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Figure 1.
The pedigree of the family, indicating the proband (arrow). Black symbols indicate affected members; white symbols, unaffected members; squares, men; and circles, women.

The pedigree of the family, indicating the proband (arrow). Black symbols indicate affected members; white symbols, unaffected members; squares, men; and circles, women.

Figure 2.
DNA sequence of exon 6 of the presenilin 1 gene. The Leu166Arg mutation is indicated by an arrow.

DNA sequence of exon 6 of the presenilin 1 gene. The Leu166Arg mutation is indicated by an arrow.

1.
Sherrington  RRogaev  ELiang  Y  et al.  Cloning a gene bearing missense mutation in early-onset familial Alzheimer's disease. Nature. 1995;375754- 760Article
2.
Levy-Lahad  EWasco  WPoorkaj  P  et al.  Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science. 1995;269973- 977Article
3.
Goate  AChartier-Harlin  MCMullan  M  et al.  Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature. 1991;349704- 709Article
4.
Saunders  AStrittmatter  WSchemechel  D  et al.  Association of apolipoprotein E allele ϵ4 with late-onset familial and sporadic Alzheimer's disease. Neurology. 1993;431467- 1472Article
5.
Corder  EHSaunders  AMStrittmatter  WJ  et al.  Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late-onset families. Science. 1993;261921- 923Article
6.
Adroer  RSantacruz  PBlesa  RLopez-Pousa  SAscaso  COliva  R Apolipoprotein E4 allele frequency in Spanish Alzheimer and control cases. Neurosci Lett. 1995;189182- 186Article
7.
Blesa  RAdroer  RSantacruz  PAscaso  CTolosa  EOliva  R High apolipoprotein E allele frequency in age-related memory decline. Ann Neurol. 1996;39548- 552Article
8.
Kamboh  MISanghera  DKFerrell  REDeKosky  ST APOE4 associated Alzheimer's disease risk is modified by α-antichymotrypsin polymorphism. Nat Genet. 1995;10486- 488Article
9.
Ezquerra  MBlesa  RTolosa  EBallesta  FOliva  R α-Antichymotrypsin and risk for Alzheimer's disease in the Spanish population. Neurosci Lett. 1998;240107- 109Article
10.
Okuizumi  KOnodera  ONamba  Y  et al.  Genetic association of the very low density lipoprotein (VLDL) receptor gene with sporadic Alzheimer's disease. Nat Genet. 1995;11207- 209Article
11.
Lehmann  DJJohnston  CSmith  AD Synergy between the genes for butyrylcholinesterase K variant and apolipoprotein E4 in late-onset, confirmed Alzheimer's disease. Hum Mol Genet. 1997;61933- 1936Article
12.
Blaker  DWilcox  MALaird  NM  et al.  Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nat Genet. 1998;19357- 360Article
13.
Kwan-Fu  RSAbraham  AMVahram  H  et al.  Modulation of the genetic risk of Alzheimer's disease in a very elderly population. Ann Neurol. 1999;4548- 53Article
14.
Yasuda  MMaeda  KHashimoto  M  et al.  A pedigree with a novel presenilin 1 mutation at a residue that is not conserved in presenilin 2. Arch Neurol. 1999;5665- 69Article
