Acta Neurol Scand 2015: 132: 430–434 DOI: 10.1111/ane.12400
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd ACTA NEUROLOGICA SCANDINAVICA
Clinical Commentary
APOE and AGT in the Finnish p.Arg133Cys CADASIL population Siitonen M, Mykk€ anen K, Pescini F, Rovio S, K€a€ari€ainen H, Baumann M, P€ oyh€ onen M, Viitanen M. APOE and AGT in the Finnish p.Arg133Cys CADASIL population. Acta Neurol Scand 2015: 132: 430–434. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Background – CADASIL is an inherited systemic small vessel disease, the affected status of brain vessels leading to subcortical vascular dementia. The defective gene is NOTCH3 in which over 230 different pathogenic mutations have been identified. The clinical course of CADASIL is highly variable even within families. Previous studies have shown that additional genetic factors modify the phenotype. Aims and methods – Altogether, 134 Finnish CADASIL patients with p.Arg133Cys mutation were analysed for possible associations between the apolipoprotein E (APOE) genotype, angiotensinogen (AGT) p.Met268Thr polymorphism or neutral p.Ala202Ala NOTCH3 polymorphism and earlier first-ever stroke or migraine. Results – We found no association between the APOE genotypes, AGT polymorphism, NOTCH3 polymorphism and earlier first-ever stroke or migraine. Conclusions – The APOE, AGT and NOTCH3 polymorphism did not modify the onset of strokes or migraine in our CADASIL sample, which is one of the largest mutationally homogenous CADASIL populations published to date. International collaboration, pooled analyses and genomewide approaches are warranted to identify the genetic factors that modify the highly variable CADASIL phenotype.
M. Siitonen1,2, K. Mykk€anen1, F. Pescini3, S. Rovio4, H. K€a€ari€ainen5, M. Baumann6, M. P€oyh€onen7,*, M. Viitanen8,9,* 1 Department of Medical Biochemistry and Genetics, Institute of Biomedicine, University of Turku, Turku, Finland; 2Department of Medical Genetics, University of Helsinki, Helsinki, Finland; 3Stroke Unit and Neurology, Azienda Ospedaliero Universitaria Careggi, Florence, Italy; 4Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku,Turku, Finland; 5National Institute for Health and Welfare, Helsinki, Finland; 6Protein Chemistry Unit, Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland; 7Department of Clinical Genetics, HUSLAB Helsinki University Central Hospital and Department of Medical Genetics, University of Helsinki, Helsinki, Finland; 8Department of Geriatrics, Turku City Hospital and University of Turku, Turku, Finland; 9Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Key words: genotype–phenotype correlation; founder effect; association study; CADASIL; NOTCH3; APOE; AGT
M. Siitonen, Institute of Biomedicine, Department of Medical Biochemistry and Genetics, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland Tel.: +358 2333 7456 Fax: +358 2230 1280 e-mail: [email protected] *Shared senior authorship. Accepted for publication March 4, 2015
Introduction
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited systemic small vessel disease with characteristic, although variable, clinical features [e.g. transient ischaemic attacks (TIA), strokes, migrainous headache, progressive cognitive decline leading finally to dementia of subcortical vascular type and psychiatric symptoms]. In magnetic resonance imaging (MRI), typical white matter alterations are common (1). The vascular pathology in the central nervous 430
system is characterized by – CADASIL-specific – electron-dense deposits of granular osmiophilic material (GOM) in close vicinity of vascular smooth muscle cells (VSMCs) with subsequent degeneration of the VSMCs and marked fibrosis and stenosis most prominent in the cerebral white matter (WM) arterioles, which together give rise to extensive leukoaraiosis and multiple lacunar infarcts (1). CADASIL is diagnosed by demonstration of GOM in a skin biopsy (2) or by genetic verification of a pathogenic mutation in the NOTCH3 gene, located on 19p13.1 (1). A vast majority of over 230 mutations responsible
APOE and AGT in the Finnish p.Arg133Cys CADASIL population for CADASIL are missense point mutations causing an uneven number of cysteine residues in one of the epidermal growth factor-like repeats in the extracellular domain of the NOTCH3 receptor molecule (2–4). The age of onset and progression of the disease are highly variable even within the same family. Some genotype–phenotype correlations have been reported (5, 6), and an association between two different mutations and shorter survival was detected in two German families, (7) but many studies have failed to show any association (8, 9). To date, at least 30 NOTCH3 polymorphisms, 19 neutral and 11 amino acid changing, have been found (4), but only a common p.Arg1560Pro NOTCH3 variant has been reported to be protective against ischaemic stroke (10), and an association between the neutral p.Ala202Ala (rs1043994) polymorphism and migraine has been reported in the general population (11). However, heritability estimates in CADASIL suggest a strong modifying influence of genetic factors distinct from the causative NOTCH3 mutation on the number