Journal of Clinical Neuroscience xxx (2018) xxx–xxx  Contents lists available at ScienceDirect Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn Clinical study Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3 Ling Yin Hung a, Tsz Ki Ling a, Nike Kwai Cheung Lau a, Wing Lan Cheung a, Yeow Kuan Chong a, Bun Sheng b, King Ming Kwok c, Chloe Miu Mak a,⇑ a Kowloon West Cluster Laboratory Genetic Service, Chemical Pathology Laboratory, Department of Pathology, Princess Margaret Hospital, Hong Kong Special Administrative Region Department of Medicine and Geriatrics, Princess Margaret Hospital, Hong Kong Special Administrative Region c Department of Diagnostic and Interventional Radiology, Kwong Wah Hospital, Hong Kong Special Administrative Region b a r t i c l e i n f o Article history: Received 29 November 2017 Accepted 24 June 2018 Available online xxxx Keywords: Adult onset hereditary stroke CADASIL Cerebral autosomal dominant arteriopathy with subcortical infarcts and Leukoencephalopathy Hong Kong Chinese NOTCH3 mutations a b s t r a c t Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset hereditary stroke syndrome characterized by recurrent stroke and progressive cognitive impairment caused by NOTCH3 mutations. We report here the clinical and molecular findings of three unrelated Hong Kong Chinese families with CADASIL syndrome. Sanger sequencing of genomic DNA revealed a novel heterozygous variant NM_000435.2(NOTCH3):c.[5903_5904insATAA];[5903_5904=] NP_000426.2:p.(Asp1969⁄);(Asp1969=) and two previously reported heterozygous mutations NM_000435.2(NOTCH3):c.[328C>T];[328C=] NP_000426.2:p.[(Arg110Cys)];[(Arg110=)] and NM_000435.2(NOTCH3):c.[580T>A];[580T=] NP_000426.2:p.(Cys194Ser);(Cys194=) in the three families respectively. Molecular basis of CADASIL in these three patients were further established. Genetic analysis provides a reliable method for confirming the diagnosis of CADASIL and enables proper genetic counseling and cascade testing. Ó 2018 Published by Elsevier Ltd. 1. Introduction Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic hereditary stroke syndrome [1] and is due to mutations in NOTCH3 [2]. The disease is characterized by adult onset, recurrent transient ischemic attacks and stroke in subcortical regions with progressive dementia. Visual disturbances, migraine and epilepsy represent other neurological symptoms [3,4]. Neurologically complicated pregnancy should also raise the suspicion of CADASIL [4]. The exact prevalence of CADASIL is not known but is estimated to be 1.98 per 100,000 adults [5]. Although an increasing number of cases have been reported in mainland Chinese [6–8], local data are still largely lacking. According to the Hong Kong Hospital Authority Genetic Testing Formulary, our group is the only laboratory providing the genetic testing for CADASIL in public hospitals. In the year 2007, we reported the first case of CADASIL identified ⇑ Corresponding author at: Room 1124, 11/F, Block G, Kowloon West Cluster Laboratory Genetic Service, Department of Pathology, Princess Margaret Hospital, Hong Kong Special Administrative Region. E-mail address: makm@ha.org.hk (C.M. Mak). in a Hong Kong Chinese patient [9]. Since then, we received ten referrals for CADASIL genetic analysis, with three more positive cases identified. Here, we describe the three cases and characterize their clinical, radiological and molecular findings. 2. Materials and method 2.1. Case history 2.1.1. Case 1 This female patient presented at the age of 54 years with 2 years of progressive memory decline since 2011 without any other neurological or psychiatric manifestations. At presentation, Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) were performed and scored 22/30 and 23/30 respectively. She had a positive family history of young onset stroke and memory impairment. The patient’s mother was reported to have dementia in her 50’s and died of stroke. Out of her five younger brothers, one had memory decline at the age of 48. The proband had no risk factors or secondary causes identified that might otherwise account for her dementia. Magnetic resonance imaging (MRI) of the brain showed T2 hyperintensity in https://doi.org/10.1016/j.jocn.2018.06.050 0967-5868/Ó 2018 Published by Elsevier Ltd. Please cite this article in press as: Hung LY et al. Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.06.050 2 L.Y. Hung et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx bilateral subcortical and deep white matter including temporal poles and external capsules (Fig. 1). Computed tomography of Fig. 2. The arrow indicates the index case of case 1. Black filled symbol indicates the family member who carries the NM_000435.2(NOTCH3):c.[5903_5904insATAA]; [5903_5904=] mutation in Notch3 with CADASIL related symptoms. Grey symbol is a relative who has the same mutation but asymptomatic. Open symbols are relatives who have not been tested and do not have known history of CADASIL symptoms. the brain showed non-specific bilateral periventricular white matter hypodensity. Neuroimaging findings were compatible with CADASIL. Skin biopsy was declined by the patient due to its invasive nature, and genetic testing was requested directly. Fig. 2 shows the family pedigree of case 1. 