ApoE ε4 and IL-6-174G/C Polymorphism may Lead to Early Onset of Alzheimer's Disease with Atypical Presentation
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.329604
Source of Support: None, Conflict of Interest: None
Background: Alzheimer's disease (AD) is the most common cause of dementia. Although genetic mutations are known in rare familial form, exact cause of neurodegeneration in sporadic AD is still unknown. While ApoE ε4 and IL-6 C-174G/C patterns have been found to increase the risk of AD in Caucasians, the results are inconsistent in other ethnic groups.
Keywords: Alzheimer disease, apolipoprotein E, interleukin-6, polymorphism, early-onset Alzheimer disease
Globally, an estimated 50 million people are suffering from dementia. In India alone, this figure was 3.7 million in 2010 and is projected to increase to 5.29 million by 2020. Alzheimer's disease (AD) a chronic neurodegenerative disease, is the most common cause of dementia worldwide.
Pathologically, AD is characterized by the presence of amyloid plaque and neurofibrillary tangles with neuronal loss. The cause of AD is poorly understood. The most accepted hypothesis is the aggregation of β amyloid and hyperphosphorylation of tau protein leading to neurodegeneration. This hypothesis came from studying rare familial cases. However, there are still many unanswered questions for common sporadic form of the disease. Sporadic AD is considered multifactorial with many environmental factors interacting with genetic factors. Genetic factors are said to increase the risk of these sporadic late onset AD. Two major genetic risk factors for AD are ApoE4 and IL-6-174G/C polymorphisms.
ApoE is produced in abundance in the brain and serves as a lipid transporter in cerebrospinal fluid. It plays an important role in neuronal repair. It is a major susceptibility gene for sporadic as well as for familial AD. ApoE ε4 increases the occurrence and lowers the age of onset of the disease. This association is high among Europeans, but low in Africans and Japanese. ApoE ε4 is considered as a risk factor not only for AD but also for vascular dementia (VaD), frontotemporal dementia, as well as for other age and lifestyle-related conditions like diabetes, atherosclerosis, hyperlipidemia, and cardiovascular conditions.,, ApoE ε4 has many detrimental effects on central nervous system as it may interact with the amyloid β peptide, which is an important factor in pathogenesis of AD. ApoE ε4 and 42 residue β-amyloid peptide (Aβ) stimulate lysosomal leakage and potentiates cell death and apoptosis in neurons. The IL-6 gene in humans is located in the short arm of chromosome 7 (7p21). Aβ peptide activates the microglia and astrocytes that secrete many inflammatory mediators like components of the complement system, acute phase proteins, and interleukin-6 (IL-6). Neuroinflammation plays a role in the pathogenesis of AD. Various inflammatory proteins including cyclooxygenase-2 (COX-2) and phospholipase A2 enzyme gene polymorphisms have been found to affect the susceptibility for AD. IL-6-174G/C polymorphism affects its transcription and thus changing the inflammatory response. Previous studies done on AD and IL-6-174 G/C polymorphism were limited and with inconsistent results. Genotype CC of IL-6-174 G/C and genotype GG plus GC of IL-6-572 C/G were found to decrease the risk of AD observed in a meta-analysis.
India is a large country with a diverse population. There are few published works on effect of ApoE ε4 on AD from Northern and Southern parts of the country.,,, Existing literature from North India is also there on IL-6 polymorphism and AD. No previous study was conducted on ApoE and IL-6 polymorphism in Eastern part of India. Our investigation envisaged the objective of correlating the genetic risk factors of ApoE ε4 and IL-6-G174 G/C polymorphism with AD for understanding the genetic basis in the patients of Eastern India.
The study was conducted in a Neurology tertiary care center in Kolkata, India for 3 years between December 2015 and September 2018 after obtaining approval from by Institutional Ethics Committee. During this period, a total 224 patients of dementia were evaluated at the Cognitive Clinic of the institute. Each patient was assessed in detail by history from a reliable caregiver, neurological examination, and cognitive functions assessment. Attention was tested by digit span and serial subtraction tests. Memory was tested by a verbal learning test of Kolkata Cognitive Battery. For language, we used the Bengali version of Western Aphasia Battery. Visuospatial domain was tested using a letter cancellation task and line bisection test, while visuoperceptual testing was done by dot counting and fragmented letters. The Frontal Assessment Battery tested the executive function. All the tools mentioned above were validated for the Bengali language, and we have used them in previous studies as well.
