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Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer's disease in Poland
Experimental Neurology 184 (2003) 991 – 996 www.elsevier.com/locate/yexnr
Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer’s disease in Poland Cezary Z˙ekanowski, a,* Maria Styczyn´ska, b Beata Peplon´ska, b Tomasz Gabryelewicz, b Dorota Religa, b,c Jan Ilkowski, d Beata Kijanowska-Haladyna, e Slawomira Kotapka-Minc, e Sanne Mikkelsen, a Anna Pfeffer, b Anna Barczak, f Elz˙bieta Luczywek, b Boguslaw Wasiak, f Malgorzata Chodakowska-Z˙ebrowska, f Katarzyna Gustaw, g Jaroslaw La˛czkowski, h Tomasz Sobo´w, i Jacek Kuz´nicki, a,j and Maria Barcikowska b b
a Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland Department of Neurodegenerative Disorders, Medical Research Centre of the Polish Academy of Sciences, 02-106 Warsaw, Poland c Department of Neurotec, Section of Experimental Geriatrics, Karolinska Institutet, Stockholm, Sweden d Neurological Department, Municipal Hospital, Poznan´, Poland e Psychogeriatric Department, Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland f Department of Neurology, MSWiA Hospital, 02-507 Warsaw, Poland g Alzheimer’s Research Unit, Institute of Agricultural Medicine, Lublin, Poland h Psychogeriatric Center WOLP, 87-100 Torun´, Poland i Department of Old Age Psychiatry and Psychotic Disorders, Medical University of Lodz, 92-216 Lo´dz´, Poland j Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland
Received 19 July 2003; revised 19 July 2003; accepted 24 July 2003
Abstract Mutations in three causative genes have been identified in patients with an autosomal-dominant form of early-onset Alzheimer’s disease (EOAD). To determine the spectrum of mutations in a group consisting of 40 Polish patients with clinically diagnosed familial EOAD and 1 patient with mild cognitive impairment (MCI) and family history of AD, we performed a screening for mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2) and amyloid precursor protein (APP) genes. Four previously recognized pathogenic mutations in PSEN1 gene (H163R, M139V) and APP gene (T714A, V715A), and three novel putative mutations in PSEN1 gene (P117R and I213F) and PSEN2 gene (Q228L) were identified. The 34 patients with no mutations detected were older than the patients with mutations. A frequency of APOE4 allele was higher in this group. Frequency of mutations is relatively low (17%), possibly due to used operational definition of a patient with familial EOAD (a patient having at least one relative with early-onset dementia). It could be concluded that screening for mutations in the three genes could be included in a diagnostic program directed at patients with a positive family history or age of onset before 55 years. D 2003 Elsevier Inc. All rights reserved. Keywords: Early-onset Alzheimer’s disease; Familial Alzheimer’s disease; Mutation; PSEN1; PSEN2; APP; Polymorphism
Introduction The genetic determinants of early-onset ( < 65 years of age) Alzheimer’s disease (EOAD) are heterogeneous. In a minority of cases, EOAD shows a clear pattern of an autosomal dominant mode of inheritance. To date, three * Corresponding author. Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Ksie˛cia Trojdena 4, PL—02109 Warsaw, Poland. Fax: +48-22-668-52-88. E-mail address: [email protected] (C. Z˙ekanowski). 0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00384-4
causative genes have been identified: amyloid precursor protein gene (APP), presenilin 1 gene (PSEN1) and presenilin 2 gene (PSEN2). Additionally, APOE4 allele is a modifying factor for age of onset in EOAD caused by the APP and PSEN2 mutations. Mutations in the PSEN1 gene accounted for 18– 55% of early-onset familial AD cases. Mutations in PSEN2 and APP genes are a much rare cause of familial AD cases, being described in few dozens of families all over the world. Few silent, polymorphic substitutions have been described so far in PSEN1, PSEN2 and APP genes.
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To determine the spectrum of mutations among Polish patients with clinically diagnosed early-onset familial AD, we performed a mutation screening of PSEN1, PSEN2 and APP genes.
Patients and methods A total of 40 unrelated patients from Poland (21 women and 19 men), who were diagnosed clinically as probable AD according to the NINCDS-ADRDA criteria and with the mean age of onset of the disease at 52.8 F 8.2 years of age, was included. One patient was diagnosed as mild cognitive impairment (MCI) according to Mayo Clinic Group criteria (Petersen et al., 1997). All of them were examined by a neurologist, a neuropsychologist [evaluation in Mini Mental State Examination (MMSE), Global Deterioration Scale, Alzheimer’s Disease Assessment Scale-cognitive subscale and Blessed Dementia Rating Scale], as well as a psychiatrist, and had obtained a CT scan of the brain. The disease was considered as familial if at least one additional first degree relative suffered from early-onset, AD-type dementia. In one case (MCI patient), there was a family history of late-onset dementia. In two cases, the family history was uninformative because of the death of patient’s parents in 5th decade of live (cause other than dementia), while other relatives were not demented. The study was approved by the Ethics Committee of the MSWiA Hospital. A written consent was obtained from all participants or their relatives after the study had been fully explained. Total RNA was isolated from peripheral blood leucocytes using RNeasy kit (Qiagen) and subsequently RT-PCR was performed using oligo-dT primer. Then the entire coding sequence of PSEN1 gene was amplified with three pairs of primers designed according to the GeneBank entries and available on request. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and directly sequenced (BIG Dye ver. 2.0 kit, Applera) with the same primers. PSEN1 gene was screened either using RT-PCR method (16 patients) or genomic DNA analysis (24 patients) as described below. Genomic DNA was isolated from peripheral blood leucocytes using salting-out procedure. Intronic primers were used to amplify exons 3 –13 of PSEN1 gene (J.R. Murrell, Indiana University Medical Center, pers. comm.) in 24 patients. A proximal promoter sequence of PSEN1 gene ( 320 + 40) was amplified in all patients using primers described previously (Theuns et al., 2000). Exons of PSEN2 and APP genes with flanking intronic sequences (at least 200 bp from both sides) were screened for mutations in all investigated groups. For PSEN2, exons 4, 5 and 7 were amplified using primers described by Campion et al. (1996). APP exons 16 and 17 were amplified using primers designed according to the GeneBank entries and available on request.
