Introduction
Alzheimer’s Disease (AD) is the most prevalent dementia in the elderly. The Hallmark of AD is the accumulation of amyloid plaques and the neurofibrillary tau protein tangles in the brain cortex [1]. Amyloid plaques are formed by the accumulation of Amyloid-Beta (Aβ) peptides resulting from the decomposition of Aβ Precursor Protein (APP). Furthermore, tau protein hyper-phosphorylation leads to the polymerization of double-helical filaments, creating neurofibrillary tau tangles [2]. Genetic changes are involved in neurodegenerative diseases such as Parkinson’s disease [3], Multiple Sclerosis [4, 5], and AD [6]. The mutations of APP and Presenilin 1 and 2 for familial AD or early-onset familial Alzheimer’s Disease and ε4 allele of Apolipoprotein E (APOE) gene (APOE ε4) for sporadic cases were demonstrated [7, 8]. Polymorphism of TERM2 [9], MS4A6A [10], d, and CD33 [11]; they were found to be associated with AD.
A defect in the Bridging Integrator 1 (BIN1) gene is also an essential genetic risk factor suggested for Late-Onset Familial Alzheimer’s Disease (LOAD) [12]. Moreover, BIN1 regulates endocytosis, inflammation, calcium homeostasis, and apoptosis [13]. The Single-nucleotide Polymorphism (SNP) rs744373 of the BIN1 gene, placed more than 25 kb before the encoding area of this gene, is the most important variation of the gene associated with AD [14]. Rs744373 polymorphism affects gene expression and interferes with tau metabolism. AD patients with this polymorphism may have high levels of tau accumulation in brain imaging studies and cerebrospinal fluid [15].
Seshadri et al. conducted a meta-analysis of more than 35000 individuals, including 8371 AD patients, and found, for the first time, the association between SNP rs744373 polymorphism of BIN1 on chromosome 2 and AD [16]. They also confirmed the association of this polymorphism with AD [Odds Ratio (OR)=1.13, P<0.05].
Gharesouran et al. studied the association between rs744373 polymorphism and LOAD in Iran’s Turkish Azeri ethnicity [17]. They investigated the distribution of 11 polymorphisms in 160 patients with LOAD and 163 healthy controls. The results revealed that alleles and genotypes of BIN1 gene rs744373 polymorphism were significantly different between LOAD and control groups.
However, Chen et al. could not validate the association of rs744373 polymorphism with AD in 17 AD cases and 34 controls from the Xinjiang Chinese population [18]. Moreover, Kaya et al. did not find an association between AD and BIN1 gene polymorphism in 53 AD patients and 56 controls from the Turkish population [19].
Given the need to identify genetic factors involved in the pathogenesis of AD in various populations, we sought to investigate the association of rs744373 polymorphism of BIN1 gene with LOAD in Guilan Province in the north of Iran.
Materials and Methods
We recruited 110 patients with LOAD and 110 unrelated healthy subjects in this case-control study. Patients over 65 years of age diagnosed with probable AD according to the National Institute on Aging and Alzheimer’s Association criteria [20] were included in the study. Patients with a history of head trauma, stroke, hereditary dementia, Central Nervous System (CNS) infection, neuropsychiatric systemic lupus erythematosus, sarcoidosis, multiple sclerosis, and other neurodegenerative diseases were excluded from the study. Moreover, 110 age- and gender-matched unrelated healthy subjects were recruited.
DNA was extracted from blood samples of study subjects using the salting-out method. Polymerase Chain Reaction (PCR) was performed using a standard protocol. The PCR products (Figure 1) were incubated with the HinfI enzyme, and the genotypes of the cut samples were determined using 3% agarose gel electrophoresis and DNA-safe stain (Figure 2). The gels were visualized under an Ultraviolet (UV) trans-illuminator.
In rs744373 polymorphism of the BIN1 gene, a nucleotide T is replaced with nucleotide C (CTCTCGG). G^ANTC is the sequence of restriction sites by the HinfI restriction enzyme. The band sizes after restriction fragment length polymorphism (RFLP) are displayed in Table 1. 


