{"id":60849,"date":"2024-09-30T10:26:30","date_gmt":"2024-09-30T10:26:30","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60849"},"modified":"2024-10-09T18:44:27","modified_gmt":"2024-10-09T18:44:27","slug":"mthfr-polymorphisms-and-plasma-homocysteine-in-early-onset-alzheimers-disease-a-case-control-study","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/mthfr-polymorphisms-and-plasma-homocysteine-in-early-onset-alzheimers-disease-a-case-control-study\/","title":{"rendered":"MTHFR Polymorphisms and Plasma Homocysteine in Early-Onset Alzheimer’s Disease: A Case-Control Study"},"content":{"rendered":"\n

Introduction <\/strong><\/p>\n\n\n\n

Incidence<\/strong><\/p>\n\n\n\n

Alzheimer\u2019s disease (AD) is an age-accelerated progressive neurodegenerative\ndisorder characterized by a decline in both behavioural and cognitive functions1,2<\/sup>.\nThe disease is the most common type of dementia in individuals aged 65 years or\nabove2,3<\/sup>, accounting for 50-70% of all cases4<\/sup>. Globally,\ndementia accounted for 3.9 % in people aged 65 years or above5<\/sup>.\nRegionally, the lowest prevalence of dementia was reported in Africa (1.6%), followed\nby China and Western Pacific regions (4.0%). In Latin America, it accounted for\n4.6% of cases, The highest prevalence was reported in North America (6-4%) and Western\nEurope (5.4%)5<\/sup>. <\/p>\n\n\n\n

Alzheimer\u2019s disease is classified as early-onset Alzheimer\u2019s disease (EOAD) and late-onset Alzheimer\u2019s disease (LOAD); based to the onset of the disease under or over 65 years old6<\/sup>, respectively. About 10-30% of individuals aged above 65 years develop late-onset Alzheimer\u2019s disease7<\/sup>. Early-onset Alzheimer\u2019s disease, on the other side, represented 5-10% of all cases of AD8<\/sup>. In other studies, however, the prevalence was as low as 3%9<\/sup>. The mean age for AD patients varied among different populations. In one study, the mean age was 72 \u00b1 5 years for LOAD and 60 \u00b1 4 years for EOAD10<\/sup>. In another study, the mean age for LOAD and EOAD was 74 \u00b1 6 years and 56 \u00b1 5 years, respectively11<\/sup>. <\/p>\n\n\n\n

Literature has already established the correlation between gender and\nAD, with highest rates detected in women than men12<\/sup>. This\ngender-related variation was attributed to hormonal and psychological factors12<\/sup>.\nIn an attempt to find an explanation, some studies have suggested a protective\nrole of the female sex hormone oestrogen against neuronal degeneration, and\nhence postmenopausal women have a greater risk of AD13<\/sup>. <\/p>\n\n\n\n

Clinically, Alzheimer\u2019s disease is characterized by a progressive\ndecline in episodic memory and cognitive function parameters such as language,\nattention, reasoning, memory, comprehension, judgement, execution, and visuospatial\nabilities14<\/sup>. The disease has three stages which take place in a\nchronological manner. The first stage is called the preclinical or asymptomatic\nstage, owing to the asymptomatic nature of the stage. The second stage is the cognitive\nimpairment stage, which is followed by the third stage of dementia2<\/sup>.\nIt is worth mentioning that the clinical presentation of early-onset and\nlate-onset disease is almost the same14<\/sup>. A notable difference is\nthat patients with the early-onset disease tend to preserve the episodic memory\nfunction rather than the cognitive function14<\/sup>. Additionally, patients\nof the early-onset disease tended to show a more aggressive course with more\nfrequent pathological patterns and a relatively shorter life span14<\/sup>.<\/p>\n\n\n\n

Generally, AD is diagnosed on clinical bases as the disease\npathology can be seen in otherwise asymptomatic individuals. Moreover, disease biomarkers\nlike plasma and cerebrospinal fluid markers2<\/sup> were unable to detect all\nabnormalities in amyloid peptide and tau protein15<\/sup>. The clinical\ncriteria for the diagnosis of dementia and Alzheimer\u2019s disease were both\nimplemented in the diagnosis of AD16<\/sup>.<\/p>\n\n\n\n

