The (Rh) blood group system (004) is the second most important blood group
after ABO. Two closely related genes, RHD and RHCE, encode over 50 different
antigens in the Rh system. Recombination, deletion, and point mutations in these
genes generate Rh allelic diversity which makes the Rh blood group the most
polymorphic blood group system. In the past decade, different DNA
microarray-based tests were introduced that enable genotyping of variant
blood group specific single nucleotide polymorphisms (SNPs). However, these
assays have limitations because they target certain nucleotides or DNA
regions through PCR, while novel ones remain unknown. Complete DNA sequencing
could be the most relevant technique to thoroughly study blood group variations.
Complete blood group genotyping could decrease Rh mistyping and eventually
minimize the adverse reactions following blood transfusion especially for
blood transfusion dependent patients.
research aims to use Next Generation Sequencing (NGS) to sequence the RHD
gene to detect RHD variants present in the population, which will help expand
the knowledge about the underlying molecular mechanism of these variants.
This research also focuses on investigating intronic SNPs that could be
linked to a specific haplotype that might be used in the future to predict Rh
Genomic DNA samples from blood donors of different phenotypes including 6
R1R1, 6 R2R2, 7 R1R2, 6 R1r, 6 R2r, and 6 R0r were sequenced using the Ion
Personal Genome MachineTM (PGMTM). All samples were tested for RHD zygosity
using digital PCR. The RHD gene was amplified in 6 overlapping amplicons
using RHD-specific primers. 200-base pair read sequencing libraries were
prepared and then sequenced on the Ion PGMTM using a 316 chip. Data was then mapped
to the hg38 human genome reference sequence and analyzed using the CLC
one R2R2 sample, one exon 9 SNP 25321889 G>C was detected resulting in the
amino acid change Gly385Ala which is linked to weak D type 2. Multiple
intronic SNPs were detected in all samples in which 15 homozygous SNPs were present
in all 37 samples, these may represent SNP variants of the DAU*0 allele which
the hg38 reference sequence encodes. Another 19 SNPs were present in all R2R2
and R2r samples as homozygous SNPs and in R1R2 samples as heterozygous SNPs.
14 intronic SNPs were present in all R1R1, R1r and R0r as homozygous SNPs and
heterozygous SNPs in all R1R2 samples. 16 heterozygous intronic SNPs were
only present in the R2R2 weak D type 2 sample. Intronic SNPs are suspected to
be linked to a specific haplotype, which could be used in the future to
establish an assay to genotype Rh antigens without the need to fully sequence
the Rh genes.
In this research, 37 samples were sequenced on the Ion PGMTM to study RHD mutations
and assess RHD variations present in the population. Further samples are
currently being sequenced using identical techniques. Intronic
used to determine their relation to specific haplotypes. They may represent
novel diagnostic approaches to investigate known and novel variants of RHD
and RHCE. The sequencing of multiple hemizygous samples results in the determination
of reference RHD genes, which we will describe.