15.
Hutton  MBusfield  FWraag  M  et al.  Complete analysis of the presenilin 1 gene in early-onset Alzheimer's disease. Neuroreport. 1996;7801- 805Article
16.
Cruts  MVan Duijn  CMBackhoven  H  et al.  Estimation of the genetic contribution of presenilin-1 and -2 mutations in a population-based study of presenile Alzheimer's disease. Hum Mol Genet. 1998;743- 51Article
17.
Wraag  MHutton  MTalbot  C  et al.  Genetic association between intronic polymorphism in presenilin-1 gene and late-onset Alzheimer's disease. Lancet. 1996;347509- 512Article
18.
Ezquerra  MBlesa  RTolosa  E  et al.  The genotype 2/2 of the presenilin-1 is decreased in Spanish early-onset Alzheimer's disease. Neurosci Lett. 1997;227201- 204Article
19.
Peña-Casanova  JAguilar  MBertrán-Serra  I  et al.  Normalización de instrumentos cognitivos y funcionales para la evaluación de la demencia (NORMACODEM), I: objetivos, contenidos y población. Neurologia. 1997;1261- 68
20.
Oliva  RTolosa  EEzquerra  M  et al.  Significant changes in the τ A0 and A3 alleles in progressive supranuclear palsy and improved genotyping by silver detection. Arch Neurol. 1998;551122- 1124Article
21.
Wenham  PRPrice  WHBlundell  G Apolipoprotein E genotyping by one-stage PCR. Lancet. 1991;3371158- 1159Article
22.
Ezquerra  MCarnero  CBlesa  RGelpí  JLBallesta  FOliva  R A presenilin 1 mutation (Ser169Pro) associated with early-onset AD and myoclonic seizures. Neurology. 1999;52566- 570Article
23.
Ramírez  MGRogaeva  EALeal  CA  et al.  A novel Leu171Pro mutation in presenilin-1 gene in a Mexican family with early-onset Alzheimer disease. Ann Genet. 1998;41149- 153
24.
Kamino  KSato  SSakaji  Y  et al.  Mutational analysis of presenilin-1 gene in early-onset Alzheimer's disease [abstract]. Am J Hum Genet. 1996;5933
25.
Campion  DFlaman  JLBrice  A  et al.  Mutations of the presenilin 1 gene in families with early-onset Alzheimer's disease. Hum Mol Genet. 1995;42373- 2377Article
26.
Kwok  JBTaddei  KHallup  M  et al.  Two novel (M233T and R278T) presenilin-1 mutations in early-onset Alzheimer's disease pedigrees and preliminary evidence for association of presenilin-1 mutations with a novel phenotype. Neuroreport. 1997;81537- 1542Article
27.
Crook  RVerkkoniemi  APerez-Tur  J  et al.  A variant of Alzheimer's disease with spastic paraparesis and unusual plaques due to deletion of exon 9 of presenilin 1. Nat Med. 1998;4452- 455Article
28.
Perez-Tur  JFroelich  SPrihar  G  et al.  A mutation in Alzheimer's disease destroying a splice acceptor site in the presenilin 1 gene. Neuroreport. 1996;7297- 301Article
29.
Tysoe  CWhittaker  JXuereb  J  et al.  A presenilin-1 truncating mutation is present in two cases with autopsy-confirmed early-onset Alzheimer disease. Am J Hum Genet. 1998;6270- 76Article
Original Contribution
April 2000

