Search for the elusive haplotype of the APOE polymorphism associated with Alzheimer’s disease

doi:10.21203/rs.3.rs-4902566/v1

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Search for the elusive haplotype of the APOE polymorphism associated with Alzheimer’s disease

https://doi.org/10.21203/rs.3.rs-4902566/v1

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The common APOE2/E3/E4 polymorphism is determined by two-site haplotypes: C112R and R158C. Due to strong linkage disequilibrium between the two sites, three of the four expected haplotypes/alleles (E2, E3, E4) have been observed. Compared to the most common haplotype of E3 (C112 – R158), the E4 (R112 – R158) and E2 (C112 – C158) haplotypes are determined by a single-point mutation at codons 112 and 158, respectively. The fourth haplotype (E5) having mutations at both sites (R112–C158) has been reported only as an incidental finding in three kindreds. To our knowledge, no systematic search has been done to determine its distribution in the general population. The objective of this study was to search for the elusive haplotype in 355 APOE 2/4 subjects derived from 14,819 genotyped subjects. A DNA fragment of 177bp from APOE 2/4 subjects was subcloned into competent bacterial cells to construct the phased haplotype clones followed by Sanger sequencing. We also used Whole-genome sequencing and RFLP assay to search for the fourth haplotype. All three strategies confirmed that the E4 and E2 alleles are present on opposite chromosomes, with no example having both alleles on the same chromosome, suggesting E5 might have minimum effect, if any, on disease risk.

Biological sciences/Biological techniques

Biological sciences/Genetics

Biological sciences/Molecular biology

APOE haplotype

Cloning

Sequencing

Restriction-fragment length polymorphism (RFLP)

Genotyping

The common APOE2/E3/E4 polymorphism is determined by two-site haplotypes at codon 112 [cysteine (C) > arginine (R)] and codon 158 (R > C), resulting in six genotypes. Due to strong linkage disequilibrium (LD) between the two sites, three of the four expected haplotypes or alleles (E2, E3, and E4) have been observed and extensively studied in relation to the risk of Alzheimer’s disease (AD) risk ^{[1, 2]}. Compared to the most common haplotype of E3 (C112 – R158), the mutant E4 (R112 – R158) and E2 (C112 – C158) haplotypes are determined by single-point mutations: T > C transition at codon 112 (TGC to CGC for E4) and C > T transition at codon158 (CGC to TGC for E2). The fourth haplotype, which we denote here as APOE5 and explained later, having amino acid substitutions at both positions (R112 – C158) has been reported only as an incidental finding in three kindreds ^[3–5]. Genetically determined structural variation in APOE was originally described using two-dimensional gel electrophoresis or isoelectric focusing (IEF) from ultracentrifuged and delipidated plasma ^[6–8]. which was subsequently simplified for APOE genotyping directly from plasma without prior ultracentrifugation and dilapidation for large-scale population screening ^[9].Gel electrophoresis that separates plasma APOE isoforms based on their size, charge, or isoelectric point differences, can also enable the identification of new APOE allelic isoforms is not possible to determine using the current TaqMan assays as they are specific for detecting genomic variations at codons 112 and 158 only.

Individuals with the APOE 2/4 genotypes are double heterozygotes at codons 112 and 158 and due to strong LD between the two sites, it is always assumed that the alleles for E4 (R112) and E2 (C158) are present on opposite chromosomes. Since TaqMan assays do not reveal the two-site haplotype phases, it is possible that some of the APOE 2/4 individuals may carry the APOE5 haplotype where both alleles (R112 and C158) are present on the same haplotype and inherited from a single parent. This could only be accomplished unequivocally by either subcloning followed by single-strand sequencing or next-generation whole-genomic sequencing (WGS). To our knowledge, no such systematic effort has been made to determine the haplotype phases and the occurrence of the APOE5 haplotype in the general population. The objective of this study was to search for the elusive APOE5 haplotype by using combination of subcloning, WGS or restriction fragment length polymorphism (RFLP) assays in a large number of APOE 2/4 subjects and determine its potential association with AD.

