Chapter 11

DNA Polymorphisms and Human Identification

Objectives
Compare and contrast different types of polymorphisms. Define restriction fragment length polymorphisms. Describe short tandem repeat structure and nomenclature. Describe gender identification using the amelogenin locus. Illustrate the use of STR for bone marrow engraftment monitoring. Define single nucleotide polymorphisms. Discuss mitochondrial DNA typing.

Polymorphism
A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 12% of a population. Polymorphisms can be single bases or thousands of bases. Polymorphisms may or may not have phenotypic effects.

Polymorphic DNA Sequences

Polymorphisms are found throughout the genome. If the location of a polymorphic sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions. Each polymorphic marker has different versions or alleles.

Types of Polymorphic DNA Sequences

RFLP: restriction fragment length polymorphisms VNTR: variable number tandem repeats (8 to >50 base pairs) STR: short tandem repeats (18 base pairs) SNP: single-nucleotide polymorphisms

Restriction Fragment Length Polymorphisms

Restriction fragment sizes are altered by changes in or between enzyme recognition sites.
GTCCAGTCTAGC GAATTC GTGGCAAAGGCT CAGGTCAGATCG CTTAAG CACCGTTTCCGA
GTCCAGTCTAGC GAA A TCCGTGGCCA AGGCT CAGGTCAGATCG CTTTAG GCACCG GTTCCGA Point mutations GTCCAGTCTAGC GA AGCGA ATTC GTGGCAAAGGCT CAGGTCAGATCG CTTCGCTTAAG CACCG TTTCCGA Insertion (duplication)

GTTCTAGC GAATTC GTGGCAAA GGCTGAATTC GTGG TCAGATCG CTTAAG CACCG TTTCCGA CTTAAG CACC
GTTCTAGC GAATTC GTGGCAAAAAA GGCTGAATTC GTGG TCAGATCG CTTAAG CACCG TTTTT T CCGA CTTAAG CACC Fragment insertion (or deletion)

Restriction Fragment Length Polymorphisms

The presence of RFLP is inferred from changes in fragment sizes.
Restriction site

+
AGATCT TCTAGA 1 A 1 + + 2 + + B 2

ATATCT TATAGA

Polymorphism
C

Size Number A,B,C 3 +/+ A, (B+C) 2 (A+B), C 2 (A+B+C) 1

+/- -/+ -/-

Gel band pattern

Restriction Fragment Length Polymorphisms

The presence of RFLP is inferred from changes in fragment sizes.
1 A B 2 C

1 + + -

2 + + -

Fragments visualized B (B+C) (A+B) (A+B+C)

Probe
+/+ +/- -/+ -/-

Genotype I ++/+II +-/-+ III ++/--

Fragments visualized B, (B+C) (A+B), (B+C) B,(A+B+C)

II III

Southern blot band patterns

Restriction Fragment Length Polymorphisms

RFLP genotypes are inherited. For each locus, one allele is inherited from each parent.
Father Locus 1 2 Mother Locus 1 2 Parents

Southern blot band patterns

Locus 1 2

Child

Parentage Testing by RFLP

Which alleged fathers genotype has the paternal alleles?
AF 1 Locus 1 2 AF2 Locus 1 2 Mother Locus 1 2

Child

Locus 1 2

Evidence Testing by RFLP

Which suspectS1 or S2was at the crime scene? (V = victim, E = crime scene evidence, M = molecular weight standard)
M S1 S2 V E M M S1 S2 V E M M S1 S2 V E M

Locus 1

Locus 2

Locus 3

Short Tandem Repeat Polymorphisms (STR)

STR are repeats of nucleotide sequences.
AAAAAA - mononucleotide ATATAT - dinucleotide TAGTAGTAG - trinucleotide TAGTTAGTTAGT - tetranucleotide TAGGCTAGGCTAGGC - pentanucleotide

Different alleles contain different numbers of repeats.

