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

Abstract

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.

Keywords

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

Ethical Statement

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

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

Abstract

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.

Keywords

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

Ethical Statement

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

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