Facial appearance reveals immunity in African men

Get Complete Project Material File(s) Now! »

Chapter 3 Determining the link between HLA heterozygosity and facial appearance in an African population.

Introduction

Human mate choice has been an important topic for human behavioural ecologists for decades. Evolutionary theory predicts that individuals should choose partners that will provide both direct (ability to supply offspring with resources; 281) and indirect benefits (“high genetic quality” 1; 5). The “genetic quality” would be inherited by offspring and confer survival and reproductive advantages (281). According to Neff and Pitcher (282) “genetic quality” could be defined as the sum of two components: additive genetic effects or “good genes” and the non-additive genetic benefits, which are referred to as compatible genes (3). The additive genetic effects are made up of specific alleles that increase fitness independently of the rest of the genome whilst heterozygote advantage, inbreeding avoidance and epitasis contribute to non-additive genetic benefits (for review see; 3).
Studies suggest the HLA or linked genes could influence human mate choice in three ways: HLA disassortative preferences, Heterozygote advantage (HLA heterozygosity preferences) and HLA frequency dependent selection. Here we focus on HLA heterozygosity and frequency dependent selection. Heterozygote advantage states that heterozygous individuals have the ability to resist a broader array of pathogens (250). The extent of the benefit depends on the amount of overlap between presented antigen peptides (283). However, not all heterozygotes are equally resistant to disease, but as a rule heterozygotes are more equally resistant than homozygotes (283).
Rare allele advantage is the most popular form of the frequency dependent selection model. It states that new mutant alleles will have a fitness advantage because the pathogens have not adapted to them yet (225). However, once the alleles become more common the pathogens would adapt to them and their frequency would decrease (284). According to the minority advantage hypothesis, once the old allele becomes rare enough the pathogens adapted to it will decrease. These alleles will then have the selective advantage and increase in frequency (285). One of the ways in which frequency dependent selection shapes HLA based mating preferences is the focus on the benefit associated with rare alleles. As previously mentioned, new rare alleles confer a selective advantage because very few pathogens are adapted to them. Due to the low frequency of these alleles they will more likely be present in a heterozygote than a homozygote, and thus favour a preference for a heterozygous mate which would be able to recognise a wider range of pathogens (47).
An alternative hypothesis for frequency dependent selection is that certain specific common alleles might be preferred because these common alleles increase the individual’s ability to resist specific pathogens. Hill et al. (286), for example, showed that common HLA alleles were associated with improved resistance to malaria. A preference for common HLA alleles may also function to avoid mates with rare alleles that exhibit gestational drive. Haig et al. (287) defined gestational drive as an instance in which a maternal allele disfavours offspring during gestation that do not inherit it. Thus gestational drive may be a property of rare female alleles because as explained by Thornhill et al. (47) any driving effects of common alleles are likely to be successfully countered by the evolution of genes that prevent driving. A mate preference in males for females who possess common HLA alleles may function to avoid mates with alleles that show gestational drive and thereby reduce the likelihood of abortion of offspring (47).
Heterozygote advantage is likely the reason that the preference for more HLA heterozygous mates has evolved (288). Indeed, studies have reported significant associations between increased HLA heterozygosity of class I loci (A, B, and C) and delayed progression to Acquired Immune Deficiency Syndrome (AIDS) (289). In addition, heterozygosity has been shown to be somewhat heritable (290), thus a HLA heterozygous mate may also provide indirect benefits in terms of HLA diverse offspring with sound immunocompetence (288). HLA heterozygosity has been associated with perceived facial health and attractiveness in some but not all studies. Roberts et al. (45) presented facial images of British men to British women to judge. They found that the faces of HLA heterozygous British men were judged as healthier and more attractive by British women than more HLA homozygous men. Similarly Lie et al. (43) used facial images of Australian men and women and estimated heterozygosity at 12 microsatellite markers (all in linkage disequilibrium with at least one HLA locus, including HLA-A,-B and –DBR1) and another 11 microsatellite markers on different chromosomes. They found that increased HLA heterozygosity, positively predicted male facial attractiveness in young Australian men (43, 44).
Coetzee et al. (46) genotyped African Tswana women for HLA-A and-B and showed the women images to African men. They found no significant relationship between HLA heterozygosity and attractiveness judgements in African women’s faces (46). HLA heterozygosity was also not significantly associated with facial attractiveness in young Australian women (43, 44). Thornhill et al. (47) found no significant relationship between HLA heterozygosity and facial attractiveness judgments in men or women faces as judged by individuals of the opposite sex. They did, however, find a significant association between HLA heterozygosity and the attractiveness of men, but not women’s, scent (17). The non-significant association between male facial attractiveness and HLA heterozygosity reported in Thornhill et al. (47) might, be explained by the wide range of ethnicities (Caucasian, Hispanic, African, American, Asian and Native American) and participant ages (18-54 years for men and 17-44 years for women) included in the study, which could have confounded the facial attractiveness judgements. Overall, HLA heterozygous men are generally judged more attractive than their homozygous counterparts, while HLA heterozygous women are not.
The relationship between the different facial cues and HLA heterozygosity has not been fully established. Roberts et al. (45) tested the relationship between HLA heterozygosity and apparent health judgements of skin patches in male faces, and found that the two are positively related, independent of facial shape information. In other words skin condition is significantly and positively associated with HLA heterozygosity in men. Lie et al. (43) found that HLA heterozygosity positively predicted male attractiveness, and specifically facial averageness, with averageness mediating the HLA heterozygosity-attractiveness relationship in male faces. In other words more heterozygous men’s faces were considered more average and attractive. Averageness was not, however, significantly associated with HLA heterozygosity in women faces (43). Facial symmetry was not significantly associated with HLA heterozygosity in either men or women faces (43, 44). Lie et al. did not find a significant relationship between masculinity and HLA heterozygosity in Australian men, nor femininity and HLA heterozygosity in Australian women.
Taking the evidence that frequency dependent selection influences HLA based mating preferences (for review see; 240). Some researchers have investigated the relationship between common/ rare alleles and attractiveness or health, since specific rare alleles might confer fitness benefits under frequency dependent selection (46). Thornhill et al. (47) found that men significantly preferred the scent of women with more common HLA alleles over women with less common alleles, but the commonness of HLA alleles were not significantly associated with the male scent attractiveness. They also found no significant association between HLA allele commonness and facial attractiveness or facial symmetry in either sex (17). On the other hand, African Tswana women with more common HLA alleles reported significantly fewer cold and flu bouts per year, fewer illnesses in the previous year and rated themselves healthier than women with rare alleles (46), although allele frequency did not significantly predict facial attractiveness in African Tswana women when rated by male volunteers (46).
To my knowledge, no previous study has tested the relationship between HLA based mating preferences and facial appearance in African men. The aim of this study is, therefore, to test the relationship between two HLA based mating preferences (preference for HLA heterozygosity and a preference for rare / common HLA alleles), facial cues (e.g. masculinity, symmetry) and overall facial appearance (attractiveness and health) in African men.

