User:Teamorigins3f03/sandbox

Introduction
The human blood consists of three primary blood group systems namely, ABO, Rh (Rhesus) and MNS. All of these blood group systems consist of genetic polymorphisms which reveal the evolutionary pathway of early primates. However, in order to do this, current anthropoid apes genetically related to Homo sapiens must be sampled for blood types, specifically the ABO blood group which is most polymorphic. Using this information, the sequential emergence of the ABO alleles can be hypothesized, which will provide insight to the evolutionary past of primates including Homo sapiens. This article aims to describe the various blood groups and their associated polymorphisms alongside the genetic basis of their development. Furthermore, the presence of these blood groups in specific primate species and the successive origins of the ABO alleles will be explored.

ABO Blood Group System
See main article ABO Blood Group

The ABO blood system was first discovered by Karl Landsteiner, an Austrian scientist, in 1900. It is the most important of the 29 blood group systems that are known. The ABO blood group consists of four antigens: A, B, O and AB. They are known as oligosaccharide antigens and are represented on the membranes of red blood cells, tissue cells, in the saliva and other body fluids. These antigens are also an important area for concern in blood transfusion to evaluate the adaptability of donor blood with bone marrow transplantations. Humans can, therefore, have the blood types A, B, O, or AB and this is a polymorphism in which two or more alleles can occur in a frequency greater than 1% in a given population (10).

RBC Antigens
See main article Antigens

The ABO group is established by the presence of the A and B antigens on the surface of red blood cells or the presence of antibodies in the serum, namely anti-A or anti-B antibodies. Thus, a person with blood type A would have red blood cells carrying antigen A and the serum having an anti-B antibody. Likewise, an individual with blood type B will possess antigen B and anti-A antibody in its serum. Also, blood type O contains no antigens but does contain the anti-A and the anti-B antibodies. With blood AB, it possesses both the A and B antigens but has no antibodies. The antibodies are not present at birth but appear after a year, quite possibly to protect against viral, bacterial or plant antigens (10).

Antigen Carrier Proteins
ABO blood group antigens are surface marker on the red blood cell and contain proteins and carbohydrates attached to lipids/proteins. These are oligosaccharide in terms of structure and are also expressed in bodily fluids. The first step that is required in the synthesis of ABO antigens it the addition of L-fucose in a 1-2 linkage on terminal galactose of a common precursor adhered to lipids, resulting in the H-antigen (O). There are six different precursors known but Type 1 and Type 2 sequences are the important ones. Type 1 is part of tissues and secretions and Type 2 is an antigen on the surface of red blood cells (10). A, B and H (O) antigen structures are characterized as carbohydrate determinants that are present in the membrane of red blood cells as well endothelial and epithelial cells.

Structure of the ABO gene locus
The human ABO genes are located on chromosome 9 and consists of 7 exons over 20 kb of genomic DNA. Exon 7 has the largest coding sequence. The ABO locus has three main allele forms: A, B (both codominant) and O alleles (recessive). Exon 6 contains the deletion found in most O alleles and these range in size from 28 to 691 base pairs in length (11). Alleles A and B contain seven nucleotide substitutions, four of which are translated in different amino acids, called glycosyltransferases, that are encoded by the alleles. The A-allele encodes A-transferase and the B allele encodes B transferase. The O allele, on the other hand, differs from the A-allele by a single deletion of guanine at position 261. This deletion leads to a frame-shift, resulting in the O-allele not coding for any transferases (11). The combination of these ABO alleles is what leads to the four major phenotypes: A, B, AB and O. The central A and B alleles, A101 and B101, differ by four amino acid changes at positions 176, 235, 266 and 268 with these being essential in determining the A and B specificity of the enzyme (11). The most frequent of all ABO alleles is the O allele, with the most common haplotypes, O01 and O02, sharing a single deletion at position 261 of exon 6. This is what leads to a premature stop codon and results in a protein with no enzymatic activity to encode transferases. However, the O01 differs from the O02 haplotype by six substitutions – which include position 297 in exon 6, which is adenine in O01 and guanine in O02 (11).

