Abstract The ability to correctly diagnose the molecular cause of genetic diseases is becoming increasingly important in medicine. This requires an efficient method for the analysis of the DNA sequence of specific genes and the detection of mutations in affected individuals. We report a method to determine the mutations responsible for tyrosinase related albinism (OCA1) using a combination of polymerase chain reaction‐single stranded conformational polymorphism (PCR‐SSCP) analysis and direct DNA cycle sequencing using fluorescently labeled oligonucleotides and an automated DNA sequencer based on infrared fluorescence technology. This method allows DNA from several individuals to be sequenced quickly and simultaneously so that the specific location of each mutation and the carrier status of family members can be determined.
OBJECTIVE:
To compare aquaporin-4 IgG assays for neuromyelitis optica spectrum disorder (NMOSD) diagnosis, 2011-2013.
BACKGROUND: Comparative performances of aquaporin-4 IgG-specific assays in a high-throughput clinical laboratory are unknown.
DESIGN/METHODS:
All patients were initially tested by service aquaporin-4-ELISA (antigen, M1-aquaporin-4). Additional testing was by cell-based assay (CBA, M1-aqupaorin-4, indirect immunofluorescence) and in-house-developed fluorescence-activated cell sorting (FACS) assays (M1 and M23 isoforms). Results were referenced to pre-test diagnoses. Patient groups comprehensively tested were: Group 1, 388 consecutive Mayo Clinic patients (January-May 2012); Group 2, 615 consecutive Mayo and non-Mayo patients (September 2012); Group 3, 36 patients physician-reported as NMOSD with negative M1-ELISA (2011-2013); Group 4, 41 patients physician-reported as not NMOSD with positive M1-ELISA (2011-2013). FACS results were compared for cells transfected with M1, M23 or both (1:1) in M23-FACS seropositive patients with and without NMOSDs.
RESULTS:
For Group 1, M1-FACS assay performance was optimum (area under the curve, 0.64 [p=0.02]). Sensitivities were: M1-FACS, 83%; M23- FACS and M1-CBA, 75%; M1-ELISA, 58% (p<0.05). M23-FACS specificity (95%) was lower than for other assays (M1-CBA and M1-FACS, 100%; M1-ELISA, 99%), p=0.004. For 24 Group 2 patients with NMOSD, positive results were yielded by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20 and M1-ELISA, 18. For 6 Group 2 patients without NMOSD, positive results were yielded by: M23-FACS only, 2; M1-ELISA only, 2; all 3 of M1-CBA, M23 FACS and M1-FACS, 2. For Group 3 patients (false negative M1-ELISA suspected) positive results were yielded by: M1-FACS, 5; M23-FACS, 3; M1-CBA, 2. For Group 4 (false positive M1-ELISA suspected) just one yielded a positive result by another assay. Non-specific IgG binding in M23-FACS was diminished using M1/ M23 co-transfected cells.
CONCLUSIONS:
Cell binding assays, particularly M1-FACS, have optimum performance and utility in evaluating potential false positive results. False positives in M23-FACS may be attributable to non-specific IgG binding to aquaporin-4 high order arrays.
Study Supported by: National Institutes of Health (R01 NS065829) and the Guthy Jackson Foundation. Disclosure: Dr. Fryer has nothing to disclose. Dr. Lennon stands to receive royalty payments for commercial assays to detect of Aquaporin 4-specific Autoantibody. Dr. Pittock9s institution has received compensation for activities with Alexion Pharmaceuticals, MedImmune, and Chugai Pharma. Dr. Pittock stands to receive royalty payments from the technology entitled Neuromyelitis Optica Autoantibodies as a Marker for Neoplasia. Dr. Pittock has received research support from Alexion Pharmaceuticals, Inc. Dr. Jenkins has nothing to disclose. Dr. Horta has nothing to disclose. Dr. Jedynak has nothing to disclose. Dr. Lucchinetti stands to receive royalty payments from Biogen Idec. Dr. Shuster has received personal compensation for activities with Prime Inc. Dr. Weinshenker has received personal compensation for activities with Novartis, Biogen Idec, and Mitsubishi Pharmaceuticals as consultant on data safety monitoring boards; and with Elan Corporation, Ono Pharmaceutical, GlaxoSmithKline Inc., Alexion and Chugai Pharmaceuticals, and Asahi Kasei Medical Company as a consultant. Dr. Weinshenker has received royalty payments from Mayo Foundation. Dr. Wingerchuk has received research support from Genentech, Inc., Genzyme Corporation, Alexion, and TerumoBCT. Dr. McKeon has nothing to disclose.