of ischaemic brain lesions (12). It has also been shown that the polygenic risk score is associated with white matter hyperintensity (WMH) volume, which suggests that multiple common variants with small effect sizes modify the WMH burden in CADASIL (13). As the main pathogenic feature of CADASIL is ischaemic lesions, the genes associated with cardiovascular diseases and stroke such as apolipoprotein E (APOE) and angiotensinogen (AGT) are obvious candidates to be involved in the variable outcome of the clinical course of CADASIL. APOE e4 carriers are shown to be at increased risk for atherosclerosis and stroke (14, 15), and AGT p.Met268Thr (rs699, previously published as p.Met235Thr) significantly contributes to stroke risk at least in East Asian populations (16, 17). It has also been shown that polymorphisms in the NOTCH3 gene itself could alter the phenotype of the disease (10, 11). Altogether, over 200 patients with CADASIL have been molecular genetically diagnosed in Finland, and the majority carry the same p.Arg133Cys mutation due to the founder effect (18). Despite the similar mutational background, the patients’ clinical pictures are highly variable. Therefore, using this unique material of 134 CADASIL patients with the same p.Arg133Cys mutation, we investigated whether APOE, AGT, seven amino acid changing and one neutral p.Ala202Ala NOTCH3 polymorphisms have an effect on the earlier occurrence of firstever stroke or migraine.
Patients and methods Clinical data
Altogether, 134 patients from 24 Finnish families with the same p.Arg133Cys mutation were included in the study. The clinical data were collected retrospectively using medical records and structured questionnaires. The incidence of, and age at incidence of, first-ever TIA and stroke and the occurrence of migrainous headache, myocardial infarction and vascular risk factors such as hypertension (>140/90 mmHg or medication), diabetes mellitus (established diagnosis) and high total serum cholesterol (>6.2 mmol/l or statin therapy) were acquired. Body mass index (BMI) was calculated and >25.0 kg/m2 was considered overweight. Patients were divided into smokers or non-smokers, and into heavy drinkers (men >280 g of alcohol/week, women >190 g/week), moderate drinkers (men A, NM_000435.2). The polymorphisms were analysed by DHLPC analysis with a WaveTM apparatus (20) for 20 patients and by sequencing for 30 patients. Neutral p.Ala202Ala NOTCH3 polymorphism was analysed from all 134 patients as described by Schwaag et al. (11). Statistical analysis
Cox proportional hazards regression was used to analyse the associations between APOE genotypes or AGT p.Met268Thr polymorphism and the first-ever stroke or TIA. The analyses were 431
Siitonen et al. conducted both unadjusted and after adjusting for age, sex, hypertension, hypercholesterolaemia, myocardial infarction, smoking and alcohol drinking. The association between the neutral NOTCH3 p.Ala202Ala polymorphism and migraine was analysed using logistic regression with sibling as a random effect. All statistical analyses were conducted with SPSS version 19.0 (IBM Corp. Armonk, NY, USA) or SAS version 9.1 (SAS Institute Inc., Cary, NC, USA.) Results
There were 68 females and 66 males in our cohort, and their mean age was 57 (range 20– 94 years), while their mean age at first-ever stroke or TIA was 45. The demographic and clinical features of our CADASIL population are presented in Table 1, and detailed genotypes are given in Table S1. The APOE allele frequencies were e2 5.4%, e3 77.5% and e4 17.1%, which is congruent with the normal population in Finland. We could not show any association between the APOE genotypes and first-ever stroke (Table 2), and the results did not change after adjusting for the traditional vascular risk factors listed in Table 1. The frequencies of the AGT p.Met268Thr genotypes were as follows: MM 23.2%, MT 56.6% and TT 20.2%. No association was found between the AGT genotypes and first-ever stroke (TT genotype and stroke, HR 0.76 and 95% CI 0.40–1.46). All of the 50 patients analysed for the seven amino acid changing NOTCH3 polymorphisms were homozygous for the common allele. Hence, the sequencing was not widened to the rest of the patient population for it was obvious that the frequency of these polymorphisms was too low Table 1 Demographics and clinical features of the Finnish CADASIL cohort Patients with the feature/with information Male sex Stroke TIA Migrainous headache Hypertension Hypercholesterolaemia Myocardial infarction Diabetes mellitus Overweight (BMI>25) Current smokers Alcohol drinking Non-drinkers Moderate drinkers Heavy drinkers
432
66/134 70/116 53/102 58/104 24/120 47/115 9/117 10/119 51/91 30/102
(49.3%) (60.3%) (52.0%) (55.8%) (20.0%) (40.9%) (7.7%) (8.4%) (56.4%) (29.4%)
31/87 (35.6%) 56/87 (64.4%) 6/87 (6.9%)
Table 2 Association between APOE e4 allele and earlier first-ever stroke, TIA or stroke/TIA
APOE e4 vs stroke APOE e4 vs stroke/TIA APOE e4 vs TIA
HR
95% CI
n
1.50 1.07 0.89
0.75–2.99 0.50–2.28 0.49–1.61
111 111 94
Values are hazard ratios (HR) with 95% confidence intervals (95% CI) for APOE e4 carriers from Cox proportional hazards regression analyses.