A Novel Presenilin 1 Mutation (Leu166Arg) Associated With Early-Onset Alzheimer Disease

Author Affiliations

From the Genetics Service IDIBAPS—Institut de investigació Biomédica Agustí Pi y Sunyer, Hospital Clínic i Provincial and University of Barcelona (Mr Ezquerra and Dr Oliva) and the Neurology Service, Hospital Clínic i Provincial (Dr Blesa), Barcelona, Spain; and the Neurology Service, Hospital Universitario Virgen de las Nieves, Granada, Spain (Dr Carnero).

Arch Neurol. 2000;57(4):485-488. doi:10.1001/archneur.57.4.485
Abstract

Background  Pathogenic mutations in the presenilin 1 (PS1) gene leading to early-onset Alzheimer disease have been described in various populations. The different mutations are not distributed randomly in the PS1 protein but are clustered in some PS1 exons.

Objective  To screen the PS1 gene in search of a potential mutation in a Spanish family with early-onset Alzheimer disease.

Methods  Single-stranded conformational polymorphism and heteroduplex analyses of all exons were used to search for a potential mutation. Subsequent sequencing of the DNA samples with an abnormal heteroduplex pattern was performed to identify the mutation in the sense strand and in the complementary strand.

Results  We found a novel mutation in exon 6 of the PS1 gene at a site that, so far, had not been described as a cluster of mutations. The mutation (an A to C change) causes a substitution of leucine for arginine at position 166 of the PS1 protein and is located adjacent to the transmembrane domain III, where few mutations have been found. In this family, the disease follows an autosomal inheritance pattern with early onset (range, 32-44 years).

Conclusion  A novel missense mutation (Leu166Arg) at an atypical site associated with early-onset Alzheimer disease has been identified in a Spanish family.

MUTATIONS IN several genes have been described as causes of Alzheimer disease (AD): the presenilin 1 (PS1) gene in chromosome 14,1 the presenilin 2 gene (PS2) in chromosome 1,2 and the amyloid precursor protein3 in chromosome 21. In addition, several risk factors associated with AD have been described, such as the presence of variants of apolipoprotein E47 in chromosome 19, α-antichymotrypsin,8,9 the low-density lipoprotein receptor,10 butyrylcholinesterase K,11 α-2 macroglobulin,12 and a subunit of the α-ketoglutarate dehydrogenase complex.13 The PS1, PS2, and amyloid precursor protein genes have been found to cause familial early-onset AD, whereas the APOE (apolipoprotein E) gene and other risk factors are involved in sporadic or familial AD with different ages at onset. PS1 gene mutations are involved in 18% to 50% of the autosomal dominant early-onset AD cases.1,1416 More than 40 different mutations of the PS1 gene have been identified in patients with AD in several populations. In addition, a polymorphism in a PS1 intron has been associated with AD being a potential risk factor for late-onset AD.17,18 In contrast, typical PS1 coding mutations result in a dominant inheritance pattern and cause early-onset AD with high penetrance. In the process of screening for mutations in the PS1 gene in patients with familial early-onset AD, we identified a novel mutation in exon 6 (Leu166Arg) in the helical end of transmembrane domain (TM) III, which causes AD in the fourth decade. So far, this domain has not been described as a cluster of mutations in the PS1 gene.

PATIENTS AND METHODS
PATIENTS

The proband (patient III-2) (Figure 1) came from a family with a history of dementia in different generations and was diagnosed at the Neurology Service of the Hospital Universitario Virgen de las Nieves, Granada, Spain. He was a 33-year-old cook who was admitted to the hospital with memory decline and inability to handle the kitchen tasks on the job. No myoclonic jerks or abnormal movements were observed. Results of neurologic examination revealed a grasping reflex, bradykinesia, and cognitive impairment. The state of dementia and memory loss were tested using the Clinical Dementia Rating Scale (patient's score, 2) and the Global Deterioration Scale (patient's score, 6). The Spanish validated version of the Mini-Mental State Examination was used.19 The initial results were 22/30 on the Mini-Mental State Examination, 1 on the Clinical Dementia Rating Scale, and 4 on the Global Deterioration Scale. Reassessment 6 months later revealed deterioration in the clinical course (Mini-Mental State Examination score, 19/30; Clinical Dementia Rating Scale score, 2; and Global Deterioration Scale score, 5). Magnetic resonance images revealed a cortical atrophy, and single photon emission computed tomographic analysis indicated the presence of parietal hypoperfusion.

The subject (patient II-5) was a 47-year-old woman admitted to the same hospital with a memory loss of 3 years of evolution and being unable to handle simple tasks. She had become irritable and depressed. She had aphasia and akinetic movements. Electroencephalograms showed that brain activity was asynchronized, and the computed tomographic scan revealed enlarged ventricles. Her mother (patient I-2) had a similar pathologic course and died at age 44 years. One brother and 2 sisters (patients II-1, II-3, and II-4) also developed early-onset AD and died at ages 38, 42, and 42 years, respectively. No consanguinity was present in this family.

