Role of Human Twin Studies to Identify Genetic Linkage of Malaria Pathogenesis and Outcomes

ABSTRACT.

Malaria remains a major public health challenge that needs attention, especially when the world is aiming at malaria elimination in the near future. It is crucial to understand the underlying genetic factors and epigenetics involved in malaria susceptibility and the dynamics of host immune responses that affect disease outcomes and relapses in Plasmodium vivax and Plasmodium ovale. Studies in newborn and adult twins can help in understanding the comparative roles of environmental and genetic factors on disease pathogenesis and outcome. These studies can help in providing insights into the factors responsible for malaria susceptibility, clinical presentation, responsiveness toward existing as well as candidate antimalarials, and even identification of novel therapeutic targets. The results and outcomes from twin studies can be further applied to the entire population. In the present manuscript, we analyze the available literature on malaria and human twins and discuss the significance and benefits of twin studies to help in better understanding malaria.

INTRODUCTION

Approximately 247 million malaria cases were reported worldwide in 2021, making it one of the leading tropical diseases and a major public health concern.¹ As per the estimation of the WHO, there was an increase in malaria incidence in 2021, as compared with 245 million cases in 2020 and 230 million cases in 2015.¹ Therefore, there is a need for more intensified efforts against malaria to achieve the intended 2030 malaria elimination target. One of the neglected aspects of malaria is malaria in pregnancy. Babies born to malaria-infected women might suffer from severe health difficulties, and the risks of low birth weight, intrauterine growth restriction, and prematurity are high.² Malaria can also be transmitted from mother to child during pregnancy or at the time of birth, leading to congenital malaria.

Malaria pathogenesis is caused by the interaction of host, parasite, and environmental factors.³ Genetic factors and innate resistance mechanisms are plausibly the key determinants of the infectious disease outcome.³ Plasmodium falciparum and Plasmodium vivax are important Plasmodium species of clinical significance. Plasmodium falciparum causes severe morbidity in humans, resulting in cerebral malaria, hypoxia, ring hemorrhages, severe anemia, and tissue lesions, whereas P. vivax leads to relapsing malaria, increasing morbidity, and disease transmission.⁴ The severity in the case of P. falciparum can be attributed to the adherence of parasitized erythrocytes to the endothelial lining of small blood vessels or cytoadherence, a characteristic not shared by the other three Plasmodium species infecting humans.⁴ Plasmodium vivax does not sequester in deep capillaries of organs during infection, unlike P. falciparum.⁵ One of the unique features of P. vivax is an invasion into reticulocytes via the Duffy antigen,⁶ and this leads to clinical infection with lower parasitemia compared with P. falciparum. The genetic basis for the varied pathogenesis and clinical presentation between different individuals and parasite species is still unclear.

In the present work, we have undertaken a review to discuss how malaria studies in twins might help improve the present understanding of the genetic basis of disease pathogenesis, leading to better disease control and management in the future.

IMPORTANCE OF TWIN STUDIES

Studies in human twins provide a solid foundation for studying genetic variations of a trait or condition and the risk factors associated with them. Studies in twins are useful to study the effect of genetics and the shared/non-shared environmental factors on phenotypic outcome of chronic and infectious diseases⁷ and associating the intra-pair similarity of dizygous (DZ) twins and monozygous (MZ) twins. Twin studies have found an important role in epigenetics, microbiota, stem cell research, and developmental studies on health and disease.⁸ Such studies might provide new insights into the role of host genetic variation on disease susceptibility and severity, disease pathogenesis, response to therapy/drugs,⁹ and even identification of new therapeutic targets.⁹ Studies in twins have led to the unraveling of common genetic and environmental factors for the trait under study. The chances of a genetic study producing significant results are enhanced in twin studies because such studies reduce genetic and/or environmental variability.¹⁰

