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Phenotypic comparison of individuals with homozygous or heterozygous mutation of NOTCH3 in a large CADASIL family
Journal of the Neurological Sciences 367 (2016) 239–243
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Phenotypic comparison of individuals with homozygous or heterozygous mutation of NOTCH3 in a large CADASIL family Hussam Abou Al-Shaar a,b, Najeeb Qadi a,b, Mohamed H. Al-Hamed c, Brian F. Meyer c,d, Saeed Bohlega a,b,⁎ a
Division of Neurology, Department of Neurosciences, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia College of Medicine, Alfaisal University, Riyadh, Saudi Arabia Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia d Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 22 September 2015 Received in revised form 10 May 2016 Accepted 31 May 2016 Available online 01 June 2016 Keywords: CADASIL NOTCH3 Homozygous Heterozygous Mutation Stroke
a b s t r a c t Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary microangiopathy caused by mutations in NOTCH3, very rarely homoallelic. Objective: To describe the clinical, radiological, and neuropsychological features in an extended CADASIL family including members with either a homozygous or heterozygous NOTCH3 R1231C mutation. Methods: The pedigree included 3 generations of a family with 13 affected individuals. The patients were examined clinically and radiologically. Neuropsychological testing was performed on the proband. Sequencing of the entire coding DNA sequence (CDS) and flanking regions of NOTCH3 was undertaken using PCR amplification and direct Sanger sequencing. Results: Homozygous C3769T mutation, predicting R1231C in exon 22 of NOTCH3 was found in 7 family members. Six other family members harbored the same in the heterozygous state. Homozygous individuals showed a slightly more severe clinical and radiological phenotype of earlier onset compared to their heterozygous counterparts. Conclusion: This study reports the largest number of patients with homozygous NOTCH3 mutation. The phenotype and imaging features of homozygous individuals is within the spectrum of CADASIL, although slightly at the severe end when compared to heterozygotes carrying the same mutation. Both genetic modifiers and environmental factors may play an essential role in modification and alteration of the clinical phenotype and white matter changes among CADASIL patients. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset inherited arteriopathy, characterized by non-hypertensive, non-arteriosclerotic, small arterial granular degeneration. CADASIL manifests as recurrent subcortical ischemic events, progressive or stepwise subcortical dementia, migraine with aura, and mood disorders, with early death [1,2]. Patients usually demonstrate prominent signal abnormalities in subcortical white matter on magnetic resonance imaging (MRI) [3,4]. The mean age of onset of clinical symptoms is the mid-forties; however, MRI abnormalities can be seen a decade before symptom onset [5].
⁎ Corresponding author at: Division of Neurology, Department of Neurosciences, King Faisal Specialist Hospital and Research Centre, P.O. Box: 3354, Riyadh 11211, Saudi Arabia. E-mail addresses: [email protected] (H. Abou Al-Shaar), [email protected] (N. Qadi), [email protected] (M.H. Al-Hamed), [email protected] (B.F. Meyer), [email protected] (S. Bohlega).
http://dx.doi.org/10.1016/j.jns.2016.05.061 0022-510X/© 2016 Elsevier B.V. All rights reserved.
CADASIL symptomatology can be attributed to the systemic vasculopathy encountered in such patients. The smooth muscles in the walls of cerebral arteries are destroyed leading to luminal obliteration and fibrosis, resulting in infarction and stroke [6]. Recurrent infarctions of the white and deep grey matter are the basis of the subsequent dementia and cognitive impairment in these patients. NOTCH3 is composed of 2321 amino acids. It is a member of a protein family involved in signaling events that control cell fate decisions during development. NOTCH3 is a single-pass transmembrane receptor with a large extracellular domain containing 34 tandem epidermal growth factor-like (EGF-like) repeats [7–9]. NOTCH3 is mainly expressed in smooth muscle cells of small arteries and in pericytes around capillaries [10,11]. There are 33 exons in NOTCH3 and mutations in this gene are considered to underlie CADASIL. Almost all mutations reported so far are missense mutations that result in a gain or loss of one cysteine residue within an EGF-like repeat domain, with a strong clustering of mutations in exons 4. CADASIL has been reported in various populations around the world [5]. However, only a few cases in the literature reported homozygous mutations of NOTCH3 [12–16].