for any statistical analysis. Interestingly, there was no association between the neutral p.Ala202Ala NOTCH3 polymorphism and migraine in our CADASIL population (AA vs GG, OR 0.71 95% CI 0.01–40.59; AG vs GG, OR 1.15 95% CI 0.27–4.86). Discussion
The great advantage of our study is the large number (134) of patients with the same p.Arg133Cys mutation. This homogeneity makes our population exceptional among other large CADASIL populations (3, 4). In addition to the same mutation, our Finnish patients can also be expected to have other similarities in their genetic background due to the founder effect (18) and the relatively small size of the Finnish population (approximately 5.4 million). When compared to other published founder populations (21, 22), our patient cohort is the largest and most profoundly characterized. Together, these facts make our population highly suitable for studying associations between genetic risk factors and their effects among patients with CADASIL. Furthermore, the frequencies of stroke, migrainous headache and vascular risk factors in our population are similar to those found in a large prospective series of patients with CADASIL (8), which increases the credibility of our study. In our CADASIL population, we could not show any association between the analysed genetic factors and first-ever stroke. This is in line with two previous CADASIL studies in which APOE genotype had no modifying effect on the clinical course (8, 23). When TIA together with stroke as the end point were considered, the results of the statistical analyses confirmed this still less. This could be due to the difficulty in distinguishing TIA from other neurological deficits. Hence, we recommend that this difficulty should be considered when using TIA as an outcome in the analyses. Only few studies have considered NOTCH3 polymorphisms in the context of cerebrovascular diseases or migraine. One study has reported an association between a NOTCH3 polymorphism
APOE and AGT in the Finnish p.Arg133Cys CADASIL population (p.Ala202Ala) and migraine (11), whereas three other studies have failed to show any association (24–26). Interestingly, the neutral p.Ala202Ala polymorphism does not associate with migraine in our mutation-homogeneous CADASIL population. CADASIL has a very restricted overall incidence, and the patient cohorts are usually fairly small, especially when focusing on one mutation. This leads to compromised statistical power, which is an inevitable limitation that also applies to our study, as reflected in the large confidence intervals. However, we could see some statistical trends in our cohort, and previous studies have shown that multiple variants with small effects influence the WMH burden in CADASIL (13). Together, these facts suggest that with a reasonably large population size these genetic variants could be found with genomewide approaches. Additional studies in collaboration with other large CADASIL populations are warranted to further clarify the genetic factors modifying the clinical course of CADASIL. Acknowledgements We are grateful to all the patients and their families for participating. We also wish to thank Prof. Hannu Kalimo for his valuable contribution and comments, Dr. Seppo Tuisku for his help with the patients and Teija Stormi and Tero Vahlberg for their valuable contribution with the statistical analyses.
Conflict of interest and sources of funding There are no conflict of interests. This work was funded by the Sigrid Juselius Foundation, Academy of Finland project number 115906, Swedish Research Council, Finnish Cultural Foundation Varsinais-Suomi Regional Fund, University of Helsinki and the EVO Research Funds of the University Hospitals of Helsinki and Turku and the City Hospital of Turku.
