Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq.

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

Monogenic cerebral small-vessel diseases: diagnosis and therapy. Consensus recommendations of the European Academy of Neurology.

BACKGROUND AND PURPOSE: Guidelines on monogenic cerebral small-vessel disease (cSVD) diagnosis and management are lacking. Endorsed by the Stroke and Neurogenetics Panels of the European Academy of Neurology, a group of experts has provided recommendations on selected monogenic cSVDs, i.e. cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), autosomal dominant High Temperature Requirement A Serine Peptidase 1 (HTRA1), cathepsin-A-related arteriopathy with strokes and leukoencephalopathy (CARASAL), pontine autosomal dominant microangiopathy and leukoencephalopathy (PADMAL), Fabry disease, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and type IV collagen (COL4)A1/2. METHODS: We followed the Delphi methodology to provide recommendations on several unanswered questions related to monogenic cSVD, including genetic testing, clinical and neuroradiological diagnosis, and management. RESULTS: We have proposed 'red-flag' features suggestive of a monogenic disease. General principles applying to the management of all cSVDs and specific recommendations for the individual forms of monogenic cSVD were agreed by consensus. CONCLUSIONS: The results provide a framework for clinicians involved in the diagnosis and management of monogenic cSVD. Further multicentre observational and treatment studies are still needed to increase the level of evidence supporting our recommendations.

Use of heavy Trypan blue in macular hole surgery.

PURPOSE: Intraocular dyes facilitate the identification of the inner limiting membrane (ILM) during surgery. Appropriate dyes should be safe, provide adequate staining, and be easy to use. Heavy Trypan blue eliminates the need for an air-fluid exchange (AFX) and appears to have little retinal toxicity. This study refers to a prospective, consecutive trial with heavy Trypan blue in macular hole surgery. PATIENTS AND METHODS: A consecutive group of 20 patients with full thickness macular holes was recruited in a single institution study. Patients were operated using conventional methods. Heavy Trypan blue was prepared by mixing isovolumetrically glucose 10% with MembraneBlue (DORC, Zuidland, The Netherlands). Patients were assessed for ease of surgery and post-operatively at 3 and 6 months (vision and ocular coherence tomography) for hole closure and vision. RESULTS: Twenty eyes were included in the study. Reapplication of dye was used in 75% of the cases, leading to improved contrast further facilitating the ILM peel. In no case was an AFX necessary to obtain sufficient staining. Macular hole closure was achieved in 19 of 20 patients with one surgery. No retinal detachment or other complication was observed in the follow-up period. CONCLUSION: Heavy Trypan blue can be delivered efficiently to the retinal surface without the need for an AFX. Staining was sufficient to help visualise and peel the ILM. Repeat applications were easily performed. The macular hole closure rate was similar to that of other series with a comparable visual improvement.

Cerebrovascular reactivity is a main determinant of white matter hyperintensity progression in CADASIL.

BACKGROUND AND PURPOSE: Basal total cerebral blood flow (TCBF) and cerebrovascular reactivity (CVR) are assumed to play an important role in the pathophysiology of small-vessel disease. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a unique monogenetic model to study the pathophysiology of arterial small-vessel disease. The aim of this study was to investigate the role of TCBF and CVR in the progression of MR imaging abnormalities in CADASIL. MATERIALS AND METHODS: Basal TCBF was measured in 25 NOTCH3 mutation carriers and 13 control subjects at baseline. CVR after administration of acetazolamide was measured in 14 NOTCH3 mutation carriers and 9 control subjects. Increase in white matter hyperintensities (WMHs), lacunar infarcts, and microbleeds on MR imaging was measured 7 years later. RESULTS: Lower CVR at baseline was associated with larger increase of WMHs (P = .001) but not with a larger increase of lacunar infarcts or microbleeds. TCBF at baseline was not associated with an increase of MR imaging abnormalities. CONCLUSIONS: Decreased CVR is a potential predictor of disease progression as indicated by increasing WMHs in CADASIL.

MRI correlates of cognitive decline in CADASIL: a 7-year follow-up study.