MOLECULAR GENETIC STUDIES

The PS1 exons have been analyzed by polymerase chain reaction amplification of genomic DNA using published primers sets.15 After 2% agarose gel electrophoresis of the polymerase chain reaction products, the bands corresponding to the amplified product were excised and purified with a microcolumn and sequenced using the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems, Warrington, England) with an automatic sequencer (Abi Prism 310 Genetic Analyzer; Perkin Elmer Applied Biosystems). The Leu166Arg mutation was identified in the sense strand and confirmed through the sequencing of the complementary strand (Figure 2) in different members of the family. We screened 60 control subjects for the presence of the Leu166Arg change using the DNA heteroduplex technique. The polymerase chain reaction product from exon 6 was denatured to 95°C for 5 minutes in a buffer containing 95% formamide, cooled for 10 minutes at 20°C, and run for 5 hours in a 7% polyacrylamide gel at 200 V at room temperature. Subsequently, the gel was silver stained as described previously.20 The mutation was detected by the presence of a double band corresponding to the heteroduplex chain (data not shown). Apolipoprotein E genotyping was performed using polymerase chain reaction amplification and Hha I restriction fragment analysis as described previously.21

RESULTS

The average age at onset of affected members in this family is 32 to 44 years. Apolipoprotein E genotyping revealed that patients III-1, III-2, and III-3 carried the 3/2 genotype; patient III-4 had the 3/3 genotype; and patient II-6 had the 4/3 genotype. The Leu166Arg mutation was present in affected member III-2 (Figure 1) and in young healthy members III-3 and III-4 (<30 years). The Leu166Arg mutation was absent in healthy older members III-1 (37 years) and II-6 (60 years), supporting the fact that the Leu166Arg change segregates with AD in this family. In addition, the Leu166Arg mutation was not detected in 60 healthy controls and in 54 unrelated patients with AD, supporting the fact that this mutation is not a common polymorphism.

COMMENT

We identified a novel mutation (Leu166Arg) in the PS1 gene associated with early-onset AD (range, 32-44 years) in a family of Spanish origin. The absence of this mutation in 1 disease-free older sibling, 1 disease-free surviving uncle, 60 unrelated controls, and 54 unrelated patients with AD supports a modestly strong correlation of this mutation with AD. The amino acid position 166 of the PS1 protein is highly conserved in different species and in different homologous proteins (PS2 and Sel12), indicating that this position is important for PS1 function. It is located in TMIII in exon 6 at a site that had not previously been described as a cluster of mutations. Many of the PS1 mutations are clustered in some regions of the PS1 protein. Two well-known clusters of mutations correspond to exon 5 (TMII and hydrophilic loop II) and exon 8 (hydrophilic loop VI). Exon 6, corresponding to TMIII and part of hydrophilic loop II, was not considered a cluster of mutations, where initially only amino acid position 163 had been found to be mutated.1 However, recently 2 additional novel mutations have been found in this exon: Ser169Pro22 and Leu171Pro.23 Thus, together with the presently reported Leu166Arg mutation, 4 different mutated positions have now been described in exon 6. In the Leu166Arg mutation, the neutral and hydrophilic amino acid leucine is mutated to positively charged amino acid (arginine) in the helical end of TMIII. Because of the marked change in the expected physical properties of the mutated residue, which in addition is adjacent to TMIII, a drastic change in function of the PS1 protein can be expected. Also, in the nearby position (His163), 3 different mutations have been found, changing to a hydrophobic residue (Ile),24 a polar residue (Tyr),1 or a charged residue (Arg).25

