Novel partial deletions, frameshift and missense mutations of CSF1R in patents with CSF1R-related leukoencephalopathy.

BACKGROUND AND PURPOSE: Colony-stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is an adult-onset leukoencephalopathy caused by mutations in CSF1R. The present study aimed to explore the broader genetic spectrum of CSF1R-related leukoencephalopathy in association with clinical and imaging features. METHODS: Mutational analysis of CSF1R was performed for 100 consecutive patients with adult-onset leukoencephalopathy. Sequence and copy number variation (CNV) analyses of CSF1R were performed. The genomic ranges of the deletions were determined by long-read sequencing. Ligand-dependent autophosphorylation of CSF1R was examined in cells expressing the CSF1R mutants identified in this study. RESULTS: CSF1R mutations were identified in 15 patients, accounting for 15% of the adult-onset leukoencephalopathy cases. Seven novel and five previously reported CSF1R mutations were identified. The novel mutations, including three missense and one in-frame 3 bp deletion, were located in the tyrosine kinase domain (TKD) of CSF1R. Functional assays revealed that none of the novel mutations in the TKD showed autophosphorylation of CSF1R. Two partial deletions of CSF1R were identified that resulted in lack of the C-terminal region, including the distal TKD, in two patients. Various clinical features including cognitive impairment, psychiatric symptoms and gait disturbance were observed. Various degrees of the white matter lesions and corpus callosum abnormalities on magnetic resonance imaging and characteristic calcifications on computed tomography were observed as imaging features. CONCLUSIONS: Our results highlight the importance of examining the CNV of CSF1R even when Sanger or exome sequencing reveals no CSF1R mutations. Genetic examination of sequences and CNV analyses of CSF1R are recommended for an accurate diagnosis of CSF1R-related leukoencephalopathy.

High frequency of HTRA1 AND ABCC6 mutations in Japanese patients with adult-onset cerebral small vessel disease.

BACKGROUND: This study aimed to clarify the frequency and clinical features of monogenic cerebral small vessel disease (mgCSVD) among patients with adult-onset severe CSVD in Japan. METHODS: This study included patients with adult-onset severe CSVD with an age of onset ≤55 years (group 1) or >55 years and with a positive family history (group 2). After conducting conventional genetic tests for NOTCH3 and HTRA1, whole-exome sequencing was performed on undiagnosed patients. Patients were divided into two groups according to the results of the genetic tests: monogenic and undetermined. The clinical and imaging features were compared between the two groups. RESULTS: Group 1 and group 2 included 75 and 31 patients, respectively. In total, 30 patients had NOTCH3 mutations, 11 patients had HTRA1 mutations, 6 patients had ABCC6 mutations, 1 patient had a TREX1 mutation, 1 patient had a COL4A1 mutation and 1 patient had a COL4A2 mutation. The total frequency of mutations in NOTCH3, HTRA1 and ABCC6 was 94.0% in patients with mgCSVD. In group 1, the frequency of a family history of first relatives, hypertension and multiple lacunar infarctions (LIs) differed significantly between the two groups (monogenic vs undetermined; family history of first relatives, 61.0% vs 25.0%, p=0.0015; hypertension, 34.1% vs 63.9%, p=0.0092; multiple LIs, 87.8% vs 63.9%, p=0.0134). CONCLUSIONS: More than 90% of mgCSVDs were diagnosed by screening for NOTCH3, HTRA1 and ABCC6. The target sequences for these three genes may efficiently diagnose mgCSVD in Japanese patients.

Patients with heterozygous HTRA1-related cerebral small vessel disease misdiagnosed with other diseases: Two case reports.

White matter hyperintensities (WMHs) on brain magnetic resonance (MR) images are characteristic of hereditary cerebral small vessel disease (CSVD), including high-temperature requirement serine peptidase A1 (HTRA1)-related CSVD. Although HTRA1-related CSVD is increasingly recognized, the diagnosis is still challenging. We encountered two patients with HTRA1-related CSVD who were misdiagnosed with other diseases, including multiple sclerosis and idiopathic normal-pressure hydrocephalus. Both patients had extended WMHs in addition to multiple lacunes and microbleeds on brain MR images, which are characteristic of CSVD. If lacunes or microbleeds are found in patients with severe WMHs, genetic tests for hereditary CSVD should be considered.

[A case of neuralgic amyotrophy with extension disturbance of fingers after Cushing's syndrome remission].