Using SPSS, the obtained data were compared and evaluated by Chi-square and logistic regression analyses.
Results
We studied 110 patients with LOAD and 110 healthy unrelated healthy control. The LOAD group consisted of 77 (70%) women and and 33 (30%) men with a mean±SD of the age of 77.4±7.8 years. Besides, the control group consisted of 69 women (62.7%) and 40 men (36.4%) with a mean±SD age of 76.9±9.6 years. There were no significant differences between age (P=0752) and gender (P=0.348) of the 2 studied groups. 
The Allelic frequency of the BIN1 gene polymorphism 
The frequency of allele T (Wild-type allele) in the control group and the LOAD group was 70.9% (n=159) and 58.6% (n=129), respectively. Additionally, the frequency of allele C in the LOAD group (41.4%) (n=91) was significantly higher than that of the control group (29.1%) (n=64). The allelic frequency of rs744373 polymorphism in the LOAD and control groups is illustrated in Figure 3. Further, there was a significant relationship between the allelic frequency of rs744373 polymorphism and LOAD [OR=1.71, 95% Confidence Interval (CI)= 1.15- 2.55, P=0.007)].
The genotypic frequency of rs744373 polymorphism in LOAD
The TT genotype was found in 65 (59.1%) and 54 (49.1%) of the control and LOAD, respectively. TC genotype was seen in 26(23.6%) and 21(19.1%) of the control and the AD, respectively. The frequency of genotype CC was higher in the LOAD group (n=35, 31.8%) than in the control group (n=19, 17.3%). Figure 4 shows the genotypic frequency of the rs744373 polymorphism in LOAD and control groups. There was a significant association between the polymorphism and LOAD (P=0.043).
Logistic regression analysis indicated that the odds of genotype CC were almost twice as high as that of genotype TT (normal homozygote) in the LOAD (Table 2).


