Molecular Human Identification and Genetic Polymorphisms Study Guide

Genetic Polymorphism and Molecular Markers

• Definition of DNA Polymorphism: A DNA polymorphism is a sequence difference compared to a specific reference standard. To be classified as a polymorphism, the difference must be present in at least $1\%-2\%$ of a population.
• General Characteristics:     - Polymorphisms can range in size from a single base to thousands of bases.     - They may or may not result in phenotypic effects.     - They are most often considered normal genetic occurrences rather than mutations.     - They are distributed throughout the entire genome.     - On autosomes, there is approximately one sequence difference for every $1000-1500$ nucleotides.
• Clinical and Mapping Utility:     - Polymorphisms facilitate the molecular tracking of clinically significant genes.     - If the location of a polymorphic sequence is established, it serves as a landmark (marker) for locating other genetic regions or genes.     - Each polymorphic marker exists in different versions known as alleles.
• Variable DNA Sequence Forms:     - HLA (Human Leukocyte Antigen) Typing.     - Transposable elements:         - LINEs: Long interspersed nucleotide sequences.         - SINEs: Short interspersed nucleotide sequences.

Major Types of Polymorphic DNA Sequences

• Restriction Fragment Length Polymorphisms (RFLP):     - These polymorphisms change the size of DNA fragments generated by restriction enzymes.     - Historically used in the Southern Blotting technique.
• Variable Number Tandem Repeats (VNTR):     - Also known as minisatellites.     - The tandemly repeated unit ranges from $10\,bp$ to $50\,bp$.     - The total length of the repeat region typically ranges from $500\,bp$ to $20,000\,bp$.
• Short Tandem Repeats (STR):     - Also known as microsatellites.     - The repeat unit is very small, ranging from $1\,bp$ to $10\,bp$.
• Single-Nucleotide Polymorphisms (SNP):     - Differences involving a single nucleotide base.

Short Tandem Repeat (STR) Principles and Nomenclature

• Inheritance: STRs are transmitted in a Mendelian fashion, characterized by independent assortment and no linkage. One allele is inherited from each parent.
• Analytical Methods:     - Adapted for automated fluorescence analysis.     - PCR primers are designed to flank the locus (the specific site) of interest.     - Alleles are labeled with fluorescent dyes to allow for multiplexing (analyzing multiple loci simultaneously).     - STR kits are standardized to define the specific loci being tested, ensuring equal amplification and minimizing artifacts.
• Structure and Repeat Types:     - STRs are repeats of specific nucleotide sequences:         - Dinucleotide: e.g., ATATAT…         - Trinucleotide: e.g., TAGTAGTAG…         - Tetranucleotide: e.g., TAGTTAGTTAGT…         - Pentanucleotide: e.g., TAGGCTAGGCTAGGC…
• Nomenclature and Genotypying:     - Alleles are designated by the number of repeats they contain.     - Example: An allele with "TTCTTCTTCTTC" has four repeats, while "TTCTTCTTCTTCTTC" has five.     - A genotype for this locus would be recorded as $4,5$ if both alleles are present in an individual.     - Homozygosity vs. Heterozygosity:         - A homozygous individual for shorter alleles will show a single high-mobility band on a gel.         - A heterozygous individual will show two distinct bands.         - A homozygous individual for longer alleles will show a single low-mobility band.

Technical Implementation of STR Analysis

• Primer Design and Amplicons:     - Primers are designed to produce amplicons between $100\,bp$ and $400\,bp$ in length, within which the STRs are embedded.
• Allelic Ladders: Used as a reference standard to identify alleles by comparing the migration of sample peaks to peaks of known repeat numbers.
• Microvariants: Alleles that contain partial repeat units (e.g., a genotype of $7/10$ or a record of a microvariant).
• Fluorescence Profiles:     - STRs can be analyzed via gel electrophoresis or capillary electrophoresis.     - In capillary electrophoresis, results are visualised as electropherograms with peaks corresponding to specific loci: e.g., D3S1358, VWA, FGA, Penta E, D8S1179, D21S11, D18S1179, Penta D, D5S818, D13S317, and D7S820.

Gender Identification: The Amelogenin Locus

• Nature of the Marker: The amelogenin locus is not an STR.
• Gene Function: The HUMAMEL gene codes for an amelogenin-like protein.
• Chromosomal Location: The gene is found on both X and Y chromosomes.     - X locus: Located at $Xp22.1\,–\,22.3$.
• Size Differentiation:     - The X allele produces a fragment of $212\,bp$.     - The Y allele produces a fragment of $218\,bp$.
• Interpretation:     - Females ($X, X$) appear homozygous (single peak/band at $212\,bp$).     - Males ($X, Y$) appear heterozygous (two peaks/bands at $212\,bp$ and $218\,bp$).