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H. Abou Al-Shaar et al. / Journal of the Neurological Sciences 367 (2016) 239–243
Herein, we report the largest number of homoallelic cases of CADASIL. We describe and compare the phenotypes of homozygous and heterozygous members within this family. 2. Methods 2.1. Patients Thirteen affected individuals from a 3 generation family were enrolled for this study. All were examined in the Department of Neurosciences, King Faisal Specialist Hospital and Research Center (KFSH&RC). The protocols for this study were approved by the institutional review board and informed consent was obtained from all participants. All subjects were enrolled under an IRB-approved protocol (RAC# 2020023) with full informed consent. History was taken and clinical examination performed on the index case and his family members. MRI was obtained for all patients. Neuropsychological analysis was performed for the index case alone. 2.2. NOTCH3 sanger sequencing DNA was isolated from whole blood using a standard salt precipitation method using a Gentra Puregene blood kit (Qiagen). Sequencing of the entire coding and flanking regions of NOTCH3 was undertaken using PCR amplification and direct Sanger sequencing using a BigDye terminator kit (Thermo Fisher, Foster City, CA). SeqScape v.2.6 software (Thermo Fisher, Foster City, CA) was used to align sequence data with NOTCH3 (ENSG00000074181). 3. Results 3.1. Clinical history The family reported in this paper originated from Kashmir in the North Eastern part of the Indian sub-continent and included 13 affected individuals spanning 3 generations as described below (Table 1). Of note, the last generation is a product of triple loop consanguinity as seen in Fig. 1. 3.1.1. IV-53 homozygous patient (as indicated in Fig. 1) The index case is a male practicing physician. He was well until the age of 53 when he had an acute onset right-sided hemiparesis, which left him with some clumsiness. Six months later, he suffered from numbness affecting the left side. Afterwards, he had problems with his vision, which he described as nocturnal blindness worse in the left eye. Ophthalmological examination showed arteriovenous nicking with moderate compression of the retinal veins as well as retinal microinfarction and chorioretinal scaring temporal to the left macula. There were tortuous narrowed retinal arterioles with mild optic disc cupping. Subsequently, multiple strokes occurred over nine years of
follow up, which left him with progressive dysarthria and a spastic gait. He became easily fatigued and markedly depressed. He was mentally slow but was able to continue his work as a Pediatrician in the Emergency Room. His MRI showed diffuse bilateral lacunar infarcts affecting basal ganglia, centrum semiovale, and periventricular white matter (Fig. 2A). Carotid angiography at the age of 55 was normal. The cognitive performance at the age of 59 (six years after the first stroke) showed a Mini-Mental Status Examination score of 24. Neuropsychology testing revealed mild problems with executive function, verbal abstraction, Wechsler Adult Intelligence Scale with visuospatial ability measured by Rey-Osterrieth Complex Figure copying and immediate reproduction and 12-memory test with free recall (12 word Rec) and recognition. Slight bipolar mood disorder was also moderately observed. The disease progressed relentlessly in a stepwise and accelerated pattern. The patient experienced difficulty swallowing associated with a pseudo bulbar affect, progressive dementia, and severe depression. He was admitted several times during this period with repeated chest infection until he became severely disabled for more than a year prior to his death. His father (III-78) died at the age of 78 after 9 years of repeated strokes, dementia, impaired vision, and moderate hearing loss. His symptoms started at the age of 62, however, he was not properly investigated. The index case's mother (III-84) died at the age of 84. She had asymmetrical coarse tremors and bradykinesia. She was diagnosed with Parkinson's disease during her last few years and she responded well to Levodopa therapy. 3.1.2. III-66 heterozygous patient The paternal uncle (Father-in-Law) of the index case was initially seen at the age of 73 with history of 3 repeated strokes, which started at the age of 66. His brain computed tomography (CT) scan was done at the age of 69 and repeated three years later during which he was diagnosed with Binswanger's disease with progression of the Leukoaraiosis. Brain MRI showed diffuse white matter hyperintense ischemic changes. He was moderately demented with hyper-reflexia and pseudo-bulbar affect. 3.1.3. III-76 heterozygous patient Paternal Aunt (Mother-in-Law) was asymptomatic at the age of 76 but brain MRI showed a few ischemic lesions in the basal ganglia and deep white matter (Fig. 2C). 3.1.4. IV-36 homozygous patient Sister of the index case started to experience recurrent episodes of migraine without aura at the age of 36. Repeated episodes of asymmetrical weakness and numbness started at the age of 42 indicating vascular insult (stroke). She also suffered from progressive visual difficulty with ocular transient ischemic attacks with retinal microinfarcts and hemorrhages. At age of 52, she started experiencing difficulty walking followed by dementia, night blindness, and seizure disorder. She
Table 1 Clinical, radiological, and exon 22 C3769T (R1231C) mutation status of the patients in relation to the index case. No.
Age of onset (years)
Sex
Relation to index case (Fig. 1)
Clinical picture
MRI
Genotype CNT
III-76 III-66 IV-35 IV-50 IV-38 IV-53 IV-36 V-7 V-11 V-13 V-22 V-30 V-31
76 66 35 50 38 53 36 7 11 13 22 30 31
F M M M F M F F M F M M F
Paternal Aunt (Mother-in-Law) Paternal Uncle (Father-in-Law) Cousin Cousin Wife Index case Sister Daughter of the second wife Son of the second wife Daughter of the second wife Son Son Daughter
Asymptomatic Stroke and dementia Migraine with aura Asymptomatic Migraine with aura Stroke Migraine without aura, stroke, dementia, seizure, and night blindness Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic
Abnormal Abnormal Abnormal Not done Abnormal Abnormal Abnormal Not done Not done Not done Abnormal Abnormal Abnormal
c/t c/t t/t c/t t/t t/t t/t c/t c/t c/t t/t t/t t/t
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Fig. 1. Family pedigree showing high degree of consanguinity, number at tab of the symbol indicates individual's age, arrow points to the index case.
required walking aid at the age of 62. MRI showed multiple sub-cortical white matter hyperintense ischemic changes.
3.1.5. IV-38 homozygous patient Cousin (Wife of the index case) had migraine with aura at the age of 38 but no stroke. Her brain MRI showed white matter hyperintense ischemic foci.
3.1.6. IV-50 heterozygous patient Cousin of the index case was asymptomatic at the age of 50. Refused to undergo MRI examination.
3.1.7. IV-35 homozygous patient Cousin of the index case, complained of migraine with aura at the age of 35. Brain MRI revealed white matter hyperintense ischemic foci.
