Methylene Tetrahydrofolate Reductase Gene

Single Nucleotide Polymorphisms and Association

with Methotrexate Toxicity Among Cancer Patients
at Uganda Cancer Institute
K .Yarsin LWASA
2018/HD13/1772U
(BSc. Biomedical Sciences)
(Makerere University)
 SUPERVISORS

1. DR. Dan ISABIRYE, PhD

(Makerere University, CoNAS, Department of Biochemistry)

2. DR. Nixon NIYONZIMA, PhD

(Uganda Cancer Institute- Mulago National Referral Hospital)
 ABBREVIATIONS & ACRONYMS
• A: Adenine Reductase
• ALL: Acute Lymphoblastic • Hcy: Homocysteine
Leukemia • IRB: Institutional Review
• BLAST: Basic Local Board
Alignment Tool
• C: Cytosine
• Ca Breast: Breast cancer
• DHF: Dihydrofolate
• DHFR: Dihydrofolate
 Cont’d
• MTHFR: Methylene
Tetrahydrofolate Reductase
• MXT: Methotrexate
• MXT-PGs: Methotrexate
polyglutamates
• NCBI: National Centre for
Biotechnology Information
• OR: Odds Ratio
• PCR: Polymerase Chain Reaction
• PPAT: Phosphoribosyl Phosphate
Amidotransferase
• SCLC: Small Cell Lung Cancer
• SNP: Single Nucleotide
Polymorphism
• T: Thymidine
• THF: Tetrahydrofolate
• TS: Thymidylate Synthase
• UCI: Uganda Cancer Institute
• UCIREC: Uganda Cancer Institute
Research and Ethics Committee
 Introduction & Background
• MXT has a multitude of therapeutic uses
• MXT competitively inhibits DHFR & DNA synthesis leading to
cytotoxicity (Friedman and Cronstein, 2019)
• DHFR-DHF-THF linked to MTHF and hence, MTHFR
• MTHFR enzyme is vital in cellular methyl donation reactions e.g
conversion of Hcy to methionine (Yuan et al., 2020)
• Sub-optimal MTHFR enzymatic activity leads to Hcy-driven toxicity
(Hernández-Preciado et al., 2019 & Araoz et al., 2015)
• C677T & A1298C are commonest SNPs (Ramirez-Pacheco et al.,
2016)
 Statement of the Problem

• MXT is essential in cancer and non-cancer therapeutics (Wang

et al., 2019 & Ulrich et al., 2001)
• MXT toxicity negatively impacts its use (Fisher and Cronstein,
2009)
• Newer (tolerable) anti-cancers are expensive (Chen et al.,
2017)
• Evidence for genetically-driven variabilities in responses
(Suthandiram et al., 2014)
 Objectives of The Study
• General Objective: To determine the association between MTHFR
gene SNPs and MXT toxicity among Cancer patients at UCI

• Specific objectives:
1). To determine the genotypes of MTHFR gene at positions 677 and
1298 of research participants
2). To determine the concentration of Hcy among research participants
 Literature Review
Burden of disease due to Cancer:
• Cancer causes significant mortality and morbidity Globally
(Hu et al., 2019, Pilleron et al., 2019)
• By 2018, 18 million new cases were expected
• Africa with the largest contribution (Bray et al., 2018)
• Prostate cancer (M), Cervical & Ca breast (F) and Colorectal (M&F)
(Jedy-Agba et al., 2012, Arnold et al., 2017)
• Irrespective of the level of development, cancer is a socio-
economically consumptive disease (Kim et al., 2015)
 Literature Review

MXT Metabolism & Toxicity

• Various SNPs affect MXT uptake toxicity and metabolism (Dervieux et al.,
2004, Li et al., 2016; Aslibekyan et al., 2014, Hernández-Preciado et al., 2019)

• SNPs on MTHFR gene show variable responses in MXT toxicity in GVHD

(Ulrich et al., 2001)

• There is scarcity of data on MTHFR SNPs in the Ugandan population

 Methodology
• Study was approved by UCIREC ( No. UCIREC-08-2020)
• A Systematic study design at UCI –targeting male in-patients
• DNA extraction, PCR & Sequencing revealed the genotypes of
study participants (MakBRC & ACTG Inc. in Wheeling, Illinois,
USA)
• MTHFR Sequence data was analyzed for SNPs using BioEdit
software
• [Hcy] was determined using chemiluminescence assay (CMIA) on
the ABBOTT applied Biosystems automated platform (Lancet Lab.
Kampala)
 Study Results
Characteristics of study participants
Characteristics of N=29
participants
Males 5
(17.3%)
Females 24 (82.7%)

SCLC 6
(20.7%)
Ca Breast 16 (55.1%)

ALL 7
(24.1%)
Participants’ age range 25-65 years

Average age 45.2 years

Results- MTHFR SNPs Vs [Hcy] in study
participants
Sample ID Age (years) C677T A1298C [Hcy] µmol/L
BB001 34 None None 7.8
BB002 30 None None 9
BB003 52 None None 15.6
BB004 46 None None 16.3
BB005 44 None None 14.9
BB006 45 None None 12
BB007 45 None None 16
BB008 28 None None 9.2
BB009 39 None None 10
BB010 51 None None 14.8
BB011 48 None NA 13.8
BB012 42 None None 12
BB013 36 None None 9.4
BB014 40 None None 10.8
BB015 29 None None 13
BB016 39 None None 11.7
BB017 62 None NA 15.3
BB018 57 None None 14.2
BB019 65 None None 14.8
BB020 62 None None 15.1
Results- Contingency Table (SNPs Vs
[Hcy])
MTHFR SNP(s) No MTHFR SNP(s) Total

[Hcy] > 15µmol/l 0 05 5

[Hcy] ≤ 15µmol/l 0 23 23

TOTAL 0 28 28
 Analysis of MTHFR Sequences

• Achieved with BioEdit software

• Poorly resolved bases at the 3’ & 5’ ends were trimmed
• All sequences underwent a BLAST search against reference
MTHFR gene from the data base
• C667T & A1298C were absent in all sequences
• Several artifacts were observed in the sequences
 Sequence Artifacts
1. Low signal intensity
Sequence Artifacts Cont’d-Secondary amplicons

S
 Discussion of Results

• This was a first of a kind study in Uganda

• Used [Hcy] as a decoy for MXT toxicity given MTHFR SNPs
• Association was not computable given nil counts for SNPs
• Subsequent normocysteinemia?
• Conformity with earlier similar findings- Blacks vs white Hispanics
(Esfahani et al., 2003)
• Low frequency of c677t & a1298c in Africans could be due to genetic
diversity & protective
(Graydon et al.,2019) & (Pereira et al., 2021)
 Conclusion & Recommendations

• No adducible association between MTHFR SNPs and MXT Toxicity

• This may explain the observed normocysteinemia
• Need for multicentric studies
• Inclusion of non-cancer patients
 Acknowledgement

• Study Supervisors (Dr. ISABIRYE & DR. NIXON)

• All faculty at CoNAS (Department of Biochemistry)
• UCI-AfDB & All Research Participants
• HEPI-MASSEU project
• Classmates
• Family & Friends
 END

THANK YOU