Neuroscience Letters 398 (2006) 53–58

A novel polymorphism in SEL1L confers susceptibility to Alzheimer’s disease Giuliana Saltini a , Roberto Dominici d , Carlo Lovati b , Monica Cattaneo c , Stefania Michelini a , Giulia Malferrari a , Andrea Caprera c , Luciano Milanesi c , Dario Finazzi f , Pierluigi Bertora b , Elio Scarpini e , Daniela Galimberti e , Eliana Venturelli e , Massimo Musicco c , Fulvio Adorni c , Claudio Mariani b , Ida Biunno c,g,∗ a

Department of Sciences and Biomedical Technologies, University of Milan Via F.lli Cervi 93, 20090 Segrate-Milan, Italy b Department of Neurology, University of Milan, Ospedale L. Sacco, Via G.B. Grassi 74, I-20157 Milan, Italy c Institute for Biomedical Technologies (National Research Council), Via F.lli Cervi 93, 20090 Segrate-Milan, Italy d Lab of Clinical Chemistry, Ospedale L. Sacco, Via G.B. Grassi 74, I-20157 Milan, Italy e Department of Neurological Sciences, “Dino Ferrari” Center and CEND, University of Milan, IRCCS Ospedale Maggiore Policlinico, Milan, Italy f Section of Chemistry, Faculty of Medicine, University of Brescia, Brescia, Italy g BioRep SrL Via Fantoli 16/15, Milano, Italy Received 14 October 2005; received in revised form 13 December 2005; accepted 14 December 2005

Abstract Alzheimer’s disease (AD) is considered to be a conformational disease arising from the accumulation of misfolded and unfolded proteins in the endoplasmic reticulum (ER). SEL1L is a component of the ER stress degradation system, which serves to remove unfolded proteins by retrograde degradation using the ubiquitin-proteosome system. In order to identify genetic variations possibly involved in the disease, we analysed the entire SEL1L gene sequence in Italian sporadic AD patients. Here we report on the identification of a new polymorphism within the SEL1L intron 3 (IVS3-88 A>G), which contains potential binding sites for transcription factors involved in ER-induced stress. Our statistical analysis shows a possible role of the novel polymorphism as independent susceptibility factor of Alzheimer’s dementia. © 2005 Published by Elsevier Ireland Ltd. Keywords: Alzheimer’s disease; SEL1L; ER-associated degradation (ERAD); Genetics; Polymorphism

Alzheimer’s disease (AD) is the most common form of dementia and its incidence is clearly age dependent and doubles every 5 years after age 65 [10]. The causes of AD are still unknown but both environmental factors and heritable predisposition most likely contribute to the development of the disease [21,34]. Increasing evidences show that protein aggregation have a critical role in AD characterized by extracellular deposition of ␤-amyloid peptides in plaques and neurofibrillary tangles within neurons, being the main neuropathological features of disease [8]. As of today, disease causing or predisposing mutations have

∗

Corresponding author. Present address: ITB-CNR, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy. Tel.: +39 02 26422712; fax: +39 02 26422770. E-mail address: [email protected] (I. Biunno). 0304-3940/$ – see front matter © 2005 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2005.12.038

been unambiguously identified in only four genes: amyloid precursor protein (APP) [11], presenilin 1 (PSEN1) [38], presenilin 2 [36] and apolipoprotein-E (APOE) [23]. Fully penetrant mutations in APP, PSEN1 and PSEN2 have been associated with the rare early onset familial forms of AD [4], whereas the carrier status for the ␧4 allele of APOE is clearly associated to the most common sporadic forms [7]. Besides the ␧4 allele, other genetic susceptibility factors have been proposed: polymorphisms on genes encoding for ␣2-macroglobulin, ␣1-anti-chymotrypsin, angiotensin I converting enzyme, though many studies did not replicate these results [2,29,35]. A stronger confirmation is available on the association between AD and a polymorphism in the interleukin-1 [12,22,30,33] and NOS3 genes [13]. During recent years several analytical strategies with different markers have been applied on different population studies, in addition to

