Multisystem proteinopathy due to a homozygous p.Arg159His VCP mutation : a tale of the unexpected

ObjectiveTo assess the clinical, radiologic, myopathologic, and proteomic findings in a patient manifesting a multisystem proteinopathy due to a homozygous valosin-containing protein gene (VCP) mutation previously reported to be pathogenic in the heterozygous state.MethodsWe studied a 36-year-old male index patient and his father, both presenting with progressive limb-girdle weakness. Muscle involvement was assessed by MRI and muscle biopsies. We performed whole-exome sequencing and Sanger sequencing for segregation analysis of the identified p.Arg159His VCP mutation. To dissect biological disease signatures, we applied state-of-the-art quantitative proteomics on muscle tissue of the index case, his father, 3 additional patients with VCP-related myopathy, and 3 control individuals.ResultsThe index patient, homozygous for the known p.Arg159His mutation in VCP, manifested a typical VCP-related myopathy phenotype, although with a markedly high creatine kinase value and a relatively early disease onset, and Paget disease of bone. The father exhibited a myopathy phenotype and discrete parkinsonism, and multiple deceased family members on the maternal side of the pedigree displayed a dementia, parkinsonism, or myopathy phenotype. Bioinformatic analysis of quantitative proteomic data revealed the degenerative nature of the disease, with evidence suggesting selective failure of muscle regeneration and stress granule dyshomeostasis.ConclusionWe report a patient showing a multisystem proteinopathy due to a homozygous VCP mutation. The patient manifests a severe phenotype, yet fundamental disease characteristics are preserved. Proteomic findings provide further insights into VCP-related pathomechanisms

Reduction of Mycobacterium avium ssp. paratuberculosis in colostrum : development and validation of 2 methods, one based on curdling and one based on centrifugation

The aim of this study was to develop and validate 2 protocols (for use on-farm and at a central location) for the reduction of Mycobacterium. avium ssp. paratuberculosis (MAP) in colostrum while preserving beneficial immunoglobulins (IgG). The on-farm protocol was based on curdling of the colostrum, where the IgG remain in the whey and the MAP bacteria are trapped in the curd. First, the colostrum was diluted with water (2 volumes colostrum to 1 volume water) and 2% rennet was added. After incubation (1 11 at 32 degrees C), the curd was cut and incubated again, after which whey and curd were separated using a cheesecloth. The curd was removed and milk powder was added to the whey. Approximately 1 log reduction in MAP counts was achieved. A reduction in total proteins and IgG was observed due to initial dilution of the colostrum. After curd formation, more than 95% of the immunoglobulins remained in the whey fraction. The semi-industrial protocol was based on centrifugation, which causes MAP to precipitate, while the IgG remain in the supernatant. This protocol was first developed in the laboratory. The colostrum was diluted with skimmed colostrum (2 volumes colostrum to 1 volume skimmed colostrum), then skimmed and centrifuged (at 15,600 x g for 30 min at room temperature). We observed on average 1.5 log reduction in the MAP counts and a limited reduction in proteins and IgG in the supernatant. To obtain a semi-industrial protocol, dairy pilot appliances were evaluated and the following changes were applied to the protocol: after 2:1 dilution as above, the colostrum was skimmed and subsequently clarified, after which the cream was heat treated and added to the supernatant. To investigate the effect of the colostrum treatment on the nutritional value and palatability of the colostrum and the IgG transfer, an animal experiment was conducted with 24 calves. Six received the dam's colostrum, 6 were given untreated purchased colostrum (control), and 2 groups of 6 calves received colostrum treated according to both of the above-mentioned methods. No significant differences were found between the test groups and the dam's colostrum group in terms of animal health, IgG uptake in the blood serum, milk; or forage uptake. Two protocols to reduce MAP in colostrum (for use on-farm or at a central location) were developed. Both methods preserve the vital IgG

Ataxia with oculomotor apraxia type 2: clinical, biological and genotype/phenotype correlation study of a cohort of 90 patients

