Worse Health Status and Higher Incidence of Health Disorders in Rhesus Negative Subjects

Rhesus-positive and Rhesus-negative persons differ in the presence-absence of highly immunogenic RhD protein on the erythrocyte membrane. The biological function of the RhD molecule is unknown. Its structure suggests that the molecular complex with RhD protein transports NH3 or CO2 molecules across the erythrocyte cell membrane. Some data indicate that RhD positive and RhD negative subjects differ in their tolerance to certain biological factors, including, Toxoplasma infection, aging and fatique. Present cross sectional study performed on 3,130 subjects) showed that Rhesus negative subjects differed in many indices of their health status, including incidences of many disorders. Rhesus negative subjects reported to have more frequent allergic, digestive, heart, hematological, immunity, mental health, and neurological problems. On the population level, a Rhesus-negativity-associated burden could be compensated for, for example, by the heterozygote advantage, but for Rhesus negative subjects this burden represents a serious problem.


Introduction
Polymorphism in the Rhesus factor, namely the existence of a large deletion in the RHD gene [1] in a substantial fraction of the human population, has been an evolutionary enigma since the discovery of this factor in the 1930's [2][3][4][5]. Theoretically, neither the RhD-negative allele can successfully spread in the RhD positive population nor the RhD-positive allele can spread in the RhD negative population [6,7]. Before the introduction of prophylactic treatment in 1968, a positive frequency dependent selection systematically penalized the less abundant allele because lots of children of RhD-negative women in the mostly RhD-positive population as well as children of RhD-positive men in the mostly RhD-positive population were dying of hemolytic anemia. It has been suggested that this polymorphism can be stabilized when the disadvantage of carriers of the locally rarer allele is counterbalanced by higher viability of their heterozygote children or by another form of frequency-dependent selection [6]. In the past seven years, several studies have demonstrated that Rhesus positive and Rhesus negative subjects differ in resistance to the adverse effects of parasitic infections, aging, fatigue and smoking [7][8][9][10][11][12][13]. A study performed on 250 blood donors has further shown that the resistance to effects of toxoplasmosis is higher in Rhesus positive heterozygotes than in Rhesus positive responders. The responders were asked to subjectively rate of their allergic, cancer, digestive, fertility, genitourinary, heart, hematological, immunity, mental health, metabolic including endocrine, musculoskeletal, neurological, respiratory organs, sense organs, and sexual life problems using 6-points Likert.scales. The second group of questions tried to collect objective information reflecting the health status of responders. We asked the responders, for example, how many drugs prescribed by doctors they currently takes per day, how many of "different herbs, food supplements, multivitamins, superfoods etc." they currently take per day, how many times they used antibiotics during the past 365 days. We also provided the responders lists of about 250 disorders (separated to 15 categories) and asked them to tick which of them they were diagnosed with. The questionnaire contained, among others, also the following questions: "What is your Rh blood group?" with three options: a) I do not know / I am not sure, b) negative (this is the less frequent variant) c) positive (the more frequent variant). Implicitly, the answer a) (I do not know/I am not sure) was checked.

Statistical methods
Before statistical analysis, suspicious data (too high or too short body height, too low or too high body mass or age, too short duration of the test etc.) were filtered out (26 cases). In the test, we also measured simple reaction times, operational, short-term and long-term memory, psychomotor performance, intelligence and personality profiles. However, here we have analyzed only data concerning health status.
SPSS v. 21. was used for all statistical tests. Ordinal and binary data were analyzed by partial Kendall´s correlation test [16,17]. This test measures strength and significance of association between binary, ordinal and continuous data regardless of their distributions. This technique enabled us to control for one confounding variable, for example the age of a responder. The Excel sheet for computing partial Kendall's Tau and the significance between variables A and B after the variable C is controlled based on Kendall Tau´s AB, AC and BC. It is available here: http://web.natur.cuni.cz/flegr/programy.php (item no. 12) and in S2 File. Certain diseases have very different incidence in men and women. Also, some biological factors, including RhD phenotype, could have different impacts on men and women. Therefore, we performed all analyses for all responders and also separately for the male and female responders.

Results
Descriptive statistics of data Among 4,286 Czech and Slovak participants of a subsequent case-control study, 3,130 subjects (840 RhD positive men, 317 RhD negative men, 1,337 RhD positive women and 636 RhD negative women) provided information about their gender and RhD phenotype. RhD negative subjects, especially women, have higher motivation to care about, and to remember, their RhD phenotype. Therefore, the frequency of RhD negative subjects (30.4%) differed from the 16% general frequencies within the Czech and Slovak populations and also between men (27.4%) and women (32.2%). The mean age of RhD positive men (37.6, S.D. 13.5) was approximately the same as that of RhD negative men (37.7, S.D. 12.7), t (1153) = -0.10, P = 0.923. RhD positive women were younger (33.6, S.D. 11.9) than RhD negative women (35.2, S.D. 12.7), t (1923) = 2.74, P = 0.006. The numbers of men and women in the particular age strata were comparable, with the exception of the 21-30 age stratum, which consisted of 363 men and 842 women (Fig 1).

