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Association between the presenilin-1 mutation Glu318Gly and complaints of memory impairment
Neurobiology of Aging 23 (2002) 55–58
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Association between the presenilin-1 mutation Glu318Gly and complaints of memory impairment Simon M. Lawsa, Roger M. Clarnettea,b, Kevin Taddeia, Georgia Martinsa, Athena Patona, Osvaldo P. Almeidab, Hans Fo¨rstlc, Ralph N. Martinsa,* a
Sir James McCusker Alzheimer’s Disease Research Unit and University of Western Australia, Department of Surgery, Hollywood Private Hospital, Nedlands, Perth 6009, Australia b University of Western Australia, Department of Psychiatry and Behavioural Science, Sir Charles Gairdner Hospital, Nedlands, Perth 6009, Australia c Department of Psychiatry and Psychotherapy, Technical University of Munich, Munich 81675, Germany Received 19 February; received in revised form 20 April 2001; accepted 10 May 2001
Abstract Presenilin-1 mutations account for nearly 50% of all early-onset familial cases of Alzheimer’s disease. Most of these mutations are completely penetrant, although the recently described Glu318Gly substitution seems to have only partial penetrance. These findings suggest that the Glu318Gly mutation may work as a genetic risk factor for Alzheimer’s disease. We designed the present study to investigate the frequency of this mutation among non-demented volunteers with subjective memory impairment (n ⫽ 58) and controls (n ⫽ 66). Four (6.8%) subjects with complaints of memory problems, but no controls, carried this mutation. The presence of the Glu318Gly mutation was associated with significantly lower cognitive performance when compared to controls (P ⫽ 0.011). However, there was no significant association between the presence of the mutation and the cognitive performance of individuals within the memory complainers group. Follow-up studies should clarify whether the Glu318Gly mutation increases the risk of cognitive decline in later life. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer’s disease; Presenilin-1; E318G; Subjective memory impairment
1. Introduction Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder that is the major cause of dementia in the elderly. Numerous highly penetrant mutations have been identified within the gene coding for Presenilin-1 (PS-1), located on chromosome 14, that account for over 50% of all early onset familial cases of AD [29]. At present 70 different missense mutations have been identified—most of which are pathogenic [26]. The pathogenicity of one PS1 mutation, namely Glu318Gly, has been questioned since it has also been found in a number of non-demented individuals [1]. This mutation does not seem to be sufficient to cause dementia but, like the ⑀4 allele of APOE, may increase the risk of AD.
* Corresponding author. Tel.: ⫹61-8-93466703; fax: ⫹61-8-93466666. E-mail address: [email protected] (R.N. Martins).
The recent availability of effective symptomatic treatment of AD and the prospect of further therapeutic advances have given impetus to the early diagnosis of the disease. The relationship between memory complaints in non-demented persons and future risk of dementia has prompted numerous studies [3–5,9,11,14 –17,20 –24,27,28,30,31]. A recent study within this cohort showed that memory complainers had worse cognitive performance than non-complainers, thus supporting the findings of other studies that suggest that subjective memory loss may be a reliable indicator of cognitive decline [6]. The association of genetic susceptibility factors such as APOE-⑀4 and the Glu318Gly mutation with cognitive decline may provide a starting point for the formulation of criteria for early diagnosis of AD. In this study, we screened at risk individuals who complained of memory impairment for the Glu318Gly mutation and assessed whether it was associated with lower CAMCOG scores. Since the ⑀4 allele of APOE is a major genetic risk factor for AD, we investigated whether there was an interaction between the ⑀4 allele, Glu318Gly mutation and
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S.M. Laws et al. / Neurobiology of Aging 23 (2002) 55–58
cognitive scores of non-demented subjects who complained of memory decline.
