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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:301-306 (2005)
© 2005 The Gerontological Society of America

Mutation Screening and Association Study of the Neprilysin Gene in Sporadic Alzheimer's Disease in Chinese Persons

Jiajun Shi1,3,4, Sizhong Zhang1,3,, Mouni Tang4, Cui Ma4, Jinghua Zhao5, Tao Li2,6, Xiehe Liu2, Yan Sun1,3, Yangbo Guo4, Haiying Han4, Yongxin Ma1,3 and Zhenhuan Zhao4

1 Department of Medical Genetics
2 Institute of Mental Health, West China Hospital, Sichuan University, Chengdu, China.
3 Division of Human Morbid Genomics, State Key Laboratory of Biotherapy of Human Diseases, Chengdu, China.
4 Guangzhou Psychiatric Hospital, Guangzhou Medical College, Guangzhou, China.
5 International Center for Health and Society, Department of Epidemiology and Public Health, London, United Kingdom.
6 Department of Psychological Medicine, Institute of Psychiatry, London, United Kingdom.

Address correspondence to Professor Sizhong Zhang, Department of Medical Genetics, West China Hospital, Sichuan University, Chengdu, 610041, China. E-mail: szzhang{at}mcwcums.com


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Neprilysin has been reported to be a major beta-amyloid peptide (Aß)-degrading enzyme. The decreased expression and activity of it may contribute to the development of Alzheimer's disease by promoting the accumulation of Aß. We used denaturing high-performance liquid chromatography to screen the neprilysin gene (NEP) for single nucleotide polymorphisms (SNPs) in 257 Chinese sporadic Alzheimer's disease patients and 242 cognitive normal controls. As a result, eight novel and one known SNP were identified. Three of them, –204G->C in the promoter region, IVS17–294C->T, and IVS22+36C->A showed a significant association with Alzheimer's disease (p =.006,.017, and.003, respectively). Subsequent haplotype analysis provided further evidence of the association (global p <.0001 for the three SNPs mentioned above, and global p <.01 for the eight SNPs with rare allele frequency >1%). These findings indicate that genetic variations within or extremely close to NEP might influence the susceptibility to Alzheimer's disease in Chinese persons.

ALZHEIMER'S disease, the most common form of dementia in elderly persons, is a genetically heterogeneous progressive neurodegenerative disorder characterized by senile plaques and neurofibrillary tangles as distinct neuropathological hallmarks in the brain. Mutations in three genes, amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PREN2), account for rare familial early-onset Alzheimer's disease (1–3), and the {epsilon}4 allele of the apolipoprotein E gene (APOE-{epsilon}4) is up to now the only widely confirmed risk factor for the sporadic form of Alzheimer's disease (4). However, about 50% of sporadic Alzheimer's disease cases do not carry any APOE {epsilon}4 allele, suggesting that other genetic risk factors may exist.

According to the most popular amyloid cascade hypothesis of Alzheimer's disease pathogenesis, accumulation and aggregation of beta-amyloid peptide (Aß) is the primary cause of Alzheimer's disease, followed by an inflammatory response, neuritic injury, hyperphosphorylation of tau protein, and formation of fibrillary tangles, leading ultimately to neuronal dysfunction and cell death (5,6). Therefore, both increased production and decreased breakdown of Aß may cause its deposition in brain tissue. Genetic variations in APP and PSENs have been shown to increase Aß biogenesis, and some genes such as APOE and {alpha}-2-macroglobulin, thought to participate in the uptake and clearance of Aß, are also associated with Alzheimer's disease (7).

Recent attention has been focused on enzymes in amyloid catabolism. Among them, neprilysin (NEP), a putative Aß-degrading enzyme, is thought to play an important role in Alzheimer's disease pathogenesis because its decreased expression and/or activity may also result in cerebral Aß accumulation (8). Thus, NEP has been become a candidate gene for Alzheimer's disease because: (a) NEP is located within the candidate chromosome 3 region (3q25.2), which is linked and associated with late-onset Alzheimer's disease (9,10); (b) previous studies have shown that NEP might be a major Aß-degrading enzyme (11,12); and (c) NEP messenger RNA and/or protein levels have been shown to be selectively reduced in Alzheimer's disease brains, particularly in areas associated with senile plaques (13–15).

