We report the fine mapping/sequencing results of promoter and regulatory regions of APOE cluster genes (APOE, APOC1, APOC4, APOC2, and TOMM40) in Alzheimer's disease (AD) risk as well as in the progression from mild cognitive impairment (MCI) to AD. Long-range sequencing in 29 MCI subjects who progressed to dementia revealed 7 novel variants. Two potentially relevant novel variants and 34 single nucleotide polymorphisms (SNPs) were genotyped in a large sample of AD, MCI, and control subjects (n = 1453). Globally, very little association signal was observed in our sample in the absence of APOE ε4. Rs5158 (APOC4 intron 1) and rs10413089 (3′ to APOC2) showed a trend toward an increase in AD risk independently from APOE ε4 associated risk though it did not survive multiple test correction (uncorrected p = 0.0099 and 0.01, respectively). Interestingly, rs10413089 showed a similar effect in an independent series. The analysis of the discovery sample showed an association of TOMM40 single nucleotide polymorphisms with progression from MCI stage to AD (rs59007384 and rs11556510), as well as with a shorter time to progression from MCI status to AD (rs10119), though these results could not be replicated in independent series. Further studies are needed to investigate the role of APOE cluster variants in AD risk.
Alzheimer's disease (AD) is the most frequent cause of dementia (Alzheimer's Association, 2010) and genetic factors account for 60%–80% of the risk (Gatz et al., 2006). Early-onset AD (EOAD) can be due to mutations in the following genes: amyloid precursor protein (APP; Mendelian Inheritance in Man [MIM]: 104760), presenilin 1 (PSEN1; MIM: 104311), and presenilin 2 (PSEN2; MIM: 600759; Ezquerra et al., 2003, Goate et al., 1991, Levy-Lahad et al., 1995, Lleó et al., 2001, Rogaev et al., 1995 and Sherrington et al., 1995). Apolipoprotein E gene (APOE; MIM: 107741; chromosome 19q13) is the most important genetic risk factor ( Strittmatter et al., 1993), though 3 recent genome-wide association studies (GWAS) suggest that other genes can also have a moderate effect on AD risk (odds ratios [ORs] approximately 1.2; Harold et al., 2009, Lambert et al., 2009 and Seshadri et al., 2010). The 3 major APOE variants are ε3, ε4 and ε2, which are distinguished by a single amino acid substitution (Arg or Cys) at positions 112 or 158. APOE ε2 protects against developing AD whereas APOE ε4 increases AD risk in a dose-dependent manner and also reduces AD age at onset (AAO; Corder et al., 1993 and Pastor et al., 2003). About 50% of AD cases are attributable to APOE ε4, suggesting that additional genetic risk factors remain unknown to us.
Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia characterized by memory complaints, and objective memory impairment, with preservation of both daily living activities and general cognitive function (Petersen et al., 1999). Depending upon the series, yearly conversion rate from MCI stage to AD varies between 6% and 15% (Petersen et al., 2009). Interestingly, we recently described how both AD risk and the rate of progression from MCI to dementia are influenced by APOE ε4 and microtubule-associated protein tau gene H1 haplotype (MAPT; MIM: 157140; Samaranch et al., 2010).
Recently, 4 GWAS have replicated the strongest AD association at the APOC1 gene (apolipoprotein C-I; MIM: 107710; Harold et al., 2009, Lambert et al., 2009, Li et al., 2008 and Seshadri et al., 2010). As chromosome 19q13 is a high linkage disequilibrium (LD) region ( Abraham et al., 2008 and Takei et al., 2009), APOC1 association with AD has usually been attributed to APOE ε4. However, recent studies suggest that APOC1 and TOMM40 (translocase of outer mitochondrial membrane 40 homolog; MIM: 608061) genes may also be involved in AD risk ( Blom et al., 2008, Hong et al., 2010, Li et al., 2008 and Lutz et al., 2010). APOE ε4 has been implicated in amyloid-β (Aβ) deposition ( Morris et al., 2010). In addition to amyloid-β metabolism impairment, mitochondrial dysfunction could also play a role in AD etiology. APP accumulates in import channels of mitochondria and could therefore interfere with cell metabolism in AD ( Anandatheerthavarada et al., 2003). TOMM40 is essential for protein trafficking into mitochondria ( Gabriel et al., 2003). Thus, certain TOMM40 variants may increase APP-induced mitochondrial dysfunction. APOC1 and APOC3 genes have also been associated with AD risk ( Drigalenko et al., 1998 and Sun et al., 2005). A linkage analysis of chromosome 19q13 in familial AD suggested that APOE ε4 only partially explains AD risk and AD AAO variation ( Blom et al., 2008). As has already been suggested ( Chartier-Harlin et al., 1994), we hypothesize that, besides APOE ε4, other variants in the APOE locus may also contribute to AD risk. Genetic variability in APOE locus could explain the increased APOE ε4 messenger RNA expression observed in AD compared with healthy subjects when considering only ε3/ε4 carriers ( Lambert et al., 1997). However, despite the interest of this region, few studies have attempted fine mapping strategies in AD to identify additional risk variants ( Bekris et al., 2008, Takei et al., 2009 and Yu et al., 2007) and only 2 studies have partially sequenced APOE regulatory region in AD series ( Belbin et al., 2007 and Takei et al., 2009). To date, no studies sequencing APOE gene cluster regulatory regions in MCI subjects have been reported.
Regulatory regions of APOE, APOC1, APOC4 (apolipoprotein C-IV; MIM: 600745), APOC2 (apolipoprotein C-II; MIM: 608083) and TOMM40 genes, as well as the codifying region of the latter, were sequenced in MCI subjects that progressed to dementia (p-MCI). We performed in silico analysis of novel variants and relevant single nucleotide polymorphisms (SNPs) to investigate which of them have a potential functional role in gene expression. Then we genotyped the candidate variants and known AD-associated SNPs ( Allan et al., 1995, Bekris et al., 2008, Belbin et al., 2007, Bullido and Valdivieso, 2000, Lambert et al., 2002, Wei et al., 1985, Xin et al., 2010 and Yu et al., 2007) in a large series of MCI, AD, and controls. We specifically wanted to know whether these variants could influence AD risk, AD onset, and progression from MCI to AD independently from APOE ε4 status.
