J Neurol (2015) 262:1369–1372 DOI 10.1007/s00415-015-7769-5
LETTER TO THE EDITORS
A novel HTRA1 exon 2 mutation causes loss of protease activity in a Pakistani CARASIL patient Zhaleh Khaleeli1 • Zane Jaunmuktane2 • Nathalie Beaufort3 • Henry Houlden4 • Christof Haffner3 • Sebastian Brandner2 • Martin Dichgans3,5 • David Werring6
Received: 26 March 2015 / Revised: 24 April 2015 / Accepted: 26 April 2015 / Published online: 10 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Dear Sirs, Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an inherited small vessel disease, reported mainly in Japan and China. We describe the clinical, radiological and pathological findings in a Pakistani patient with a novel mutation (c.517G[A P.A173T), and the impact of the mutation on protease activity. A 35-year-old Pakistani woman developed acute left leg weakness. She partially improved, but after 2 years her mobility deteriorated. She developed bladder urge incontinence, headaches, cognitive slowing and low mood. Her parents were first cousins, and her maternal grandfather had a stroke at a young age (Fig. 1). Blood pressure was Z. Khaleeli, Z. Jaunmuktane, N. Beaufort, M. Dichgans and D. Werring have contributed equally to the manuscript. & David Werring [email protected] 1
National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, England, UK
2
Division of Neuropathology, UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, England, UK
3
Institute for Stroke and Dementia Research (ISD), Klinikum der Universita¨t Mu¨nchen, Heiglhofstr. 55, 81377 Munich, Germany
4
UCL Institute of Neurology, Queen Square, London WC1N 3BG, England, UK
5
Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
6
Stroke Research Group, Department of Brain Repair and Rehabilitation, Institute of Neurology, UCL, Queen Square, London WC1N 3BG, England, UK
normal. She had alopecia (Fig. 2). Neurological examination showed moderate executive dysfunction, reduced cognitive speed and attention, saccadic intrusions, slow tongue movements, brisk jaw jerk, mild spastic paraparesis, and extensor plantars. Full blood count, urea and electrolytes, liver function, clotting, antineuronal antibodies, ANCA, ENA, HIV, treponemal and anti-cardiolipin antibodies were normal or negative. CSF was unremarkable (negative for oligoclonal bands). MRI brain showed confluent T2 white matter hyperintensities, multiple T1 hypointensities and multiple cerebral microbleeds; MRI spine showed non-compressive spondylosis (Fig. 2a–c). Due to a further acute deterioration in her cognitive function and mobility, we performed non-dominant frontal brain biopsy to rapidly exclude potentially reversible causes, including cerebral vasculitis. The biopsy demonstrated widespread concentric hyaline arteriolosclerosis in the leptomeninges, neocortex and white matter (Fig. 2e–h). There was focal perivascular lymphocytic inflammation in the white matter, but no features of vasculitis. Notch-3 gene mutations in exons 2, 3, 4, 5, 6, 8, 11, 18 and 19 were absent. Sequencing of the High Temperature Requirement protease A1 (HTRA1) gene revealed a novel homozygous mutation in Exon 2 at (c.517G[A P.A173T); her parents were heterozygous carriers of the same mutation. We transfected human embryonic kidney cells with plasmids encoding either wild-type (WT) HtrA1, an inactive variant obtained by replacing catalytic serine with alanine (S328A); the A173T mutant. Protein expression and secretion of all HtrA1 forms was confirmed by Western blot of cell culture medium (Fig. 2i). Protease activity assays were performed against bovine serum albumin (BSA) and casein (Fig 2j, k). HtrA1-WT was active in both assays, but neither HtrA1-S328A nor A173T mutants
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J Neurol (2015) 262:1369–1372
Fig. 1 Family tree
showed protease activity, establishing the A173T mutation’s pathogenicity. About 50 CARASIL families have been reported, mostly from Japan [1], but also from China [2, 3], Spain [4], Italy [5] and Turkey [6]. Although we are not aware of reports from the Indian subcontinent, the clinico-radiological phenotype of our patient is typical [1]. Unlike CADASIL [7] and amyloid angiopathies, brain tissue reveals no pathognomonic features. CARASIL is caused by HTRA1 gene mutations in the 10q26 region, coding for HtrA1, a primarily secreted serine protease. HtrA1 mediates protein degradation, cell signalling, skeletal development and osteogenesis. Furthermore, HtrA1 modulates transforming growth factor-b (TGF-b) signalling [8, 9], a pathway associated with vascular angiogenesis and remodelling [7], perhaps explaining the alopecia and lumbar degenerative disease in CARASIL [1, 8].
