Molecular – genetic variance of RH blood group system within human population of Bosnia and Herzegovina

Th ere are two major theories for inheritance of Rh blood group system: Fisher – Race theory and Wiener theory. Aim of this study was identifying frequency of RHDCE alleles in Bosnian – Herzegovinian population and introduction of this method in screening for Rh phenotype in B&H since this type of analysis was not used for blood typing in B&H before. Rh blood group was typed by Polymerase Chain Reaction, using the protocols and primers previously established by other authors, then carrying out electrophoresis in - agarose gel. Percentage of Rh positive individuals in our sample is ., while the percentage of Rh negative individuals is .. Inter-rater agreement statistic showed perfect agreement (K=) between the results of Rh blood system detection based on serological and molecular-genetics methods. In conclusion, molecular – genetic methods are suitable for prenatal genotyping and specifi c cases while standard serological method is suitable for high-throughput of samples. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Th e Rh blood group system (commonly known as Rh factor) is considered to be the most important blood group system in the world.Th is blood group system is involved in several life-threatening diseases like hemolytic disease of the newborns and autoimmune hemolytic anemia [].Rh antigens have major clinical impact in transfusiology, since they can produce hemolytic transfusion reactions.Molecular testing has shown that the Rh-locus of human genome, which is located on the short arm of chromosome  (p.-p),consists of two closely related and structurally homologous genes, RHD and RHCE.RHD and RHCE genes consist of  exons and they stretch along kb of DNA sequence [].So far, the only observed diff erence between these two genes is a partial deletion of RHD gene in the fourth exon of this gene [].In cases of deletion of RHD gene in individual genotypes, there is an Rh (D-) phenotype on erythrocytes, which is diff erent if there is no deletion on RHD gene, since in that case there is an Rh (D+) phenotype.In previously described studies [, ] it is reported that the diff erence between the Rh E and e alleles encoding these polypeptides is in only one nucleotide at position  of RHCE gene [].Comparing the Rh C and c alleles there are six nucleotides diff erences, four of which result in amino acid substitutions.These substitutions are  G>C,  C>A,  A>G and  C>T [, ].Th ere are two major theories that try to explain inheritance of Rh blood group system: Fisher -Race theory (gene complex theory) and Wiener theory (multiple -allele theory).Fisher and Race used letter symbols, which make CDE nomenclature, for antigen and gene marking.In this theory, there are  antigens in Rh blood group (C, D, E, c, d, e) inherited through  gene alleles, located on  gene loci.Gene alleles (C/c, E/e, D/d) are labeled with same letters as antigens.Every gene complex or haplotype is inherited from each parent.With simple serological blood tests, it is possible to determine hetero-or homozygosity of Rh blood group, regarding antigens C, c, E and e. Serological test for antigen D will show only if erythrocytes contains antigen D or not.On the other hand, molecular-genetic typing of RHD gene will identify exact genetic variance regarding heterozygosity or homozygosity.Prenatal testing for fetal RHD status was the first application of RHD DNA genotyping, and this method is now in use in many different laboratories.In Bosnia and Herzegovina, there is mostly serological method of determining RhD phenotype in use, so we tried to introduce RHD and RHCE genotyping with use of PCR-based method.

Subjects
Total of  ml of peripheral blood from  healthy volunteers of diff erent age and sex were collected.A number of these subjects () were submitted to the routine blood group determination, using the serology method for comparison with DNA genotyping.All subjects were introduced to aim of this study and signed letter of acknowledgment.Ethical aspects of use of healthy volunteers were considered and approved by Science Council of Institute for Genetic Engineering and Biotechnology, Sarajevo.DNA was isolated from  ml of whole blood, with the standard salting -out method [].After isolation, DNA concentration was determined, and DNA was diluted to the concentration of  ng/μl.

Procedures
To perform RHD specific PCR, we used previously described primer sets [], forward primer A '-AC-GATACCCAGTTTGTCT -' and reverse primer A '-TGACCCTGAGATGGCTGT-', which generate  amplicons, one control fragment of  bp and Rh positive phenotype specific fragment of  bp.PCR reaction was carried out in  μl of total volume, with , μl RED-Taq® ReadyMix™ PCR reaction mix (Sigma, Saint Louis, Missouri, USA),  pM of each primer (Biotez, Berlin, Germany) and  μg/μl of DNA in each reaction.PCR procedure consisted of  cycles of  o C for  minute,  o C for . minutes and  o C for . minutes, with initial denaturation at  o C for  minutes and fi nal extension at  o C for  minutes.PCR products were separated on  agarose gel ad visualized with ethidium bromide.For fragments sizing we used kb DNA Ladder (Fermentas, Maryland, USA).For RHCE genotyping we use also previously described primer sets, for RH E/e genotyping we used two allele specific PCR-reactions with following primers: set A (for E genotyping) ' -CCAAGTGTCAACTCTC -' and ' -TGACCCTGAGATGGCTGT -'; and set C (for e genotyping) : ' -ACAGACTACCACATGAAC -' and ' -GCTTTGGCAGGCACCAGGCCAC -' [].These primer sets generated diff erent size amplicons - bp for set A and  bp for set C. PCR reaction was carried out in  μl of total volume, with . μl REDTaq® ReadyMix™ PCR reaction mix (Sigma, Saint Louis, Missouri, USA) with addition of . mM MgCl,  pM of each primer (Biotez, Berlin, Germany

Statistical analysis
Haplotype frequencies and gene diversity [] was estimated for all observed Rh loci, since allele and genotype frequencies, PIC (Polymorphism Information Content) [] and exact p-value test for Hardy-Weinberg equilibrium departure test [] were calculated for C and E loci separately.In order to achieve these estimations POW-ERMARKER software v.. [] was implemented.Allele frequencies were calculated with FreqSim software [].Inter-rater agreement statistic (K, Kappa) with  confidence interval was calculated to estimate accordance between results based on serological and molecular-genetics methods.Total of  samples among  was included in this comparison.For this analysis MedCalc ver.. (MedCalc software, Mariakerke, Belgium) has been used.