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full-genome screens using linkage or association-based methodologies; the results obtained revealed a number of locus-specific and AD candidate genes [9]. However, no single study has yet emerged as being reproducible and APOE still remains the only gene clearly associated to AD [3]. Accumulating evidences suggest that a high level of endoplasmic reticulum (ER) stress and consequently defective ER stress signalling can cause chronic human diseases such as Alzheimer’s and Parkinson’s disease, prion-related encephalopathies, type I and II diabetes [1,4,19]. High ER-stress elicits the unfolded protein response (UPR), an adaptive response of the cell. A component of UPR is the ER-associated degradation (ERAD) system [20], which requires retrograde transport of unfolded proteins from the ER back to the cytosol for degradation by the ubiquitin-proteosome system

[6,39]. SEL1L is a component of the ERAD system [15,24,41], thus we screened a population of AD patients and matched controls for genomic mutations and here we report on the identification of a new intronic polymorphism significantly associated with AD dementia. A total of 180 consecutive patients (122 women and 58 men, age range 76.96 ± 7.87 years; mean age years) clinically diagnosed as AD were enrolled at the Neurology Units of Ospedale Maggiore Policlinico (Milan) and Ospedale L. Sacco (Milan). All patients underwent a standard battery of examinations, including familiarity and medical history, physical and neurological examination, screening laboratory tests, neurocognitive evaluation, brain magnetic resonance imaging (MRI) or computed tomography (CT). Dementia severity was assessed by the

Table 1 Primer sequences and relative product sizes used for PCR and sequencing reactions SEL1L region Promoter

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 + 8 Exon 9 Exon 10 Exon 11 Exon 12 Exon 13 Exon 14 Exon 15 Exon 16 Exon 17 Exon 18 Exon 19 Exon 20 Exon 21

Primer name

Primer nucleotide sequence

PCR products size (bp)

To11586F IB32R IB31F PROM6R IB11R PROM5F SEL1LProEx01R SEL1LEx02F SEL1LEx02R SEL1LEx03F SEL1LEx03R SEL1LEx04F SEL1LEx04R SEL1LEx05F SEL1LEx05R SEL1LEx06F SEL1LEx06R SEL1LEx0708F SEL1LEx0708R SEL1LEx09F SEL1LEx09R SEL1LEx10F SEL1LEx10R SEL1LEx11F SEL1LEx11R SEL1LEx12F SEL1LEx12R SEL1LEx13F SEL1LEx13R SEL1LEx14F SEL1LEx14R SEL1LEx15F SEL1LEx15R SEL1LEx16F SEL1LEx16R SEL1LEx17F SEL1LEx17R SEL1LEx18F SEL1LEx18R SEL1LEx19F SEL1LEx19R SEL1LEx20F SEL1LEx20R SEL1LEx21F SEL1LEx21R

5 -GCTCTTATCACTATGCCCAAT

674

5 -GCACACAGCAGCAGCGTCAG 5 -CCAAAGTCCTTCACTTGGC 5 -GTGCGGGTGACTGTTGAAGT 5 -GACCCGCATCCTCCTCTCGG 5 -GACCATGGTGATTGGACCAGA 5 -GACAGCCCAGTGCGAGAT 5 -CTAAACCCCCAACCCATTTT 5 -CAGACCTCATATCCTTTCTAA 5 -GTAAAAATAACGGTCAAATAGG 5 -CAGTCTTCTTCAGCCTCTTT 5 -CAGGCATCATGAATCTTTGC 5 -TGTGGAAAAAGGATGGCTAG 5 -CATTTTGTTTGTATGACTGCC 5 -CTTCTGCGTCTCTGGCAG 5 -AGGAAATTATTTATGTTGCGTTT 5 -ATGGTCAGCCACAAAATTGG 5 -TGTCTGTCTGTGTCTGTCTT 5 -GAAAAAGTTGTGGCCGCTAT 5 -TTTGTTCCACGAAGAATGACA 5 -TGAGTCCACATAAAAGAGCC 5 -GCAGCTGGAATTGTACTTCT 5 -GCTCAGGGCAATGAAAATGT 5 -CAGAAGCCATTTTACGACCA 5 -GCTCATTTGTGATGGGTGTT 5 -TTTGGCTGAAAGTCAAAAGCA 5 -GAAAATGCCTCAGAACAGCA 5 -GTAATTGGAGAGAGGGTTAC 5 -CATGAATCATACTTAATATGCAG 5 -GTCTGATGAGAAGAAGAAAGT 5 -GGCTGACTGGAAGCTAATTA 5 -TCCCCCTATGACTTAATGTAT 5 -CAGAATTTGATAGCACCCATT 5 -ACTCAGTAAGAATTCTTTCATTA 5 -TAGGCTGAGTCTAGAACTAC 5 -AGCTGCCAGAAATTGAAGGA 5 -CCTGTGTGTTCAGTTCCAG 5 -AACTTATGCTTCACCTATCCT 5 -AGTACTCGGCTTCATGATTTA 5 -AGCATGTCAATGGGAGGAG 5 -GTGGCCTGAAATCCAACTG 5 -GTTTAGGTGAGGGAAACTCA 5 -TACCTGCAGGACCACTCTT 5 -TCTGTACTTACTTCTGGTGAT 5 -CATATGGCACTGAAACAAACA