The concept of studying twins to understand heritability runs back to 1875 in the works of Sir Francis Galton titled The History of Twins, which was further perfected in the 1920s.¹¹ Walter Jablonski, in 1922, studied human optical refraction and the effect heredity has on it.¹¹ Bouchard et al.¹² published that up to 70% of the variation in intelligence quotient described in identical twins was associated with genetic variation. A large-scale follow-up twin study found a high risk of cancer heredity in the case of multiple types of cancers: prostate, breast, ovary, uterus, and melanoma.¹³ Apart from cancer, twin studies and analyses of several twin databases across the world have found the genetic basis for multiple other common chronic diseases, including asthma, osteoarthritis, cataract, eating disorders, and even obesity and back pain.^7,14 A review by Polderman et al.¹⁵ concluded that all human traits are heritable up to variable extents, and the relative combination of genetics and environmental factors shape the final phenotypic trait.

Thus, it would be important to do follow-up clinical sequelae in newborn twins to study the long-term effects of congenital malaria and its genetic linkage. Also, the genetic and immunological studies in adult twins, and correlating the results with malaria susceptibility and severity, might help in the improved understanding of malaria pathogenesis and disease outcome and its association with host genetic constitution.

METHODOLOGY

For the current review, all the original published and registered studies from PubMed, Google Scholar, and Cochrane were searched for the terms “malaria” (as MESH term) and “twins” using filters like “human studies” and “languages English.” After de-duplication, a total of 84 studies were retrieved from the databases. After manually screening the title and abstract of these 84 studies, 28 research studies that had worked on twins and malaria were found to be relevant. The full texts of these 28 studies were then reviewed for the eligibility of their inclusion in the study. After the full-text screening of these 28 twin studies (both epidemiological and case studies), only 19 relevant studies including epidemiological twin studies on malaria (Table 1) and congenital malaria case studies (Table 2) were included in this review. The rest of the nine studies were excluded based on the exclusion criteria, which include text in languages other than English, full text not available, and studies with missing information. The data were extracted from the final dataset based on “author,” “location,” “Plasmodium species,” “sample size,” “age,” “sex,” “treatment provided,” “zygosity, and “findings.” The PRISMA flowchart for the selection of studies is given in Figure 1. The summary and important findings of all these studies are provided in Tables 1 and 2.

Table 1.

Summary and important findings from malaria epidemiological studies in adult twins

Study no.	References	Location/year	Pv/Pf	Sample size/zygosity	Age (years)	Sex of the twins	Findings
1	Troye-Blomberg et al.23	Liberia and Madagascar, 1990	Pf	12 MZ	NA	NA	Antibody response to Pf155/RESA-derived peptides is genetically regulated
2	Jepson et al.16	Gambia, 1991	Pf	34 MZ, 186 DZ	1–10	NA	Not malaria susceptibility but the clinical presentation of malaria including fever is genetically regulated
3	Sjöberg et al.36	Liberia and Madagascar, 1992	Pf	5 MZ, 3 DZ; 18 MZ, 8 DZ	6–25, 2–35	12 M, 4 F	Antibody response to the intact Pf155/RESA antigen was more concordant in MZ twins than in DZ
4	Riley et al.24	Gambia, 1993	Pf	6 MZ, 15 DZ	18	NA	The anti-Pfs 230 response is concordant in both DZ and MZ twin pairs
5	Troye-Blomberg et al.21	Madagascar, 1994	Pf	14 MZ, 6 DZ	2–35	NA	The intensity of the T-cell responses might be genetically regulated
6	Troye-Blomberg et al.26	Gambia, 1997	Pf	10 MZ, 9 DZ	15–50	MZ: 12 M, 8 F; DZ: 7 M, 11 F	Non-HLA class II genetic factors have a profound effect on the shaping of the T-cell repertoire
7	Taylor et al.25	Gambia, 1996	Pf	15 MZ, 21 DZ	NA	NA	The selective recognition of malaria antigens by serum antibodies is not genetically regulated
8	Jepson et al.18	Gambia, 1997	Pf	22 DZ	5.3 (mean)	NA	Genetic factors including MHC genes affect the risk of mild malaria
9	Perlmann et al.29	Gambia, Madagascar, 1999	Pf	15 MZ, 18 DZ	NA	NA	Serum IgE levels vary more in DZ twins as compared with MZ twins
10	Duah et al.30	Gambia, 2009	Pf	Adult: 58 MZ, 155 DZ; children: 32 MZ, 167 DZ	14–92, 1–10	NA	The genetic regulation of all antigen-specific isotype responses is mediated by non-HLA genes
11	Goncalves et al.17	Mali, 2022	Pf	2 MZ, 23 DZ	0–5	25 F, 25 M	Along with genetics, environmental factors also play a crucial role in malaria phenotype variations
12	Starck et al.28	Sub-Saharan Africa, 2022	Pf	4,314 MZ, 2,994 DZ	0.5–5	136,718 M, 134,011 F	Malaria infection induces a reduction in hemoglobin levels and affects population level hemoglobin