Supporting Information Additional Supporting Information may be found in the online version of this article. Table S1. Detailed clinical information about the patients and their APOE, AGT p.Met268Thr (rs 699) and NOTCH3 p.Ala202Ala (rs1043994) genotypes.
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3. DOTTI MT, FEDERICO A, MAZZEI R et al. The spectrum of Notch3 mutations in 28 Italian CADASIL families. J Neurol Neurosurg Psychiatry 2005;76:736–8. 4. JOUTEL A, VAHEDI K, CORPECHOT C et al. Strong clustering and stereotyped nature of Notch3 mutations in CADASIL patients. Lancet 1997;350:1511–5. 5. JOUTEL A, CHABRIAT H, VAHEDI K et al. Splice site mutation causing a seven amino acid Notch3 in-frame deletion in CADASIL. Neurology 2000;54:1874–5. 6. ARBOLEDA-VELASQUEZ JF, LOPERA F, LOPEZ E et al. C455R notch3 mutation in a Colombian CADASIL kindred with early onset of stroke. Neurology 2002;59:277– 9. 7. OPHERK C, PETERS N, HERZOG J, LUEDTKE R, DICHGANS M. Long-term prognosis and causes of death in CADASIL: a retrospective study in 411 patients. Brain 2004;127 (Pt 11):2533–9. 8. SINGHAL S, BEVAN S, BARRICK T, RICH P, MARKUS HS. The influence of genetic and cardiovascular risk factors on the CADASIL phenotype. Brain 2004;127(Pt 9):2031– 8. 9. DICHGANS M, HERZOG J, GASSER T. NOTCH3 mutation involving three cysteine residues in a family with typical CADASIL. Neurology 2001;57:1714–7. 10. ROSS OA, SOTO-ORTOLAZA AI, HECKMAN MG et al. NOTCH3 variants and risk of ischemic stroke. PLoS ONE 2013;8:e75035. 11. SCHWAAG S, EVERS S, SCHIRMACHER A, STOGBAUER F, RINGELSTEIN EB, KUHLENBAUMER G. Genetic variants of the NOTCH3 gene in migraine–a mutation analysis and association study. Cephalalgia 2006;26:158–61. 12. OPHERK C, PETERS N, HOLTMANNSPOTTER M, GSCHWENDTNER A, MULLER-MYHSOK B, DICHGANS M. Heritability of MRI lesion volume in CADASIL: evidence for genetic modifiers. Stroke 2006;37:2684–9. 13. OPHERK C, GONIK M, DUERING M et al. Genome-wide genotyping demonstrates a polygenic risk score associated with white matter hyperintensity volume in CADASIL. Stroke 2014;45:968–72. 14. BANGEN KJ, BEISER A, DELANO-WOOD L et al. APOE genotype modifies the relationship between midlife vascular risk factors and later cognitive decline. J Stroke Cerebrovasc Dis 2013;22:1361–9. 15. LOPEZ MF, KRASTINS B, NING M. The role of apolipoprotein E in neurodegeneration and cardiovascular disease. Expert Rev Proteomics 2014;11:371–81. 16. WANG B, GUO Q, PENG Y, LU J, SINGH B, HUA B. Association of AGT M235T and ACE I/D polymorphisms with the risk of ischemic stroke: meta-analysis in Han Chinese population. J Neurol Sci 2012;320:79– 84. 17. LIANG X, QIU J, LIU X et al. Polymorphism of angiotensinogen gene M235T in myocardial infarction and brain infarction: a meta-analysis. Gene 2013;529:73–9. € K, SAVONTAUS ML, JUVONEN V et al. Detec18. MYKKANEN tion of the founder effect in Finnish CADASIL families. Eur J Hum Genet 2004;12:813–9. 19. RUSS AP, MAERZ W, RUZICKA V, STEIN U, GROSS W. Rapid detection of the hypertension-associated Met235– >Thr allele of the human angiotensinogen gene. Hum Mol Genet 1993;2:609–10. 20. ESCARY JL, CECILLON M, MACIAZEK J, LATHROP M, TOURNIER-LASSERVE E, JOUTEL A. Evaluation of DHPLC analysis in mutational scanning of Notch3, a gene with a high G-C content. Hum Mutat 2000;16:518–26. 21. LEE YC, LIU CS, CHANG MH et al. Population-specific spectrum of NOTCH3 mutations, MRI features and
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© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd ACTA NEUROLOGICA SCANDINAVICA
Clinical Commentary
APOE and AGT in the Finnish p.Arg133Cys CADASIL population Siitonen M, Mykk€ anen K, Pescini F, Rovio S, K€a€ari€ainen H, Baumann M, P€ oyh€ onen M, Viitanen M. APOE and AGT in the Finnish p.Arg133Cys CADASIL population. Acta Neurol Scand 2015: 132: 430–434. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Background – CADASIL is an inherited systemic small vessel disease, the affected status of brain vessels leading to subcortical vascular dementia. The defective gene is NOTCH3 in which over 230 different pathogenic mutations have been identified. The clinical course of CADASIL is highly variable even within families. Previous studies have shown that additional genetic factors modify the phenotype. Aims and methods – Altogether, 134 Finnish CADASIL patients with p.Arg133Cys mutation were analysed for possible associations between the apolipoprotein E (APOE) genotype, angiotensinogen (AGT) p.Met268Thr polymorphism or neutral p.Ala202Ala NOTCH3 polymorphism and earlier first-ever stroke or migraine. Results – We found no association between the APOE genotypes, AGT polymorphism, NOTCH3 polymorphism and earlier first-ever stroke or migraine. Conclusions – The APOE, AGT and NOTCH3 polymorphism did not modify the onset of strokes or migraine in our CADASIL sample, which is one of the largest mutationally homogenous CADASIL populations published to date. International collaboration, pooled analyses and genomewide approaches are warranted to identify the genetic factors that modify the highly variable CADASIL phenotype.