Correlation of RhD phenotype with self-reported health problems (ordinal variables)
Twenty-two dependent variables (mostly ratings of particular health problems on a scale from 1-6, 1: "no problems at all", 6: "frequent or serious") were ordinal and had a highly skewed distribution. Therefore, the nonparametric partial Kendall's correlation test (which enables to control one confounding variable, here the age) was used to search for an association between the RhD phenotype and the intensity of fifteen categories of health problems (allergic, cancer, digestive, fertility, genitourinary, heart, hematological, immunity, mental health, metabolic including endocrine, musculoskeletal, neurological, respiratory organs, sense organs, and sexual life problems) and also another six health-related variables, namely the numbers of drugs prescribed by doctors that the subject currently takes per day, numbers of "different herbs, food supplements, multivitamins, superfoods etc." the subject currently takes per day, how many times the subject has used antibiotics during the past 365 days, how many times the subject was required to seek acute medical care for a serious illness (not injury) that lasted more than 3 days during the past 5 years, how many specialized medical doctors the subject had to regularly attend (not for prevention) at least once in the past two years, how often the subject felt tired (not after exertion, e.g. sports) and how often the subject has experienced a headache. The results showed that the RhD negative subjects had more serious health problems in 6 of 22 analyzed variables than the RhD positive subjects (Table 1).

Association between RhD phenotype and incidence of particular diseases (binary variables)
After rating each particular category of health problems, the subjects were asked to identify which specific disorders they suffered from on the lists of 225 disorders. They were also asked to identify which specialized medical doctors they had to visit regularly (not for prevention) at  least once in the past two years from a list of 10 types of specialists. The associations were analyzed with the partial Kendall's Tau correlation test with age being a covariate. One hundred fifty four (154) of 225 diseases/disorders were reported by at least 10 subjects. Within this subset, 31 significant associations with RhD negativity (21 positive and 10 negative) were expressed in all subjects. In male subjects, the number of significant association was 35 (19 positive and 16 negative) while in female subjects the number of significant associations was 30 (18 positive and 12 negative). The expected number of false significant results for 462 statistical tests was not 96 but 23. For example, RhD negative men more often reported certain mental health disorders including panic disorders, antisocial personality disorders and attention deficits, ticks, fasciculation, thyroiditis, immunity disorders, allergies, especially skin allergies, excessive bleedings, anemia, osteoporosis, liver disease, infectious diseases and acute diarrhea diseases, while they less often reported gall bladder attacks, coeliac disease, maldigestion, malabsobtion, warts, some types of cancers and prostate hypertrophy. RhD negative women reported more frequently psoriasis, constipation and diarrheas, ischemic diseases, type 2 diabetes, some types of cancers, lymphatic nodes swelling, vitamin B deficiency, thrombosis, tonsil stones, too high sex desire, precocious puberty, urinary tract infections, scoliosis and they less often reported hearing loss, weight loss, hypoglycemia, glaucoma, fasciculation and warts. RhD negative subjects had to make more frequent visits to medical professionals specializing in otolaryngology (P = 0.021), psychiatry (P = 0.008), gynecology (P < 0.001), and dermatology (P = 0.014) (the theoretical number of false positive results was 0.5). Table 2 shows the associations between RhD negativity and disease incidences while Table 3 shows the associations between RhD negativity and visiting specialized doctors.

