Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy Family Members With a Pathogenic NOTCH3 Variant Can Have a Normal Brain Magnetic Resonance Imaging and Skin Biopsy Beyond Age 50 Years.

BACKGROUND: To determine whether extremely mild small vessel disease (SVD) phenotypes can occur in NOTCH3 variant carriers from Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) pedigrees using clinical, genetic, neuroimaging, and skin biopsy findings. METHODS: Individuals from CADASIL pedigrees fulfilling criteria for extremely mild NOTCH3-associated SVD (mSVDNOTCH3) were selected from the cross-sectional Dutch CADASIL cohort (n=200), enrolled between 2017 and 2020. Brain magnetic resonance imaging were quantitatively assessed for SVD imaging markers. Immunohistochemistry and electron microscopy was used to quantitatively assess and compare NOTCH3 ectodomain (NOTCH3ECD) aggregation and granular osmiophilic material deposits in the skin vasculature of mSVDNOTCH3 cases and symptomatic CADASIL patients. RESULTS: Seven cases were identified that fulfilled the mSVDNOTCH3 criteria, with a mean age of 56.6 years (range, 50-72). All of these individuals harbored a NOTCH3 variant located in one of EGFr domains 7-34 and had a normal brain magnetic resonance imaging, except the oldest individual, aged 72, who had beginning confluence of WMH (Fazekas score 2) and 1 cerebral microbleed. mSVDNOTCH3 cases had very low levels of NOTCH3ECD aggregation in skin vasculature, which was significantly less than in symptomatic EGFr 7-34 CADASIL patients (P=0.01). Six mSVDNOTCH3 cases had absence of granular osmiophilic material deposits. CONCLUSIONS: Our findings demonstrate that extremely mild SVD phenotypes can occur in individuals from CADASIL pedigrees harboring NOTCH3 EGFr 7-34 variants with normal brain magnetic resonance imaging up to age 58 years. Our study has important implications for CADASIL diagnosis, disease prediction, and the counseling of individuals from EGFr 7-34 CADASIL pedigrees.

NOTCH3 variant position is associated with NOTCH3 aggregation load in CADASIL vasculature.

AIMS: CADASIL, the most prevalent hereditary cerebral small vessel disease, is caused by cysteine-altering NOTCH3 variants (NOTCH3cys ) leading to vascular NOTCH3 protein aggregation. It has recently been shown that variants located in one of NOTCH3 protein epidermal growth-factor like repeat (EGFr) domains 1-6, are associated with a more severe phenotype than variants located in one of the EGFr domains 7-34. The underlying mechanism for this genotype-phenotype correlation is unknown. The aim of this study was to analyse whether NOTCH3cys variant position is associated with NOTCH3 protein aggregation load. METHODS: We quantified vascular NOTCH3 aggregation in skin biopsies (n = 25) and brain tissue (n = 7) of CADASIL patients with a NOTCH3cys EGFr 1-6 variant or a EGFr 7-34 variant, using NOTCH3 immunohistochemistry (NOTCH3 score) and ultrastructural analysis of granular osmiophilic material (GOM count). Disease severity was assessed by neuroimaging (lacune count and white matter hyperintensity volume) and disability (modified Rankin scale). RESULTS: Patients with NOTCH3cys EGFr 7-34 variants had lower NOTCH3 scores (P = 1.3·10-5 ) and lower GOM counts (P = 8.2·10-5 ) than patients with NOTCH3cys EGFr 1-6 variants in skin vessels. A similar trend was observed in brain vasculature. In the EGFr 7-34 group, NOTCH3 aggregation levels were associated with lacune count (P = 0.03) and white matter hyperintensity volume (P = 0.02), but not with disability. CONCLUSIONS: CADASIL patients with an EGFr 7-34 variant have significantly less vascular NOTCH3 aggregation than patients with an EGFr 1-6 variant. This may be one of the factors underlying the difference in disease severity between NOTCH3cys EGFr 7-34 and EGFr 1-6 variants.

The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation.

INTRODUCTION: CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model. RESULTS: We generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the 'NOTCH3 score', a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing. CONCLUSIONS: This novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker.

SOX antibodies in small-cell lung cancer and Lambert-Eaton myasthenic syndrome: frequency and relation with survival.

PURPOSE: SOX1 antibodies are common in small-cell lung carcinoma (SCLC) with and without paraneoplastic syndrome (PNS) and can serve as serological tumor marker. Addition of other antibodies might improve its diagnostic power. We validated an enzyme-linked immunosorbent assay (ELISA) to assess the diagnostic value of serum antibodies in SCLC and Lambert-Eaton myasthenic syndrome (LEMS). Clinical outcome with respect to SOX antibodies was evaluated, as the SOX-related antitumor immune response might help to control the tumor growth. PATIENTS AND METHODS: We used recombinant SOX1, SOX2, SOX3, SOX21, HuC, HuD, or HelN1 proteins in an ELISA to titrate serum samples and validated the assay by western blot. We tested 136 consecutive SCLC patients, 86 LEMS patients (43 with SCLC), 14 patients with SCLC and PNS (paraneoplastic cerebellar degeneration or Hu syndrome), 62 polyneuropathy patients, and 18 healthy controls. RESULTS: Our ELISA was equally reliable as western blot. Forty-three percent of SCLC patients and 67% of SCLC-LEMS patients had antibodies to one of the SOX or Hu proteins. SOX antibodies had a sensitivity of 67% and a specificity of 95% to discriminate between LEMS with SCLC and nontumor LEMS. No difference in survival was observed between SOX positive and SOX negative SCLC patients. CONCLUSION: SOX antibodies are specific serological markers for SCLC. Our assay is suitable for high throughput screening, detecting 43% of SCLC. SOX antibodies have diagnostic value in discriminating SCLC-LEMS from nontumor LEMS, but have no relation to survival in patients with SCLC.

Hypocretin (orexin) loss in Parkinson's disease.

The hypothalamic hypocretin (orexin) system plays a central role in the regulation of various functions, including sleep/wake regulation and metabolism. There is a growing interest in hypocretin function in Parkinson's disease (PD), given the high prevalence of non-motor symptoms such as sleep disturbances in this disorder. However, studies measuring CSF hypocretin levels have yielded contradictory results. In PD patients and matched controls, we (i) estimated the number of hypocretin neurons in post-mortem hypothalami using immunocytochemistry and an image analysis system (ii) quantified hypocretin levels in post-mortem ventricular CSF and (iii) prefrontal cortex using a radioimmunoassay. Furthermore, presence of Lewy bodies was verified in the hypothalamic hypocretin cell area. Data are presented as median (25th-75th percentile). We showed a significant decrease between PD patients and controls in (i) the number of hypocretin neurons (PD: 20 276 (13 821-31 229); controls: 36 842 (32 546-50 938); P = 0.016); (ii) the hypocretin-1 concentration in post-mortem ventricular CSF (PD: 365.5 pg/ml (328.0-448.3); controls: 483.5 (433.5-512.3); P = 0.012) and (iii) the hypocretin-1 concentrations in prefrontal cortex (PD: 389.6 pg/g (249.2-652.2); controls: 676.6 (467.5-883.9); P = 0.043). Hypocretin neurotransmission is affected in PD. The hypocretin-1 concentration in the prefrontal cortex was almost 40% lower in PD patients, while ventricular CSF levels were almost 25% reduced. The total number of hypocretin neurons was almost half compared to controls.