2. Materials and methods With approval from our institute’s ethics committee, we investigated the frequency of the Glu318Gly mutation among 58 individuals with subjective memory impairment (age; 62.5 ⫾ 1.3 years) and 66 age and education matched controls (age; 61.6 ⫾ 2.1 years). The 66 healthy controls were recruited from individuals referred to The Memory and Capacity Evaluation Unit at Osborne Park Hospital between 1996 and 1998. Additionally, advertisements were placed in local newspapers asking people with memory complaints to attend for assessment. Control subjects were recruited through advertisements in local papers and by asking spouses of complainers to volunteer for the study. All volunteers were reviewed clinically by an experienced physician (RC) who took the history and completed a physical and neurological examination. Those with dementia [2], Mini-Mental State Examination (MMSE) score lower that 24 [13], or prior history of stroke were excluded. A trained nurse (AP) interviewed all subjects using the Cambridge Examination for Mental Disorders of the Elderly (CAMDEX) [25]. The CAMDEX includes standardised patient and informant interviews, and a cognitive examination. The cognitive component (CAMCOG) includes tests of orientation, memory, language, praxis, attention, tactile perception, calculation, abstract thinking and visual perception. Scores can range from 0 to 107. A score of 80 or greater is considered to exclude dementia in most people [27]. The CAMCOG also includes questions that allow computation of the MMSE and the Abbreviated Mental Test Score (AMTS) [19]. In addition, the interviews gather demographic information and recent and remote medical history. Genomic DNA was extracted from collected peripheral blood samples. Glu318Gly variation was determined via polymerase chain reaction (PCR) assay using the forward ‘mismatched’ primer, 5⬘-ATCCAAAAATTCCAAGTATAATCCAG-3⬘ and the reverse primer 5⬘-CTGGGCATTATCATAGTTCTCAAG-3⬘ to generate a BstnI digestion site as described [7]. The amplified product was digested using the restriction enzyme BstnI and electrophoresed using 8% non-denaturing polyacrylamide gels. Gels were then stained with ethidium bromide and viewed on an UV transilluminator. For the determination of APOE alleles, genomic DNA was extracted from the collected peripheral blood samples and genotyped after being subjected to PCR amplification as described [18]. The oligonucleotide primers, (P1) 5⬘-TCCAAGGAGCTGCAGGCGGCGCA-3⬘ and (P2) 5⬘-ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3⬘, were used as described [32]. The amplified product was digested using the restriction enzyme HhaI and electrophoresed using 8% non-denaturing polyacrylamide gels. Gels were then stained
Table 1 CAMCOG scores for GLU318GLY (⫹) and GLU318GLY (⫺) controls and memory complainers
Controls GLU318GLY(⫹) GLU318GLY (⫺) Memory Complainers GLU318GLY (⫹) GLU318GLY (⫺)
n
CAMCOG
66
101.48 ⫾ 0.37
— 66
— 101.48 ⫾ 0.37
58
96.64 ⫾ 0.73**
4 54
97.50 ⫾ 1.04* 96.57 ⫾ 0.78
All scores are Mean ⫾ Standard Error of Mean * P ⬍ 0.05 compared to controls ** P ⬍ 0.0001 compared to controls CAMCOG ⫽ Cambridge Cognition Examination
with ethidium bromide and viewed on an UV transilluminator to reveal DNA fragments with electrophoretic migration patterns unique to each allele [18].
3. Results Memory complainers had significantly lower CAMCOG scores (96.64 ⫾ 0.73) than controls (101.48 ⫾ 0.37; t ⫽ 6.15, P ⬍ 0.00001; Table 1). Four of the 58 subjects with memory complaints (6.8%) displayed the Glu318Gly mutation, whereas this mutation was not observed in any of the 66 control individuals. Glu318Gly subjects had significantly lower CAMCOG scores (97.50 ⫾ 1.04) than controls (101.48 ⫾ 0.37; t ⫽ 2.61; P ⫽ 0.011), however there was no significant difference between carriers and non-carriers within the memory complainers group. One of the four carriers of the Glu318Gly mutation was also homozygous for the ⑀4 allele of APOE; three were homozygous for the ⑀3 allele. There was no obvious interaction between Glu318Gly/⑀4⑀4 and the scores on the CAMCOG (Table 2). Eight subjects were homozygous for the ⑀4 allele —seven complained of memory difficulties. They tended to show lower CAMCOG scores than complainers with other genotypes (93.86 ⫾ 2.82 vs. 97.47 ⫾ 0.72; t ⫽ 1.62; P ⫽ 0.11).