The aetiology of Alzheimer\u2019s disease is not fully understood16<\/sup>\nand is thought to have a multifactorial nature17<\/sup>. This is applied,\nin particular; to cases of LOAD18<\/sup>. The main pathological abnormalities\nin AD included the extracellular accumulation of amyloid-beta (A\u03b2) plaques and\nthe intraneuronal deposition of tau proteins (p-tau)19<\/sup>. Other abnormalities\nincluded the synaptic and neuronal loss20<\/sup>, the vascular dysfunction9<\/sup>,\nand the brain atrophy21<\/sup>. Furthermore, infection22<\/sup>,\ngenetic susceptibility23<\/sup>, and mutations23<\/sup> were all incriminated\nin the disease pathology. Other factors included abnormal acetylcholine in the\nbrain, insulin resistance, vascular dysfunction, oxidative stress,\nmitochondrial dysfunction and inflammation9<\/sup>. In addition, several\nrisk factors for the disease were identified, including advanced age18<\/sup>,\nethnicity24<\/sup>, diabetes9<\/sup>, Down syndrome9<\/sup>, cardiovascular\nand cerebral diseases9<\/sup>, environmental factors9<\/sup>, smoking25<\/sup>\nand alcohol consumption25<\/sup>.<\/p>\n\n\n\n

Genetically, the \u03b54 allele of the APOE gene is the most common identified mutation associated with the late onset disease8<\/sup>. For the early-onset disease, almost all cases have familial nature9<\/sup>, with 30-60% of cases have at least one affected first-degree relative26<\/sup>, and 10% have autosomal dominant pattern of inheritance8<\/sup>. The familial pattern of the disease was attributed to mutations affecting A\u03b2 precursor protein (APP) gene, Presenilin 1 (PSEN1) gene, and Presenilin 2 (PSEN2) gene9<\/sup>. Those genes are affected by over 400 known mutations8<\/sup>. <\/p>\n\n\n\n

Pathologically, the mutations are thought to increase not only the\nproduction of \u03b2-amyloid proteins23<\/sup> but also its deposition8<\/sup>.\nOther suggested mutations include those affecting APOE, BIN1, SORL1, CLU, TREM29<\/sup>,\nand ABCA727<\/sup> genes. The methylene\ntetrahydrofolate reductase (MTHFR) gene has three polymorphisms associated with\nAD: C677T, A1298C, and A1793G28<\/sup>. MTHFR C677T was reported as the\nmost significant of these mutations28<\/sup>. Regarding the other two\npolymorphisms, it was hypothesized that they protect against the development of\nAD28<\/sup>. Furthermore, MTHFR C677T was proposed as a risk factor for AD\nin a study on Asians29<\/sup>, and in a meta-analysis study30<\/sup>. <\/p>\n\n\n\n

Both the heterozygous (CT) and homozygous alleles (TT) of MTHFR C677T polymorphism were associated with an increased risk for AD31<\/sup>. Interestingly, a study on Chinese population concluded that both MTHFR C677T and A1298C polymorphisms were associated with the late onset disease28<\/sup>. This conclusion was supported by a case study which detected both C677T and A1298C polymorphisms in a patient diagnosed with early-onset Alzheimer\u2019s disease32<\/sup>. Moreover, another study found that A1298C polymorphism, but not MTHFR C677T; was associated with the disease33<\/sup>. Among the theories that explained the role of MTHFR C677T polymorphism in the development of AD were the white matter signal abnormalities caused by hyperhomocysteinemia34<\/sup>, the impaired function and deposition of the neuronal amyloid-\u03b2 protein precursor (A\u03b2PP) caused by the polymorphism-induced phosphorylation of amyloid- \u03b2 protein precursor35<\/sup>, and the impaired cognitive function caused by the low folate levels secondary to MTHFR C677T polymorphism36<\/sup>.<\/p>\n\n\n\n

Methylene tetrahydrofolate reductase (MTHFR) is an enzyme that catalyses the biochemical conversion of\n5,10-methylenetetrahydrofolate to 5-methyltrahydrofolate37<\/sup>. The 5-methyltetrahydrofolate\nis a coenzyme in the formation of methionine from homocysteine37<\/sup>.  The gene encoding the enzyme is located on\nthe short arm of\nchromosome 1 (1p36.3)37<\/sup>. More than 40 single nucleotide\npolymorphisms in MTHFR gene were described38<\/sup>, the most common being\nC677T and A1298C39<\/sup>. MTHFR C677T polymorphism results in the\nformation of a thermolabile enzyme40<\/sup>.  Accordingly, enzyme activity will be\ndiminished, resulting in elevated levels of homocysteine, a condition known as\nhyperhomocysteinemia41<\/sup>. MTHFR C677T polymorphism and the resulting\nhyperhomocysteinemia were significantly associated with the development of several\ndiseases and syndromes including subacute combined degeneration of the spinal\ncord42<\/sup>, cardiac syndrome X43<\/sup>, hypertrophic cardiomyopathy44<\/sup>,\ncleft lip\/palate45<\/sup>, \u03b2-thalassemia46<\/sup>, preeclampsia47<\/sup>, diabetes\nmellitus48<\/sup>, rectal cancer49<\/sup>, stroke50<\/sup>, neural\ntube defects51<\/sup>, psoriasis52<\/sup>, late-onset Alzheimer’s53<\/sup>,\nand bipolar disorders54<\/sup>. Literature has reported a global frequency\nof MTHFR 677CT of about 25%. Regionally, the highest frequency was 47%,\nreported among Hispanics. The frequency dropped to 36% in Europeans, 30% in\nEast Asians, 12% in South Asians, and finally 9% among Africans55<\/sup>. Additionally,\nthe frequency of the homozygous and heterozygous alleles in the general population\nwas 8.5% and 2.5%, respectively56<\/sup>.<\/p>\n\n\n\n