704

379 323 393 369 303 389 349 267 345 280 292 266 302 364 373 416 264 368 364 517

G. Saltini et al. / Neuroscience Letters 398 (2006) 53–58

Clinical Dementia Rating (CDR) and the Mini Mental Scale Examination (MMSE) at the time of sampling. The diagnosis of probable AD was made according to NINCDS-ADRDA criteria [27]. A total of 153 ethnicity-matched healthy individuals (94 women and 59 men, mean age 66.8 ± 11.93 years) were also enrolled by the previously mentioned hospitals. Healthy control cases (HC) were selected from a non-demented population having a normal score at the Mini Mental State Examination (score > 24). An informed consent was obtained from all subjects enrolled in the study. A database was created, hosted at the Institute of Biomedical Technologies of the CNR, Segrate (MI) and contains clinical and bio-molecular data directly inserted by investigators involved in the project, to support statistical studies, and collects patients’ data while maintaining their complete anonymity. Whole blood samples were stored at −20 ◦ C until used for DNA extraction and genomic DNA was purified using a silica-based method previously described [25]. SEL1L (AF052059) promoter region (−1190 to start codon) and exons (including exon–intron junctions) were amplified by standard PCR. The primer sequences and relative product sizes are listed in Table 1. PCR reactions were carried out using the DYnaZYme DNA Polymerase (Finnzymes) with standard amplification conditions; the PCR products were purified using ExoSAP treatment (GE Healthcare), according to the manufacturer’s instructions, and sequenced with the DYEnamic ET Dye Terminator Cycle Sequencing Kit (GE Healthcare). Sequence products were purified using Montage SEQ96 Sequencing Reaction Cleanup KIT (Millipore). All PCR, purification and sequencing reactions were prepared using the Multiprobe II HT EX Robotic Liquid Handling System (Perkin Elmer) [26]. All sequences were performed on the MegaBACE 1000 DNA Analysis System (GE Healthcare). Cimarron 3.12 was used as the base caller. The strength of association between Alzheimer’s disease and the allelic configuration for SEL1L was quantified with the odds ratio and its statistical significance was calculated with chi-square as described by Manthel–Haenszel.

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The estimates of the risk were also calculated adjusting for the presence of the APOE ␧4 allele by means of a logistic regression analysis. For each risk estimate the 95% confidence interval was derived from the standard errors of the regression coefficients. DNA sequencing of the SEL1L gene (minimal promoter, exons and exon–intron junctions) was initially performed on 24 AD Italian patients. Sequence analysis of PCR amplified genomic DNA revealed a novel polymorphism located in the intron located between exons 3 and 4: here the adenine (A) at position −88 from intron/exon junction is substituted by a guanine (G) residue (IVS3-88A>G). Table 2 describes the statistical analysis performed in our case–control association study on a total of 180 patients diagnosed as having sporadic AD patients and 153 healthy controls. The novel intronic polymorphism was observed in hetero- and homozygosis with different frequency in each group of subjects. AD patients analysed were older than controls but equally distributed in the two sexes. ␧4 allele of APOE was more common among cases both in hetero and homozygosis. Also A/G and G/G SEL1L polymorphisms were more common in Alzheimer disease cases than in controls. When the strength of association was quantified with the calculation of odds ratios as estimates of the relative risk, a significant risk increase was observed for age, number of ␧4 alleles and for the A/G or G/G polymorphism. The crude estimates of AD risk were more than five folds increased in subjects presenting one or two ␧4 alleles and were about 1.9 times increased in subjects with the A/G or G/G polymorphism with respect to those with the A/A configuration. When the multivariable analysis was carried out age was still significant, the risk associated with ␧4 allele was about six times increased in those subjects with one allele and more than nine times increased in those with two ␧4 alleles. The risk increase associated with ␧4 were statistically significant. The risk associated with the polymorphisms of SEL1L was statistically significant (p = 0.045) with an OR of 1.8 for carriers of one or two G alleles when compared to subjects without G alleles. Table 3 describes the genotype frequencies