Neprilysin (Mendelian Inheritance in Man No. 120520), the human membrane-associated neutral endopeptidase, also known as encephalinase, common acute lymphocytic leukemia antigen, and membrane metallopeptidase, is a 100-kd type II transmembrane glycoprotein, which can degrade Aß in vitro and in vivo (8). The gene contains 24 exons, spans more than 80 kilobases (kb), and is mapped to 3q21–q27 (16,17). Previous association analysis of several polymorphisms in the promoter or 3' untranslated region of NEP with Alzheimer's disease produced conflicting results (18–22). Moreover, a variation screen of the whole NEP gene and association analysis between the gene and sporadic Alzheimer's disease in Chinese persons has not yet been reported. Therefore, using a combination of polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC), PCR-restriction fragment length polymorphism (RFLP), and DNA sequencing, we screened for variants in NEP and then investigated their possible association with sporadic Alzheimer's disease in Chinese persons.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients
The patient group (n = 257) included 104 men and 153 women, diagnosed with probable Alzheimer's disease according to the National Institute of Neurological and Communicative Disorders and Stroke–AD and Related Disorders Association criteria (23). The patients were recruited from Guangzhou Psychiatric Hospital and from three retirement homes in suburbs of Guangzhou, Guangdong province, South China. The average age of the patients at examination was 76.7 ± 8.8 years, ranging from 58 to 98 years (mean age of onset = 73.3 ± 8.3 years). Among the patients, 48 (18.7%) were < 65 years at age of onset. All patients had no family history of dementia. Meanwhile, the age-matched control group, which consisted of 242 individuals, including 114 males and 128 females, was enrolled either from staff of the Psychiatric Hospital or from the communities nearby. Any insidious cognitive deficit was ruled out after clinical, mental, and neurological examinations and the Mini-Mental Status Examination (MMSE) (24). The mean age of the controls was 80.0 ± 7.6 years, ranging from 59 to 99 years (4 patients were < 65 years old at examination). All patients were genetically unrelated, and written informed consent was obtained from them or their relatives.

DNA Preparation and PCR Amplification
Genomic DNA was extracted from peripheral blood lymphocytes by using a standard phenol-chloroform procedure. Fragments containing the putative promoter region and individual exons including all intron-exon boundaries were amplified by PCR. The designed oligonucleotide primers (Table 1) for PCR were based on the alignments of the messenger RNA (GenBank accession No. NM_000902) and human genomic sequence.


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  		Table 1. Details of Region of Amplified, Primer Sequence, Annealing Temperature, Length of Amplicon, and Melting Temperature of Denaturing High-Performance Liquid Chromatography (DHPLC) for Screening Mutations in the NEP Gene.

 
Each PCR amplification mixture contained 0.1 µg of genomic DNA, 20 pmol of each primer, 30 pmol of dNTPs, 1 unit of Taq polymerase (TaKaRa), and standard PCR buffer in a total volume of 50 µl. Annealing temperature for the PCR was listed in Table 1. After being melted for 5 minutes at 95°C, 30 cycles of PCR amplification were performed with a temperature profile consisting of denaturing for 30 seconds at 94°C, annealing for 30 seconds at a temperature between 54°C and 62°C, extension for 1 minute at 72°C, and a final extension for 5 minutes at 72°C. Amplicons were resolved by agarose gel electrophoresis to confirm the presence of specific amplified products. The unpurified PCR products were also injected into the HPLC system under a nondenaturing, sizing gradient at 50°C to further verify the presence of a single PCR product. The remaining unpurified PCR products were then analyzed using the DHPLC method as described below.

DHPLC
Variant screening by DHPLC was carried out on the fully automated WAVE 3500HT Nucleic Acid Fragment Analysis System (Transgenomic Inc., Omaha, NE). Briefly, we first analyzed the predicted melting profile of the fragments by the supplied WAVEMAKER4.1 (Transgenomic) software to determine the optimal melting temperatures (Table 1) and the gradient conditions to be tested. Then 5 µL of each DNA sample was injected into a high-throughput DNASep column (Transgenomic) and eluted within 3 minutes through a 260 nm detector, with concentrations of buffer A (0.1 M triethylammonium acetate [TEAA]) and buffer B (0.1 M TEAA and 25% acetonitrile in ultrapure water) adjusted automatically as calculated by the Navigator software package (Transgenomic). Heterozygous fragments with aberrant DHPLC patterns were reamplified and purified for subsequent cloning and sequencing.