DZ = dizygous; HLA = human leukocyte antigen; MHC = major histocompatibility complex; MZ = monozygous; NA = information not available.

Table 2.

Congenital malaria case studies in twins

Study no.	Study	Pv/Pf	Zygosity	Sex (M/F)	Diagnosis	Infected individual	Treatment	Mother on antimalarial	Findings
1.	Bradbury34; England, 1977	Pv	DZ	NA	Detected at birth, positive by microscopy	2nd twin	Chloroquine	No	Early placental separation was probably responsible for the infection of the second twin
2.	Devlin and Bannatyne38; Canada, 1977	Pv	NA	NA	Detected in the mother during the 3rd month of pregnancy and postpartum by microscopy. Detected in twins at 3rd week of birth by microscopy	Both twins, mother	Chloroquine	Chloroquine + primaqine	Developed jaundice on the 4th day, slow weight gain, and fever on days 21–22
3.	Cummins et al.32; England 1990	Pv	MZ	NA	Detected 1 month after birth, positive by microscopy	1st twin	Chloroquine	No	Probable twin-to-twin transfusion. Both twins were treated
4.	Balatbat et al.31; USA, 1995	Pv	DZ	F	Detected 1 month after birth, positive by microscopy	2nd twin	Chloroquine	No	A probable case of perinatal transmission with an uninfected mother and twin sibling
5.	Opare33; Ghana, 2012	Pf	MZ	M	Detected post-delivery, Mother, and twins both positive by microscopy	Both twins, mother	Quinine	SP as prophylaxis	Congenital malaria in both twins. Evaluation of newborns for congenital malaria is recommended
6.	Mudji et al.35; Democratic Republic of the Congo, 2017	Pf	NA	F	Detected on 35 weeks of gestation, mother and child both positive by microscopy	Both twins, mother	Quinine; 1st twin recovered, 2nd died	SP as prophylaxis	Gestational and congenital malaria, premature birth with the possibility of bacterial co-infection
7.	Conroy et al.37; Uganda, 2019	Pf	DZ	NA	Detected on 14 weeks 6 days of gestation, mother negative by microscopy	Mother, 2nd twin	DP	DP	Differences in neurodevelopmental outcomes in placental malaria-discordant dizygotic twins

DP = dihydroartemisinin–piperaquine; DZ = dizygous; MZ = monozygous; NA = information not available; SP = sulphadoxine pyrimethamine.

Figure 1.

Prisma flowchart of review.

