Methylene tetrahydrofolate reductase gene mutation in sickle cell anaemia patients in Lagos, Nigeria

Introduction The significant causes of mortality among individuals with sickle cell anaemia (SCA) such as acute chest syndrome and cerebrovascular disease are related to vascular occlusion. Polymorphisms of the methylene tetrahydrofolate reductase (MTHFR) gene in persons with sickle cell anaemia have been suggested as a potential risk for vaso-occlusive events, with the C677T and A1298C polymorphisms being the commonest. This study therefore aimed to establish the pattern of MTHFR C677T and A1298C gene mutations among adults with HbSS phenotype attending the Haematology Clinic in Lagos State University Teaching Hospital Lagos, Nigeria. Methods A cross-sectional study was done among SCA patients attending the Haematology Clinic of the Lagos State University Teaching Hospital (LASUTH), using age and sex matched HbAA controls. DNA extraction and gene analysis were done. The selective amplification of a particular segment of the DNA by polymerase chain reaction (PCR) was done and subsequent digestion of the amplified MTHFR gene into its various fragments. Results The overall prevalence of the C677T mutation among participants was 19.3% (37 of 192), while the prevalence of A1298C was 15% (29 of 192). Conclusion The prevalence of MTHFR C677T was higher than A1298C mutations among sickle cell anaemia subjects.


Introduction
Sickle cell anaemia has been recognized as a problem of major public health significance by the World Health Organization [1]. The highest frequency of the disease is found in tropical regions particularly sub-Saharan Africa, India and the Middle East. More than 75% of sickle cell anaemia cases occur in sub-Saharan Africa [1]. Nigeria has the highest burden of sickle cell anaemia worldwide, with prevalence values ranging from 2% to 3% of the 140 million population [2][3][4][5].   [37][38][39][40][41]. Furthermore, avascular necrosis of the femoral and humeral head is a common musculoskeletal complication seen in adults with sickle cell anaemia which reduces their quality of life [42]. MTHFR mutations have also been implicated as a probable risk factor for avascular necrosis in sickle cell anaemia [43][44][45]

Study area and population
The study was conducted among adult patients with sickle cell anaemia attending the Haematology Clinic of LASUTH, Lagos, Nigeria, while apparently healthy individuals with no known medical condition served as controls. LASUTH is a major tertiary healthcare facility, located in the south-western part of Nigeria, within the Lagos metropolis.

Study design
This was a cross-sectional study.

Study period
The study was carried out over a period of eight months from May 2016 to December 2016.

Sampling technique
Purposive  [46]; haemoglobin AA persons (confirmed by cellulose acetate electrophoresis at pH 8.6) who served as the control group; those who met the above criteria and agreed to participate in the study by signing the informed consent form. The participants were classified as follows: adults with sickle cell anaemia in steady state; HbAA controls who were age and sex-matched with the adults with sickle cell anaemia.
The exclusion criteria were: confirmed phenotype HbAC, HbAS; history of acute or chronic illness e.g. any febrile illness, hypertension, diabetes, epilepsy, asthma; intravenous drug users; non consenting participants.

Sample size determination
Sample size was determined using the formula for prevalence study by Daniel [47,48].
Where: n = sample size; Z = statistic for a level of confidence of 95%, which is conventional, Z value is 1.96; p = expected prevalence; q = 1-p; d = precision. Therefore, considering Z = 1.96; p = prevalence of MTHFR gene mutation among sub-Saharan Africans is 6% [49,50]; However, in order to accommodate possible attrition or unforeseen errors in completing the study questionnaire or blood sample processing, an additional 10% (9 patients) of the calculated figure was added. Therefore, a total of 192 participants (96 in each group) were recruited into this study.

Ethical consideration and clearance
Ethical approval was obtained from the Health Research and Ethics Committee of LASUTH prior to commencement of the study.

Participants' informed consent
A written informed consent was obtained from all the participants before being recruited into the study. No participant was in any way coerced or cajoled to participate in this study. Study was done at no cost to the participants.

Confidentiality
The names and initials of all participants were not recorded or used to guarantee confidentiality. Participants were instead given code numbers. Paper records were secured in a cabinet inside a secured room. Electronic data was protected using a password known only to the researchers.

Questionnaire administration and history taking
Each participant was interviewed to obtain relevant demographic and clinical data with the use of a questionnaire. The questionnaire was administered to each participant by a member of the research team.
Information on the HbSS subjects and their medical history including disease complications were retrieved from their case notes.

Specimen collection and storage
Seven milliliters of venous blood was drawn from each subject. Five milliliters of this was dispensed into sodium ethylene diamine tetraacetate (EDTA) specimen bottles. This sample was used for a full blood count (FBC). This was analyzed within 2 hours of collection.
Some of the blood collected in the EDTA specimen bottle was used for   Table 1).

MTHFR C677T and A1298C allelic frequency distribution
The relative frequencies of the 677T alleles for the cases and controls were 8.9% and 10.9% respectively while that of the 1298C alleles was 8.3% in both groups (Table 3).

Ages and genders of participants with C677T and A1298C mutations
Both mutations in the two groups were not statistically significant when correlated with the ages and genders of the participants except age vs A1298C in controls which was statistically significant (Table 4). Africans from four tribes in sub-Saharan Africa, the frequency of the homozygous recessive TT genotype was also not reported. The frequency of the TT genotype among Blacks living outside Africa was also noted to be low [51]. This conflicts with a frequency of TT genotype ranging from 8% among Germans, 18% among Italians and 10-14% among whites living outside Europe to about 21% in Hispanics. These estimates suggest that the homozygous recessive TT variant is less common in Africans than in some other populations.

Discussion
Regarding the A1298C mutation, the homozygous recessive (CC) genotype frequency was 1% in cases and 2.1% in the controls which was not found to be statistically significant. This differs from a CC

Conclusion
The prevalence of MTHFR C677T was higher than A1298C mutations among sickle cell anaemia subjects. This research was self-funded, and cost was a major limiting factor. A larger study using more than 96 HbSS and 96 HbAA could be more representative of the aims and objectives of this study.

What is known about this topic
• MTHFR gene mutation reduces the activity of the MTHFR enzyme and thus leads to hyperhomocysteinaemia; • This is particularly heightened in patients with sickle cell anaemia who have an increased demand for folate due to their high red cell turnover or shortened red cell lifespan; • Hyperhomocysteinaemia has been implicated with a number of complications in sickle cell anaemia including avascular necrosis of the femur head, acute chest syndrome etc.

What this study adds
• The prevalence of MTHFR C677T is higher than A1298C mutations among sickle cell anaemia subjects in Lagos, Nigeria.