The CC genotype was associated with LOAD (P=0.019).
Investigating the relationship between different genotypes of polymorphism and AD according to the inheritance model and through nominal logistic regression indicated that the codominant and recessive models could play roles in the inheritance of polymorphism rs744373. Both models yielded similar associations between genotype and AD.
Discussion
The present study examined the association between rs744373 polymorphisms of the BIN1 gene and LOAD in a population in Guilan Province in the north of Iran.
The only study on the association between this polymorphism and LOAD in Iran was conducted by Gharesouran et al. [17] on a population of Iranian Azeris. The authors examined 11 polymorphisms, including rs744373, rs11554585, and rs7561528 polymorphisms in the BIN1 gene in 160 patients with LOAD and 163 healthy controls. The frequency of allele C of rs744373 in the patient group (12.8%) was significantly greater than that in the control group (5%), and the polymorphism was associated with LOAD (OR = 2.847, P<0.001). However, the results of these studies should be interpreted with caution due to their limited sample sizes.
The odds of genotype CC were almost twice as high as that of genotype TT (normal homozygote) in the LOAD in the present study. CC genotype was also associated with LOAD (P=0.019). 
BIN1 plays a role mainly through interaction with protein tau in AD [21]. Tau protein, a highly-soluble protein in the cytoplasm that maintains the stability of microtubules, is hyper-phosphorylated in AD, precipitates, and creates a dual string helix structure that ultimately leads to the creation of neurofibrillary tangles. Accumulation of these tangles inside the cell is neurotoxic and considered a mechanism of neurodegeneration [22]. Recent studies hypothesized that polymorphisms of BIN1 increase the neurotoxicity of the hyper-phosphorylated protein tau by interfering with the interaction between proteins tau and bin1, thereby changing the synapses [23].
Seshadri et al. conducted an international three-stage meta-analysis in which more than 35,000 individuals (including 8371 AD patients) participated. For the first time, they found the association between single nucleotide rs744373 polymorphism near the BIN1 gene on chromosome 2. They also confirmed the association of this polymorphism with AD [16].
In a study in 3 contrasting European populations from Finland, Italy, and Spain, Lambert et al. evaluated the association between BIN1 gene rs744373 polymorphism (OR=1.26, 95%CI=1.15-1.38, P=2.9×10-7) [14].
Moreno et al. conducted a case-control study in Colombia and found a significant association between polymorphism rs744373 in the BIN1 gene and AD (OR=1.42, 95%CI=1.07-1.88, P=0.015) [8].
Wang et al. found that rs744373 was significantly associated with AD in a population in East China (OR=1.256, 95%CI=1.028-1.535, P=0.038) [24]. The author found that such an association was not present in a population in Southwest China (OR=1.024, 95%CI =0.820-1.281, P=0.874). However, the relationship between rs744373 of the BIN1 gene with AD was confirmed after meta-analysis (OR=1.14, 95% CI=1.05-1.24, P=0.001).
Dos Santos et al. further conducted a case-control study on rs744373 polymorphism of the BIN1 gene in a Brazilian population [25]. Their findings indicated no association between rs744373 and AD in the Brazilian population (CC genotype of BIN1; OR=0.79, 95%CI=0.28-2.26, P=0.660), TT genotype of BIN1 (OR=1.20, 95%CI=0.66-2.19, P=0.547).
Carrasquillo et al. genotyped rs744373 and rs597668 variants in a large (3,287 LOAD, 4,396 controls) series from the USA and Europe [26]. They outlined a significant association between BIN1 and LOAD (OR=1.17, 95%CI=1.08-1.26, P=1.1×10−4).
Miyashita et al. conducted a 3-stage genome-wide association study using three Japanese, Koreans, and Caucasians [27]. They reported a significant association between rs744373 of BIN1 gene with LOAD in Japanese (OR=1.25, 95%CI=1.11-1.4, P=1.39×10 -4) and Korean (OR=0.98, 95%CI=0.81-1.18, P=8.05×10-1).
Chen et al. investigated the association of the five AD‑associated variants, 8‑oxoguanine DNA glycosylase 1 rs1052133, BIN1 rs744373, sortilin-related receptor 1, rs1133174, presenilin 2 rs8383, and nerve growth factor rs6330, in the Xinjiang Chinese population [18]. They recruited 17 AD cases and 34 controls from the Xinjiang Chinese population. The authors were unable to validate the association of rs744373 polymorphism with AD. They declared this might be because of the limited sample size.
Kaya et al. examined the polymorphism and allele frequency of the APOE and BIN1 genes in 53 AD patients and 56 controls in a Turkish population [19]. There was no significant difference in CC genotype prevalence of the BIN1 gene between patients and controls (P>0.05). The authors concluded no association between AD and the BIN1 gene polymorphism.
Han et al. conducted a meta-analysis with 71,168 samples (22,395 AD cases & 48773 controls, from 37 studies of 19 articles) [28]. They identified a significant association between rs744373 polymorphism with AD in pooled populations (OR=1.12; 95%CI=1.07-1.17, P=5×10-07) and in Caucasian populations (OR=1.16, 95%CI=1.10-1.22, P=3.38×10-08). However, the association was not identified in the East Asian populations (OR=1.057, 95%CI= 0.95-1.15, P=0.393).
The association between rs744373 and LOAD has presented to be heterogeneous results. Its presence in the European populations (white), Colombia, Japan, China, and Iran was associated with an increased risk of LOAD [8, 14, 25, 26, 27]. Still, such an association did not exist in Brazil, Turkey, and the East Asian mixed population [18, 19, 25, 28].
Conclusion
The present study findings suggested that polymorphism rs744373 of the BIN1 gene was significantly associated with LOAD in the studied population. It is helpful to conduct further studies of this polymorphism with larger sample sizes. Further studies on the expression of the BIN1 gene are recommended.

Ethical Considerations
Compliance with ethical guidelines
All study procedures were done in compliance with the ethical guidelines of the Declaration of Helsinki, 2013. The present study was approved by the Ethics Committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1398.518).

Funding
Guilan University of Medical Sciences financially supported the study.

Authors contributions
All of the authors helped shape this collaborative research study and contributed to the project.

Conflict of interest
The authors declared no conflicts of interest.

Acknowledgements
We would like to thank the Clinical Research Development Unit of Poursina Hospital, Guilan University of Medical Sciences, Rasht, Iran, for its support.


