Alzheimer's disease associated with mutations in presenilin 2 is rare and variably penetrant
Alzheimer's disease associated with mutations in presenilin 2 is rare and variably penetrantR. Sherrington1, S. Froelich2, S. Sorbi3, D. Campion4, H. Chi1, E. A. Rogaeva1, G. Levesque1, E. I. Rogaev1, C. Lin1, Y. Liang1, M. Ikeda1, L. Mar5, A. Brice6, Y. Agid6, M. E. Percy7, F. Clerget-Darpoux8, S. Piacentini3, G. Marcon9, B. Nacmias3, L. Amaducci3, T. Frebourg4, L. Lannfelt2, J. M. Rommens5 and P. H. St George-Hyslop1,*
1Centre for Research in Neurodegenerative Diseases, Department of Medicine, University of Toronto; Department of Medicine, Division of Neurology, The Toronto Hospital, Toronto, Ontario M5S 1A8, Canada, 2Karolinska Institute, Department of Clinical Neurosciences, Geriatric Medicine, Novum, KFC, 141 86 Huddinge, Sweden, 3Department of Neurology and Psychiatry, University of Florence, Viale Morgagni 65, Florence, Italy, 4Laboratoire de Genetique Moleculaire, CHU Rouen, 76031 Rouen, France, 5Department of Medical and Molecular Genetics, University of Toronto, and Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, 6Department of Neurology, Hopital de la Salpetriere, and INSERM U289, 75013 Paris, France, 7Surrey Place Centre, Department of Obstetrics and Gynaecology and Dept. of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and 8INSERM U155, Chateau de Longchamp, 75016, Paris, France and 9Department of Neurology, University of Udine, Udine, Italy
Received February 20, 1996;Revised and Accepted April 17, 1996
Missense mutations in the presenilin 2 (PS-2) gene on chromosome 1 were sought by direct nucleotide sequence analysis of the open reading frame of 60 pedigrees with familial Alzheimer's disease (FAD). In the majority of these pedigrees, PS-1 and [beta]-amyloid precursor protein ([beta]APP) gene mutations had been excluded. While no additional PS-2 pathogenic mutations were detected, four silent nucleotide substitutions and alternative splicing of nucleotides 1338-1340 (Glu325) were observed. Analysis of additional members of a pedigree known to segregate a Met239Val mutation in PS-2 revealed that the age of onset of symptoms is highly variable (range 45-88 years). This variability is not attributable to differences in ApoE genotypes. These results suggest (i) that, in contrast to mutations in PS-1, mutations in PS-2 are a relatively rare cause of FAD; (ii) that other genetic or environmental factors modify the AD phenotype associated with PS-2 mutations; and (iii) that still other FAD susceptibility genes remain to be identified.
Missense mutations associated with early onsetAlzheimer's disease (AD) have been discovered in two related genes termed presenilin I on chromosome 14 (1 ) and presenilin II on chromosome 1 (2 ,3 ). Analysis of a number of large data sets have revealed that missense mutations in the presenilin I (PS-1) gene are a frequent cause of early onset familial Alzheimer's disease (FAD), accounting for perhaps as much as 50% of all cases of early onset FAD (1 ,2 ,4 -6 ). In order to determine the frequency of mutations in the presenilin II (PS-2) gene, we undertook a survey for mutations in the open reading frame (ORF) of the PS-2 gene in 60 pedigrees with FAD. The disease in these families was characterized by the occurrence of AD consistent with the NINDS-ADRDA criteria in at least three closely related subjects, often in more than one generation. In these pedigrees, the age at onset (range: 35-82 years; 36 with mean age at onset <= 64 years; 14 with mean age at onset >= 65 years; 10 with insufficient reliable data on age at onset) overlapped that observed in the pedigrees with known PS-2 mutations (onset between 45 and 72 years). To determine the penetrance of PS-2 mutations, we also examined several additional members of the FLO10 pedigree previously described with the Met239Val missense mutation in the PS-2 gene (2 ). Our data suggest that missense mutations in the PS-2 gene are a rare cause of AD and that these missense mutations may be subject to the modifying action of other genes or environmental influences.
Fibroblasts, transformed lymphoblasts or peripheral blood samples were obtained from at least one affected member of 60 kindreds with multiple (>3) family members affected by FAD that were ascertained through the familial Alzheimer's disease registry at the University of Toronto, through clinics at the University of Florence and the Massachusetts Alzheimer's Disease Research Centre, from the Karolinska Institute, Stockholm, Sweden, from French University Hospitals through the National INSERM network of France, and from the Indiana University Alzheimer's Disease DNA Bank and the NIA sponsored repository at the Coriell Institute. Diagnosis of AD in affected members used the NINDS-ADRDA criteria or the similar CERAD criteria. Mutations in the PS-1 and [beta]-amyloid precursor protein ([beta]APP) genes had previously been excluded in the majority of these pedigrees by sequencing of reverse transcriptase-polymerase chain reaction (RT-PCR) products corresponding to the PS-1 gene and genomic PCR products corresponding to exons 16 and 17 of [beta]APP.
Analysis of the nucleotide sequence of RT-PCR products corresponding to the ORF of PS-2 in these 60 FAD pedigrees failed to uncover any additional mutations beyond the two initial mutations (Asn141Ile mutation in three related kindreds of Volga-German origin, and the Met239Val mutation in the FLO10 pedigree of Italian origin) previously reported in this data set (2 ). Specific screening just for the Asn141Ile and Met239Val mutations in a supplemental set of genomic DNA samples from affected probands of 81 Swedish FAD pedigrees also failed to identify further instances of either mutation. However, although no pathogenic nucleotide sequence differences were observed, four `silent' nucleotide substitutions were detected (T -> C at 626 bp/codon 23; C -> T at 806 bp/codon 147; C -> T at 1571 bp/codon 402; and G -> A at 1670 bp/codon 435-sequence numbering from Accession no. L44577). All of these substitutions occurred at the third nucleotide position of a codon, and would therefore be predicted not to cause a change in the amino acid sequence. A fifth sequence variant-the variable presence of nucleotides 1338-1340 encoding the Glu325 residuewas found in transcripts from most tissues. Transcripts with and without nucleotides 1338-1340, which reflects the use of an alternate splice donor sequence, were of approximately equal abundance in brain, fibroblasts and leukocytes.
