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The ε4 allele of apolipoprotein E (ApoE) accounts for an estimated 45–60% of the genetic risk for late onset sporadic Alzheimer’s disease, suggesting that it may be possible to identify other genetic loci that could account for the remaining risk associated with this disease. Recently, a biallelic polymorphism (G/A) in the 3′ untranslated region (UTR) of the transcription factor LBP-1c/CP2/LSF (for brevity, CP2) has been implicated in Alzheimer’s disease susceptibility, with the 3′-UTR A allele being associated with a reduction in the risk of sporadic Alzheimer’s disease.1–3 The CP2 gene is a plausible candidate for influencing Alzheimer’s disease risk: it is located near the LDL receptor related protein gene within the Alzheimer’s disease linkage region on chromosome 12; it controls the expression of several genes (α2 macroglobulin, glycogen synthase kinase-3β); and it interacts with different proteins (serum amyloid A3, interleukin 1α, tumour necrosis factor α, and Fe65 protein) and viruses (herpes simplex virus type I or human immunodeficiency virus) that are probably linked to Alzheimer’s disease pathogenesis.14 In the present study, we investigated the potential association of the CP2 polymorphism in a sample of sporadic early onset and late onset cases along with age and sex matched control subjects from southern Italy.
The Alzheimer’s disease group consisted of 166 patients (62 men and 104 women) from the Apulia region with a mean (SD) actual age of 69.4 (10.3) years, including 95 patients with sporadic late onset disease (age at onset ⩾70 years; mean age 78.1 (4.9) years; 64 women and 31 men), and 71 patients with sporadic early onset disease (age at onset <70 years; mean age 63.7 (4.3) years; 50 women and 21 men). A clinical diagnosis of probable Alzheimer’s disease was made according to the NINCDS/ADRDA criteria.5 The age at onset of Alzheimer’s disease symptoms was estimated by semistructured interviews with the patients’ caregivers.
The non-demented age, sex, and ethnically matched control group comprised 225 unrelated caregivers (72 men and 153 women), spouses, friends, neighbours, or volunteers, consecutively examined between June 1998 and October 2002 in our centre. Their mean age at the time of the study was 71.3 (10.4) years. The healthy subjects included 193 individuals of ⩾70 years of age (130 women and 63 men) and 32 of <70 years (23 women and nine men).
The ascertainment, diagnosis, and collection of cases and controls has been described in detail elsewhere.6 The study protocol was approved by the ethics committee of the University of Bari. Informed written consent was obtained from all subjects or their relatives before blood samples were collected. Genomic DNA was extracted from peripheral blood samples using Cod 1796828 (Roche Diagnostics kit). APOE genotypes were determined as previously described.7 CP2 polymorphism was analysed on a Lightcycler system using specifically designed hybridisation probes (sensor probe: 5′-GCGTTTCATGCCCAGTGGC-fluorescein; anchor probe: red 640–GCTCCTTCCTTCACCTCTGAAAACGG-phosphate; TIB Molbiol). Polymerase chain reaction (PCR) amplification was undertaken using 200 ng of genomic DNA, 50 pmol each primer (5′- GACAGAATTTCGCTCTTGTTGCC-3′, reverse primer; 5′-TCAGGTTCTTGCAGACCTTCAA-3′ forward primer), 3 pmol each probe, 2.5 mM MgCl2, 1×DNA master hybridisation probes (Roche Diagnostics). The amplification conditions were 95°C for two minutes, and 38 cycles of 94°C for five seconds, 58°C for 15 seconds, and 72°C for 10 seconds. After amplification, the temperature was raised to 94°C for 30 seconds, lowered to 40°C at 20°C/s of temperature transition rate, and held at 40°C for one minute. A melting curve analysis profile was obtained by raising the temperature to 80°C at 0.05°C/s while collecting fluorescence data continuously. The melting temperatures were 60°C for the 3′-UTR G allele and 66°C for the 3′-UTR A allele.
Statistical analysis was done using Pearson χ2 tests to make genotype and allele comparisons, and a test for data agreement using Hardy-Weinberg principles. Allele frequencies were determined by allele counting. To express variances of allele and genotype frequencies, we used 95% confidence intervals (CI), calculated by Wilson’s formulae. Differences among age at onset of Alzheimer’s disease symptoms in relation to different CP2 genotypes were calculated using the Mann–Whitney test. To evaluate whether the association between Alzheimer’s disease and CP2 genotypes was homogeneous in all ApoE strata we used a permutation based exact logistic model by LogXact procedure implemented in the SAS system. (Proc-LogXact 5 by CYTEL Software Corporation, Cambridge, Massachusetts, USA). The odds ratios and the 95% CI between Alzheimer subjects with and without at least one A or G allele were calculated. In most cases χ2 (by SAS FREQ procedure, version 8.2) or z tests were calculated by asymptotic p values, while exact p values (by Proc-StatXact version 5.0) were used when the data in comparisons were smallest. Any statistics were calculated for the AA genotype, because they cannot be computed when the number of non-empty rows or columns in 2×2 contingency tables is 1. The threshold of significance was set at p<0.05.