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The HTRA1 gene consists of nine exons, which encode an insulin-like growth factor binding protein domain (ILGFBPD), a Kazal-type serine protease inhibitor domain, a trypsin-like serine protease domain and a PDZ domain. CARASIL-associated mutations described previously mostly target exons 3, 4, and 6, and affect the protease domain (i.e., A252T, R274Q, P285L, G295R, V297M, A321T, P364L) [2, 4, 5]. Other allelic variants include c.126delG (exon 1; p.G42fs; ILGFBPD), resulting in a premature stop codon and the expression of a truncated form lacking the entire protease domain [5], and c.1108C[T (exon 6; p.R370X; PDZ domain), which impairs mRNA stability and thus protein expression [8]. We provide the first description of a pathogenic HTRA1 mutation in exon 2. Although this affects an amino acid residue outside the protease domain (i.e., in a short interdomain region connecting the Kazal-type and protease
J Neurol (2015) 262:1369–1372
1371
Fig. 2 Clinical, radiological, pathological and biochemical features: a T2-weighted MRI image of the brain showing extensive areas of high signal in the cerebral white matter and thalamus (arrowed), b susceptibility-weighted MRI of the brain showing multiple cerebral microbleeds (arrowed), c T2-weighted MRI of the lumbar spinal cord showing spondylotic changes with loss of lordosis, d alopecia of the scalp, e haematoxylin and eosin (H&E) stained section of white matter reveals concentric hyaline thickening of the walls of small calibre vessels, f periodic acid Schiff preparation (PAS) accentuates the smooth thickening of the walls with no evidence of granular deposits, g Van Gieson’s stain showed increased connective tissue, reduplication and fragmentation of the elastic lamina within the vessel walls. h Immunostaining for smooth muscle actin confirms the loss of this specific smooth muscle cell marker in affected vessel walls. A normal age-matched arteriole is shown for comparison. i Proteolytic
activity of the A173T HtrA1 mutant: cells were transfected with a control vector (Vec) or with plasmids encoding Myc-tagged wild-type (WT), active site mutant (S328A), or CARASIL mutant (A173T) HtrA1. After 48 h culturing, serum-free culture media were collected and analysed by Western blot using an anti-Myc Ab. j BSA (10 lg) was slightly denatured by addition of 1.5 mM dithiothreitol, then exposed to transfected cell culture medium for 24 h at 37 °C, before its degradation was evaluated by SDS-PAGE followed by Coomassie staining. k Fluorescein-labelled (FTC)-casein (5 lg) was exposed to transfected cell culture medium and released fluorescence was monitored over 10 h at room temperature. Histograms represent the initial rate of casein hydrolysis with that produced by HtrA1-WT being set to 1. Results are expressed as the mean ?SEM of 4 independent assays
domains), we demonstrate that it prevents protease activity, indicating a loss-of-function mechanism [10] in CARASIL. Further work will elucidate the contribution of the targeted residue, and this domain, to HtrA1 activity.
Excellence on the Pathogenesis of Small Vessel Disease of the Brain), the Vascular Dementia Research Foundation (to M.D.), and the Jackstaedt Foundation. Dr. Werring receives research support from the Department of Health/Higher Education Funding Council for England (Clinical Senior Lectureship Award), the Stroke Association and the British Heart Foundation. Part of this work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme.
Conflicts of interest Dr. Khaleeli reports no disclosures. Dr. Jaunmuktane reports no disclosures. Dr. Beaufort reports no disclosures. Prof Houlden reports no disclosures. Dr. Haffner reports no disclosures. Prof Brandner reports no disclosures. Prof Dichgans is funded by the Fondation Leducq (Transatlantic Network of
Informed consent Informed consent was obtained from the individual included in the study.
