□ CASE REPORT □ Two Japanese CADASIL Families Exhibiting Notch3 Mutation R75P Not Involving Cysteine Residue Toshiki Mizuno 1, Manabu Muranishi 1, Torusunjian Torugun 1, Hiromi Tango 1, Yoshinari Nagakane 1, Tukasa Kudeken 3, Yuji Kawase 3, Kiyokazu Kawabe 3, Fumiko Oshima 4, Takeshi Yaoi 2, Kyoko Itoh 2, Shinji Fushiki 2 and Masanori Nakagawa 1 Abstract Most previously reported mutations in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) result in an odd number of cysteine residues within the epidermal growth factor (EGF)-like repeats in Notch3. We report here R75P mutation in two Japanese CADASIL families not directly involving cysteine residues located within the first EGF-like repeats. Probands in both families had repeated episodes of stroke, depression, dementia as well as T2 high-intensity lesions in the basal ganglia and periventricular white matter, but fewer white matter lesions in the temporal pole on MRI. These families provide new insights into the diagnosis and pathomechanisms of CADASIL. Key words: CADASIL, Notch3, EGF-like repeat, cysteine, white matter lesion, GOM (Inter Med 47: 2067-2072, 2008) (DOI: 10.2169/internalmedicine.47.1391) Introduction Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial adult-onset progressive vascular disorder characterized by recurrent ischemic attacks leading to vascular dementia (1). Joutel et al identified the defective gene in CADASIL as Notch3, which consists of 33 exons encoding a protein of 2321 amino acids, including 34 epidermal growth factor (EGF)-like repeats in the extracellular amino-terminal region (2). CADASIL is caused by single missense mutations (2-5), small in-frame deletions (6, 7), or splice site mutations (8) in the Notch3 gene. Most previously reported mutations have resulted in an odd number of cysteine residues within the first five EGF-like repeats (4, 9). Each missense mutation is predicted to replace the wild-type amino acid with a cysteine residue or to replace one of the six highly conserved cysteine residues with another amino acid in the EGF-like repeats. These mutations are thought to change the conformation of EGF-like repeats due to altered covalent bonding between cysteine residues (9). Brain magnetic resonance imaging (MRI) shows diffuse white matter abnormalities and small subcortical cystic lesions suggestive of infarcts in CADASIL patients (10-14). These lesions are predominantly located in the centrum semiovale, thalamus, basal ganglia and pons. Several reports have focused on temporopolar white matter lesions as a diagnosis marker for CADASIL, which allows the condition to be distinguished from other cerebrovascular diseases, such as Binswanger disease (12-15). Therefore, a clinician may suspect CADASIL in hereditary young stroke patients with these specific MRI findings. Here, we report two interesting Japanese CADASIL families exhibiting Notch3 mutation R75P that does not directly involve cysteine residues, and fewer white matter lesions in the temporal pole on MRI. 1 Department of Molecular Neurology, Kyoto Prefectural University of Medicine, Kyoto, 2Department of Pathology & Applied Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, 3Department of Neurology, Oomori Hospital, Toho University, Tokyo and 4Department of Neurology, Kyoto First Red Cross Hospital, Kyoto Received for publication June 6, 2008; Accepted for publication August 18, 2008 Correspondence to Dr. Toshiki Mizuno, mizuno@koto.kpu-m.ac.jp 2067 Inter Med 47: 2067-2072, 2008 DOI: 10.2169/internalmedicine.47.1391 abnormalities. MMSE and Hamilton’s scale were normal. Case Family B (Fig. 1B) A 64-year-old woman in Family B (BII-3) visited a nearby hospital due to right thalamic hemorrhage in 1996. Hypertension and diabetes mellitus were noted. Her MRI revealed multiple, old cerebral infarctions (Fig. 3A-C). Repeated cerebral infarctions were seen over a 7-year period. She became bedridden and could not eat meals. Generalized tonic seizure occasionally occurred and she was transferred to hospital. Her parents and elderly brother (II-1) suffered from dementia. Her elderly brother (BII-2) and sister (BII-4) suffered from cerebral hemorrhage and infarctions, and her sister died of pneumonia at age 70 years. Neurological examination showed akinetic mutism, pseuFi g ur e1 . Pe di g r e eo ft wof a mi l i e swi t ht heR7 5 Pmut a t i o n. Ar r o wsi ndi c a t et hepr o ba ndi ne a c hf a mi l y .Bl a c kf i l l e ds y m- dobalbar palsy, tetraplegia and contraction of the four extremities. Electroencephalography revealed paroxymal spikes bo l si ndi c a t et ho s ewi t hc l i ni c a ls y mpt o mso fCADASI L. and waves, predominantly in the left hemisphere. MRI in 2003 (Fig. 3D-F) revealed diffuse cerebral atrophy, multiple cerebral infarction at the midbrain, bilateral basal ganglia, Case Family A (Fig. 1A) corona radiata and diffuse white matter lesions, but these The proband of Family A was a 52-year-old woman (AII- did not spread to the temporal pole. Ultrastructural analysis 1). She was diagnosed as having depression after accouche- of the skin revealed granular osmophilic materials (GOM) ment and received medication. At 50 years of age, she en- within the basal lamina in the artery (Fig. 4). Ultimately, she tered the hospital due to reduced activities of daily life and developed pneumonia and died at 73 years of age. MRI revealed multiple cerebral infarctions. She entered hospital again for pneumonia at 51 years of age, and she beMutation Analysis came bedridden. At 52 years of age, she entered hospital for generalized convulsions and consulted the neurological deGenomic DNA was extracted from peripheral blood lympartment. Her father had suffered from repeated episodes of phocytes using a DNA extraction kit (Quiagen) after incerebral infarction and died at age 52 years. Neurological formed consent was obtained. All exons and the promoter examination showed akinetic mutism, pseudobalbar palsy, region of the Notch3 gene (16) were directly sequenced usand tetraplegia. EEG showed spike waves in the whole cere- ing an ABI Prism 377 (Applied Biosystems, Foster City, brum. MRI revealed mild frontal and temporal lobe atrophy, CA). Mutations were confirmed by PCR, followed by remultiple cerebral infarctions at the bilateral basal ganglia, striction fragment length polymorphism (RFLP) assay, and and diffuse white matter lesions, which did not extend to the were compared with 100 healthy Japanese controls. temporal pole (Fig. 2A-C). The proband (AII-1) of Family A possessed a 302 G>C A 49-year-old woman in Family A (AII-2) noticed shoul- missense mutation of the Notch3 mRNA, which resulted in der stiffness and headaches beginning at age 40 years. She an arginine to proline amino acid change at codon 75. While also experienced periods of tinnitus and vertigo. She visited this mutation was also seen in her sisters, who had abnora neurosurgeon because she sustained a head injury at age malities on MRI (AII-2 and 4), the mutation was not seen in 49 years. Brain MRI revealed multiple infarctions at the bi- her healthy brother, who had no abnormalities on MRI (AIIlateral basal ganglia and corona radiate (Fig. 2D-F). White 3). This mutation results in the loss of an Aci 1 recognition matter high intensity was also noted on T2 and FLAIR site in exon 3. RFLP analysis revealed this mutation in the (fluid attenuated inversion recovery) imaging, but it did not three sisters, but not in the brother. The same mutation was extend to the temporal pole. Neurological findings revealed seen in the proband of Family B (BII-3), but not in her no paralysis, but deep tendon reflex increased bilaterally and daughter (BIII-7), who had no abnormalities on MRI. This Babinski signs were bilaterally positive. MMSE and Hamil- mutation was not seen on PCR-RFLP analysis in 100 Japaton scale were normal. nese controls (data not shown). The silent heterozygous 381 A 44-year-old woman in Family A (AII-4) visited a neu- C>T mutation, which results in the loss of the Aci 1 recogrosurgeon with head injury, and brain atrophy was noted. nition site, was seen in both Family A (AII-1) and Family B She had experienced headache since age 39 years, and had (BII-3). This polymorphism was also seen in 32% of healthy experienced vertigo and dizziness since age 41 years. MRI Japanese controls (16). revealed multiple infarctions in the right basal ganglia and corona radiata (Fig. 2G-I). Neurological findings revealed no 2068 Inter Med 47: 2067-2072, 2008 DOI: 10.2169/internalmedicine.47.1391 Fi g ur e2 . T2 we i g ht e di ma g e sf r o m Fa mi l yA.Mul t i pl el a c una ea ndwhi t ema t t e rl e s i o nswe r eo bs e r v e di ne a c hpa t i e nt ,butnol e s i o nswe r ede t e c t e di nt het e mpo r a lpo l e .ACa r ef r o mt hepr o ba nd( AI I 1 ) ,DFa r ef r o m AI I 2a ndGIa r ef r o m AI I 4 . Discussion R75P mutation is a rare mutation of Notch3 previously only reported in 4 unrelated Korean families (17). Most previous mutations in CADASIL patients were related a gain or loss of cysteine residues within an EGF-like repeat domain. An odd number of cysteine residues within an EGF-like repeat domain is thought to induce abnormal folding or dimerization, and aberrant interactions with other proteins. However, the R75P mutation does not directly involve the cysteine residues within the extracellular domain of the Notch3 as well as Italian case (7). We confirmed cosegregation of R75P mutation with development of CADASIL and found no causative mutations in other exons of the Notch3. In addition, we observed GOMs in the basal lamina of the skin vascular wall in Family B; GOMs in extracerebral arteries, including skin arterioles, are a specific hallmark of CADASIL (18). This suggests that CADASIL is caused by the substitution of non-cysteine residues in Notch3. The 3D structure of the first EGF-like repeat in the Notch 3 protein is predicted