APOE-TOMM40 in the Pharmacogenomics of Dementia

Ramón Cacabelos1*, Dmitry Goldgaber2, Alexander Vostrov2, Hideyuki Matsuki2, Clara Torrellas1, Dolores Corzo1, Juan Carlos Carril1 and Allen D Roses 1Chair of Genomic Medicine, Camilo José Cela University, Madrid; EuroEspes Biomedical Research Center, Institute for Medical Science and Genomic Medicine, Corunna, Spain 2Department of Psychiatry and Behavioral Science, Stony Brook University, New York, USA 3Zinfandel Pharmaceuticals, Inc., Durham, NC; Duke University Medical Center, Department of Neurology, Durham, NC, USA

Multiple polymorphic risk variants can increase neuronal vulnerability to premature death. Among these susceptibility genes, the apolipoprotein E (APOE) gene (19q13.2) (AD2) is the most prevalent as a risk factor for AD, especially in those subjects harboring the APOE-4 allele [12,13], whereas carriers of the APOE-2 allele are prone to longevity [14] and might be protected against dementia [15][16][17].
Linnertz et al. [55] investigated the genomic region spanning the TOMM40 and APOE genes, to determine whether intronic poly T (rs10524523) within this region affects expression of the APOE and TOMM40 genes in the brain of patients with late-onset Alzheimer's disease (LOAD). The expression of both genes was significantly increased with disease. Mean expression of APOE and TOMM40 mRNA levels was higher in VL homozygotes compared with S homozygotes in the temporal and occipital cortexes from normal and LOAD cases. The 523 VL poly T resulted in significantly higher expression than the S poly T. These results suggest that the 523 locus may contribute to LOAD susceptibility by modulating the expression of TOMM40 and/or APOE transcription [55]. Recent studies also suggest that the TOMM40 gene rs10524523 ("523") variable length poly T repeat polymorphism is associated to a certain extent with similar AD phenotypes as those reported for APOE, such as brain white matter changes [56,57] or different biomarkers [58][59][60][61]. In addition, the TOMM40 rs2075650 G allele may be a risk factor for the development of depression [62] and sporadic inclusion body myositis [63]. Different markers at the 19q13-q13.2 chromosomal region, including the rs2075650 and rs157590 (TOMM40), rs1064725 (APOC1), and rs429358 and rs7412 (APOE) SNPs also show association with primary progressive aphasia and the behavioural variant frontotemporal dementia [64].
In the present study we have investigated the structure of the APOE-TOMM40 region in Spanish patients with dementia, and the influence of polymorphic variants in this genomic segment on the therapeutic response to a multifactorial treatment adapted to the pathogenic profile of the patients. The main aims of the study were: (i) structural analysis of the APOE-TOMM40 region (distribution and frequency of major genotypes, with special emphasis on TOMM40 poly T variants) in the Spanish population with dementia; and (ii) APOE-and TOMM40 poly T1/T2-related therapeutic response to a multifactorial therapy in AD.

APOE genotyping
Genomic DNA was extracted from EDTA-treated blood samples factors on cognition [27,28]. Patients with hypertension (>150/85 mmHg) (28.04%) received enalapril (5-20 mg/day, p.o.); patients with hypercholesterolemia (>220 mg/dL) (43.48%) received atorvastatin (10-20 mg/day); patients with diabetes (glucose >105 mg/dL) (24.24%) received metformin (850-1700 mg/day, p.o.); and patients (<3%) with other ailments (e.g. hypothyroidism, hyperuricemia, etc.) received the appropriate treatment according to their medical condition. Psychotropic drugs (antidepressants, neuroleptics, hypnotics, sedatives) were avoided, and less than 5% of the patients required a transient treatment with benzodiazepines for short periods of  Data: mean ± SD using a QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany). To determine the APOE ε2, ε3 and ε4 alleles, SNPs rs7412 and rs429358 were analyzed by allelic discrimination using the TaqMan realtime PCR system. Validated SNP genotyping assays (ID number C_3084793_20 for rs429358 and C_904973_10 for rs7412) were purchased from Applied Biosystems (Applied Biosystems, CA, USA). Both SNP genotyping reactions were optimized in a total volume of 15 μl using 20 ng of DNA, and fluorescence was detected in a StepOne Plus Real-Time PCR system (Applied Biosystems, CA, USA). Control samples with known APOE genotypes were included in each PCR run, which were analyzed by the alternative PCR-RFLP method.

