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ORIGINAL ARTICLE
Year : 2022 | Volume
: 21
| Issue : 3 | Page : 231-236
Haptoglobin genotypes and malaria comorbidity in breast cancer and healthy Nigerian women
Titilope M Dokunmu1, Patience O Obi1, Omolara A Fatiregun2, Oluwakemi A Rotimi1, Sulaiman O Agodirin3, Solomon O Rotimi1
1 Department of Biochemistry, Covenant University, Ota, Nigeria
2 Oncology Unit, Department of Radiology, Lagos State University Teaching Hospital, Lagos, Nigeria
3 Department of Surgery, University of Ilorin, Ilorin, Nigeria
Correspondence Address:
Titilope M Dokunmu
Department of Biochemistry, KM 10 Idiroko Road, Canaanland, Ota
Nigeria
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/1596-3519.356811
Abstract
Background: Breast cancer is the leading cause of mortality among women, with over a million cases recorded globally. Haptoglobin (Hp) protein and genotypes play important roles in cancer predisposition and progression, but studies have reported varying outcomes in populations. Aim: The association of Hp genotypes in breast cancer patients with malaria has not been investigated in Nigerians, which is the aim of our study. In healthy women (control; n = 279) and clinically diagnosed breast cancer patients (breast cancer; n = 70). Methods: Haptoglobin genotypes and Plasmodium falciparum cyclooxygenase III genes were detected by polymerase chain reaction (PCR). Proportions were compared, and the test of association was carried out with a significance level set at P < 0.05. Results: Overall, 311 of 349 (89%) individuals had malaria infection with similar proportions in breast cancer (63 of 70) and healthy control group (248 of 279); malaria incidence was, however, lower in Hp 2-2 breast cancer patients (P = 0.04). The prevalence of Hp genotypes was Hp 1-1 (78.2%), Hp 2-1 (7.2%), and 2-2 (14.6%). In breast cancer groups, Hp 2-2 genotype was significantly lower with 3 (4.2%) of 70 vs. 48 (17.2%) of 279 in control group (P = 0.006). Conclusions: The results of the study show low Hp 2-2 genotype relative to other genotypes in breast cancer patients; we conclude that low Hp 2-2 genotype is associated with lower malaria risk in breast cancer Nigerian women. It is important to further understand the roles malaria, Hp, and other genotypes play in the pathogenesis of aggressive breast cancer commonly seen in Nigerian women.
Abstract in French Résumé
Contexte: Le cancer du sein est la principale cause de mortalité chez les femmes, avec plus d'un million de cas enregistrés dans le monde. La protéine et les génotypes de l'haptoglobine (Hp) jouent un rôle important dans la prédisposition et la progression du cancer, mais des études ont rapporté des résultats variables dans les populations. Objectif: L'association des génotypes d'haptoglobine chez les patientes atteintes d'un cancer du sein et atteintes de paludisme n'a pas été étudiée chez les Nigérians, ce qui est l'objectif de notre étude. Chez les femmes en bonne santé (témoin ; nombre = 279) et les patientes atteintes d'un cancer du sein diagnostiqué cliniquement (cancer du sein ; nombre = 70). Méthodologie: Les génotypes de l'haptoglobine et les gènes de la cyclooxygénase-III de Plasmodium falciparum ont été détectés par PCR. Les proportions ont été comparées et le test d'association a été réalisé avec un seuil de signification fixé à P < 0,05. Résultats: Dans l'ensemble, 311 personnes sur 349 (89 %) avaient une infection palustre avec des proportions similaires dans le groupe du cancer du sein (63 sur 70) et dans le groupe témoin sain (248 sur 279); l'incidence du paludisme était cependant plus faible chez les patientes atteintes d'un cancer du sein Hp 2-2 (p = 0,04). La prévalence des génotypes Hp était : Hp 1-1 (78,2 %), Hp 2-1 (7,2 %) et 2-2 (14,6 %). Dans les groupes de cancer du sein, le génotype Hp 2-2 était significativement plus faible avec 3 (4,2 %) sur 70 contre 48 (17,2 %) sur 279 dans le groupe témoin (p = 0,006). Conclusions: Les résultats de l'étude montrent un faible génotype Hp 2-2 par rapport aux autres génotypes chez les patientes atteintes d'un cancer du sein; nous concluons qu'un faible génotype Hp 2-2 est associé à un risque de paludisme plus faible chez les femmes nigérianes atteintes d'un cancer du sein. Il est important de mieux comprendre les rôles que jouent le paludisme, l'haptoglobine et d'autres génotypes dans la pathogenèse du cancer du sein agressif couramment observé chez les femmes nigérianes.