HISTORY OF STUDIES ON TWINS IN MALARIA AND THEIR ROLE IN MALARIA SUSCEPTIBILITY, PATHOGENESIS, AND OUTCOME

Clinically, patients present almost identically in all malaria infections (i.e., with fever plus a constellation of other possible symptoms). Reports suggest that the susceptibility and clinical presentation of malaria might be genetically regulated.¹⁶ Although susceptibility to malaria is attributed to the host genetics, only limited gene polymorphisms with causal effects are known. Monozygous twins share the same intrauterine conditions and genetic composition, either partially or entirely. Moreover, most of the time, twins share the same environment throughout childhood, suggesting exposure to the same malaria vectors.¹⁷ These factors make twins the ideal subjects to explore the extent to which host genetics affect malaria susceptibility and clinical response.¹⁶

There has been growing evidence that susceptibility to malaria is determined by the genes eliciting a variety of immune responses. Of these genes, a few may even precisely affect susceptibility to and clinical presentation with specific strains of the malaria parasite. One such important gene complex is the major histocompatibility complex (MHC). Major histocompatibility complexes are one of the major deciding factors for malaria pathogenesis as mild or severe malaria.^18–20 A study by Troye-Blomberg et al.²¹ compared the consonance of immune responses to malaria antigens in human leukocyte antigen (HLA)-typed DZ and MZ twins. The study found that nonproliferative T cells, as well as the antibody responses, are attributed to non-MHC genes.²¹ The blood stage antigen Pf155/RESA of P. falciparum induces genetically regulated T-cell and B-cell responses due to the presence of multiple T-cell– and B-cell–immunodominant epitopes.^19–21 This genetic restriction was found to be independent or weakly associated with the MHC system.²² However, the concordant antibody and T-cell response in MZ twins suggests the role of factors other than MHC or MHC-independent pathways in generating Pf155/RESA-specific immune responses.^21–23 The antibody response against another P. falciparum antigen, Pfs 230, is not induced against all P. falciparum infections. Riley et al.²⁴ found that selective antibody response to the P. falciparum surface antigen Pfs 230 was genetically independent because the response was found to be concordant in both MZ and DZ twins. Similarly, Taylor et al.²⁵ conducted twin studies to explore the role of host genetics, specifically HLA II, in selective response against Pfs 230 along with other two antigens, including P. falciparum merozoite surface protein (MSP)1 and MSP2. The study concluded that antibody responses against these three antigens (Pfs 230, MSP1, and MSP2) were not genetically regulated and that clonal imprinting might be one of the probable reasons for the selective antibody response. Also, a study conducted in West Africa by Troye-Blomberg et al.²⁶ in MZ and DZ twins in the P. falciparum–endemic region showed that the expression of T-cell receptor repertoire is independent of the MHC haplotype. However, similar to the antibody response against other antigens, the expression of the repertoire is more consistent among MZ pairs than among DZ pairs.²⁶

Another study conducted by Goncalves et al.¹⁷ followed 25 pairs of twins. In 16 pairs of twins, both children tested positive for P. falciparum and often were symptomatic. In eight pairs, only one of the twins was infected. An interesting finding in their study came from the fact that they were able to observe siblings with dissimilar hemoglobin types, hence comparing parasitemia during participants’ first infections with hemoglobin S mutation status. Of 50 participants in this study, the AA genotype was shown by 36, the heterozygous hemoglobin C mutation (AC genotype) was seen in nine, and five showed the AS genotype. The study reported low parasitemia associated with hemoglobin genotype AS, whereas the AA genotype was associated with multiple infections with high parasite load, providing further proof that hemoglobin S heterozygosity has a protective effect against malaria.²⁷ The study concluded that, along with genetics, environmental factors also play a significant role in phenotypic variations of malaria infection. Recently an analysis of 7,384 twins from 23 sub-Saharan African countries by Starck et al.²⁸ studied the malaria-induced effect on hemoglobin levels. The study found a decrease in hemoglobin levels of the infected twin as compared with its healthy uninfected counterpart, thus verifying the impact of malaria on anemia at the population level.