Table 2 CAMCOG scores in memory complainers stratified by APOE Genotype
Memory Complainers
GLU318GLY (⫹) GLU318GLY (⫺)
3/3 4/4 3/3 4/4
All scores are Mean ⫾ Standard Deviation CAMCOG ⫽ Cambridge Cognition Examination
n
CAMCOG
3 1 26 6
97.67 ⫾ 2.52 97 97.35 ⫾ 6.60 93.33 ⫾ 8.02
S.M. Laws et al. / Neurobiology of Aging 23 (2002) 55–58
4. Discussion Fifty-eight of the 124 subjects included in the study complained of memory difficulties. They had significantly lower cognitive scores than non-complainers, even though none of them met clinical criteria for dementia. Four of the memory complainers carried the Glu318Gly mutation of PS1 (4/58; 6.8%) compared to none in the corresponding control group. This increased frequency, of the Glu318Gly mutation in the memory complainers group, is consistent when compared to larger control populations from Australia (2.8%; Taddei and Martins, unpublished data) and Holland (3.2%; 8). Furthermore, in the Australian group the frequency of Glu318Gly is increased in AD (Taddei and Martins, unpublished data). However, this value of 6.8% for the memory complainers group is not increased when compared to control groups in the Finnish (6.8%) and the Spanish (5.3%) populations [1]. The CAMCOG scores of the four Glu318Gly carriers were lower than the scores recorded for the control subjects. However, it is unclear at this stage whether this result was directly influenced by the presence of this mutation as when CAMCOG scores were compared between Glu318Gly carriers and non-carriers in the memory complainers group there was no significant differences observed. This lack of difference in cognitive scores between Glu318Gly carriers and non-carriers in the latter group indicates that other genetic or environmental factors may be involved in the non-Glu318Gly carriers. However, whether they play any role in promoting cognitive decline in the four Glu318Gly carriers remains to be determined by follow-up studies. One prime candidate is the well-established genetic risk factor for AD-the ⑀4 allele of APOE. Our results suggest that subjects homozygous for the ⑀4 allele, with memory complaints, have impaired cognitive abilities when compared with complainers who lack the ⑀4 allele —this supports the findings of previous studies indicating that the presence of these alleles are associated with cognitive decline [10,12,33]. When the data were investigated to determine any cumulative effect of APOE genotype and Glu318Gly it was found that in non-carriers there was a trend for APOE-⑀4 homozygotes to have decreased scores in the CAMCOG whilst in Glu318Gly carriers there was no significant difference in the CAMCOG scores between subjects homozygous for APOE-⑀4 and APOE-⑀3. However, this analysis is based on very small numbers (n ⫽ 1 versus n ⫽ 3) and as such, no conclusion can be drawn on the possibility of a cumulative effect. In support of a possible cumulative effect of APOE-⑀4 and Glu318Gly, it should be noted that one subject with memory complaints who was excluded from the analysis due to the development of clinical symptoms compatible with the diagnosis of dementia (CAMCOG score ⫽ 70 and MMSE score ⫽ 19) was positive for both APOE-⑀4 homozygosity and the presence of the Glu318Gly mutation. Follow-up studies should clarify whether the
57
Glu318Gly mutation increases the risk of cognitive decline in later life and whether or not it does impart any increased risk for the development of AD. We conclude that the frequency of the PS1 mutation, Glu318Gly, is relatively increased (6.8%) in subjects complaining of memory impairment when compared to controls (0%, 0/66) in the Australian population. However, whether this mutation, per se, directly contributes to cognitive decline remains to be determined. No significant interaction with the ⑀4 allele of APOE was observed, but the latter tended to be associated with decreased cognitive scores in memory complainers when compared to other genotypes.