Cloning and Sequencing
The location and chemical nature of the mismatch were determined by DNA sequencing of the reamplified product. The purified heterozygous DNA was cloned into the pGEM-T Easy Vector System (Promega, Madison, WI), then the ligated products were transformed into the Escherichia coli JM109 competent cells (TaKaRa) according to a heat-shock protocol recommended by the manufacturer. After transformation, cells were grown overnight at 37°C on duplicate LB/Ampicillin/IPTG/X-Gal (Sigma and Bio101, Carlsbad, CA) plates. White colonies were harvested and grown in 5 mL of SOC medium (20 g/L tryptone, 5 g/L yeast extract, 10 mmol/L NaCl, and 20 mmol/L glucose) at 37°C for 18 hours. Plasmid DNA was extracted using an EZNA Plasmid Mini Kit (Omega Bio-Tek, Doraville, GA). The presence of inserts of appropriate length in the extracted plasmids was confirmed by digestion at 37°C for 1 hour with restriction endonuclease EcoRI (Roche, Basel, Switzerland), and the digests were subjected to electrophoresis through a 1% agarose gel stained with ethidium bromide. Finally, the purified plasmid DNA was sequenced in both directions on the ABI PRISM 377 DNA Sequencer using the Big-Dye terminator cycle sequencing kit (Sangon Co., Shanghai, China).

Genotyping
All participants were genotyped for SNPs identified in the present study, either by digestion with corresponding restriction enzymes or by re-DHPLC analysis of the mixed amplicons of homozygous samples shown by previous DHPLC analysis with the reference sample of known sequence. In genotyping by re-DHPLC, for individuals homozygous with the known sequence, a single elution peak is predicted, whereas for individuals different from the known sequence, a complex DHPLC pattern was predicted (25).

Statistical Analysis
Deviation from Hardy-Weinberg equilibrium for alleles at individual loci as well as differences in genotype between Alzheimer's disease patients and controls was assessed using the chi-square test. Logistic regression analysis was used to examine the interaction between NEP and APOE polymorphisms on the risk for Alzheimer's disease. Pairwise linkage disequilibrium with D' and r2 was calculated using R package genetics (cran.r-project.org).

To test for association of estimated haplotypes with Alzheimer's disease, we used C program GENECOUNTING (26) and R package haplo.score (27). GENECOUNTING performs model-free analysis and permutation tests for global association of all SNPs, as well as individual haplotypes, using an expectation-maximization algorithm (26). Haplo.score was used to test for association with or without adjustment for age, sex, and APOE {epsilon}4 status; it assigns the probability for each haplotype pair in each individual and then directly models an individual's phenotype as a function of each inferred haplotype pair, weighted by their estimated probability, to account for haplotype ambiguity (27,28). This program has the advantage that adjustment for covariates such as age, sex, and APOE {epsilon}4 status, and computation of simulation p values for each haplotype can be performed. The number of simulations for empirical p values was set at 10,000.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Using PCR primers flanking each exon and putative promoter region, we screened exons 1–23, the coding part of exon 24, and 625 bp of the 5' region (totally 10,416 bp) of the NEP gene in 257 Alzheimer's disease patients and 242 controls by DHPLC. Then, each candidate single nucleotide polymorphism was confirmed by DNA sequencing. As a result, nine SNPs were found, of which one was located in the promoter, one in exon 6 resulting in a Gln134Arg mutation, and two in the 3' untranslated region; the remaining five were in introns (Table 2). Except for SNP 8, all the SNPs (that were identified) have not been reported before.


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  		Table 2. Characteristics of Identified Single Nucleotide Polymorphisms (SNPs) in the NEP Gene in Alzheimer's Disease Patients and Controls.