Detection of clusters of PS1 mutations is important to determine which of the different domains are important for PS1 normal function. In addition, age at onset (range, 28-68 years) and severity and characteristics of the clinical picture associated with each mutation might help determine the importance of each residue in the normal and pathologic function of PS1. However, in this family, the Leu166Arg mutation causes AD, with an age at onset of 32 to 44 years, suggesting that other environmental or genetic factors might modify the effect of this PS1 mutation. The clinical symptoms associated with AD are also different in the different mutations. For instance, some PS1 mutations cause AD with myoclonus seizures.21 Moreover, 2 PS1 mutations have been associated with AD copresenting with familial spastic paraparesis.26,27 The role of the PS1 protein in AD remains unclear, but it is proposed that it acts through a gain of function or through dominant loss of function.1 Most PS1 mutations are inherited in an autosomal dominant fashion and are either missense16 or in-frame splice mutations,28 but not nonsense mutations or deletions that would be expected to cause a loss of function. However, recently it has been reported a deletion, in a splice donor consensus sequence of 2 patients with AD, associated with 2 short transcripts with premature termination codons probably leading a nonfunctional protein,29 suggesting, at least in this case, that the pathogenic mechanism might be caused by haploinsufficiency.

The pathogenic mechanism of the PS1 protein is not completely understood. Detection of the Leu166Arg mutation in exon 6—together with the mutations found in PS1 protein positions 163, 169, and 171—suggests that this site of the PS1 protein should be reassessed as an additional cluster of pathogenic mutations. The opportunity is now open to perform functional studies of the mutated residues.

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Article Information

Accepted for publication November 9, 1999.

This work was supported by grant FIS 96-0658 from the Fondo de Investigaciones Sanitarias, Barcelona, Spain (Dr Oliva).

Corresponding author: Rafael Oliva, MD, PhD, Genetics Service IDIBAPS, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain (e-mail: oliva@medicina.ub.es).

