Journal of the Neurological Sciences 246 (2006) 123 – 130 www.elsevier.com/locate/jns The R110C mutation in Notch3 causes variable clinical features in two Turkish families with CADASIL syndrome Z.O. Uyguner a,c,*,1, A. Siva b,*,1, H. Kayserili c, S. Saip b, A. AltNntaş b, M.Y. Apak c, S. Albayram d, N. IşNk e, G. Akman-Demir f, M. Taşyürekli g, B. Öz h, B. Wollnik a,c,i a Child Health Institute, Division of Medical Genetics, Istanbul University, Turkey b Cerrahpaşa Medical School, Department of Neurology, Turkey c Istanbul Medical School, Department of Medical Genetics, Turkey d Cerrahpaşa Medical School, Department of Neuroradiology, Turkey e Social Security Hospital, Department of Neurology Göztepe – Istanbul, Turkey f Istanbul Medical Faculty, Department of Neurology, Turkey g Cerrahpaşa Medical School, Department of Histology, Turkey h Cerrahpaşa Medical Faculty, Department of Pathology, Turkey i Center for Molecular Medicine (CMMC), University of Cologne, Germany Received 21 October 2005; received in revised form 9 February 2006; accepted 24 February 2006 Abstract Mutations in Notch3 gene are responsible for the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). It is a late onset neurological disorder recognized by recurrent strokes and dementia. We describe here the clinical and molecular findings of three unrelated Turkish families with CADASIL syndrome. Two of the families were identified to have the same mutation, p.R110C (c.C328T), located in exon 3 of the Notch3 gene. Interestingly, the phenotypic expression of the disease in these two families was markedly different in severity and age of onset implicating additional genetic and/or non-genetic modulating factors involved in the pathogenesis. In addition, we identified the novel p.C201R (c.T601C) mutation in exon 4 of the Notch3 gene in a proband of the third family with two consecutive stroke-like episodes and typical MRI findings. Mutations described here cause an odd number of cysteines in the N-terminal of the EGF domain of Notch3 protein, which seems to have an important functional effect in the pathophysiology of CADASIL. The phenotypic variability in families carrying the same molecular defect as presented here makes the prediction of prognosis inconceivable. Although DNA analysis is effective and valuable in diagnosing approximately 90% of the CADASIL patients, lack of genotype – phenotype correlation and prognostic parameters makes the presymptomatic genetic counseling very difficult. D 2006 Elsevier B.V. All rights reserved. Keywords: CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy); Notch3; Mutation; Migraine * Corresponding authors. Uyguner is to be contacted at Istanbul University, Child Health Center, Department of Medical Genetics, Milllet Cad. Çapa, Istanbul – Turkey. Tel./fax: +90 212 631 1363. Siva, Istanbul University, Cerrahpaşa Medical Faculty, Department of Neurology, Cerrahpaşa, Istanbul – Turkey. Tel.: +90 212 529 0886; fax: +90 212 249 2106. E-mail addresses: o.uyguner@istanbul.edu.tr (Z.O. Uyguner), asiva@tnn.net (A. Siva). 1 Co-senior authors who contributed equally to this research. 0022-510X/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2006.02.021 1. Introduction CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an adult onset inherited neurological disorder characterized by recurrent strokes and progressive dementia, with or without migrainelike headaches, seizures, and pseudobulbar palsy. The absence 124 Z.O. Uyguner et al. / Journal of the Neurological Sciences 246 (2006) 123 – 130 of hypertension or any other known vascular risk factors is essential for the clinical diagnosis [1–3]. MRI of the brain is always abnormal in symptomatic patients and subtle changes may be detected in affected individuals prior to the onset of any symptoms [4]. Clinically affected and asymptomatic patients with minimal MRI abnormalities had characteristic granular electron-dense deposits in or near the external side of the thickened basal lamina of dermal and muscle vessels. Thus electron microscopy of the skin biopsy specimen is one of the methods used for the diagnosis of CADASIL [5]. Positional cloning strategies identified Notch3 as the responsible gene for the disorder [6]. The name Notch derives from a concavity occurring in the wing of Drosophilia melanogaster carrying only one functioning copy of the gene. It plays a prominent role in development by contributing to the determination of cell fate in many different tissues [7]. Heterozygous mutations in Notch3 have been presented in more than 90% of CADASIL patients, and two-thirds of these