Currently, there is no treatment available for AD19<\/sup>. The life expectancy of the disease depends on age, gender, and genetics19<\/sup> and is relatively shorter in EOAD than LOAD14<\/sup>. Furthermore, the early-onset disease has a poorer prognosis than the late-onset disease as its course is more progressive14<\/sup>. Accordingly, EOAD has a greater socioeconomic burden14<\/sup> given that the associated responsibilities like raising children and working53<\/sup>. Moreover, EOAD is associated with a considerable delay in diagnosis54<\/sup>, reflected as higher rates of premature mortality6<\/sup>.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Study Type and Population<\/strong><\/p>\n\n\n\n

This case-control study was conducted among Alzheimer\u2019s patients in Khartoum, Sudan, during the period from May 2021-May 2022. Participants were recruited from different neuropsychiatric and healthcare facilities in Khartoum. The sample size was calculated based on a 95% confidence interval, a 5% margin of error, an estimated 5% population proportion, and a population size of 6000 citizens, assuming a normal sample distribution57<\/sup>. The population proportion was taken as 5% based on previous well-designed review study8<\/sup>. The following formula was used to calculate the sample size58<\/sup>:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

whereas:<\/em><\/p>\n\n\n\n

n<\/em> = sample size <\/p>\n\n\n\n

N<\/em> = population\nsize<\/p>\n\n\n\n

p<\/em> = expected\npercentage of the variable<\/p>\n\n\n\n

q<\/em> = 1-p<\/p>\n\n\n\n

e<\/em> = accepted\nmargin of error<\/p>\n\n\n\n

t\u03b1<\/em> = 1.96 for 95%\nconfidence interval. <\/p>\n\n\n\n

Accordingly, a total of 73 patients; clinically diagnosed with EOAD,\nwere enrolled using simple random sampling. All patients diagnosed by expert\nneurologists and met the clinical criteria for diagnosis of dementia15<\/sup>,\nand AD clinical stages15<\/sup> during the study period were included in\nthe study. The control group included 73 healthy subjects recruited from individuals\nattending the same facilities for other reasons. <\/p>\n\n\n\n

Data Collection<\/strong><\/p>\n\n\n\n

Sociodemographic data and medical data related to Alzheimer\u2019s disease were collected from the hospital\u2019s medical records after obtaining an ethical approval.<\/p>\n\n\n\n

DNA Extraction and PCR<\/strong><\/p>\n\n\n\n

Whole blood was collected, and DNA was extracted using Qiagen kits (FlexiGene DNA Kit)59<\/sup>.  DNA was amplified by polymerase chain reaction using GeneAmp PCR kit (PerkinElmer Cetus)60<\/sup>. The 198 bp amplified fragments were digested with HinfI endonuclease which identifies C-T substitution. Accordingly, fragments were cut into 175 and 23 bp fragments. Fragments were then electrophoresed in polyacrylamide gel and stained with ethidium bromide43<\/sup>. The wildtype (CC) allele appeared as a 198 bp band whereas the homozygous (TT) allele appeared as a 175 bp band. The heterozygous (CT) allele appeared as two bands (198 and 175 bp)43<\/sup>. For A1298C polymorphism, the amplified 163 bp was digested with MboII. The AA genotype produced 5 fragments of 56, 31, 30, 28 and 18 bp. The homozygous (CC) allele gives 4 fragments of 84, 31, 30 and 18 bp whereas the heterozygous (AC) allele produced 6 fragments of 84, 56, 31, 30, 28 and 18 bp43<\/sup>.<\/p>\n\n\n\n

Plasma Homocysteine<\/strong><\/p>\n\n\n\n

Plasma homocysteine level was measured using Human Homocysteine (HCY) ELISA Kit61<\/sup> . EDTA tube was used to collect blood. sample was immediately Centrifuged for 15 minutes at 1000 x g, 2-8\u00b0C. Plasma homocysteine levels over 15 micromole\/l were considered as hyperhomocysteinemia62<\/sup>. <\/p>\n\n\n\n