Fig. 2. Axial brain fluid attenuated inversion recovery (FLAIR) MRI sequence examples of the homozygous and heterozygous patients. Index case (IV-53: homozygous) (A) demonstrating diffuse confluent bilateral hyperintensities in the periventricular region associated with microinfarcts (arrows). V-31: homozygous (B) depicting non-confluent bilateral hyperintensities and mild brain atrophy. III-76: heterozygous (C) showing scattered hyperintense patches.
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3.1.8. V-31 homozygous patient Daughter of the index case was asymptomatic at the age of 31. Brain MRI showed few white matter hyperintense ischemic changes (Fig. 2B). 3.1.9. V-22 and V-30 homozygous patients Sons of the index case are asymptomatic at the age of 22 and 30, respectively. Brain MRI depicted white matter hyperintense ischemic foci. 3.1.10. V-7, V-11, and V-13 heterozygous patients Son and daughters of the second wife are clinically asymptotic. MRI was not done for them. 3.2. Molecular genetic analysis Direct sequencing of the 33 exons of NOTCH3 in the index case showed a homozygous C NT transition at nucleotide 3769 (C3769T) predicting an amino acid change from arginine N cysteine at position 1231 (R1231C) as previously reported [17]. Segregation of this mutation was studied in 12 other individuals of this family identifying 6 homozygotes and 6 heterozygotes as shown in Table 1 and Fig. 3. 4. Discussion The extensive consanguinity and segregation of the C3769T (R1231C) in both homoallelic and heteroallelic states within this family is consistent with Mendelian transmission of a single mutation. Based on the consensus of dominance, homozygous mutations should not aggravate the clinical phenotype in autosomal dominant disorders [18–20]. However, this is not always the case, as homozygous mutations in some diseases have been reported to have a more clinically severe phenotype than heterozygous mutations [21]. Initially, homozygous mutations in NOTCH3 had been observed to result in a more severe clinical phenotype than heterozygous mutations [12,16]. This observation was questioned later when some homozygous mutations resulted in a mild or only slightly more severe clinical phenotype than heterozygous mutations [13–15]. These contradictory results led to the premise that the phenotype of CADASIL patients is influenced and modified by both genetic modifiers and environmental factors, not yet discovered [14,22], that alleviate or aggravate the clinical phenotype. Interestingly, in one study the authors observed a striking variability in measuring the volume of cerebral ischemic lesions in 151 CADASIL individuals suggesting a strong modifying influence of some genetic factors distinct from the causative
Fig. 3. Sequencing result of the homozygous and heterozygous mutations in different patients of this family. The homozygous patients have mutated “t” in both alleles and heterozygous patients have both “c” and “t” at nucleotide 3769 at exon 22 of the NOTCH3 gene, c.3691CNT (p. Arg1231Cys).
NOTCH3 mutation on the amount of ischemic brain lesions [22]. Those genetic modifiers could exert their effects on different pathways affected among CADASIL patients including smooth muscle cells, vascular endothelial cells, and others. They also can act trough different mechanisms interfering with autoregulation and neuronal response and repair to ischemia [23,24]. Therefore, CADASIL was considered an example of diseases where zygosity does not play a role in influencing the severity of the disease. In our cohort, the index case (homozygous mutation) experienced his first ischemic episode at the age of 53. The patient's father, paternal uncle, and paternal aunt suffered from neurological disorders, consistent with CADASIL that manifested around 70 years of age. The index case had mild cognitive and memory problems and retinal defects, as evident from his neuropsychological and ophthalmological examinations, respectively. The index case parents are most likely heterozygous, based on the increased homozygosity in the offspring, however, homozygosity cannot be entirely rule out. Moreover, five of the seven homozygous patients (aged between 22–38 years) demonstrated no symptoms or exhibited migraine with aura, while the remaining two suffered from stroke and cognitive decline with an age of onset of 42 and 53 years, similar to the previous notion of stroke onset among CADASIL patients (range from 20–70 years of age) [5]. On the other hand, the six heterozygous individuals included three children under 13 years of age as well as two subjects (50 and 76 years old), all of whom were asymptomatic. The last heterozygous patient had dementia and exhibited his first stroke at 66 years of age. It is known that there is marked unexplained phenotypic variation both intra-familially and between the families with the same mutation as well as between families with different mutations among CADASIL patients [5]. Our homozygous patients' disease fits well within the clinical and radiological phenotype of other reported CADASIL patients. Our homozygous patients did not exhibit an extraordinarily more severe phenotype compared to the heterozygous patients. Chabriat et al. and Opherk et al. noted variable white matter hyperintensity variation among 19 and 151 patients with CADASIL despite their clinical course. They concluded that a strong contribution of unknown genetic modifiers to interindividual differences might exist, which could explain the variability in the volume of ischemic brain lesions and clinical severity among these patients [22,25]. Therefore, one of the shortcomings of our report that we did not do a quantitative assessment, which correlate with the disease progression (e.g. brain atrophy) nor we did a quantitative measurement of the