M. Siitonen1,2, K. Mykk€anen1, F. Pescini3, S. Rovio4, H. K€a€ari€ainen5, M. Baumann6, M. P€oyh€onen7,*, M. Viitanen8,9,* 1 Department of Medical Biochemistry and Genetics, Institute of Biomedicine, University of Turku, Turku, Finland; 2Department of Medical Genetics, University of Helsinki, Helsinki, Finland; 3Stroke Unit and Neurology, Azienda Ospedaliero Universitaria Careggi, Florence, Italy; 4Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku,Turku, Finland; 5National Institute for Health and Welfare, Helsinki, Finland; 6Protein Chemistry Unit, Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland; 7Department of Clinical Genetics, HUSLAB Helsinki University Central Hospital and Department of Medical Genetics, University of Helsinki, Helsinki, Finland; 8Department of Geriatrics, Turku City Hospital and University of Turku, Turku, Finland; 9Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden Key words: genotype–phenotype correlation; founder effect; association study; CADASIL; NOTCH3; APOE; AGT
M. Siitonen, Institute of Biomedicine, Department of Medical Biochemistry and Genetics, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland Tel.: +358 2333 7456 Fax: +358 2230 1280 e-mail: [email protected] *Shared senior authorship. Accepted for publication March 4, 2015
Introduction
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited systemic small vessel disease with characteristic, although variable, clinical features [e.g. transient ischaemic attacks (TIA), strokes, migrainous headache, progressive cognitive decline leading finally to dementia of subcortical vascular type and psychiatric symptoms]. In magnetic resonance imaging (MRI), typical white matter alterations are common (1). The vascular pathology in the central nervous 430
system is characterized by – CADASIL-specific – electron-dense deposits of granular osmiophilic material (GOM) in close vicinity of vascular smooth muscle cells (VSMCs) with subsequent degeneration of the VSMCs and marked fibrosis and stenosis most prominent in the cerebral white matter (WM) arterioles, which together give rise to extensive leukoaraiosis and multiple lacunar infarcts (1). CADASIL is diagnosed by demonstration of GOM in a skin biopsy (2) or by genetic verification of a pathogenic mutation in the NOTCH3 gene, located on 19p13.1 (1). A vast majority of over 230 mutations responsible
APOE and AGT in the Finnish p.Arg133Cys CADASIL population for CADASIL are missense point mutations causing an uneven number of cysteine residues in one of the epidermal growth factor-like repeats in the extracellular domain of the NOTCH3 receptor molecule (2–4). The age of onset and progression of the disease are highly variable even within the same family. Some genotype–phenotype correlations have been reported (5, 6), and an association between two different mutations and shorter survival was detected in two German families, (7) but many studies have failed to show any association (8, 9). To date, at least 30 NOTCH3 polymorphisms, 19 neutral and 11 amino acid changing, have been found (4), but only a common p.Arg1560Pro NOTCH3 variant has been reported to be protective against ischaemic stroke (10), and an association between the neutral p.Ala202Ala (rs1043994) polymorphism and migraine has been reported in the general population (11). However, heritability estimates in CADASIL suggest a strong modifying influence of genetic factors distinct from the causative NOTCH3 mutation on the number of ischaemic brain lesions (12). It has also been shown that the polygenic risk score is associated with white matter hyperintensity (WMH) volume, which suggests that multiple common variants with small effect sizes modify the WMH burden in CADASIL (13). As the main pathogenic feature of CADASIL is ischaemic lesions, the genes associated with cardiovascular diseases and stroke such as apolipoprotein E (APOE) and angiotensinogen (AGT) are obvious candidates to be involved in the variable outcome of the clinical course of CADASIL. APOE e4 carriers are shown to be at increased risk