Discussion
The RhD negative subjects expressed many indices of a worse health status. Men, women or both sexes reported more frequent allergic, digestive, heart, hematological, immunity, mental health and neurological problems. They also reported the usage of more drugs prescribed by doctors per day, attended more specialized doctors, namely, dermatologists, gynecologists, internal medicine doctors, neurologists, and psychiatrists (men) in the past two years, a higher frequency of headaches and being tired more often than RhD positive subjects. Incidence of various diseases and disorders also differed between RhD negative and RhD positive subjects, mostly being higher in the former. RhD negative subjects have increased the risk of developing of certain heart diseases, respiratory diseases and some immunity and autoimmunity related diseases, for example rheumatoid arthritis. The general pattern suggests that RhD negative subjects could have problems with autoimmunity, could be more resistant to infections of viral origin and could be less resistant to infections of bacterial origin.
The mechanism of the effect of the RhD phenotype on human health status is not clear. RhD protein together with strongly homologous RhCE protein and with also homologous RhAG glycoprotein are all components of a membrane complex of which the function is not quite clear. It is most probably involved in NH 3 transport and possibly also in CO 2 transport [18,19]. This complex is associated with spectrin-based cytoskeleton and therefore plays an important role in maintaining the typical shape (biconcave discoid) of human erythrocytes [20]. The biological functions of complexes containing the RhD protein are unknown. However, they might be involved in NH 3 /NH 4 + detoxification of organs. Ammonia, the product of protein catabolism is extremely toxic, especially for brain cells and must be quickly removed from the sensitive organs. It was observed that the concentration of ammonium is three times higher in red cells than in plasma [20] and it was further suggested that the RhD containing complex plays a key role in its capturing and its transport to the kidneys and the liver [20]. It was also suggested that the complex might participate in intracellular pH regulation [20] and consequently also in the regulation of local oxygen tension. It was suggested that RhD-negativity-associated anoxia in certain parts of the nervous system could be responsible for physiological (and also behavioral) effects of the RhD phenotype [21]. The variation of the oxygen tension in various organs and tissues could, of course, influence also other biological functions, including the functions of the immune system. This could explain why RhD negativity seems to be associated with neurological, mental health and immunological disorders. The probable roles of the RhD-containing complex in keeping the normal morphology and adhesiveness of red cells (for review see [20]) could be responsible for the observed associations of RhD negativity with some haematological and inflammation-related diseases, including arthritis.
Limitations and strength of present study: Using very effective Facebook-based snow-ball method we obtained data from a large number of subjects. However, most of them were relatively young people (mean age was 35.4). Most of the diseases and disorders with the largest public health impact (but possibly not the largest economic impact) start at a higher age in developed European countries such as the Czech and Slovak Republics. This can largely distort the whole picture of the RhD negativity impacts on public health. Future studies (which could   be easily done in countries with available national databases of medical records of all citizens) should aim to recruit middle age and senior subjects. Our study compared the health status of RhD negative subjects (16% in general population of Czech and Slovak Republics) with RhD positive subjects, i.e., with the health status of mixed population of RhD positive homozygotes (36% of the general populations within the Czech and Slovak Republics) and heterozygotes (48% the general populations in the Czech and Slovak Republics). The results of the published case-control studies on the effects of the RhD genotype on psychomotor performance [22,23], as well as the heterozygote advantage hypothesis, however, suggest that the health status of RhD positive homozygotes and heterozygotes differs. In further studies concentrated on particular disorders, smaller populations of subjects should be RhD genotyped using molecular biology techniques and then the health status of all three RhD genotypes have to be compared. In Numbers of RhD negative subjects without particular disorders, RhD negative subjects with particular disorders, RhD positive subjects without particular disorders, RhD positive subjects with particular disorders, partial Kendall´s Tau and statistical significance, respectively, are shown in six columns of each section. The effect of age on health status was controlled in partial Kendall's correlation (non-parametric) test. Positive Tau corresponds to a positive association and negative B to a negative association of RhD negativity with incidence of particular disorder. Significant results (p < 0.05) and trends (p < 0.10) are printed in bold. Asterisks indicate results significant in two-sided tests. p values < 0.0005 are coded as 0.000. The effect size is shown as the present study, the health status data were collected using a questionnaire. This enabled to study of the effects of the RhD phenotypes on rarer disorders using a large population sample. Of course, more precise and more detailed data could be obtained from medical records. Primarily, we have run the study to confirm or disprove the alarming results of a previous small scale studies performed on non-typical populations. However, we had no a priory hypotheses which health-related variables should correlate with RhD phenotype or which disorders should Table 3. Differences between RhD positive and RhD negative participants in specialised medical doctors the subject had to regularly visit at least once in the past two years. occur more frequently in RhD negative subjects. Therefore, the present study had a more or less explorative character. Hence, we have reported the results of statistical tests without formal correction to multiple tests. It should be noted, however, that, for example, we have obtained 41% positive results for the ordinal health status variables and 20% positive results for binary health status variables. Theoretically, only 5% of false positive results should be expected in multiple tests. The main strength of the present study is the absence of any sieve effect, which could result in publication bias in other types of studies. Positive results of particularly observational or experimental studies and partly also meta-analytic studies, could be an artefact of intentional or unintentional "cherry-picking"; i.e. preferential or even exclusive publication of positive results. In our study we have searched for the effects of the RhD phenotype on all diseases and all disorders having high enough incidences in the Czech population (n = 154) and we have reported all, both positive and negative results.

Conclusions
Some of the associations observed the present study were relatively strong and some of them concerned rather frequent disorders. Therefore, the total impact of frequency of RhD negative homozygotes in the general population on public health could be large.
The aim of the present study was to search for indices of validity of the heterozygote advantage hypothesis, namely for the indices of impaired health status of RhD negative subjects. It must be reminded, however, that the observed specific disease burden of the RhD negative subpopulation is in an agreement with predictions of this hypothesis but does not prove its validity. The higher disease burden in RhD negative homozygotes could be compensated either by increased fitness of heterozygotes (heterozygote advantage hypothesis) or by still unknown selection pressure in favor of RhD negative subjects. In this context, the shorter reaction times of RhD negative, Toxoplasma-free blood donors [7] and university students [8] and higher intelligence in RhD negative, Toxoplasma-infected soldiers [11] should be remembered. It could be speculated to what extent the highly uneven distributions of RHD minus alleles in world populations might be the result of a founder event and a gene flow [24] and to what extent it is also modulated by specific selection pressures caused by differences in the geographical distribution of a disease or diseases.
Supporting Information S1 File. Excel file containing the data set.