Acknowledgements This project was supported by a research grant from the NHMRC (Australia) and from Sir James McCusker. S.M. Laws is a recipient of a Dora Lush (Biomedical) postgraduate research scholarship from the NHMRC (Australia).
References [1] Aludo J, Bullido MJ, Frank A, Valdivieso F. Missense mutation E318G of the Presenilin-1 gene appears to be a nonpathogenic polymorphism. Ann Neurol 1998;44(6):985– 6. [2] American Psychiatric Association. Diagnostic, and Statistical Manual of Mental Disorders. 4th ed. Washington DC: DSM-IV, 1994. [3] Bassett SS, Folstein MF. Memory complaint, memory performance and psychiatric diagnosis: a community study. J Geriatr Psychiatry Neurol 1993;6:105–11. [4] Bolla KI, Lindgren KN, Bonaccorsy C, Bleecker ML. Memory complaints in older adults: fact or fiction. Arch Neurol 1991;48:61– 4. [5] Christensen H. The validity of memory complaints by elderly persons. Internat J Geriat Psychiat 1991;6:307–12. [6] Clarnette RM, Almeida OP, Forstl H, Paton A, Martins RN. Clinical characteristics of individuals with subjective memory loss in Western Australia: results from a cross-sectional survey. Internat J Geriat Psychiat 2001;16:168 –74. [7] Dermaut B, Cruts M, Slooter AJ, Van Gestel S, De Jonghe C, Vanderstichele H, Vanmechelen E, Breteler MM, Hofman A, van Duijn CM, Van Broeckhoven C. The Glu318Gly substitution in presenilin-1 is not causally related to Alzheimer’s disease. Am J Hum Genet 1999;64:290 –92. [8] Dermaut B, Cruts M, Slooter AJC, Van Gestel S, De Jonghe C, Backhovens H, Vanderstichele H, Vanmechelen E, Breteler MMB, Hofman A, et al. In: Iqbal K, Swaab DF, Winblad B, Wisniewski HM. (ed). Alzheimer’s Disease and Related Disorders-Glu318Gly in presenilin-1 is a neutral mutation in relation to dementia: The Rotterdam study. John Wiley and Sons Ltd. 1999:87–92. [9] Derouesne C, Alperovitch A, Arvay N, Migeon P, Moulin F, Vollant M, Rapin JR, Le Poncin M. Memory complaints in the elderly: a study of 367 community dwelling individuals from 50 to 80 years old. Arch Gerontol Geriatr 1989;8(suppl 1):151– 63. [10] Dik MG, Jonker C, Bouter LM, Geerlings MI, van Kamp GJ, Deeg DJ. APOE-epsilon4 is associated with memory decline in cognitively impaired elderly. Neurology 2000;54(7):1492–7. [11] Flicker C, Ferris SH, Reisberg B. A longitudinal study of cognitive function in elderly persons with subjective memory complaints. J Am Geriatr Soc 1993;41:1029 –32.