PCR reaction was done using temperature and time profiles designed according to the primer’s sequences with MasterMix Kit or Hot Start Master Mix Kit (Qiagen). Resulting PCR fragments were purified with QIAquick PCR Purification Kit (Qiagen) before sequencing. Direct fluorescent sequencing was then performed using amplification forward primer. In the case of novel substitution, sequencing from both primers was performed. In all the cases mentioned above, exons were screened. APOE genotype was determined using PCR-RFLP method (Chapman et al., 1996). Confirmation of a novel mutation in PSEN2 gene was performed in a control group of 100 late-onset, sporadic AD patients (SAD) and 100 healthy subjects. The diagnosis of probable AD was made according to the NINCDS-ADRDA criteria. The mean age at onset of the 100 patients was 71.5 F 4.2. The group consisted of 64 women and 36 men. Patients were compared with a control subject of the same age range (mean 71.2 F 5.9 years old) and sex distribution as AD. The control group consisted of volunteers recruited from the Department of Internal and Surgery Medicine and the Third Age University. All subjects were screened on cognitive function. Analysis was done using single stranded conformational analysis (SSCA) with MDE gel (BMA) in standard conditions and direct fluorescent sequencing of amplimers with changed gel mobility. Screening for the E318G substitution was performed in the same group of 100 SAD patients and 100 healthy subjects using ACRS strategy as described previously (Taddei et al., 2002). Statistical analysis was done using Statistica ver. 6.0 software.
Results Seven mutations were detected in the group (see: AD Mutation Database, http://molgen-www.uia.ac.be/ADMutations). Four disease-causing mutations were detected in PSEN1 gene (P117R, M139V, H163R, and I213F). In two cases, the same mutation was found in affected sibling of probands. In APP gene, two mutations were detected (T714A, V715A). In one MCI patient, a novel mutation in exon 7 of PSEN2 gene was detected (Q228L). This sequence variant was absent from 100 unrelated patients with sporadic AD and 100 unrelated healthy controls. All identified mutations and a brief clinical characteristics of patients are presented in Table 1. A novel mutation P117R (CCA ! CGA) in PSEN1 gene was identified in a female patient, who has had complaints of memory loss since she was 36 years old. Initially it was treated as a neurosis, but the cognitive deficits progressed and 1 year later, she showed marked deficits in memory and learning. After 3 years, the MMSE score was 10 and neuropsychological examination revealed generalized cognitive impairment especially in memory, learning, visuospatial tasks as well as marked apathy and stereotyped
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Table 1 Characteristics of identified mutations, main clinical features and family history of the patients Gene
Mutation
Substitution
Exon
Protein domain
Age of onset (years)
Main clinical features
Family members with probable AD (age of onset)
PSEN1
P117Ra
(CCA ! CGA)
E5
HL-I
36
PSEN1
M139V
(ATG ! GTG)
E5
TM-II
40
Memory and learning impairment, apathy, stereotyped behaviour Memory impairment, aphasia, apraxia, epilepsy, dystonia
PSEN1
H163R
(CAT ! CGT)
E6
HL-II
50
Typical AD
PSEN1
I213Fa
(ATT ! TTT)
E7
TM-IV
33
Memory impairment, disorientation, anxiety, bradykinesia
PSEN2 APP
Q228La T714A
(CAG ! CTG) (ACA ! GCA)
E7 E17
TM-V TM-I
60 44
APP
V715A
(GTG ! GCC)
E17
TM-I
42
Memory impairment, irritability Memory impairment, disorientation, behavioural changes, aphasia Impairment of recent verbal memory and visuospatial functions
Data not available (Father: suicide approximately 50 years) Brother (PSEN1 M139 V) (40 years), Mother’s brother (50 years) (mother died in 3rd decade), Grandmother and two brothers (approximately 50 years) Sister (PSEN1 H163 R) (51 years), Mother (50 years) Sister, brother (before 35 years), Mother and her sister (before 35 years) Mother—late onset AD (80 years) Mother (approximately 45 years)
a
Data not available (Father—sudden death before 50 years)
Novel mutations.