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive disease due to mutations in the senataxin gene, causing progressive cerebellar ataxia with peripheral neuropathy, cerebellar atrophy, occasional oculomotor apraxia and elevated alpha-feto-protein (AFP) serum level. We compiled a series of 67 previously reported and 58 novel ataxic patients who underwent senataxin gene sequencing because of suspected AOA2. An AOA2 diagnosis was established for 90 patients, originating from 15 countries worldwide, and 25 new senataxin gene mutations were found. In patients with AOA2, median AFP serum level was 31.0 mu g/l at diagnosis, which was higher than the median AFP level of AOA2 negative patients: 13.8 mu g/l, P = 0.0004; itself higher than the normal level (3.4 mu g/l, range from 0.5 to 17.2 mu g/l) because elevated AFP was one of the possible selection criteria. Polyneuropathy was found in 97.5% of AOA2 patients, cerebellar atrophy in 96%, occasional oculomotor apraxia in 51%, pyramidal signs in 20.5%, head tremor in 14%, dystonia in 13.5%, strabismus in 12.3% and chorea in 9.5%. No patient was lacking both peripheral neuropathy and cerebellar atrophy. The age at onset and presence of occasional oculomotor apraxia were negatively correlated to the progression rate of the disease (P = 0.03 and P = 0.009, respectively), whereas strabismus was positively correlated to the progression rate (P = 0.03). An increased AFP level as well as cerebellar atrophy seem to be stable in the course of the disease and to occur mostly at or before the onset of the disease. One of the two patients with a normal AFP level at diagnosis had high AFP levels 4 years later, while the other had borderline levels. The probability of missing AOA2 diagnosis, in case of sequencing senataxin gene only in non-Friedreich ataxia non-ataxia-telangiectasia ataxic patients with AFP level >= 7 mu g/l, is 0.23% and the probability for a non-Friedreich ataxia non-ataxia-telangiectasia ataxic patient to be affected with AOA2 with AFP levels >= 7 mu g/l is 46%. Therefore, selection of patients with an AFP level above 7 mu g/l for senataxin gene sequencing is a good strategy for AOA2 diagnosis. Pyramidal signs and dystonia were more frequent and disease was less severe with missense mutations in the helicase domain of senataxin gene than with missense mutations out of helicase domain and deletion and nonsense mutations (P = 0.001, P = 0.008 and P = 0.01, respectively). The lack of pyramidal signs in most patients may be explained by masking due to severe motor neuropathy

Efficacy and safety of rozanolixizumab in moderate to severe generalized myasthenia gravis : a phase 2 randomized control trial

OBJECTIVE: To explore the clinical efficacy and safety of subcutaneous (SC) rozanolixizumab, an anti-neonatal Fc receptor humanized monoclonal antibody, in patients with generalized myasthenia gravis (gMG). METHODS: In this phase 2a, randomized, double-blind, placebo-controlled, 2-period, multicenter trial (NCT03052751), patients were randomized (1:1) in period 1 (days 1-29) to 3 once-weekly (Q1W) SC infusions of rozanolixizumab 7 mg/kg or placebo. In period 2 (days 29-43), patients were re-randomized to either rozanolixizumab 7 mg/kg or 4 mg/kg (3 Q1W SC infusions), followed by an observation period (days 44-99). Primary endpoint was change from baseline to day 29 in Quantitative Myasthenia Gravis (QMG) score. Secondary endpoints were change from baseline to day 29 in MG-Activities of Daily Living (MG-ADL) and MG-Composite (MGC) scores and safety. RESULTS: Forty-three patients were randomized (rozanolixizumab 21, placebo 22 [period 1]). Least squares (LS) mean change from baseline to day 29 for rozanolixizumab vs placebo was as follows: QMG (LS mean -1.8 vs -1.2, difference -0.7, 95% upper confidence limit [UCL] 0.8; p = 0.221; not statistically significant), MG-ADL (LS mean -1.8 vs -0.4, difference -1.4, 95% UCL -0.4), and MGC (LS mean -3.1 vs -1.2, difference -1.8, 95% UCL 0.4) scores. Efficacy measures continued to improve with rozanolixizumab 7 mg/kg in period 2. The most common adverse event in period 1 was headache (rozanolixizumab 57%, placebo 14%). CONCLUSION: Whereas change from baseline in QMG was not statistically significant, the data overall suggest rozanolixizumab may provide clinical benefit in patients with gMG and was generally well tolerated. Phase 3 evaluation is ongoing (NCT03971422). CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with gMG, rozanolixizumab is well-tolerated, but did not significantly improve QMG score

Dirac's Observables for the Rest-Frame Instant Form of Tetrad Gravity in a Completely Fixed 3-Orthogonal Gauge

We define the {\it rest-frame instant form} of tetrad gravity restricted to Christodoulou-Klainermann spacetimes. After a study of the Hamiltonian group of gauge transformations generated by the 14 first class constraints of the theory, we define and solve the multitemporal equations associated with the rotation and space diffeomorphism constraints, finding how the cotriads and their momenta depend on the corresponding gauge variables. This allows to find quasi-Shanmugadhasan canonical transformation to the class of 3-orthogonal gauges and to find the Dirac observables for superspace in these gauges. The construction of the explicit form of the transformation and of the solution of the rotation and supermomentum constraints is reduced to solve a system of elliptic linear and quasi-linear partial differential equations. We then show that the superhamiltonian constraint becomes the Lichnerowicz equation for the conformal factor of the 3-metric and that the last gauge variable is the momentum conjugated to the conformal factor. The gauge transformations generated by the superhamiltonian constraint perform the transitions among the allowed foliations of spacetime, so that the theory is independent from its 3+1 splittings. In the special 3-orthogonal gauge defined by the vanishing of the conformal factor momentum we determine the final Dirac observables for the gravitational field even if we are not able to solve the Lichnerowicz equation. The final Hamiltonian is the weak ADM energy restricted to this completely fixed gauge.Comment: RevTeX file, 141 page