RH (Rhesus) Blood Group System
See main article RH Blood Group System

The Rh blood group system, which is sometimes referred to as the Rhesus, comes right after the ABO blood system. It is most significant in transfusion medicine. It is highly polymorphic, consisting of over 45 different antigens that are located on variant forms of RhD and RhCcEe proteins (8). These proteins form a core complex along with a glycosylated homolog (RhAG) and are only expressed when it this is present. The most substantial of these polymorphisms is the presence or absence of the RhD antigen present on red cells. An incompatibility between an RhD antigen of a mother, who has given birth to more than one child, and her unborn child can lead to an austere immune reaction leading to hemolytic disease – which is the rupture or destruction of red blood cells, or even intrauterine death (8). Severe problems also arise when there is an incompatibility for the RhD between a transfusion donor and a recipient. Variance in the other RH products such as the C and E antigen series can also lead to hemolytic reactions.

The Rh antigens are located on three non-glycosylated transmembrane proteins encoded onto the two RHD and RHCE genes. Absence of the D antigen expression is most likely due to the loss of the entire RHD gene from the genome of Rh-D individuals. C and E antigen series are formed from the single RHCE gene and alternative mRNA splicing is thought to have been responsible for the productions of these two distinct polypeptides (9). Divergent forms of the C and E antigens are produced by base substitutions in RHCE. Alleles that express the C antigen in RhCE differ from alleles expressing the c antigen at six nucleotide positions, five in exon 2 and one in exon 1. Only four of these result in amino acid substitutions, at nucleotide 307. The four alleles of RHCE make up all the possible haplotypes, although there are differences in their frequencies (9).

RhD and RhCcEe proteins
The RhD protein possesses the D antigen, whereas the RhCcEe protein expresses either C or c antigens along with E or e antigens on the same protein. These two proteins have a high degree of homology between them. Primary analysis of the amino acid sequences has shown that the first 41 N-terminal amino acids of RhD and RhCE/e are matching and that RhD differs from the common forms of rhCE by only 30-35 amino acids along the entire protein (9). Despite such a high degree of similarity, the RhD does not express any c or e antigens and the RhCcEe protein does not carry any D isotopes.

D-Antigen
The D-antigen is the most important antigen in the Rhesus blood group and is found on the RhD gene. It is responsible for the + and – that is seen beside the ABO blood group. The sorting of Rh-positive and Rh-negative individuals is based on the presence or absence of the RhD protein on erythrocytes (9). The D-negative phenotype is associated with a deletion of RhD but in some populations, the D-negative is associated with a normal RhD gene. Analysis of these genes showed a normal RhD sequence but a severely limited expression of the RHD mRNA, suggesting a deficiency at the level of processing or pre-mRNA processing. Also, RHD transcripts from people expressing a weak configuration of the D-antigen were found to have missense mutations within the trans-membrane of RhD (9).

CcEe Antigen
The RhCcEe polymorphism is caused by substitutions of the nucleotide in the RHCE gene. Six nucleotide substitutions causing 4 amino acid changes are responsible for the C to c polymorphism although only one of this (Ser103Pro) polymorphism equates with the C/c antigenicity. The substitution at Pro102 is also a crucial part of the c antigen (9). The presence of adjacent proline residues (102 and 103) forms a rigid structure that opposes any changes in nearby amino acids. This might explain why there are a relatively low number of c variants when compared with other Rh antigens. The polymorphism of E to e is said to be possible by a single nucleotide substitution (Pro226Ala). Many more variants of the e antigen have been described, but the requirements for these variants of e have not been clearly understood (9).

MNS Blood Group System
The MNS blood group system is right behind the Rh blood group in terms of its complexity. The MNS antigens are expressed on glychophorin A (GPA), glycophorin B (GPB) and combinations that arise from single nucleotide substitutions, gene conversion between the glycophorin genes and also crossing over (12). Refer to MNS Blood System

Antigens of M/N
The MNS antigens, GPA and GPB, are fully developed at birth. GPA and GPB are both Type I transmembrane sialoglycoproteins that traverse the red blood cell lipid bilayer and are positioned witht heir amino-termini to the outide of the red blood cell membrane (12). Both the terminal domains of GPA and GPB carry O-glycans, but GPA also carries an N-glycan (asparagine-linked glycan) attached to amino acid residue 26. The M and N group polymorphism is the result of a separate amino acid sequence at the terminus of GPA, while the S and s blood group polymorphism begins with a single amino acid substitution at residue 29 of GPB (12).