The CP2 genotype and allele frequencies in the whole Alzheimer’s disease sample and age and sex matched non-demented controls are shown in table 1. The genotype distributions were in Hardy-Weinberg equilibrium in both Alzheimer’s disease and control subjects (cases: Pearson χ2 = 0.61, p = 0.43; controls: χ2 = 0.09, p = 0.761). Statistically significant differences were found in CP2 genotype frequencies between cases and controls (GG v GA and AA, and GA v GG and AA: Pearson χ2 = 7.97, Bonferroni p<0.05, df = 1). A statistically significant increase in A allele frequency was found in the Alzheimer’s disease sample compared with the controls (Pearson χ2 = 7.670, p = 0.006). In particular, the presence of the A allele was associated with Alzheimer’s disease with an odds ratio of 2.97 (95%CI, 6.66 to 1.33). When we subdivided the whole Alzheimer’s disease sample into early onset and late onset groups, no statistically significant differences were found in CP2 genotype frequencies between the Alzheimer patients and the controls, while the A allele showed a statistically significant increase only in the Alzheimer patients who were less than 70 years old (Pearson χ2 = 4.740, exact p = 0.03). Furthermore, the Alzheimer patients bearing the A allele had a mean age of onset lower than those carrying the G allele (mean age at onset: A allele, 64.8 (12.2) years; G allele, 68.0 (9.4) years), although this difference was not statistically significant (z = 0.9, p>0.05). We did not find any significant differences in rates between CP2 alleles and Alzheimer’s disease among ApoE allele strata.
The major finding of the present study is that the A allele of the 3′-UTR CP2 gene polymorphism increases the risk of sporadic Alzheimer’s disease (OR = 2.97), without interaction with ApoE alleles. After stratification for age at onset, this effect was statistically significant only in patients with early onset disease (<70 years), whereas in late onset disease (⩾70 years) there was a difference in the A allele frequency between affected subjects and controls (though this did not reach statistical significance). Lambert et al reported an association between the CP2 polymorphism and sporadic Alzheimer’s disease in French and British populations, and a similar trend in a north American population.1 The combined analysis of the three independent populations suggested a protective effect of the A allele (OR = 0.58), that decreased with age (OR = 0.43 before 70 years; OR = 0.52 between 70 and 80 years; OR = 0.83 after 80 years). More recently, Taylor and colleagues found similar results, detecting a significant protective effect of the A allele (OR = 0.59) in 216 neuropathologically confirmed patients with late onset disease and 301 controls from the United Kingdom.2 Finally, Luedecking-Zimmer et al found that the frequency of the A allele was higher in controls than in cases (0.07 v 0.05), suggesting a moderate protective effect of the CP2 polymorphism against the risk of Alzheimer’s disease (OR = 0.65).3
To the best of our knowledge, this is the first report suggesting a risk of Alzheimer’s disease linked to the CP2 A allele, and the contrasting results of our study are, at present, difficult to explain. However, Lambert et al did not observe a significant protective effect of the A allele in the US population,1 and we recently provided a novel finding that the ApoE ε4 allele frequency decreases according to a geographic trend from northern to southern Europe.7 We hypothesise that the variability in the association between the A allele and Alzheimer’s disease can be related to ethnic and geographical variations: from 0.09 to 0.07 of A allele frequency in healthy controls from the UK, France, and north America, to only 0.02 in southern Italy.12 It is also possible that a moderate effect associated with the CP2 polymorphism is caused by its non-random association with a functional mutation present somewhere in the gene. Finally, it is possible that there is linkage disequilibrium with another biologically relevant locus on chromosome 12. The possible role of the A allele as a risk factor for sporadic Alzheimer’s disease is supported by the lower mean age at onset of Alzheimer’s disease in patients with the A allele than those carrying the G allele, though this difference was not significant. We found no interaction between CP2 polymorphism and ApoE alleles in relation to Alzheimer’s disease risk, and this finding is consistent with previous results.12
In conclusion, our data support CP2 as a candidate gene for sporadic Alzheimer’s disease, suggesting further studies on larger, ethnically and geographically different populations to clarify the role of this gene in Alzheimer’s disease pathogenesis.