RESULTS
Total number of subjects in this study was , of both sex and different age.Individuals were from different regions in Bosnia and Herzegovina.We amplifi ed two amplicons from each subject using PCR method, one of  bp and other from  bp. bp amplicon was reaction control fragment (exon  of RHD gene), while the  bp fragmet was specifi c RHD +/-fragment.In case of amplifi cation of this fragment, we concluded that individual was RhD positive, and in case of non-amplifi cation of this fragment, individual was RhD negative (  .Estimated gene diversity values are between . for D, . for C and . for E loci.Signifi cant deviation from Hardy-Weinberg equilibrium was observed for C and E loci (p<.).The highest polymorphism information content was detected for E locus (.).We also calculated frequency of every possible genotype ( genotypes) and the number of samples corresponding to those genotypes.Genotype frequency are shown in Table .Inter-rater agreement statistic showed perfect agreement (K=) between the results of Rh blood system detection based on serological and molecular -genetics methods.

DISCUSSION
In blood group systems diagnostics (ABO and Rh system) there is rising demand for molecular -genetic methods (genotyping) of these blood group systems, apart from serological method which is routinely used in transfusion laboratories in Bosnia and Herzegovina, as well as around the world.The question that rises from employment of different methods is: is it really necessary to use molecular -genetic methods for RH blood group typing?One of the advantages of PCR allele specifi c method is that this method does not require blood sample for determina-     tion of blood group haplotype.For example, this is of extreme importance in prenatal diagnostics for determination of fetal D antigen status, were fetal blood does not need to be collected, because PCR can be performed on DNA isolated from amniotic fl uid.Also, there is a new trend in prenatal diagnostics for screening of fetal Rh phenotype from mother's blood.Allele specifi c PCR also has major impact in transfusion medicine because of inability of determining D weak and D partial antigens with routine serological method.Th is method is used in case of Rh negative blood transfusion to Rh phenotype negative person with D weak or D partial antigen.In these cases there is present immunization of receptor and late transfusion reaction in individuals with formed anti-D antigen in blood serum.
On the other hand, serological typing is faster and cost -effective.For serological typing pearson only needs drop of blood and commercially available serums.Reaction time is measured in minutes (as opposed to days for molecular -genetic typing), but, as we already suggested, serological typing is not as sensitive as molecular -genetic typing and only material for serological typing is blood (as opposed to molecular -genetic typing where is possible to use every type of cells in humans, including fetal cells).
In conclusion, molecular -genetic methods are more expensive and more complicated than standard serological method, but they are also very precise and reliant compared to serological methods of blood group diagnostics.For highthroughput samples it is enough to use serological methods, but in case of special needs for typing (fetal typing, partial D and weak D typing, in case of other materials then blood) we suggest employment of molecular -genetic methods.
) and  μg/μl of DNA in each reaction.PCR procedure consisted of  cycles of  o C for  minute,  o C for . minutes and  o C for , minutes, with initial denaturation at  o C for  minutes and fi nal extension at  o C for  minutes.PCR products were separated on .  agarose gel and visualized with ethidium bromide.For fragments sizing we used bp DNA Ladder (Fermentas, Maryland, USA).For RH C/c genotyping multiplex PCR primer sets were used: Rh C set -' -CAGGGCCACCACCATTTGAA -' and ' -GAACATGCCACTTCACTCCAG -', Rh c set -' -TCGGCCAAGATCTGACCG -' and ' -TGAT-GACCACCTTCCCAGG -' [].Th ese sets generated PCR products of  bp for C allele and  bp for c allele.Multiplex PCR reaction was carried out in μl of total volume, with PCR Master Mix (X) (Fermentas, Maryland, USA),  pM of Rh C primer sets and  pM of Rh c primer sets, with  μg/μl of DNA in each reaction.Touch -down PCR procedure consisted of  cycles overall -initial denaturation at  o C for  minutes, then  cycles of  o C for  seconds and  o C for  minute,  cycles at  o C for  seconds,  o C for  minute and  o C for  seconds and fi nal extension at  o C for  minutes.PCR products were separated on   agarose gel and visualized with ethidium bromide.For fragments sizing we used bp DNA Ladder (Fermentas, Maryland, USA).
Figure .).For the RHC genotyping we used multiplex PCR reactions which generated two amplicons of  base pairs for C allele and  base pairs for c allele (shown in Figure ).RHE genotyping was performed in two separated PCR reactions for each allele.Size of the PCR product for allele E was  bp, and for allele e was  bp.With the RHE genotyping, agarose gel was loaded in following order: PCR mix with primer set for E allele and in next lane PCR mix with primer set for e allele from the same sample.Percentage of Rh positive individuals in our sample is ., while the percentage of Rh negative individuals is ..Regarding homozygote/heterozygote for RHCE, we observed following results: dominant homozygote CC -., heterozygote Cc -., recessive homozygote cc -.; dominant homozygote EE -., heterozygote Ee -. and recessive homozygote ee -..Allele frequencies for D, d, C, c, E and e allele and haplotype frequencies -percentages are shown in Table

FIGURE 1 .
FIGURE 1. PCR reaction result.In position 1 is 1 kb DNA ladder.RhD positive individual is in lane 1, and RhD negative individual is shown in lane 2.

TABLE 1 .
Allele and haplotype frequencies in B-H population

TABLE 2 .
RHDCE genotype frequencies in B-H population