mechanism were causative remained elusive [14]. 261 Histologically, MMD and MMS are characterized by focally thickened vascular intima 262 due to proliferation of endothelial and smooth muscle cells, loss of muscle cells of the 263 media, and dilated or irregular-shaped vessel lumen [11–13]. Novel neuroradiological 264 techniques allow to consider not only the vessel lumen but also the vessel wall 265 [15,16]. However, the role of contrast-enhancement of the arterial wall in MMS is not 266 uniformly agreed on. With high resolution vessel wall imaging (HR-VWI) Ryoo et al. 267 found diffuse and circular enhancement in the arterial walls of the stenotic segments 268 in 90% of 32 MMD cases, whereas Kim et al. observed focal enhancement in only 2 269 of 7 cases [17,18]. Diffuse contrast enhancement associated with swelling of the 270 arterial wall is the radiological hallmark of acute vasculitis, as opposed to 271 cerebrovascular atherosclerosis which is characterized by focal and eccentric 272 deposition of plaques, hemorrhage and contrast enhancement [16]. Contrast 273 enhancement in cases of MMS could be an unspecific sign of an inflammatory 274 pathophysiology, and following recent reviews only the lack of swelling of the whole 275 vascular wall with increase of the vascular diameter could be an argument to 276 differentiate MMS from acute vasculitis [19–21]. It should be emphasized that 277 development of collaterals is not pathognomonic for idiopathic MMD and could also 278 represent MMS due to an unspecific reaction of the stenosis of the proximal 279 intracerebral arteries in progressive large vessel CNS vasculitis. In our patient 280 however, systemic vasculitis work-up was negative, including repeatedly normal 281 serum markers and lack of inflammatory changes in the brain- and meningeal biopsy. 282 In line with these findings, aggressive immunosuppressant treatment failed to control 283 the 284 thromboembolic events, but finally failed also to prevent the recurrent infarction in the 285 final stage of the disease because of progressive stenosis of the cerebral arteries. 286 Eventually, these investigations as well as the clinical and neuroradiological course 287 led to the diagnosis of MMS, which is now post-mortemally explained by CD59 288 deficiency 289 Concerning a possible pro-thrombotic component, intravascular hemolysis resulting 290 in depletion of nitric oxygen might have activated the platelets and endothelium [1]. 291 However, the neuroradiological and haemostaseological investigations were not AC C EP TE D M AN U SC RI PT 259 disease. Antiplatelet therapy may have extended the time without Klemann et al. 11 ACCEPTED MANUSCRIPT compatible with local thrombotic disease which would be the hallmark in PNH. MMS 293 is known to be associated with intimal and endothelial dysfunction [13]. In CD59 294 deficiency chronic or acute hemolysis might trigger leukocyte- and endothelial 295 dysfunction promoting an inflammatory component in the vasculopathy which could 296 give rise to the observed contrast enhancement of vessel walls. Furthermore, it 297 seems possible that the cerebral events are a direct consequence of the lack of 298 CD59 on the cerebral endothelium making it vulnerable to disruption by the 299 complement system (fig. 3). 