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[12] Flory JD, Manuck SB, Ferrell RE, Ryan CM, Muldoon MF. Memory performance and the apolipoprotein E polymorphism in a community sample of middle-aged adults. Am J Med Genet. 2000;96(6):707–11. [13] Folstein MF, Folstein SF, McHugh PR. Mini-Mental State. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12:189 –98. [14] Gagnon M, Dartigues JF, Mazaux JM, Dequae L, Letenneur L, Giroire JM, Barberger-Gateau P. Self reported memory complaints and memory performance in elderly French community residents: results of the PAQUID research program. Neuroepidemiology 1994; 13:145–54. [15] Geerlings MI, Jonker C, Bouter LM, Ader HJ, Schmand B. Association between memory complaints incident Alzheimer’s Disease in elderly people with normal baseline cognition. Am J Psychiatry 1999;156:531–37. [16] Grut M, Jorm AF, Fratiglioni L, Forsell Y, Viitanen M, Winblad B. Memory complaints of elderly people in a population survey: variation according to dementia stage and depression. J Am Geriat Soc 1993;41:1295– 00. [17] Hanninen T, Reinikainen KJ, Helkala EL, Koivisto K, Mykkanen L, Laakso M, Pyorala K, Riekkinen PJ. Subjective memory complaints and personality traits in normal elderly subjects. J Am Geriatr Soc 1994;42:1– 4. [18] Hixson JE, Vermier DT. Restriction of human apolipoprotein E by gene amplification and cleavage with Hla1. J Lipid Res 1990;31:545– 48. [19] Hodkinson HM. Evaluation of a mental test score for the assessment of mental impairment in the elderly. Age Ageing 1972;1:233– 8. [20] Jonker C, Launer LJ, Hooijer C, Lindeboom J. Memory complaints and memory impairment in older individuals. J Am Geriatr Soc 1996;44:44 –9. [21] Jorm AF, Christensen H, Henderson AS, Korten AE, Mackinnon AJ, Scott R. Complaints of cognitive decline in the elderly: a comparison of reports by subjects and informants in a community survey. Psychol Med 1994;24:365–74. [22] Jorm AF, Christensen H, Korten AE, Henderson AS, Jacomb PA, MacKinnon A. Do cogntive complaints either predict future cognitive decline or reflect past cognitive decline? A longitudinal study of an elderly community sample Psychol Med 1997;27:91– 8.
[23] O’Brien JT, Beats B, Hill K, Howard R, Sahakian B, Levy R. Do subjective memory complaints precede dementia? A three year follow up of patients with supposed “benign senescent forgetfulness” Int J Geriatr Psychiatry 1992;7:481– 6. [24] O’Connor DW, Pollitt PA, Roth M, Brook PB, Reiss BB. Memory complaints and impairment in normal, depressed and demented elderly persons identified in a community survey. Arch Gen Psychiatry 1990;47:224 –7. [25] Roth M, Tym E, Mountjoy CQ, Huppert FA, Hendrie H, Verma S, Goddard R. CAMDEX: A standardised instrument for the diagnosis of mental disorder in the elderly with special reference to the early detection of dementia Brit J Psychiat 1986;149:698 –709. [26] Sandbrink R, Zhang D, Schaeffer S, Masters CL, Bauer J, Forstl H, Beyreuther K. Missense mutations of the PS-1/S182 gene in German early-onset Alzheimer’s disease patients. Ann Neurol 1996;40(2): 265– 6. [27] Schmand B, Jonker C, Hooijer C, Lindeboom J. Subjective memory complaints may announce dementia. Neurology 1996;46:121–5. [28] Schofield PW, Marder K, Dooneief G, Jacobs DM, Sano M, Stern Y. Association of subjective memory complaints with subsequent cognitive decline in community-dwelling elderly individuals with baseline cognitive impairment. Am J Psychiatry 1997;154:609 –15. [29] Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, et al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease. Nature 1995;375(6534):754 – 60. [30] Sunderland A, Watts K, Baddeley AD, Harris JE. Subjective memory assessment and test performance in elderly adults. J Gerontol 1986; 41:376 – 84. [31] Taylor JL, Miller TP, Tinklenberg JR. Correlates of memory decline: a 4 year longitudinal study of older adults with memory complaints. Psychol Aging 1992;7:185–93. [32] Wenham PR, Price WH, Blundell G. Apolipoprotein E genotyping by one-stage PCR. Lancet 1991;337:1158 –59. [33] Yaffe K, Cauley J, Sands L, Browner W. Apolipoprotein E phenotype, and cognitive decline in a prospective study of elderly community women. Arch Neurol 1997;54(9):1110 – 4.