Data Presentation and Analysis<\/strong><\/p>\n\n\n\n

Descriptive data were presented as means and standard deviations for quantitative variables and frequencies for qualitative variables. Results were statistically analysed using the SPSS (18th version). Chi-Square test was used to compare gender and the distribution of MTHFR C677T genotypes between EOAD patients and controls. The two-tailed t-test was used to compare means of plasma homocysteine between AD cases and control subjects. Analysis of variance (ANOVA) test was used to correlate plasma homocysteine with the distribution of MTHFR C677T genotypes. The p-value was considered significant when \u2264 0.05.<\/p>\n\n\n\n

Ethical approval<\/strong><\/p>\n\n\n\n

The study was ethically approved by the local ethical committee at Faculty of Medicine, El-Neelain University, and the Ministry of Health, Sudan. <\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Demographic Data<\/strong><\/p>\n\n\n\n

The current study recruited 73 Sudanese patients with early-onset\nAlzheimer\u2019s and other 73 control subjects. The mean age of participants was 57.9\nyears (table 1). With regards to gender, males represented 27.3% and 53 females\nrepresented 72.4% (table 2).  The\ndifference between cases and control subjects was significant (p=0.000). <\/p>\n\n\n\n

Table 1: Mean age of EAOD patients<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Count, n<\/strong><\/p>\n<\/td>\n

\n

73<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Sum, \u03a3x<\/p>\n<\/td>\n

\n

4221<\/p>\n<\/td>\n<\/tr>\n

\n

Mean, x\u0304<\/p>\n<\/td>\n

\n

57.821917808219<\/p>\n<\/td>\n<\/tr>\n

\n

Variance, s2<\/p>\n<\/td>\n

\n

9.9539573820396<\/p>\n<\/td>\n<\/tr>\n

\n

Standard deviation<\/p>\n<\/td>\n

\n

3.1549892839817<\/p>\n<\/td>\n<\/tr>\n

\n

Standard error of mean (SEM)<\/p>\n<\/td>\n

\n

0.36926356518872<\/p>\n<\/td>\n<\/tr>\n

\n

Confidence of interval<\/p>\n<\/td>\n

\n

95%, 1.960sx\u0304<\/p>\n<\/td>\n<\/tr>\n

\n

Margin of error (confidence interval 95%)<\/p>\n<\/td>\n

\n

57.8219 \u00b10.724 (\u00b11.25%)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 2: Sex distribution among EOAD cases and controls<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Cases <\/strong><\/p>\n<\/td>\n

\n

Observed <\/strong><\/p>\n<\/td>\n

\n

20 (27.4%)<\/strong><\/p>\n<\/td>\n

\n

53 (72.6%)<\/strong><\/p>\n<\/td>\n

\n

73<\/strong><\/p>\n<\/td>\n

\n

df = 1<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

33<\/p>\n<\/td>\n

\n

40<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

p-value = 0.000<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

5.1212<\/p>\n<\/td>\n

\n

4.225<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

Chi Square = 18.6924<\/p>\n<\/td>\n<\/tr>\n

\n

Controls<\/p>\n<\/td>\n

\n

Observed<\/p>\n<\/td>\n

\n

46 ()<\/p>\n<\/td>\n

\n

27<\/p>\n<\/td>\n

\n

73<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

33<\/p>\n<\/td>\n

\n

40<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

5.1212<\/p>\n<\/td>\n

\n

4.225<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

Column totals<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

66<\/p>\n<\/td>\n

\n

80<\/p>\n<\/td>\n

\n

146<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Plasma Homocysteine<\/strong><\/p>\n\n\n\n

Table 3 shows that the mean plasma homocysteine level was 15.5\nmicromole\/l among cases and 13.01 micromole\/l among control subjects. The\ndifference in the mean plasma homocysteine level between cases and control\nsubjects was significant (p<\/em>=0.0001).<\/p>\n\n\n\n

Table 3: Mean plasma homocysteine levels among EOAD and controls<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

AD<\/strong><\/p>\n<\/td>\n

\n

Count, N:<\/strong><\/p>\n<\/td>\n

\n

73<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/p>\n

df = 144<\/p>\n

Standard error of difference = 0.172<\/p>\n

t = 14.221F<\/p>\n

two-tailed p value = 0.0001<\/p>\n

95% CI = 2.1112572999529675-2.792852<\/p>\n

2890890335.<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/p>\n

 <\/strong><\/p>\n<\/td>\n

\n

Sum, \u03a3x:<\/p>\n<\/td>\n

\n

1129<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Mean, x\u0304:<\/p>\n<\/td>\n

\n

15.465753424658<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Variance, s2:<\/p>\n<\/td>\n

\n

1.8356164383562<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Standard deviation<\/p>\n<\/td>\n

\n

1.3548492308579<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Standard error of mean (SEM)<\/p>\n<\/td>\n

\n

0.1585731082574<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Confidence of interval<\/p>\n<\/td>\n

