Linkage Disequilibrium between ABO and Rh Loci in Barak Valley Populations vis-à-vis a Few Exotic Populations

From a study of 28 human populations, linkage disequilibrium (LD) between ABO and Rh blood group loci in human genome were estimated using two different statistic of population genetics viz. D and Q . In general, the haplotypes iRh+ and iRh - were, on average, 2 to 3-fold and 1.1 to 1.8-fold more frequent respectively than the corresponding expected haplotype frequency in the populations under study. Expected haplotype frequencies were calculated for each population assuming equal frequencies of alleles at ABO and Rh loci. The observed frequency of I A Rh+ haplotype was more or less close to the expected frequency (0.165). The haplotypes I A Rh-, I B Rh+ and I B Rh- were less frequent than expected in the populations. The D value for haplotype iRh+ was positive for all the populations suggesting preponderance of this haplotype and hence non-random association between ABO and Rh loci. Q is a single measure of LD and suitable for estimation of LD between loci having multiple alleles. Q value ranged from 2.18 (Finland) to 8.07 (Hongkong). High LD, indicated by high Q , between ABO and Rh loci was observed in the populations of Hongkong, BV Muslims, Saudi Arabia, BV Hindus, India and Iceland. But the populations of Finland, Austria, Estonia, Poland and Israel showed low LD revealed by low Q between ABO and Rh loci.


Introduction
The study of linkage disequilibrium (LD) between two or more loci in any species is a candidate of contemporary research in population genetics. LD is the non-random association of alleles at two or more loci, not necessarily on the same chromosome. It is not the same as linkage, which depicts the association of two or more loci on a chromosome with limited recombination between them. Smaller the distance between two loci on a chromosome, greater is the linkage. The phenomenon of linkage actually interferes with Mendel's law of independent segregation.
On the other hand, LD describes a situation in which some combinations of alleles or genetic markers occur more or less frequently in a population than would be expected from a random formation of haplotypes from alleles based on their frequencies. LD is also called gametic disequilibrium. Haplotype is a combination of alleles at multiple linked loci that are transmitted together. Nonrandom associations between polymorphisms at different loci are measured by the degree of linkage disequilibrium. Numerically, it is the difference between observed and expected (assuming random distributions) allelic frequencies.
The term LD is one of the most unfortunate terms that do not reveal its meaning. The term dates back to Lewontin and Kojima (1960). Detecting LD does not ensure either linkage or lack of disequilibrium. The term LD is somewhat misleading because the loci involved may be unlinked i.e. on different chromosomes and still be in LD. The level of LD between two or more loci located on same or different chromosomes is influenced by several factors viz. linkage, selection, rate of recombination, rate of mutation, genetic drift, non-random mating, age and sex, and population structure. Linkage disequilibrium can occur for a number of reasons namely random drift in allele frequencies, natural selection for or against a particular combination of alleles, non-random mating, mixtures of subpopulations with different allele frequencies and mutation (Thomas, 2004). It tends to decay over time at a rate that depends on the recombination rate. In fact, it is possible that there might be a population association (LD) between alleles at loci that are not linked -possibly not even on the same chromosome-as a result of admixture or chance.
ABO and Rh blood group systems in humans are two important genetic markers and routinely analyzed prior to blood transfusion and medical treatment. ABO blood group system is governed by a single gene with three alleles (I A , I B and i), of which I A and I B alleles are codominant but both of them are dominant over the recessive allele 'i' in intra-allelic interaction in diploid condition. The gene for ABO blood group is located on chromosome 9 of human genome (Povey et al., 1976).
Rh blood group types were discovered in 1940 by Karl Landsteiner and Alexander Weiner. This blood group system may be the most complex one genetically of all blood type systems since it involves 45 different antigens on the rium between ABO and Rh blood group loci in Hindu and Muslim populations of Barak valley in comparison to a few exotic populations.