We describe a 57-year-old female patient who experienced hypercortisolemia caused by adrenal Cushing's syndrome. Two months post-adrenalectomy, she developed acute severe bilateral pain starting in her fingers and spreading up her arms. In the subsequent two weeks, the patient presented upper extremity patchy paralysis with extension disturbance of fingers. In the following two months, she experienced atrophy of the muscles in the hands and joint contracture. Consequently, we diagnosed her with neuralgic amyotrophy. Nerve conduction studies showed low compound muscle action potential of all the peripheral nerves in the forearms, suggesting motor neuron axonopathy. Gadolinium-enhanced MRI and ultrasound studies did not reveal any abnormalities in the brachial plexus and peripheral nerves of the forearms. The patient tested positive for anti-GalNAc-GD1a-IgM antibodies and received intravenous immunoglobulin 6 months after the onset of symptoms, which resulted in reduction of pain, muscle weakness, and contractures. This rare case of potentially immune-mediated bilateral patchy paralysis may have important implications in the understanding of clinical and pathological heterogenicity of neuralgic amyotrophy.

Candesartan prevents arteriopathy progression in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy model.

Cerebral small vessel disease (CSVD) causes dementia and gait disturbance due to arteriopathy. Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary form of CSVD caused by loss of high-temperature requirement A1 (HTRA1) serine protease activity. In CARASIL, arteriopathy causes intimal thickening, smooth muscle cell (SMC) degeneration, elastic lamina splitting, and vasodilation. The molecular mechanisms were proposed to involve the accumulation of matrisome proteins as substrates or abnormalities in transforming growth factor ß (TGF-ß) signaling. Here, we show that HTRA1-/- mice exhibited features of CARASIL-associated arteriopathy: intimal thickening, abnormal elastic lamina, and vasodilation. In addition, the mice exhibited reduced distensibility of the cerebral arteries and blood flow in the cerebral cortex. In the thickened intima, matrisome proteins, including the hub protein fibronectin (FN) and latent TGF-ß binding protein 4 (LTBP-4), which are substrates of HTRA1, accumulated. Candesartan treatment alleviated matrisome protein accumulation and normalized the vascular distensibility and cerebral blood flow. Furthermore, candesartan reduced the mRNA expression of Fn1, Ltbp-4, and Adamtsl2, which are involved in forming the extracellular matrix network. Our results indicate that these accumulated matrisome proteins may be potential therapeutic targets for arteriopathy in CARASIL.

Corrigendum: HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade.

[This corrects the article DOI: 10.3389/fneur.2019.00693.].

Heterozygous Cysteine-sparing NOTCH3 Variant p.Val237Met in a Japanese Patient with Suspected Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy.

A 64-year-old Japanese man with recurrent cerebral ischemic events and cognitive impairment was suspected of having cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) because of a family history and brain magnetic resonance imaging findings of cerebral white matter hyperintensities. The cysteine-sparing variation p.Val237Met was identified in NOTCH3. An intensive skin biopsy showed negative results (no granular osmiophilic material or positive NOTCH3 immunostaining), suggesting that the patient's definite diagnosis and pathogenicity of p.Val237Met were uncertain. We additionally reviewed previous reports of two Japanese families with p.Val237Met.

A Nationwide Survey and Multicenter Registry-Based Database of Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy in Japan.

OBJECTIVES: Clinical characteristics of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) include migraine, recurrent stroke, white matter lesions, and vascular dementia. CADASIL is one of the most common hereditary cerebral small vessel diseases. Clinical presentation of CADASIL varies and a racial gap may exist between the Asian and Caucasian populations. This is the first nationwide epidemiological survey which aimed to elucidate the clinical features of CADASIL in Japan. Moreover, the registration database of CADASIL was constructed. METHODS: Subjects included CADASIL patients who visited the hospitals (totally 1,448 hospitals) certified by the Japanese Society of Neurology and/or Japan Stroke Society in 2016. This study consisted of a two-step survey; patients with CADASIL were identified genetically by the first questionnaire, and their clinical features were assessed by the second questionnaire. Selected 6 hospitals registered the data of all CADASIL patients using a Research Electronic Data Capture (REDCap) system for the second questionnaire. RESULTS: Based on the criteria, 88 patients (50 male and 38 female) with CADASIL were enrolled. The mean age of symptom onset was 49.5 years. Sixteen (18.2%) patients had an elderly onset (>60 years). Thirteen patients (13.6%) had history of migraine with aura and 33 patients (37.5%) had vascular risk factor(s). From among the 86 patients who were examined using magnetic resonance imaging, abnormal deep white matter lesions were detected in 85 patients (98.8%), WMLs extending to anterior temporal pole in 73 patients (84.9%), and cerebral microbleeds in 41 patients (47.7%). Anti-platelet therapy was received by 65 patients (73.9%). Thirty-eight patients (43.2%) underwent treatment with lomerizine hydrochloride. Thirty-four different mutations of NOTCH3 were found in exons 2, 3, 4, 5, 6, 8, 11, 14, and 19. Most of the mutations existed in exon 4 (n = 44, 60.3%). The prevalence rate of CADASIL was 1.20 to 3.58 per 100,000 adults in Japan. CONCLUSION: This questionnaire-based study revealed clinical features and treatment status in Japanese CADASIL patient, although it may not be an exhaustive search. We have constructed the REDCap database for these CADASIL patients.