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References 1. Fukutake T (2011) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL): from discovery to gene identification. J Stroke Cerebrovasc Dis 20:85–93 2. Wang XL, Li CF, Guo HW, Cao BZ (2012) A novel mutation in the HTRA1 gene identified in Chinese CARASIL pedigree. CNS Neurosci Ther 18:867–869 3. Chen Y, He Z, Meng S, Li L, Yang H, Zhang X (2013) A novel mutation of the high-temperature requirement A serine peptidase 1 (HTRA1) gene in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). J Int Med Res 41:1445–1455 4. Mendioroz M, Fernandez-Cadenas I, Del Rio-Espinola A et al (2010) A missense HTRA1 mutation expands CARASIL syndrome to the Caucasian population. Neurology 75:2033–2035 5. Bianchi S, Di Palma C, Gallus GN et al (2014) Two novel HTRA1 mutations in a European CARASIL patient. Neurology 82:898–900
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J Neurol (2015) 262:1369–1372 6. Bayrakli F, Balaban H, Gurelik M, Hizmetli S, Topaktas S (2014) Mutation in the HTRA1 gene in a patient with degenerated spine as a component of CARASIL syndrome. Turk Neurosurg 24:67–69 7. Yamamoto Y, Craggs L, Baumann M, Kalimo H, Kalaria RN (2011) Review: molecular genetics and pathology of hereditary small vessel diseases of the brain. Neuropathol Appl Neurobiol 37:94–113 8. Hara K, Shiga A, Fukutake T et al (2009) Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N Engl J Med 360:1729–1739 9. Beaufort N, Scharrer E, Kremmer E, Lux V, Ehrmann M, Huber R, Houlden H, Werring D, Haffner C, Dichgans M (2014) The cerebral small vessel disease-related protease HtrA1 processes latent TGF-ß binding protein 1 and facilitates TGF-ß signalling. Proc Natl Acad Sci USA 111:16496–16501 10. Aarts N, Akoudad S, Noordam R et al (2014) Inhibition of serotonin reuptake by antidepressants and cerebral microbleeds in the general population. Stroke J Cereb Circ 45:1951–1957
LETTER TO THE EDITORS
A novel HTRA1 exon 2 mutation causes loss of protease activity in a Pakistani CARASIL patient Zhaleh Khaleeli1 • Zane Jaunmuktane2 • Nathalie Beaufort3 • Henry Houlden4 • Christof Haffner3 • Sebastian Brandner2 • Martin Dichgans3,5 • David Werring6
Received: 26 March 2015 / Revised: 24 April 2015 / Accepted: 26 April 2015 / Published online: 10 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Dear Sirs, Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an inherited small vessel disease, reported mainly in Japan and China. We describe the clinical, radiological and pathological findings in a Pakistani patient with a novel mutation (c.517G[A P.A173T), and the impact of the mutation on protease activity. A 35-year-old Pakistani woman developed acute left leg weakness. She partially improved, but after 2 years her mobility deteriorated. She developed bladder urge incontinence, headaches, cognitive slowing and low mood. Her parents were first cousins, and her maternal grandfather had a stroke at a young age (Fig. 1). Blood pressure was Z. Khaleeli, Z. Jaunmuktane, N. Beaufort, M. Dichgans and D. Werring have contributed equally to the manuscript. & David Werring [email protected] 1
National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, England, UK
2
Division of Neuropathology, UCL Institute of Neurology and National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, England, UK
3
Institute for Stroke and Dementia Research (ISD), Klinikum der Universita¨t Mu¨nchen, Heiglhofstr. 55, 81377 Munich, Germany
4
UCL Institute of Neurology, Queen Square, London WC1N 3BG, England, UK
5
Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
6
Stroke Research Group, Department of Brain Repair and Rehabilitation, Institute of Neurology, UCL, Queen Square, London WC1N 3BG, England, UK