TTCTTCTTCTTC - four repeat allele TTCTTCTTCTTCTTC - five repeat allele

Short Tandem Repeat Polymorphisms

STR alleles can be analyzed by fragment size (Southern blot).
One repeat unit Restriction site

Allele 1
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCT GCTGGGCCGTGG CAAGATCG CCGGCACCG TCGATCGATCGATCGA CGACCCGGCACC tandem repeat

Allele M 1 2 M

Allele 2
GTTCTAGCGGCCGTGGCAGCTAGCTAGCT GCTGGGCCGTGG CAAGATCG CCGGCACCG TCGATCGATCGA CGACCCGGCACC

Short Tandem Repeat Polymorphisms

STR alleles can also be analyzed by amplicon size (PCR).
Allele 1 ....TCATTCATT CATT CATT CATTCATT CAT.... ....AGTAAGTAAGTAAGTAAGTAAGTAAGTA.... Allele 2 ....TCAT TCATT CATTCATT CATT CATTCATTCAT.... ....AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA....

PCR products: Allele 1 187 bp (7 repeats) Allele 2 191 bp (8 repeats)

(Genotype)
7/8

Short Tandem Repeat Polymorphisms

Allelic ladders are standards representing all alleles observed in a population.
11 repeats

(Allelic ladder)

5 repeats

Genotype: 7,9

Genotype: 6,8

Short Tandem Repeat Polymorphisms

Multiple loci are genotyped in the same reaction using multiplex PCR. Allelic ladders must not overlap in the same reaction.

Short Tandem Repeat Polymorphisms by Multiplex PCR

FGA PentaE

TPOX

D18S51

D2S11 D8S1179 THO1

vWA

D3S1358

Amelogenin Locus, HUMAMEL

The amelogenin locus is not an STR. The HUMAMEL gene codes for amelogenin-like protein. The gene is located at Xp22.122.3 and Y.
X allele = 212 bp Y allele = 218 bp Females (X, X) - homozygous Males (X, Y) - heterozygous

Analysis of Short Tandem Repeat Polymorphisms by PCR

STR genotypes are analyzed using gel or capillary gel electrophoresis.
11 repeats

11 repeats 5 repeats

5 repeats (Allelic ladder)

Genotype: 7,9

STR-PCR
STR genotypes are inherited.
Childs alleles

Mothers alleles

Fathers alleles

One allele is inherited from each parent.

Parentage Testing by STR-PCR

Which alleged fathers genotype has the paternal alleles?
Locus D3S1358 vWA FGA TH01 TPOX CSF1PO D5S818 D13S317 Child 16/17 14/18 21/24 6 10/11 11/12 11/13 9/12 Mother 16 16/18 20/21 6/9.3 10/11 12 10/11 9 1 17 14/15 24 6/9 8/11 11 13 12/13 2 17 16/17 24 6/7 8/9 11/13 9/13 11/12

Evidence Testing by STR-PCR

Which suspectS1 or S2was at the crime scene? (V = victim, E = crime scene evidence, AL = Allelic ladder)
AL S1 S2 V E M AL S1 S2 V E M AL S1 S2 V E M

Locus 1

Locus 2

Locus 3

Short Tandem Repeat Polymorphisms: Y-STR

The Y chromosome is inherited in a block without recombination. STR on the Y chromosome are inherited paternally as a haplotype. Y haplotypes are used for exclusion and paternal lineage analysis.

Chimerism Testing Using STR

Allogeneic bone marrow transplants are monitored using STR.
Autologous transplant
Recipient receives his or her own purged cells

Allogeneic transplant

Recipient receives donor cells

A recipient with donor marrow is a chimera.

Chimerism Testing Using STR

There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis
Before transplant
Donor Recipient

After transplant

Complete (Full)

Mixed

Graft failure

Chimerism Testing Using STR: Informative Analysis

STR are scanned to find informative loci (donor alleles differ from recipient alleles). Which loci are informative?
Locus: 1 2 3 4 5

Chimerism Testing Using STR: Informative Analysis

There are different degrees of informativity. With the most informative loci, recipient bands or peaks do not overlap stutter in donor bands or peaks.
Stutter is a technical artifact of the PCR reaction in which a minor product of n-1 repeat units is produced.