This study was approved by the University of Pittsburg institutional Review Board and informed consent was obtained from all the subjects prior to their participation in the study. All the experimental protocols were reviewed and approved by Department of Human Genetics, School of Public Health University of Pittsburgh and were performed in accordance with the approved guidelines and regulations of the dpartemnt. APOE genotype data on 14,819 subjects (mean age = 72.7 years; female = 56.6%; Table 1) derived from multiple studies ^[10–13] was used in this study. All subjects previously classified as APOE 2/4 were re-genotyped by TaqMan assays for the two APOE SNPs: rs429358 (E4) and rs7412 (E2) to confirm the APOE 2/4 genotype. For subcloning, A 177 bp product with single deoxyadenosine (A) overhang on 3’ end was PCR amplified from genomic DNA (0.5-1.0 µg) using primers [5’GCGGACATGGAGGACGTG-3’; 5’GGCCTGGTACACTGCCAG-3’] ^[14]. and confirmed by running on agarose gel with 1X TBE buffer. The PCR product was purified using QIAGEN purification kit and ligated into 4kb PCR™ II-TOPO linearized vector with single 3’ deoxythymidine(T) residue and Ampicillin resistance ORF (bases 2173–3033), then transformed into chemically competent bacterial cells (E. coli DH5αTM -T1^R)) to construct the phased haplotype clones of 177 bp derived from the maternal or paternal chromosome. The clones were cultured on Luria-Bertani (LB) agar containing ampicillin to get multiple copies of the constructed clone. To differentiate the bacterial colonies from the successful clone constructs, 40mg/ml X-gal was spread onto the plates before culturing the clone. To create a stock of each colony, the colonies with successful constructs were incubated separately overnight at 37°C into 3mL LB broth containing ampicillin. DNA was isolated from the cultured colony cells using Biolab miniprep plasmid DNA kit followed by sequencing with M13 forward and reverse primers. The sequencher (v5.4.6) software was used to analyze the inserted phased haplotype.

Table 1

A. Demographic and *APOE* Genotype/Allele data in study participants
	Total Sample (N = 14819)	AD Cases (N = 3280)	Controls (N = 11535)	P-value between cases and controls
Mean age ± SD	72.7 ± 14.2	72.1 ± 9.8	72.9 ± 15.2	4.94E-04
Female, N (%)	8142 (56.6)	2000 (61.0)	6141 (55.4)	1.20E-08
White/Black/ Others (%)	86.8/12.7/0.5	93.4/6.3/0.3	84.8/14.6/0.6	1.36E-36
APOE Genotype N (%)				1.95E-238
2/2	88 (0.6)	8(0.2)	80 (0.7)
2/3	1557 (10.5)	163(5.0)	1394(12.1)
2/4	355 (2.4)	88(2.7)	267(2.3)
3/3	8254 (55.7)	1280(39.0)	6973(60.5)
3/4	3948 (26.6)	1415(43.1)	2531(21.9)
4/4	617 (4.2)	326(10.0)	290(2.5)
APOE Allele Frequency (%)				7.08E-254
E2	7.1	4.1	7.9
E3	74.2	63.1	77.5
E4	18.7	32.8	14.6

Table 1

B. Odds ratios (ORs) for *APOE* Genotypes using *APOE* 3/3 as reference
APOE Genotype	ORs (95% confidence interval)	P-Value
2/2	0.73 (0.32–1.45)	0.41
2/3	0.65(0.54–0.77)	1.64E-06
2/4	2.01(1.55–2.58)	8.92E-08
3/4	3.26(2.97–3.58)	2.72E-137
4/4	6.87(5.70–8.30)	4.50E-90

We also employed an RFLP assay, utilizing two different enzymes to discern the APOE5 haplotype (Murrell et al., 2006). We amplified a 221 bp PCR product using forward (5’CTGTCCAAGGAGCTGCAG 3’) and reverse (5’ GCCCCGGCCTGGTACACTGCCAG 3’) primers followed by overnight digestion at 37°C with a 3:2 ratio of smartcut enzymes AflIII (for codon 112 digestion) and HaeII (for codon 158 digestion). The samples were loaded onto a 3.5% metaphor GEL using 1XTBE buffer alongside a 50 bp ladder. The AflIII enzyme cleaves at codon112 only in the presence of nucleotide TGC (C112), while the HaeII enzyme cleaves only if there is nucleotide CGC (R158) at codon158. Whereas the cutting sites at both positions would be abolished in the E5 haplotype (CGC/R112 and TGC/C158).