Studies focused on recurrent longitudinally extensive transverse myelitis (rLETM) are lacking.To determine the aquaporin 4 (AQP4) IgG detection rate using recombinant human APQ4-based assays in sequential serum specimens collected from patients with rLETM categorized as negative by first-generation tissue-based indirect immunofluorescence (IIF) assay and to define the clinical characteristics and motor disability outcomes in AQP4-IgG-positive rLETM.A search of the Mayo Clinic computerized central diagnostic index (October 1, 2005, through November 30, 2011), cross-linked with the Neuroimmunology Laboratory database, identified 48 patients with rLETM, of whom 36 (75%) were positive and 12 (25%) negative for neuromyelitis optica (NMO) IgG (per IIF of serial serum specimens). Stored serum specimens from "seronegative" patients were retested with recombinant human AQP4-based assays, including enzyme-linked immunosorbent, transfected cell-based, and fluorescence-activated cell-sorting assays. Control patients included 140 AQP4-IgG-positive patients with NMO, of whom a subgroup of 20 initially presented with 2 attacks of transverse myelitis (rLETM-onset NMO).AQP4-IgG serostatus, clinical characteristics, and Expanded Disability Status Scale score.Six patients with negative IIF results were reclassified as AQP4-IgG positive, yielding an overall AQP4-IgG seropositivity rate of 89%. Fluorescence-activated cell-sorting, cell-based, and enzyme-linked immunosorbent assays improved the detection rate to 89%, 85%, and 81%, respectively. The female to male ratio was 2:3 for AQP4-IgG-negative rLETM and 5:1 for AQP4-IgG-positive patients. The AQP4-IgG-positive patients with rLETM or rLETM-onset NMO were similar in age at onset, sex ratio, attack severity, relapse rate, and motor disability. From Kaplan-Meier analyses, 36% of AQP4-IgG-positive patients with rLETM are anticipated to need a cane to walk within 5 years after onset. For patients with rLETM-onset NMO, the median time from onset to first optic neuritis attack (54 months) was similar to the median disease duration for AQP4-IgG-positive patients with rLETM (59 months). The median number of attacks was 3 for AQP4-IgG-positive patients with rLETM (range, 2-22), and the first optic neuritis attack for those with rLETM-onset NMO followed a median of 3 myelitis attacks (range, 2-19). Immunosuppressant therapy reduced the relapse rate in both AQP4-IgG-positive and AQP4-IgG-negative patients with rLETM.Recombinant antigen-based assays significantly increase AQP4-IgG detection in patients with rLETM, and AQP4-IgG-negative adults with rLETM are rare. Evolution to NMO can be anticipated in AQP4-IgG-positive patients. Early initiation of immunotherapy may result in a more favorable motor outcome.
Objective: To determine glial autoantibody serostatus in rLETM patients. Background: Autoimmune AQP-4 channelopathy is the commonest cause of immune-mediated recurrent longitudinally extensive transverse myelitis (rLETM). In 2014, our group reported AQP4-IgG detection in 89% rLETM cases [JAMA Neurol 2014]. Autoimmune myelin oligodendrocyte glycoproteinopathy (MOG-opathy) has recently been reported in AQP4-IgG seronegative LETM. The frequency of MOG-IgG1 antibodies in rLETM is unknown. The autoimmune GFAP astrocytopathy phenotype may include myelitis as a component of encephalomyelitis. Design/Methods: Patients (identified at Mayo Clinic 2005–2017) fulfilling the following criteria were included: 1) initial 2 attacks LETM, 2) serum available (for at least 1 glial antibody test), 3) alternative etiologies (e.g. infectious, rheumatologic, neoplastic, vascular) excluded. AQP4-IgG and MOG-IgG1 serostatus was determined using a validated flow cytometry assay utilizing live M1-AQP4-transfected and full length MOG-transfected HEK293 cells. GFAP-IgG serostatus was determined using indirect immunofluorescence assay. Results: AQP4-IgG was detected in 56/63 (89%) patients with rLETM. Serum was available on 58 of 63 rLETM patients for MOG-IgG1 testing: only 1 patient tested positive for MOG-IgG1 (subsequently developed optic neuritis). None (including 13 cerebrospinal fluid samples) tested positive for GFAP-IgG. Thus overall, AQP4-IgG+, MOG-IgG1+ and double negative (DN) groups accounted for 89%, 1.7% and 9% respectively (AQP4-IgG vs MOG-IgG1+/DN, p Conclusions: Accepting potential confounding by referral bias, autoimmune AQP4 channelopathy is the etiology of almost half of single LETM and almost 90% of rLETM. Autoimmune MOG-opathy can present with LETM, but recurrent sequential LETM is rarely associated with this condition. Inflammation, mainly restricted to spinal cord, as in these LETM cases is likely not within the Autoimmune GFAP astrocytopathy spectrum. Disclosure: Dr. Jitprapaikulsan has nothing to disclose. Dr. Lopez has nothing to disclose. Dr Flanagan has nothing to disclose. Dr. Fryer has nothing to disclose. Dr. McKeon has received research support from Medimmune, Euroimmun, Grifols and Alexion. Dr. Yujuan has nothing to disclose. Dr. Weinshenker has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Novartis, Alexion, MedImmune, Caladrius Biosciences, Brainstorm Therapeutics. Dr. Majed has nothing to disclose. Dr. Tobin has nothing to disclose. Dr. Keegan has nothing to disclose. Dr. Lucchinetti has nothing to disclose. Dr. Lennon has received royalty, license fees, or contractual rights payments from RSR, royalties from sale kits for AQP4 IgG detetction and from clinical service assays performed outside Mayo clinic. Dr. Sagen has nothing to disclose. Dr. Pittock has nothing to disclose.