Examples of Informative Loci

(Type 5) [Thiede et al., Leukemia 18:248 (2004)]

Recipient

Stutter
Donor

Recipient

Donor

Examples of Noninformative Loci (Type 1)

Recipient

Donor

Chimerism Testing Using STR: Informative Analysis

Which loci are informative?

vWA

TH01

Amel

TPOX

CSF1PO

Chimerism Testing Using STR: Engraftment Analysis

Using informative loci, peak areas are determined in fluorescence units or from densitometry scans of gel bands.
A(R) + A(D)

A(D)

A(R)

A(R) = area under recipient-specific peaks A(D) = area under donor-specific peaks

Chimerism Testing Using STR: Engraftment Analysis

Formula for calculation of % recipient or % donor (no shared alleles).
% Recipient DNA = A(R) A(R) + A(D) 100

% Donor DNA =

A(D) A(R) + A(D)

100

Chimerism Testing Using STR: Engraftment Analysis

Calculate % recipient DNA in post-1 and post-2:

Use the area under these peaks to calculate percentages.

Chimerism Analysis of Cellular Subsets

Cell subsets (T cells, granulocytes, NK cells, etc.) engraft with different kinetics. Analysis of cellular subsets provides a more detailed description of the engrafting cell population. Analysis of cellular subsets also increases the sensitivity of the engraftment assay.

Chimerism Analysis of Cellular Subsets

T cells (CD3), NK cells (CD56), granulocytes, myeloid cells (CD13, CD33), myelomonocytic cells (CD14), B cells (CD19), stem cells (CD34) Methods
Flow cytometric sorting Immunomagnetic cell sorting Immunohistochemistry + XY FISH

Chimerism Analysis of Cellular Subsets

Detection of different levels of engraftment in cellular subsets is split chimerism.
R D T G

R = Recipient alleles D = Donor alleles T = T-cell subset (mostly recipient) G = Granulocyte subset (mostly donor)

Single Nucleotide Polymorphisms (SNP)

Single-nucleotide differences between DNA sequences. One SNP occurs approximately every 1,250 base pairs in human DNA. SNPs are detected by sequencing, melt curve analysis, or other methods. 99% have no biological effect; 60,000 are within genes.

SNP Detection by Sequencing

T/T

T/A

A/A

5 AGTCTG

5 AG(T/A)CTG

5 AGACTG

SNP Haplotypes
SNPs are inherited in blocks or haplotypes.
haplotype ~10,000 bp

Applications of SNP Analysis

SNPs can be used for mapping genes, human identification, chimerism analysis, and many other applications. The Human Haplotype Mapping (HapMap) Project is aimed at identifying SNP haplotypes throughout the human genome.

Mitochondrial DNA Polymorphisms

Sequence differences in the hypervariable regions (HV) of the mitochondrial genome.
HV 1 (342 bp) PH1 PH2 PL HV 2 (268 bp)

Mitochondrial genome 16, 600 bp

Mitochondrial DNA Polymorphisms

Mitochondria are maternally inherited. There are an average of 8.5 base differences in the mitochondrial HV sequences of unrelated individuals. All maternal relatives will have the same mitochondrial sequences. Mitochondrial typing can be used for legal exclusion of individuals or confirmation of maternal lineage.

Summary
Four types of polymorphisms are used for a variety of purposes in the laboratory: RFLP, VNTR, STR, and SNP. Polymorphisms are used for human identification and parentage testing. Y-STR haplotypes are paternally inherited. Polymorphisms are used to measure engraftment after allogeneic bone marrow transplants.

Summary
Single-nucleotide polymorphisms are detected by sequencing, melt curve analysis, or other methods. SNPs can be used for the same applications as other polymorphisms. Mitochondrial DNA typing is performed by sequencing the mitochondrial HV regions. Mitochondrial types are maternally inherited.