In Focus
Go for the breakpoint!
Diagnostic relevance of DNA breakpoints
The DNA sequences of humans are dynamic in nature. During the formation of egg and sperm cells, the 2 homologous chromosomes align.
Breakpoint
Similar DNA sequences from one chromosome merge with those from the other chromosome, creating a new DNA sequence. This process occurs between non-sister chromatids during prophase I of meiosis when a crossing over event creates a Holliday junction. The resolution of this junction can result in the merger of genetic material, called “recombination”, generating a new variant allele. The spot between 2 nucleotides where the 2 chromosomal DNA sequences are joined is called the “breakpoint” (Figure 1).
In most instances, the exact position of a breakpoint cannot be known because DNA sequence exchanges generally occur in long chromosomal regions that are highly similar, known as homologous DNA sequences. In these cases, the breakpoint is defined by the DNA stretch bordered by the 2 closest nucleotide variations between the 2 parental DNA sequences. Such DNA stretches are the recombination regions, also known as the “breakpoint regions”. An illustrative example is depicted for a 3-nucleotide breakpoint region (Figure 1). In clinical diagnostics, however, the breakpoint regions are more typically 10s or 100s of nucleotides long.
The exact diagnosis
Ideally, the breakpoint region or, if possible, the 2 nucleotides straddling the exact breakpoint will be determined for clinical diagnostics. If the breakpoints remain uncertain, even by just a few nucleotides, the clinical recommendation may be incorrect. The precise position is particularly important when the breakpoint is close to a splice-site junction, involves a gene regulation site, or overlaps exons. The exact diagnostic of breakpoints enables the classification and annotation of the DNA sequences associated with distinct gene variants (alleles). Eventually, their functional impact will be evaluated to enable reliable clinical recommendations.
Figure 1. Illustration of a breakpoint and a breakpoint region. The schematic figure exemplifies an exact breakpoint between 2 nucleotides (vertical bar, green) and a breakpoint region of 3 nucleotides length (box, blue and yellow blend). The recombination occurred somewhere within the breakpoint region, which is bordered and can be defined by the 2 closest nucleotide variations between the 2 parental DNA sequences.
Gene conversion
Among the 47 blood group systems currently recognized by the International Society of Blood Transfusion (ISBT), recombination events frequently occur between the 2 genes of the RH, the 2 genes of CH/RG, or the 3 genes of the MNS blood group systems. Such recombination is facilitated by the close chromosomal proximity and high degree of sequence similarity (homology) between the RHD and RHCE, the C4A and C4B, or the GYPA, GYPB and GYPE genes.
These special gene loci with adjacent homologous genes allow a process known as “gene conversion”, which has been best described for the RH gene locus.1-3 It mediates the unidirectional transfer of genetic information from one gene to the adjacent other gene on the same chromosome.4 This possibility of frequent gene conversions (a special from of recombination) may be the reason why RH and MNS are the 2 most polymorphic blood group systems with the largest set of antigens.5 While recombined DNA sequences with breakpoints in the exons are relatively easy to characterize and annotate, breakpoints within long intronic sequences pose greater challenges.
Recombined RHD allele
We recently sequenced a recombined RHD allele in a Pacific Islander individual where >60% of the RHD gene (Figure 2A, blue) was replaced by a sequence from the RHCE gene (Figure 2B, yellow).6 The exact breakpoint regions of the recombined RHD allele were determined, represented by 281 and 73 nucleotides at the 5’ and 3’ breakpoint regions, respectively (Figure 2B, red). We, however, could not determine the D antigen expression, such as D-positive, weak D, DEL or D-negative, with our sample, because its recombined RHD allele occurred in trans to a normal RHD*01 allele with the D-positive phenotype.
When the Pacific Islander’s DNA sequence (Figure 2B)6 was aligned with 2 similar structures, both published independently in 2009 (Figure 2C and 2D),7,8 all 3 structures looked identical based on their exon sequences. However, differences in their D antigen expressions suggested differences in the location of the breakpoints. We were unable to compare the nucleotide sequences of the breakpoint between the 3 hybrid alleles,6-8 because no breakpoint sequence information was available for the other 2 hybrid alleles.7,8 Despite the existence of 67 known RHD alleles composed of RHD-RHCE-RHD recombinations,9,10 the recombined RHD allele in the Pacific Islander donor is one of the few hybrid alleles involving more than 1 exon where both breakpoint regions have been exactly defined. The first had been the RHD*03N.01 allele in 2001 with breakpoint regions of 699 and 1,000 nucleotides.11
Figure 2. Breakpoint regions in 3 examples of RHD gene variants (alleles) created by distinct gene conversion events. The schematic figure illustrates the wild type RHD gene structure (a), the RHD gene structure in the Pacific Islander (b) with known breakpoint regions (red),6 and the 2 published RHD gene structures (c and d)7,8 with unknown breakpoint regions (blue and yellow blend).
Personalized medicine
The exact description of breakpoint regions will allow researchers to design assays for their targeted screening. Breakpoints within similar-looking sequences in hybrid alleles need to be distinguished, because they often differ in the phenotype that they express.
Commercial kits for red cell genotyping still have limitations in correctly predicting the antigens expressed by hybrid alleles, which are caused by recombination and gene conversions. Characterization of their breakpoints will assist clinicians in identifying the clinically relevant gene sequences and develop personalized medicine programs to improve patient care and patient safety. If you encounter a patient harboring blood group genes with breakpoints, consider sharing the sample for red cell genotyping. It’s worth the research!
References
- Wagner FF, Flegel WA. RHD gene deletion occurred in the Rhesus box. Blood 2000;95:3662-8.
- Wagner FF, Moulds JM, Flegel WA. Genetic mechanisms of Rhesus box variation. Transfusion 2005;45:338-44.
- Grootkerk-Tax MG, Maaskant-van Wijk PA, van Drunen J, van der Schoot CE. The highly variable RH locus in nonwhite persons hampers RHD zygosity determination but yields more insight into RH-related evolutionary events. Transfusion 2005;45:327-37.
- Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet 2001;2:10.
- Denomme GA. Defining the breakpoints of hybrid blood group alleles. Blood Transfus 2024;22:185-6.
- Srivastava K, Bueno MU, Flegel WA. Breakpoint regions of an RHD-CE(4-9)-D allele and a rare JK allele in a Pacific Islander individual. Blood Transfus 2023.
- Li Q, Hou L, Guo ZH, Ye LY, Yue DQ, Zhu ZY. Molecular basis of the RHD gene in blood donors with DEL phenotypes in Shanghai. Vox Sang 2009;97:139-46.
- Pereira J, Salvado R, Martins N, Ribeiro ML. RHD null alleles in the Portuguese population (abstract). Transfus Med 2009;19:25.
- Wagner FF, Flegel WA. The Rhesus Site. Transfus Med Hemother 2014;41:357-63.
- Floch A, Téletchéa S, Tournamille C, de Brevern AG, Pirenne F. A review of the literature organized into a new database: RHeference. Transfus Med Rev 2021;35:70-7.
- Flegel WA, Wagner FF. Two molecular polymorphisms to detect the (C)ce(s) type 1 haplotype. Blood Transfus 2014;12:136-7.