mutations were identified in exons 3 and 4 followed by of lesser intensity in exons 2, 5 and 11 [8–10]. This clustering is important to expedite the genetic diagnosis by testing these exons before analyzing the remaining 31 exons of the gene. CADASIL shows a true dominancy and most of the mutations are missense mutations leading to an odd number of cysteine residues within 1–34 EGF-like repeats in the Notch3 protein. In addition, the mutational spectrum also includes splice site mutations and small in-frame deletions [11–13]. CADASIL’s true dominancy, which means that homozygosity has no significant effect on the severity, was further supported by a single report on a patient with homozygous mutation presenting clinically indistinguishable features from heterozygous patients [14]. However, inter- and intra-variability of the onset and the severity of the disease in families are very intriguing [15]. Reports of de novo missense mutations identified in CADASIL patients without any family history emphasize that clinical diagnostic criteria for CADASIL are essential in the absence of a familial inheritance and therefore should be carefully re-assessed [10,16]. Therefore, the understanding of the molecular pathology behind the causative mutations is very important to elucidate the phenotypic variability of the disease. It has been suggested that aberrant interactions of Notch3 mutants may multimerize and eventually cause ineffective signaling and/or accumulate at extracellular space due to impaired clearance eventually causing a toxic effect at expressed tissues [17]. Further support to this finding came from the bioinformatic analysis of the Notch paralogs and orthologs, which showed that CADASIL mutations might result in a gain-of-function [18]. Gain-of-function can be generated in several ways: overexpressing of a normal gene product, expressing an overreactive mutant, or by ectopic expression where a gene is expressed outside the normal spatial or temporal domains of the corresponding endogenous gene. The results of a molecular study, which showed an identical mRNA level of Notch3 in CADASIL patients over control individuals, may point that formation of overreactive Notch3 in CADASIL is more likely [17]. The abnormal accumulation of protein is also seen in other progressive late onset neurological diseases like Parkinson, Alzheimer and Huntington [17,19]. We describe here the clinical and molecular findings of three Turkish families with CADASIL syndrome and discuss the clinical features of R110C mutation. 2. Subjects and methods 2.1. Family evaluations Three families, with at least three affected individuals in each, were clinically diagnosed as having the CADASIL Syndrome by typical MRI findings accompanied by migraine Table 1 Clinical findings of three Turkish families with cerebral autosomal dominant arteriopathy with subcortical infarcts Cases Family 1 I-2 II-2 II-8 Family 2 I-2 II-1 II-4 III-3 III-5 Family 3 I-2 II-2 II-3 III-1 III-2 III-7 Onset of symptoms Ischemic episodes Cognitive deficits Migraine Mood disorders MRI Age at last examination / (age at death) 57 49 49 + + + + ? + + + + + NE/(64) 63/still living 50/still living ? 30 30 32 28 + + + + + + + + + + ? ? ? + + + ? + + + + + NE/(unknown) NE/(37) NE/(39) 36/still living 30/still living 46 62 48 54 55 50 + + + + + + ? + + + ? + ? ? + + + + + + + + NE/(48) NE/(64) NE/(62) NE/(65) 66/still living 60/still living (+): present; ( ): absent; (NE): Not examined. + + + + Z.O. Uyguner et al. / Journal of the Neurological Sciences 246 (2006) 123 – 130 125 A. Family 1 cys I 1 2 T G * II III 3 1 4 5 6 7 C C 9 10 8 2 3 4 II-2 and II-8 B. Family 2 I II C C arg110cys C G T T * 2 1 pro 1 1 2 2 cys pro arg110cys T G C C C C 3 4 5 * C G T T * III 1 2 3 5 4 6 7 8 IV 1 2 3 III-3 and III-5 C. Family 3 val I II 1 1 2 G T G 3 2 4 5 6 9 10 pro C C C cys201arg T G T C 7 * III 1 2 3 2 3 4 4 5 6 7 8 IV 1 5 6 7 8 III-7 Fig. 1. Pedigrees and their molecular findings of the three Turkish CADASIL families. Arrow shows the index patients of each family; an asterisk indicates the patients, which were clinically evaluated in our clinic. Molecular analysis was performed on individuals marked by a black bar. attacks and/or stroke like episodes (Table 1). These individuals were studied extensively and followed over a number of years. Medical and neurological histories were obtained for up to 3 – 4 generations and detailed clinical history was obtained by interviewing one of the other healthy members of the family. Genetic analyses were performed on family members (Fig. 1A,B,C). 