Genotyping Apolipoprotein E (APOE) Isoforms with Sequence-Specific-Primer (SSP)-PCR in Early‑Onset Alzheimer’s Disease Patients: A Rapid and Revised Methodology

Yıl 2024, Cilt: 50 Sayı: 2, 157 - 163, 08.10.2024

Işıl Ezgi Eryılmaz , Mustafa Bakar , Ünal Egeli , Gülşah Çeçener

https://doi.org/10.32708/uutfd.1468449

Öz

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive damage to brain cells, leading to impairment in cognitive functions. The Apolipoprotein E (APOE) variants play a significant role in the genetic basis of AD, especially late-onset AD (LOAD), and increase the disease risk at an earlier age. Although controversial, some studies reveal the association between APOE genotype and early-onset AD (EOAD) regardless of family history. Therefore, diagnostic laboratories widely perform routine tests to determine the APOE genotype. In the present study, we implemented a revised methodology for the Sequence-Specific-Primer-PCR (SSP-PCR) test for rapid APOE genotyping in 67 EOAD patients. Then, the findings were validated using automatic sequencing with newly designed primers for the related region of the APOE. We state clearly that the applicability of the SSP-PCR method was improved when the primer concentrations of control genes were increased 2-fold, as we reported. All data obtained from SSP-PCR were consistent with Sanger sequencing confirmations. Based on the genotyping results, the four different APOE genotypes were detected: E2/E4, E3/E3, E3/E4, and E4/E4. The frequencies were 1.5% (n=1) for E2/E4, 76.1% (n=51) for E3/E3, 16.4% (n=11) for E3/E4, and 6% (n=4) for E4/E4. In the study group, 23.9% (n=16) of the patients had homozygous or heterozygous APOE E4. However, we detected no significant association between the clinical features and the APOE genotype. As a result, this method is reliable, cost-effective, and rapid for performing genotyping analysis of the APOE for routine tests and research studies with larger EOAD cohorts.

Anahtar Kelimeler

APOE, early-onset Alzheimer’s disease, SSP-PCR, Sanger sequencing

Etik Beyan

This study was approved by the Medical Ethics Committee (2023-19/37) and complied with the ethical standards of the Helsinki Declaration.

Erken Başlangıçlı Alzheimer Hastalarında Sekans-Spesifik-Primer (SSP)-PCR ile Apolipoprotein E (APOE) İzoformlarının Genotiplendirilmesi: Hızlı ve Revize Bir Metodoloji

Öz

Alzheimer hastalığı (AD), beyin hücrelerinde ilerleyici hasara neden olan ve bilişsel işlevlerde bozulmaya yol açan nörodejeneratif bir hastalıktır. Apolipoprotein E (APOE) varyantları, özellikle geç başlangıçlı AD (LOAD) olmak üzere AD'nin genetik temelinde önemli bir rol oynar ve hastalık riskini daha erken yaşta artırır. Tartışmalı olmasına rağmen, bazı çalışmalar APOE genotipi ile erken başlangıçlı AD (EOAD) arasındaki ilişkiyi aile geçmişinden bağımsız olarak ortaya koymaktadır. Bu nedenle, tanı laboratuvarları APOE genotipini belirlemek için yaygın olarak rutin testler yapmaktadır. Mevcut çalışmada, 67 EOAD hastasında hızlı APOE genotiplemesi için Sekans-Spesifik-Primer-PCR (SSP-PCR) testi için revize edilmiş bir metodoloji uyguladık. Ardından, bulgular APOE'nin ilgili bölgesi için yeni tasarlanmış primerlerle otomatik sekanslama kullanılarak doğrulandı. Bildirdiğimiz gibi, kontrol genlerinin primer konsantrasyonları 2 kat artırıldığında SSP-PCR yönteminin uygulanabilirliğinin arttığını açıkça belirtiyoruz. SSP-PCR'den elde edilen tüm veriler Sanger dizileme doğrulamalarıyla tutarlıydı. Genotipleme sonuçlarına dayanarak dört farklı APOE genotipi tespit edildi: E2/E4, E3/E3, E3/E4 ve E4/E4. Sıklıklar E2/E4 için %1.5 (n=1), E3/E3 için %76.1 (n=51), E3/E4 için %16.4 (n=11) ve E4/E4 için %6 (n=4) idi. Çalışma grubunda hastaların %23.9'unda (n=16) homozigot veya heterozigot APOE E4 vardı. Ancak klinik özellikler ile APOE genotipi arasında anlamlı bir ilişki tespit etmedik. Sonuç olarak bu yöntem, rutin testler ve daha büyük EOAD kohortlarıyla yapılan araştırma çalışmaları için APOE'nin genotipleme analizini gerçekleştirmek için güvenilir, uygun maliyetli ve hızlıdır.

Anahtar Kelimeler

APOE, early-onset Alzheimer’s disease, SSP-PCR, Sanger sequencing

Etik Beyan

This study was approved by the Medical Ethics Committee (2023-19/37) and complied with the ethical standards of the Helsinki Declaration.

