Background: Molecular bases of blood group systems, including Rh blood group, have been poorly studied in the Indian population so far, while specificities of Europeans, East Asians and Africans have been well known for years. In order to gain insights into the molecular bases of this population, we sought to characterize the RHD allele in D- Indian donors expressing C and/or E antigen(s). Methods:RHD gene was analyzed in 171 serologically D-, C/E+ samples by standard molecular methods such as quantitative, multiplex PCR of short fluorescent fragments (QMPSF) and direct sequencing when necessary. Results:RHD whole gene deletion at the homozygous state was found to be the most common genotype associated with D- phenotype (118/171, 69.0%). Nonfunctional, negative hybrid genes with reported molecular backgrounds were observed in approximately one-third of the samples, while only four samples carry single-nucleotide variations, including one novel nonsense (RHD(Y243X)), one novel frameshift (RHD(c.701delG)), and two missense (RHD(T148R) and RHD(T148R, T195M)) alleles. Conclusion: Overall we report for the first time the molecular bases of D antigen negativity in the D-, C/E+ Indian population, which appears to be qualitatively similar to other populations, but with a population-specific, quantitative distribution of D-- alleles.
D antigen in the Rh blood group system is of critical importance due to its involvement in hemolytic disease of the fetus and newborns and in hemolytic transfusion reactions . Anti-D immunization may occur in D- recipients. Frequency of D- varies widely with ethnic-specific distribution: typically 11-17% in Caucasians, 3-7% in Africans, and <1% in East Asians [2,3,4,5]. The most frequent cause of D- phenotype in Caucasians is the absence of functional RhD protein, which results from the whole deletion of the RHD gene [3,5]. In D- Black Africans, 66% and 15% carry the nonfunctional RHD pseudogene (RHDΨ) and RHD-CE-Ds hybrid alleles, respectively, while 18% lacked the RHD gene . Although the main RHD alleles resulting in a D- phenotype, i.e. RHDΨ and (C)ces, are most frequently cis-associated with RHCE*ce, other RHD gene variants in D- individuals, including several hybrid genes, have been shown to segregate with RHCE alleles expressing C and/or E (C/E+) antigens in Caucasian, Asian, African, and mixed populations [4,7,8,9,10,11,12,13,14,15,16,17,18,19,20].
Several studies have reported that 3-7% of Indians harbor a D- phenotype [21,22,23]. Expression of C and/or E antigen(s) has already been observed in this subset of population (personal unpublished data). As opposed to the aforementioned populations, the underlying mechanism responsible for RhD negativity has not been elucidated in the Indian population. Hence we then sought to systemically analyze and characterize the RHD locus in D-, C/E+ Indian blood donors by using standard molecular approaches in an effort to gain insights into the specific molecular basis of D antigen negativity in this population.
Material and Methods
Samples and Serological Analysis
Blood samples from 171 individuals of Indian origin, typed as D- but C+ and/or E+ by routine serological analyses, were taken for the study. Serologic typing for D, C/c, and E/e antigens was performed using the commercial anti-D, anti-C, anti-c, anti-E and anti-e antisera (MS24, MS33, MS260, MS16, MS21, MS63). All tests were performed in accordance with the manufacturer's instructions (Bio-Rad Laboratories, Diamed GmbH, Cressier, Switzerland) by tube technique. In addition, an indirect antiglobulin test and adsorption/elution tests  were performed for all samples to detect weak D and DEL phenotype.
DNA was isolated from 5 ml of blood by phenol chloroform method. All D-, C+ and/or E+ samples were first screened for the presence of all ten RHD exons by quantitative multiplex PCR of short fluorescent fragments (i.e. RHD QMPSF) for exon copy number variation assessment in conditions previously described . When hybrid genes were suspected, RHCE QMPSF was carried out . Sanger sequencing was performed as previously described when QMPSF analysis was inconclusive .
171 individuals typed as D- but C+ and/or E+ by serology (phenotype ddCcee: n = 150; ddCCee: 1; ddccEe: 20) were analyzed. Weak D and DEL phenotypes were not detected in these samples.