behaviour. Neurological examination showed bradykinesia and myoclonus of the upper limbs. There was also an evidence of two epileptic seizures. A second novel mutation I213F (ATT ! TTT) in PSEN1 gene was identified in a male patient, with the age of onset at 33 years. First symptom he had experienced was progressive impairment of memory. After 2 years, his mood became labile and he was disoriented to time and space. Neurological examination revealed bradykinesia and rigidity. After 5 years from the onset of the disease, the MMSE score was 10. Four members of his family (sister, brother, mother and aunt) had died before the age of 40 with probable AD, but no postmortem examinations were obtained in those cases. A recognized mutation M139V (ATG ! GTG) in PSEN1 gene was observed in siblings (man and woman, with age of onset at 40 years). There were some differences between siblings observed in clinical symptoms and the course of the disease. However, in both cases, the course of the disease was rapid and devastating. The sister had progressive memory, language and praxis deficits leading to severe dementia and aphasia 2 years after the beginning of symptoms. Her brother had prominent memory, visuospatial and behavioural disturbances, although the language functions were relatively preserved. After 2 years, he was also severely demented. A pathogenic mutation H163R (CAT ! CGT) in PSEN1 gene was detected in two sisters, with age of onset at 50 and 51 years of age. The clinical course was typical for AD and the older of the sisters developed severe dementia 5 years after the onset of the disease. The younger was mildly demented 2 years after the onset of symptoms. A novel mutation Q228L (CAG ! CTG) in PSEN2 gene was identified in one patient with a positive familial history
of EOAD and the diagnosis of MCI. Symptoms were initiated at the age of 60, and 2 years later, she reported slowly but significantly intensifying irritability, verbal aggressivity, abrasiveness and insomnia along with mild deficits in memory and short episodes of disorientation. The MMSE score was 28. After following 2 years of observation, she was still diagnosed as MCI, but the slight progression in cognitive impairment was shown. Her 88 years old mother has probable AD and is a resident in the nursing home. Due to ethical reasons, no segregation analysis of the mutation was performed in the risk family. Two previously recognized mutations in APP gene were detected in two patients. An Iranian mutation T714A (ACA ! GCA) was detected in a man with the age of onset at 44 years of age. After 4 years, he has developed severe memory impairment, disorientation to space and time, behavioral changes and aphasia. The same mutation was identified in a second family living in the same city, and was recognized as a result of probable consanguinity. The age of onset in the examined male patient from the second family was similar (45 years old) and he had memory impairment, spatial disorientation and aphasia. A German mutation V715A (GTG ! GCG) was identified in a female patient with the onset at 42 years old. Neuropsychological examination at the age of 44 showed severe cognitive impairment, especially in recent verbal memory and visuospatial tasks. Nine silent, common polymorphic substitutions in the analyzed regions of three genes were identified in the familial AD group. Table 2 lists all identified polymorphisms. Only one silent, intronic substitution was detected in analyzed regions of APP gene (IVS16 nt + 47a ! g). In the same person, E318G substitution in exon 9 of PSEN1 gene was detected. As the possible role of this substitution is
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Table 2 Polymorphisms identified in the examined group Gene
Region
Substitution
Comments
PSEN1 PSEN1 PSEN1 PSEN2 PSEN2 PSEN2 PSEN2 PSEN2 APP
5VUTR E9 I9 E3 I3 I3 I3 I5 I16
48c ! t E318G (GAA ! GGA) nt + 17t ! g H87H (CAT ! CAC) nt 42a ! g nt 41t ! c nt 29c ! t nt + 30g ! c nt + 47a ! g
rare rarea very common very common very common very common common rare rarea
a
Both polymorphisms detected in one patient.
controversial, a panel of 100 Polish patients with sporadic AD and 100 age-matched control subjects were screened for E318G. It was found that E318G was present in SAD patients, as well as in controls with a frequency of about 3.4%. The difference in E318G frequency between SAD patients and controls was statistically insignificant. Additionally, two apparently silent polymorphisms in PSEN2 gene were identified in a group of sporadic AD patients: S236S (AGT/AGC), A252A (GCG/GCC). Frequency of APOE4 allele was higher in SAD patients, compared with the FAD cases (v2 test, P = 0.0458). There were no differences in frequencies of a common PSEN1 promoter polymorphisms genotypes ( 48 CC and 48 CT) between patients with FAD and SAD. (v2 test, P = 0.5910). Additionally, there was no interaction between this polymorphism and APOE4 allele in the reported group. However, in a group of 100 SAD patients, there is a statistically significant association between APOE4 allele and CT genotype compared with CC genotype (v2 test, P = 0.0016, data not shown). In other studies, CC genotype was associated with increased risk for EOAD (but not late-onset AD), independent of the APOE4 genotype (Theuns et al., 2003). Presented results could be due to a statistical bias; however, it could reflect population differences.
Discussion In the reported group, mutations were detected in all three analyzed genes. Frequency of mutations is relatively low (17%), possibly due to used definition of a patient with familial AD (a patient having at least one relative with presenile dementia). In the reported group, the only two affected relatives of patients have had identified PSEN1 mutations. In highly selected group of patients, with two or more relatives in two or more generations affected, similar screening gave a prevalence of pathogenic mutations of 68% (Cruts et al., 1998; Janssen et al., 2003; Kamimura et al., 1998). However, the detection rate reported previously ranges from 18% to 56%, depending on the method of patient selection, population screened and the mutation detection technique used (Campion et al., 1999). No differences in utility and sensitivity of mutation detection in