\n

95%, 1.960sx\u0304<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Margin of error<\/p>\n<\/td>\n

\n

15.4658 \u00b10.311 (\u00b12.01%)                   <\/p>\n<\/td>\n<\/tr>\n

\n

CONTROL<\/strong><\/p>\n<\/td>\n

\n

Count, N:<\/strong><\/p>\n<\/td>\n

\n

73<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Sum, \u03a3x:<\/p>\n<\/td>\n

\n

950<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Mean, x\u0304:<\/p>\n<\/td>\n

\n

13.013698630137<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Variance, s2:<\/p>\n<\/td>\n

\n

0.33453196347032<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Standard deviation<\/p>\n<\/td>\n

\n

0.57838738183878<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Standard error of mean (SEM)<\/p>\n<\/td>\n

\n

0.067695122694177<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Confidence of interval<\/p>\n<\/td>\n

\n

95%, 1.960sx\u0304<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Margin of error<\/p>\n<\/td>\n

\n

13.0137 \u00b10.133 (\u00b11.02%)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

MTHFR C677T Genotypes<\/strong><\/p>\n\n\n\n

Regarding MTHFR C677T genotype distribution among cases of EOAD, we\nfound that CC, CT, and TT frequencies were 24.7%, 27.4% and 47.9% respectively.\nGenotype frequencies among control subjects were 78%, 12.3%, and 9.6%,\nrespectively (table 4). The difference in genotype frequencies between cases\nand control subjects was statistically significant (p<\/em>=0.0001). Table 5\nshows the mean age among different MTHFR C677T genotypes of EOAD patients. Statistical\nanalysis was significant (p<\/em>= 0.000).  The gender distribution of MTHFR C677T\ngenotypes among cases of EOAD was shown in table 6. The difference among MTHFR\nC677T genotypes was significant (p<\/em>= 0.000). Moreover, we compared the\nplasma homocysteine levels among MTHFR C677T genotypes (table 7). Statistical\nanalysis found the difference between genotypes to be significant (p<\/em>=\n0.000).<\/p>\n\n\n\n

Table 4: MTHFR C677T genotype distribution among EOAD cases and controls<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

CT<\/strong><\/p>\n<\/td>\n

\n

TT<\/strong><\/p>\n<\/td>\n

\n

CC<\/strong><\/p>\n<\/td>\n

\n

Raw Totals<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Cases<\/p>\n<\/td>\n

\n

Observed<\/p>\n<\/td>\n

\n

20 (27.4%)<\/p>\n<\/td>\n

\n

35 (47.9%)<\/p>\n<\/td>\n

\n

18 (24.7%)<\/p>\n<\/td>\n

\n

73<\/p>\n<\/td>\n

\n

df = 2<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

14.5<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

37.5<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

p-value = 0.0001<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

2.0862      <\/p>\n<\/td>\n

\n

9.3333<\/p>\n<\/td>\n

\n

10.14         <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

Chi Square = 43.1191<\/p>\n<\/td>\n<\/tr>\n

\n

Controls<\/p>\n<\/td>\n

\n

Observed<\/p>\n<\/td>\n

\n

9 (12.3%)<\/p>\n<\/td>\n

\n

7 (9.6%)<\/p>\n<\/td>\n

\n

57 (78%)<\/p>\n<\/td>\n

\n

73<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

14.5<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

37.5<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

2.0862<\/p>\n<\/td>\n

\n

9.3333<\/p>\n<\/td>\n

\n

10.14<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

Column Totals<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

29<\/p>\n<\/td>\n

\n

42<\/p>\n<\/td>\n

\n

75<\/p>\n<\/td>\n

\n

146<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 5: Mean age among MTHFR C677T genotypes of cases<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

G<\/strong>roup<\/strong><\/p>\n<\/td>\n

\n

n<\/strong><\/p>\n<\/td>\n

\n

M<\/strong>ean age<\/strong><\/p>\n<\/td>\n

\n

SD<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

CT<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

57.95<\/p>\n<\/td>\n

\n

0.6863<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

TT<\/p>\n<\/td>\n

\n

35<\/p>\n<\/td>\n

\n

57.5143<\/p>\n<\/td>\n

\n

0.7425<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

CC<\/p>\n<\/td>\n

\n

18<\/p>\n<\/td>\n

\n

59.0556<\/p>\n<\/td>\n

\n

0.8726<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

df<\/p>\n<\/td>\n

\n

SS<\/p>\n<\/td>\n

\n

F-statistic<\/p>\n<\/td>\n

\n

p<\/em>-value<\/p>\n<\/td>\n<\/tr>\n

\n

Variation among samples<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

28<\/p>\n<\/td>\n

\n

24.4160<\/p>\n<\/td>\n

\n

0.000<\/p>\n<\/td>\n<\/tr>\n

\n

Variation within samples<\/p>\n<\/td>\n

\n

70<\/p>\n<\/td>\n

\n

42<\/p>\n<\/td>\n<\/tr>\n

\n

Total<\/p>\n<\/td>\n

\n

72<\/p>\n<\/td>\n

\n

69<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 6: Gender distribution of MTHFR C677T genotypes among cases<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Males<\/strong><\/p>\n<\/td>\n