volume of the lacunar infarcts, which also correlate with the disease severity. Another limitation of this study is the absence of objective assessment of cognitive decline and disability, which are better clinical markers of disease severity, among all members in the family, due to their immigration back to their country. Therefore, due to these shortcomings and due to the well-known intra- and interfamilial clinical variability in CADASIL, we cannot generalize our observation to all CADASIL patients with homozygous mutations. Further studies to delineate the effects of zygosity on the severity of white matter changes and clinical course using objective measures are therefore needed. In dominantly inherited disorders, the mutation may cause loss- or gain-of-function phenomena. This results in either a single functional allele that cannot maintain the required function (haploinsufficiency) or an accentuated function that has a pathological effect. Interestingly, homozygous mutations in NOTCH3 have been proposed to result in a gain-of-function mechanism that results in more accumulation of the harmful proteins and the resultant smooth muscle damage [12,14]. This is consistent with observation of more diffuse granular osmiophilic material (GOM) deposition around smooth muscle cells in patients with homozygous mutations [12,15]. It has been postulated that if a mutation were to cause haploinsufficiency or display a dominant-negative effect, then it would cause a more severe or lethal phenotype [13,14]. It has been thought that in loss-of-function mutations, the fact that the double dose of gene defect does not appear to aggravate the symptoms
H. Abou Al-Shaar et al. / Journal of the Neurological Sciences 367 (2016) 239–243
compared to gain-of-function mutations, may indicate that either the mutated NOTCH3 has some retained function or other (NOTCH?) molecules and genetic modifiers can compensate for the loss [12]. Only scarce reports of homozygous mutations in NOTCH3 are present in the literature, with single cases of homozygosity in the majority of the studies [12,13,15,16]. To the best of our knowledge, only 6 homozygous patients and one cell-line from a homozygous patient have been reported in the literature [12–16,26]. Our study is the first report of a large number of patients harboring a homozygous mutation in NOTCH3. Finally, longitudinal studies on the progression of the disease are lacking. Thus, it is unknown whether early onset homozygous CADASIL patients are associated with more rapid or slower progression. More studies on homozygous NOTCH3 mutations are therefore needed to further answer this question. 5. Conclusion We report the largest number of patients with homozygous NOTCH3 mutation in a single family. The phenotype and imaging features of our homozygous individuals is within the spectrum of CADASIL, albeit slightly at the severe end when compared to heterozygous patients. It is likely that both genetic modifiers and environmental factors may play an essential role in the modification and alteration of the clinical phenotype and white matter changes among CADASIL patients. Author contributions Conception and design: Abou Al-Shaar, Qadi, Bohlega. Acquisition of data: Abou Al-Shaar, Qadi, Bohlega, Meyer. Analysis and interpretation: Abou Al-Shaar, Bohlega, Meyer. Management of the patients: Bohlega, Qadi. Genetic testing: Meyer, Al-Hamed. Drafting the article: Abou AlShaar. Critically revising the article: all authors. Final approval of the submitted version: all authors. Disclosure The authors declare no conflicts of interest regarding the production of this article. The authors have no personal financial or institutional interest in any of the drugs, materials, or devices described in this article. References [1] H. Chabriat, K. Vahedi, M.T. Iba-Zizen, et al., Clinical Spectrum of CADASIL: a study of 7 families. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Lancet 346 (1995) 934–939. [2] M. Baudrimont, F. Dubas, A. Joutel, E. Tournier-Lasserve, M.G. Bousser, Autosomal dominant syndrome leukoencephalopathy and subcortical ischemic stroke. A clinicopathological study, Stroke 24 (1993) 122–125. [3] H.S. Markus, R.J. Martin, M.A. Simpson, et al., Diagnostic strategies in CADASIL, Neurology 59 (2002) 1134–1138.
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[4] D. Herve, O. Godin, C. Dofouil, et al., Three-dimensional MRI analysis of individual volume of Lacunes in CADASIL, Stroke 40 (2009) 124–128. [5] H. Chabriat, A. Joutel, M. Dichgans, E. Tournier-Lasserve, M.G. Bousser, CADASIL review, Lancet Neurol. 8 (2009) 643–653. [6] A. Viswanathan, F. Gray, M.G. Bousser, M. Baudrimont, H. Chabriat, Cortical neuronal apoptosis in CADASIL, Stroke 37 (2006) 2690–2695. [7] H. Karlstrom, P. Beatus, K. Danneus, G. Chapman, U. Lendahl, J. Lundkvist, A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 17119–17124. [8] R. Kopan, M.X. Ilagan, The canonical Notch signaling pathway: unfolding the activation mechanism, Cell 137 (2009) 216–233. [9] A. Joutel, C. Corpechot, A. Ducros, et al., Notch3 mutation in CADASIL, a hereditary adult-onset condition causing stroke and dementia, Nature 383 (1996) 707–710. [10] A. Joutel, M. Monet-Leprêtre, C. Gosele, et al., Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease, J. Clin. Invest. 120 (2010) 433–445. [11] E. Lewandowska, G.M. Szpak, T. Wierzba-Bobrowicz, et al., Capillary vessel wall in CADASIL angiopathy, Folia Neuropathol. 48 (2010) 104–115. [12] S. Tuominen, V. Juvonen, K. Amberla, et al., Phenotype of a homozygous CADASIL patient in comparison to 9 age-matched heterozygous patients with the same R133C NOTCH3 mutation, Stroke 32 (2001) 1767–1774. [13] M.K. Liem, S.A. Lesnik-Oberstein, H.A. Middelkoop, J. van der Grond, A.T. Heldermanvan den enden, Homozygosity for a NOTCH3 mutation in a 65-year-old CADASIL patient with mild symptoms: a family report, J. Neurol. 