Table 2 Demographic characteristics, number of ␧4 alleles and SEL1L polymorphisms in patients with Alzheimer disease and controls Controls

Alzheimer’s disease

Crude odds ratio 95% CI

Multivariable odds ratio 95% CI

Gender Men Women

59 (38,8) 93 (61,2)

58 (32,2) 122 (67,8)

1.3 (0.8–2.1), p = 0.211

1.2 (0.7–2.1), p = 0.577

Mean age (range)

66.8 (18,0–93,0)

76.9 (52,0–93,0)

1.1 (1.07–1.13), p = 0.000

1.1 (1.1–1.12), p = 0.000

Number of ␧4 alleles 0 1 2

133 (86,9) 18 (11,8) 2 (1,3)

98 (54,4) 74 (41,1) 8 (4,4)

5.6 (3.1–9.9), p = 0.000 5.4 (1.1–26.1), p = 0.035

5.8 (2.8–11.8), p = 0.000 9.7 (1.2–81.4), p = 0.036

SEL1L polymorphism A/A A/G or G/G

116 (75,8) 37 (24,2)

114 (63,3) 66 (36,7)

1.9 (1.2–3.0), p = 0.010

1.8 (1.0–3.3), p = 0.045

Total

153

180

Odds ratios are presented crude and adjusted for all the other variables of the table. Numbers in parentheses are column percentages when not otherwise specified. CI 95% stands for confidence interval at 95%.

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G. Saltini et al. / Neuroscience Letters 398 (2006) 53–58

Table 3 Genotype frequencies and raw p-values for AA, AG and GG comparisons Genotype frequencies AD

Controls

SeL1L Pol-88

A/A A/G G/G

114 (63,3) 58 (32,2) 8 (4,4)

116 (75,8) 30 (19,6) 7 (4,6)

Total

180

153

333

Total

230 88 15

p = 0.033 (chi-square test). Number in parenthesis are percentages and the pvalue is calculated using the chi-square test.

for the A and G comparisons showing a statistically significant association (p = 0.033). The accumulation of proteins in neurons seems to be influenced by the ERAD system [17,18] which plays an important function in the survival of stressed cells [15]. The ERAD system serves to remove unfolded proteins by retrograde degradation using the ubiquitin-proteosome system [6,39]. Components of the ERAD system are HRD1 and SEL1L; HRD1 is a bona fide homolog of the yeast Hrd1 [14] and SEL1L a candidate homolog of the yeast Hrd3 [32]. Both HRD1 and SEL1L co-localize in the endoplasmic reticulum, physically interact to each other and are induced in response to UPR [14,19,24]. SEL1L forms a 1:1 complex with HRD1 and together links dislocation, ubiquitination and extraction of misfolded proteins from the ER membrane [24]. We focused our attention on the gene SEL1L due to its sub-cellular location and induced expression upon ER stress (Cattaneo, personal communication) with the aim to find sequence alterations which could be associated to AD. We performed mutation scanning of the entire gene (intron/exon boundaries included) and the minimal promoter on an Italian population of AD patients as well as controls and found a pre-

viously undetected polymorphism (IVS3-88A>G). The risk of AD associated with this polymorphism is statistically significant even though the control population analysed was somewhat younger than the patients, but equally distributed by gender. When multivariate analysis was carried out, age was still significant and the risk associated with ␧4 allele was about six times increased in those subjects with one allele and more than nine times in those with two ␧4 alleles. The risk increase associated with ␧4 was statistically significant. To our knowledge, this is the first report on a study of AD patients and controls using a gene known to play a function in the response of cells to ER stress. The polymorphism lies within the longest intron of the gene in which, extensive computeraided analysis (MatInspector program), revealed the presence of consensus sequences for transcription factors known to be activated in ER stress (Table 4). Although no similarity to known transcription factor consensus sequences were found neither in subjects with A nor with the G transition genotype, several eukaryotic regulatory elements were identified within this large intronic region. In particular we found the presence of several potential binding sites for transcription factors that are involved in ER-induced stress such as: HSF-1, ATF6 and CHOP [37,40,43], among them, the heat shock factor 1 which is the first factor responsible for the transcriptional response to heat stress in mammalian cells [31]. The heat shock response protects proteins from the deleterious effects of acute or chronic stress by stabilizing and refolding protein-folding intermediates or facilitating protein degradation, a central issue to neurodegenerative diseases. It was reported that activation of HSF-1 is highly sensitive to oxidative stress, a condition associated with neurodegenerative disorders and that its DNA binding activity is lower in AD than control cybrids [5]. The CHOP protein, also known as growth arrest and DNA damage-inducible gene