During the investigation of the PS-2 gene in several newly ascertained members of the FLO10 pedigree segregating the Met239Val mutation in PS-2, we identified an additional carrier of this mutation (subject III-20 in Table 1 ). Surprisingly, this subject was aged 87 years and had had no evidence for cognitive or memory impairment on serial follow-up over several years until age 84 years when she was noted to become mildly demented, and is now profoundly demented (Mini Mental Status Examination score 4/30) with clinical features that sustain a diagnosis of probable AD by the NINDS-ADRDA criteria. A review of clinical data on additional members of the extended pedigree also uncovered two other subjects in the direct lineage of the family who had either historical evidence for late onset AD (subject I-2, the affected grandfather of III-20 who died at 75 years with dementia) or who currently have clinical features consistent with probable AD using the NINDS-ADRDA criteria (subject III-39 onset at 88 years). We and others have previously reported that the genotype at the Apolipoprotein E gene can modulate the age of onset of Alzheimer's disease in subjects with the Val717Ile mutation in [beta]APP (7 ,8 ). However, in the FLO10 family with the Met239Val mutation in PS-2, ApoE genotypes do not correlate with age of onset (Table 1 ). Thus, the very elderly symptomatic subject III-20 (onset 84 years) has the same ApoE genotype ([epsilon]3/[epsilon]3) as three other affected carriers of the Met239Val mutation, all of whom developed symptoms at a younger age (onset 45-72 years). Moreover, the affected carrier IV-21, with the ApoE genotype of [epsilon]3/[epsilon]4 developed symptoms at an age (48 years) similar to that of his affected sibling IV-18 with an ApoE genotype of [epsilon]3/[epsilon]3 (onset = 45 years) (Table 1 , Fig. 1 ). Our sample size is too small to draw reliable conclusions about the presence or absence of interactions between the ApoE genotype and the Asn141Ile mutation (Table 1 ).
Subjects with familially clustered Alzheimer's disease (as defined by the occurrence of at least three first or second degree relatives with AD) were recruited from patients referred for the investigation of dementia at the University of Toronto, University of Florence, the Massachusetts Alzheimer's Disease Research Centre, the Huddinge Hospital of the Karolinska Institute, and French University Hospitals. All subjects provided informed consent using institutionally approved protocols. The diagnosis of AD was made in affected pedigree members by direct clinical examination and standard laboratory procedures which met or exceeded the stringency of the NINCDS-ADRDA criteria (11 ,12 ). Evidence of AD in deceased family members was ascertained through the investigation of medical and family records as previously described (13 ). Additional FAD pedigree samples were obtained from the Cornell Institute (Camden, NJ) (N = 4) and from the Indiana University Alzheimer's Disease DNA Bank (Indianapolis, IN) (N = 6).
Mutations were sought in the ORF of the PS-1 gene (47/60 pedigrees) and in exon 16 and 17 of the [beta]APP gene (51/60 pedigrees) using the methods previously described (1 ,14 ,15 ). The PS-2 gene ORF was investigated in the North American and Italian pedigrees by isolating complimentary DNA fragments by amplification using RT-PCR and the oligonucleotide primer sequences and reaction conditions as previously described (2 ). In the French families, RT-PCR was performed using primers F1: 5'-caggaaacagctatgaccgagctgaccctcaaatacgg and F2: 5'-tgtaaaacgacggccagtgagatcatacacagagatgg to recover codons 85-257, and primers F3: 5'-caggaaacagctatgacctcaagtacctcccagagtgg and F4: 5'-tgtaaaacgacggccagtagcctgtggcacaccatgtc to recover codons 251-448. The nucleotide sequence of the RT-PCR products for each affected pedigree member was determined using fluorescent dye terminator or dye primer cycle sequencing. Mutations were detected by analysis of the resultant chromatograms for heterozygous nucleotidesubstitutions using both direct inspection and the Factura (ver 1.2.0) and the Sequence Navigator (ver 1.0.1b15) software packages ABI, Foster City, CA.
The presence of the T -> C substitution at base pair 1624 of the PS-2 cDNA sequence which gives rise to the Met239Val mutation was assessed in genomic DNA as previously described (2 ).
Genotypes at ApoE were determined by PCR amplification and HhaI digestion as previously described (16 ,17 ).
This work was supported by the Alzheimer Association of Ontario, Canadian Genetic Diseases Network, Medical Research Council of Canada, American Health Assistance Foundation, National Institute of Health, the Scottish Rite Charitable Foundation, the Eva Gordon Memorial Trust, Lifebeat, the Swedish Medical Research Council (project 10819)(LL), INSERM (network 492002), Consiglio Nazionale delle Richerche (PF Aging 952631) (SS), Telethon Italy (E.304) (BN), Glaxo Canada (RS), Helen B. Hunter Foundation (GL and HC), Peterborough-Burgess Fellowship (EAR), Ontario Mental Health Foundation (MEP). PStGH and JMR are Scholars of the Medical Research Council of Canada. Cell lines were obtained from the Indiana University Alzheimer's Disease DNA bank (P30 AG10133).
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*To whom correspondence should be addressed
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