Materials and methods
The data on the phenotypic distribution of ABO and Rh blood group system of exotic populations (Tab. 1) were collected from http://en.wikipedia.org/wiki/ABOblood-group-system and were used for the estimation of allelic frequencies of ABO and Rh blood group loci. The allelic frequencies for ABO and Rh loci in Hindu and Muslim populations of Barak Valley were collected from a study of the ABO and Rh blood group distribution carried out by Chakraborty (2010).
Since ABO system is governed by multiple allelic system and Rh by biallelic system, the LD between these two loci in all the populations were estimated as per Hedrick (2000) as: where ij x is the estimate of the frequency of gamete A i B j in the population and i p and i q are the estimates of the frequencies of alleles A i and B i , respectively. The LD between these two loci for all the populations were also calculated by estimating the Q statistic proposed by Hill (1975)  here k and m are number of alleles at loci A and B, respectively and n is the number of gametes. If all Dij=0, then Q approximates a chi-square distribution with (k-1) (m-1) degrees of freedom.
Linkage disequilibrium (D) varies in size as a function of the frequencies of the constituent alleles. D has a maximum and a minimum value of 0.25 and -0.25, respectively only when all the alleles have equal frequencies i.e. 0.5 (in the case of biallelic loci). When the allelic frequencies are not equal, the D value is smaller and falls between 0.25 and -0.25.

Results and discussion
Estimates of the allelic frequency for ABO locus in different populations revealed that i allele was the most frequent one among the three alleles in all the populations (Tab. 1). The frequency of i allele was more than 3-fold and 5-fold the frequency of dominant alleles I A and I B , respectively. Highest frequency of i allele was found in the populations of Iceland (0.75) followed by Ireland (0.74) and Australia (0.70). The lowest frequency of i allele was found in Finland (0.56), Israel (0.58) and Turkey (0.58) amongst all the populations. The frequency of I A allele ranged from 0.18 (BV Muslims, Iceland, Hongkong) to 0.31 (Finland). On the other hand, the frequency of I B allele varied from the lowest value 0.06 (Portugal) to the highest value 0.22 (India).
surface of red cells that are controlled by 2 closely linked genes on chromosome 1. The Rh system was named after rhesus monkeys since these monkeys were initially used in the research to make the antiserum for typing blood samples. If the antiserum agglutinates the red cells of a blood sample, the sample is typed as Rh+ and if it doesn't, the blood sample is typed as Rh-. Despite actual genetic complexity, the inheritance of this trait can usually be predicted by a simple conceptual model in which there are two alleles (Rh+ and Rhand Rh+ is completely dominant over Rh-). Individuals who are either dominant homozygote (Rh+Rh+) or dominant heterozygote (Rh+Rh-) have Rh antigen and hence they are typed as Rh+ individuals. But recessive homozygotes (Rh-Rh-) do not have Rh antigen and hence typed as Rhindividuals.
Barak valley (BV), named after the mighty river Barak flowing through the region, is located in southern part of Assam state in North Eastern region of India and shares to its west the international border with Bangladesh. Geographically BV lies between 24°15' and 25°9' N latitude and between 92°16' and 93°15' E longitude. The zone is characterized by an undulating topography with wide plain area, low lying water logged area and hillocks. The climate of Barak Valley is sub-tropical, warm and humid with an average annual rainfall of 318 cm and 146 rainy days per annum. The mean minimum temperature ranges from 12.2°C in January to 25.4°C in August and the mean maximum temperature ranges from 24.3°C in January to 32°C in August. Two major communities of this region are Hindus (BV Hindus) and Muslims (BV Muslims) and they have coinhabited this region for quite a few centuries.
Study of linkage disequilibrium is of importance in evolutionary biology and human genetics because many factors affect it and are affected by it. LD provides information about past events and it constrains the potential response to both natural and artificial selection (Slatkin, 2008). It has been clear for many years that LD is prevalent across much of the human genome (Conrad et al., 2006;Sved, 2009). The importance of LD to evolutionary biology and human genetics was unrecognized outside population genetics till late 1970s. But interest in LD grew rapidly in the 1980s once the usefulness of LD for gene mapping became evident and large-scale surveys of closely linked loci became feasible. LD now finds important applications in mapping gene, detecting natural selection and estimating allele age. LD throughout the genome reflects the population history, the breeding system and the pattern of geographic subdivision, whereas LD in each genomic region reflects the history of natural selection, gene conversion, mutation and other forces that cause gene-frequency evolution. These factors affect LD in a genomic region and it depends on local recombination rate. No information is available on the linkage disequilibrium in the populations of Barak valley. The present study was, therefore, undertaken to estimate the linkage disequilib-For Rh locus, the frequency of Rh+ allele varied from the lowest value 0.56 (Australia, Austria) to the highest value 0.86 (BV Muslims). But the frequency of Rhallele ranged from 0.14 (BV Muslims) to 0.44 (Australia, Austria, Belgium and Spain). In general, Rh+ allele was more frequent than Rhallele in all the populations.

