Non-invasive comparative study of HLA genotyping between urinary and blood DNA using sequencing-based typing and...

Area of Science:

• Genetics and Molecular Biology
• Immunology
• Clinical Diagnostics

Background:

• High-resolution human leukocyte antigen (HLA) genotyping is crucial for transplantation and autoimmune disease research.
• Conventional methods rely on blood samples, which can be invasive and challenging for certain patient populations.
• Exploring noninvasive alternatives like urinary DNA is essential for improving accessibility and patient comfort.

Purpose of the Study:

• To assess the feasibility of using urinary DNA for high-resolution HLA genotyping.
• To validate the concordance of HLA genotyping results from urine DNA compared to traditional blood DNA methods.
• To establish urine as a clinically viable source for HLA profiling.

Main Methods:

• Collected matched urine and blood samples from 11 healthy volunteers.
• Optimized a column-based protocol for urinary DNA extraction and used an automated system for blood DNA.
• Performed high-resolution HLA typing (HLA-A, -B, -C, -DRB1, -DQB1) using sequencing-based typing (SBT) and third-generation sequencing (TGS).

Main Results:

• Urinary DNA concentrations were lower than blood DNA (9.74 vs. 33.13 ng/μL) but had comparable purity (OD 260/280 ratio: 1.65 vs. 1.81).
• Both SBT and TGS demonstrated 100% concordance between urine- and blood-derived HLA genotypes across five classical loci.
• TGS successfully resolved full-length HLA sequences (5'UTR-3'UTR) with high coverage (≥30×).

Conclusions:

• Urinary DNA provides blood-comparable accuracy for noninvasive HLA genotyping.
• An optimized protocol effectively addresses DNA yield and purity limitations.
• Urine is established as a clinically viable alternative for HLA profiling, particularly when blood sampling is difficult.