Analysis of allelic drop-out at autosomal short tandem repeat loci in composite amplification

Abstract

Objective To investigate the characteristics and causes of allelic drop-out at the autosomal short tandem repeat (STR) loci, and to explore the confirmation strategy of suspected mutations at the STR loci alleles in paternity. Methods From January to June 2017, nine family members from 3 families of three-banded patterns at Shenzhen Blood Center were selected as subjects. Subject inclusion criteria: subjects′ initial test results of the STR loci alleles did not conform to the Mendels laws of genetics, which were suspected allelic drop-out at STR loci. GlobalFiler™ Express kits from ABI, USA were initially used to detect genotype of 21 autosomal STR loci alleles from all blood spot samples. All blood spot samples suspected for having allelic drop-out were verified with the PowerPlex® 21 kits from Promega, USA. Supplemental experiment was used human leukocyte antigen (HLA)-A/-B/-C/-DR/-DQB1 loci genotype test results with PCR-sequence-based typing (SBT) method, and then the haplotype of the subjects was deduced by the family analysis to complete the individual identification verification. This study met the requirements of World Medical Association Declaration of Helsinki revised in 2013. Informed consent was obtained from every subject. Results ① In this study, the allelic drop-out in 6 subjects occurred in 3 STR loci of CSF1PO, D1S165 and TH01, respectively, and the number of mutation steps were different. These allelic drop-out could be either paternal or maternal. ② In family 1, the initial test result of CSF1PO locus was homozygous in both mother and child. The verification test result of CSF1PO locus was heterozygous, and allelic drop 10-locus were confirmed. ③ In family 2, the initial test result of D1S165 locus was homozygous in both child and father. The verification test result of D1S165 locus was heterozygous, and allelic drop-out 16-locus was confirmed. ④ In family 3, the initial test result of TH01 locus was homozygous in both the mother and child. The verification test result of TH01 locus was heterozygous, and allelic drop-out 9-locus was confirmed. ⑤ Twelve HLA-A/-B/-C/-DRB1/-DQB1 loci haplotypes were detected in the DNA samples of 9 subjects, and the HLA locus haplotypes were detected in accordance with Mendels laws of genetics. Conclusions For the suspected STR allelic mutations in STR composite amplification, a single test has the possibility of allelic drop-out risk. Different kits could be used to verify the authenticity of allele mutations, and more reliable results could be obtained. Key words: Loss of heterozygosity; Alleles; Tandem repeat sequences; HLA antigens; Paternity