Recognizing the characteristics of myelin oligodendrocyte glycoprotein autoantibody (MOG-IgG) myelitis is essential for early accurate diagnosis and treatment.
Objective
To evaluate the clinical, radiologic, and prognostic features of MOG-IgG myelitis and compare with myelitis with aquaporin-4–IgG (AQP4-IgG) and multiple sclerosis (MS).
Design, Setting, and Participants
We retrospectively identified 199 MOG-IgG–positive Mayo Clinic patients from January 1, 2000, through December 31, 2017, through our neuroimmunology laboratory. Fifty-four patients met inclusion criteria of (1) clinical myelitis; (2) MOG-IgG positivity; and (3) medical records available. We excluded 145 patients without documented myelitis. Myelitis of AQP4-IgG (n = 46) and MS (n = 26) were used for comparison.
Main Outcomes and Measures
Outcome variables included modified Rankin score and need for gait aid. A neuroradiologist analyzed spine magnetic resonance imaging of patients with MOG-IgG and control patients blinded to diagnosis.
Results
Of 54 included patients with MOG-IgG myelitis, the median age was 25 years (range, 3-73 years) and 24 were women (44%). Isolated transverse myelitis was the initial manifestation in 29 patients (54%), and 10 (19%) were initially diagnosed as having viral/postviral acute flaccid myelitis. Cerebrospinal fluid–elevated oligoclonal bands occurred in 1 of 38 (3%). At final follow-up (median, 24 months; range, 2-120 months), 32 patients (59%) had developed 1 or more relapses of optic neuritis (n = 31); transverse myelitis (n = 7); or acute disseminated encephalomyelitis (n = 1). Clinical features favoring MOG-IgG myelitis vs AQP4-IgG or MS myelitis included prodromal symptoms and concurrent acute disseminated encephalomyelitis. Magnetic resonance imaging features favoring MOG-IgG over AQP4-IgG or MS myelitis were T2-signal abnormality confined to gray matter (sagittal line and axial H sign) and lack of enhancement. Longitudinally extensive T2 lesions were of similar frequency in MOG-IgG and AQP4-IgG myelitis (37 of 47 [79%] vs 28 of 34 [82%];P = .52) but not found in MS. Multiple spinal cord lesions and conus involvement were more frequent with MOG-IgG than AQP4-IgG but not different from MS. Wheelchair dependence at myelitis nadir occurred in one-third of patients with MOG-IgG and AQP4-IgG but never with MS, although patients with MOG-IgG myelitis recovered better than those with AQP4-IgG.
Conclusions and Relevance
Myelitis is an early manifestation of MOG-IgG–related disease and may have a clinical phenotype of acute flaccid myelitis. We identified a variety of clinical and magnetic resonance imaging features that may help clinicians identify those at risk in whom MOG-IgG should be tested.
Background: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory central nervous system disorder characterized, despite immunotherapy treatments, by life-long, severe, and disabling attacks of optic neuritis and myelitis. The aim is to determine if autologous non-myeloablative hematopoietic stem cell transplantation could be an alternative treatment option.
To compare the sensitivity and specificity of immunofluorescence (IF) and immunoprecipitation (IP) assays using green fluorescent protein–tagged aquaporin-4 (AQP4) in 6335 patients for whom serological evaluation was requested on a service basis.
Design
Case-control study.
Setting
Mayo Clinic Neuroimmunology Laboratory (Rochester, Minnesota) and Departments of Neurology (Rochester, Minnesota; Scottsdale, Arizona; and Jacksonville, Florida).
Patients
Group 1, 835 Mayo Clinic patients, 100 with a neuromyelitis optica (NMO) spectrum disorder diagnosis and 735 without NMO spectrum disorder; group 2, 5500 non–Mayo Clinic patients.
Main Outcome Measure
Sensitivity and specificity of each assay for NMO or NMO spectrum disorder, individually and combined.
