High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

Abstract

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodiumsp., Babesiasp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.

Keywords: GYPB deletion; Glycophorins; Plasmodium; invasion; malaria; red blood cell.

Figure 1.

Schematic diagram of the human reference GYP gene region on chromosome 4. The three GYP segmental duplication units (SDUs) are indicated by different colors. The GYP gene-region boundary-locations and genes are shown with respect to GRCh37. The approximate locations of the DEL1 and DEL2 deletions are shown. Intergenic region names used in the main text are shown.

Figure 2.

Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.

Figure 3.

GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.

Figure 4.

GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4. Sample designations identified from Leffler et al.

Figure 5.

Breakpoint sequences identified for GYPB DEL1 and DEL2 from Sanger sequencing. (a) The GYPB DEL1 highlighted regions correspond to 4:143914016-143914126 (GYPE-GYPB) and 4:144024254-144024364 (GYPB-GYPA) in GRCh38; XXXXXXXX; identifies the 5ʹ boundary for the GYPB DEL1 breakpoint, Y identifies the 3ʹ end, and ZZ identifies 2 further distinguishing bases 62 bases downstream. (b) The GYPB DEL2 highlighted regions correspond to 4:143870925-143871054 (5ʹ-GYPE), 4:143991718-143991848 (GYPE-GYPB) and 4:144094974-144095103 (GYPB-GYPA) in GRCh38. Bases marked with X's identify the group of distinguishing bases at the 5ʹ end of the GYPB DEL2 breakpoint, while the Y's identify the 3ʹ end of the GYPB DEL2 breakpoint. Supplementary Files 1–5 provide further detail and Sanger Sequencing pile-ups. The GYPE sequence was included to confirm that the amplicons were not amplifying this gene region.

Figure 6.

Location of sampling site in Ghana. Boarders for level 2 administrative districts are shown within Ghana with the sampling districts filled in blue. The major towns where sampling was conducted are shown and named. The offset Africa map shows the location of Ghana in red. The map was generated using R (https://www.r-project.org/) using a shape file downloaded from GADM (https://gadm.org/download_country_v3.html). Town GPS coordinates were identified from Google Maps (https://www.google.com/maps).