for atherosclerosis and stroke (14, 15), and AGT p.Met268Thr (rs699, previously published as p.Met235Thr) significantly contributes to stroke risk at least in East Asian populations (16, 17). It has also been shown that polymorphisms in the NOTCH3 gene itself could alter the phenotype of the disease (10, 11). Altogether, over 200 patients with CADASIL have been molecular genetically diagnosed in Finland, and the majority carry the same p.Arg133Cys mutation due to the founder effect (18). Despite the similar mutational background, the patients’ clinical pictures are highly variable. Therefore, using this unique material of 134 CADASIL patients with the same p.Arg133Cys mutation, we investigated whether APOE, AGT, seven amino acid changing and one neutral p.Ala202Ala NOTCH3 polymorphisms have an effect on the earlier occurrence of firstever stroke or migraine.
Patients and methods Clinical data
Altogether, 134 patients from 24 Finnish families with the same p.Arg133Cys mutation were included in the study. The clinical data were collected retrospectively using medical records and structured questionnaires. The incidence of, and age at incidence of, first-ever TIA and stroke and the occurrence of migrainous headache, myocardial infarction and vascular risk factors such as hypertension (>140/90 mmHg or medication), diabetes mellitus (established diagnosis) and high total serum cholesterol (>6.2 mmol/l or statin therapy) were acquired. Body mass index (BMI) was calculated and >25.0 kg/m2 was considered overweight. Patients were divided into smokers or non-smokers, and into heavy drinkers (men >280 g of alcohol/week, women >190 g/week), moderate drinkers (men A, NM_000435.2). The polymorphisms were analysed by DHLPC analysis with a WaveTM apparatus (20) for 20 patients and by sequencing for 30 patients. Neutral p.Ala202Ala NOTCH3 polymorphism was analysed from all 134 patients as described by Schwaag et al. (11). Statistical analysis
Cox proportional hazards regression was used to analyse the associations between APOE genotypes or AGT p.Met268Thr polymorphism and the first-ever stroke or TIA. The analyses were 431
Siitonen et al. conducted both unadjusted and after adjusting for age, sex, hypertension, hypercholesterolaemia, myocardial infarction, smoking and alcohol drinking. The association between the neutral NOTCH3 p.Ala202Ala polymorphism and migraine was analysed using logistic regression with sibling as a random effect. All statistical analyses were conducted with SPSS version 19.0 (IBM Corp. Armonk, NY, USA) or SAS version 9.1 (SAS Institute Inc., Cary, NC, USA.) Results
There were 68 females and 66 males in our cohort, and their mean age was 57 (range 20– 94 years), while their mean age at first-ever stroke or TIA was 45. The demographic and clinical features of our CADASIL population are presented in Table 1, and detailed genotypes are given in Table S1. The APOE allele frequencies were e2 5.4%, e3 77.5% and e4 17.1%, which is congruent with the normal population in Finland. We could not show any association between the APOE genotypes and first-ever stroke (Table 2), and the results did not change after adjusting for the traditional vascular risk factors listed in Table 1. The frequencies of the AGT p.Met268Thr genotypes were as follows: MM 23.2%, MT 56.6% and TT 20.2%. No association was found between the AGT genotypes and first-ever stroke (TT genotype and stroke, HR 0.76 and 95% CI 0.40–1.46). All of the 50 patients analysed for the seven amino acid changing NOTCH3 polymorphisms were homozygous for the common allele. Hence, the sequencing was not widened to the rest of the patient population for it was obvious that the frequency of these polymorphisms was too low Table 1 Demographics and clinical features of the Finnish CADASIL cohort Patients with the feature/with information Male sex Stroke TIA Migrainous headache Hypertension Hypercholesterolaemia Myocardial infarction Diabetes mellitus Overweight (BMI>25) Current smokers Alcohol drinking Non-drinkers Moderate drinkers Heavy drinkers
432
66/134 70/116 53/102 58/104 24/120 47/115 9/117 10/119 51/91 30/102
(49.3%) (60.3%) (52.0%) (55.8%) (20.0%) (40.9%) (7.7%) (8.4%) (56.4%) (29.4%)
31/87 (35.6%) 56/87 (64.4%) 6/87 (6.9%)
Table 2 Association between APOE e4 allele and earlier first-ever stroke, TIA or stroke/TIA
APOE e4 vs stroke APOE e4 vs stroke/TIA APOE e4 vs TIA
HR
95% CI
n
1.50 1.07 0.89
0.75–2.99 0.50–2.28 0.49–1.61
111 111 94
Values are hazard ratios (HR) with 95% confidence intervals (95% CI) for APOE e4 carriers from Cox proportional hazards regression analyses.