 
To assess whether these SNPs are associated with Alzheimer's disease risk, SNP 1, 6, and 8 were genotyped by PCR-RFLP analysis with enzymes Mbo I, BsrD I, and Xba I, respectively, whereas the others were genotyped by re-DHPLC analysis. The polymorphic distribution of SNPs is presented in Table 3, except for SNP 9 with a rare allele frequency < 0.01, which was not included in the subsequent analysis. As observed in Table 3, the allele frequency of –204 C decreased significantly, and that of IVS17–294T and IVS22+36A increased significantly in the patients, giving odds ratios (ORs) of 0.33 (95% confidence interval [CI], 0.17–0.75), 1.86 (95% CI, 1.11–3.10), and 2.23 (95% CI, 1.29–3.83), respectively. In addition, there was no interaction between NEP and APOE polymorphisms in affecting the risk of Alzheimer's disease (results not shown).


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  		Table 3. Distribution of Single Nucleotide Polymorphisms (SNPs) in the NEP Gene in Patients With Alzheimer's Disease (AD) and Controls*.

 
The pair-wise measure of linkage disequilibrium in the control sample is reported in Table 4. Most of the SNPs showed a significant moderate but not complete linkage disequilibrium, except that SNP 3 was not in linkage disequilibrium with SNP 1, 2, 5, and 8, and SNP 8 was not in linkage disequilibrium with SNP 2 and 5.


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  		Table 4. D' Values (Below Diagonal) and r2 Values (Above Diagonal) for Linkage Disequilibrium Between Single Nucleotide Polymorphisms (SNPs) Spanning the NEP Gene Included in This Study*.

 
GENECOUNTING haplotype analysis showed that the haplotype "GTA" of –204G/C, IVS17–294C->T, and IVS22+36C->A was more frequent in patients (5.42%) than in unaffected controls (1.05%), and that the global test also revealed a significant association of haplotypes with Alzheimer's disease (Table 5). Seventeen of the 28 possible 2-SNP haplotypes yielded globally significant evidence for association, as did 41 of 56 of the 3-SNP haplotypes, 54 of 70 of the 4-SNP haplotypes, 42 of 56 of the 5-SNP haplotypes, and 4 of 28 of the 6-SNP haplotypes. In total, approximately two thirds of all the possible haplotypes revealed results that gave global significance at p <.05, and most of them contained one to three of SNP 1, 6, and 7.


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  		Table 5. Haplotype Frequency Estimates and Significance Levels in Patients With Alzheimer's Disease and Controls Using GENECOUNTING Program*.

 
Results from haplo.score analysis of eight SNPs, comparable to those from GENECOUNTING, further confirmed the association between some haplotypes and Alzheimer's disease (global-stat = 35.12, df = 14, p <.01). Table 6 shows the results of statistical analysis of haplotype-specific score adjusted for age, sex, and APOE {epsilon}4 status. The table shows that haplotype 1 was associated with decreasing risk for Alzheimer's disease, whereas haplotypes 13 and 14 were associated with increasing risk for Alzheimer's disease. Moreover, haplotype 2 showed a trend to protection from Alzheimer's disease (haplotype-specific score statistic = –1.93, simulation p =.051).


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  		Table 6. Haplo.score Analysis of Eight SNPs in the NEP Gene in Patients With Alzheimer's Disease (AD)*.

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
As the first systematic screening of single nucleotide polymorphisms in the NEP gene and their haplotype analysis in sporadic Alzheimer's disease, the present investigation identified eight novel SNPs, three of which (–204G->C, IVS17–294C->T, and IVS22+36C->A) revealed significant association with the disease. Subsequent haplotype analyses also provided further evidence for the association between these NEP polymorphisms and Alzheimer's disease.

The –204G->C polymorphism seemed to play a protective role in the development of Alzheimer's disease, because not only the frequency of the –204C allele decreased significantly in the patients, but because the GC genotype was also associated with decreased risk of Alzheimer's disease (OR = 0.36, 95% CI, 0.15–0.83, p =.01, Table 3). Although it is not known whether this polymorphism is functional, transcription factor binding-site analysis by the programme Consite (http://forkhead.cgr.ki.se/cgi-bin/consite) showed that the –204G->C polymorphism was located within the putative transcription repressor factor Pax-4 binding region. Therefore, it could be postulated that the NEP –204C allele might have a lower transcription repressor activity than the G allele, which could in turn lead to an enhanced expression of neprilysin and possibly a reduced accumulation of Aß in the brain. To test the transcription activity of this predicated promoter SNP, reporter gene assays and gel shift assays are ongoing.