BACKGROUND: Cognitive decline is one of the clinical hallmarks of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a cerebrovascular disease caused by NOTCH3 mutations. In this 7-year follow-up study, we aimed to determine whether there are associations between the different radiologic hallmarks in CADASIL and decline in specific cognitive domains. METHODS: Twenty-five NOTCH3 mutation carriers and 13 controls had standardized neuropsychological testing and MRI examinations at baseline and after a follow-up of 7 years. To identify longitudinal associations between MRI abnormalities and cognitive decline, correlation analysis was used. RESULTS: At follow-up, mutation carriers showed a decline in global cognitive function (CAMCOG, p < 0.01) and in the cognitive domains language, memory, and executive function, compared to controls. Cognitive decline, especially executive dysfunction, was associated with increase in lacunar infarcts, microbleeds, and ventricular volume. In contrast, WMHs and brain atrophy were not associated with cognitive decline. CONCLUSION: Increase in lacunar infarcts, microbleeds, and ventricular volume, but not white matter lesions or atrophy, are associated with cognitive decline in the process of CADASIL in younger-aged, mildly affected patients with CADASIL.

Ganglion cell neurites in human idiopathic epiretinal membranes.

AIM: To identify and confirm the presence of neural elements in idiopathic epiretinal membranes removed from patients' eyes during vitrectomy with epiretinal membrane peeling. METHODS: Human epiretinal membranes from patients with no other known eye disease and of varying durations were labelled immunohistochemically with antibodies for neurofilament protein, laminin and either vimentin or GFAP; proteins expressed in ganglion cells, the inner limiting membrane (ILM), and Muller cells, respectively. RESULTS: Anti-neurofilament labelled neurites, presumed to originate from ganglion cells, were found in all 32 idiopathic epiretinal membranes examined. The neurites were only observed in regions of anti-vimentin or -GFAP labelled glial cells, both of which were observed embedded in anti-laminin labelled material assumed to originate from the ILM. CONCLUSIONS: We show that neurofilamentous processes, presumed to originate from retinal ganglion cells, are found universally in idiopathic epiretinal membranes, suggesting that the presence of these membranes is sufficient to stimulate neurite growth in the absence of trauma or disease. In addition, since neurites were invariably found in association with glial cells, the glia may play a permissive role in neurite growth both within the retina and into extra-retinal glial membranes.

Cerebral white matter abnormalities in 6p25 deletion syndrome.

Submicroscopic deletion of the terminal part of the short arm of chromosome 6, including 6p25, leads to developmental retardation, hearing impairment, ocular dysgenesis, and dysmorphic features. We diagnosed 3 patients referred because of white matter abnormalities of unknown origin. MR imaging showed multifocal areas of abnormal signal and enlarged perivascular spaces in the cerebral white matter that were stable during follow-up. Multifocal white matter abnormalities are most commonly seen in static, nonmetabolic encephalopathies, including chromosomal abnormalities.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset hereditary syndrome characterized by recurrent TIAs and strokes, cognitive decline and dementia, migraine with aura (+/-40% of patients), and psychiatric disturbances (+/-30% of patients). Affected individuals have prominent signal abnormalities on brain MRI. Symmetrical white matter abnormalities are invariably seen and often small subcortical infarcts are also present. The extent of the MRI lesions increases with age, from subtle white matter abnormalities in the anterior temporal poles in the early 20 years to confluent white matter lesions with subcortical infarcts and microbleeds in the 6(th) decade. A typical arteriopathy with electron dense granular depositions in the media of small cerebral arteries underlies this disorder. These arterial lesions can be found, to a lesser extent, in extra-cerebral arteries such as skin arterioles. In 1996, the defective gene in CADASIL was discovered to be NOTCH3. NOTCH3 encodes a 300-kd transmembrane protein with a receptor and cell signal transduction function. Mutations are almost always missense mutations causing the loss or gain of a cysteine residue and are detected in over 90% of patients. How alterations in NOTCH3 lead to the CADASIL phenotype has yet to be elucidated.

Corticosteroid-induced osteoporosis in patients with uveitis.