coding regions were observed between RT-PCR method and exon-screening method. However, sequencing of genomic DNA could potentially reveal clinically important polymorphisms. Mutations identified in PSEN1 and APP genes showed almost complete penetrance by the age of 55 years. A lower age of onset was observed in a patient with a novel mutation I213F (before 35 years) and the sib pair bearing M139V mutation (40 years of age). In the latter case, a slightly different course of the disease in both siblings was observed. This is in accordance with the previously published data, indicating that M139V mutation can be presented differently between kindreds (Finckh et al., 2000b; Fox et al., 1997; Rippon et al., 2003; Sandbrink et al., 1996). The clinical course of a patient with Q228L substitution resembles that of other patients with known PSEN2 mutations. Q2228L mutation was absent in a group of 100 Polish patients with sporadic AD and 100 age-matched healthy controls. Analysis using ConSeq program (http://conseq. bioinfo.tau.ac.il/) suggests that Q228 residue is functional, highly conserved in evolution and exposed in the protein structure. The substitution affects the residue conserved between PS2 and PS1 and also affects the amino acid residue located in the transmembrane domain V, near two other known PSEN2 mutations (M239V, M239I) (Finckh et al., 2000a). A mutation in homologous position in PSEN1 gene (Q222R) was observed in a German population, indicating a functional relevance of Q228 residue in PS2 (Rogaeva et al., 2001). Collected information strongly suggests that Q228L substitution could be considered as a disease-causing mutation. A novel mutation P117R (CCA ! CGA) in PSEN1 gene is located at the same codon as two other highly pathogenic mutations (P117S and P117L) detected in Polish population previously (Dowjat et al., 2002; Wisniewski et al., 1998). One of the lowest age of onset in the presented group (36 years old) and the first characteristic symptoms (i.e. memory loss) resemble phenotypic features of patients with P117S and P117L mutations. ConSeq program analysis suggests that codon 117 is also a conserved one (however, the normalized conservation score is lower than in the case of codon 228 of PS2). On this basis, mutation P117R was annotated as pathogenic one. A novel mutation I213F (ATT ! TTT) in PSEN1 gene is located at the same codon as two other pathogenic mutations (I213T and I213L) detected previously (Kamino et al., 1996; Rogaeva et al., 2001). The patient is characterized with the lowest age of onset in the presented group (33 years old). ConSeq program analysis suggests that codon 213 is a highly conserved one, and is located in transmembrane domain IV. On this basis, mutation I213F was annotated as pathogenic one. APP mutation T714A was detected previously in an Iranian family (Pasalar et al., 2002). The age of onset was 11 years higher than in the presented cases. The second APP mutation V715A was identified in different European
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countries, and seems to be a relatively common mutation, with the mean age of onset at 47 years of age (Beck et al., 2002; Cruts et al., 2002; De Jonghe et al., 2001). A substitution E318G in exon 9 of PSEN1 gene was detected in one patient. The role of this mutation is controversial. Initially it has been reported to cause AD with a variable age of onset (Cruts and van Broeckhoven, 1998). However, the mutation was found in a number of non-demented control individuals, as well as patients of early- and late-onset AD with or without a known family history (Arango et al., 2001; Helisalmi et al., 2000). E318G substitution is regarded as either a rare polymorphism, a neutral mutation or a mutation of incomplete penetrance (Taddei et al., 2002). It is possible, however, that the putative pathogenetic effect depends on the ethnic background. In Polish subjects, however, the difference in E318G frequency between SAD patients and controls was statistically insignificant. This could suggest that E318G is a silent polymorphism in Polish population. No mutations in PSEN1, PSEN2 and APP genes have been found in 34 probands. In this group, the mean age of onset was 54.7 F 6.7 years old as compared to patients with identified mutations (43.57 F 9.08 years). The difference in mean age of onset between the groups was statistically significant (t test; P < 0.05). The APOE4 frequency in the group without mutation was higher than the one in the control Polish population, and lower than the one in lateonset sporadic AD cases (19% vs. 11% and 32%, respectively; v2 test, P = 0.2, NS) (Styczynska et al., 2003). Three patients are APOE4 homozygotes. It is possible that some of the patients have AD caused by the presence of one or two APOE4 alleles. An increased APOE4 allele frequency among early-onset AD patients without identified mutations was observed in other studies (Lleo et al., 2002). It could be also speculated that in these families, disease-causing mutations fall outside the analyzed regions or that large gene rearrangement is missed. A rare intronic polymorphism in APP gene (IVS16 nt + 47a ! g) was found in one patient with E318G substitution. This patient bears also A117A polymorphism in PRNP gene, rare among Polish AD patients (data not shown). This could suggest a distinct genetic background in this case. Other silent substitutions in PSEN1 and PSEN2 genes were common ones, present at least in 10 FAD alleles, and also found in healthy control people. Exonic PSEN2 polymorphisms (S236S and A262A) were detected in SAD patients only. Additionally, there was no interaction between PSEN1 48c ! t polymorphism and APOE4 allele in the reported group. Nevertheless, in a group of 100 SAD patients, there is a statistically significant association between APOE4 allele and CT genotype compared with CC genotype (v2 test, P = 0.0016, data not shown). In other studies, CC genotype was associated with increased risk for EOAD (but not late-onset AD), independent of the APOE4 genotype (Araria-Goumidi et al., 2002; Lambert et al., 2001;
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van Duijn et al., 1999). Presented results could be due to a statistical bias; however, it could reflect a population difference. It is concluded that molecular diagnosis could be used to confirm familial AD in a well-designed clinical setting (Croes et al., 2000). Currently, the absence of effective prophylactic treatments for AD limits presymptomatic and antenatal testing for ethical reasons (Hedera, 2001). However, if any of the experimental prevention or treatment strategies proves to be effective, molecular diagnosis in risk families will become very important. This study suggests a possible screening strategy in Poland.
Acknowledgment This work was supported by State Committee for Scientific Research (KBN) grant no. PBZ-KBN/002/CD/ P05/2000.