Mots-clés: Cancer du sein, génotypes, haptoglobine, paludisme, Nigeria
Keywords: Breast cancer, genotypes, haptoglobin, malaria, Nigeria
Introduction 
Globally, breast cancer is the most commonly diagnosed cancer in women and accounts for 24.2% of all cancers. [1,2] Although, there is a declining rate of breast cancer mortality globally, the highest age-standardized breast cancer mortality rate continues to be recorded among African women; which is due in part to several underlying genetic factors.[3] In Nigeria, breast cancer is the leading cancer type with an increasing prevalence of 22.7% and mortality of 16.4% in 2018.[3] Higher rates of aggressive molecular subtypes like triple-negative breast cancer (TNBC) and loss of function mutations in tumor suppressor genes including BRCA1, BRCA2, TP53, PALB2, and BRIP1 are underlying causes of higher breast cancer disparity in Nigerian women.[4],[5]
In addition, additional genetic factors such as typical chemokine receptor 1 (DARC/ACKR1) negativity that are associated with adaptation to malaria in tropical West Africa have been linked to the aggressiveness of TNBC in black women.[6] Despite the continuous effort to eradicate malaria, asymptomatic malaria is common in Africa, and it contributes to the high endemicity of malaria in the region with the highest prevalence reported from Nigeria.[7],[8] Chronic inflammation and infections predispose to the development and progression of different cancers, by activation of inflammatory cytokines that are implicated in cancers.[9],[10] Large-scale studies investigating the molecular epidemiology of malaria comorbidity in the breast cancer population have not been evaluated. The understanding of the role of malaria-associated adaptive molecular factors in breast cancer risk is essential to devising precision strategies to further reduce the high breast cancer burden and improve clinical outcomes within the African race. One of such malaria-associated adaptive molecular factors is haptoglobin (Hp),[11],[12],[13] which is an acute-phase glycoprotein in the serum,[14] produced primarily in the liver as well as other tissues.[15] The physiological function of Hp includes serving as an antioxidant that inhibits oxidative damage induced by hemoglobin breakdown from hemolysis of red cells, which is a hallmark of malaria pathogenesis.[16]
A major role of Hp is eliminating free hemoglobin from circulation by binding to them, and it does so with the aid of hepatocytes or by attaching to the monocyte CD163. It is also involved in various processes such as angiogenesis, endothelial function, and innate immune response.[16] Two distinct forms of Hp alleles can be found in humans: Hp 1 found in exon 5 and Hp 2 which is believed to have developed from exons 3 and 4 of Hp 1 allele over 100,000 years ago in Southeast Asia.[17] The Hp 1 allele is more prevalent in South America, East Africa, and West Africa, while the Hp 2 allele is more prevalent in Asia, Europe, Australia, and North America.[17] A selective advantage is believed to be associated with Hp 2 allele having resulted from Hp 1 allele.[17] These alleles are further divided into three genotypes: Hp 1-1, Hp 2-1, and Hp 2-2.[17],[18],[19] The prevalence of Hp genotypes varies between populations from different countries and ethnic groups.[17],[20] Hp levels and genotypes have been associated with increased risk of certain types of cancer, such as breast cancer,[21],[22] ovarian cancer,[23],[24] pancreatic cancer,[25] colorectal cancer,[26] and lung cancer.[27] Although previous studies have reported the association of different Hp genotypes with breast cancer, aggressiveness, and early death,[21],[28],[29] there is still a dearth of empirical information on the role of Hp in breast cancer in malaria-endemic regions like Nigeria.