to be a two stranded beta-sheet followed by double (Fig. 5) (6). The six cysteines form three disulfide bonds and the double hairpin is fixed by the 5th and 6th cysteines. Substitution of the 6th cysteine in the first EGF repeat with tryptophan at codon 76 is reported to be the cause of CADASIL (19). In the 3-dimendional studies of β-hairpin and antiparallel β-sheet structure formed by water soluble peptides, proline residues contributed to stabilize βhairpin because proline fits well into the topography of βturn (20). In addition, the presence of proline residues combined with disulfide bonds can lead to different conformational isoforms of both the proline and the cysteine bonds (21, 22). The mutation of bovine pancreatic trypsion inhibitor at the 13th proline replaced by isoleucine adjacent disulfide bridge showed conformational change and enzyme in- 2069 Inter Med 47: 2067-2072, 2008 DOI: 10.2169/internalmedicine.47.1391 Fi g ur e3 . MR i ma g e sf r o mt hepr o ba ndi nFa mi l yB.Mul t i pl el a c una ea ndwhi t ema t t e rl e s i o ns we r eo bs e r v e d,butnol e s i o nswe r ede t e c t e di nt het e mpo r a lpo l e .ACa r eT1 we i g ht e di ma g e sa t 6 4y e a r so fa g e ,a ndDFa r eT2 we i g ht e di ma g e sa t7 1y e a r so fa g e . Fi g ur e4 . A.El e c t r o n mi c r o g r a ph o ft hes ki nv e s s e lr e v e a l e dg r a nul a ro s mi o phi l i cma t e r i a l s ( GOM)( * )wi t hi nt hei r r e g ul a rba s e me ntme mbr a neo fv a s c ul a rs mo o t hmus c l ec e l l .Thea r r o wsi n di c a t et heba s e me ntme mbr a ne .B.Hi g he rma g ni f i c a t i o nde mo ns t r a t e dGOM a ndpr e s e nc eo fpi no c y t i cv a c uo l e si nt hes mo o t hmus c l ec e l lc y t o pl a s m.Theba r=4 1 0nm i nAa nd2 0 0nm i nB. hibitory activity (23). Dichgans et al (6) predicted that the cysteine at codon 76 would be related to fixation of the double hairpin in the EGF-like repeat of the Notch3 (Fig. 5). These previous data about the cysteine bond and proline residues support the possibility of conformational change in the EGF-like repeat by the substitution of basic arginine for the proline at codon 75. Interestingly, the R75P mutation has only been reported in the Korean and Japanese populations, although most mutations related to cysteine residues in CADASIL have been reported worldwide. CADASIL patients on the western coast of Finland carry the same R133C mutation, and a founder effect is suspected, as similar haplotypes are linked to the mutation in all 18 pedigrees (24). These reports suggest that a founder effect related to the R75P mutation might exist in far-eastern Asian families. On the other hand, a founder effect for other mutations in Notch3 is unlikely, as no similar hapotypes are seen in unrelated European CADASIL patients with the same missense mutations (9, 25). Therefore, haplotype analysis is required in order to confirm a founder effect in R75P families. We have focused on these families for the clinical diagnosis of CADASIL because fewer white matter lesions were seen in the temporal pole on MRI. O’Sullivan et al (15) discussed the significance of temporopolar lesions as a diagnosis marker for CADASIL, and Auer et al confirmed that 2070 Inter Med 47: 2067-2072, 2008 DOI: 10.2169/internalmedicine.47.1391 (13). Tomimoto et al also reported that temporopolar lesions are diagnostic markers distinguishing CADASIL from Binswanger disease in Japanese patients, even at early stages (14). Thus, possible CADASIL patients are routinely selected for these MRI features. However, Kim et al reported less frequent white matter lesions in the anterior temporal lobe in Korean families with the R75P mutation which was true as well in the present cases (17). These cases suggested that a neurologist should pay attention to the familiar stroke patients with leukoencephalopathy in the far-eastern Asian family as a candidate for CADASIL even if MRI reveals no white matter lesion in the temporal pole. In conclusion, the R75P mutation may provide new insight into the pathogenesis of CADASIL, particularly with regard to the role of temporopolar lesions. Fi g ur e5 . Thr e e di me ns i o na lmo de lo ft hepr e di c t e dma i n c ha i nt r a c eo ft hef i r s tNo t c h3EGFl i ker e pe a tdo ma i n[ mo di - Acknowledgement The authors are grateful to Dr. Kagami for the skin biopsy, f i e df r o m Di c hg a nse ta l( 6 ) ] .TheCo do n7 5a nd7 6r e s i due s Ms. Uehori for her technical support and Professor Akaji for his a r ei ndi c a t e dbya r r o ws . suggestion. This work was supported in part by a grant from the temporopolar lesions were specific to CADASIL in Europe Ministry of Health, Labour and Welfare of Japan. References 1. Tournier-Lasserve E, Joutel A, Melki J, et al. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy maps to chromosome 19q12. Nat Genet 3: 256-259, 1993. 2. Joutel A, Corpechot C, Ducros A, et al. 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