HTRA1-Related Cerebral Small Vessel Disease: A Review of the Literature.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is clinically characterized by early-onset dementia, stroke, spondylosis deformans, and alopecia. In CARASIL cases, brain magnetic resonance imaging reveals severe white matter hyperintensities (WMHs), lacunar infarctions, and microbleeds. CARASIL is caused by a homozygous mutation in high-temperature requirement A serine peptidase 1 (HTRA1). Recently, it was reported that several heterozygous mutations in HTRA1 also cause cerebral small vessel disease (CSVD). Although patients with heterozygous HTRA1-related CSVD (symptomatic carriers) are reported to have a milder form of CARASIL, little is known about the clinical and genetic differences between the two diseases. Given this gap in the literature, we collected clinical information on HTRA1-related CSVD from a review of the literature to help clarify the differences between symptomatic carriers and CARASIL and the features of both diseases. Forty-six symptomatic carriers and 28 patients with CARASIL were investigated. Twenty-eight mutations in symptomatic carriers and 22 mutations in CARASIL were identified. Missense mutations in symptomatic carriers are more frequently identified in the linker or loop 3 (L3)/loop D (LD) domains, which are critical sites in activating protease activity. The ages at onset of neurological symptoms/signs were significantly higher in symptomatic carriers than in CARASIL, and the frequency of characteristic extraneurological findings and confluent WMHs were significantly higher in CARASIL than in symptomatic carriers. As previously reported, heterozygous HTRA1-related CSVD has a milder clinical presentation of CARASIL. It seems that haploinsufficiency can cause CSVD among symptomatic carriers according to the several patients with heterozygous nonsense/frameshift mutations. However, the differing locations of mutations found in the two diseases indicate that distinct molecular mechanisms influence the development of CSVD in patients with HTRA1-related CSVD. These findings further support continued careful examination of the pathogenicity of mutations located outside the linker or LD/L3 domain in symptomatic carriers.

Excessive Production of Transforming Growth Factor ß1 Causes Mural Cell Depletion From Cerebral Small Vessels.

It is increasingly becoming apparent that cerebrovascular dysfunction contributes to the pathogenic processes involved in vascular dementia, Alzheimer's disease, and other neurodegenerative disorders. Under these pathologic conditions, the degeneration of cerebral blood vessels is frequently accompanied by a loss of mural cells from the vascular walls. Vascular mural cells play pivotal roles in cerebrovascular functions, such as regulation of cerebral blood flow and maintenance of the blood-brain barrier (BBB). Therefore, cerebrovascular mural cell impairment is involved in the pathophysiology of vascular-related encephalopathies, and protecting these cells is essential for maintaining brain health. However, our understanding of the molecular mechanism underlying mural cell abnormalities is incomplete. Several reports have indicated that dysregulated transforming growth factor ß (TGFß) signaling is involved in the development of cerebral arteriopathies. These studies have specifically suggested the involvement of TGFß overproduction. Although cerebrovascular toxicity via vascular fibrosis by extracellular matrix accumulation or amyloid deposition is known to occur with enhanced TGFß production, whether increased TGFß results in the degeneration of vascular mural cells in vivo remains unknown. Here, we demonstrated that chronic TGFß1 overproduction causes a dropout of mural cells and reduces their coverage on cerebral vessels in both smooth muscle cells and pericytes. Mural cell degeneration was also accompanied by vascular luminal dilation. TGFß1 overproduction in astrocytes significantly increased TGFß1 content in the cerebrospinal fluid (CSF) and increased TGFß signaling-regulated gene expression in both pial arteries and brain capillaries. These results indicate that TGFß is an important effector that mediates mural cell abnormalities under pathological conditions related to cerebral arteriopathies.

HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade.