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sanova, J., Rojo, A., Oliva, R., 2002. Frequency of mutations in the presenilin and amyloid precursor protein genes in early-onset Alzheimer disease in Spain. Arch. Neurol. 59, 1759 – 1763. Petersen, R.C., Smith, G.E., Waring, S.C., Ivnik, R.J., Kokmen, E., Tangelos, E.G., 1997. Aging, memory and mild cognitive impairment. Int. Psychogeriatr. 9 (Suppl. 1), 65 – 69. Pasalar, P., Najmabadi, H., Noorian, A.R., Moghimi, B., Jannati, A., Soltanzadeh, A., Krefft, T., Crook, R., Hardy, J., 2002. An Iranian family with Alzheimer’s disease caused by a novel APP mutation (Thr714Ala). Neurology 58, 1574 – 1575. Rippon, G.A., Crook, R., Baker, M., Halvorsen, E., Chin, S., Hutt, M., Hardy, J., Lynch, T., 2003. Presenilin 1 mutation in an African American family presenting with atypical Alzheimer dementia. Arch. Neurol. 60, 884 – 888. Rogaeva, E.A., Fafel, K.C., Song, Y.Q., Medeiros, H., Sato, C., Liang, Y., Richard, E., Rogaev, E.I., Frommelt, P., Sadovnick, A.D., Meschino, W., Rockwood, K., Boss, M.A., Mayeux, R., St George-Hyslop, P., 2001. Screening for PS1 mutations in a referral-based series of AD cases: 21 novel mutations. Neurology 57, 621 – 625. Sandbrink, R., Zhang, D., Schaeffer, S., Masters, C.L., Bauer, J., Forstl, H., Beyreuther, K., 1996. Missense mutations of the PS-1/S182 gene in German early-onset Alzheimer’s disease patients. Ann. Neurol. 40, 265 – 266. Styczynska, M., Religa, D., Pfeffer, A., Luczywek, E., Wasiak, B., Styczynski, G., Peplonska, B., Gabryelewicz, T., Golebiowski, M., Kobrys, M., Barcikowska, M., 2003. Simultaneous analysis of five genetic risk factors in Polish patients with Alzheimer’s disease. Neurosci. Lett. 344, 99 – 102. Taddei, K., Fisher, C., Laws, S.M., Martins, G., Paton, A., Clarnette, R.M., Chung, C., Brooks, W.S., Hallmayer, J., Miklossy, J., Relkin, N., St George-Hyslop, P.H., Gandy, S.E., Martins, R.N., 2002. Association between presenilin-1 Glu318Gly mutation and familial Alzheimer’s disease in the Australian population. Mol. Psychiatry 7, 776 – 781. Theuns, J., Del-Favero, J., Dermaut, B., van Duijn, C.M., Backhovens, H., van den Broeck, M.V., Serneels, S., Corsmit, E., Van Broeckhoven, C.V., Cruts, M., 2000. Genetic variability in the regulatory region of presenilin 1 associated with risk for Alzheimer’s disease and variable expression. Hum. Mol. Genet. 9, 325 – 331. Theuns, J., Remacle, J., Killick, R., Corsmit, E., Vennekens, K., Huylebroeck, D., Cruts, M., Van Broeckhoven, C., 2003. Alzheimer-associated C allele of the promoter polymorphism 22C > T causes a critical neuron-specific decrease of presenilin1 expression. Hum. Mol. Genet. 12, 869 – 877. Wisniewski, T., Dowjat, W.K., Buxbaum, J.D., Khorkova, O., Efthimiopoulos, S., Kulczycki, J., Lojkowska, W., Wegiel, J., Wisniewski, H.M., Frangione, B., 1998. A novel Polish presenilin-1 mutation (P117L) is associated with familial Alzheimer’s disease and leads to death as early as the age of 28 years. NeuroReport 9, 217 – 221. van Duijn, C.M., Cruts, M., Theuns, J., Van Gassen, G., Backhovens, H., van den Broeck, M., Wehnert, A., Serneels, S., Hofman, A., Van Broeckhoven, C., 1999. Genetic association of the presenilin-1 regulatory region with early-onset Alzheimer’s disease in a population-based sample. Eur. J. Hum. Genet. 7, 801 – 806.
Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer’s disease in Poland Cezary Z˙ekanowski, a,* Maria Styczyn´ska, b Beata Peplon´ska, b Tomasz Gabryelewicz, b Dorota Religa, b,c Jan Ilkowski, d Beata Kijanowska-Haladyna, e Slawomira Kotapka-Minc, e Sanne Mikkelsen, a Anna Pfeffer, b Anna Barczak, f Elz˙bieta Luczywek, b Boguslaw Wasiak, f Malgorzata Chodakowska-Z˙ebrowska, f Katarzyna Gustaw, g Jaroslaw La˛czkowski, h Tomasz Sobo´w, i Jacek Kuz´nicki, a,j and Maria Barcikowska b b
a Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland Department of Neurodegenerative Disorders, Medical Research Centre of the Polish Academy of Sciences, 02-106 Warsaw, Poland c Department of Neurotec, Section of Experimental Geriatrics, Karolinska Institutet, Stockholm, Sweden d Neurological Department, Municipal Hospital, Poznan´, Poland e Psychogeriatric Department, Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland f Department of Neurology, MSWiA Hospital, 02-507 Warsaw, Poland g Alzheimer’s Research Unit, Institute of Agricultural Medicine, Lublin, Poland h Psychogeriatric Center WOLP, 87-100 Torun´, Poland i Department of Old Age Psychiatry and Psychotic Disorders, Medical University of Lodz, 92-216 Lo´dz´, Poland j Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland
Received 19 July 2003; revised 19 July 2003; accepted 24 July 2003
Abstract Mutations in three causative genes have been identified in patients with an autosomal-dominant form of early-onset Alzheimer’s disease (EOAD). To determine the spectrum of mutations in a group consisting of 40 Polish patients with clinically diagnosed familial EOAD and 1 patient with mild cognitive impairment (MCI) and family history of AD, we performed a screening for mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2) and amyloid precursor protein (APP) genes. Four previously recognized pathogenic mutations in PSEN1 gene (H163R, M139V) and APP gene (T714A, V715A), and three novel putative mutations in PSEN1 gene (P117R and I213F) and PSEN2 gene (Q228L) were identified. The 34 patients with no mutations detected were older than the patients with mutations. A frequency of APOE4 allele was higher in this group. Frequency of mutations is relatively low (17%), possibly due to used operational definition of a patient with familial EOAD (a patient having at least one relative with early-onset dementia). It could be concluded that screening for mutations in the three genes could be included in a diagnostic program directed at patients with a positive family history or age of onset before 55 years. D 2003 Elsevier Inc. All rights reserved. Keywords: Early-onset Alzheimer’s disease; Familial Alzheimer’s disease; Mutation; PSEN1; PSEN2; APP; Polymorphism
Introduction The genetic determinants of early-onset ( < 65 years of age) Alzheimer’s disease (EOAD) are heterogeneous. In a minority of cases, EOAD shows a clear pattern of an autosomal dominant mode of inheritance. To date, three * Corresponding author. Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Ksie˛cia Trojdena 4, PL—02109 Warsaw, Poland. Fax: +48-22-668-52-88. E-mail address: [email protected] (C. Z˙ekanowski). 0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00384-4
causative genes have been identified: amyloid precursor protein gene (APP), presenilin 1 gene (PSEN1) and presenilin 2 gene (PSEN2). Additionally, APOE4 allele is a modifying factor for age of onset in EOAD caused by the APP and PSEN2 mutations. Mutations in the PSEN1 gene accounted for 18– 55% of early-onset familial AD cases. Mutations in PSEN2 and APP genes are a much rare cause of familial AD cases, being described in few dozens of families all over the world. Few silent, polymorphic substitutions have been described so far in PSEN1, PSEN2 and APP genes.
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To determine the spectrum of mutations among Polish patients with clinically diagnosed early-onset familial AD, we performed a mutation screening of PSEN1, PSEN2 and APP genes.