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Association between the presenilin-1 mutation Glu318Gly and complaints of memory impairment Simon M. Lawsa, Roger M. Clarnettea,b, Kevin Taddeia, Georgia Martinsa, Athena Patona, Osvaldo P. Almeidab, Hans Fo¨rstlc, Ralph N. Martinsa,* a
Sir James McCusker Alzheimer’s Disease Research Unit and University of Western Australia, Department of Surgery, Hollywood Private Hospital, Nedlands, Perth 6009, Australia b University of Western Australia, Department of Psychiatry and Behavioural Science, Sir Charles Gairdner Hospital, Nedlands, Perth 6009, Australia c Department of Psychiatry and Psychotherapy, Technical University of Munich, Munich 81675, Germany Received 19 February; received in revised form 20 April 2001; accepted 10 May 2001
Abstract Presenilin-1 mutations account for nearly 50% of all early-onset familial cases of Alzheimer’s disease. Most of these mutations are completely penetrant, although the recently described Glu318Gly substitution seems to have only partial penetrance. These findings suggest that the Glu318Gly mutation may work as a genetic risk factor for Alzheimer’s disease. We designed the present study to investigate the frequency of this mutation among non-demented volunteers with subjective memory impairment (n ⫽ 58) and controls (n ⫽ 66). Four (6.8%) subjects with complaints of memory problems, but no controls, carried this mutation. The presence of the Glu318Gly mutation was associated with significantly lower cognitive performance when compared to controls (P ⫽ 0.011). However, there was no significant association between the presence of the mutation and the cognitive performance of individuals within the memory complainers group. Follow-up studies should clarify whether the Glu318Gly mutation increases the risk of cognitive decline in later life. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer’s disease; Presenilin-1; E318G; Subjective memory impairment
1. Introduction Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder that is the major cause of dementia in the elderly. Numerous highly penetrant mutations have been identified within the gene coding for Presenilin-1 (PS-1), located on chromosome 14, that account for over 50% of all early onset familial cases of AD [29]. At present 70 different missense mutations have been identified—most of which are pathogenic [26]. The pathogenicity of one PS1 mutation, namely Glu318Gly, has been questioned since it has also been found in a number of non-demented individuals [1]. This mutation does not seem to be sufficient to cause dementia but, like the ⑀4 allele of APOE, may increase the risk of AD.
* Corresponding author. Tel.: ⫹61-8-93466703; fax: ⫹61-8-93466666. E-mail address: [email protected] (R.N. Martins).
The recent availability of effective symptomatic treatment of AD and the prospect of further therapeutic advances have given impetus to the early diagnosis of the disease. The relationship between memory complaints in non-demented persons and future risk of dementia has prompted numerous studies [3–5,9,11,14 –17,20 –24,27,28,30,31]. A recent study within this cohort showed that memory complainers had worse cognitive performance than non-complainers, thus supporting the findings of other studies that suggest that subjective memory loss may be a reliable indicator of cognitive decline [6]. The association of genetic susceptibility factors such as APOE-⑀4 and the Glu318Gly mutation with cognitive decline may provide a starting point for the formulation of criteria for early diagnosis of AD. In this study, we screened at risk individuals who complained of memory impairment for the Glu318Gly mutation and assessed whether it was associated with lower CAMCOG scores. Since the ⑀4 allele of APOE is a major genetic risk factor for AD, we investigated whether there was an interaction between the ⑀4 allele, Glu318Gly mutation and
0197-4580/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 7 - 4 5 8 0 ( 0 1 ) 0 0 2 5 4 - 8
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S.M. Laws et al. / Neurobiology of Aging 23 (2002) 55–58
cognitive scores of non-demented subjects who complained of memory decline.