Background: Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Most carriers for HTRA1 mutations are asymptomatic, but more than 10 mutations have been reported in symptomatic carriers. The molecular differences between the mutations identified in symptomatic carriers and mutations identified only in CARASIL patients are unclear. HTRA1 is a serine protease that forms homotrimers, with each HTRA1 subunit activating the adjacent HTRA1 via the sensor domain of loop 3 (L3) and the activation domain of loop D (LD). Previously, we analyzed four HTRA1 mutant proteins identified in symptomatic carriers and found that they were unable to form trimers or had mutations in the LD or L3 domain. The mutant HTRA1s with these properties are presumed to inhibit trimer-dependent activation cascade. Indeed, these mutant HTRA1s inhibited wild-type (WT) protease activity. In this study, we further analyzed 15 missense HTRA1s to clarify the molecular character of mutant HTRA1s identified in symptomatic carriers. Methods: We analyzed 12 missense HTRA1s identified in symptomatic carriers (hetero-HTRA1) and three missense HTRA1s found only in CARASIL (CARASIL-HTRA1). The protease activity of the purified recombinant mutant HTRA1s was measured using fluorescein isothiocyanate-labeled casein as substrate. Oligomeric structure was evaluated by size-exclusion chromatography. The protease activities of mixtures of WT with each mutant HTRA1 were also measured. Results: Five hetero-HTRA1s had normal protease activity and were excluded from further analysis. Four of the seven hetero-HTRA1s and one of the three CARASIL-HTRA1s were unable to form trimers. The other three hetero-HTRA1s had mutations in the LD domain. Together with our previous work, 10 of 11 hetero-HTRA1s and two of six CARASIL-HTRA1s were either defective in trimerization or had mutations in the LD or L3 domain (P = 0.006). By contrast, eight of 11 hetero-HTRA1s and two of six CARASIL-HTRA1 inhibited WT protease activity (P = 0.162). Conclusions: HTRA1 mutations identified in symptomatic carriers have the property of interfering the trimer-dependent activation cascade of HTRA1.

Retinal Vasculopathy With Cerebral Leukodystrophy: Clinicopathologic Features of an Autopsied Patient With a Heterozygous TREX 1 Mutation.

Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an autosomal-dominant disorder involving the cerebral, retinal, renal, and other systemic microvessels due to frameshift mutations in the TREX1 gene. Under physiological conditions, the TREX1 protein is localized in the cellular cytoplasm and perinuclear area, but translocates into the nucleus in response to oxidative DNA damage. It has been speculated that aberrant localization of the protein may be associated with systemic microangiopathy in patients with RVCL. However, cellular expression of TREX1 in the brain and visceral organs of patients with RVCL has been unclear. Here, we report the clinicopathologic features of an autopsied patient with a heterozygous T249fs mutation in TREX1. The patient showed the clinical phenotype of vasculopathy with retinopathy, nephropathy, and stroke. CT with contrast enhancement demonstrated a tumorous lesion in the subcortical white matter. Histologically, the lesion consisted of confluent foci of necrosis with calcification and fibrous thickening of small vessel walls. TREX1 immunohistochemistry demonstrated positivity in the nuclei of cells in the CNS and visceral organs, indicating aberrant localization of the truncated protein, and the expression was remarkable in oligodendrocytes within the lesion, suggesting possible involvement of the protein in the pathomechanism of vasculopathy leading to white matter degeneration.

Identification and functional characterization of novel mutations including frameshift mutation in exon 4 of CSF1R in patients with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia.

OBJECTIVE: Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is caused by mutations in CSF1R. Pathogenic mutations in exons 12-22 including coding sequence of the tyrosine kinase domain (TKD) of CSF1R were previously identified. We aimed to identify CSF1R mutations in patients who were clinically suspected of having ALSP and to determine the pathogenicity of novel CSF1R variants. METHODS: Sixty-one patients who fulfilled the diagnostic criteria of ALSP were included in this study. Genetic analysis of CSF1R was performed for all the coding exons. The haploinsufficiency of CSF1R was examined for frameshift mutations by RT-PCR. Ligand-dependent autophosphorylation of CSF1R was examined in cells expressing CSF1R mutants. RESULTS: We identified ten variants in CSF1R including two novel frameshift, five novel missense, and two known missense mutations as well as one known missense variant. Eight mutations were located in TKD. One frameshift mutation (p.Pro104LeufsTer8) and one missense variant (p.His362Arg) were located in the extracellular domain. RT-PCR analysis revealed that the frameshift mutation of p.Pro104LeufsTer8 caused nonsense-mediated mRNA decay. Functional assay revealed that none of the mutations within TKD showed autophosphorylation of CSF1R. The p.His362Arg variant located in the extracellular domain showed comparable autophosphorylation of CSF1R to the wild type, suggesting that this variant is not likely pathogenic. CONCLUSIONS: The detection of the CSF1R mutation outside of the region-encoding TKD may extend the genetic spectrum of ALSP with CSF1R mutations. Mutational analysis of all the coding exons of CSF1R should be considered for patients clinically suspected of having ALSP.