TOMM40 genotyping
The chr19:50094819+50094997 (NCBI36/hg18 human genome assembly) fragment containing rs10524523 was amplified from genomic DNA samples (10-20 ng) with 5'-fluorescently labeled forward primer ACCTCAAGCTGTCCTCTTGC and unlabeled reverse primer GAGGCTGAGAAGGGAGGATT (each at 0.5 µM concentration). The PCR reaction was performed with FailSafe PCR kit (Epicentre Biotechnologies, Madison, WI) using buffer G from the kit. The PCR reaction was carried out in 20 µl volumes under the following conditions: 3 min at 95°C, 40 s at 98°C, then 10 cycles with 30 s at 95°C, 90 s at 63°C, then 18 cycles with 30 s at 95°C, 1 min at 61°C, then final extension 7 min at 72°C. One microliter of each PCR product was diluted with 15 µl of HiDi Formamide (Applied Biosystems, Foster city, CA) and supplemented with 0.3 µl of 600 LIZ size standard (Applied Biosystems, Foster city, CA). The size analysis was performed by Cornell University Genomics Core Laboratory (Ithaca, NY) on an ABI 3730 DNA analyzer (Applied Biosystems, Foster city, CA). ABI 3730 data were analyzed using GeneMarker software (SoftGenetics, State College, PA). The presence of poly T stretch in the fragment causes "stuttering" during the PCR reaction [74]. Therefore, an additional procedure was implemented in order to interpret complex peak patterns and to determine the genotypes with precision. Briefly, PCR products from several patients were subcloned and the length of the poly T stretch in each clone was determined by sequencing. A clone library that covered poly T lengths from 14 to 38 was generated. Plasmid DNA from each clone was diluted and PCR amplified under exactly the same conditions as described above. Peak patterns from PCR amplification of individual cloned DNA were compared to peak patterns obtained from each individual patient's DNA. The accuracy of the method was verified by determination of poly T lengths in coded samples of patients' DNA that were previously deep sequenced and coded by an independent group. Our method was found to be 100% accurate.

Statistical analysis
Statistical analysis (paired t-test, Analysis of Variance, χ 2 and Fisher exact test, Mann-Withney Rank Sum test, Linear and Non-linear Regression analysis, Durbin-Watson statistic, Pearson correlation, Spearman rank) were performed by using the IBM SPSS Statistics 20 and Sigma Stat 3.5 programs, where appropriate. Results are expressed as mean ± SD in the text and as mean ± SEM in Figs. 2, 3 and 5 for clarity. A 2-tailed p<0.05 (or p value (χ 2 ) ≤ 0.05) was considered statistically significant.

Sex-related therapeutic response
In the whole group Figure 2, there was a clear pattern in terms of cognitive performance, with a significant improvement in MMSE scores at 1 (p<0.001), 3 (p<0.001), 6 (p=0.008), and 9 months (p<0.05) of treatment. The peak effect was observed during the first 3 months of treatment with a gradual cognitive decline thereafter. This trend was almost identical in females and males Figure 2, with a response rate (% of cases with a MMSE score superior or equal to baseline values after 12 months of treatment) of 60% in females and 59% in males (59% responders (R) and 41% non-responders (NR) in the global sample), despite substantial gender-related differences in biochemical and hematological parameters Table 1; however, the degree of significance appeared to be more apparent in females than in males Figure 2.