The antibody-mediated response also forms an important component of antimalarial response and disease presentation. IgE antibodies are elevated during Plasmodium infections, and their levels vary between mild and severe malaria infections.²⁹ This antimalarial response via IgE is shown to be genetically regulated because the variation in the overall levels of IgE in monozygotic twins with malaria infection is found to be more coherent when compared with dizygotic twins.²⁹ This IgE also induces the release of tumor necrosis factor, which further mediates fever and tissue lesions, thus determining disease outcome. IgG4a has been reported as the most heritable antibody subclass.³⁰ A comparative genome-wide screening approach can be adopted in malaria-exposed twins to identify antibody subclass/subtype activated in response to infection and its effect on the disease phenotype.

There have been reports of congenital malaria in the case of P. vivax in the form of new infection as well as relapse.^31–33 These infections are reported from both identical and non-identical twins, which showed that, apart from host genetics, environmental factors and epigenetics might play role in P. vivax susceptibility and relapse.^32,34 A case study of congenital and gestational malaria with low birth weight, prematurity, and bacterial co-infection was done in the Democratic Republic of the Congo where probable drug resistance to sulfadoxine-pyrimethamine was what caused treatment failure in the mother,³⁵ leading to congenital malaria in both twins. One of the twins died, whereas the other one survived, showing different responses toward infection by each twin. The genetic studies in twins thus might help to understand the underlying reasons for the development of severe infections with fatal outcomes in some and mild clinical symptoms in others.

DISCUSSION

In the present review, we have summarized and reviewed the information available through twin studies on malaria. We also discussed the scope of these studies in exploring the genetic basis of malaria susceptibility, pathogenesis, the outcome as well as long-term clinical effects, as in the case of congenital malaria. Previously, twin epidemiological studies explored the role of underlying genetics and environmental factors in multiple aspects of malaria infection, including host immune responses; disease outcome; and even the effect of malaria on other major public health issues, like malaria-induced anemia. These studies concluded that the antibody responses against Pf 155/RESA and Pfs 230 are genetically regulated,^23,24,36 whereas the selective recognition and response against other antigens, including MSP1, MSP2, and Ps 260/230, are independent of host genetics.^24,25 Although the MHC genes affect the risk of mild and severe malaria,^18–20 variable antibody isotype and subclass response are attributed to non-MHC genes.³⁰ Also, the shaping of the T-cell repertoire and the intensity of the T-cell response against malaria is found to be genetically regulated.^23,26

Similarly, the case studies on twins and malaria highlight important issues worth exploring. One such issue is relapse malaria due to P. vivax.^32–35 Malaria relapse might prove a serious concern in newborns because it often goes unnoticed and might hamper the overall mental and physical growth of the infant. The long-term effect of malaria on infant health and growth and its genetic linkage need due attention. Congenital malaria studies in twins can help elucidate multiple genetic aspects of the disease, including the correlation between malaria and placental health, birthweight, stillbirths, and co-infections in newborns.³⁴ Congenital malaria epidemiological studies need to be conducted, and newborns should be screened for malaria along with other infectious diseases, especially in high malaria-endemic regions. Also, it is important to check the likelihood of other co-infections in newborns, even in the cases of confirmed malaria infections. The follow-up of twins with congenital malaria can help explore the long-term effects, including neurodevelopment and its genetic basis in infants and children.³⁷ One very crucial role these twin studies can play is to explore the role of host genetics if any in emerging artemisinin resistance.

Twin studies are an important tool to study the genetic basis of disease development, progression, and outcome. However, most of the studies on malaria and twins in the past are mere reports of congenital malaria without actually looking at the underlying reasons for the disparity in susceptibility to malaria infection and variable disease progression within twin pairs.^31,32,34,38 A few planned studies in children and adult twins exposed to malaria have addressed the genetic basis of variable immune responses, suggesting that the immune response in malaria might be genetically regulated. Thus, twin studies can help identify and explore the underlying factors for the concordance in immune response in monozygotic twins. Also, these twin studies might help correlate malaria with already known as well as newly discovered genetic factors responsible for host immune responses, drug pharmacokinetics and efficacy, G6PD deficiency spectrum (in the case of female twins), and even antimalarial resistance.