\n

Observed <\/strong><\/p>\n<\/td>\n

\n

2 (10%)<\/strong><\/p>\n<\/td>\n

\n

5 (25%)<\/strong><\/p>\n<\/td>\n

\n

13 (65%)<\/strong><\/p>\n<\/td>\n

\n

20<\/strong><\/p>\n<\/td>\n

\n

df = 2<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

5.4795<\/p>\n<\/td>\n

\n

9.5890<\/p>\n<\/td>\n

\n

4.9315<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

p-value = 0.000<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

2.2095<\/p>\n<\/td>\n

\n

2.1962<\/p>\n<\/td>\n

\n

13.2010<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

Chi Square = 25.2506<\/p>\n<\/td>\n<\/tr>\n

\n

females<\/p>\n<\/td>\n

\n

Observed<\/p>\n<\/td>\n

\n

18 (33.9%)<\/p>\n<\/td>\n

\n

13 (24.5%)<\/p>\n<\/td>\n

\n

5 (9.4%)<\/p>\n<\/td>\n

\n

53<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

14.5205<\/p>\n<\/td>\n

\n

25.4110<\/p>\n<\/td>\n

\n

13.0685<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

0.8338<\/p>\n<\/td>\n

\n

0.8287<\/p>\n<\/td>\n

\n

4.9815<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Column Totals<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

35<\/p>\n<\/td>\n

\n

18<\/p>\n<\/td>\n

\n

73<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 7: Mean plasma homocysteine levels among MTHFR C677T genotypes<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

CT<\/p>\n<\/td>\n

\n

20<\/strong><\/p>\n<\/td>\n

\n

15.1<\/strong><\/p>\n<\/td>\n

\n

(14.8565, 15.3435)<\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

TT<\/p>\n<\/td>\n

\n

35<\/p>\n<\/td>\n

\n

15.5714<\/p>\n<\/td>\n

\n

(15.3874, 15.7555)<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

CC<\/p>\n<\/td>\n

\n

18<\/p>\n<\/td>\n

\n

13.8333<\/p>\n<\/td>\n

\n

(13.5767, 14.09)<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

df<\/p>\n<\/td>\n

\n

SS<\/p>\n<\/td>\n

\n

F-statistic<\/p>\n<\/td>\n

\n

p-value<\/p>\n<\/td>\n<\/tr>\n

\n

Variation among samples<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

36<\/p>\n<\/td>\n

\n

60.5622<\/p>\n<\/td>\n

\n

0.000<\/p>\n<\/td>\n<\/tr>\n

\n

Variation within samples<\/p>\n<\/td>\n

\n

70<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n<\/tr>\n

\n

Total<\/p>\n<\/td>\n

\n

72<\/p>\n<\/td>\n

\n

57<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

MTHFR A1298C Genotypes<\/strong><\/p>\n\n\n\n

Regarding MTHFR 1298C, we reported frequencies of 38.4%, 27.4%, and\n34.2% for AA, AC, and CC genotypes, respectively. Genotype frequencies among\ncontrol subjects were 35.6%, 32.9%, and 31.5%, respectively (table 8). There\nwas no statistical difference between cases and control subjects (p<\/em>= 0.6228).\n<\/p>\n\n\n\n

Table 8: MTHFR A1298C genotype distribution among EOAD cases and controls<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Cases <\/strong><\/p>\n<\/td>\n

\n

Observed <\/strong><\/p>\n<\/td>\n

\n

20 (27.4%)<\/strong><\/p>\n<\/td>\n

\n

25 (34.2%)<\/strong><\/p>\n<\/td>\n

\n

28 (38.4%)<\/strong><\/p>\n<\/td>\n

\n

73<\/strong><\/p>\n<\/td>\n

\n

df = 3<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n

\n

24<\/p>\n<\/td>\n

\n

27<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

p-value = 0.6228<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

0.1818<\/p>\n<\/td>\n

\n

0.0417<\/p>\n<\/td>\n

\n

0.037<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

Chi Square = 1.764<\/p>\n<\/td>\n<\/tr>\n

\n

Controls<\/p>\n<\/td>\n

\n

Observed<\/p>\n<\/td>\n

\n

24 (32.9%)<\/p>\n<\/td>\n

\n

23 (31.5%)<\/p>\n<\/td>\n

\n

26 (35.6%)<\/p>\n<\/td>\n

\n

73<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Expected<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n

