Molecular Human Identification and Genetic Polymorphisms

Genetic Polymorphisms and Markers

• Definition of DNA Polymorphism: A DNA sequence difference compared to a reference standard that is present in at least 1\text{%}--2\text{%} of a population.     - Polymorphisms can range in size from a single base to thousands of bases.     - They may or may not result in phenotypic effects; usually, they are considered normal genetic occurrences.     - These differences facilitate the molecular tracking of genes with clinical importance.     - They are distributed throughout the genome; on autosomes, there is approximately one sequence difference for every $1000$--$1500$ nucleotides.
• Polymorphic Sequences as Landmarks: If the location of a polymorphic sequence is known, it serves as a landmark or marker for locating other genes or genetic regions.
• Alleles: Each polymorphic marker has different versions, known as alleles.
• Variable DNA Sequence Forms:     - HLA Typing: Human Leukocyte Antigen typing.     - Transposable Elements:         - LINES: Long interspersed nucleotide sequences.         - SINES: Short interspersed nucleotide sequences.
• Primary Types of Polymorphic DNA Sequences:     - RFLP: Restriction fragment length polymorphisms. These change the fragment size of DNA and are typically detected using Southern Blotting techniques.     - VNTR: Variable number tandem repeats. These units are $10$ to $50$ bp in length and are referred to as minisatellites. The total length of a VNTR region ranges from $500$ to $20,000$ bp.     - STR: Short tandem repeats. These units are $1$ to $10$ bp in length and are referred to as microsatellites.     - SNP: Single-nucleotide polymorphisms.

Short Tandem Repeats (STR) and Microsatellites

• Inheritance: STRs are transmitted in a Mendelian fashion, characterized by independent random assortment without linkage. One allele is inherited from each parent.
• Analysis Methodology: Adapted for automated fluorescence analysis.     - PCR primers flank the specific locus of interest.     - Alleles are labeled with fluorescent dyes for multiplexing (testing multiple loci simultaneously).     - Commercial STR kits provide defined loci, minimal artifacts, and equal amplification across alleles.
• STR Structure and Nomenclature:     - STRs consist of repeat nucleotide sequences:         - Dinucleotide: e.g., ATATAT…         - Trinucleotide: e.g., TAGTAGTAG…         - Tetranucleotide: e.g., TAGTTAGTTAGT…         - Pentanucleotide: e.g., TAGGCTAGGCTAGGC…     - Allele Designation: Alleles are identified by the number of repeats they contain.         - Example: A trinucleotide sequence repeated four times (TTCTTCTTCTTC) is a "$4$" allele.         - Example: The same sequence repeated five times (TTCTTCTTCTTCTTC) is a "$5$" allele.         - A heterozygous individual with both would have a genotype of $4,5$ at that locus.
• Visualizing VNTR/STR via Digestion and Blotting:     - Allele 1: Contains $3$ repeat units, resulting in a $200\,bp$ restriction product.     - Allele 2: Contains $6$ repeat units, resulting in a $230\,bp$ restriction product.     - Allele 3: Contains $9$ repeat units, resulting in a $260\,bp$ restriction product.
• Primer Design and Amplicons:     - PCR primers allow for multiplexing by targeting amplicons between $100$ to $400\,bp$.     - Allelic Ladders: Used as benchmarks to identify alleles.     - Microvariants: Represent alleles with partial repeat units (e.g., genotype $7/8$ and $7/10$).     - STR Resolution: Can be performed via Polyacrylamide Gel Electrophoresis or Capillary Electrophoresis. Capillary electrophoresis displays peaks on an electropherogram corresponding to specific loci like D3S1358, VWA, FGA, and others mapped against a molecular weight standard.

Forensic and Identity Applications

• Gender Identification (Amelogenin Locus):     - The Amelogenin locus is not an STR.     - The HUMAMEL gene codes for an amelogenin-like protein.     - It is located at $Xp22.1$--$22.3$ and on the Y chromosome.         - X allele: $212\,bp$.         - Y allele: $218\,bp$.     - Interpretation: Females (X, X) appear homozygous (one peak/band); Males (X, Y) appear heterozygous (two peaks/bands).
• Paternity Testing:     - Comparisons are made between the child, the mother, and the alleged father (AF).     - Paternity Index (PI): The likelihood ratio of paternity for a specific locus.     - Combined Paternity Index (CPI): The product of all individual PIs.     - Example Data (Inclusion):         - Locus D16S539: Child (8, 9), Alleged Father (9, 10), Shared Allele (9), $PI = 5.719$         - Locus D5S818: Child (10, 12), Alleged Father (7, 12), Shared Allele (12), $PI = 8.932$         - Locus FESFPS: Child (9, 13), Alleged Father (13, 14), Shared Allele (13), $PI = 15.41$
• CODIS (Combined DNA Index System):     - Inspired by the work of Sir Alec Jeffreys (RFLP and DNA Fingerprinting).     - Utilized by the Armed Forces Institute of Pathology.     - Hierarchy:         - LDIS: Local DNA Index System.         - SDIS: State DNA Index System.         - NDIS: National DNA Index System.     - Used by organizations like The Innocence Project to exonerate the wrongly convicted.