2.2. Genetic analysis DNA from peripheral blood leukocytes of the tested individuals was isolated by standard methods. Exons 3 and 4 of Notch3 gene were screened by a previously established strategy that is based on one-step amplification and sequencing on both strands (Macrogen, Seoul, Korea) [20]. As a control group, 44 DNA samples from healthy Turkish controls were used and screened by direct sequence analysis. 3. Results All the affected probands had normal electrocardiography and echocardiography. Their biochemical tests and blood serology were also normal. 3.1. Case report and molecular analysis of family 1 The proband (Fig. 1A, II-2) had a past history of migraine with aura and had symptoms that consisted of visual phenomena and cherio-oral paresthesias. She was hospitalized in 1989, with a severe migraine attack and a transient brainstem syndrome at age of 48. A cranial computerized tomography done at that time was reported to be insignificant. However, an MRI done at follow-up showed bilateral white matter lesions (Fig. 2A), that were interpreted as probably related to migraine after an extensive clinical and laboratory work-up ruled out any other explanation [21]. According to the family history, her father died from stroke but no neurological examination was done. At age 61, she was readmitted to the hospital with her sister (Fig. 1A, II-8) who was 48 and had symptoms consistent with migraine with aura, generalized anxiety and essential tremor. The follow-up MRI of the proband did not show a significant change (Fig. 2B), and her sisters’ MRI was found to display abnormalities almost identical to hers (Fig. 3). The appearance and distribution of the lesions were interpreted as suggestive of the CADASIL syndrome (Figs. 2 and 3). The proband reported to have only mild migraine attacks over the last years, had a complete cessation of her auras and did not experience any other neurological events until mid-2002, 126 Z.O. Uyguner et al. / Journal of the Neurological Sciences 246 (2006) 123 – 130 Fig. 2. (A) Proband of family I (II-2), T2W MR images showing characteristic hyperintense lesions in anterior temporal lobes and frontal lobes, lacunar like infarcts within the corona radiata, and widespread subcortical lacunar lesions, consistent with the diagnosis of CADASIL. Study was done on December 18, 1990. (B) Proband of family I (II-2), MR images showing bilateral subcortical lesions consistent with the diagnosis of CADASIL. Study was done on January 28, 2004. Changes are minimal when compared with the 1990 scans. when she started to have some difficulty with her gait and balance. A neurological examination at that time disclosed bilateral corticospinal tract signs prominent in the lower extremities. On her last follow-up in June 2005, she showed only mild progression of her spastic gait. Her cognition was normal. The neurological examination was and remained normal with the exception of tremor in the sibling. EMG did not yield any abnormality of the neuro-muscular junction in either one. Mutation analysis of the Notch3 gene of the siblings revealed heterozygous p.R110C (c.C328T) mutation in exon 3 (Fig. 1A). The results of the skin biopsy were consistent with CADASIL. This mutation was described earlier in patients with CADASIL [8]. A haplotype analysis was done using markers D19S841, D19S923, and D19S432 located 5V, intragenic and 3Vof the Notch3 gene, respectively in order to compare it with the haplotype of family 2, carrying the same mutation. ProbandVs mother was from The Marmara Region and father was from The Northern Black Sea Region. Proband’s allele showed homozygosity for all markers. 3.2. Case report and molecular analysis of family 2 Medical history of the proband (Fig. 1B, III-5) showed that his first symptoms started at age of 28 with double Fig. 3. Younger sister of proband of family I (II-8). MR images showing hyperintense subcortical lesions consistent with CADASIL, more prominent in both anterior temporal and frontal lobes, and in capsula extrema with lacunar like infarcts. Study done on June 7, 2002. MR images are almost identical with her affected sister (Fig. 2A and B). Z.O. Uyguner et al. / Journal of the Neurological Sciences 246 (2006) 123 – 130 127 Fig. 4. (A) Proband of family III (III-7). T2W MR images showing characteristic hyperintense lesions in the right anterior temporal lobe and frontal lobes, lacunar like infarcts within the corona radiata, and widespread subcortical lacunar lesions. Study done on September 17, 1994. (B) Proband of family II (III-7). T2W MR images show an increase in the number and volume of hyperintense ‘‘CADASIL’’ lesions