2.1.2. Case 2 The patient, an adopted child with unknown family history, presented at the age of 43 with dizziness and vertigo. MRI of the brain performed in 2002 showed multiple patchy T2 intense areas in subcortical and deep white matter of bilateral cerebral hemispheres and was initially diagnosed as multiple sclerosis. On repeated MRI of the brain in 2012, the imaging findings were reviewed and the suspicion of CADASIL was then raised. Skin biopsy was subsequently arranged and revealed the presence of granular osmiophilic globules at the basement membrane of pericytes, in keeping with the diagnosis of CADASIL. Genetic testing was thus requested. 2.1.3. Case 3 The patient presented at the age of 59 with acute onset of leftsided weakness and slurring of speech in 2017. She had a family history of minor stroke in her elder and younger brother at their 50’s. MRI of the brain revealed acute infarct over right corona radiata, as well as extensive confluenting T2 hyperintense signal over bilateral fronto-parieto-temporal subcortical white matter and ventricular white matter, patchy T2 hyperintense signal over anterior aspect of bilateral temporal lobes and bilateral external capsules, suggestive of small vessel disease or CADASIL. Skin biopsy was not performed in this case and genetic testing was requested directly. 2.2. Genetic analysis Genetic analysis of all the coding exons and their 10-bp flanking regions of NOTCH3 was performed by polymerase chain reaction followed by Sanger sequencing of genomic DNA extracted from lymphoblastoid cells in EDTA blood samples. 3. Results Fig. 1. T2 FLAIR MRI of the brain showed widespread confluent white matter hyperintensities at periventricular region (A), involving the external capsule (B, arrows) and anterior temporal lobes (C, arrow heads). Table 1 shows a summary of the clinical, radiological and genetic findings of the three CADASIL patients. A novel heterozygous NM_000435.2(NOTCH3):c.[5903_5904insATAA];[5903_5904=] variant was detected in Case 1 and his affected younger brother, whereas, two reported disease-causing mutations NM_00435.2 (NOTCH3):c.[328C>T];[328C=] and NM_000435.2(NOTCH3):c. [580T>A];[580T=] were detected in Case 2 and Case 3 respectively. Please cite this article in press as: Hung LY et al. Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.06.050 4. Discussion ExAC: Exome Aggregation Consortium; gnomAD: Genome Aggregation Database; N/A: not available. Absent Extracellular – EGF-like NM_000435.2(NOTCH3):c.[580T>A]; [580T=] NP_000426.2:p.(Cys194Ser); (Cys194=) in exon 4 N/A F/59 3 Acute stroke + Acute infarct over right corona radiata; extensive confluent T2 hyperintense signal over bilateral fronto-parietotemporal subcortical white matter and ventricular white matter; patchy T2 hyperintense signal over anterior aspect of bilateral temporal lobes and bilateral external capsules Absent Extracellular – EGF-like NM_000435.2(NOTCH3):c.[328C>T]; [328C=] NP_000426.2:p.(Arg110Cys); (Arg110=) in exon 3 Granular osmiophilic globules seen at basement membrane of pericytes Patchy T2 intense area in subcortical and deep periventricular white matter. N/A On-and-off vertigo, severe giddiness and poor memory F/56 2 3 In case 1, the novel frameshift variant is absent from controls in Genome Aggregation Database and Exome Aggregation Consortium. In addition, given the diagnostic MRI findings and family history with compatible mode of inheritance. The variant is considered to be likely pathogenic. Fig. 3 shows the Sanger sequencing electrophoretogram of the novel variant. Pathogenic Likely pathogenic Frameshift causing premature protein termination SIFT: damaging MetaSVM: damaging PROVEAN: deleterious PolyPhen2: Probably damaging SIFT: damaging MetaSVM: damaging PROVEAN: deleterious Polyphen2: Probably damaging Absent Ankyrin repeat Prominent small artery disease pattern with periventricular hypodensities F/54 1 Cognitive decline + N/A NM_000435.2(NOTCH3):c. [5903_5904insATAA];[5903_5904=] NP_000426.2:p.(Asp1969*);(Asp1969=) in exon 32 Pathogenic Variant classification MRI Family history Manifestations Sex/Age at diagnosis Case Table 1 Clinical and genetic findings of the three CADASIL patients. Skin biopsy Genetic findings/Exons Domain Allele frequency in ExAC/ gnomAD In silico predictions L.Y. Hung et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx We report here the three unrelated families of CADASIL identified amongst Hong Kong Chinese and characterize the novel variant NM_000435.2(NOTCH3):c.[5903_5904insATAA];[5903_5904=]. NOTCH3 codes for one of the four Notch receptors found in mammals. These receptors are single-pass transmembrane heterodimers produced from cleavage of a single polypeptide during protein maturation. The dimer associates non-covalently between the larger extracellular domain and smaller intracellular domain [10]. More than 300 mutations in NOTCH3 has been reported according to Human Gene Mutation Database Ò (as of Jun 2018) with 98.8% being missense/nonsense substitutions, splicing substitutions, small deletions, insertions, duplications or indels [11]. More than 95% of the known mutations of NOTCH3 in CADASIL involve addition or removal of cysteine residues in the EGF repeats of extracellular domains. The resulting odd number of cysteine residues may promote aberrant multimerization with unpaired thiol groups during maturation, leading to abnormal accumulation of Notch3 extracellular domain in vascular wall [10]. The mutations identified in the second and third case, NM_00435.2(NOTCH3):c. [328C>T];[328C=] NP_000426.2:p.