Results
In group 1, the sensitivity rates for NMO were IF, 58%; IP, 33%; and combined assays, 63%. The sensitivity rates for relapsing longitudinally extensive transverse myelitis were IF, 29%; IP, 6%; and combined assays, 29%. The specificity rates for NMO and relapsing longitudinally extensive transverse myelitis were IF, 99.6%; IP, 99.3%; and combined assays, 99.2%. In group 2, NMO-IgG was detected by IF in 498 of 5500 patients (9.1%) and by IP in 331 patients (6.0%); 76 of the 331 patients seropositive by IP (23%) were negative by IF. Clinical information was available for 124 patients (including 16 of those seropositive by IP only); 123 had a definite NMO spectrum disorder and 1 was at risk for NMO (monophasic optic neuritis).
Conclusions
In this large, clinical practice-based study, NMO-IgG detected by IF or IP was highly specific for NMO spectrum disorders. The IP assay was significantly less sensitive than IF. Combined testing improved sensitivity by 5%.
OBJECTIVE: 1) Investigate in rTM patients the AQP4-IgG detection rate in recombinant antigen-based assays for sera scored negative by 1st generation tissue-based indirect-immunofluorescence assay (IIF); 2) Compare clinical characteristics of seropositive rTM and NMO. BACKGROUND: Seropositive rTM is classified as an NMO-spectrum disorder. Evolving serological assay sensitivity has precluded comparison of the clinical characteristics of rTM and NMO. DESIGN/METHODS: From 2005-2011, 5349 samples from Mayo Clinic patients were tested by IIF assay. Clinical records revealed 48 patients had rTM (pediatric onset in 3). Of those, 36 (75%) were IIF-positive and 12 (25%) IIF-negative; 11 of the 12 scored negative were available for testing by ELISA (RSR/Kronus) and AQP4-transfected cell-binding assays (CBA slides [EUROIMMUN] and in-house fluorescence-activated-cell-sorting [FACS]). Clinical characteristics were compared based on finally assigned serostatus. 138 AQP4-IgG-positive NMO patients served as control cases. RESULTS: Overall, the AQP4-IgG detection rate improved to 91% by FACS, 85% by CBA, and 83% by ELISA. Only 4 cases remained seronegative: 2 boys (onset ages 3 and 6) and 2 adults (1 male, 1 female). Among 42 seropositive adult rTM cases (excluding single female with onset age 15) the female:male ratio = 5:1. rTM cases required a cane sooner (median 19.5 months) than NMO cases (48 months; p=0.04).Two findings suggest seropositive rTM may progress to NMO: 1) rTM cases had a shorter median disease duration (first attack to last follow-up, 61 months) than control NMO cases (106 months); 2) For the NMO subgroup with initial rTM course, time from onset to 1st optic neuritis (54 months) was similar to rTM cases9 median disease duration. CONCLUSIONS: Adult rTM is rarely AQP4-IgG-seronegative. NMO evolution can be anticipated in seropositive cases. The 2 seronegative boys may have MS because ∼ 14% of pediatric MS cases have longitudinally-extensive lesions [Banwell et al, Neurology, 2008]; adult MS cases do not. Supported by: Mayo Clinic Foundation, Guthy-Jackson Charitable Foundation (VL, CL, SP, BW, DW) and NIH (R01-NS65829, SP). Disclosure: Dr. Jiao has nothing to disclose. Dr. Fryer has nothing to disclose. Dr. Lennon stands to receive royalties for commercial assays to detect of aquaporin 4-specific autoan. Dr. McKeon has nothing to disclose. Ms. Smith has received research support from Abbott Laboratories. Dr. Jenkins has nothing to disclose. Dr. Iorio has nothing to disclose. Dr. Costanzi has nothing to disclose. Dr. Quek has nothing to disclose. Dr. Weinshenker has received personal compensation for activities with Novartis, Biogen Idec, Elan Corporation, GlaxoSmithKline Inc., Asahi Kasei Kuraray Medical Company as a consultant and/or participant on a data safety monitoring board. Dr. Weinshenker has received (royalty or license fee or contractual rights) payments from Mayo Medical Ventures. Dr. Wingerchuk has received research support from Genentech, Genzyme, Alexion, TerumoBCT and the Guthy-Jackson Charitable Foundation. Dr. Shuster has nothing to disclose. Dr. Lucchinetti stands to receive royalties for commercial assays to detect aquaporin 4-specific autoantibody. Dr. Pittock and Mayo Clinic have a financial interest in the technology entitled Neuromyelitis Optica Autoantibodies as a Marker for Neoplasia. This technology has been licensed to a commercial entity but no royalties have been received. Dr Sean Pittock has received research support from Alexion Pharmaceuticals, Inc.
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