This study analyzed the four main polymorphisms of the genes in homocysteine metabolism in malaria patients. Forty-two randomly selected subjects, diagnosed positive for
Homocysteine is an intermediate amino acid in the metabolism of methionine. The plasma level in homocysteine depends on many factors, including the folate, B12, and B6 vitamins status. In addition, the genetic polymorphisms of Methylene Tetrahydrofolate Reductase (MTHFR) play a crucial role. Hyperhomocysteinaemia is a risk factor for several pathologies such as cardiovascular disease [
Depending on the polymorphisms of the genes responsible for the synthesis of these enzymes, the route of the remethylation can be promoted over that of the transsulfuration and vice versa. The development of
Studies on the possible association of the polymorphisms of genes involved in homocysteine metabolism and malaria in Burkina Faso are missing. However, Chillemi et al. (2005) suggested that the determination of plasma homocysteine levels could be used as a measure of the severity of malaria infection [
The present study was a prospective study conducted from September to November 2014 in Koubri. This is a rural municipality located in the southern Ouagadougou at approximately 25 km, with the following geographical coordinates: 12°10′ north and 1°24′ west. Koubri has an average annual temperature of 28.2°C and it falls on average 780 mm of rain per year. However, the locality has numerous reservoirs of water that make the presence of mosquitoes vectors persistent throughout the year, so explaining the endemicity of malaria in the area.
The study initially included 182 patients (1 to 79 years) who underwent malaria rapid diagnosis test, SD BIOLINE Malaria Ag P.f/Pan, which enables the detection of the
From the 42 selected patients, 5 mL of venous blood was collected in two EDTA impregnated tubes. The first tube was used for the hematological analysis; the second was centrifuged to discard plasma and the pellets were stored at −80°C until use for nucleic acid extractions.
Platelet count and hemoglobin were determined with total venous blood using the ABX micro 60 automate. Hemoglobin electrophoresis was performed at alkaline pH (tris-glycine buffer pH 9.5) on cellulose acetate strip (CELLOGEL
The extraction of DNA from the blood pellet was carried out using the salting out method [
The samples genotyping with real time PCR was carried out using “Real Time PCR kit for detection of MTHFR 677C>T, MTHFR 1298A>C, MTR 2756A>G, and MTRR 66A>G” (Sacace Biotechnologies, REF: T01002-96-S). SaCycler-96 (Sacace Biotechnologies®; Como, Italy) was used for amplification. Thermocycling was performed at 80°C for 2 min and 94°C for 3 min and then 40 cycles at 94°C for 15 sec and 64°C (annealing temperature) for 40 sec.
The data was processed using Excel 2010 (Microsoft) and SPSS® software version 20 (SPSS Inc., Chicago, USA). The Pearson Chi test was used for comparisons and any
The study was approved by the Ethics Committee for Health Research in Burkina Faso (Deliberation number 2014-9-128). Adults have given their free and informed consent for their participation in the study while the parents or guardians of the minors have given their approval for the participation of the minor.
The study involved 69.05% (29/42) men and 30.95% (13/42) women, the majority of whom were children with 14.28% (6/42) over 20 years of age (Table
Sociodemographic and biological data.
Characteristics |
|
(%) |
---|---|---|
Treatment | ||
Yes | 22 | 52.38 |
No | 20 | 47.62 |
Parasitaemia (parasite/ |
||
<3,000 | 14 | 33.33 |
[3,000–10,000[ | 5 | 11.90 |
>10,000 | 23 | 54.76 |
Hemoglobin (g⋅L−1) | ||
<10 | 10 | 23.81 |
10–12 | 15 | 35.71 |
>12 | 17 | 40.48 |
Platelets count | ||
<15,000 | 14 | 33.33 |
≥15,000 | 28 | 66.67 |
Hemoglobin genotypes | ||
AA | 24 | 57.14 |
AC | 14 | 33.33 |
AS | 2 | 4.76 |
CC | 1 | 2.38 |
SC | 1 | 2.38 |
Sex | ||
M | 29 | 69.05 |
F | 13 | 30.95 |
Age groups | ||
≤5 | 11 | 26.19 |
5–20 | 25 | 59.52 |
>20 | 6 | 14.28 |
With regard to the anemia, 40.48% patients were not anemic (Hb > 12 g L−1), while 35.72% (15/42) had moderate anemia (10 < Hb < 12 g L−1) and 23.80% (10/42) had anemia (Hb < 10 g L−1). It should also be noted that 33.33% (14/32) of patients had thrombocytopenia compared to 66.67% (28/42) with normal platelet count. The frequency of the HbAA genotype was 57.14% (27/42) with one of the patients (0.02%) presenting major sickle cell syndrome (SC).