300 Recognizing the combination of early onset hemolysis, CIDP and stroke would have 301 prompted further diagnostics including PNH flow cytometry. With this, lack of CD59 302 expression while other GPI-anchored proteins are normally present would have 303 enabled a timely diagnosis [7]. Therapeutically, targeted complement inhibition with 304 the anti-C5 monoclonal antibody eculizumab appears to be a promising treatment 305 option also for CD59 deficiency [6,7]. 306 In summary, this case expands the clinical spectrum of autosomal-recessive CD59 307 deficiency syndrome to radiologic findings of MMS. We conclude, that our case 308 emphasizes the need for early diagnosis of CD59 deficiency in young children with 309 hemolysis and chronic neuropathy as treatment with eculizumab could positively 310 influence the clinical course. TE D M AN U SC RI PT 292 EP 311 Acknowledgements 313 The authors would like to thanks the patient´s family as well as all physicians 314 involved. We thank Dr. Christian Schulz-Huotari, Institute for Transfusion Medicine, 315 University of Freiburg and Dr. Christof Weinstock, Institute for Transfusion Medicine, 316 University of Ulm, Germany for helpful discussion. We are grateful to the other team 317 members of the Center for Pediatrics and CCI Advanced Diagnostic Unit for their 318 excellent work. This study was supported by the German Federal Ministry of 319 Education and Research (BMBF01EO1303). The authors declare no conflicting 320 interests. 321 AC C 312 Klemann et al. 322 References: 323 [1] Höchsmann B, Schrezenmeier H. Congenital CD59 Deficiency. Hematol Oncol Clin North Am 2015;29:495–507. doi:10.1016/j.hoc.2015.01.006. 324 325 12 ACCEPTED MANUSCRIPT [2] Motoyama N, Okadan N, Yamashinam M, Okada H, Yamashina M, Okada N, et al. Paroxysmal nocturnal hemoglobinuria due to hereditary nucleotide 327 deletion in the HRF20 (CD59) gene. Eur J Immunol 1992;22:2669–73. 328 doi:10.1002/eji.1830221029. 329 [3] RI PT 326 Yamashina M, Ueda E, Kinoshita T, Takami T, Ojima A, Ono H, et al. Inherited complete deficiency of 20-kilodalton homologous restriction factor (CD59) as a 331 cause of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1990;323:1184– 332 9. doi:10.1056/NEJM199010253231707. [4] Nevo Y, Ben-Zeev B, Tabib A, Straussberg R, Anikster Y, Shorer Z, et al. M AN U 333 SC 330 334 CD59 deficiency is associated with chronic hemolysis and childhood relapsing 335 immune-mediated polyneuropathy. Blood 2013;121:129–35. 336 doi:10.1182/blood-2012-07-441857. 337 [5] Ben-Zeev B, Tabib A, Nissenkorn A, Garti B-Z, Gomori JM, Nass D, et al. Devastating recurrent brain ischemic infarctions and retinal disease in pediatric 339 patients with CD59 deficiency. Eur J Paediatr Neurol 2015;19:688–93. 340 doi:10.1016/j.ejpn.2015.07.001. 341 [6] TE D 338 Höchsmann B, Dohna-Schwake C, Kyrieleis H a, Pannicke U, Schrezenmeier H. Targeted therapy with eculizumab for inherited CD59 deficiency. N Engl J 343 Med 2014;370:90–2. doi:10.1056/NEJMc1308104. [7] Early-onset chronic axonal neuropathy, strokes, and hemolysis: Inherited CD59 345 deficiency. Neurology 2015;84:1220–4. doi:10.1212/WNL.0000000000001391. 346 347 Haliloglu G, Maluenda JJJ, Sayinbatur B, Aumont C, Temucin C, Tavil B, et al. AC C 344 EP 342 [8] DeZern AE, Brodsky RA. Paroxysmal nocturnal hemoglobinuria: a 348 complement-mediated hemolytic anemia. Hematol Oncol Clin North Am 349 2015;29:479–94. doi:10.1016/j.hoc.2015.01.005. 350 [9] Kolev M V, Tediose T, Sivasankar B, Harris CL, Thome J, Morgan BP, et al. 351 Upregulating CD59: a new strategy for protection of neurons from complement- 352 mediated degeneration. Pharmacogenomics J 2010;10:12–9. 