Intron 4 of the RH Gene in Han Chinese,Tibetan,and Mongol Populations

Introduction

RH is the most clinically complex and important blood group system, and it comprises two highly homologous genes (i.e., RHD and RHCE). The RHD and RHCE genes are tail-to-tail toward the end of the short arm of chromosome 1 (p34-36). Some genetic complexities are explained by their close locus proximity and unusual orientation (Flegel, 2011). A previous study of RHD and RHCE introns in primates demonstrated that the RH common ancestor gene gave rise to two genes, one differing from the other by a 654-bp deletion encompassing an Alu element in intron 4 (Apoil and Blancher, 2000). Okuda et al. (1997) demonstrated that the RHD gene intron 4 is missing about 649 bp compared with the RHCE gene, and the nondeleted sequence of intron 4 in the RHD gene is identical to the RHCE gene except for three nucleotides; therefore, the introns of RHCE and RHD genes have four sequence length polymorphisms (Okuda et al., 2000). In recent years, studies on the RHD and RHCE have mainly focused on the coding region, but the noncoding region, especially the introns, is rarely reported. The characteristics of the nucleotide insertions, substitutions, and deletions in noncoding regions, such as introns, must be investigated to elucidate the mechanisms of recombinations, mutations, polymorphisms, and evolution of RHD and RHCE genes, as well as to analyze RH genotyping. The present study aimed to investigate whether RHD and RHCE intron 4 exhibit certain differences between various ethnic groups. Furthermore, the role that intron polymorphisms play during recombination and mutation in the evolution of the RHD and RHCE genes was determined. A comparative study for RHD and RHCE intron 4 polymorphisms among Han Chinese, Tibetans, and Mongols was conducted.

Materials and Methods

Amplification of RHD and RHCE intron 4

As described by Okuda et al. (1997), the amplification strategy of RHD and RHCE intron 4 is shown in Figure 1.

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FIG. 1.

Primers Intron4F/Intron4R amplified a 1228-bp fragment of the RHCE gene and a 576-bp fragment of the RHD gene in intron 4.

To investigate intron 4 in the RHD and RHCE genes, polymerase chain reaction (PCR) amplification was performed by a set of primers: Intron4F (5′-TGTTCGCAGCCTATTTTGGG-3′) and Intron4R (5′-TGACACTTGGCCAGAACATC-3′). Primer Intron4F for a sequence located in exon 4 and primer Intron4R for a sequence located in exon 5, respectively. The final concentration of the PCR mixture consisted of 1× buffer (Mg²⁺-free), 1.5 mM MgCl₂, 320 mM dNTPs, 2 U Taq DNA polymerase (Promega Co., Ltd., Shanghai, China), 50-200 ng of genomic DNA, and 0.24 mM of both primers Intron4F and Intron4R in a total volume of 50 μL. PCR was conducted as follows: denaturation at 95°C for 5 min; 35 cycles of 40 s at 95°C, 40 s at 55°C, and 2 min at 72°C; and final cycle at 72°C for 5 min. The reaction products were electrophoresed on 1% agarose gel containing 0.5 μg/mL ethidium bromide.

Results

Detection of RHD gene intron 4

All 32 RHD-positive samples showed a 576-bp PCR product; however, the 1228-bp PCR product did not appear due to PCR inhibition (Fig. 2).

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FIG. 2.

To evaluate the results of special amplification by polymerase chain reaction, the products were electrophoresed on 1% agarose gel and stained with ethidium bromide. Intron 4 was amplified in both the RHD and RHCE genes with a size of 576 (lanes 1-6) and 1228 bp (lanes 7-10), respectively. M shows the marker.

Sequence contrast of RHD and RHCE intron 4 among Chinese and other ethnic groups

According to the report of Okuda et al. (1997), the sequence of RHCE gene intron 4 among Chinese was found to be consistent with that among Caucasians, but different from that among Japanese in five bases. However, we noted five nucleotide differences: three insertions (A at nucleotide 413, G at nucleotide 418, and A at nucleotide 497) and two substitutions (C→T at nucleotide 644, G→A at nucleotide 802) in intron 4 of the RHCE gene (Fig. 3). The sequences of Chinese PCR products were different from those of French PCR products. The size of the deletion sequence in intron 4 of the RHD gene was 652 and 654 or 651 bp in Chinese and France populations, respectively (Apoil et al., 1999; Apoil and Blancher, 2000). RHD and RHCE intron 4 of Han Chinese and Tibetan populations are shown in Figure 4.