Kaynakça

• 1. McKhann GM, Knopman DS, Chertkow H, et al. The diagnosis of dementia due to Alzheimer’s disease: recommendations from the National Institute on Aging- Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 2011;7:263–9.
• 2. Cacace R, Sleegers K, Van Broeckhoven C. Molecular genetics of early-onset Alzheimer's disease revisited. Alzheimers Dement 2016;12:733-48.
• 3. Reitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. Nat Rev Neurol 2011;7:137-52.
• 4. Licht EA, McMurtray AM, Saul RE, Mendez MF. Cognitive differences between early- and late-onset Alzheimer’s disease. Am J Alzheimer’s Dis Other Dement 2007;22:218–222.
• 5. Warren JD, Fletcher PD, Golden HL. The paradox of syndromic diversity in Alzheimer's disease. Nat Rev Neurol 2012;8:451–464.
• 6. Strittmatter WJ. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci USA 1993;90:1977–1981.
• 7. Mahley RW, Nathan BP, Pitas RE. Apolipoprotein E. Structure, function, and possible roles in Alzheimer’s disease. Ann N Y Acad Sci 1996;777:139–45.
• 8. Mahley RW, Rall Jr SC. Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet 2000;1:507–37.
• 9. Farrer LA, Cupples LA, Haines JL, et al. Effects of age, sex, and ethnicity on the association between Apolipoprotein E genotype and Alzheimer's disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium. JAMA 1997;278:1349–56.
• 10. Saunders AM, Strittmatter WJ, Schmechel D, et al. Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer’s disease. Neurology 1993;43:1467–72.
• 11. Zhong L, Xie YZ, Cao TT, et al. A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism. Mol Neurodegener 2016;11:2.
• 12. Corder EH, Saunders AM, Risch NJ, et al. Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nat Genet 1994;7:180–4.
• 13. Bertram L, Tanzi RE. Thirty years of Alzheimer’s disease genetics: the implications of systematic meta-analyses. Nat Rev Neurosci 2008;9:768–78.
• 14. van der Vlies AE, Pijnenburg YA, Koene T, et al. Cognitive impairment in Alzheimer's disease is modified by APOE genotype. Dement Geriatr Cogn Disord 2007;24:98-103.
• 15. Smits LL, Pijnenburg YA, van der Vlies AE, et al. Early onset APOE E4-negative Alzheimer's disease patients show faster cognitive decline on non-memory domains. Eur Neuropsychopharmacol 2015;25:1010-7.
• 16. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res 1990;31:545–548.
• 17. Somsen GW, Welten HT, Mulder FP, Swart CW, Kema IP, de Jong GJ. Capillary electrophoresis with laser-induced Genotyping APOE isoforms with SSP-PCR in EOAD fluorescence detection for fast and reliable apolipoprotein E genotyping. J Chromatogr B Analyt Technol Biomed Life Sci 2002;775:17-26.
• 18.Wenham PR, Newton CR, Price WH. Analysis ofapolipoprotein E genotypes by the Amplification RefractoryMutation System. Clin Chem 1991;37:241-4.
• 19.Pantelidis P, Lambert-Hammill M, Wierzbicki AS. Simplesequence-specific-primer-PCR method to identify the threemain apolipoprotein E haplotypes. Clin Chem 2003;49:1945-8.
• 20.Eryilmaz IE, Bakar M, Egeli U, et al. Evaluation of the ClinicalFeatures Accompanied by the Gene Mutations: The 2 NovelPSEN1 Variants in a Turkish Early-onset Alzheimer DiseaseCohort. Alzheimer Dis Assoc Disord 2021;35:214-222.
• 21.Yang L, Yan J, Jin X, et al. Estimation of diagnosticperformance of dementia screening tests: Mini-Mental StateExamination, Mini-Cog, Clock Drawing test and AscertainDementia 8 questionnaire. Aging Ment Health 2018;22:942-946.
• 22.Younis M, Ali MAM, Ghareeb DA, Youssef R, Fathy SA.Maternal Thrombophilic and Hypofibrinolytic Genetic Variantsin Idiopathic Recurrent Pregnancy Loss: a Continuing Mystery.Reprod Sci 2023;30:656-666.
• 23.Al Harthi F, Huraib GB, Zouman A, Arfin M, Tariq M, Al-Asmari A. Apolipoprotein E gene polymorphism and serumlipid profile in Saudi patients with psoriasis. Dis Markers2014;2014:239645.
• 24.Calero O, García-Albert L, Rodríguez-Martín A, Veiga S,Calero M. A fast and cost-effective method for apolipoproteinE isotyping as an alternative to APOE genotyping for patientscreening and stratification. Sci Rep 2018;8:5969.
• 25.Cilingir O, Ozbabalik Adapinar BD, Durak Aras B, et al. Association Between Alzheimer Disease and APOE GenePolymorphisms in Turkish Population, Osmangazi Journal ofMedicine 2020;42:222-230.