References
1.
Sherrington  RRogaev  ELiang  Y  et al.  Cloning a gene bearing missense mutation in early-onset familial Alzheimer's disease. Nature. 1995;375754- 760Article
2.
Levy-Lahad  EWasco  WPoorkaj  P  et al.  Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science. 1995;269973- 977Article
3.
Goate  AChartier-Harlin  MCMullan  M  et al.  Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature. 1991;349704- 709Article
4.
Saunders  AStrittmatter  WSchemechel  D  et al.  Association of apolipoprotein E allele ϵ4 with late-onset familial and sporadic Alzheimer's disease. Neurology. 1993;431467- 1472Article
5.
Corder  EHSaunders  AMStrittmatter  WJ  et al.  Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late-onset families. Science. 1993;261921- 923Article
6.
Adroer  RSantacruz  PBlesa  RLopez-Pousa  SAscaso  COliva  R Apolipoprotein E4 allele frequency in Spanish Alzheimer and control cases. Neurosci Lett. 1995;189182- 186Article
7.
Blesa  RAdroer  RSantacruz  PAscaso  CTolosa  EOliva  R High apolipoprotein E allele frequency in age-related memory decline. Ann Neurol. 1996;39548- 552Article
8.
Kamboh  MISanghera  DKFerrell  REDeKosky  ST APOE4 associated Alzheimer's disease risk is modified by α-antichymotrypsin polymorphism. Nat Genet. 1995;10486- 488Article
9.
Ezquerra  MBlesa  RTolosa  EBallesta  FOliva  R α-Antichymotrypsin and risk for Alzheimer's disease in the Spanish population. Neurosci Lett. 1998;240107- 109Article
10.
Okuizumi  KOnodera  ONamba  Y  et al.  Genetic association of the very low density lipoprotein (VLDL) receptor gene with sporadic Alzheimer's disease. Nat Genet. 1995;11207- 209Article
11.
Lehmann  DJJohnston  CSmith  AD Synergy between the genes for butyrylcholinesterase K variant and apolipoprotein E4 in late-onset, confirmed Alzheimer's disease. Hum Mol Genet. 1997;61933- 1936Article
12.
Blaker  DWilcox  MALaird  NM  et al.  Alpha-2 macroglobulin is genetically associated with Alzheimer disease. Nat Genet. 1998;19357- 360Article
13.
Kwan-Fu  RSAbraham  AMVahram  H  et al.  Modulation of the genetic risk of Alzheimer's disease in a very elderly population. Ann Neurol. 1999;4548- 53Article
14.
Yasuda  MMaeda  KHashimoto  M  et al.  A pedigree with a novel presenilin 1 mutation at a residue that is not conserved in presenilin 2. Arch Neurol. 1999;5665- 69Article
15.
Hutton  MBusfield  FWraag  M  et al.  Complete analysis of the presenilin 1 gene in early-onset Alzheimer's disease. Neuroreport. 1996;7801- 805Article
16.
Cruts  MVan Duijn  CMBackhoven  H  et al.  Estimation of the genetic contribution of presenilin-1 and -2 mutations in a population-based study of presenile Alzheimer's disease. Hum Mol Genet. 1998;743- 51Article
17.
Wraag  MHutton  MTalbot  C  et al.  Genetic association between intronic polymorphism in presenilin-1 gene and late-onset Alzheimer's disease. Lancet. 1996;347509- 512Article
18.
Ezquerra  MBlesa  RTolosa  E  et al.  The genotype 2/2 of the presenilin-1 is decreased in Spanish early-onset Alzheimer's disease. Neurosci Lett. 1997;227201- 204Article
19.
Peña-Casanova  JAguilar  MBertrán-Serra  I  et al.  Normalización de instrumentos cognitivos y funcionales para la evaluación de la demencia (NORMACODEM), I: objetivos, contenidos y población. Neurologia. 1997;1261- 68
20.
Oliva  RTolosa  EEzquerra  M  et al.  Significant changes in the τ A0 and A3 alleles in progressive supranuclear palsy and improved genotyping by silver detection. Arch Neurol. 1998;551122- 1124Article
21.
Wenham  PRPrice  WHBlundell  G Apolipoprotein E genotyping by one-stage PCR. Lancet. 1991;3371158- 1159Article
22.
Ezquerra  MCarnero  CBlesa  RGelpí  JLBallesta  FOliva  R A presenilin 1 mutation (Ser169Pro) associated with early-onset AD and myoclonic seizures. Neurology. 1999;52566- 570Article
23.
Ramírez  MGRogaeva  EALeal  CA  et al.  A novel Leu171Pro mutation in presenilin-1 gene in a Mexican family with early-onset Alzheimer disease. Ann Genet. 1998;41149- 153
24.
Kamino  KSato  SSakaji  Y  et al.  Mutational analysis of presenilin-1 gene in early-onset Alzheimer's disease [abstract]. Am J Hum Genet. 1996;5933
25.
Campion  DFlaman  JLBrice  A  et al.  Mutations of the presenilin 1 gene in families with early-onset Alzheimer's disease. Hum Mol Genet. 1995;42373- 2377Article
26.
Kwok  JBTaddei  KHallup  M  et al.  Two novel (M233T and R278T) presenilin-1 mutations in early-onset Alzheimer's disease pedigrees and preliminary evidence for association of presenilin-1 mutations with a novel phenotype. Neuroreport. 1997;81537- 1542Article
27.
Crook  RVerkkoniemi  APerez-Tur  J  et al.  A variant of Alzheimer's disease with spastic paraparesis and unusual plaques due to deletion of exon 9 of presenilin 1. Nat Med. 1998;4452- 455Article
28.
Perez-Tur  JFroelich  SPrihar  G  et al.  A mutation in Alzheimer's disease destroying a splice acceptor site in the presenilin 1 gene. Neuroreport. 1996;7297- 301Article
29.
Tysoe  CWhittaker  JXuereb  J  et al.  A presenilin-1 truncating mutation is present in two cases with autopsy-confirmed early-onset Alzheimer disease. Am J Hum Genet. 1998;6270- 76Article
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