Materials and methods

Ethics statement

This study was approved in writing by the ethics committee at the University of Pretoria (EC141002-083).

Participants

Seventy one African men (mean age=20.4, SD=2.8) were recruited from the University of Pretoria. The data was collected from the same participants used in chapter two, however an extra blood sample was collected for DNA extraction. Each participant provided written informed consent and completed a short questionnaire, including questions on gender and ethnicity. Full colour frontal facial photographs were taken with a Canon EOS 40D digital camera under standardized conditions. Participants were asked to maintain a neutral expression. The facial photographs were standardised for orientation and size using Psychomorph and other in-house software.

Image Ratings

Twenty African women (mean age =22.5; SD= 2.2) were recruited from the University of Pretoria to rate all the male facial photographs for attractiveness, health, symmetry, masculinity, distinctiveness and facial adiposity. The male facial images were presented in a randomised order on a computer screen. The females participants were asked to rate each image (How attractive is this person?; How healthy is this person?; How masculine is this person?; How symmetric is this person?; Please indicate this persons weight?; How distinctive is this face?) on separate 7-point Likert scales (1=very unattractive, 7=very attractive etc.) and for weight (1= very underweight, 7= very overweight). Distinctiveness was reverse coded for the analyses to indicate averageness. Each female participant provided informed consent and completed a short questionnaire including questions on age and gender.