Table 4 Consensus sequences for transcription factors within the SEL 1L intron 3 Transcription factor

Positiona

Core simb

Matrix simc

Sequenced

Heat shock factor 1

820–830 1667–1677 2627–2637 5298–5308 9876–9886 12240–12250 14605–14615

1.000 1.000 1.000 1.000 1.000 1.000 1.000

0.977 0.987 0.970 0.966 0.952 0.935 0.950

AGAAtattctc AGAAtgttctt AGAAttttctc AGAAttttcct AGAAgcttcca AGAAaaatcgg AGAAacttcca

1891–1903 9036–9048 9434–9446

1.000 1.000 1.000

0.907 0.976 0.918

gagTGCAatggca agaTGCAatagcc accTGCAatacac

Member of b-zip family induced by ER damage/stress binds to the ERSE in association with NF-Y

7222–7236 18118–18132

1.000 1.000

0.946 0.936

ctgCCACtccccttt ctgCCACacccagct

Activating transcription factor 6 member of b-zip family, induced by ER stress

5702–5722 10525–10545 12802–12822

1.000 1.000 1.000

0.878 0.987 0.893

atcataaGACGtgagcggggt gggtgatGACGtgagctggcg cttgggcGACGtgacttcgag

Heterodimers of CHOP and C/EBP alpha

a

The coordinates refer to the beginning of the third intron. Core similarity: the core sequence of a matrix is defined as the (usually four) consecutive highest conserved positions of the matrix. The maximum core similarity of 1.000 is only reached when the highest conserved bases of a matrix match exactly in the sequence. c Matrix similarity: a perfect match to the matrix gets a score of 1.000, a good match to the matrix usually has a similarity of >0.8. d Base pairs in capital letters describe the core sequence used by MatInspctor. Underlined base pairs show a high information content, i.e. the matrix exhibits a high conservation (ci-value > 60) at this position. b

G. Saltini et al. / Neuroscience Letters 398 (2006) 53–58

153 (GADD153), is a leucine zipper transcription factor that is activated in response to induced ER stress [42]. It was reported that the basal protein levels of CHOP are markedly increased by PS1 mutations which are responsible for early onset familial Alzheimer’s disease by a mechanism believed to involve perturbed endoplasmic reticulum function and altered proteolytic processing of the amyloid precursor protein [28]. The activating transcription factor 6 (ATF6) is a basic leucine zipper transcription factor that during ER stress binds to DNA on the ER stress response element (ERSE) in association with NF-Y and activates the unfolded protein response target genes [43]. It was reported that SEL1L is induced by ER stress via the signal transducers ATF6 and IRE1 [15]. Moreover, the familial Alzheimer’s disease-linked presenilin-1 mutation leads to vulnerability to ER stress by inhibiting the activation of ER stress transducers ATF6, IRE1 and PERK [16]. We hypothesize, based on the reported observations, that the G substitution may cause a marked change in the secondary structure of DNA, thus affecting the access of the transcription apparatus and possibly down regulating SEL1L signalling and the ERAD response. In fact, the presence of G configuration may form a more potential palindrome, thermodynamically more stable, which may interfere with protein/DNA interaction leading to SEL1L down-modulation. In conclusion, here we describe a novel polymorphism in the SEL1L gene which could be a risk factor in AD development. So far, association studies with candidate genes have been widely used for the study of complex diseases, however, this approach has been criticized because results are sometimes not replicated. Therefore, further association studies, using larger populations together with functional analysis are indeed necessary to confirm and reproduce these preliminary data.

[6]

[7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

Acknowledgements

[16]

This work was supported by grants from Associazione Amici del “Centro Dino Ferrari”, CARIPLO and Monzino Foundations, “Associazione per la Ricerca sulle Demenze (ARD)”, and IRCCS Ospedale Maggiore Milano; MIUR-Cofin 2002 to M.C. and D.F.; MIUR (Bioinformatics, LITBIO) and EU SSH BIOINFOGRID to L.M. and A.C.

[17]

[18]

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