Function of GPA and GPB
GPA and GPB provide the carbohydrate on the red blood cell membrane. The O-glycans on these glycophorins carry the sialic acid and add to the net charge of the red blood cell. These protect the RBC from sticking to each other and protect it from invasion by pathogens. GPA-deficient RBCs are resistant to invasion to pathogens as sialic acid is crucial for the attachment of the parasite to the RBC. GPA also is responsible in chaperoning band 3 transport to the RBC membrane. An absence of GPA or GPB on the RBCs does not result in a significantly different morphology and the individuals with this do not show any abnormal phenotypes (12).

Why Anthropoid Apes?
See main article Anthropoid Apes

Human and anthropoid ape blood groups display similarities in reactions of respective blood cells with specific typing reagents and in general structures of some main blood group systems that have been defined. During studies of blood groups in non-human primates, anthropoid apes have garnered the most attention as they most closely resemble humans physiologically. The study of anthropoid apes provides information about blood types in these species as well as insight into the evolution of human blood groups (13).

A-B-O Blood Groups
A-B-O blood groups have been most deeply examined in apes out of all human type blood groups. All four groups, including O, A, B, and AB, are seen in apes in general, but they are not present in a single species at once. This suggests that the apes have not reached the high level of A-B-O polymorphism that is found in humans.

According to Table 1, Orangutans and Gibbons had three of the four blood groups, chimpanzees had two, and gorillas appeared to be monomorphic. The B substance in gorillas was not found on red blood cells (only in antibody secretions) which more closely resembles the Old World monkeys rather than anthropoid apes. The total number of apes tested pales in comparison to the millions of human A-B-O blood type tests. However, it is safe to conclude that the high level of A-B-O red blood cell polymorphism is exclusively found in humans (15).

H substance
The central problem of how the H substance (or antigen) is related to A and B groups was somewhat clarified with the study of A-B-O blood types in anthropoid apes. H is a precursor to the A and B blood group antigens on red blood cells as H must be attached to the precursor substance before it accepts A or B determinants at their respective sites (14). This precursor theory is supported by the idea that humans with the O group (homozygous for amorphic O gene) have red blood cells and antibody secretions that react most strongly for H. On the contrary, the data in Table 1 shows that this precursor theory may not apply to apes because the reciprocal relationship between H and A-B is not maintained. The red blood cells of group O or A2 do not react with the anti-H lectin (reacts strongly with human cells). On the other hand, B group red blood cells in gibbons react as strongly as human group O cells. Further exploration of the H substance in different human races and other primates spawned an alternative theory about how the H and AB antigens are linked. This alternative theory proposes that H acts in parallel to the A-B gene when attaching determinant groups on the red blood cell surface (13).

M-N Blood Groups
Investigation results on the homologues of human M-N blood types in various anthropoid apes are shown in Table 2. Gibbons are the only apes that have all three M-N types similar to the ones found in humans. Chimpanzees were M-Positive, with only about 40% found to be also N-positive, which leads to chimpanzees only displaying two M-N blood types: M or MN. In contrast, all gorillas were found to be N-positive and 76% were also M-positive, which results in only two types: N or MN. Half of the orangutan population were M-positive, while another half were M or N-negative. Thus, orangutans have only two defined M-N types including M or m (13).

M-N Genetics
The classic theory of Landsteiner and Levine can explain the distribution of M-N types in humans and gibbons by employing a pair of allelic genes M and N; this theory cannot account for the occurrence of M-N blood groups in other anthropoid species. Furthermore, Hardy-Weinberg laws cannot explain M and N types found in chimpanzees and gorillas if the same allelic gene pair was to be used. For these reasons, a new theory was proposed which could explain M-N blood types in all anthropoid apes (13).