Figure
Correlation between genotypes and selected patient characteristics.
Genes | Genotypes | Hemoglobin genotypes |
|
Parasitaemia |
|
Sex |
| |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
AA | AC | AS | CC | SC | <3000 | 3000–10000 | >10000 | M | F | |||||
MTHFR C677T | CC | 20 | 12 | 0 | 2 | 2 |
|
16 | 6 | 14 |
|
22 | 14 |
|
CT | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 4 | 0 | ||||
TT | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||
|
||||||||||||||
MTHFR A1298C | AA | 22 | 8 | 2 | 1 | 0 |
|
11 | 3 | 19 |
|
26 | 7 |
|
AC | 2 | 5 | 0 | 0 | 1 | 3 | 2 | 3 | 2 | 6 | ||||
CC | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | ||||
|
||||||||||||||
MTR A2756G | AA | 17 | 8 | 1 | 0 | 0 |
|
4 | 3 | 19 |
|
19 | 7 |
|
AG | 6 | 5 | 1 | 1 | 1 | 9 | 2 | 3 | 10 | 4 | ||||
GG | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 2 | ||||
|
||||||||||||||
MTRR A66G | AA | 14 | 6 | 0 | 0 | 0 |
|
4 | 2 | 14 |
|
16 | 4 |
|
AG | 8 | 8 | 1 | 1 | 1 | 9 | 2 | 8 | 10 | 9 | ||||
GG | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 |
Frequencies of mutations studied in different populations.
Country (reference) | Burkina Faso | Pakistan [ |
China [ |
South Korea [ |
Sweden [ |
Brazil [ | |
---|---|---|---|---|---|---|---|
Genes | Genotypes | % | % | % | % | % | % |
MTHFR C677T | CC |
|
71.3 | 32.8 | 28.5 | 55.9 | 53.8 |
CT |
|
26.2 | 43.9 | 52.9 | 35.8 | 40.4 | |
TT |
|
2.5 | 23.3 | 18.6 | 8.4 | 5.7 | |
|
|||||||
MTHFR A1298C | AA |
|
20.8 | 66.8 | 67.8 | — | — |
AC |
|
48.7 | 29.3 | 30.3 | — | — | |
CC |
|
30.5 | 3.9 | 1.9 | — | — | |
|
|||||||
MTR A2756G | AA |
|
52.4 | — | — | 64.8 | — |
AG |
|
38.8 | — | — | 30.9 | — | |
GG |
|
8.8 | — | — | 4.3 | — | |
|
|||||||
MTRR A66G | AA |
|
— | 55.2 | — | 16.7 | — |
AG |
|
— | 38.2 | — | 50.8 | — | |
GG |
|
— | 6.6 | — | 32.4 | — |
Polymorphisms distribution among screened people.
This study described the MTHFR C677T, MTHFR A1298C, MTR A2756G, and MTRR A66G mutations. The genotypic frequencies found for the MTHFR C677T mutation are similar to those already reported in Burkina Faso [
Genotypic frequencies for this gene (78.6% AA, 19% AC, and 2.4% CC) in our study are also similar to those found in 20–45 years of age (79.27% AA, 17.07% AC, and 3.66% CC) in Burkina Faso [
Our results, however, differ from the genotypic frequencies (66.8% AA, 29.3% AC, and 3.9% CC) reported in Chinese Han adults [
The results of our study for the mutation MTR A2756G (61.9% AA, 33.3% AG, and 4.8% GG) are also different from the genotypic frequencies (52.4% AA, 38.8% AG, and 8.8% GG) reported in Pakistan [
A correlation between MTRR A66G and hemoglobin genotypes was observed in this study. A2756G MTR genotype and parasitaemia showed a statistically significant association; MTR 2756AA individuals with predominantly high parasitaemia (>10,000 trophozoites/
The small size of our study population because of limited financial resources therefore suggests the need for confirmation of these observations on a sufficiently large study population. The NFS results were comparable to those found in the general population. Only platelet and hemoglobin levels were used in this study to evaluate the correlations with the different mutations.
The effect of the mutations was studied on certain parameters such as variations in the number of white blood cells that may be due to other pathologies outside malaria which were difficult to assess. Despite the small sample size, this is the first time that all four polymorphisms have been studied in the same group of individuals in Ouagadougou, Burkina Faso.
Burkina Faso is a malaria endemic country with a high frequency of host resistance polymorphisms to
The authors declare that they have no conflicts of interest.
The authors express their sincere gratitude to the entire staff of CERBA/LABIOGENE for their assistance to carry out this study. Their thanks also go to the Italian Episcopal Conference (CEI) and West African Economic and Monetary Union (UEMOA) (through the PACER2 program) for their financial support.