Population Genetics and Probability

• Hardy-Weinberg Equilibrium: Describes the population frequency of two alleles, $p$ and $q$, using the formula:     - $p^2 + 2pq + q^2 = 1.0$     - Assumptions: Large population, random mating, no immigration, no emigration, no mutation, and no natural selection.     - Allows for the approximation of true allele frequencies in a population given sufficient assessment size.
• The Product Rule: Used to calculate the matching probability of STR genotypes across multiple loci.     - 8 loci: African American ($1/274,000,000$); White American ($1/114,000,000$); Hispanic American ($1/145,000,000$).     - 9 loci: African American ($1/5.18 \times 10^9$); White American ($1/1.03 \times 10^9$); Hispanic American ($1/1.84 \times 10^9$).     - 14 loci: African American ($1/6.11 \times 10^{17}$); White American ($1/9.96 \times 10^{17}$); Hispanic American ($1/1.31 \times 10^{17}$).
• Inclusion/Exclusion criteria: A profile is considered different (excluded) if at least one locus genotype is different.

Uniparental Inheritance: Y-STR and Mitochondrial DNA

• Y-STRs:     - Follow Paternal Inheritance.     - The profile is referred to as a Haplotype.     - Uses: Forensic analysis of mixed samples (male/female), lineage studies (e.g., the Hemings & Jefferson case), and population studies.
• Mitochondrial DNA (mtDNA) Polymorphisms:     - Follow Maternal Inheritance.     - All maternal relatives share the same mitochondrial sequence.     - Mitochondrial Genome: Total size is $16,569\,bp$.     - Hypervariable Regions: HV1 ($268\,bp$) and HV2 ($342\,bp$).     - Unrelated individuals have an average of $8.5$ base differences in these regions.     - Applications: Legal exclusion of individuals or confirmation of maternal lineage (e.g., Anastasia of Russia case).

Single Nucleotide Polymorphisms (SNPs) and HapMap

• SNP Characteristics: Single-nucleotide differences between DNA sequences.
• Frequency: One SNP occurs approximately every $1250\,bp$ in the human genome.
• Detection Methods: Sequencing, melt curve analysis, and other molecular techniques.
• Biological Impact: 99\text{%} have no biological effect; approximately $60,000$ are located within genes.
• Inheritance: SNPs are inherited in organized blocks called haplotypes.
• HapMap Project: The Human Haplotype Mapping Project aims to identify SNP haplotypes throughout the human genome for mapping genes, identification, and chimerism analysis.

Clinical Monitoring: Engraftment and Chimerism Testing

• Monitoring Bone Marrow Transplants:     - Autologous Transplant: Recipient receives their own purged cells.     - Allogeneic Transplant: Recipient receives donor cells.
• Chimerism: A recipient with donor marrow is considered a chimera.
• Analysis Phases:     - Pre-transplant Informative Analysis: STRs are scanned to find "informative loci" where donor alleles differ from recipient alleles.     - Post-transplant Engraftment Analysis: Monitoring for complete chimerism, mixed chimerism, or graft failure.
• Calculations for Engraftment:     - Peak areas are measured in fluorescence units or densitometry.     - $A(R)$ = Area under recipient-specific peaks.     - $A(D)$ = Area under donor-specific peaks.     - %% Recipient DNA Formula:         - \text{% Recipient DNA} = \frac{A(R)}{A(R) + A(D)} \times 100     - %% Donor DNA Formula:         - \text{% Donor DNA} = \frac{A(D)}{A(R) + A(D)} \times 100

Clinical Molecular Disorders: Nucleotide Repeat Expansions

• Fragile X Syndrome:     - Associated with the FMR-1 gene and mental retardation.     - Expansion: CGG repeat.         - Normal: $5$--$55$ repeats.         - Carrier (Premutation): $56$--$200$ repeats.         - Full Mutation: $> 200$ repeats (often associated with methylation).     - Detection: PCR can detect premutations ($50$--$90$ repeat range), but because of the large size of full mutations, Southern Blot is required for definitive detection.
• Huntington Disease:     - Neurological degenerative disorder associated with the Huntingtin gene.     - Manifests typically in the $40\text{'s}$--$50\text{'s}$.     - 50\text{%} chance of inheritance from an affected parent.     - Expansion: CAG repeat.         - Normal: $10$--$29$ repeats ($80$--$170\,bp$ amplicon).         - Huntington Disease: $> 40$ repeats.     - Detection: Labeled PCR primers and autoradiogram of polyacrylamide gels.