(Arg110Cys);(Arg110=) and NM_000435.2(NOTCH3):c.[580T>A];[580T=] NP_000426.2:p.(Cy s194Ser);(Cys194=), belong to the group of mutations causing alteration in the number of cysteine residues [12,13]. Evidence of aberrant Notch3 multimerization, as suggested by granular osmiophilic material (GOM) deposits seen in skin biopsy, was also observed in this patient carrying the known disease-causing mutation. Here, we report a novel mutation with the insertion of 4 nucleotides before the 5th ankyrin repeat at the C-terminal of the intracellular domain of Notch3 receptor (Fig. 4). Such mutation, located within the last 50 base pairs of the penultimate exon, is predicted to cause premature termination of the protein without nonsense mediated mRNA decay. Its effect on protein turnover or signaling is not known. Since the intracellular domain is responsible for downstream signaling [10], a CADASIL-causing mutation within such domain (and presumably independent of the extracellular domain) may suggest that CADASIL pathogenicity acts from a more downstream part of the Notch signaling pathway. Although there have been isolated reports of loss-of-function NOTCH3 mutations reported in CADASIL cases [14], it is generally accepted that CADASIL-causing mutations are gain-of-function mutations [15,16]. It is thus logical to postulate that such mutation within the intracellular domain also exerted its effects via a gain-offunction mechanism. Furthermore, as there is no involvement of the extracellular domains, the dimerization of NOTCH3 receptors should in theory not be affected, and the typical GOM should not be seen in skin biopsy. However, such postulations have not been confirmed as skin biopsy findings of patients with rare mutations sparing the extracellular domain has been scarcely reported. The patient and her affected family members carrying this novel variant presented mainly with progressive cognitive decline in midadulthood. Compared with the phenotypes of the patients carrying mutation affecting the cysteine residues, there were no apparent subcortical ischaemic events, migraines and mood disturbances seen in this group of patients [3]. Further functional characterization of this mutant may shed light on the pathogenetic mechanisms of CADASIL. Please cite this article in press as: Hung LY et al. Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.06.050 4 L.Y. Hung et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx Fig. 3. (A) Exon 32 sequence analysis (reverse compliment) of case 1 shows heterozygous insertion of four bases ATAA as indicated in the box. (B) Exon 32 sequence analysis of a control subject, arrow indicating the site of insertion. Fig. 4. Distribution of known pathogenic NOTCH3 mutation causing CADASIL. Red ovals represent EGF-like repeat; green ovals represent LIN-12/notch repeats (LNR) domains; blue ovals represent ankyrin repeats. Red arrow indicates the position of the novel NM_000435.2(NOTCH3):c.[5903_5904insATAA];[5903_5904=] NP_000426.2:p. (Asp1969*);(Asp1969=) identified in the current study. Data collected from uniprot.org with 114 missense/deletion represented on this diagram as of 6 May 2016. CADASIL causing mutations included only. According to an analysis of 33 mainland Chinese families with CADASIL by Wang et al. 2011, most common clinical features amongst Chinese is transient ischemic attack/stroke, accounting for 82% [7]. Cognitive decline comes second, accounting for 60% of presentation [7]. Studies have also shown that CADASIL may present with intracerebral hemorrhage, expanding the spectrum of neurologic features [17]. Up to 25% of symptomatic CADASIL patients had intracerebral hemorrhage in a Korean study [18]. In Please cite this article in press as: Hung LY et al. Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3. J Clin Neurosci (2018), https://doi.org/10.1016/j.jocn.2018.06.050 L.Y. Hung et al. / Journal of Clinical Neuroscience xxx (2018) xxx–xxx the subsequent study of 73 CADASIL patients by the same group, a significant association between hypertension and intracerebral hemorrhage was demonstrated, with an odds ratio of 10.3 compared to CADASIL patients without hypertension [19]. Progressive cognitive decline is the major clinical presentation in the first case, while the typical neurological features of CADASIL being absent at the time of diagnosis for the second case. There is poor genotypephenotype correlation. The expression of the disease is variable in terms of age of onset, the variety of clinical symptoms, as well as disease severity and progression, even among carriers of the same mutation. Phenotype of homozygous mutations were also found to lie within