All patient underwent detailed investigations including complete blood count, erythrocyte sedimentation rate, blood biochemical tests like thyroid function test, serum Vitamin B12, fasting and postprandial blood sugar, glycosylated hemoglobin, lipid profile, liver and renal function tests, cardiological evaluation including electrocardiogram, 2D echocardiogram, and carotid Doppler study. Additional tests like serology for HIV and syphilis, antinuclear antibody and other autoantibody profile, serum homocysteine, prothrombotic profile, and others were performed depending upon the clinical condition. A detailed magnetic resonance imaging (MRI) scan of brain in a Siemens 3Tesla scanner was performed in almost all patients. Those who had contraindication for MRI or could not complete MR scanning a cranial CT scan was performed. (18F)-2–fluro-deoxy-D-glucose positron emission tomography (FDG-PET) scan of the brain was performed in Siemens Biograph True point 16 with HD PET and a multislice computerized tomography scanner in selected patients. During this period, detailed evaluation could not be performed in 18 patients as they did not turn up in subsequent visits. Of the 204 patients completing full evaluation, 122 (59.22%) were diagnosed as probable AD. Probable AD was diagnosed using NIA-AA criteria. Those with mixed dementia, mild cognitive impairment, and other form of dementia were excluded. Family members of seven AD patients refused to participate in the study. Thus, we recruited a total of 115 AD patients during this period. We also included 162 healthy age and sex matched controls. The control subjects were selected from healthy relatives of patients attending general neurology outpatient department of the institute. They were screened to exclude any cognitive impairment before including them as controls. A written informed consent was taken from every patient and their family members, and also from control subjects. Ten milliliters of venous blood was collected from patients and controls.
Genomic DNA was extracted from the EDTA blood by the phenol chloroform method. APOE and IL-6 genes were amplified by polymerase chain reaction (PCR). The primers used for APOE and IL-6 amplification are forward 5'-ACAGAATTCGCCCCGGCCTACAC-3' and reverse 5'-TAAGCTTGGCACGGCTGTCCAAGGA-3' and forward 5'- CAGAAGAACTCAGATGACTGG-3' and reverse 5'- GCTGGGCTCCTGGAGGGG-3', respectively. The PCR amplification was carried out in a final reaction volume of 25 μL containing ~ 100 ng of genomic DNA, 1X PCR buffer, 2mM MgCl2, 0.2 mM dNTPs, 10 picomoles forward and reverse primer each, and 1.25 units Taq polymerase. Both the PCRs were hot start processes. PCR conditions for ApoE consist of initial denaturation for 6 min and then 30 cycles of amplification at 95°C for 1 min, 60°C for 1 min, and 70°C for 2 min. The final extension was at 72°C for 7 min. The initial denaturation for IL-6 PCR was done at 95°C for 5 min followed by 25 cycles of 95°C for 1 min, 63°C for 1 min, and 72°C for 2 min. The final extension was at 72°C for 10 min. PCR products were stored at -20°C. The amplified fragments were digested with restriction enzymes Hhal (for ApoE) and SfaNl (for IL-6). For determination of ApoE polymorphism, restriction digests containing 10 μL of PCR product and 2U HhaI were incubated overnight at 37°C. Similarly, for determination of IL-6-174 polymorphism, restriction digests containing 10 μL of PCR product and 1.5U SfaNI were incubated overnight at 37°C. The products of both the reactions were analyzed on a 4% agarose gel. Serum IL-6 levels were determined by ELISA using human IL-6 ELISA Kit (Raybiotech, USA).
Data were analyzed using the Statistical Package for Social Sciences (SPSS) software version 25.0 (SPSS, Chicago, IL). The demographic variable of the participants were expressed in the terms of mean (SD) and frequency (percentage). Comparison of ApoE and IL-6-174 G/C polymorphism pattern between patients and control and of various clinical and polymorphism patterns between young-onset (50Y) and late-onset patients (>50Y) were analyzed using Mann–Whitney U test. A P value < 0.05 was considered significant.