observed in earlier scans. Study done on June 26, 2003. vision, emotional lability and speech difficulty. These symptoms were followed by weakness on the left side. His laboratory work-up did not reveal any abnormality but MRI disclosed bilateral periventricular and subcortical white matter lesions, which were interpreted as suggestive of a vasculitic disorder or multiple sclerosis (MS) in another hospital. He was given pulse i.v. methyprednisolone, without any improvement. He experienced two generalized seizures in the following two years and then was referred to our center. His neurological symptoms and signs at that time were diplopia, dysarthria, left-sided mild weakness of the upper motor neuron type and emotional lability. Interviews with the family members revealed that his brother (Fig. 1B, III-3) was also hospitalized at the age of 32 with a probable diagnosis of MS with intermittent numbness. Re-evaluation of the MRIs of both brothers and their positive family history were in agreement with CADASIL syndrome. The clinical histories of the 3 deceased individuals are shown in Table 1. Mutation analysis of Notch3 gene in the two brothers showed that both carried the heterozygous p.R110C (c.C328T) mutation in exon 3 (Fig. 1B). Again, haplotype analysis of markers D19S841, D19S923 and D19S432 was performed (data not shown). Proband’s parents were both from the same city from the Northern Black Sea Region, but from different villages. Haplotype analysis showed heterozygosity for this region, sharing same haplotype with the proband of family one. 3.3. Case report and molecular analysis of family 3 Medical history of the proband of family 3 (Fig. 1C, III-7) revealed two stroke-like episodes, one at age of 49, and 51, respectively. The second episode was right-sided hemiparesis in 1994, when he was referred with an initial diagnosis of MS, but the pattern of neurological involvement, his MRI findings (Fig. 4A) and positive family history of neurological disease were all suggestive of a hereditary vascular syndrome. His sister and a maternal cousin also had neurological disorders with stroke-like episodes at that time. The family history revealed that his maternal grandmother, mother and maternal aunt all had died after undiagnosed longstanding neurological disorders which consisted of recurrent strokes and dementia, at ages 48, 62 and 60, respectively (Fig. 1C, I-1, II-3, III-1). A skin biopsy done in 1996 was consistent with CADASIL. Some behavioral changes were observed in early 2002, when he developed a syndrome that consisted of psychomotor retardation, which was followed by progressive dementia. In June 2003, he was admitted to the emergency room with chest pain, but a workup for ischemic heart disease did not reveal any coronary pathology. His final cranial MRI done in June 2003 showed a progression of the white matter changes and acute ischemic lesions (Fig. 4B). Currently, he is severely demented. Mutation analysis of the Notch3 receptor gene identified the novel heterozygous p.C201R (c.T601C) mutation in 128 Z.O. Uyguner et al. / Journal of the Neurological Sciences 246 (2006) 123 – 130 exon 4 in proband. This novel mutation was not found in 88 normal control chromosomes. 4. Discussion We report here the clinical and molecular findings of 3 Turkish CADASIL families, with one family carrying a novel mutation, and two others displaying distinct clinical features despite showing the same known mutation in Notch3. Systematic arterial microvascular changes were found, as shown by the skin biopsy results in two of the probands, but the documented disease expression was mostly confined to the central nervous system, with a wide variety of neurological manifestations and different mutations. Two families carried the R110C mutation and one had the novel C201R mutation in Notch3. These mutations cause an odd number of cysteines in the Nterminal EGF-domain of the Notch3 protein that represents the main pathophysiological mechanisms of the disease, for which the resulting effect is not yet well understood. Together with another Turkish case carrying the R90C mutation published recently [20], a total of three different mutations in four CADASIL families are now reported from Turkey. Although the parents of probands of family 1 and 2 originated from different regions in Turkey but they are carrying the same mutation (R110C) and sharing one putative disease associated haplotype may show that this mutation is likely to be a founder mutation. These two families showed a dramatic interfamilial phenotypic variation. The difference in the onset and the