Demographic characteristics of the subjects:

Demographics information along with the APOE genotype/allele frequencies in the total sample and sample stratified by case-control are provided in Table 1. Of 14,819 individuals four subject lacked case control status. As expected, the frequency of APOE4 carriers (2/4, 3/4, and 4/4 genotypes) was higher in AD cases than in controls, the difference of which is also reflected in the APOE4 allele frequency (32.8% vs 14.6%). Likewise, the frequency of APOE2 carriers (2/2 and 2/3 genotypes) was lower in AD cases than in controls, as also reflected in the APOE2 allele frequency difference (4.1% vs 7.9%). Although the 2/4 genotype carries opposite protective and risk alleles, it was a risk factor for AD (odds ratio (OR) = 2.01, p = 8.92E-08), which was similar to the OR of 2.6 reported earlier in more than 17,000 cases and controls ^[15]. Since the E2 and E4 alleles in the 2/4 genotype are assumed to be inherited from both parents on different haplotypes, it would be intriguing to investigate the effect of the elusive E5 haplotype on AD risk where both alleles are inherited from a single parent. Of the 355 subjects with the 2/4 genotype (see Table 1), DNA was not available from 15 controls and thus we proceeded with the 340 samples (17% Black) for the search for the elusive haplotype via subcloning, WGS or RFLP assays.

Subcloning:

Results from subcloning followed by sequencing are depicted in Fig. 1. The plasmid DNA sequence from each clone revealed the presence of the following combination of nucleotide bases corresponding to the 1st base of codon 112 and 1st base of codon 158: T – T for the E2 haplotype or C – C for the E4 haplotype (Fig. 1). In no instance, the C – T combination corresponding to the E5 haplotype was observed. Identical results were obtained with WGS (results not shown).

RFLP:

The RFLP results are shown in Fig. 2. The full-length gel image of the RFLP assay is provided in the supplementary data(Supplementary fig). In the RFLP analysis of the 221 bp PCR amplified fragment followed by double enzyme digestion, the tested samples with the APOE 2/4 genotype gave the expected diagnostic bands of 168 bp and 198 bp (Fig. 2). No sample revealed the expected uncut band of 221 bp corresponding to the E5 haplotype due to loss of restriction sites at codon 112 and codon 158. It is noteworthy that in some samples with the 2/4 genotype, we visualized a faint band at position 221 bp, which was due to incomplete digestion (Fig. 2) as we confirmed it upon sequencing. One study also reported such an undigested band in their one subject with the 2/4 genotype and suggested it to be due to the formation of heterodimers during amplification ^[4]. However, we think this vague band is most likely due to incomplete digestion rather than being heterodimers because this band was observed only in few 2/4 subjects. This cautionary note may be helpful for those seeking to detect the E5 haplotype using the RFLP assay alone.

Previously, the E5 haplotype has been reported in only 5 subjects from three unrelated kindreds. The first case was found in an autistic Italian child and his unaffected mother while investigating the potential association of APOE alleles with primary autism in trios ^[3]. The authors named this haplotype as E3r because it possesses reverse arrangement of the cysteine and arginine residues at codons 112 and 158 (R112 - C158) compared to the common E3 haplotype (C112 - R158). The second case was reported in a 70-year-old healthy Yoruban female with normal lipid profile and in her 34-year-old son from Ibadan, Nigeria and this was named as E1Y ^[4] to differentiate it from the previously rare E1 isoform (Asp127 – Cys158)^[16]. The third unrelated case of E5 was observed in a 77-year-old Caucasian patient with motor neuron disease but with normal cognition and lipid profile ^[5]. This haplotype was not inherited by his two children.