255 (2008) 1978–1980. [14] B.W. Soong, Y.C. Liao, P.H. Tu, et al., A homozygous NOTCH3 mutation p.R544C and a heterozygous TREX1 variant p.C99MfsX3 in a family with hereditary small vessel disease of the brain, J. Chin. Med. Assoc. 76 (2013) 319–324. [15] M. Ragno, L. Pianese, M. Morroni, et al., “CADASIL coma” in an Italian homozygous CADASIL patient: comparison with clinical and MRI findings in age-matched heterozygous patients with the same G528C NOTCH3 mutation, Neurol. Sci. 34 (2013) 1947–1953. [16] C. Vinciguerra, A. Rufa, S. Bianchi, et al., Homozygosity and severity of phenotypic presentation in a CADASIL family, Neurol. Sci. 35 (2014) 91–93. [17] A. Joutel, K. Vahedi, C. Corpechot, et al., Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients, Lancet 350 (1997) 1511–1515. [18] N.S. Wexler, A.B. Young, R.E. Tanzi, et al., Homozygotes for Huntington's disease, Nature 326 (1987) 194–197. [19] A. Dürr, V. Hahn-Barma, A. Brice, C. Pêcheux, C. Dodé, J. Feingold, Homozygosity in Huntington's disease, J. Med. Genet. 36 (1999) 172–173. [20] R. Gabizon, H. Rosenmann, Z. Meiner, et al., Mutation and polymorphism of the prion protein gene in Libyan Jews with Creutzfeldt-Jakob disease (CJD), Am. J. Hum. Genet. 53 (1993) 828–835. [21] F.G. Sturtz, P. Latour, Y. Mocquard, et al., Clinical and electrophysiological phenotype of a homozygously duplicated Charcot-Marie-Tooth (type 1A) disease, Eur. Neurol. 38 (1997) 26–30. [22] C. Opherk, N. Peters, M. Holtmannspötter, A. Gschwendtner, B. Müller-Myhsok, M. Dichgans, Heritability of MRI lesion volume in CADASIL: evidence for genetic modifiers, Stroke 37 (11) (2006) 2684–2689. [23] A. Hassan, B.J. Hunt, M. O'Sullivan, et al., Homocysteine is a risk factor for cerebral small vessel disease, acting via endothelial dysfunction, Brain 127 (Pt 1) (2004) 212–219. [24] A. Hoy, B. Leininger-Muller, O. Poirier, et al., Myeloperoxidase polymorphisms in brain infarction. Association with infarct size and functional outcome, Atherosclerosis 167 (2) (2003) 223–230. [25] H. Chabriat, E. Tournier-Lasserve, K. Vahedi, et al., Autosomal dominant migraine with MRI white-matter abnormalities mapping to the CADASIL locus, Neurology 45 (6) (1995) 1086–1091. [26] S. Tikka, Y.P. Ng, G. Di Maio, et al., CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells, J. Cereb. Blood Flow Metab. 32 (2012) 2171–2180.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Phenotypic comparison of individuals with homozygous or heterozygous mutation of NOTCH3 in a large CADASIL family Hussam Abou Al-Shaar a,b, Najeeb Qadi a,b, Mohamed H. Al-Hamed c, Brian F. Meyer c,d, Saeed Bohlega a,b,⁎ a
Division of Neurology, Department of Neurosciences, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia College of Medicine, Alfaisal University, Riyadh, Saudi Arabia Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia d Saudi Human Genome Program, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia b c
a r t i c l e
i n f o
Article history: Received 22 September 2015 Received in revised form 10 May 2016 Accepted 31 May 2016 Available online 01 June 2016 Keywords: CADASIL NOTCH3 Homozygous Heterozygous Mutation Stroke
a b s t r a c t Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary microangiopathy caused by mutations in NOTCH3, very rarely homoallelic. Objective: To describe the clinical, radiological, and neuropsychological features in an extended CADASIL family including members with either a homozygous or heterozygous NOTCH3 R1231C mutation. Methods: The pedigree included 3 generations of a family with 13 affected individuals. The patients were examined clinically and radiologically. Neuropsychological testing was performed on the proband. Sequencing of the entire coding DNA sequence (CDS) and flanking regions of NOTCH3 was undertaken using PCR amplification and direct Sanger sequencing. Results: Homozygous C3769T mutation, predicting R1231C in exon 22 of NOTCH3 was found in 7 family members. Six other family members harbored the same in the heterozygous state. Homozygous individuals showed a slightly more severe clinical and radiological phenotype of earlier onset compared to their heterozygous counterparts. Conclusion: This study reports the largest number of patients with homozygous NOTCH3 mutation. The phenotype and imaging features of homozygous individuals is within the spectrum of CADASIL, although slightly at the severe end when compared to heterozygotes carrying the same mutation. Both genetic modifiers and environmental factors may play an essential role in modification and alteration of the clinical phenotype and white matter changes among CADASIL patients. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset inherited arteriopathy, characterized by non-hypertensive, non-arteriosclerotic, small arterial granular degeneration. CADASIL manifests as recurrent subcortical ischemic events, progressive or stepwise subcortical dementia, migraine with aura, and mood disorders, with early death [1,2]. Patients usually demonstrate prominent signal abnormalities in subcortical white matter on magnetic resonance imaging (MRI) [3,4]. The mean age of onset of clinical symptoms is the mid-forties; however, MRI abnormalities can be seen a decade before symptom onset [5].
⁎ Corresponding author at: Division of Neurology, Department of Neurosciences, King Faisal Specialist Hospital and Research Centre, P.O. Box: 3354, Riyadh 11211, Saudi Arabia. E-mail addresses: [email protected] (H. Abou Al-Shaar), [email protected] (N. Qadi), [email protected] (M.H. Al-Hamed), [email protected] (B.F. Meyer), [email protected] (S. Bohlega).
http://dx.doi.org/10.1016/j.jns.2016.05.061 0022-510X/© 2016 Elsevier B.V. All rights reserved.