for any statistical analysis. Interestingly, there was no association between the neutral p.Ala202Ala NOTCH3 polymorphism and migraine in our CADASIL population (AA vs GG, OR 0.71 95% CI 0.01–40.59; AG vs GG, OR 1.15 95% CI 0.27–4.86). Discussion
The great advantage of our study is the large number (134) of patients with the same p.Arg133Cys mutation. This homogeneity makes our population exceptional among other large CADASIL populations (3, 4). In addition to the same mutation, our Finnish patients can also be expected to have other similarities in their genetic background due to the founder effect (18) and the relatively small size of the Finnish population (approximately 5.4 million). When compared to other published founder populations (21, 22), our patient cohort is the largest and most profoundly characterized. Together, these facts make our population highly suitable for studying associations between genetic risk factors and their effects among patients with CADASIL. Furthermore, the frequencies of stroke, migrainous headache and vascular risk factors in our population are similar to those found in a large prospective series of patients with CADASIL (8), which increases the credibility of our study. In our CADASIL population, we could not show any association between the analysed genetic factors and first-ever stroke. This is in line with two previous CADASIL studies in which APOE genotype had no modifying effect on the clinical course (8, 23). When TIA together with stroke as the end point were considered, the results of the statistical analyses confirmed this still less. This could be due to the difficulty in distinguishing TIA from other neurological deficits. Hence, we recommend that this difficulty should be considered when using TIA as an outcome in the analyses. Only few studies have considered NOTCH3 polymorphisms in the context of cerebrovascular diseases or migraine. One study has reported an association between a NOTCH3 polymorphism
APOE and AGT in the Finnish p.Arg133Cys CADASIL population (p.Ala202Ala) and migraine (11), whereas three other studies have failed to show any association (24–26). Interestingly, the neutral p.Ala202Ala polymorphism does not associate with migraine in our mutation-homogeneous CADASIL population. CADASIL has a very restricted overall incidence, and the patient cohorts are usually fairly small, especially when focusing on one mutation. This leads to compromised statistical power, which is an inevitable limitation that also applies to our study, as reflected in the large confidence intervals. However, we could see some statistical trends in our cohort, and previous studies have shown that multiple variants with small effects influence the WMH burden in CADASIL (13). Together, these facts suggest that with a reasonably large population size these genetic variants could be found with genomewide approaches. Additional studies in collaboration with other large CADASIL populations are warranted to further clarify the genetic factors modifying the clinical course of CADASIL. Acknowledgements We are grateful to all the patients and their families for participating. We also wish to thank Prof. Hannu Kalimo for his valuable contribution and comments, Dr. Seppo Tuisku for his help with the patients and Teija Stormi and Tero Vahlberg for their valuable contribution with the statistical analyses.
Conflict of interest and sources of funding There are no conflict of interests. This work was funded by the Sigrid Juselius Foundation, Academy of Finland project number 115906, Swedish Research Council, Finnish Cultural Foundation Varsinais-Suomi Regional Fund, University of Helsinki and the EVO Research Funds of the University Hospitals of Helsinki and Turku and the City Hospital of Turku.
Supporting Information Additional Supporting Information may be found in the online version of this article. Table S1. Detailed clinical information about the patients and their APOE, AGT p.Met268Thr (rs 699) and NOTCH3 p.Ala202Ala (rs1043994) genotypes.
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