2. Materials and methods With approval from our institute’s ethics committee, we investigated the frequency of the Glu318Gly mutation among 58 individuals with subjective memory impairment (age; 62.5 ⫾ 1.3 years) and 66 age and education matched controls (age; 61.6 ⫾ 2.1 years). The 66 healthy controls were recruited from individuals referred to The Memory and Capacity Evaluation Unit at Osborne Park Hospital between 1996 and 1998. Additionally, advertisements were placed in local newspapers asking people with memory complaints to attend for assessment. Control subjects were recruited through advertisements in local papers and by asking spouses of complainers to volunteer for the study. All volunteers were reviewed clinically by an experienced physician (RC) who took the history and completed a physical and neurological examination. Those with dementia [2], Mini-Mental State Examination (MMSE) score lower that 24 [13], or prior history of stroke were excluded. A trained nurse (AP) interviewed all subjects using the Cambridge Examination for Mental Disorders of the Elderly (CAMDEX) [25]. The CAMDEX includes standardised patient and informant interviews, and a cognitive examination. The cognitive component (CAMCOG) includes tests of orientation, memory, language, praxis, attention, tactile perception, calculation, abstract thinking and visual perception. Scores can range from 0 to 107. A score of 80 or greater is considered to exclude dementia in most people [27]. The CAMCOG also includes questions that allow computation of the MMSE and the Abbreviated Mental Test Score (AMTS) [19]. In addition, the interviews gather demographic information and recent and remote medical history. Genomic DNA was extracted from collected peripheral blood samples. Glu318Gly variation was determined via polymerase chain reaction (PCR) assay using the forward ‘mismatched’ primer, 5⬘-ATCCAAAAATTCCAAGTATAATCCAG-3⬘ and the reverse primer 5⬘-CTGGGCATTATCATAGTTCTCAAG-3⬘ to generate a BstnI digestion site as described [7]. The amplified product was digested using the restriction enzyme BstnI and electrophoresed using 8% non-denaturing polyacrylamide gels. Gels were then stained with ethidium bromide and viewed on an UV transilluminator. For the determination of APOE alleles, genomic DNA was extracted from the collected peripheral blood samples and genotyped after being subjected to PCR amplification as described [18]. The oligonucleotide primers, (P1) 5⬘-TCCAAGGAGCTGCAGGCGGCGCA-3⬘ and (P2) 5⬘-ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3⬘, were used as described [32]. The amplified product was digested using the restriction enzyme HhaI and electrophoresed using 8% non-denaturing polyacrylamide gels. Gels were then stained
Table 1 CAMCOG scores for GLU318GLY (⫹) and GLU318GLY (⫺) controls and memory complainers
Controls GLU318GLY(⫹) GLU318GLY (⫺) Memory Complainers GLU318GLY (⫹) GLU318GLY (⫺)
n
CAMCOG
66
101.48 ⫾ 0.37
— 66
— 101.48 ⫾ 0.37
58
96.64 ⫾ 0.73**
4 54
97.50 ⫾ 1.04* 96.57 ⫾ 0.78
All scores are Mean ⫾ Standard Error of Mean * P ⬍ 0.05 compared to controls ** P ⬍ 0.0001 compared to controls CAMCOG ⫽ Cambridge Cognition Examination
with ethidium bromide and viewed on an UV transilluminator to reveal DNA fragments with electrophoretic migration patterns unique to each allele [18].
3. Results Memory complainers had significantly lower CAMCOG scores (96.64 ⫾ 0.73) than controls (101.48 ⫾ 0.37; t ⫽ 6.15, P ⬍ 0.00001; Table 1). Four of the 58 subjects with memory complaints (6.8%) displayed the Glu318Gly mutation, whereas this mutation was not observed in any of the 66 control individuals. Glu318Gly subjects had significantly lower CAMCOG scores (97.50 ⫾ 1.04) than controls (101.48 ⫾ 0.37; t ⫽ 2.61; P ⫽ 0.011), however there was no significant difference between carriers and non-carriers within the memory complainers group. One of the four carriers of the Glu318Gly mutation was also homozygous for the ⑀4 allele of APOE; three were homozygous for the ⑀3 allele. There was no obvious interaction between Glu318Gly/⑀4⑀4 and the scores on the CAMCOG (Table 2). Eight subjects were homozygous for the ⑀4 allele —seven complained of memory difficulties. They tended to show lower CAMCOG scores than complainers with other genotypes (93.86 ⫾ 2.82 vs. 97.47 ⫾ 0.72; t ⫽ 1.62; P ⫽ 0.11).