353 doi:10.1038/tpj.2009.52. Klemann et al. 13 ACCEPTED MANUSCRIPT 354 [10] Miwa T, Zhou L, Maldonado MA, Madaio MP, Eisenberg RA, Song W-C. 355 Absence of CD59 exacerbates systemic autoimmunity in MRL/lpr mice. J 356 Immunol 2012;189:5434–41. doi:10.4049/jimmunol.1201621. 357 [11] Kim T, Oh CW, Bang JS, Kim JE, Cho W-S. Moyamoya Disease: Treatment 358 and Outcomes. J Stroke 2016;18:21–30. doi:10.5853/jos.2015.01739. 361 362 363 364 Stroke 2016;18:2–11. doi:10.5853/jos.2015.01627. RI PT 360 [12] Kim JS. Moyamoya Disease: Epidemiology, Clinical Features, and Diagnosis. J [13] Bang OY, Fujimura M, Kim S-K. The Pathophysiology of Moyamoya Disease: An Update. J Stroke 2016;18:12–20. doi:10.5853/jos.2015.01760. SC 359 [14] Lin HC, Chen RL, Wang PJ. Paroxysmal nocturnal hemoglobinuria presenting as moyamoya syndrome. Brain Dev 18:157–9. [15] Kaku Y, Morioka M, Ohmori Y, Kawano T, Kai Y, Fukuoka H, et al. Outer- 366 diameter narrowing of the internal carotid and middle cerebral arteries in 367 moyamoya disease detected on 3D constructive interference in steady-state 368 MR image: Is arterial constrictive remodeling a major pathogenesis? Acta 369 Neurochir (Wien) 2012. doi:10.1007/s00701-012-1472-4. M AN U 365 [16] Yuan M, Liu Z qiang, Wang Z qiang, Li B, Xu L jun, Xiao X lan. High-resolution 371 MR imaging of the arterial wall in moyamoya disease. Neurosci Lett 2015. 372 doi:10.1016/j.neulet.2014.10.021. 373 TE D 370 [17] Ryoo S, Cha J, Kim SJ, Choi JW, Ki CS, Kim KH, et al. High-resolution magnetic resonance wall imaging findings of moyamoya disease. Stroke 375 2014;45:2457–60. doi:10.1161/STROKEAHA.114.004761. EP 374 [18] Kim YJ, Lee DH, Kwon JY, Kang DW, Suh DC, Kim JS, et al. High resolution 377 MRI difference between moyamoya disease and intracranial atherosclerosis. 378 AC C 376 Eur J Neurol 2013. doi:10.1111/ene.12202. 379 [19] Küker W, Gaertner S, Nägele T, Dopfer C, Schöning M, 380 Fiehler J, et al. Vessel Wall Contrast Enhancement: A Diagnostic Sign of 381 Cerebral Vasculitis. Cerebrovasc Dis 2008;26:23–9. doi:10.1159/000135649. 382 [20] Alexander MD, Yuan C, Rutman A, Tirschwell DL, Palagallo G, Gandhi D, et al. 383 High-resolution intracranial vessel wall imaging: imaging beyond the lumen. J 384 Neurol Neurosurg Psychiatry 2016;87:589–97. doi:10.1136/jnnp-2015-312020. 385 [21] Choi YJ, Jung SC, Lee DH. Vessel Wall Imaging of the Intracranial and Klemann et al. 14 ACCEPTED MANUSCRIPT 386 Cervical Carotid Arteries. J Stroke 2015;17:238–55. 387 doi:10.5853/jos.2015.17.3.238. 388 AC C EP TE D M AN U SC RI PT 389 Klemann et al. Figures: RI PT 390 15 ACCEPTED MANUSCRIPT SC 391 Figure 1 393 A) Pedigree of the family with CD59 deficiency: The index patient (arrow) was 394 homozygous (black) for a frameshift mutation in CD59. His sister and parents 395 proofed to be heterozygous (grey). 396 B) CD59 p.Asp49Valfs*31 mutation: Forward genomic DNA sequence of a control 397 and the index patient.The homozygous deletion of A introduces a shift of the open 398 reading frame. AC C EP TE D M AN U 392 Klemann et al. Figure 2 M AN U SC RI PT 399 16 ACCEPTED MANUSCRIPT AC C EP TE D 400 401 402 Legend Figure 2: 403 A-C) MR imaging at age of 6 yrs.: A) An axial T2-weighted image shows multiple 404 lesions in the left centrum semiovale. B) 3D-TOF-MRA shows severe stenosis of the Klemann et al. 17 ACCEPTED MANUSCRIPT carotid-T on the left and moderate stenosis on the right side. C) The arrows in C 406 point to enlarged lenticulostriate arteries indicating the Moyamoya pattern 407 D-E) MR imaging at age of 7 yrs.: D) 3D TOF-MRA shows progressive stenosis on 408 both sides. E) 3Tesla vessel wall imaging shows distinct diffuse contrast 409 enhancement of the distal carotid arteries with shrinkage of vascular diameter. 