Hiroaki Nozaki1, Taisuke Kato1, Megumi Nihonmatsu1, Yohei Saito1, Ikuko Mizuta1, Tomoko Noda1, Ryoko Koike1, Kazuhide Miyazaki1, Muichi Kaito1, Shoichi Ito1, Masahiro Makino1, Akihide Koyama1, Atsushi Shiga1, Masahiro Uemura1, Yumi Sekine1, Ayuka Murakami1, Suzuko Moritani1, Kenju Hara1, Akio Yokoseki1, Ryozo Kuwano1, Naoto Endo1, Takeshi Momotsu1, Mari Yoshida1, Masatoyo Nishizawa1, Toshiki Mizuno1, Osamu Onodera2. 1. From the Department of Medical Technology, School of Health Sciences, Faculty of Medicine (H.N.), Department of Molecular Neuroscience, Resource Branch for Brain Disease, Brain Research Institute (T.K., A.Y., O.O.), Department of Neurology, Clinical Neuroscience Branch, Brain Research Institute (M. Nihonmatsu, Y. Saito, A.K., A.S., M.U., Y. Sekine, M. Nishizawa), Department of Regenerative and Transplant Medicine, Division of Orthopedic Surgery (N.E.), and Department of Molecular Genetics, Bioresource Science Branch, Brain Research Institute (R. Kuwano), Niigata University, Niigata City; Department of Neurology (I.M., T. Mizuno), Kyoto Prefectural University of Medicine; Department of Neurology (T.N.), Ichinomiya Municipal Hospital, Aichi; Department of Neurology (R. Koike), Nishi-Niigata Chuo National Hospital, Niigata; Department of Neurology (K.M.), Shiseikai-Daini Hospital, Tokyo; Department of Neurology (M.K.), Kanazawa Medical University, Ishikawa; Department of Neurology (S.I.), Chiba University; Department of Neurology (M.M.), Nantan General Hospital, Kyoto; Departments of Neurology (A.M.) and Advanced Diagnosis (S.M.), Nagoya Medical Center, Aichi; Department of Neurology (K.H.), Japanese Red Cross Akita Hospital; Department of Internal Medicine (T. Momotsu), Sado General Hospital, Niigata; and Institute for Medical Science of Aging (M.Y.), Aichi Medical University, Japan. 2. From the Department of Medical Technology, School of Health Sciences, Faculty of Medicine (H.N.), Department of Molecular Neuroscience, Resource Branch for Brain Disease, Brain Research Institute (T.K., A.Y., O.O.), Department of Neurology, Clinical Neuroscience Branch, Brain Research Institute (M. Nihonmatsu, Y. Saito, A.K., A.S., M.U., Y. Sekine, M. Nishizawa), Department of Regenerative and Transplant Medicine, Division of Orthopedic Surgery (N.E.), and Department of Molecular Genetics, Bioresource Science Branch, Brain Research Institute (R. Kuwano), Niigata University, Niigata City; Department of Neurology (I.M., T. Mizuno), Kyoto Prefectural University of Medicine; Department of Neurology (T.N.), Ichinomiya Municipal Hospital, Aichi; Department of Neurology (R. Koike), Nishi-Niigata Chuo National Hospital, Niigata; Department of Neurology (K.M.), Shiseikai-Daini Hospital, Tokyo; Department of Neurology (M.K.), Kanazawa Medical University, Ishikawa; Department of Neurology (S.I.), Chiba University; Department of Neurology (M.M.), Nantan General Hospital, Kyoto; Departments of Neurology (A.M.) and Advanced Diagnosis (S.M.), Nagoya Medical Center, Aichi; Department of Neurology (K.H.), Japanese Red Cross Akita Hospital; Department of Internal Medicine (T. Momotsu), Sado General Hospital, Niigata; and Institute for Medical Science of Aging (M.Y.), Aichi Medical University, Japan. onodera@bri.niigata-u.ac.jp.
Abstract
OBJECTIVE: To elucidate the molecular mechanism of mutant HTRA1-dependent cerebral small vessel disease in heterozygous individuals. METHODS: We recruited 113 unrelated index patients with clinically diagnosed cerebral small vessel disease. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. RESULTS: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. The mean age at cognitive impairment onset was 51.1 years. Spondylosis deformans was observed in all cases, whereas alopecia was observed in 3 cases; an autopsied case with p.G283E showed arteriopathy in their cerebral small arteries. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (A252T and V297M) did not inhibit wild-type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319I HTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer-associated HTRA1 activation. CONCLUSIONS: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.
OBJECTIVE: To elucidate the molecular mechanism of mutant HTRA1-dependent cerebral small vessel disease in heterozygous individuals. METHODS: We recruited 113 unrelated index patients with clinically diagnosed cerebral small vessel disease. The coding sequences of the HTRA1 gene were analyzed. We evaluated HTRA1 protease activities using casein assays and oligomeric HTRA1 formation using gel filtration chromatography. RESULTS: We found 4 heterozygous missense mutations in the HTRA1 gene (p.G283E, p.P285L, p.R302Q, and p.T319I) in 6 patients from 113 unrelated index patients and in 2 siblings in 2 unrelated families with p.R302Q. The mean age at cognitive impairment onset was 51.1 years. Spondylosis deformans was observed in all cases, whereas alopecia was observed in 3 cases; an autopsied case with p.G283E showed arteriopathy in their cerebral small arteries. These mutant HTRA1s showed markedly decreased protease activities and inhibited wild-type HTRA1 activity, whereas 2 of 3 mutant HTRA1s reported in cerebral autosomal-recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (A252T and V297M) did not inhibit wild-type HTRA1 activity. Wild-type HTRA1 forms trimers; however, G283E and T319IHTRA1, observed in manifesting heterozygotes, did not form trimers. P285L and R302Q HTRA1s formed trimers, but their mutations were located in domains that are important for trimer-associated HTRA1 activation; in contrast, A252T and V297M HTRA1s, which have been observed in CARASIL, also formed trimers but had mutations outside the domains important for trimer-associated HTRA1 activation. CONCLUSIONS: The mutant HTRA1s observed in manifesting heterozygotes might result in an impaired HTRA1 activation cascade of HTRA1 or be unable to form stable trimers.
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