Forensic Statistics and Population Genetics

• Matching Probability of STR Genotypes:     - The probability of a random match decreases as more loci are tested (Product Rule).     - 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 vs. Exclusion: Two samples are considered different if the genotype at even a single locus does not match.
• Hardy-Weinberg Equilibrium:     - Used to calculate the population frequency of two alleles ($p$ and $q$):     - $p^2 + 2pq + q^2 = 1.0$     - Assumptions:         - Large population size.         - Random mating.         - No immigration or emigration.         - No mutation.         - No natural selection.
• CODIS (Combined DNA Index System):     - Developed following the work of Sir Alec Jeffreys (RFLP and DNA Fingerprinting; Case of "The Blooding").     - Agencies involved: Armed Forces Institute of Pathology.     - Hierarchy: NDIS (National), SDIS (State), LDIS (Local).     - Application: Used by the Innocence Project to exonerate wrongfully convicted individuals.

Paternity Testing

• Paternity Index (PI): A likelihood ratio comparing the probability that the alleged father is the biological father versus a random man.
• Combined Paternity Index (CPI): The product of all individual PIs from the loci tested.
• Inclusion Example Data:     - Locus D16S539: Child alleles $9, 10$, Alleged Father alleles $8, 9$; Shared allele is $9$ (PI: $5.719$).     - Locus D5S818: Child alleles $7, 12$, Alleged Father alleles $10, 12$; Shared allele is $12$ (PI: $8.932$).     - Locus FESFPS: Child alleles $13, 14$, Alleged Father alleles $9, 13$; Shared allele is $13$ (PI: $15.41$).

Lineage Markers: Y-STR and Mitochondrial DNA

• Y-STRs:     - Inheritance: Paternally inherited (passed from father to son).     - Haplotype: The set of alleles on the Y chromosome is inherited as a single unit.     - Uses:         - Forensic analysis of mixed samples (e.g., male DNA in a female background).         - Lineage studies (e.g., the Hemings & Jefferson case).         - Population studies.
• Mitochondrial DNA (mtDNA) Polymorphisms:     - Inheritance: Maternally inherited.     - Structure: Circular genome of $16,569\,bp$.     - Polymorphic Regions: Sequence differences are concentrated in hypervariable regions:         - HV 1: Approximately $268\,bp$.         - HV 2: Approximately $342\,bp$.     - Variation: On average, there are $8.5$ base differences in the HV regions between unrelated individuals.     - Applications: Legal exclusion or confirmation of maternal lineage (e.g., the case of Anastasia of Russia). All maternal relatives share the same sequence.

Single Nucleotide Polymorphism (SNP) Detail

• Frequency: One SNP occurs approximately every $1250\,bp$ in human DNA.
• Detection: Sequencing, melt curve analysis, and other molecular methods.
• Biological Impact: $99\%\%$ have no biological effect; however, approximately $60,000$ are located within genes.
• Inheritance: SNPs are often inherited in blocks known as haplotypes.
• HapMap Project: The Human Haplotype Mapping Project aims to identify SNP haplotypes across the entire human genome for mapping genes, identification, and chimerism analysis.

Engraftment Monitoring Using STR

• Context: Monitoring allogeneic bone marrow transplants.     - Autologous transplant: Recipient receives their own purged cells.     - Allogeneic transplant: Recipient receives donor cells.
• Chimerism: A recipient who carries donor marrow is a chimera.
• Testing Phases:     - Pre-transplant Informative Analysis: STRs of both donor and recipient are scanned to find "informative loci" (loci where the donor alleles differ from the recipient alleles).     - Post-transplant Engraftment Analysis: Monitoring the ratio of donor to recipient DNA.
• Analysis of Results:     - Results may show Complete Chimerism (full donor DNA), Mixed Chimerism (ratio of donor and recipient), or Graft Failure.     - Quantitation: Based on peak areas from fluorescence units or densitometry scans: $A(R)$ = area under recipient-specific peaks; $A(D)$ = area under donor-specific peaks.
• Calculations (No Shared Alleles):     - $\text{\% Recipient DNA} = \frac{A(R)}{A(R) + A(D)} \times 100$     - $\text{\% Donor DNA} = \frac{A(D)}{A(R) + A(D)} \times 100$

Trinucleotide Repeat Expansion Disorders

• Mechanism: Dynamic mutations where the number of repeats increases during gametogenesis.
• Fragile X Syndrome:     - Condition: Mental retardation caused by expansion in the FMR-1 gene.     - Repeat Unit: CGG.     - Thresholds:         - Normal: $5\,–\,55$ repeats.         - Premutation (Carrier): $56\,–\,200$ repeats.         - Full Mutation (Affected): $> 200$ (up to $2000+$) repeats.     - Detection:         - Premutations can be detected by PCR.         - Full mutations require Southern Blot due to large expansion size and methylation. In females, the inactive X is cleaved by methylation-specific restriction enzymes.
• Huntington Disease:     - Condition: Neurological degenerative disorder involving the Huntingtin gene.     - Repeat Unit: CAG.     - Inheritance: $50\%\%$ chance of inheritance from an affected parent; usually manifests in the $40s\,–\,50s$.     - Thresholds:         - Normal: $10\,–\,29$ repeats ($80\,–\,170\,bp$ PCR product).         - Affected: $> 40$ repeats.     - Detection: Labeled PCR primers followed by autoradiogram of polyacrylamide gel electrophoresis.