Patients and methods A total of 40 unrelated patients from Poland (21 women and 19 men), who were diagnosed clinically as probable AD according to the NINCDS-ADRDA criteria and with the mean age of onset of the disease at 52.8 F 8.2 years of age, was included. One patient was diagnosed as mild cognitive impairment (MCI) according to Mayo Clinic Group criteria (Petersen et al., 1997). All of them were examined by a neurologist, a neuropsychologist [evaluation in Mini Mental State Examination (MMSE), Global Deterioration Scale, Alzheimer’s Disease Assessment Scale-cognitive subscale and Blessed Dementia Rating Scale], as well as a psychiatrist, and had obtained a CT scan of the brain. The disease was considered as familial if at least one additional first degree relative suffered from early-onset, AD-type dementia. In one case (MCI patient), there was a family history of late-onset dementia. In two cases, the family history was uninformative because of the death of patient’s parents in 5th decade of live (cause other than dementia), while other relatives were not demented. The study was approved by the Ethics Committee of the MSWiA Hospital. A written consent was obtained from all participants or their relatives after the study had been fully explained. Total RNA was isolated from peripheral blood leucocytes using RNeasy kit (Qiagen) and subsequently RT-PCR was performed using oligo-dT primer. Then the entire coding sequence of PSEN1 gene was amplified with three pairs of primers designed according to the GeneBank entries and available on request. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and directly sequenced (BIG Dye ver. 2.0 kit, Applera) with the same primers. PSEN1 gene was screened either using RT-PCR method (16 patients) or genomic DNA analysis (24 patients) as described below. Genomic DNA was isolated from peripheral blood leucocytes using salting-out procedure. Intronic primers were used to amplify exons 3 –13 of PSEN1 gene (J.R. Murrell, Indiana University Medical Center, pers. comm.) in 24 patients. A proximal promoter sequence of PSEN1 gene ( 320 + 40) was amplified in all patients using primers described previously (Theuns et al., 2000). Exons of PSEN2 and APP genes with flanking intronic sequences (at least 200 bp from both sides) were screened for mutations in all investigated groups. For PSEN2, exons 4, 5 and 7 were amplified using primers described by Campion et al. (1996). APP exons 16 and 17 were amplified using primers designed according to the GeneBank entries and available on request.
PCR reaction was done using temperature and time profiles designed according to the primer’s sequences with MasterMix Kit or Hot Start Master Mix Kit (Qiagen). Resulting PCR fragments were purified with QIAquick PCR Purification Kit (Qiagen) before sequencing. Direct fluorescent sequencing was then performed using amplification forward primer. In the case of novel substitution, sequencing from both primers was performed. In all the cases mentioned above, exons were screened. APOE genotype was determined using PCR-RFLP method (Chapman et al., 1996). Confirmation of a novel mutation in PSEN2 gene was performed in a control group of 100 late-onset, sporadic AD patients (SAD) and 100 healthy subjects. The diagnosis of probable AD was made according to the NINCDS-ADRDA criteria. The mean age at onset of the 100 patients was 71.5 F 4.2. The group consisted of 64 women and 36 men. Patients were compared with a control subject of the same age range (mean 71.2 F 5.9 years old) and sex distribution as AD. The control group consisted of volunteers recruited from the Department of Internal and Surgery Medicine and the Third Age University. All subjects were screened on cognitive function. Analysis was done using single stranded conformational analysis (SSCA) with MDE gel (BMA) in standard conditions and direct fluorescent sequencing of amplimers with changed gel mobility. Screening for the E318G substitution was performed in the same group of 100 SAD patients and 100 healthy subjects using ACRS strategy as described previously (Taddei et al., 2002). Statistical analysis was done using Statistica ver. 6.0 software.
Results Seven mutations were detected in the group (see: AD Mutation Database, http://molgen-www.uia.ac.be/ADMutations). Four disease-causing mutations were detected in PSEN1 gene (P117R, M139V, H163R, and I213F). In two cases, the same mutation was found in affected sibling of probands. In APP gene, two mutations were detected (T714A, V715A). In one MCI patient, a novel mutation in exon 7 of PSEN2 gene was detected (Q228L). This sequence variant was absent from 100 unrelated patients with sporadic AD and 100 unrelated healthy controls. All identified mutations and a brief clinical characteristics of patients are presented in Table 1. A novel mutation P117R (CCA ! CGA) in PSEN1 gene was identified in a female patient, who has had complaints of memory loss since she was 36 years old. Initially it was treated as a neurosis, but the cognitive deficits progressed and 1 year later, she showed marked deficits in memory and learning. After 3 years, the MMSE score was 10 and neuropsychological examination revealed generalized cognitive impairment especially in memory, learning, visuospatial tasks as well as marked apathy and stereotyped
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Table 1 Characteristics of identified mutations, main clinical features and family history of the patients Gene
Mutation
Substitution
Exon
Protein domain
Age of onset (years)
Main clinical features
Family members with probable AD (age of onset)
PSEN1
P117Ra
(CCA ! CGA)
E5
HL-I
36
PSEN1
M139V
(ATG ! GTG)
E5
TM-II
40
Memory and learning impairment, apathy, stereotyped behaviour Memory impairment, aphasia, apraxia, epilepsy, dystonia
PSEN1
H163R
(CAT ! CGT)
E6
HL-II
50
Typical AD
PSEN1
I213Fa
(ATT ! TTT)
E7
TM-IV
33
Memory impairment, disorientation, anxiety, bradykinesia
PSEN2 APP
Q228La T714A
(CAG ! CTG) (ACA ! GCA)
E7 E17
TM-V TM-I
60 44
APP
V715A
(GTG ! GCC)
E17
TM-I
42
Memory impairment, irritability Memory impairment, disorientation, behavioural changes, aphasia Impairment of recent verbal memory and visuospatial functions
Data not available (Father: suicide approximately 50 years) Brother (PSEN1 M139 V) (40 years), Mother’s brother (50 years) (mother died in 3rd decade), Grandmother and two brothers (approximately 50 years) Sister (PSEN1 H163 R) (51 years), Mother (50 years) Sister, brother (before 35 years), Mother and her sister (before 35 years) Mother—late onset AD (80 years) Mother (approximately 45 years)
a
Data not available (Father—sudden death before 50 years)
Novel mutations.