Several studies have proposed that Hp 2-2 genotype, in particular, affords a protective role against Plasmodium falciparum infection.[12],[13] Both Hp 1 and Hp 2 alleles have been linked to increased susceptibility to some diseases, based on different distributions of the genotypes in a population. Based on this, we aim to answer two research questions: (1) what is the distribution of Hp genotypes in Nigerian women with breast cancer and (2) does breast cancer women with Hp 2-2 have protection against malaria? So far, the role of malaria in cancer susceptibility in Africa has been limited to Burkitt lymphoma, for which a recent study from Kenya, Tanzania, and Uganda showed that subtle malaria infection increases the risk of Burkitt lymphoma in children.[30] It is therefore the aim of this study to investigate the association of Hp genotypes with asymptomatic malaria comorbidity in Nigerian breast cancer patients.
Materials and Methods The study was a molecular epidemiological survey of Hp genotype and malaria comorbidity in breast cancer patients >30 years and age-matched healthy women. The study was carried out as part of a larger community health awareness outreach at two field sites located in southwest states in Nigeria for screening for breast cancer and infection with malaria parasites. A population of clinically diagnosed breast cancer patients undergoing oncology care at Lagos State Teaching Hospital and University of Ilorin, Nigeria, were also recruited. Blood samples of a total of 70 breast cancer and 279 healthy women consented participants were included for analysis. The study procedures are in accordance with the ethical standards of conducting experiments in humans. Ethical approval was sought under the Covenant University Health Research Ethics Committee (approval number CHREC/028/2019). Women of reproductive age who volunteered to participate in epidemiological screening for breast cancer and malaria or have already been diagnosed with breast cancer were included in the study. Women aged <20 years and men were excluded from the study.
Sample processing and analysis
Genomic deoxyribonucleic acid (DNA) was extracted from blood using Qiagen® kit. Hp genotyping was done using oligonucleotide primers set: 5'-GAGGGGAGCTTGCCTTTCCATTG-3' and 5'-GAGATTTTTGAGCCCTGGCTGGT-3', which were used to amplify a 1757-base pair (bp) Hp 1 allele-specific sequence and a 3481-bp Hp 2 allele-specific sequence.[30] Additional primers 5'-CCTGCCTCGTATTAACTGCACCAT-3' and 5'-CCGAGTGCTCCACATAGCCATGT-3' were used for the amplification of a 349-bp Hp 2 allele-specific sequence.[31] Primers were synthesized by Integrated DNA Technologies (Iowa, USA). PCR reaction was carried out in a final volume of 25 μL containing 5–10 ng of genomic DNA following the established method.[30],[31]
The presence of P. falciparum (malaria) was determined by amplification of cyclooxygenase III (COX III) gene using specific primers as described by Echeverry et al.[32] Oligonucleotide primers set 5'-AGCGGTTAACCTTTCTTTTTCCTTACG-3' and 5'-AGTGCATCATGTATGACAGCATGTTTACA were used to amplify a 504-bp COX III-specific gene sequence. PCR reaction was carried out in a final volume of 25 μL containing 5–10 ng of genomic DNA, PCR products were resolved by electrophoresis in a 1% agarose gel stained with ethidium bromide and visualized under ultraviolet transilluminator.
Statistical analysis
The sample size for the study was based on a ratio of 1:5 for cases and control, this is assumed based on the prevalence of 1 in 4 cases of breast cancer in Nigerian women, and a convenient sample size of 362 individuals was recruited. Data were represented as proportions or frequencies using Microsoft Excel®. Frequencies were reported and test of proportions (Chi-square test) was carried out using SPSS to determine the association between the covariables in cancer patients and healthy control. The significance difference was set at P < 0.05.
