Identification of two novel HLA-B null alleles using next generation sequencing

Abstract

Aim Typing of two patients (one of Asian and one of Hispanic descent) using SSOP and SBT resulted in the detection of two new HLA-B null alleles. Detailed analysis of the probe and sequence data showed the new null alleles to be generated by single nucleotide point mutations. To confirm these results, the DNA samples were tested by next generation sequencing (NGS). Methods DNA was extracted from whole blood using the Promega Maxwell 16 Blood DNA Purification kit and Promega Maxwell 16 IVD instrument. Low resolution HLA typing was obtained by performing DNA amplification and probe hybridization using the One Lambda LABType SSO HLA-B Locus kit. High resolution HLA typing was achieved by DNA amplification and subsequent sequencing of the amplified product using the Conexio Genomics SBT Resolver kits. Confirmation testing of the novel HLA-B null alleles was performed by amplifying the DNA samples using the Scisco Genetics NGS HLA kit. Results Results from SSOP, SBT and NGS testing confirmed the presence of two new null alleles. The first null allele from the Hispanic individual is similar to B∗35:01:01:01 with a mutation at position 295 (codon 75) in exon 2. This position appears to be a common polymorphic site for HLA Class I null alleles: A∗02:366N, A∗03:178N, A∗24:60N, B∗08:72N, B∗15:94N, B∗15:294N, B∗44:58N, B∗51:41N, C∗01:69N, C∗03:277N, C∗07:152N, C∗12:84N and C∗17:27N. The nucleotide change (GAG → TAG) results in the creation of a stop codon. The second null allele from the Asian individual is similar to B∗15:01:01:01 with a mutation at position 862 (codon 264) in exon 4. The nucleotide change (GGA → TGA) results in the creation of a stop codon. There are no documented polymorphisms at this position for any other allele. However, A∗02:43N has an insertion at codon 236 that causes a frameshift and premature stop at codon 264. Conclusions Identification of null alleles is important in determining what donors should be evaluated and selected for hematopoietic stem cell transplants. Misidentification could have an impact on graft outcome and lead to complications such as graft-versus-host disease. NGS has shown to be a valuable method for the identification of these kinds of new variants. Furthermore, the variant in exon 4 may suggest that typing outside of the HLA Class I peptide binding domains (exon 2 and 3) should be performed.