The demographic details of the patients and controls are given in [Table 1]. There was no significant difference between patients and controls in any of the demographic parameters. Out of 115 AD patients, seven were below the age of 50.
From the data generated, it was evident that ApoE ε3 allele was common in comparison to ApoE ε4 allele in our AD patients and control subjects. Similarly, in IL-6-174 G/C polymorphism, GG was the commonest followed by GC and CC in our AD patients as well as in control subjects [Table 2]. No association was found between the IL-6 levels in sera of patients and controls with either G or C at position-174.
In comparison with control, there was no difference in the pattern of ApoE and IL-6 polymorphism in our AD patients. However, when we looked into the younger patients with age of onset below 50 years an interesting pattern emerged [Table 3]. They had a significantly higher percentage of ε4 variant than the patient group of above 50. All seven patients with onset below 50 years were ε3/ε4 as compared to 12.04% of patients with age of onset above 50 years (P = 0.00).
The IL-6-174 variant also showed a very unique pattern. The most common type of IL-6 variant found in our study was GG, followed by GC. The least frequent allele was found to be CC. Only one patient had ApoE ε4/ε4 genotype had IL-6-174 GG genotype. However, in patients below 50 years of age, the percentage of C allele was found to be very high. Five patients who were below 50 years of age (age of onset 43, 45, 47, 43, and 27 years), had ε3/ε4 polymorphism for ApoE and CC polymorphism for IL-6-174, and only two had (age of onset 42 and 41 years) had ε3/ε4 polymorphism for ApoE and GC polymorphism for IL-6-174. These seven patients live far away from each other, even not related to each other and the patients were from both the genders. None of our controls had both ε4 and C allele in this study.
Though the overall distributive pattern of ApoE and IL-6-174 was found to be statistically not significant, the findings in the young onset AD patients were significant. The CC polymorphism of IL-6-174 was significantly more associated with patients with age of onset below 50 (P = 0.00). While 71.43% of younger patients had CC polymorphism in IL-6-174, none had this in older patients. Frequencies of GC polymorphism were not significantly different in two groups (P = 0.262).
Clinical parameters were also significantly different in patients with age of onset below 50 [Table 4]. While two of the seven patients (28.57%) with onset below 50 years had a positive family history, only four (3.7%) patients with age of onset more than 50 years had family history (P = 0.004). One of them (Patient 3: age of onset - 47 years) had a younger sibling with similar condition and another patient (Patient 5: age of onset - 27 years) had a family history of similar illness, suggesting autosomal dominant inheritance [Figure 1]. His siblings (both were elder sisters) were genotyped and found to be ε3/ε3 and GC polymorphism and they did not have any symptoms.
The atypical clinical presentation was observed in three (42.85%) young patients and in only one (0.92%) older patients (P = 0.002). The youngest patient (Patient 5: age of onset 27 years) presented with executive dysfunction at the beginning with subsequent involvement of other cognitive domains like episodic memory, visuospatial dysfunction, and MRI, showing global cortical atrophy and FDG-PET consistent with AD with prominent hypometabolism in bilateral frontal, medial temporal, posterior cingulate, and precuneus. Another patient (Patient 7: age of onset 41 years) presented with visual symptoms and features of Balint's syndrome with MRI and FDG-PET features suggesting posterior cortical atrophy (PCA). The third patient (Patient 3: age of onset 47 years) presented with apraxia and visuospatial dysfunction and MRI features of biparietal atrophy. While only one patient (with age of onset 56 years) beyond 50 years of age range with ApoE (ε3/ε4) and IL-6-174 (GC), polymorphism presented with atypical presentation and imaging correlation with initial presentation of PCA. During an average of two year follow-up, none of them showed any parkinsonian feature or myoclonus. Their symptoms progressed to involve recent episodic memory, language, and executive dysfunction, suggesting atypical variant of AD. All other patients had typical multidomain amnestic presentation.