Results Study population
The overall results are for 349 participants (70 in breast cancer group and 279 in healthy control group). Women with breast cancer were aged between 37 and 80 years and had been diagnosed with cancer prior to sample collection, while healthy subjects were age-matched women who presented with no symptoms of disease but participated in a community health screening for breast cancer and malaria. In breast cancer patients, tumor was mostly reported in the left (60.9%) than the right (39.1%) breast and very few reported family history of cancer. Most cases were invasive ductal carcinoma (Stage III to advanced stage) and had undergone treatment for cancer (surgery and/or chemotherapy).
Malaria incidence and breast cancer
Overall, 311 of 349 (89.1%) individuals had asymptomatic malaria detected by PCR amplification of COX III-specific primers with the prevalence of 90% (63 of 70) in breast cancer versus 88.9% (248 of 279) in the control group. The proportion of participants with malaria was not significantly different between breast cancer and control, (χ2 = 0.07, P = 0.78). [Figure 1] shows the gel image for COX III gene amplification.
Figure 1: Gel image of PCR of Plasmodium falciparum Cox III gene in healthy control and breast cancer subjectsHaptoglobin alleles
Hp gene was detectable in all 349 participants; Hp 1 and Hp 2 alleles were confirmed by fragment size of 1757-bp and 3481-bp, respectively, this is shown in [Figure 2]. [Table 1] shows the distribution of Hp genotypes in the cohort. Hp 1 was detected in 60 and 213, while Hp 2 was present in 10 and 66 individuals in breast cancer and control groups, respectively, this proportion was similar in breast cancer and control groups (χ2 = 2.88, P = 0.08). Overall in the study cohort, Hp 1 allele frequency was 273 (78.2%) and this was significantly higher compared to 76 (21.8%) Hp 2 allele. The prevalence of Hp 1-1, Hp 2-2, and Hp 2-2 genotypes in the cohort was 273, 25, and 51 respectively, this was significantly different (χ2 = 8.0, P = 0.018). [Figure 2] shows the representative gel image for Hp amplification. The prevalence of Hp 1-1 genotype was similar (χ2 with Yates correction = 2.88, P = 0.08), as well as Hp 2-1 in breast cancer and control groups (χ2 = 1.05, P = 0.30). In breast cancer, there was a significantly lower proportion of Hp 2-2 genotype (3 of 70) compared to 48 of 279 individuals in control group (χ2 with Yates correction = 7.48, P = 0.006).
Figure 2: Haptoglobin genotyping in study cohort. Hp genotypes are characterized by single band size of 1757-bp (Hp 1_1), both 1757 and 3481bp (Hp 2-1) and Hp 2-2 genotype was characterized by single band size of 3481bp
Table 1: Distribution of haptoglobin genotypes in breast cancer and healthy womenHaptoglobin genotypes and malaria incidence
[Table 2] shows the distribution of Hp and malaria prevalence in breast cancer and control groups. Malaria was predominant in Hp 1-1 genotype, and malaria prevalence in Hp 1-1 and Hp 2-1 was similar in breast cancer and control groups (P > 0.05); however, in the control group, the prevalence of malaria in Hp 2-2 genotype was 14.1%, this was significantly higher compared to breast cancer group (4.7%), (χ2 = 4.09, P = 0.04).
Table 2: Distribution of haptoglobin alleles and malaria incidence in control group and breast cancer groupAssociation between malaria, haptoglobin genotypes, and breast cancer
There was no significant association between breast cancer and malaria infection (relative risk [RR] = 1.02, 95% confidence interval [CI]: 0.83–1.24, P = 0.94); however, there was an inverse relationship between Hp 2-2 genotype and malaria infection in breast cancer (RR = 2.72, 95% CI: 0.86–8.57, P = 0.04).
