Chapter 23: Mitochondrial DNA Profiling

23.1: Human Mitochondrial Genome

  • Mitochondria: Are cellular organelles that serve as the energy-generating components of cells.

  \    Mitochondrion. mtDNA, mitochondrial DNA.

Genetic Contents of Mitochondrial Organelle Genomes

  • Cambridge reference sequence (CRS): The sequence was largely derived from a placental sample from an individual of European descent and also partially from HeLa cells and bovine cells.
  • It was later discovered, by resequencing the original mtDNA sample, that the CRS contains substitution errors at 10 nucleotide positions.   * The revised Cambridge reference sequence (rCRS) was published in 1999 and presented corrections to these substitution errors.
  • Control Region: also known as Displacement Loop; it contains the origin of replication for one of the mtDNA strands but does not code for any gene products.

\  Human circular mitochondrial genome.

 Pedigree of a human family showing inheritance of mtDNA. Females and males are denoted by circles and squares, respectively. Red symbols indicate individuals who inherited the same mtDNA.

Maternal Inheritance of mtDNA

  • Maternal inheritance is typically observed for the mtDNA genome, which is inherited differently from nuclear genes.
  • The inheritance of the mtDNA genome does not obey the rules of Mendelian inheritance and is thus called non-Mendelian inheritance.
  • Mitotype: Considered a a haplotype treated as a single locus.

23.2: mtDNA Polymorphic Regions

Hypervariable Regions

  • The most polymorphic region of mtDNA is located within the D-loop.
  • The three hypervariable regions in the D-loop are designated: Hypervariable Region I, Hypervariable Region II and, Hypervariable Region III
  • The most common polymorphic regions of the human mtDNA genome analyzed for forensic purposes are HV1 and HV2.

 A model of uniparental mtDNA inheritance in humans.

 Hypervariable regions of the D-loop in mtDNA (with nucleotide positions).

Heteroplasmy

  • Heteroplasmy occurs when an individual carries more than one mtDNA haplotype.
  • Sequence heteroplasmy: The presence of two different nucleotides at a single position shown as overlapping peaks in a sequence electropherogram.
  • Light heteroplasmy: Are often observed at the uninterrupted C stretches in sequencing, in which sequencing products with various lengths of polymeric cytosine residues are present.

 Electropherogram showing mtDNA sequence heteroplasmy at position 234R (A/G) as indicated by an arrow. N, unresolved sequence.

23.3: Forensic mtDNA Testing

General Considerations

  • mtDNA analysis is often used on samples derived from skeletal or decomposed remains.
  • The surface of the sample should be cleaned to remove any adhering debris or contaminants.
  • Bones and teeth are pulverized to facilitate extraction of the mtDNA Assay.
  • mtDNA-specific quantization methods using real-time PCR can also be used to directly obtain measurements of mtDNA extracted.

mtDNA Screen Assay

  • ASO Assay: It allows initial screening of mtDNA sequence polymorphisms and has the potential to reduce the number of samples required for mtDNA sequencing.
  • Linear Array™ mtDNA HV1/HV2 region sequence typing kit: It utilizes reverse ASO configuration with a panel of immobilized ASO probes that detect common polymorphic sites.

mtDNA Sequencing

  • A combination of PCR amplification and DNA sequencing techniques is utilized to reduce the time and labor needed to obtain DNA sequences from genomic DNA templates.
  • mtDNA sequencing usually consists of:   * PCR amplification;   * DNA sequencing reactions;   * Separation using electrophoresis; and   * Data collection of Sequence analysis.
  • DNA Sequencing Reactions   * Chain-Termination or Sanger Method: An oligonucleotide primer that can anneal to a single stranded DNA template is utilized.   * Cycle Sequencing: It utilizes thermal cycling to generate a single-stranded template for chain-termination sequencing reactions.
  • Electrophoresis, Sequence Analysis, and Mitotype Designations   * Reporting Format: Sequence differences relative to the rCRS are listed in data format.   * Insertions: Described by noting the position followed by a decimal point and a number.   * Deletions: These site designation is followed by letter d.   * Hetero-plasmic Sites: The IUPAC codes for base calling can be applied to hetero-plasmic sites.

\  Reverse blot assay employed in mtDNA screen.

 Linear array mtDNA assay results (top) and negative control (bottom).

\  Diagram of the Sanger sequencing products.

Interpretation of mtDNA Profiling Results

  • Interpretation guidelines are used when comparing sequencing results between evidence and reference samples.
  • Exclusion: If the sequences of questioned and known samples are different, then the samples can be excluded as originating from the same source.   * It should be taken into account that higher mutation rates are found with the mtDNA genome than are found with the nuclear genome.
  • Cannot Exclude: If the sequences are the same, the reference sample and evidence cannot be excluded as arising from the same source.   * When an mtDNA profile cannot be excluded, it is desirable to evaluate the weight of the evidence.
  • Inconclusive Result: If the questioned and known samples differ by a single nucleotide, and no heteroplasmy is present, the results are considered to be inconclusive.

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