Wiener Theory
This theory proposes that all humans are homozygous for gene N, while gene M is transmitted independently with amorphic gene m as its allele instead of gene N. The following genotypes would result from the three M-N types: M (genotype MMNN), N (genotype mmNN); and MN (genotype MmNN). The Wiener theory postulates that the M-m genotype affects the expression of the always present N antigen. The independently transmitted genes M and N interact phenotypically depending on proximity of the M and N antigens. In genotype mmNN, N would be maximally expressed, due to the lack of determinant group for M, resulting in the type N individual. In individuals with genotype MMNN (type M), the M determinant group would be expressed fully, masking the N determinant group (which may not be accessed by anti-N reagents due to proposed deeper location within antigen molecule). In the genotype MmNN, a fewer number of M groups are present, leading to N groups being accessed more readily (intermediary reactivity with anti-N reagents), which results in the MN blood type (13). This theory successfully accommodates M-N type genetics of all anthropoid ape species.

The Fallout
According to the Wiener theory, the two blood types observed in chimpanzees (M and MN) can be attributed to genotypes MMNN or MMNn which result in type MN and genotype MMnn which leads to type M. Chimpanzees are missing the N type blood group because of the complete lack of gene m. In contrast, gorillas lack the n gene with genotypes mmNN (type N), MMNN or MmNN (type MN). Orangutans are postulated to have genotypes MMnn or Mmnn (type M) and mmnn (M-negative type) as the N gene is totally absent.

Rh-Hr Blood Groups
As seen in Table 3, no anthropoid ape possesses the complete set of human Rh-Hr blood group specificities (15).

Simian-type Blood Groups
The analysis of these groups is conducted by reagents that are prepared with immunization against red blood cell antigens of primate animals. The majority of data on simian-type specificities is available for chimpanzees because chimpanzees are the only anthropoid apes available for large scale immunization and intense blood grouping investigation.

V-A-B-D Blood Group
During the study conducted by Socha and Moor-Jankowski (1979), it was evident that the V-A-B system in chimpanzees was comparable to the human M-N-S blood group system. This was due to the finding that the Nv lectin detected both the Vc antibody in chimpanzees and N antibody from human red blood cells (13).

R-C-E-F System
The chimpanzee R-C-E-F blood group system greatly resembles the human Rh-Hr system. The main antigen (Rc) is very close in identity to the principal antigen from the human Rh-Hr system known as Rh0 (13).

Theories on the emergence of ABO alleles
As discussed earlier, the human ABO histo-blood group system is controlled by alleles at a single locus on chromosome 9 (1). The A and B antigens reside on oligosaccharide chains which are of variable length and can exist in free form (milk, urine), bound to proteins, and as lipids. The discovery of the ABO system in primates has led to a debate whether the A and B blood group genes were subject to convergent evolution, such that A and B reactivity was generated independently from each other in the hominoids and Old World monkey lineages, or a trans- species polymorphism (5).

Convergent Evolution
See main article Convergent evolution

Is a process whereby organisms that are not related to each other, independently evolve similar traits in order to adapt to similar environmental pressures (2).

Evidence for convergent evolution
Arguably the best studied case of convergent evolution in the ABO blood group comes from a study done by Saitou and Yamamoto in 1997. The figure on the right is one of 10 equally parsimonious trees found using PAUP. The tree indicates that the common ancestral gene for hominoid and Old World monkey blood group is the A type, and three B alleles evolved independently in the human, gorilla and baboon populations. The emergence of B alleles corresponds to two amino acid changes on positions 266 and 268 .This is particularly due to two amino acid substitutions on position 266 and 268 (5).

Trans Species Polymorphism (TSP)
Is the occurrence of similar alleles in related species, excluding the chances in which the similarity arose due to convergent evolution. TSPs are generated by the passage of alleles from ancestral to descendant species, and only remain in a population as long as there is balancing selection present in the genetic systems. One thoroughly investigated example is of the major histocompatibility complex of jawed vertebrates (3).

Evidence for trans-species polymorphism in the ABO blood group
In 2012 Sègurel and his team provided concrete evidence which shows that genetic variation in humans, gibbons and old world monkeys are inconsistent with a model of convergent evolution. Instead, the data supports the hypothesis of an ancient polymorphism. The results seem to indicate that the trans-species polymorphism has remained under balancing selection for millions of years (4). The researchers sequenced exon 7 of the ABO gene in bonobos, western chimpanzees, lowland gorillas, orangutans (hominoid species), colobus, velvet monkeys (Old World monkeys), marmosets and howler monkeys (New World monkeys). Among all of these species, the ABO polymorphism is present (4).