Discussion In our study, we explored Hp genotypes and its association with malaria comorbidity in breast cancer to understand its role in pathogenesis and genetics of breast cancer in patients living where both diseases are endemic. Majority (>65%) of patients presented with Stages III and IV breast cancer and had invasive ductal carcinoma histology subtype. Two patients had familial breast cancer and two patients had TNBC (data not shown). The proportion of Hp 1 and 2 alleles detected in our population was 78.2% and 21.8%, respectively, and this was similar in healthy and breast cancer women. However, in the breast cancer group, a significantly lower proportion of Hp 2-2 relative to the proportion in healthy women was detected. Our findings on Hp 1 allele support a previous study which reported a higher frequency of Hp 1 allele in Nigerians.[33],[34]
Hp plays important prognostic roles and functionality in breast and other cancers; however, there are variations between the association of Hp serum levels, allele distribution, and breast cancer phenotypes in different ethnicities.[21],[28],[29],[33],[34],[35],[36],[37],[38],[39],[40],[41],[42],[43],[44],[45] Elevated levels of Hp are linked with an increased risk of early death and aggressive TNBC in Swedish and Indian patients.[37],[38],[39] Hp 1 allele is predominant in breast cancer patients[28] or in familial breast cancer, while Hp 2 allele is associated with nonfamilial breast cancer.[21] This study results show a high frequency of Hp 1-1 genotype in breast cancer population as seen in Greece, India, Jordan, and Germany;[21],[28],[40] Furthermore, the observation of nonsignificant association with breast cancer is similar to reports on Sudanese population.[41],[42] Hp expression is generally low in breast and several other cancers compared to normal control,[36] and Hp 2-2 genotype is associated with reduced serum Hp, increased angiogenic ability,[43] and may be linked to breast cancer since angiogenesis is known to be involved in proliferation, tumor growth, and metastasis.[29] Our study reported significantly lower Hp 2-2 genotype in breast cancer relative to the control group. There was no apparent association between Hp genotype and survival in our population.
We detected malaria infection in 89.1%, and an inverse association of malaria with Hp 2-2 genotype in the breast cancer group was observed. Chronic inflammation is associated with breast cancer through the generation of reactive oxygen species and pro-inflammatory cytokines that may promote cancer and affect overall survival in breast cancer patients.[44],[45] This implies chronic inflammation and selection pressure of malaria may account for genetic variance in the breast cancer population in this area. Nigeria is highly endemic for malaria and submicroscopic malaria infection is common in endemic countries.[46],[47] The overall high prevalence of malaria is rather alarming in an era of global malaria eradication. Evolution of genetic variants that protect against malaria and other diseases in endemic countries is well known,[48] as Hp 1 is predominant in malaria-endemic areas, where other hemoglobinopathies also prevail.[17],[20],[49],[50] Cancer and malaria burdens in Nigeria are high; hence, predominant Hp 1-1 genotype can serve protective roles in these diseases. The prevalence of Hp 2-2 was consistently lower in the breast cancer population than in the control group, in the presence or absence of malaria infection. This finding in breast cancer patients is similar to another report of Hp 2-2 protection against malaria in another endemic country.[13] Since Hp 2-2 is less efficient in mopping off free hemoglobin to protect from oxidative effects,[37] protecting against cancer progression and other sequelae in the presence of malaria needs to be evaluated in breast cancer.
There are limitations of this study: the proportion of cases versus control in our study was relatively lower and may influence some outcomes, and history of previous malaria treatment or impact of malaria on breast cancer disease was not evaluated. It is recommended that associations of other genotypes and common diseases in this area be evaluated in the cohort to fully understand their contributions to the pathogenesis of aggressive breast cancer in Nigerian women. We investigated for the first time in Nigeria the association between Hp genotypes in breast cancer patients and malaria comorbidity and we conclude that a lower risk of malaria is associated with low hp 2-2 genotype found in breast cancer participants.
Financial support and sponsorship
We acknowledge the funding support of Covenant University Centre for Research, Innovation, and Discovery for the publication of this article.
Conflicts of interest
There are no conflicts of interest.
References 
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