We name this elusive haplotype as APOE5 because its earlier designation as E3r or E1Y is confusing and gives the misleading impression that this may not be part of the APOE2/E3/E4 polymorphism. The original nomenclature of three APOE isoforms was based on their structure and isoelectric focusing (IEF) point differences on gel electrophoresis where IEF point of E2 isoform was more acidic and the IEF point of E4 was more basic compared to the common E3 isoform ^[6]. The E1 isoform (G127D, R158C) differs from E2 at amino acid position 127 where glycine is replaced with aspartic acid, causing one negative charge difference from E2 ^[16]. Thus, the genetic determinant of E1 is a point mutation at codon 127 and its designation represents its relative IEF position to E2 on gel electrophoresis. This situation is like a rare APOE4Pittsburgh variant (L28P, C112R) which differs from E4 at amino acid position 28 where leucine is replaced with proline ^[17]. Most importantly, E1 and APOE4Pittsburgh do not correspond to the elusive E5 haplotype of the common APOE polymorphism determined by variation at codons 112 and 158. On the other hand, E5 is a part of the well-known APOE polymorphism due to point mutations at both codons.

The most likely explanation for the observation of four two-site APOE haplotypes, E2, E3, E4, and E5, is due to intragenic crossover between the nucleotide sequence of codons 112 and 158 (Fig. 3). The ultra rarity of the E5 haplotype may be explained due to the small distance of only 138 nucleotides between the E4 and E2 mutant sites that may prevent frequent recombination between the two sites. An alternative explanation may be that the E2 mutation arose recently on the E4 haplotype after the split of human races, like the example of the APOE4Pittsburgh mutation that occurred on the E4 background ^[17]. However, the observation of E5 haplotype in one African kindred along with two kindreds of European descent belies this hypothesis.

Although E3 is considered as the parent haplotype or allele in humans because of its common occurrence followed by E4 and E2, E4 has been postulated as the ancestral allele because all the great apes code for arginine with the identical codon sequence (CGC) at positions 112 and 158 corresponding to the human E4 haplotype ^[18]. Accordingly, it has been hypothesized that human E3 evolved from primate E4 by a C to T point mutation coding for cysteine at codon 112 (TCG) and then E2 evolved from E3 by a C to T transition coding for cysteine at codon 158 (TCG). As above, we hypothesize that the E5 haplotype was formed most likely due to crossover between the E4 and E2 haplotypes (Fig. 3).

In conclusion, we have performed a systematic and focused search to identify the elusive E5 haplotype in the general population by cloning and sequencing a large number of subjects heterozygous for the APOE 2/4 genotype but found no such example. For this reason, we could not examine its role in AD risk. Our data suggests that the occurrence of E5 is extremely rare, and it might have a minimum effect, if any, on disease risk.

Supplementary data

Supplementary figure

Additional information

The authors declare no conflict of interest.

Acknowledgements

The study was supported in part by NIH grants R01 AG064877 and P30 AG066468

Funding Sources

NIH grants R01 AG064877 and P30 AG066468

Author Contributions

Conceptualization, Funding acquisition, Supervision: M. Ilyas Kamboh; Data curation: Asma Naseer Cheema, Elizabeth Lawrence, Narges Zafari, Kang-Hsien Fan, Ruyu Shi, Muaaz Aslam, Vibha Acharya, Alayna Jean Holderman, Annie Bedison, Eleanor Feingold Formal analysis: M. Ilyas Kamboh, Asma Naseer Cheema, Ruyu Shi , Kang-Hsien Fan; Methodology and Investigation: Asma Naseer Cheema, Elizabeth Lawrence, Narges Zafari, Kang-Hsien Fan; Writing – original draft: Asma Naseer Cheema Writing – review and editing: M. Ilyas Kamboh , Asma Naseer Cheema; Ruyu Shi, Muhammad Muaaz Aslam, Vibha Acharya

Data Availability Sequencing data has been submitted to the National Institute on Aging Genetics of Alzheimer’s Disease Data Storage Site (NIAGADS) and is available at https://dss.niagads.org/studies/sa000012/. The principal investigator (corresponding author) of the study may be contacted if someone needs to acquire the data.

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No competing interests reported.

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Search for the elusive haplotype of the APOE polymorphism associated with Alzheimer’s disease

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Abstract

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Introduction

Methods

Results

Demographic characteristics of the subjects:

Subcloning:

RFLP:

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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