behaviour. Neurological examination showed bradykinesia and myoclonus of the upper limbs. There was also an evidence of two epileptic seizures. A second novel mutation I213F (ATT ! TTT) in PSEN1 gene was identified in a male patient, with the age of onset at 33 years. First symptom he had experienced was progressive impairment of memory. After 2 years, his mood became labile and he was disoriented to time and space. Neurological examination revealed bradykinesia and rigidity. After 5 years from the onset of the disease, the MMSE score was 10. Four members of his family (sister, brother, mother and aunt) had died before the age of 40 with probable AD, but no postmortem examinations were obtained in those cases. A recognized mutation M139V (ATG ! GTG) in PSEN1 gene was observed in siblings (man and woman, with age of onset at 40 years). There were some differences between siblings observed in clinical symptoms and the course of the disease. However, in both cases, the course of the disease was rapid and devastating. The sister had progressive memory, language and praxis deficits leading to severe dementia and aphasia 2 years after the beginning of symptoms. Her brother had prominent memory, visuospatial and behavioural disturbances, although the language functions were relatively preserved. After 2 years, he was also severely demented. A pathogenic mutation H163R (CAT ! CGT) in PSEN1 gene was detected in two sisters, with age of onset at 50 and 51 years of age. The clinical course was typical for AD and the older of the sisters developed severe dementia 5 years after the onset of the disease. The younger was mildly demented 2 years after the onset of symptoms. A novel mutation Q228L (CAG ! CTG) in PSEN2 gene was identified in one patient with a positive familial history
of EOAD and the diagnosis of MCI. Symptoms were initiated at the age of 60, and 2 years later, she reported slowly but significantly intensifying irritability, verbal aggressivity, abrasiveness and insomnia along with mild deficits in memory and short episodes of disorientation. The MMSE score was 28. After following 2 years of observation, she was still diagnosed as MCI, but the slight progression in cognitive impairment was shown. Her 88 years old mother has probable AD and is a resident in the nursing home. Due to ethical reasons, no segregation analysis of the mutation was performed in the risk family. Two previously recognized mutations in APP gene were detected in two patients. An Iranian mutation T714A (ACA ! GCA) was detected in a man with the age of onset at 44 years of age. After 4 years, he has developed severe memory impairment, disorientation to space and time, behavioral changes and aphasia. The same mutation was identified in a second family living in the same city, and was recognized as a result of probable consanguinity. The age of onset in the examined male patient from the second family was similar (45 years old) and he had memory impairment, spatial disorientation and aphasia. A German mutation V715A (GTG ! GCG) was identified in a female patient with the onset at 42 years old. Neuropsychological examination at the age of 44 showed severe cognitive impairment, especially in recent verbal memory and visuospatial tasks. Nine silent, common polymorphic substitutions in the analyzed regions of three genes were identified in the familial AD group. Table 2 lists all identified polymorphisms. Only one silent, intronic substitution was detected in analyzed regions of APP gene (IVS16 nt + 47a ! g). In the same person, E318G substitution in exon 9 of PSEN1 gene was detected. As the possible role of this substitution is
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Table 2 Polymorphisms identified in the examined group Gene
Region
Substitution
Comments
PSEN1 PSEN1 PSEN1 PSEN2 PSEN2 PSEN2 PSEN2 PSEN2 APP
5VUTR E9 I9 E3 I3 I3 I3 I5 I16
48c ! t E318G (GAA ! GGA) nt + 17t ! g H87H (CAT ! CAC) nt 42a ! g nt 41t ! c nt 29c ! t nt + 30g ! c nt + 47a ! g
rare rarea very common very common very common very common common rare rarea
a
Both polymorphisms detected in one patient.
controversial, a panel of 100 Polish patients with sporadic AD and 100 age-matched control subjects were screened for E318G. It was found that E318G was present in SAD patients, as well as in controls with a frequency of about 3.4%. The difference in E318G frequency between SAD patients and controls was statistically insignificant. Additionally, two apparently silent polymorphisms in PSEN2 gene were identified in a group of sporadic AD patients: S236S (AGT/AGC), A252A (GCG/GCC). Frequency of APOE4 allele was higher in SAD patients, compared with the FAD cases (v2 test, P = 0.0458). There were no differences in frequencies of a common PSEN1 promoter polymorphisms genotypes ( 48 CC and 48 CT) between patients with FAD and SAD. (v2 test, P = 0.5910). Additionally, there was no interaction between this polymorphism and APOE4 allele in the reported group. However, in a group of 100 SAD patients, there is a statistically significant association between APOE4 allele and CT genotype compared with CC genotype (v2 test, P = 0.0016, data not shown). In other studies, CC genotype was associated with increased risk for EOAD (but not late-onset AD), independent of the APOE4 genotype (Theuns et al., 2003). Presented results could be due to a statistical bias; however, it could reflect population differences.