progression of the disease was especially striking. In family 1, it is interesting in having only two symptomatic cases in the same generation with migraine-like headaches with similar auras as the major manifestation of the disease for decades. The MRIs of the affected individuals were identical with only mild changes. Furthermore, proband developed a late and gradual onset of isolated progressive mild spastic paraparesis after age 60, an unusual manifestation for CADASIL. Her MRI studies did not show significant changes after 12 years, and this is noteworthy as an increase in the number and type of MRI lesions with advanced age is described and would be expected [22]. In family 2, the disease was manifested in more than one generation. The neurological signs consisted of stroke-like episodes, pseudobulbar palsy, seizures and paresthesias, and an onset at a much younger age. Similarly, phenotypic variation was shown both within families and between families of Finnish origin for R133C mutation and the lack of genotype –phenotype relationships in CADASIL was acknowledged by previous studies [15,23 –25]. Phenotypic heterogeneity of CADASIL might be modulated by additional genetic factors such as functional polymorphisms in genes encoding proteins involved in the Notch3 pathway of development or acquired by environmental risk factors not yet established. Analysis for modifier genes in our two families could provide further insights into the genetic basis of phenotypic variations in CADASIL patients. In two of the three studied families, the electron microscopy of the skin arteriole revealed numerous aggregates of granular osmophilic material (GOM) adjacent to smooth muscle cells. Electron microscopy of skin biopsy specimen is one of the powerful tools for the diagnosis of CADASIL. However, this is not found adequate in all cases and possible false-negative results should be kept in mind when peripheral tissue biopsy is used alone [26,27]. It has been reported that immunostaining of skin biopsies with a Notch3 specific monoclonal antibody is an accurate technique with 96% sensitivity and 100% specificity for the diagnosis of CADASIL, depending upon the availability of the antibody [28]. Although CADASIL can be diagnosed by a combination of clinical and non-molecular methods, such as the skin biopsy and a certain extent with MRI, the molecular diagnosis represents the final proof of diagnosis. Furthermore, mutation detection in CADASIL families could subsequently offer presymptomatic testing in family members at risk. However, the presymptomatic testing for a non-treatable and lethal disorder such as CADASIL requires an accurate genetic counseling and professional psychological support prior to the decision. The most common neurological manifestations of CADASIL reported include stroke-like episodes before age 60 years, cognitive and behavioral disturbances migraine with aura, dementia and pseudobulbar palsy, and to lesser extent, seizures and acute encephalopathies [29,30]. However, recently other neurological symptoms such as acute unilateral visual loss, sudden sensorineural hearing loss, and peripheral neuropathy have been reported to be part of the CADASIL phenotype [31 – 33]. Our experience in Turkey is further evidence that CADASIL is prevalent all over the world and that the phenotypic spectrum is large with a variable expression even among carriers of the same mutation. The expanding spectrum of CADASIL, as well as the increasing number of reported cases from different geographical regions and ethnic backgrounds, suggests that the disorder may be more common than previously recognized. In a recent study the prevalence of CADASIL was found to be 1.98 per 100.000 adults in the west of Scotland, with a probable mutation prevalence of 4.14 per 105 [34]. It is quite likely that these figures are at the lower rate of the syndrome with the increasing awareness of the expanding clinical spectrum of CADASIL and more widely used molecular testing an increase in the number of cases will be diagnosed. Besides, it has been reported that a Notch3 mutation underlying CADASIL can arise de novo [35]. These findings are also suggestive that CADASIL may be much more prevalent than previously appreciated and is likely to be responsible for a proportion of a large number of strokes, migraine headaches, and psychiatric complaints and as well as some other neurological manifestations for which no Z.O. Uyguner et al. / Journal of the Neurological Sciences 246 (2006) 123 – 130 definitive etiology is identified. 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