Related Experiment Video
Updated: May 26, 2026
Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
Published on: August 23, 2024
Identification of breakpoint regions and single nucleotide variations of RHD hybrid alleles by long-read sequencing
Ti-Cheng Chang1, Jing Yu2, Bensheng Ju3
1Center for Applied Bioinformatics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
Insights
New genetic markers, specifically three intronic single nucleotide variations (SNVs), accurately identify the RHD*DIIIa-CEVS(4-7)-D hybrid allele. This aids in preventing C alloimmunization by ensuring correct blood type matching for at-risk individuals.
Area of Science:
- Genetics
- Immunology
- Molecular Biology
Background:
- Hybrid RHD alleles RHD*DIIIa-CEVS(4-7)-D and RHD*D-CEVS(4-7)-D encode a partial C antigen, increasing alloimmunization risk.
- Accurate detection of these hybrid alleles is challenging, complicating the provision of C-negative blood to at-risk individuals.
Purpose of the Study:
- To identify breakpoint regions and intronic single nucleotide variations (SNVs) of RHD hybrid alleles.
- To establish reliable genetic markers for accurate identification of RHD*DIIIa-CEVS(4-7)-D.
Main Methods:
- Pacific Bioscience (PacBio) long-read sequencing was used to analyze RHD hybrid alleles in patients.
- Single nucleotide variations (SNVs) associated with RHD*DIIIa-CEVS(4-7)-D were evaluated in a cohort of 910 sickle cell disease patients.
Main Results:
- Identified 5' and 3' breakpoint regions for RHD*DIIIa-CEVS(4-7)-D and RHD*D-CEVS(4-7)-D.
- Discovered three intronic SNVs (chr1:25293603 G>A, chr1:25293891 A>G, chr1:25310934 T>C) for RHD*DIIIa-CEVS(4-7)-D.
- These SNVs demonstrated high sensitivity (94.83%-96.55%) and specificity (99.88%) in detecting RHD*DIIIa-CEVS(4-7)-D.
Conclusions:
- Three intronic SNVs serve as accurate markers for identifying the RHD*DIIIa-CEVS(4-7)-D allele.
- These markers facilitate improved blood transfusion strategies for individuals at risk of C alloimmunization.
Background And Objectives:
RHD*03N.01 (RHD*DIIIa-CEVS(4-7)-D) and RHD*01N.06(RHD*D-CEVS(4-7)-D) are hybrid alleles that encode a partial C antigen, resulting in increased risk of C alloimmunization in individuals without a conventional C antigen. As detection of the hybrid alleles remains challenging, new markers can aid accurate identification, ensuring provision of C-negative blood to individuals at risk.
Materials And Methods:
To identify breakpoint regions and distinct intronic single nucleotide variations (SNVs) of the hybrid alleles, Pacific Bioscience (PacBio) long-read sequencing was performed in two patients with RHD*DIIIa-CEVS(4-7)-D and one patient with RHD*D-CEVS(4-7)-D; two patients without hybrid alleles served as controls. Association of the identified SNVs with RHD*DIIIa-CEVS(4-7)-D was subsequently evaluated in 910 patients with sickle cell disease.
Results:
The 5' breakpoint region of RHD*DIIIa-CEVS(4-7)-D was found in intron 3 at chr1:25293827-25293837 (RHD c.486+3036-3046) and chr1:25293907-25293980 (RHD c. 486+3116-3189) for the two patients. The 5' breakpoint region of RHD*D-CEVS(4-7)-D was at chr1:25294633-25295072 (RHD c.486+3842-4281). Both hybrid alleles had identical 3' breakpoint region in intron 7 at chr1:25310252-25310377 (RHD c.1073+3523-3648). Three intronic SNVs were found in RHD*DIIIa-CEVS(4-7)-D: chr1:25293603 (RHD c.486+2812) G>A, chr1:25293891 (RHD c.486+3100) A>G in intron 3; chr1:25310934 (RHD c.1073+4205) T>C in intron 7. In 910 patients, the SNVs demonstrated high sensitivities (94.83%, 94.83%, 96.55%) and specificities (99.88%, 99.88%, 99.88%) in detecting RHD*DIIIa-CEVS(4-7)-D. One SNV, chr1:25294894 (RHD c.486+4103) T>C in intron 3, was identified in RHD*D-CEVS(4-7)-D, but not confirmed due to lack of additional carriers.
Conclusion:
Three intronic SNVs can be used to accurately identify RHD*DIIIa-CEVS(4-7)-D.