Haplotype frequency
Considering 3 alleles for ABO locus and 2 alleles for Rh locus, the frequency of 6 haplotypes (iRh+, iRh-, I A Rh+, I A Rh-, I B Rh+ and I B Rh-) for each population were calculated from the estimated allele frequencies (Tab. 2). In our study, the expected frequencies of 6 haplotypes for each population were calculated assuming the occurrence of all alleles in equal frequencies for ABO and Rh loci. All the populations, therefore, had the same expected frequencies for 6 haplotypes i.e. iRh+(0.170), iRh-(0.170), I A Rh+(0.165), I A Rh-(0.165), I B Rh+(0.165) and I B Rh-(0.165). The comparison of observed and expected haplotype frequencies for all the populations , in general, revealed that the frequencies of observed iRh+ and iRh-haplotypes were, on average, 2 to 3-fold and 1.1 to 1.8-fold higher than corresponding expected haplotype frequency (i.e. 0.170). It suggested that the alleles of ABO and Rh loci, although located on separate chromosomes, did not show random association in gametes resulting in linkage disequilibrium between two loci during evolution.
The observed frequency of haplotype I A Rh+ was more or less close to expected frequency (0.165) whereas the observed frequency of haplotype I A Rhwas lower than the expected haplotype frequency (0.165). On the other hand, the observed frequency of haplotypes I B Rh+ and I B Rhwere much lower than the expected frequency (0.165).

Linkage disequilibrium
Linkage disequilibrium between ABO and Rh loci was estimated for all haplotypes using the statistic D on the basis of observed and expected haplotype frequency in each population (Tab. 3). The D estimate for the haplotype iRh+ was positive for all the populations and it varied from the lowest value of 0.172 (Austria) to the highest value of 0.410 (Hongkong). D normally ranges from -0.25 to 0.25. In this study, seven populations namely Hongkong,  than the Hindus (0.498). For the haplotype iRh-, the D estimate was positive for almost all the populations except Hongkong, India, BV Hindus and BV Muslims. Negative D estimate indicated that expected haplotype frequency was more than observed frequency. For the remaining three haplotypes i.e. I A Rh-, I B Rh+ and I B Rh-, the D estimate was negative for all the populations suggesting that these haplotypes occurred fewer than expected in all the populations. This further indicated non-random association of alleles between ABO and Rh loci and hence occurrence of linkage disequilibrium between the two loci.
The statistic Q is a single, combined measure of linkage disequilibrium unlike D and it takes into account all the D values of all haplotypes in a population. In our study, the sum of all D values for each population was zero; hence calculated Q was compared with chi-square value (5.99) for 2 degrees of freedom at p=0.05 for significance. Only two populations namely Hongkong (8.07) and BV Muslims (6.53) had Q estimate higher than the table value of chi-square (5.99) indicating that the observed haplotype frequency was significantly different (p=0.05) from expected haplotype frequency in these populations.
In general, the Q estimate ranged from 2.18 (Finland) to 8.07 (Hongkong) (Tab. 3). Higher the value of Q, greater is the magnitude of linkage disequilibrium. High linkage disequilibrium indicated by high Q between ABO and Rh loci was observed in the populations of Hongkong, BV Muslims, Saudi Arabia, BV Hindus, India and Iceland. But populations like Finland, Austria, Estonia, Poland, Israel showed low linkage disequilibrium i.e. Q between ABO and Rh loci. In Barak valley, the Muslims (6.53) recorded higher linkage disequilibrium than the Hindus (5.21) between ABO and Rh loci. In the present study, the LD observed between ABO and Rh blood group genes in different populations could be due to natural selection of i and Rh+ allele resulting in higher observed frequency of the haplotype iRh+ than expected. Further study could be taken up to understand the cause behind high LD between ABO and Rh genes in human populations.
Iceland, India, Saudi Arabia, Ireland, BV Hindus and BV Muslims outranged the maximum value of 0.25. It could be due to very high frequency of the Rh+ allele in these populations. In Barak valley zone, the Muslims (0.542) showed higher frequency of observed haplotype iRh+