normal. She had alopecia (Fig. 2). Neurological examination showed moderate executive dysfunction, reduced cognitive speed and attention, saccadic intrusions, slow tongue movements, brisk jaw jerk, mild spastic paraparesis, and extensor plantars. Full blood count, urea and electrolytes, liver function, clotting, antineuronal antibodies, ANCA, ENA, HIV, treponemal and anti-cardiolipin antibodies were normal or negative. CSF was unremarkable (negative for oligoclonal bands). MRI brain showed confluent T2 white matter hyperintensities, multiple T1 hypointensities and multiple cerebral microbleeds; MRI spine showed non-compressive spondylosis (Fig. 2a–c). Due to a further acute deterioration in her cognitive function and mobility, we performed non-dominant frontal brain biopsy to rapidly exclude potentially reversible causes, including cerebral vasculitis. The biopsy demonstrated widespread concentric hyaline arteriolosclerosis in the leptomeninges, neocortex and white matter (Fig. 2e–h). There was focal perivascular lymphocytic inflammation in the white matter, but no features of vasculitis. Notch-3 gene mutations in exons 2, 3, 4, 5, 6, 8, 11, 18 and 19 were absent. Sequencing of the High Temperature Requirement protease A1 (HTRA1) gene revealed a novel homozygous mutation in Exon 2 at (c.517G[A P.A173T); her parents were heterozygous carriers of the same mutation. We transfected human embryonic kidney cells with plasmids encoding either wild-type (WT) HtrA1, an inactive variant obtained by replacing catalytic serine with alanine (S328A); the A173T mutant. Protein expression and secretion of all HtrA1 forms was confirmed by Western blot of cell culture medium (Fig. 2i). Protease activity assays were performed against bovine serum albumin (BSA) and casein (Fig 2j, k). HtrA1-WT was active in both assays, but neither HtrA1-S328A nor A173T mutants
123
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J Neurol (2015) 262:1369–1372
Fig. 1 Family tree
showed protease activity, establishing the A173T mutation’s pathogenicity. About 50 CARASIL families have been reported, mostly from Japan [1], but also from China [2, 3], Spain [4], Italy [5] and Turkey [6]. Although we are not aware of reports from the Indian subcontinent, the clinico-radiological phenotype of our patient is typical [1]. Unlike CADASIL [7] and amyloid angiopathies, brain tissue reveals no pathognomonic features. CARASIL is caused by HTRA1 gene mutations in the 10q26 region, coding for HtrA1, a primarily secreted serine protease. HtrA1 mediates protein degradation, cell signalling, skeletal development and osteogenesis. Furthermore, HtrA1 modulates transforming growth factor-b (TGF-b) signalling [8, 9], a pathway associated with vascular angiogenesis and remodelling [7], perhaps explaining the alopecia and lumbar degenerative disease in CARASIL [1, 8].
123
The HTRA1 gene consists of nine exons, which encode an insulin-like growth factor binding protein domain (ILGFBPD), a Kazal-type serine protease inhibitor domain, a trypsin-like serine protease domain and a PDZ domain. CARASIL-associated mutations described previously mostly target exons 3, 4, and 6, and affect the protease domain (i.e., A252T, R274Q, P285L, G295R, V297M, A321T, P364L) [2, 4, 5]. Other allelic variants include c.126delG (exon 1; p.G42fs; ILGFBPD), resulting in a premature stop codon and the expression of a truncated form lacking the entire protease domain [5], and c.1108C[T (exon 6; p.R370X; PDZ domain), which impairs mRNA stability and thus protein expression [8]. We provide the first description of a pathogenic HTRA1 mutation in exon 2. Although this affects an amino acid residue outside the protease domain (i.e., in a short interdomain region connecting the Kazal-type and protease
J Neurol (2015) 262:1369–1372
1371
Fig. 2 Clinical, radiological, pathological and biochemical features: a T2-weighted MRI image of the brain showing extensive areas of high signal in the cerebral white matter and thalamus (arrowed), b susceptibility-weighted MRI of the brain showing multiple cerebral microbleeds (arrowed), c T2-weighted MRI of the lumbar spinal cord showing spondylotic changes with loss of lordosis, d alopecia of the scalp, e haematoxylin and eosin (H&E) stained section of white matter reveals concentric hyaline thickening of the walls of small calibre vessels, f periodic acid Schiff preparation (PAS) accentuates the smooth thickening of the walls with no evidence of granular deposits, g Van Gieson’s stain showed increased connective tissue, reduplication and fragmentation of the elastic lamina within the vessel walls. h Immunostaining for smooth muscle actin confirms the loss of this specific smooth muscle cell marker in affected vessel walls. A normal age-matched arteriole is shown for comparison. i Proteolytic