CADASIL symptomatology can be attributed to the systemic vasculopathy encountered in such patients. The smooth muscles in the walls of cerebral arteries are destroyed leading to luminal obliteration and fibrosis, resulting in infarction and stroke [6]. Recurrent infarctions of the white and deep grey matter are the basis of the subsequent dementia and cognitive impairment in these patients. NOTCH3 is composed of 2321 amino acids. It is a member of a protein family involved in signaling events that control cell fate decisions during development. NOTCH3 is a single-pass transmembrane receptor with a large extracellular domain containing 34 tandem epidermal growth factor-like (EGF-like) repeats [7–9]. NOTCH3 is mainly expressed in smooth muscle cells of small arteries and in pericytes around capillaries [10,11]. There are 33 exons in NOTCH3 and mutations in this gene are considered to underlie CADASIL. Almost all mutations reported so far are missense mutations that result in a gain or loss of one cysteine residue within an EGF-like repeat domain, with a strong clustering of mutations in exons 4. CADASIL has been reported in various populations around the world [5]. However, only a few cases in the literature reported homozygous mutations of NOTCH3 [12–16].
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Herein, we report the largest number of homoallelic cases of CADASIL. We describe and compare the phenotypes of homozygous and heterozygous members within this family. 2. Methods 2.1. Patients Thirteen affected individuals from a 3 generation family were enrolled for this study. All were examined in the Department of Neurosciences, King Faisal Specialist Hospital and Research Center (KFSH&RC). The protocols for this study were approved by the institutional review board and informed consent was obtained from all participants. All subjects were enrolled under an IRB-approved protocol (RAC# 2020023) with full informed consent. History was taken and clinical examination performed on the index case and his family members. MRI was obtained for all patients. Neuropsychological analysis was performed for the index case alone. 2.2. NOTCH3 sanger sequencing DNA was isolated from whole blood using a standard salt precipitation method using a Gentra Puregene blood kit (Qiagen). Sequencing of the entire coding and flanking regions of NOTCH3 was undertaken using PCR amplification and direct Sanger sequencing using a BigDye terminator kit (Thermo Fisher, Foster City, CA). SeqScape v.2.6 software (Thermo Fisher, Foster City, CA) was used to align sequence data with NOTCH3 (ENSG00000074181). 3. Results 3.1. Clinical history The family reported in this paper originated from Kashmir in the North Eastern part of the Indian sub-continent and included 13 affected individuals spanning 3 generations as described below (Table 1). Of note, the last generation is a product of triple loop consanguinity as seen in Fig. 1. 3.1.1. IV-53 homozygous patient (as indicated in Fig. 1) The index case is a male practicing physician. He was well until the age of 53 when he had an acute onset right-sided hemiparesis, which left him with some clumsiness. Six months later, he suffered from numbness affecting the left side. Afterwards, he had problems with his vision, which he described as nocturnal blindness worse in the left eye. Ophthalmological examination showed arteriovenous nicking with moderate compression of the retinal veins as well as retinal microinfarction and chorioretinal scaring temporal to the left macula. There were tortuous narrowed retinal arterioles with mild optic disc cupping. Subsequently, multiple strokes occurred over nine years of
follow up, which left him with progressive dysarthria and a spastic gait. He became easily fatigued and markedly depressed. He was mentally slow but was able to continue his work as a Pediatrician in the Emergency Room. His MRI showed diffuse bilateral lacunar infarcts affecting basal ganglia, centrum semiovale, and periventricular white matter (Fig. 2A). Carotid angiography at the age of 55 was normal. The cognitive performance at the age of 59 (six years after the first stroke) showed a Mini-Mental Status Examination score of 24. Neuropsychology testing revealed mild problems with executive function, verbal abstraction, Wechsler Adult Intelligence Scale with visuospatial ability measured by Rey-Osterrieth Complex Figure copying and immediate reproduction and 12-memory test with free recall (12 word Rec) and recognition. Slight bipolar mood disorder was also moderately observed. The disease progressed relentlessly in a stepwise and accelerated pattern. The patient experienced difficulty swallowing associated with a pseudo bulbar affect, progressive dementia, and severe depression. He was admitted several times during this period with repeated chest infection until he became severely disabled for more than a year prior to his death. His father (III-78) died at the age of 78 after 9 years of repeated strokes, dementia, impaired vision, and moderate hearing loss. His symptoms started at the age of 62, however, he was not properly investigated. The index case's mother (III-84) died at the age of 84. She had asymmetrical coarse tremors and bradykinesia. She was diagnosed with Parkinson's disease during her last few years and she responded well to Levodopa therapy. 3.1.2. III-66 heterozygous patient The paternal uncle (Father-in-Law) of the index case was initially seen at the age of 73 with history of 3 repeated strokes, which started at the age of 66. His brain computed tomography (CT) scan was done at the age of 69 and repeated three years later during which he was diagnosed with Binswanger's disease with progression of the Leukoaraiosis. Brain MRI showed diffuse white matter hyperintense ischemic changes. He was moderately demented with hyper-reflexia and pseudo-bulbar affect. 3.1.3. III-76 heterozygous patient Paternal Aunt (Mother-in-Law) was asymptomatic at the age of 76 but brain MRI showed a few ischemic lesions in the basal ganglia and deep white matter (Fig. 2C). 3.1.4. IV-36 homozygous patient Sister of the index case started to experience recurrent episodes of migraine without aura at the age of 36. Repeated episodes of asymmetrical weakness and numbness started at the age of 42 indicating vascular insult (stroke). She also suffered from progressive visual difficulty with ocular transient ischemic attacks with retinal microinfarcts and hemorrhages. At age of 52, she started experiencing difficulty walking followed by dementia, night blindness, and seizure disorder. She
Table 1 Clinical, radiological, and exon 22 C3769T (R1231C) mutation status of the patients in relation to the index case. No.