High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

Adaptor protein complex 4-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of adaptor protein complex 4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here, we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by adaptor protein complex 4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z'-factor robust >0.3, strictly standardized mean difference >3). The `ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 +/- 0.13 versus 1.21 +/- 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia

Prepubertal unilateral gynecomastia: a report of two cases

Item does not contain fulltextBACKGROUND: Gynecomastia is defined as the presence of excessive breast tissue in males, which can appear unilateral or bilateral. Bilateral gynecomastia is frequently found in the neonatal period, early in puberty, and with increasing age. Prepubertal unilateral gynecomastia in the absence of endocrine abnormalities is extremely rare, with only a few cases in literature. METHODS: We report the cases of two otherwise healthy boys of 8 and 11 years old with unilateral breast masses. No abnormalities were found on ultrasonography and all endocrine parameters were within normal limits. Treatment consisted of peripheral liposuction followed by subcutaneous partial resection of the gland, conducted through an infra-areolar incision. Results : Microscopy of the subcutaneous mastectomy specimen revealed gynecomastia without signs of malignancy. Postoperative course of both patients was uncomplicated, with no signs of recurrence of breast tissue. CONCLUSIONS: Atypical presentations of gynecomastia are often not recognized, with little attention to breast development in prepubertal non-obese children. Since prepubertal gynecomastia could be a sign of possible underlying diseases, a thorough examination and further research is recommended. If there is no causal treatment, surgical resection is the therapy of first choice. Peripheral liposuction and surgical resection of the gland tissue are the mainstay of treatment. In summary, we describe two cases of prepubertal unilateral gynecomastia with a normal endocrine workup. Further research is needed to establish the pathophysiologic mechanisms of prepubertal gynecomastia, since underlying etiology in most cases remains unclear

Long-term safety and efficacy of eculizumab in generalized myasthenia gravis

Introduction: Eculizumab is effective and well tolerated in patients with antiacetylcholine receptor antibody-positive refractory generalized myasthenia gravis (gMG; REGAIN; NCT01997229). We report an interim analysis of an open-label extension of REGAIN, evaluating eculizumab's long-term safety and efficacy. Methods: Eculizumab (1,200 mg every 2 weeks for 22.7 months [median]) was administered to 117 patients. Results: The safety profile of eculizumab was consistent with REGAIN; no cases of meningococcal infection were reported during the interim analysis period. Myasthenia gravis exacerbation rate was reduced by 75% from the year before REGAIN (P < 0.0001). Improvements with eculizumab in activities of daily living, muscle strength, functional ability, and quality of life in REGAIN were maintained through 3 years; 56% of patients achieved minimal manifestations or pharmacological remission. Patients who had received placebo during REGAIN experienced rapid and sustained improvements during open-label eculizumab (P < 0.0001). Discussion: These findings provide evidence for the long-term safety and sustained efficacy of eculizumab for refractory gMG. Muscle Nerve 201

FAHN/SPG35 : a narrow phenotypic spectrum across disease classifications

The endoplasmic reticulum enzyme fatty acid 2-hydroxylase (FA2H) plays a major role in the formation of 2-hydroxy glycosphingolipids, main components of myelin. FA2H deficiency in mice leads to severe central demyelination and axon loss. In humans it has been associated with phenotypes from the neurodegeneration with brain iron accumulation (fatty acid hydroxylase-associated neurodegeneration, FAHN), hereditary spastic paraplegia (HSP type SPG35) and leukodystrophy (leukodystrophy with spasticity and dystonia) spectrum. We performed an in-depth clinical and retrospective neurophysiological and imaging study in a cohort of 19 cases with biallelic FA2H mutations. FAHN/SPG35 manifests with early childhood onset predominantly lower limb spastic tetraparesis and truncal instability, dysarthria, dysphagia, cerebellar ataxia, and cognitive deficits, often accompanied by exotropia and movement disorders. The disease is rapidly progressive with loss of ambulation after a median of 7 years after disease onset and demonstrates little interindividual variability. The hair of FAHN/SPG35 patients shows a bristle-like appearance; scanning electron microscopy of patient hair shafts reveals deformities (longitudinal grooves) as well as plaque-like adhesions to the hair, likely caused by an abnormal sebum composition also described in a mouse model of FA2H deficiency. Characteristic imaging features of FAHN/SPG35 can be summarized by the WHAT' acronym: white matter changes, hypointensity of the globus pallidus, ponto-cerebellar atrophy, and thin corpus callosum. At least three of four imaging features are present in 85% of FA2H mutation carriers. Here, we report the first systematic, large cohort study in FAHN/SPG35 and determine the phenotypic spectrum, define the disease course and identify clinical and imaging biomarkers

High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

Adaptor protein complex 4-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of adaptor protein complex 4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here, we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by adaptor protein complex 4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z'-factor robust >0.3, strictly standardized mean difference >3). The 'ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 ± 0.13 versus 1.21 ± 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia