| Mitochondrial DNA (mtDNA) typing has found an important niche in the forensic testing of degraded samples and shed hairs. Currently, most forensic mtDNA laboratories focus on sequence information within the two hypervariable regions (HV1 and HV2) of about 600+ bases within the control region. The distribution of mtDNA types is highly skewed toward rare types, making the significance of a match for a mtDNA type previously unseen in a database quite substantial. There are also a number of common types observed in various populations. One limitation of mtDNA testing is the low power of discrimination associated with common HV1/HV2 types. For example, in the European Caucasian forensic database, there are approximately twenty common HV1/HV2 types that occur at a population frequency of 0.5% or greater, for an aggregate frequency of about twenty-one percent of the population.; We have sequenced the entire mtDNA genome (mtGenome) of 241 Caucasian individuals who match one of eighteen common HV1/HV2 types in order to identify single nucleotide polymorphisms (SNPs) in the coding region useful for additional discrimination. Focusing on SNPs that were shared, neutral and non-redundant, we have developed a set of eight multiplex panels containing 59 informative sites suitable for SNP typing assays. Each panel contains seven to eleven SNPs, and is specific to discriminating one or more of the common HV1/HV2 types in the Caucasian population. The discrimination provided by the multiplex panels provides maximal discrimination while preserving limited DNA extract from forensic casework. Applying all eight multiplex panels to the 241 sequences resolved the individuals into 106 haplotypes, 56 of which were unique, a nearly 6-fold improvement over the initial 18 common HV1/HV2 types.; We have also investigated evolutionary properties of the 59 discriminating SNPs by characterizing the mutation rates in the mtDNA coding region using phylogenetic trees constructed by parsimony. Most of the SNPs that discriminated among the 18 common HV1/HV2 types in Caucasians (51/58. 88%) can be classified as having relatively slow mutation rates, indicating that these sites are narrowly useful for resolving within these specific common HV1/HV2 types. The remaining SNPs in the multiplex assay could be classified as having relatively fast rates. The decision to use brute-force whole mtGenome sequencing was necessary to discover sites specific for resolving common HV1/HV2 types in Caucasians. The strategy of mtGenome sequencing for identifying discriminatory SNPs will be required to resolve common HV1/HV2 types in other forensically important groups (African Americans, Hispanics). |