410 F-G) MRI imaging at age of 8 yrs. 1 month: F) 3D TOF show complete obliteration of 411 the distal internal carotid and proximal MCA on both sides and extra-intracranial 412 anastomoses after multiple synangiosis operations. G) DWI-MRT shows severe 413 atrophy on the left after repeated infarctions and a recent complete media infarction 414 on the right. 415 Figure 3 AC C EP TE D M AN U SC RI PT 405 416 417 Legend Figure 3: 418 Serial sections were stained immunohistochemically for CD34 and CD61. 419 (A) Blood vessels with endothelial damage (arrow) show a lack of immunoreactivity 420 of CD34. Klemann et al. 18 ACCEPTED MANUSCRIPT 421 (B) Detection of platelet aggregates within the same vessel in the CD61 reaction. 422 (C) The brain biopsy tissue shows a complete lack of CD59 (arrows) compared to a 423 healthy control (D). 424 Report Yamashina 1990, Motoyama 1992 [2,3] Nevo 2013 [4] Ben-Zeev 2015 [5] Höchsmann 2014 [6] Patient # Ethnic Origin 1 Japanese yes compound heterozygous p.Val42Serfs*38; p.Ala121Glnfs 13 yrs yes, chronic and acute not reported 11/12 North African Jewish, 1 family no homozygous c.266 G>A p.Cys89Tyr 7 Turkish Consanguinity Mutation 2-6 North African Jewish, 4 families 1/5 yes homozygous c.266 G>A p.Cys89Tyr 3-7 mo Yes 3-5 mo yes 7 mo yes not reported Coombs test none reported Central neurological disease cerebral infarction 1/5 HUS like disease w/ renal failure not reported AC C Treatment EP Others: Outcome Motor-sensory, recurrent, 4/5 demyelinated secondary axonal (CIPD), 1/5 primary axonal, sMRT dorsal root enhancement 2/3 None reported n/a IVIG, plasmapheresis, steroids, rituximab, cyclosporin 4 alive ~5 yrs of age, 1 deceased at 3.5 yrs. Current report 8-10 Turkish, 1 family 13 Turkish yes homozygous c.146delA, p.Asp49Valfs*31 Yes homozygous c.146delA, p.Asp49Valfs*31 6-11 mo no/ yes, chronic/yes acute & chronic negative 1 mo yes, chronic and acute intermittingly positive Recurrentremitting, steroidresponsive demyelinated with conduction block not reported not reported recurrent peripheral demyelinating neuropathy with conduction block (CIDP) Demyelinating neuropathy with secondary axonal damage 3/3 primary recurrent axonal polyneuropathy, 1/3 secondary demyelination in later course, 2/3 gadolinium enhancement of dorsal roots Recurrent cerebral infarctions from age 18 mo and 5 y, retinal and optic nerve involvement Possible brainstem infarction versus encephalitis (MRI) at age 18 mo 1/3 supratentorial stroke at age 5y; 1/3 infratentorial hemorrhagic stroke and occlusion of PICA at age 18 mo; 1/3 no stroke up to age 4.5 y TE D Peripheral neurological disease yes homozygous c.146delA, p.Asp49Valfs*31 Haliloglu 2015 [7] SC Onset of disease Hemolysis 426 RI PT Table 1: M AN U 425 atypical glomerulonephritis, necrotic fingers IVIG, steroids deceased at age of 8 and 2.5 yrs. 2/3 phototherapy newborn necessary Recurrent supratentorial infarctions from age 6 y, small vessel endothelial damage, large vessel enhancement, moyamoya as IVIG, plasmapheresis, eculizumab not reported/IVIG/IVIG & eculizumab IVIG, steroids, azathioprin, MMF, rituximab, cyclophosphamid 5 yrs, improvement under eculizumab therapy deceased at age 16 /alive ~10 yrs with mild cognitive impairment /alive ~5 yrs with global developmental delay Deceased at age 8 years 427 Table legend: 428 Table 1: Summary of reported patients with CD59 deficiency.