Discussion
The present results appear to indicate that the combination therapy used in this study, probably together with the lipid-lowering effects and anti-atherosclerotic properties of sardilipin [18,24,73] and the prometabolic activity of animon complex, exert a positive synergistic effect on brain function accompanied by a transient improvement in mental performance, lasting for about 9-12 months Figure 2. The effect of CDP-choline, either alone or in combination with donepezil, as well as the effects of sardilipin, are APOE-dependent [3,[18][19][20][21][22][23][24][25][26][27][28]. In our study, the administration of cholinesterase inhibitors was avoided in   order to eliminate distorting variability and side-effects associated with CYP2D6-related factors [25,27,75].
Our data also illustrate that polymorphic variants in the APOE-TOMM40 region are determinant for the therapeutic response to conventional drugs in patients with AD Figure 3-7. The role of APOE in this endeavor seems to be fundamental due to the influence of the APOE gene on the AD phenotype [76]; however, the impact of the TOMM40 gene on AD pathogenesis and pharmacogenetics is a novelty [33][34][35][36]. From studies designed to define APOE-related AD phenotypes, several conclusions can be drawn: (i) the age-at-onset is 5-10 years earlier in approximately 80% of AD cases harboring the APOE-4/4 genotype;  Figure 5 at baseline (BL) and after 1, 3, 6, 9, and 12 months of treatment.
brain atrophy and AD neuropathology is markedly increased in APOE-4/4>APOE-3/4>APOE-3/3; (xi) brain mapping activity shows a significant increase in slow wave activity in APOE-4/4 from early stages of the disease; (xii) brain hemodynamics, as reflected by reduced brain blood flow velocity and increased pulsatility and resistance indices, is significantly worse in APOE-4/4 (and in APOE-4 carriers in general, as compared with APOE-3 carriers); brain hypoperfusion and neocortical oxygenation is also more deficient in Linnertz et al. [39] defined 3 allele groups for rs10524523 ('523'), based on the number of 'T'-residues: 'Short' (S, T ≤ 19), 'Long' (L, 20 ≤ T ≤ 29) and 'Very Long' (VL, T ≥ 30). Roses et al. [33][34][35][36] reported that longer lengths of rs10524523 are associated with a higher risk for LOAD; for APOE-3/4 patients who developed LOAD after 60 years of age, individuals with long poly T repeats (19-39 nucleotides) linked to APOE-3 develop LOAD on an average of 7 years earlier than individuals with shorter poly T repeats (11-16 nucleotides) linked to APOE-3 [33,34,37]. Apparently, these results could not be replicated by other authors [46][47][48][49]. In our case, we clearly found that patients harboring the APOE-4/4-L/L cluster developed dementia at an earlier age (<70 yrs) than their counterparts with other genotypes. In fact, L/L carriers were the youngest at age of onset, followed by S/S carriers. In addition, virtually 100% of L/L carriers were exclusively associated with APOE-4/4 Figure 1, representing the worst responders to our combination therapy Figure 6. The APOE-3/3-VL/VL cluster, with an earlier age at onset (mean age ~70 yrs), was present in approximately a quarter of APOE-3/3 carriers Figure 1.
Bernardi et al. [45] studied the association between TOMM40 rs10524523, age of onset, and memory performance in patients with the PSEN1 M146L mutation in a large familial AD Calabrian kindred, and found that APOE33/TOMM40VL/VL patients showed a tendency for an earlier age at onset compared to those with APOE33/TOMM40VL/S and APOE33/TOMM40S/S. TOMM40VL/VL patients had better memory performance, when compared to TOMM40S/S but not to TOMM40VL/S patients. For Li et al. [46], TOMM40 intron 6 poly T length may explain some of the variation in age at onset in PSEN2 familial AD and may be associated with AD neuropathology in persons with APOE-3/3.
The allele distribution of TOMM40 poly T repeats in the Spanish population reflects a high proportion of heterozygous S/VL (39%), followed by homozygous VL (27%) and homozygous S (19%). Homozygous L/L represents 1.52% of the Spanish population, and both S/VL and L/VL genotypes conform a group of about 7-8% of the population Figure 6. Potential dissimilarities with other White and Hispanic populations [39], might be due to the ancestral admixture of different cultures in the Iberian peninsula. The linkage pattern between TOMM40-'523' and APOE alleles in Whites and Hispanics reflects that the L is primarily linked to APOE-4, while the majority of the VL and S are linked to APOE-3. In African-Americans, Ghanaians and Japanese, there is an increased frequency of the '523'S-APOE-4 haplotype [39].
Several reports suggest that both APOE and TOMM40 influence memory performance in normal [53] and pathological conditions [54,80]. For some authors, both TOMM40 and APOE significantly influence age-related memory performance, but they appear to do so independently of each other [80]. Others suggest important APOEindependent associations between the TOMM40 '523' polymorphism and specific cognitive domains of memory and executive control that are preferentially affected in early-stage AD, with S homozygotes performing better than the S/L-S/VL and the VL/L-L/VL-VL/VL genotype groups on measures associated with memory and executive function [54]. According to our data, the best mental performance (and response rate to treatment) is observed in patients harboring the APOE-3/3-S/S haplotype (R~70%), followed by those with the APOE-3/3-S/ VL haplotype (R~60%) (Figs. 3-6). In general, S/S carriers are the best responders > S/VL (61%) > VL/VL (57%) > L/VL (51%) > S/L (45%) > L/L (35%) Figure 6. The presence of the L allele appears to contribute to a poor therapeutic outcome, and when the L/L genotype associates with the APOE-4/4 genotype, carriers of the APOE-4/4-S/S haplotype (30% of APOE-4/4 carriers) are converted into the worst responders.
Although a retrospective study such as this, designed to assess the potential influence of the APOE-TOMM40 region on cognitive performance in response to a heterogeneous multifactorial therapy in a natural setting, has its limitations, interpretation of the obtained results appears to indicate that the APOE-TOMM40 cluster is important in both the clinical course of the disease and the biological conditions of AD patients in their response to the different drugs necessary to protect brain function, transiently halting disease progression. The relevance of the APOE-TOMM40 locus in the complex phenotype of dementia might be due to the pleiotropic properties of these genes. Although a large number of pharmacogenetic studies in AD used APOE as a reference gene with plural results [3,76], more specific studies exploring the influence of APOE, TOMM40, CYP, and ABCB1 variants on the pharmacogenetic outcome are needed to optimize therapeutics in AD [3,35,81].

A multifactorial treatment with conventional neuroprotectants
and other drugs or metabolic factors to treat concomitant pathologies o metabolic deficiencies, respectively, seems to be useful for patients with dementia in approximately 50% of the cases, stabilizing or improving cognitive deterioration for a transient period of time (<12 months).
2. APOE-4 carriers are the worst responders and APOE-3 carriers are the best responders to conventional treatments.
3. TOMM40 poly T-S/S carriers are the best responders, VL/VL and S/VL carriers are intermediate responders, and L/L carriers are the worst responders to treatment.