Variant alleles, triallelic patterns, and point mutations observed in nuclear short tandem repeat typing of populations in Bosnia and Serbia

Aim

To present a compendium of off-ladder alleles and other genotyping irregularities relating to rare/unexpected population genetic variation, observed in a large short tandem repeat (STR) database from Bosnia and Serbia.

Methods

DNA was extracted from blood stain cards relating to reference samples from a population of 32 800 individuals from Bosnia and Serbia, and typed using Promega’s PowerPlex®16 STR kit.

Results

There were 31 distinct off-ladder alleles were observed in 10 of the 15 STR loci amplified from the PowerPlex®16 STR kit. Of these 31 alleles, 3 have not been previously reported. Furthermore, 16 instances of triallelic patterns were observed in 9 of the 15 loci. Primer binding site mismatches that affected amplification were observed in two loci, D5S818 and D8S1179.

Conclusion

Instances of deviations from manufacturer’s allelic ladders should be expected and caution taken to properly designate the correct alleles in large DNA databases. Particular care should be taken in kinship matching or paternity cases as incorrect designation of any of these deviations from allelic ladders could lead to false exclusions.Commercial kits for forensic short tandem repeat (STR) multiplexes include allelic ladders that assist the user in allele designation by co-migrating with any commonly expected allelic variants in the population (1). In the selection of loci and other steps in the development of commercial STR kits, huge amounts of effort are put into screening population genetic variation to ensure that the locus varies in a simple and easy-to-interpret manner, and that allele frequencies are known for statistical interpretation. Allelic ladders are based on variation observed in the development process, but, inevitably, when large population samples are typed, variants are encountered that were not represented in the developmental screening. This is particularly true when the populations under testing differ from those that were studied during kit development.Practitioners of forensic STR typing need to be aware of the possibility of rare variants, so the variants can be recognized and dealt with properly in casework interpretation. If designated correctly, variant alleles can sometimes greatly increase the power of discrimination in DNA comparisons (2). In this regard, it is important that the forensic science community share information on the occurrence of these variants. In some contexts, these variants may actually occur rather commonly, and public documentation of their occurrence can save individual investigators much time and effort (3).Unexpected or “anomalous” genetic variation that can complicate STR typing takes a number of forms, with a variety of consequences on the testing results. New length variants not represented on the allelic ladder can be due to insertion/deletions of full repeat units, or to “microvariants” differing due to the insertion/deletion of single bases or partial repeats (2,4-6). In some cases, larger or smaller off-ladder alleles may fall within the allelic ladder of an adjacent locus (5,6), with the potential for significant confusion. Triallelic patterns (7-9) can be due to length mutations that occur and segregate during an individual’s development, or to localized duplication of a locus, or to chromosomal trisomy. Sequence variation, rather than length variation, can also have effects, particularly in the case of sequence differences in the amplification primer binding sites. Primer mismatches can result in complete amplification failure and cause null alleles, or can lower the peak height of affected alleles (10-18). Any of these anomalies can cause problems during interpretation of results if the analysts are not familiar with these occurrences.The International Commission on Missing Persons (ICMP) was founded in 1996 to address the issue of persons missing as a result of the conflicts that occurred during the breakup of the former Yugoslavia during the 1990s. DNA profiles are obtained from reference samples collected from living relatives which are entered into a DNA reference database (currently greater than 80 000 unique profiles). Likewise, DNA profiles generated from skeletal remains recovered from grave sites are entered into a missing persons DNA database (currently containing more than 12 000 unique profiles). Both databases are screened against each other on a daily basis, resulting in possible kinship matches.We report here a new compendium of all off-ladder variants/microvariants, primer binding site mutations, and instances of triallelic patterns that have been observed in a subset of the ICMP blood sample reference database representing ~ 32 800 individuals from Bosnia and Serbia, including Kosovo.