Refrences

1. Andalib S, Ghayeghran A, Moadabi Y, Asadi K, Mohammadpour M, Ghorbani-Shirkouhi S. Association of diabetes mellitus type 2 and alzheimer’s disease. Caspian J Health Res. 2019; 4(4):86-9. [DOI:10.29252/cjhr.4.4.86]
2. Alexander AG, Marfil V, Li C. Use of Caenorhabditis elegans as a model to study Alzheimer’s disease and other neurodegenerative diseases. Front Genet. 2014; 5:279. [DOI:10.3389/fgene.2014.00279] [PMID] [PMCID]
3. Andalib S, Vafaee MS, Gjedde A. Parkinson’s disease and mitochondrial gene variations: A review. J Neurol Sci. 2014; 346(1-2):11-9. [DOI:10.1016/j.jns.2014.07.067] [PMID]
4. Andalib S, Emamhadi M, Yousefzadeh-Chabok S, Salari A, Sigaroudi AE, Vafaee MS. MtDNA T4216C variation in multiple sclerosis: A systematic review and meta-analysis. Acta Neurol Belg. 2016; 116(4):439-43. [DOI:10.1007/s13760-016-0675-5] [PMID]
5. Andalib S, Talebi M, Sakhinia E, Farhoudi M, Sadeghi-Bazargani H, Motavallian A, et al. Multiple sclerosis and mitochondrial gene variations: A review. J Neurol Sci. 2013; 330(1-2):10-5. [DOI:10.1016/j.jns.2013.04.018] [PMID]
6. Karch CM, Cruchaga C, Goate AM. Alzheimer’s disease genetics: From the bench to the clinic. Neuron. 2014; 83(1):11-26. [DOI:10.1016/j.neuron.2014.05.041] [PMID] [PMCID]
7. Huang X, Liu H, Li X, Guan L, Li J, Tellier LCAM, et al. Revealing Alzheimer’s disease genes spectrum in the whole-genome by machine learning. BMC Neurol. 2018; 18(1):5. [DOI:10.1186/s12883-017-1010-3] [PMID] [PMCID]
8. Moreno DJ, Ruiz S, Ríos Á, Lopera F, Ostos H, Via M, et al. Association of GWAS top genes with late-onset Alzheimer’s disease in Colombian population. Am J Alzheimers Dis Other Demen. 2017; 32(1):27-35. [DOI:10.1177/1533317516679303] [PMID]
9. Jonsson T, Stefansson H, Steinberg S, Jonsdottir I, Jonsson PV, Snaedal J, et al. Variant of TREM2 associated with the risk of Alzheimer’s disease. N Engl J Med. 2013; 368(2):107-16. [DOI:10.1056/NEJMoa1211103] [PMID] [PMCID]
10. Antúnez C, Boada M, González-Pérez A, Gayán J, Ramírez-Lorca R, Marín J, et al. The membrane-spanning 4-domains, subfamily A (MS4A) gene cluster contains a common variant associated with Alzheimer’s disease. Genome Med. 2011; 3(5):33. [DOI:10.1186/gm249] [PMID] [PMCID]
11. Mehdizadeh E, Khalaj-Kondori M, Shaghaghi-Tarakdari Z, Sadigh-Eteghad S, Talebi M, Andalib S. Association of MS4A6A, CD33, and TREM2 gene polymorphisms with the late-onset Alzheimer’s disease. BioImpacts. 2019; 9(4):219-25. [DOI:10.15171/bi.2019.27] [PMID] [PMCID]
12. Franzmeier N, Rubinski A, Neitzel J, Ewers M; Alzheimer’s Disease Neuroimaging Initiative (ADNI). The BIN1 rs744373 SNP is associated with increased tau-PET levels and impaired memory. Nat Commun. 2019; 10(1):1766. [DOI:10.1038/s41467-019-09564-5] [PMID] [PMCID]
13. Tan MS, Yu JT, Tan L. Bridging integrator 1 (BIN1): Form, function, and Alzheimer’s disease. Trends Mol Med. 2013; 19(10):594-603. [DOI:10.1016/j.molmed.2013.06.004] [PMID]
14. Lambert JC, Zelenika D, Hiltunen M, Chouraki V, Combarros O, Bullido MJ, et al. Evidence of the association of BIN1 and PICALM with the AD risk in contrasting European populations. Neurobiol Aging. 2011; 32(4):756.e11-5. [DOI:10.1016/j.neurobiolaging.2010.11.022] [PMID]