As both RHD whole gene deletion and hybrid RHD-RHCE genes have been commonly reported, DNA samples were first screened for the presence of all ten RHD exons by QMPSF analysis, a quantitative method that has proven useful for assessment of exon copy number variation (fig. 1) . Total deletion of the gene was observed in 118/171 samples (69.0%), of which 99 and 19 were C+ (65.6% of C+ samples) and E+ (95.0% of E+ samples), respectively (table 1). In the other 53 samples, hybrid genes were identified and confirmed by RHCE QMPSF in 49 samples at the hemizygous state, and in one sample at the compound heterozygous state (table 1). This latter sample as well as the remaining three hemizygous samples with no variation identified by QMPSF analysis were further analyzed by Sanger sequencing. The compound heterozygous sample was identified to carry a D-CE(4-9)-D hybrid allele in trans with the novel RHD(c.701delG) allele (GenBank accession number: KY229721) (fig. 2A). In the other three D-, C+ samples, the previously reported RHD(T148R) allele (HE861895)  as well as two novel variant alleles, including the nonsense RHD(Y243X) and complex RHD(T148R, T195M) alleles (KY229722 and KY229723, respectively), were found (fig. 2B-D).
We investigated the genetic bases of D- phenotype in D-, C/E+ donors of Indian origin by standard molecular analysis of the RHD gene. Homozygous deletion of the whole RHD gene, i.e. RHD*01N.01, was found to be the most common D- allele (allele frequency = 0.842) in Indians (table 1) as typically observed in Europeans. This frequency appears to be intermediate between Caucasians and East Asians (table 2).
Apart from RHD deletion, two other mutational mechanisms causing D negativity have been identified in the present study: hybrid genes and single-point mutations (table 1; allele frequency = 0.146 and 0.012, respectively). Three different hybrid genes were deduced from QMPSF analysis (table 1). First a RHD-CE(3-9)-D hybrid allele was identified. On direct sequencing of RHD exon 2, the c.203G>A variation was not present (data not shown), suggesting that this allele is RHD*01N.03. On the basis of our data we assume that RHD-CE(3-8)-D is likely to be similar to RHD*01N.05, also referred to as RHD-CE(2-7)-D and RHD-CE(3-7)-D (RhesusBase; www.rhesusbase.info/). Indeed investigating the origin of exons 2 and 8, which share common sequences in both RHD and RHCE , may yield different results as location of PCR primers in intronic regions varies depending on the original locus, while exonic sequences are the same. To our knowledge this allele has been reported in Caucasians only to date [7,9,16]. In 6 individuals, RHD-CE(4-9)-D allele was identified (table 1: Ccee, 5; ccEe, 1). Li and colleagues  reported an apparently similar allele in a single Chinese individual, officially registered as RHD*DEL44, expressing a DEL phenotype. However in the present study, adsorption-elution studies showed absence of DEL phenotype in these individuals. This observation is somehow intriguing. From a strict molecular point of view, as RHD*01N.07, a RHD-CE(4-7)-D hybrid allele lacking four RHD-specific exons critical for the expression of the D antigen, results in the expression of a D- phenotype ; same phenotype may be expected from RHD-CE(4-9)-D, which even lacks a higher number of RHD-specific exons. Additional functional analyses as well as investigation of antigen density will help to conclude definitely.
Contrary to hybrid genes found in almost one-third of samples (i.e. 31.0%), single-nucleotide variations are rare in D-, C/E+ Indians, as only four individuals each harboring a different allele were found. While genotype-phenotype correlation is obvious in individuals carrying either a nonsense or frameshift variation, the molecular mechanism(s) driving D negativity in the other two missense alleles is (are) still to be determined. Interestingly, these latter alleles share the c.443C>G variation, which results in the replacement of a threonine amino acid by an arginine residue at position 148 (p.Thr148Arg) of the RhD protein. It is actually the second time this amino acid substitution is reported to be associated to D negativity , confirming that this missense variation completely impairs the membrane expression of the RhD protein.
Overall we report the molecular bases of D antigen negativity in the D-, C/E+ Indian population, which appears to be qualitatively similar to other populations, but with a specific quantitative distribution of D-- alleles. On the basis of our data it may be valuable to design a simple molecular test to analyze presence of RHD gene in D- individuals.
This work was supported by the Indo French Center for the Promotion of Advanced Research (IFCPAR/CEFIPRA, project n°5203-1), the National Institute of Immunohematology (NIIH, ICMR), the Etablissement français du sang (EFS) Bretagne and the Institut National de la Santé et de la Recherche Médicale (Inserm), France.
The authors declare that they have no conflict of interest.