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FIG. 3.

Three insertions (A at nucleotide 413, G at nucleotide 418, and A at nucleotide 497 (a)) b4C and two substitutions (C/T at nucleotide 644 (b), G/A at nucleotide 802 (c)) in intron 4 of the RHCE gene. Color images available online at www.liebertpub.com/gtmb

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FIG. 4.

The intron 4 sizes of the RHCE and RHD genes of a Chinese population were 1078 and 426 bp, respectively. The underlined letters indicate the 652-bp deletion of the RHD gene, in which the double-underlined fragment indicates the Alu sequence. The letters surrounded with an oblong frame demonstrate the three nucleotide insertions in Chinese, whereas the two italicized letters show the nucleotide substitutions between the Chinese and Japanese.

Discussion

Systematic studies and sporadic observations indicated that the RH is one of the most complex blood group systems. Apoil and Blancher (2000) studied RHD and RHCE evolution in primates. Their research group demonstrated that chimpanzees and gorillas possess two types of RH intron 4, one differing from the other by a 654-bp deletion, encompassing an Alu element. One type has a size similar to that of human RHD intron 4, whereas the other displays a 12-bp insertion (Apoil and Blancher, 2000). A number of chromosomal polymorphisms of RHD and RHCE genes in primates suggested that unequal crossing over most probably arose after the original duplication of the RH ancestor gene. The RHCE and RHD genes differ in their coding regions and noncoding sequence, including the length polymorphisms of intron 4 (Arce, 1993). In humans, intron 4 of the RHCE gene differs from intron 4 of the RHD gene in the presence of an approximate 650-bp fragment that encompasses an Alu element (Westhoff and Wylie, 1996; Huang, 1997; Okuda et al., 1997). However, various deletions in the RHD gene among different regions and populations exist. Sequencing analysis of RHD and RHCE intron 4 indicated that Chinese, Caucasian, Japanese, French, and British populations have similarities and differences. The data obtained clearly demonstrated that RHD and RHCE intron 4 were exactly the same among Han Chinese, Tibetan, and Mongol populations. However, despite the geographical proximity of Japan and China, similarities and differences were present. A large sample size should be used in future studies to learn more about the RHD and RHCE among populations of Chinese, Japanese, and other regions for constructing a genetic tree.

Our study observed the Han Chinese, Tibetan, and Mongol populations. Compared with the RHCE gene, the RHD gene intron 4 is missing an entire Alu sequence. The genetics and biological significance of the difference between the two genes remain unclear. On cytogenetic levels, the Alu sequence is often concentrated in the active chromosome segments of gene transcription; it may help in inserting and restructuring mutagenesis in primate genomes, and various potential mechanisms may mediate gene deletions (Deininger and Batzer, 1999). Studies on RHD and RHCE intron 4 of a variety of primates showed that the Alu sequence exists in the RHD and RHCE of the common ancestor of human, apes, Old World monkeys, and New World monkeys (Apoil and Blancher, 2000). Deininger and Batzer (1999) pointed out that the Alu sequence not only contributes to some genetic evolution but also, its range-mediated homologous recombination, may promote human genetic diseases. The Alu sequence, a molecular fossil, can provide DNA recombination and phylogenetic information of primates (Andreassen, 2004). However, the Alu sequence in RHCE gene intron 4 has real functions and its influence remains unclear. We speculated that the Alu sequence in the RHCE genes plays a certain important role in the process of RH genetic evolution (Rudiger et al., 1995), such as mediating about 650-bp deletion of the RHD gene, and it may be associated with alternative splicing of exons.

In conclusion, the numerical genetic polymorphism of RHD and RHCE genes in primates suggested that unequal crossing over arose most probably after the duplication of the ancestor RHD and RHCE genes. Various mechanisms of intergenic exchanges, such as crossing over, gene conversion, or homologous recombination between RHD and RHCE genes, have occurred in humans and other species (i.e., gorillas and chimpanzees). The major difference between the RHD and RHCE genes is intron 4. The polymorphisms of intron 4 and whether it is influenced by natural selection need further evolutionary studies.