Table 2 CAMCOG scores in memory complainers stratified by APOE Genotype
Memory Complainers
GLU318GLY (⫹) GLU318GLY (⫺)
3/3 4/4 3/3 4/4
All scores are Mean ⫾ Standard Deviation CAMCOG ⫽ Cambridge Cognition Examination
n
CAMCOG
3 1 26 6
97.67 ⫾ 2.52 97 97.35 ⫾ 6.60 93.33 ⫾ 8.02
S.M. Laws et al. / Neurobiology of Aging 23 (2002) 55–58
4. Discussion Fifty-eight of the 124 subjects included in the study complained of memory difficulties. They had significantly lower cognitive scores than non-complainers, even though none of them met clinical criteria for dementia. Four of the memory complainers carried the Glu318Gly mutation of PS1 (4/58; 6.8%) compared to none in the corresponding control group. This increased frequency, of the Glu318Gly mutation in the memory complainers group, is consistent when compared to larger control populations from Australia (2.8%; Taddei and Martins, unpublished data) and Holland (3.2%; 8). Furthermore, in the Australian group the frequency of Glu318Gly is increased in AD (Taddei and Martins, unpublished data). However, this value of 6.8% for the memory complainers group is not increased when compared to control groups in the Finnish (6.8%) and the Spanish (5.3%) populations [1]. The CAMCOG scores of the four Glu318Gly carriers were lower than the scores recorded for the control subjects. However, it is unclear at this stage whether this result was directly influenced by the presence of this mutation as when CAMCOG scores were compared between Glu318Gly carriers and non-carriers in the memory complainers group there was no significant differences observed. This lack of difference in cognitive scores between Glu318Gly carriers and non-carriers in the latter group indicates that other genetic or environmental factors may be involved in the non-Glu318Gly carriers. However, whether they play any role in promoting cognitive decline in the four Glu318Gly carriers remains to be determined by follow-up studies. One prime candidate is the well-established genetic risk factor for AD-the ⑀4 allele of APOE. Our results suggest that subjects homozygous for the ⑀4 allele, with memory complaints, have impaired cognitive abilities when compared with complainers who lack the ⑀4 allele —this supports the findings of previous studies indicating that the presence of these alleles are associated with cognitive decline [10,12,33]. When the data were investigated to determine any cumulative effect of APOE genotype and Glu318Gly it was found that in non-carriers there was a trend for APOE-⑀4 homozygotes to have decreased scores in the CAMCOG whilst in Glu318Gly carriers there was no significant difference in the CAMCOG scores between subjects homozygous for APOE-⑀4 and APOE-⑀3. However, this analysis is based on very small numbers (n ⫽ 1 versus n ⫽ 3) and as such, no conclusion can be drawn on the possibility of a cumulative effect. In support of a possible cumulative effect of APOE-⑀4 and Glu318Gly, it should be noted that one subject with memory complaints who was excluded from the analysis due to the development of clinical symptoms compatible with the diagnosis of dementia (CAMCOG score ⫽ 70 and MMSE score ⫽ 19) was positive for both APOE-⑀4 homozygosity and the presence of the Glu318Gly mutation. Follow-up studies should clarify whether the
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Glu318Gly mutation increases the risk of cognitive decline in later life and whether or not it does impart any increased risk for the development of AD. We conclude that the frequency of the PS1 mutation, Glu318Gly, is relatively increased (6.8%) in subjects complaining of memory impairment when compared to controls (0%, 0/66) in the Australian population. However, whether this mutation, per se, directly contributes to cognitive decline remains to be determined. No significant interaction with the ⑀4 allele of APOE was observed, but the latter tended to be associated with decreased cognitive scores in memory complainers when compared to other genotypes.
Acknowledgements This project was supported by a research grant from the NHMRC (Australia) and from Sir James McCusker. S.M. Laws is a recipient of a Dora Lush (Biomedical) postgraduate research scholarship from the NHMRC (Australia).
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