15. Wang HF, Wan Y, Hao XK, Cao L, Zhu XC, Jiang T, et al. Bridging integrator 1 (BIN1) genotypes mediate Alzheimer’s disease risk by altering neuronal degeneration. J Alzheimers Dis. 2016; 52(1):179-90. [DOI:10.3233/JAD-150972] [PMID]
16. Seshadri S, Fitzpatrick AL, Ikram MA, DeStefano AL, Gudnason V, Boada M, et al. Genome-wide analysis of genetic loci associated with Alzheimer disease. JAMA. 2010; 303(18):1832-40. [DOI:10.1001/jama.2010.574] [PMID] [PMCID]
17. Gharesouran J, Rezazadeh M, Khorrami A, Ghojazadeh M, Talebi M. Genetic evidence for the involvement of variants at APOE, BIN1, CR1, and PICALM loci in risk of late-onset Alzheimer’s disease and evaluation for interactions with APOE genotypes. J Mol Neurosci. 2014; 54(4):780-6. [DOI:10.1007/s12031-014-0377-5] [PMID]
18. Chen W, Zhou X, Duan Y, Zou T, Liu G, Ying X, et al. Association of OGG1 and DLST promoter methylation with Alzheimer’s disease in Xinjiang population. Exp Ther Med. 2018; 16(4):3135-42. [DOI:10.3892/etm.2018.6524] [PMID] [PMCID]
19. Kaya G, Gündüz E, Acar M, Hatipoğlu ÖF, Acar B, Ilhan A, et al. Potential genetic biomarkers in the early diagnosisof Alzheimer disease: APOE and BIN1. Turk J Med Sci. 2015; 45(5):1058-72. [PMID]
20. McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR Jr, Kawas CH, 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(3):263-9. [DOI:10.1016/j.jalz.2011.03.005] [PMID] [PMCID]
21. Gao P, Ye L, Cheng H, Li H. The mechanistic role of bridging integrator 1 (BIN1) in Alzheimer’s disease. Cell Mol Neurobiol. 2021; 41(7):1431-40. [DOI:10.1007/s10571-020-00926-y] [PMID]
22. Ropper AH SM, Klein J, Prasad S. Degenerative diseases of the nervous system. In: Ropper AH, Brown RH, editors. Adams and victor’s principles of neurology 11th Edition. New York: McGraw-Hill Education; 2019. p. 1082-155. https://www.google.com/books/edition/Adams_and_Victor_s_Principles_of_Neurolo/gAtWvQEACAAJ?hl=en
23. Glennon EB, Lau DH, Gabriele RMC, Taylor MF, Troakes C, Opie-Martin S, et al. Bridging integrator 1 protein loss in Alzheimer’s disease promotes synaptic tau accumulation and disrupts tau release. Brain Commun. 2020; 2(1):fcaa011. [DOI:10.1093/braincomms/fcaa011] [PMID] [PMCID]
24. Wang HZ, Bi R, Hu QX, Xiang Q, Zhang C, Zhang DF, et al. Validating GWAS-identified risk loci for Alzheimer’s disease in Han Chinese populations. Mol Neurobiol. 2016; 53(1):379-90. [DOI:10.1007/s12035-014-9015-z] [PMID]
25. Ramos Dos Santos L, Belcavello L, Camporez D, Iamonde Maciel de Magalhães C, Zandonade E, Lírio Morelato R, et al. Association study of the BIN1 and IL-6 genes on Alzheimer’s disease. Neurosci Lett. 2016; 614:65-9. [DOI:10.1016/j.neulet.2015.12.046] [PMID]
26. Carrasquillo MM, Belbin O, Hunter TA, Ma L, Bisceglio GD, Zou F, et al. Replication of BIN1 association with Alzheimer’s disease and evaluation of genetic interactions. J Alzheimers Dis. 2011; 24(4):751-8. [DOI:10.3233/JAD-2011-101932] [PMID] [PMCID]
27. Miyashita A, Koike A, Jun G, Wang LS, Takahashi S, Matsubara E, et al. SORL1 is genetically associated with late-onset Alzheimer’s disease in Japanese, Koreans and Caucasians. PLoS One. 2013; 8(4):e58618. [PMID]
28. Han Z, Wang T, Tian R, Zhou W, Wang P, Ren P, et al. BIN1 rs744373 variant shows different association with Alzheimer’s disease in Caucasian and Asian populations. BMC Bioinformatics. 2019; 20(Suppl 25):691. [DOI:10.1186/s12859-019-3264-9] [PMID] [PMCID]