As a result of recombination, it is expected that the diversity among ABO alleles should be highly heterogeneous. Therefore, the researchers proceeded to focus on two sections of exon 7 flanking the AB gene. The pairwise synonymous diversity was then compared to the synonymous divergence between the same allele (A or B) from different species.

The following results were obtained and provide evidence that the ancestral polymorphism was stably maintained for millions of years and inherited across many species (4).
 * It was found that there is tremendous synonymous diversity between A and B alleles in macaques, colobus monkeys, and baboons (4).
 * Two synonymous polymorphisms are shared between macaques and baboons. This is unlikely to occur in convergent evolution (4).
 * In hominoids, divergence between chimpanzee A and human B alleles exceeds the divergence among African ape lineages. This can only occur if the A/B polymorphism predates the species split (4).
 * A gene of humans is more closely related to the A gene of gibbons than to the human B variant. This is true for other species as well (4).

These results indicate that the ABO polymorphism originated at least 20 million years ago and has been evolving trans-specifically such that it arose before the divergence of Old World and New World monkeys (4).

In another study, researchers investigated whether the four critical nucleotide substitutions (positions 523, 700, 793, and 800) believed to differentiate the human A and B alleles, also occur in the genes of non-human primates (7). Two oligonucleotide primers, complementary to nucleotide positions 412-432 and 867-847 of the A allele were synthesized. The primers were then used in PCR to amplify DNA derived from chimpanzees, Gorillas, Orangutans. The DNA was then sequenced. There were nucleotide substitutions at 20 positions among the ape sequences. Out of these, eight were synonymous ( does not alter amino acid sequence) and 13 were non synonymous (alters amino acid sequence). The non synonymous substitutions at positions 793 and 800 is also critical for A and B allele differentiation in primates. Because of this similarity between primates and humans for the differentiation of A and B alleles, it can be established that these two substitutions (positions 793 and 800) are responsible for the enzymatic activities of the A and B transferases. Additionally, identical substitution patterns in humans and primates indicated at sites 793 and 800, lead to the conclusion that the A and B genes must have arisen before the separation of the lineages (7).

Evidence for balancing selection
It was found that there is unusually high large coalescence times for human ABO genes. To compare these numbers with coalescence times for non-human primate ABO genes, numbers of nucleotide substitutions from the most recent ancestor sequence to the sequences in each species were first estimated. Once the values were obtained, a calibration of the evolutionary rate (1.4 x 10 -9) based on the human-chimpanzee comparison was used. It is interesting to note that alongside for humans, chimpanzees, orangutans, and baboon also show large coalescence times. However, these primates also showed large coalescence times for DNA region under neutral evolution (238 bp HOX2). This is in stark contrast to humans. Therefore, long coalescence times estimated for the human ABO gene, both from the complete cDNA region and from a partial cDNA region suggest the possibility of the existence of a balancing selection on the ABO loci(5).

Other theories
Emerging research, although agreeing somewhat with the theory of convergence evolution, has found that major A allele ( A101) is actually a recombination between exon 6 from the B101 allele and exon 7 from the O01 allele (6). If figure 2 is observed it can be seen that the upstream portion of A101 is similar to B101, whereas the downstream portion is similar to O016, providing proof of recombination. Interestingly, the O01 allele also seems to be a recombination of B101 and O026. This can be seen in figure 3 (6).

Significance
At present, modern humans have three allele types, while several primates have fewer ones. Chimpanzees, for example, have only A and O alleles while all gorillas have only the B allele. Thus, it is possible that human ancestors only had B and O alleles at some point in time. As mentioned before, evidence seem to specify that the A type allele was resurrected by recombination around 260, 000 years ago (6).

Therefore it is possible that Homo erectus had only the B and O allele. Surprisingly, O01 allele types were also found in two Neandertal individuals. Thus, the emergence of the A allele must have occurred before anatomically modern humans were present (6).