The prevalence of ε4 allele in the AD population worldwide is not consistent. Studies from Norway and Spain, reported a higher prevalence of ε4 allele, whereas lower percentage was reported from Hungary and Iran., Similarly, various studies from Italy, Japan, and Spain showed a positive association of G allele of IL-6-174 with AD.,, Some studies revealed a role of C allele in AD pathogenesis, while others found this protective. Indian population has high frequency of ApoE ε3 allele and IL-6-174 G allele. Studies from different parts of India showed varying results in ApoE and IL-6-174 polymorphism and their effect in AD. In Northern India, a report suggested that the presence of ApoE ε4 and IL-6-174 C allele increased the chances of having AD and VaD. A study from Kerala showed that a positive family history of dementia was more likely in AD patients than VaD patients. In Southern India, it was reported that there was a positive association of ApoE ε4 polymorphism with dementia. A meta-analysis showed that all genotypes of ApoE ε4 allele were associated with increased risk of AD. In our study, we found that the IL-6-174 level in blood is not associated with its G/C polymorphism. This is consistent with a few previous reports, although some data suggested the contrary.
None of our controls had both ε4 and C allele in this study. The findings indicate a somewhat protective function for some variants like ε3/ε3 for ApoE and GG for IL-6-174. It seems that both the presence of one ε4 allele in ApoE gene and one C allele in case of IL-6-174 gene make a person susceptible to develop AD at an early age.
Early-onset AD is clinically different and often have genetic basis. The initial and most common presentation of AD is the recent episodic memory impairment, most aptly called as amnestic presentation. While majority of AD patients present with such multidomain amnestic presentation, around one-third of AD present with initial symptoms pertaining to nonamnestic domains like language, visuospatial, and executive dysfunction. These atypical presentation are mostly seen in young-onset AD, although they are not always having genetic mutation. Three of our younger patients whose age of onset were below 50 years had atypical presentation, while only one (onset at 56 years of age) with onset above 50 years of age had atypical presentation. By standard definition, all of them were young-onset AD with onset before 65 years of age. However, those with very early onset, i.e., before 50 years of age had some odd features in our cohort. Two of them had positive family history, with one having autosomal dominant pattern. We could not analyze his genetic mutation, but he had dysexecutive presentation. That he had probable AD was established by his global cognitive deficit at presentation and MRI as well as FDG-PET study. The other patient (Patient 3) had one of his siblings having similar illness. He had clinical and radiological features of biparietal variant of PCA. The third patient (Patient 7) had typical presentation of PCA with imaging correlation. During an average of 2-year follow-up, all three did not show any parkinsonian feature or myoclonus, rather their symptoms progressed to involve other cognitive domains suggesting atypical variant of AD. The only other patient having atypical presentation was having onset at the age of 56 years.
The ApoE and IL-6-174 polymorphism pattern of our patients having onset before 50 was very characteristic that lead us to infer that the presence of ε4 and C pattern make a person susceptible to develop the disease at a very early age with atypical presentation. These two are not causal, but susceptibility genes and with presence of genetic and epigenetic factors may facilitate the development of AD pathology and clinical manifestation of the disease.
In our cohort of AD, ε3/ε3 in ApoE and GG in IL-6-174 were the commonest polymorphism pattern observed and we did not find any difference from the control subjects. However, in patients with age of onset below 50 years, ApoE ε3/ε4 and IL-6-174 CC and GC polymorphism were common and these young patients were more commonly having atypical clinical presentation. Our preliminary observations need to be validated with more number of patients of early onset AD from diverse ethnic population. We think these alleles together could be potential biomarkers for early onset AD.
Limitation and strength of the study
Our study was not without limitations. First, we could analyze only seven patients of early onset AD in the study period. Second, we could not perform assessment of biomarker in our patients. The strength of our study was detailed evaluation of demographic and clinical profile and correlating these with ApoE and IL-6-174 status.
The authors acknowledge financial support from the Department of Science and Technology, Government of West Bengal, India for this study. We also acknowledge Mr. Sourav Banerjee for his help in carrying out statistical analysis of the study. Infrastructural facilities of the Neurogenetic unit (laboratory) provided by the Director, Bangur Institute of Neurosciences are gratefully acknowledged.
Financial support and sponsorship
The study received research grant from the Department of Biotechnology, Government of West Bengal.
Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2], [Table 3], [Table 4]