Age of onset (years)
Sex
Relation to index case (Fig. 1)
Clinical picture
MRI
Genotype CNT
III-76 III-66 IV-35 IV-50 IV-38 IV-53 IV-36 V-7 V-11 V-13 V-22 V-30 V-31
76 66 35 50 38 53 36 7 11 13 22 30 31
F M M M F M F F M F M M F
Paternal Aunt (Mother-in-Law) Paternal Uncle (Father-in-Law) Cousin Cousin Wife Index case Sister Daughter of the second wife Son of the second wife Daughter of the second wife Son Son Daughter
Asymptomatic Stroke and dementia Migraine with aura Asymptomatic Migraine with aura Stroke Migraine without aura, stroke, dementia, seizure, and night blindness Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic Asymptomatic
Abnormal Abnormal Abnormal Not done Abnormal Abnormal Abnormal Not done Not done Not done Abnormal Abnormal Abnormal
c/t c/t t/t c/t t/t t/t t/t c/t c/t c/t t/t t/t t/t
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Fig. 1. Family pedigree showing high degree of consanguinity, number at tab of the symbol indicates individual's age, arrow points to the index case.
required walking aid at the age of 62. MRI showed multiple sub-cortical white matter hyperintense ischemic changes.
3.1.5. IV-38 homozygous patient Cousin (Wife of the index case) had migraine with aura at the age of 38 but no stroke. Her brain MRI showed white matter hyperintense ischemic foci.
3.1.6. IV-50 heterozygous patient Cousin of the index case was asymptomatic at the age of 50. Refused to undergo MRI examination.
3.1.7. IV-35 homozygous patient Cousin of the index case, complained of migraine with aura at the age of 35. Brain MRI revealed white matter hyperintense ischemic foci.
Fig. 2. Axial brain fluid attenuated inversion recovery (FLAIR) MRI sequence examples of the homozygous and heterozygous patients. Index case (IV-53: homozygous) (A) demonstrating diffuse confluent bilateral hyperintensities in the periventricular region associated with microinfarcts (arrows). V-31: homozygous (B) depicting non-confluent bilateral hyperintensities and mild brain atrophy. III-76: heterozygous (C) showing scattered hyperintense patches.
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3.1.8. V-31 homozygous patient Daughter of the index case was asymptomatic at the age of 31. Brain MRI showed few white matter hyperintense ischemic changes (Fig. 2B). 3.1.9. V-22 and V-30 homozygous patients Sons of the index case are asymptomatic at the age of 22 and 30, respectively. Brain MRI depicted white matter hyperintense ischemic foci. 3.1.10. V-7, V-11, and V-13 heterozygous patients Son and daughters of the second wife are clinically asymptotic. MRI was not done for them. 3.2. Molecular genetic analysis Direct sequencing of the 33 exons of NOTCH3 in the index case showed a homozygous C NT transition at nucleotide 3769 (C3769T) predicting an amino acid change from arginine N cysteine at position 1231 (R1231C) as previously reported [17]. Segregation of this mutation was studied in 12 other individuals of this family identifying 6 homozygotes and 6 heterozygotes as shown in Table 1 and Fig. 3. 4. Discussion The extensive consanguinity and segregation of the C3769T (R1231C) in both homoallelic and heteroallelic states within this family is consistent with Mendelian transmission of a single mutation. Based on the consensus of dominance, homozygous mutations should not aggravate the clinical phenotype in autosomal dominant disorders [18–20]. However, this is not always the case, as homozygous mutations in some diseases have been reported to have a more clinically severe phenotype than heterozygous mutations [21]. Initially, homozygous mutations in NOTCH3 had been observed to result in a more severe clinical phenotype than heterozygous mutations [12,16]. This observation was questioned later when some homozygous mutations resulted in a mild or only slightly more severe clinical phenotype than heterozygous mutations [13–15]. These contradictory results led to the premise that the phenotype of CADASIL patients is influenced and modified by both genetic modifiers and environmental factors, not yet discovered [14,22], that alleviate or aggravate the clinical phenotype. Interestingly, in one study the authors observed a striking variability in measuring the volume of cerebral ischemic lesions in 151 CADASIL individuals suggesting a strong modifying influence of some genetic factors distinct from the causative
Fig. 3. Sequencing result of the homozygous and heterozygous mutations in different patients of this family. The homozygous patients have mutated “t” in both alleles and heterozygous patients have both “c” and “t” at nucleotide 3769 at exon 22 of the NOTCH3 gene, c.3691CNT (p. Arg1231Cys).