Although IVS17–294T and IVS22+36A were associated with increased risk for Alzheimer's disease (Table 3), their corresponding SNPs seemed unlikely to influence neprilysin expression levels or activity, because both of them are located in introns and are 36 bp or approximately 300 bp away from the splice sites. In contrast, it cannot be excluded that both SNPs are in linkage disequilibrium with other "true" Alzheimer's disease risk variant(s) within or near the NEP gene. For instance, SNP 3 (c.401A->G), which causes Gln->Arg mutation, might be such a candidate, although it did not reveal any association with Alzheimer's disease in the present study (p >.05).

The NEP gene was assessed for patterns of linkage disequilibrium to identify SNPs for haplotype analysis (Table 4). Because no complete or very strong linkage disequilibrium was observed among eight SNPs, all of them were included in the haplotype analysis. First of all, using the GENECOUNTING program, we identified a haplotype involving three SNPs (–204G->C, IVS17–294C->T, and IVS22+36C->A; see Tables 3 and 5) associated with Alzheimer's disease (global p <.0001), and the haplotype GTA was most strongly associated with the disease (p =.01). Then the haplotype score analysis of all eight SNPs revealed a global significant association and several haplotype-specific associations (Table 6). These results further supported that the NEP polymorphisms might contribute to Alzheimer's disease risk.

Recently, two reports also implied that the NEP gene might confer susceptibility to Alzheimer's disease. In contrast to results by other Japanese teams and a Swedish group (18–20), Sakai and colleagues (22) found a GT-repeat polymorphism in the promoter region of the NEP gene significantly associated with late-onset Alzheimer's disease, and the 22-repeat allele interacted with the APOE {epsilon}4 allele in raising risk for late-onset Alzheimer's disease. Because of the high variability in short tandem repeat loci (29), these findings need to be replicated using both larger samples and different ethnic populations. The other positive finding was that the 159C/T polymorphism in the 3' untranslated region of the NEP gene was associated with Alzheimer's disease in persons less than 75 years old, and the C/C genotype was a risk factor independent of APOE {epsilon}4 status in a Spanish population (21). However, the present study failed to find any genotype or allele association between this SNP and Alzheimer's disease in Chinese persons either younger or older than 75 years (data not shown). The most plausible explanation of the discrepant results might be the NEP*159C/T in linkage disequilibrium with certain functional variant(s) within the gene or a locus nearby.

Caution should be taken when interpreting our findings. Although the significant difference in allele and haplotype frequencies between patients and unaffected control subjects supported the association of the polymorphisms in NEP with Alzheimer's disease, it could result from other reasons, such as a type I error or population stratification. Regarding a type I error, all the reported significance levels were nominal p values and were not adjusted using the Bonferroni correction because we did not know how many multiple tests were conducted. Regarding population stratification, the results, as a case–control study, are always subject to this potential confounder. For instance, the different genotype frequencies observed may partially reflect different genetic backgrounds in patients and controls (30,31). <--?1-->Although great attention was paid to the study design and analysis, including the restriction of the study to Han Chinese only, we cannot rule out the possible population stratification. Finally, a limitation of our study might be the sample size. For example, a case–control sample of 277 patients and 833 controls would be required to provide 80% power to detect the association between the NEP –204G->C polymorphism and Alzheimer's disease (alpha = 0.05).

Summary
Our results indicated that the NEP gene might either contribute to the susceptibility to Alzheimer's disease or be in linkage disequilibrium with another functional locus on 3q25.2. More independent studies as well as further identification and functional analysis of the variants in the neprilysin gene are needed to completely clarify the possible role of the NEP gene in Alzheimer's disease.


   Acknowledgments
 
This work was supported by grants 2001AA224021-03 and 2002BA711A08 from the National High Technology Research and Development Program, 39993240 from the National Natural Science Foundation of China, AG13196 from the National Institute on Aging (USA), and JB02-2000202601 from the Science and Technology Committee of Guangzhou.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received August 14, 2004

Accepted November 9, 2004


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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