Discussion In the reported group, mutations were detected in all three analyzed genes. Frequency of mutations is relatively low (17%), possibly due to used definition of a patient with familial AD (a patient having at least one relative with presenile dementia). In the reported group, the only two affected relatives of patients have had identified PSEN1 mutations. In highly selected group of patients, with two or more relatives in two or more generations affected, similar screening gave a prevalence of pathogenic mutations of 68% (Cruts et al., 1998; Janssen et al., 2003; Kamimura et al., 1998). However, the detection rate reported previously ranges from 18% to 56%, depending on the method of patient selection, population screened and the mutation detection technique used (Campion et al., 1999). No differences in utility and sensitivity of mutation detection in
coding regions were observed between RT-PCR method and exon-screening method. However, sequencing of genomic DNA could potentially reveal clinically important polymorphisms. Mutations identified in PSEN1 and APP genes showed almost complete penetrance by the age of 55 years. A lower age of onset was observed in a patient with a novel mutation I213F (before 35 years) and the sib pair bearing M139V mutation (40 years of age). In the latter case, a slightly different course of the disease in both siblings was observed. This is in accordance with the previously published data, indicating that M139V mutation can be presented differently between kindreds (Finckh et al., 2000b; Fox et al., 1997; Rippon et al., 2003; Sandbrink et al., 1996). The clinical course of a patient with Q228L substitution resembles that of other patients with known PSEN2 mutations. Q2228L mutation was absent in a group of 100 Polish patients with sporadic AD and 100 age-matched healthy controls. Analysis using ConSeq program (http://conseq. bioinfo.tau.ac.il/) suggests that Q228 residue is functional, highly conserved in evolution and exposed in the protein structure. The substitution affects the residue conserved between PS2 and PS1 and also affects the amino acid residue located in the transmembrane domain V, near two other known PSEN2 mutations (M239V, M239I) (Finckh et al., 2000a). A mutation in homologous position in PSEN1 gene (Q222R) was observed in a German population, indicating a functional relevance of Q228 residue in PS2 (Rogaeva et al., 2001). Collected information strongly suggests that Q228L substitution could be considered as a disease-causing mutation. A novel mutation P117R (CCA ! CGA) in PSEN1 gene is located at the same codon as two other highly pathogenic mutations (P117S and P117L) detected in Polish population previously (Dowjat et al., 2002; Wisniewski et al., 1998). One of the lowest age of onset in the presented group (36 years old) and the first characteristic symptoms (i.e. memory loss) resemble phenotypic features of patients with P117S and P117L mutations. ConSeq program analysis suggests that codon 117 is also a conserved one (however, the normalized conservation score is lower than in the case of codon 228 of PS2). On this basis, mutation P117R was annotated as pathogenic one. A novel mutation I213F (ATT ! TTT) in PSEN1 gene is located at the same codon as two other pathogenic mutations (I213T and I213L) detected previously (Kamino et al., 1996; Rogaeva et al., 2001). The patient is characterized with the lowest age of onset in the presented group (33 years old). ConSeq program analysis suggests that codon 213 is a highly conserved one, and is located in transmembrane domain IV. On this basis, mutation I213F was annotated as pathogenic one. APP mutation T714A was detected previously in an Iranian family (Pasalar et al., 2002). The age of onset was 11 years higher than in the presented cases. The second APP mutation V715A was identified in different European
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countries, and seems to be a relatively common mutation, with the mean age of onset at 47 years of age (Beck et al., 2002; Cruts et al., 2002; De Jonghe et al., 2001). A substitution E318G in exon 9 of PSEN1 gene was detected in one patient. The role of this mutation is controversial. Initially it has been reported to cause AD with a variable age of onset (Cruts and van Broeckhoven, 1998). However, the mutation was found in a number of non-demented control individuals, as well as patients of early- and late-onset AD with or without a known family history (Arango et al., 2001; Helisalmi et al., 2000). E318G substitution is regarded as either a rare polymorphism, a neutral mutation or a mutation of incomplete penetrance (Taddei et al., 2002). It is possible, however, that the putative pathogenetic effect depends on the ethnic background. In Polish subjects, however, the difference in E318G frequency between SAD patients and controls was statistically insignificant. This could suggest that E318G is a silent polymorphism in Polish population. No mutations in PSEN1, PSEN2 and APP genes have been found in 34 probands. In this group, the mean age of onset was 54.7 F 6.7 years old as compared to patients with identified mutations (43.57 F 9.08 years). The difference in mean age of onset between the groups was statistically significant (t test; P < 0.05). The APOE4 frequency in the group without mutation was higher than the one in the control Polish population, and lower than the one in lateonset sporadic AD cases (19% vs. 11% and 32%, respectively; v2 test, P = 0.2, NS) (Styczynska et al., 2003). Three patients are APOE4 homozygotes. It is possible that some of the patients have AD caused by the presence of one or two APOE4 alleles. An increased APOE4 allele frequency among early-onset AD patients without identified mutations was observed in other studies (Lleo et al., 2002). It could be also speculated that in these families, disease-causing mutations fall outside the analyzed regions or that large gene rearrangement is missed. A rare intronic polymorphism in APP gene (IVS16 nt + 47a ! g) was found in one patient with E318G substitution. This patient bears also A117A polymorphism in PRNP gene, rare among Polish AD patients (data not shown). This could suggest a distinct genetic background in this case. Other silent substitutions in PSEN1 and PSEN2 genes were common ones, present at least in 10 FAD alleles, and also found in healthy control people. Exonic PSEN2 polymorphisms (S236S and A262A) were detected in SAD patients only. Additionally, there was no interaction between PSEN1 48c ! t polymorphism and APOE4 allele in the reported group. Nevertheless, in a group of 100 SAD patients, there is a statistically significant association between APOE4 allele and CT genotype compared with CC genotype (v2 test, P = 0.0016, data not shown). In other studies, CC genotype was associated with increased risk for EOAD (but not late-onset AD), independent of the APOE4 genotype (Araria-Goumidi et al., 2002; Lambert et al., 2001;
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van Duijn et al., 1999). Presented results could be due to a statistical bias; however, it could reflect a population difference. It is concluded that molecular diagnosis could be used to confirm familial AD in a well-designed clinical setting (Croes et al., 2000). Currently, the absence of effective prophylactic treatments for AD limits presymptomatic and antenatal testing for ethical reasons (Hedera, 2001). However, if any of the experimental prevention or treatment strategies proves to be effective, molecular diagnosis in risk families will become very important. This study suggests a possible screening strategy in Poland.
Acknowledgment This work was supported by State Committee for Scientific Research (KBN) grant no. PBZ-KBN/002/CD/ P05/2000.
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