NOTCH3 mutation on the amount of ischemic brain lesions [22]. Those genetic modifiers could exert their effects on different pathways affected among CADASIL patients including smooth muscle cells, vascular endothelial cells, and others. They also can act trough different mechanisms interfering with autoregulation and neuronal response and repair to ischemia [23,24]. Therefore, CADASIL was considered an example of diseases where zygosity does not play a role in influencing the severity of the disease. In our cohort, the index case (homozygous mutation) experienced his first ischemic episode at the age of 53. The patient's father, paternal uncle, and paternal aunt suffered from neurological disorders, consistent with CADASIL that manifested around 70 years of age. The index case had mild cognitive and memory problems and retinal defects, as evident from his neuropsychological and ophthalmological examinations, respectively. The index case parents are most likely heterozygous, based on the increased homozygosity in the offspring, however, homozygosity cannot be entirely rule out. Moreover, five of the seven homozygous patients (aged between 22–38 years) demonstrated no symptoms or exhibited migraine with aura, while the remaining two suffered from stroke and cognitive decline with an age of onset of 42 and 53 years, similar to the previous notion of stroke onset among CADASIL patients (range from 20–70 years of age) [5]. On the other hand, the six heterozygous individuals included three children under 13 years of age as well as two subjects (50 and 76 years old), all of whom were asymptomatic. The last heterozygous patient had dementia and exhibited his first stroke at 66 years of age. It is known that there is marked unexplained phenotypic variation both intra-familially and between the families with the same mutation as well as between families with different mutations among CADASIL patients [5]. Our homozygous patients' disease fits well within the clinical and radiological phenotype of other reported CADASIL patients. Our homozygous patients did not exhibit an extraordinarily more severe phenotype compared to the heterozygous patients. Chabriat et al. and Opherk et al. noted variable white matter hyperintensity variation among 19 and 151 patients with CADASIL despite their clinical course. They concluded that a strong contribution of unknown genetic modifiers to interindividual differences might exist, which could explain the variability in the volume of ischemic brain lesions and clinical severity among these patients [22,25]. Therefore, one of the shortcomings of our report that we did not do a quantitative assessment, which correlate with the disease progression (e.g. brain atrophy) nor we did a quantitative measurement of the volume of the lacunar infarcts, which also correlate with the disease severity. Another limitation of this study is the absence of objective assessment of cognitive decline and disability, which are better clinical markers of disease severity, among all members in the family, due to their immigration back to their country. Therefore, due to these shortcomings and due to the well-known intra- and interfamilial clinical variability in CADASIL, we cannot generalize our observation to all CADASIL patients with homozygous mutations. Further studies to delineate the effects of zygosity on the severity of white matter changes and clinical course using objective measures are therefore needed. In dominantly inherited disorders, the mutation may cause loss- or gain-of-function phenomena. This results in either a single functional allele that cannot maintain the required function (haploinsufficiency) or an accentuated function that has a pathological effect. Interestingly, homozygous mutations in NOTCH3 have been proposed to result in a gain-of-function mechanism that results in more accumulation of the harmful proteins and the resultant smooth muscle damage [12,14]. This is consistent with observation of more diffuse granular osmiophilic material (GOM) deposition around smooth muscle cells in patients with homozygous mutations [12,15]. It has been postulated that if a mutation were to cause haploinsufficiency or display a dominant-negative effect, then it would cause a more severe or lethal phenotype [13,14]. It has been thought that in loss-of-function mutations, the fact that the double dose of gene defect does not appear to aggravate the symptoms
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compared to gain-of-function mutations, may indicate that either the mutated NOTCH3 has some retained function or other (NOTCH?) molecules and genetic modifiers can compensate for the loss [12]. Only scarce reports of homozygous mutations in NOTCH3 are present in the literature, with single cases of homozygosity in the majority of the studies [12,13,15,16]. To the best of our knowledge, only 6 homozygous patients and one cell-line from a homozygous patient have been reported in the literature [12–16,26]. Our study is the first report of a large number of patients harboring a homozygous mutation in NOTCH3. Finally, longitudinal studies on the progression of the disease are lacking. Thus, it is unknown whether early onset homozygous CADASIL patients are associated with more rapid or slower progression. More studies on homozygous NOTCH3 mutations are therefore needed to further answer this question. 5. Conclusion We report the largest number of patients with homozygous NOTCH3 mutation in a single family. The phenotype and imaging features of our homozygous individuals is within the spectrum of CADASIL, albeit slightly at the severe end when compared to heterozygous patients. It is likely that both genetic modifiers and environmental factors may play an essential role in the modification and alteration of the clinical phenotype and white matter changes among CADASIL patients. Author contributions Conception and design: Abou Al-Shaar, Qadi, Bohlega. Acquisition of data: Abou Al-Shaar, Qadi, Bohlega, Meyer. Analysis and interpretation: Abou Al-Shaar, Bohlega, Meyer. Management of the patients: Bohlega, Qadi. Genetic testing: Meyer, Al-Hamed. Drafting the article: Abou AlShaar. Critically revising the article: all authors. Final approval of the submitted version: all authors. Disclosure The authors declare no conflicts of interest regarding the production of this article. The authors have no personal financial or institutional interest in any of the drugs, materials, or devices described in this article. References [1] H. Chabriat, K. Vahedi, M.T. Iba-Zizen, et al., Clinical Spectrum of CADASIL: a study of 7 families. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Lancet 346 (1995) 934–939. [2] M. Baudrimont, F. Dubas, A. Joutel, E. Tournier-Lasserve, M.G. Bousser, Autosomal dominant syndrome leukoencephalopathy and subcortical ischemic stroke. A clinicopathological study, Stroke 24 (1993) 122–125. [3] H.S. Markus, R.J. Martin, M.A. Simpson, et al., Diagnostic strategies in CADASIL, Neurology 59 (2002) 1134–1138.
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