Sample collection

Blood (5ml) was collected by a qualified phlebotomist using EDTA vacutainer® tubes (to avoid clotting) and transported at ambient conditions to the laboratory. The blood was used for DNA extraction following the Quick-DNA™ Universal Kit as per manufacturer’s instructions (ZYMO Research). In short, a 200 μl sample was mixed with biofluid, cell buffer and proteinase K and incubated. The digested sample was bound using genomic binding buffer, cleaned and eluted into clean microcentrifuge tubes. The purified DNA quality and concentration was measured using a Spec Nanodrop; the 260/230 ratio, 260/280 ratio and the DNA concentrations in were recorded in ng/μl were recorded. The gDNA was analysed on a 1% agarose gel to ensure the DNA was intact. The DNA was diluted into 50 ng/μl solutions and stored at -20 °C.

SNP genotyping

Twelve Single Nucleotide Polymorphisms (SNPs) that were associated with a range of different HLA loci, some of which have been investigated in previous HLA mating preference studies (e.g. HLA-A,B, DRB1,DRB2 (44, 46, 116, 244) were selected for SNP genotyping (Table 3.1). The SNPs were included on a custom designed TaqMan® OpenArray® Plate (Applied Biosystems®) and quantified using the Quantstudio 12K flex system (Life Technologies) according to manufacturer’s instructions for Genotyping (Applied Biosystems™ QuantStudio™ 12KFlex Real-Time PCR System user guide).

Analysis

SNP quality control was performed using the Taqman® Genotyper Software. SNP calls were analysed for all samples. The assay call rates, minor allele frequencies (MAF) and Hardy-Weinberg Equilibrium p-values (HWE*) are reported in Table 3.1. One SNP (rs3129720) had a poor amplification rate (< 50%) and was discarded and not used for further analysis. An overall HLA heterozygosity score was calculated for each participant by summing all the heterozygous SNP genotypes for that participant. To calculate the overall “common alleles” score we first calculated which of the two alleles were most common in the dataset for each SNP and then summed all the common alleles for each participant. All further analyses were performed in SPSS version 23.

Declaration
Preface
Acknowledgments
Note to the reader
List of abbreviations 
List of Figures
List of Tables 
Chapter 1: Literature review Facial appearance, health and Immunity
1. Introduction
1.1. Facial appearance
1.1.1. Associations between Facial Attractiveness and Health
1.2. Facial Cues that play a role in Attractiveness
1.2.1. Facial Symmetry
1.2.2. Associations between Facial Symmetry and Attractiveness
1.2.3. Association between Facial Symmetry and Health
1.2.4. Averageness
1.2.5. Associations between Averageness and Attractiveness
1.2.6. Associations between Averageness and Health
1.2.7. Sexual Dimorphism
1.2.8. Associations between Sexual Dimorphism and Attractiveness
1.2.9. Associations between Sexual Dimorphism and Health
1.2.10. Skin Colour /texture
1.2.11. Associations between Skin Colour and Attractiveness
1.2.12. Associations between Skin Colour and Health
1.2.13. Facial Adiposity
1.2.14. Associations between Facial adiposity and Attractiveness
1.2.15. Associations between Facial Adiposity and Health
1.3. Immunity
1.3.1. Peripheral Blood Mononuclear cells (PBMCs)
1.3.2. Cytokines
1.3.3. Pro-inflammatory cytokines
1.3.4. Anti-inflammatory cytokines
1.3.5. C – reactive protein (CRP)
1.3.6. Human Leukocyte Antigen Complex
1.3.7. HLA Diversity
1.3.8. HLA based mating preferences
1.4. Consolidating the gaps in the current literature
Chapter 2 Facial appearance reveals immunity in African men
2. Introduction
2.1. Materials and methods
2.1.1. Ethics statement
2.1.2. Participants
2.1.3. Image Ratings
2.1.4. Immunological analysis
2.1.5. Statistical analysis
2.2. Results
2.3. Discussion
Chapter 3  Determining the link between HLA heterozygosity and facial appearance in an African population. 
3. Introduction
3.1. Materials and methods
3.2. Result
3.3. Discussion
Chapter 4 Conclusions
 References
GET THE COMPLETE PROJECT