\n

24<\/p>\n<\/td>\n

\n

27<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Chi Square contribution<\/p>\n<\/td>\n

\n

0.1818<\/p>\n<\/td>\n

\n

0.0417<\/p>\n<\/td>\n

\n

0.037<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

Column totals<\/p>\n<\/td>\n

\n

44<\/p>\n<\/td>\n

\n

48<\/p>\n<\/td>\n

\n

54<\/p>\n<\/td>\n

\n

146<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

In the current study, we explored the relationship between MTHFR C677T and A1298C polymorphisms and early (young)-onset Alzheimer’s disease among Sudanese population. We recruited seventy-three subjects clinically diagnosed with EOAD who were attending or admitted to different neuropsychiatric healthcare facilities in Khartoum, Sudan. EOAD is defined as dementia that occurs before the age 65 years6<\/sup>. This entity of AD is of particular clinical and socioeconomic importance owing to its poor prognosis, early presentation, and tremendous consequences on life quality14<\/sup>. Clinically, EOAD differs from LOAD in some respects. Firstly, EOAD tends to preserve the episodic memory function but not the cognitive impairment14<\/sup>. Secondly, EOAD tends to have more aggressive course and relatively shorter life span than LOAD14<\/sup>. Thirdly, the disease is often underestimated and misdiagnosed54<\/sup>, which was reflected as relatively higher mortality rates6<\/sup>. The global frequency of EOAD was 24.2 in 100000 (0.2%)6<\/sup>. Despite the low frequency, EOAD is considered was considered as the most common dementia before the age 65 years6<\/sup>. Because the study belonged to the case-control entity, we were not able to compute the prevalence of EOAD in Sudanese population.<\/p>\n\n\n\n

The mean age of EOAD patients in our study was 57.9 \u00b13 years, which\nwas within the range given in the definition of EOAD6<\/sup>. The literature\nreview in this regard suggested a variation in the mean age of EOAD even within\nthe same population. For an instance, the mean age in one Dutch study was 60 \u00b1\n4 years10<\/sup>. In another Dutch study, the mean age was 56 \u00b1 5 years11<\/sup>.\nThis variation in our opinion, depended on the period after which an\nestablished diagnosis of EOAD was given. We usually expect a relatively later\nonset for the disease in developing countries due to the lack of modern\ndiagnostic tools. However, the mean age for EOAD in our study did not vary much\nwith literature in this context. <\/p>\n\n\n\n

Our findings regarding gender suggested that females were more\naffected than males (72.4% vs. 27.6%). Our findings agreed with the general\nassumption that Alzheimer\u2019s disease occurs primarily in women as suggested by Mary12<\/sup>\n, despite the fact that some studies did not discriminate between males and\nfemales12<\/sup>. In her study, Mary has attributed this variation in\ngender to hormonal and psychological factors and pregnancy-related morbidities12<\/sup>.\nIt was established that the female hormones oestrogen and progesterone increased\nthe risk for dementia12<\/sup>. Mental disorders related to pregnancy such\nas psychosis and post-partum depression were also known to aggravate the\ncognitive impairment in AD12<\/sup>. <\/p>\n\n\n\n

When we looked into the mean age and gender together, we could see\nthat our patients were mainly post-menopausal women. Therefore, our results supported\nthe study by Geeske13<\/sup> who suggested that oestrogen may protect\nagainst neuronal degeneration, and hence postmenopausal women have a greater\nrisk of AD than perimenopausal women or women taking hormone replacement\ntherapy. From our point of view, the lack of oestrogen in our patients might\nhave played a role in the acceleration of the neuronal degeneration seen in AD\npatients. Our findings confirmed that age and gender have altered the\ncorrelation between MTHFR C677T polymorphism and EOAD to some degree.<\/p>\n\n\n\n

The frequency of MTHFR C677T in the current study was 39.7% for the\nheterozygous allele and 57.5% for the homozygous allele. Both frequencies were\nwithin the proposed range for the t<\/em> alle prevalence of 12-57%28<\/sup>.\nThe current study concluded that both the homozygous (TT) and the heterozygous\nalleles (CT) of MTHFR C677T polymorphism were associated with EOAD in Sudanese\npopulation. Because the mutation was associated with the early onset disease, our\nfindings supported the conclusion drawn by Yaling28<\/sup> that MTHFR C677T\npolymorphism might advance the onset of the disease. The current study,\nhowever, did not find a significant association between the disease and A1298C polymorphism,\nsupporting the fact that MTHFR C677T is the most clinically significant polymorphism\nassociated with AD among the three polymorphisms28<\/sup>. Our study,\nhowever, could not finalize whether this polymorphism might be considered as a\nrisk factor for AD or not29,30<\/sup>. Our results also confirmed the\nfindings of the study by Ye Hu31<\/sup> who concluded that both the\nheterozygous and homozygous alleles of MTHFR C677T were associated with\nAD.  <\/p>\n\n\n\n