the CADASIL spectrum and indistinguishable from that of heterozygous mutations. Neuroimaging is essential for the evaluation of suspected CADASIL. In the above three cases, genetic analysis was initiated only after suggestive MRI findings were correctly identified. A review of imaging characteristics of CADASIL published in 2015 suggested three groups of features on neuroimaging [20]: (1) White matter changes, which may be observed as bilateral, and typically symmetrical, hyperintensities over MRI T2-weighted images or hypodensities on CT. Commonly involved regions include anterior temporal pole, external capsules and periventricular regions. (2) Lacunar infarcts in pons, thalamus and basal ganglia. (3) Cerebral microbleeds, which may be defined as focal areas of signal loss with <10 mm in MRI T2-weighted imaging [21]. Higher number of microbleeds have proven association with worse clinical outcomes defined with modified Rankin scale [19]. This further adds to the value of neuroimaging in CADASIL patients. These imaging abnormalities may develop as early as the second decade of life, before the emergence of clinical symptoms. These lesions increase progressively with age and are almost always abnormal in all patients by the age of 35. Therefore, absence of such features may not reliably rule out possibility of underlying CADASIL, especially in young patients. Moreover, as findings are often misinterpreted as multiple sclerosis, Alzheimer’s disease or other neurodegenerative diseases, the sensitivity of picking up suspected CADASIL cases with neuroimaging poses further diagnostic challenge and would often rely on the experience of the radiologist. Historically, the conventional diagnosis of CADASIL is based on skin biopsy findings [22]: either by immunostaining with monoclonal antibody against Notch3 receptor or followed by examination under electron microscopy for granular osmiophilic material [23]. These tests rely on the observation that abnormal Notch3 proteins accumulating around the vascular smooth muscle cells both within cerebral arterioles and extracerebral arterioles. However, the sensitivity of skin biopsy has been questioned. In the study evaluating the diagnostic utility of immunostaining, out of the 22 NOTCH3 extracellular domain mutation-positive CADASIL cases, one case was negative for skin biopsy immunostaining [22]. Sensitivity of identification of GOM in skin biopsy was also variable with only 45% in the study by Markus HS et al. in 2002 [23]. All in all, diagnosis by either way of histological examination of skin biopsy is likely to be a highly specific test but may not be as sensitive as previously suggested, and serves only as an adjunctive but nonessential role in making the diagnosis. According to a study analyzing data from 1999 to 2007, the crude total incidence of recurrent stroke in Hong Kong is estimated to range from 47.6 to 117.2 cases per 100,000 adults/ year, with up to 30.4 cases per 100,000/year occurring in the 35–54 age group [24]. According to the Hong Kong Hospital Authority genetic test formulary, our laboratory is the only centre providing genetic testing service for CADASIL in public hospitals in Hong Kong. Given the large case load of recurrent stroke, we have only received 10 referrals for CADASIL testing over the last decade. This highlights the possible problem of underdiag- 5 nosis of CADASIL. Active case detection with MRI and skin biopsy in relatively young patients presenting with recurrent stroke and early cognitive impairment or patients with strong family history of similar neurological symptoms may identify more suspected CADASIL cases. However, as the common screening modalities with neuroimaging and skin biopsy have variable sensitivity as discussed in previous sections, it would ultimately require the astuteness on the clinician’s part for referral of genetic testing, which is currently the gold-standard of diagnosis of CADASIL. 5. Conclusion In this study, we presented our 10-year experience on genetic diagnosis of CADASIL in Hong Kong. Three positive cases were identified, including a novel heterozygous mutation NM_000435.2 (NOTCH3):c.[5903_5904insATAA];[5903_5904=]. This mutation differs from most other CADASIL-causing mutations in two aspects: (1) the mutation only involved the intracellular domain of NOTCH3 receptor and (2) there was no alteration in the cysteine residues in the extracellular domain. As the only local laboratory providing this test, there were only ten referrals over the past ten years. This likely reflects a lack of awareness of this disease in patients presenting with the typical CADASIL features. We recommend a more active approach in screening and case detection such that these patients and their at-risk family members may be diagnosed at an earlier, or even pre-symptomatic stage. 6. Sources of support Nil. References [1] Chabriat H et al. Cadasil. Lancet Neurol 2009;8(7):643–53. [2] Joutel A et al. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 1996;383(6602):707–10. [3] Dichgans M et al. The phenotypic spectrum of CADASIL: clinical findings in 102 cases. 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