Histopathologic features of an autopsied patient with cerebral small vessel disease and a heterozygous HTRA1 mutation.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary cerebral small vessel disease (CSVD) caused by homozygous or compound heterozygous mutations of the high temperature requirement A serine peptidase 1 gene (HTRA1). Affected patients suffer from cognitive impairment, recurrent strokes, lumbago and alopecia. Recently, clinical studies have indicated that some patients with heterozygous mutations in HTRA1 may also suffer CSVD. Here, we report the histopathologic features of an autopsied 55-year-old male patient who had shown cognitive impairment and multiple cerebral infarcts, and was found to have a heterozygous missense mutation (p.R302Q) in the HTRA1 gene. Histologically, small vessels in the brain and spinal cord showed intimal proliferation, splitting of the internal elastic lamina, and degeneration of smooth muscle cells in the tunica media. Thus, although less severe, the features were quite similar to those of patients with CARASIL, indicating that patients with heterozygous mutations develop CSVD through underlying pathomechanisms similar to those of CARASIL.

Case Report: A patient with spinocerebellar ataxia type 31 and sporadic Creutzfeldt-Jakob disease.

We report a Japanese patient with spinocerebellar ataxia type 31 (SCA31) and sporadic Creutzfeldt-Jakob disease (sCJD). A 52-year-old man developed progressive cognitive impairment after the appearance of cerebellar symptoms. Brain MR diffusion-weighted imaging (DWI) demonstrated a slowly expanding hyperintense lesion in the cerebral cortex. The patient was finally diagnosed as having both SCA31 and sCJD by identification of genetic mutations and by real-time quaking-induced conversion (RT-QUIC) analysis of the cerebrospinal fluid (CSF), respectively. Here, we report the clinical details of this rare combined case, with particular reference to the association between prion protein and the early onset of SCA31.

Renal histopathological findings of retinal vasculopathy with cerebral leukodystrophy.

Retinal vasculopathy with cerebral leukodystrophy (RVCL) is a rare autosomal dominant systemic microvascular disease. Neurological disorders and visual disturbance are highlighted as manifestations of RVCL; however, there are few reports focused on nephropathy. Herein, we describe detailed renal histopathological findings in a daughter and father with RVCL, proven by TREX1 genetic analysis. A kidney biopsy of the daughter, 35-year-old with asymptomatic proteinuria, revealed unique and various glomerular changes. Atypical double contour (not tram track-like) of the capillary wall was widely found, an apparent characteristic finding. Glomerular findings were varied due to a combination of new and old segmental mesangial proliferative changes, mesangiolysis, and segmental glomerulosclerosis-like lesions; these changes may be related to endothelial cell damage. Collapsed tufts were also found and thought to be the result of ischemia due to arterial changes. Glomerular findings in a kidney biopsy of the father revealed similarity to the daughter's glomerulus at a relatively advanced stage, but the degree of variety in the glomerular findings was much less. Kidney biopsy findings suggesting endothelial cell damage of unknown etiology need to be considered for possible RVCL.

Duplication and deletion upstream of LMNB1 in autosomal dominant adult-onset leukodystrophy.

OBJECTIVE: To characterize the genetic and clinical features of patients with autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) carrying duplication and deletion upstream of lamin B1 (LMNB1). METHODS: Ninety-three patients with adult-onset leukoencephalopathy of unknown etiology were genetically analyzed for copy numbers of LMNB1 and its upstream genes. We examined LMNB1 expression by reverse transcription-qPCR using total RNA extracted from peripheral leukocytes. Clinical and MRI features of the patients with ADLD were retrospectively analyzed. RESULTS: We identified 4 patients from 3 families with LMNB1 duplication. The duplicated genomic regions were different from those previously reported. The mRNA expression level of LMNB1 in patients with duplication was significantly increased. The clinical features of our patients with LMNB1 duplication were similar to those reported previously, except for the high frequency of cognitive impairment in our patients. We found 2 patients from 1 family carrying a 249-kb genomic deletion upstream of LMNB1. Patients with the deletion exhibited relatively earlier onset, more prominent cognitive impairment, and fewer autonomic symptoms than patients with duplication. The presence of cerebellar symptoms and lesions may be characteristic in our patients with the deletion compared with the previously reported family with the deletion. Magnetic resonance images of patients with the deletion exhibited a widespread distribution of white matter lesions including the anterior temporal region. CONCLUSIONS: We identified 4 Japanese families with ADLD carrying duplication or deletion upstream of LMNB1. There are differences in clinical and MRI features between the patients with the duplication and those with the deletion upstream of LMNB1.