Considering A1298C polymorphisms, we could not totally agree with\nthe case study by Leila32<\/sup> who detected both C677T and A1298C\npolymorphisms in a patient with EOAD32<\/sup>. Furthermore, we could not support\nor deny whether A1298C polymorphism has a causative33<\/sup>, or a\nprotective28<\/sup> role in the pathogenesis of AD28<\/sup>. Because\nEOAD is basically multifactorial, we could not conclude that C677T polymorphism\nwas the sole etiology behind AD4<\/sup>.<\/p>\n\n\n\n

The current study has confirmed a significant elevation in plasma\nhomocysteine levels among cases, suggesting a link between MTHFR 677T mutation\nand AD. It was already proven that MTHFR C677T polymorphism causes hyperhomocysteinemia41<\/sup>,\nwhich is associated with AD53<\/sup>. Furthermore, it was already established\nthat elevated homocysteine levels might induce white matter signal\nabnormalities34<\/sup> that can be responsible for the impaired cognitive\nfunction in patients with AD34<\/sup>. This assumption, however, was not\nconclusive because of the multifactorial nature of AD and the fact that both\ngenetic and environmental factors together decide who will get the disease4<\/sup>.\nThe low folate probably due to MTHFR C677T polymorphism was thought to ameliorate\nthe cognitive impairment seen in patients with AD36<\/sup>. It was suggested\nthat hyperhomocysteinemia might be caused by factors other than MTHFR C677T\npolymorphism. These factors included low folate and vitamin B12<\/sub>\nlevels, aging, smoking and renal impairment34<\/sup>. Because we did not\nmeasure folate and B12<\/sub> levels in our participants, we could not confirm\nwhether low folate level has contributed to the cognitive impairment or not.\nMoreover, we could not exclude low folate as a cause of hyperhomocysteinemia. Interestingly, our findings clashed\nwith one study by Luchsinger40<\/sup> who found no significant association\nbetween high plasma homocysteine levels and AD.  We concluded that individuals with elevated plasma\nhomocysteine levels were more likely to develop EOAD than individuals with\nnormal homocysteine levels.<\/p>\n\n\n\n

We have also looked into the means of plasma homocysteine levels\nwithin patients having CC, CT, and TT genotypes. Our results suggested that\nhomocysteine levels were significantly elevated in the group with the\nhomozygous allele, followed by the heterozygous allele, whereas levels were\nwithin the normal range in the group having the wildtype allele. This finding supported\nthe conclusion drawn by Munshi38<\/sup> who reported higher plasma homocysteine\nlevels in the TT genotype, followed by the CT then the CC genotype. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In conclusion, the frequency of EOAD among Sudanese population was\nas low as 0.0012%. The mean age of EOAD patients was 57.9 \u00b13 years. Females were\nmore commonly affected than males due to hormonal and psychological variation. The\nfrequency of MTHFR C677T in the current study was 39.7% for the heterozygous\nallele and 57.5% for the homozygous allele. Both the homozygous and\nheterozygous alleles were associated with EOAD. It appears that the mutation might\nadvance the onset of the disease and the elevated plasma homocysteine levels might\nbe partially responsible for the neurodegenerative abnormalities seen in those patients.\nOur results suggested that homocysteine levels were significantly increased in\nthe group with the homozygous allele, followed by the heterozygous allele,\nwhereas levels were within the normal range in the group having the wildtype\nallele. MTHFR A1298C polymorphism was not associated with EOAD in the study. Individuals\nwith family history of Alzheimer\u2019s disease may need to be screened for this\nmutation as early as possible, so that proper preventive and therapeutic measures\ncould be implemented once the polymorphism is detected. MTHFR C677T-positive\nindividuals should be considered at-risk for progression to Alzheimer’s\ndisease. <\/p>\n\n\n\n

The study was without limitations. We could not exclude all factors\nthat might elevate plasma homocysteine levels like diet, low folate and B12<\/sub>\nlevels. Additionally, we were unable to establish a direct effect or causation as\na case-control study. Moreover, the study might be less useful for examining\nthe risk factors for such a rare disease as in cohort studies. <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors would like to thank the staff at Khartoum Teaching Hospital, Al-Shaab Teaching Hospital, Al-Tijani Al-Mahi Neuropsychiatric Hospital and the Research Centre at the Faculty of Medicine, El-Neelain University for their kind help in recruiting study subjects and collecting samples.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article <\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors do not have any conflict of\ninterest <\/p>\n\n\n\n

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