activity of the A173T HtrA1 mutant: cells were transfected with a control vector (Vec) or with plasmids encoding Myc-tagged wild-type (WT), active site mutant (S328A), or CARASIL mutant (A173T) HtrA1. After 48 h culturing, serum-free culture media were collected and analysed by Western blot using an anti-Myc Ab. j BSA (10 lg) was slightly denatured by addition of 1.5 mM dithiothreitol, then exposed to transfected cell culture medium for 24 h at 37 °C, before its degradation was evaluated by SDS-PAGE followed by Coomassie staining. k Fluorescein-labelled (FTC)-casein (5 lg) was exposed to transfected cell culture medium and released fluorescence was monitored over 10 h at room temperature. Histograms represent the initial rate of casein hydrolysis with that produced by HtrA1-WT being set to 1. Results are expressed as the mean ?SEM of 4 independent assays
domains), we demonstrate that it prevents protease activity, indicating a loss-of-function mechanism [10] in CARASIL. Further work will elucidate the contribution of the targeted residue, and this domain, to HtrA1 activity.
Excellence on the Pathogenesis of Small Vessel Disease of the Brain), the Vascular Dementia Research Foundation (to M.D.), and the Jackstaedt Foundation. Dr. Werring receives research support from the Department of Health/Higher Education Funding Council for England (Clinical Senior Lectureship Award), the Stroke Association and the British Heart Foundation. Part of this work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centres funding scheme.
Conflicts of interest Dr. Khaleeli reports no disclosures. Dr. Jaunmuktane reports no disclosures. Dr. Beaufort reports no disclosures. Prof Houlden reports no disclosures. Dr. Haffner reports no disclosures. Prof Brandner reports no disclosures. Prof Dichgans is funded by the Fondation Leducq (Transatlantic Network of
Informed consent Informed consent was obtained from the individual included in the study.
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References 1. Fukutake T (2011) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL): from discovery to gene identification. J Stroke Cerebrovasc Dis 20:85–93 2. Wang XL, Li CF, Guo HW, Cao BZ (2012) A novel mutation in the HTRA1 gene identified in Chinese CARASIL pedigree. CNS Neurosci Ther 18:867–869 3. Chen Y, He Z, Meng S, Li L, Yang H, Zhang X (2013) A novel mutation of the high-temperature requirement A serine peptidase 1 (HTRA1) gene in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). J Int Med Res 41:1445–1455 4. Mendioroz M, Fernandez-Cadenas I, Del Rio-Espinola A et al (2010) A missense HTRA1 mutation expands CARASIL syndrome to the Caucasian population. Neurology 75:2033–2035 5. Bianchi S, Di Palma C, Gallus GN et al (2014) Two novel HTRA1 mutations in a European CARASIL patient. Neurology 82:898–900
123
J Neurol (2015) 262:1369–1372 6. Bayrakli F, Balaban H, Gurelik M, Hizmetli S, Topaktas S (2014) Mutation in the HTRA1 gene in a patient with degenerated spine as a component of CARASIL syndrome. Turk Neurosurg 24:67–69 7. Yamamoto Y, Craggs L, Baumann M, Kalimo H, Kalaria RN (2011) Review: molecular genetics and pathology of hereditary small vessel diseases of the brain. Neuropathol Appl Neurobiol 37:94–113 8. Hara K, Shiga A, Fukutake T et al (2009) Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N Engl J Med 360:1729–1739 9. Beaufort N, Scharrer E, Kremmer E, Lux V, Ehrmann M, Huber R, Houlden H, Werring D, Haffner C, Dichgans M (2014) The cerebral small vessel disease-related protease HtrA1 processes latent TGF-ß binding protein 1 and facilitates TGF-ß signalling. Proc Natl Acad Sci USA 111:16496–16501 10. Aarts N, Akoudad S, Noordam R et al (2014) Inhibition of serotonin reuptake by antidepressants and cerebral microbleeds in the general population. Stroke J Cereb Circ 45:1951–1957