AIMS: To estimate the prevalence of low bone density and osteoporosis in a population of patients with uveitis taking systemic steroid treatment; to clarify the risks of steroid-induced fracture and to suggest a protocol for the prevention and management of bone loss in patients with ophthalmic inflammatory disease. METHODS: Bone densitometry was performed on 129 adult patients with prednisolone-treated uveitis from four centres. Information on uveitis diagnosis, associated risk factors, steroid dosage and treatment duration, prophylaxis and management, was collected. Juveniles, patients with scleritis and those who had used deflazacort, were excluded. RESULTS: Steroid treatment time varied from 13 weeks to 31 years, and the total dosage from 1.29 g to 166.5 g. Twenty-six percent of patients also used one or more immunosuppressives. Forty-eight percent had additional risk factors for bone loss. Bone density was abnormally low in 44.2%, and 15.5% had osteoporosis. Osteoporosis was substantially more common in males (20.6%, all under 60 yrs) than in females (9.8%). Seven symptomatic fractures occurred in patients on treatment. Bone loss correlated with total steroid dose, mean dose, duration of treatment and the presence of pre-existing risk factors. CONCLUSIONS: The prevalence of steroid-induced osteoporosis and fracture is low for patients with uveitis but young males are at risk. Patients at high risk should be identified, and prophylaxis and treatment should be used as required. The guideline of the National Osteoporosis Society is recommended as a management protocol.

Cerebral microbleeds in CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary arteriopathy leading to recurrent cerebral infarcts and dementia. Intracerebral hemorrhage (ICH) has been described sporadically in patients with CADASIL, suggesting that the affected arteries in CADASIL are not bleed-prone. However, the presence of cerebral microbleeds, which often remain undetected on conventional MRI, has not been determined in CADASIL. OBJECTIVE: To determine whether cerebral vessels in patients with CADASIL are prone to microbleeding. METHODS: T2*-weighted gradient echo MRI, which is highly sensitive for visualizing microbleeds, was performed in patients with CADASIL and their family members (n = 63). Known risk factors for ICH were determined for all individuals. On an exploratory basis, the presence of cerebral microbleeds was correlated with demographic variables, vascular risk factors, disease progression, ischemic MR lesions, and genotype. RESULTS: Cerebral microbleeds were present in 31% of symptomatic CADASIL mutation carriers, predominantly in the thalamus. Vascular risk factors such as hypertension did not account for the microbleeds in these patients. Factors associated with microbleeds were age (p = 0.008), Rankin disability score (p = 0.017), antiplatelet use (p = 0.025), number of lacunae on MRI (p = 0.009), and the Arg153Cys Notch3 mutation (p = 0.017). After correction for age, only the Arg153Cys mutation remained significantly associated with the presence of microbleeds. CONCLUSION: Patients with CADASIL have an age-related increased risk of intracerebral microbleeds. This implies that they may have an increased risk for ICH, which should be taken into account in CADASIL diagnosis and patient management.

[Congenital ptosis of the upper eyelid: indication for early ophthalmological examination]. / Congenitale ptosis van het bovenooglid: reden voor vroegtijdig oogheelkundig onderzoek.

OBJECTIVE: Description of the results of clinical investigation and surgical correction in patients with congenital upper eyelid ptosis. DESIGN: Descriptive. SETTING: The Eye Hospital, Rotterdam. METHOD: Evaluation of clinical findings and results of operative treatment in 114 consecutive patients with congenital ptosis. RESULTS: Amblyopia was present in 20% of the 114 patients, and was caused by stimulus deprivation due to the ptotic eyelid (22%), strabismus (48%), and refraction disorder (30%). Treatment of amblyopia did not induce visual improvement in 7% of the patients (8/114). These patients were all older than 7 years, meaning they were probably too old for treatment. Of the 114 patients, 14% showed strabismus without amblyopia, 8% showed abnormalities of eye movement without squint, and 6% showed torticollis due to the ptosis. At time of referral 28% were younger than 6 years and 35% had already been treated surgically for the ptosis elsewhere. Operative correction of ptosis induced symmetry within 1 mm in 62%, and symmetry within 2 mm in 22%. Reoperation was performed in 16%. Previous operation elsewhere had no influence on the results. CONCLUSIONS: Patients with congenital ptosis frequently show amblyopia, strabismus, and abnormalities of eye movement. In the patients referred to us, amblyopia had not always been diagnosed and treated in time. Ophthalmological examination in the first year of life is indicated in these patients. If the ptotic eyelid induces deprivation amblyopia or torticollis, direct ptosis correction is indicated. It is advised for psychological reasons to correct elective cases at the age of 4 or 5 years. For this reason, also, patients should be referred sooner.