part 2: DNA polymorphisms

What percent of the human genome does not code for genes?

98%

Polymorphism

The presence of two or more inherited variant forms of a specific DNA sequence that can occur among different individuals or populations

Do polymorphisms generally affect phenotype?

No, they are generally considered inherited DNA sequence features that do not adversely affect phenotype

Why are polymorphisms clinically relevant?

Variations may underlie differences in health; they can be used as landmarks/markers in the genome; the more often one is present in people with a disease phenotype, the more likely an affected gene is close to it

Useful polymorphism

A genetic variation occurring in >1% of the population

RFLP: structure and detection method

Restriction fragment length polymorphism: one or more nucleotide changes that affect the size of restriction enzyme products; detected by Southern blot

VNTR: structure and detection method

Variable-number tandem repeat: repeats of 10–50 base sequences in tandem; detected by Southern blot or PCR

STR: structure and detection method

Short tandem repeat: repeats of 1–10 base sequences in tandem; detected by PCR

SNP: structure and detection method

Single-nucleotide polymorphism/variant: alteration of a single nucleotide; detected by sequencing, sequence-specific (primer) PCR, or array technology

RFLP typing

Detecting differences in the sizes and number of fragments from restriction enzyme digestion of DNA; a change in nucleotide sequence can alter the restriction site

Type II restriction enzymes

Recognize palindromic sequences and cut within the sequence

BamH1 recognition site

GGATCC

How can sequence changes alter a BamH1 RFLP pattern?

A single base change can destroy the site (larger fragment); duplications, deletions, and insertions also change fragment sizes

Steps of RFLP typing

Create a restriction map, compare number and sizes of fragments; polymorphisms are detected by observing fragment numbers and fragment sizes

RFLP: if both Bgl II sites are present (+/+)

3 fragments: A, B, C

RFLP: if site 1 present, site 2 absent (+/-)

2 fragments: A and B+C (B+C is the large fragment)

RFLP: if both Bgl II sites absent (-/-)

1 fragment: A+B+C

RFLP with Southern blot: why only some fragments are seen

A probe (e.g. binding region B) only visualizes fragments containing the probe sequence (B, B+C, A+B or A+B+C)

RFLP use: parentage testing

Offspring inherit a combination of parental polymorphisms; band patterns represent RFLPs inherited from each parent

How many loci are typically needed for comprehensive RFLP parentage analysis?

8 loci

RFLP use: linkage analysis

Since RFLPs are inherited via Mendelian mechanisms, they serve as markers to locate other genes; a disease's genetic component is confirmed by close linkage to a known RFLP marker (requires rigorous statistical testing)

Is the linked RFLP the gene causing the disease?

Not necessarily; they are closely linked and inherited together

Examples of RFLP linkage analysis

BRCA1 mapping (17q21); cystic fibrosis near CFTR gene on chromosome 7; Fragile X syndrome near FRAXA gene

STR typing is a powerful tool used in…

Forensics

How many core STR loci are used to identify individuals in the US?

13 core STR loci (CODIS, the STR database of the FBI)

STR allele

The number of repeats within an STR

D7S820 example

STR on chromosome 7 with GATA repeat units (12 different alleles)

STR loci named D#S### are usually located in…

Introns (noncoding)

How were STRs historically detected?

By RFLP mapping and Southern blotting

STR typing by PCR

Primers complementary to sequences flanking the STR are used; after PCR, the number of repeats is determined by resolution and comparison to a molecular standard (allelic ladder)

STR TH01 example

TCAT repeat linked to the human tyrosine hydroxylase gene on chromosome 11p15.5; allele 1 = 187 bp (7 repeats), allele 2 = 191 bp (8 repeats)

AMEL locus

Amelogenin locus, used to identify male individuals

AMEL Y vs X allele size

The Y-allele is 6 bp larger than the X-allele

AMEL results for males vs females

Males (XY): two bands/peaks; females (XX): one band/peak

Microvariant allele

Allele containing a partial repeat unit; written as number of complete repeats, a decimal point, then the number of bases in the partial repeat

e.g 9.3 locus, indicates 9 full TCAT repeats and one 3bp repeat (TCA)

• 3.2 allele - microvariant

9.3 allele

9 full TCAT repeats plus one 3 bp partial repeat (TCA)

How is a microvariant seen on electrophoresis?

As a second band/peak near the full-length allele (e.g. 15 and 15.2)

Heterozygous vs homozygous on STR results

Heterozygous shows two peaks/bands (two different alleles); homozygous shows one band (same allele from both parents)

How are STR alleles resolved?

By capillary electrophoresis

Genetic concordance

All alleles from two sources are the same; indicates (does not confirm) inclusion of a single individual as donor of both genotypes (could be identical twins)

Exclusion

Two samples are considered different if at least one locus differs

Match probability

The more loci tested, the higher the probability the locus genotype positively identifies an individual

Product rule

The frequency of a set of alleles/genotype in a population is the product of the frequency of each allele separately: OF = F1 x F2 x F3 x … Fn

Overall frequency (OF) example: Penta D (1 in 10) and D7S829 (1 in 50)

OF = 1/10 x 1/50 = 1/500, one in every 500 individuals

Important consideration when matching profiles to a database

Whether the database is representative of the population or subpopulation

NDIS

National DNA Index System; contains ~13 million offender profiles (2018); fed by CODIS

CODIS

Combined DNA Index System; national (NDIS), state (SDIS), and local (LDIS) levels

H0 in paternity testing

The test subject is NOT the father of the tested individual

H1 in paternity testing

The test subject IS the father of the tested individual

How many alleles does a child share with the father at each locus?

One allele of every locus

Paternity index (PI)

Likelihood of paternity calculated for each locus where father and child share an allele; how many times more likely the child's allele came from the alleged father than another man

PI and allele frequency

An allele that occurs frequently in the population has a low PI; a rare allele has a high PI

PI of 5.719 for D16S539 means…

The child is 5.719 times more likely to have inherited the 9 allele from the alleged father than from a random man

Combined paternity index (CPI)

The product of the PI of each locus tested

CPI example calculation

CPI = 5.719 x 8.932 x 15.41 x 10.22 = 8,044.931 (odds of not being the father and sharing alleles by chance ≈ 1 in 8,045)

Prior odds in paternity testing

0.50 (50/50 chance the test subject is the father)

Probability of paternity formula

(CPI x prior odds) / [(CPI x prior odds) + (1 − prior odds)] = (CPI x 0.5) / [(CPI x 0.5) + 0.5]

Probability of paternity for CPI = 8,044.931

0.99988, or 99.988% probability of paternity

How often does human DNA sequence differ between individuals?

Every 1,000 to 1,500 bases (mostly SNPs, mostly in noncoding regions); NGS shows SNPs are more frequent, every ~300 bp

How many SNPs does each individual have?

~11 million (genome is 3 billion bp)

What percent of SNPs have no biological effect?

99%; ~6,000 SNPs are within genes and associated with disease

Type I SNP

Coding region, nonconservative alteration

Type II SNP

Coding region, conservative alteration

Type III SNP

Coding region, silent alteration

Type IV SNP

Noncoding 5' UTR

Type V SNP

Noncoding 3' UTR

Type VI SNP

Noncoding, other

Why do Type IV–VI SNPs cause no phenotypic change?

They are in noncoding regions

Clinical use of polymorphisms: bone marrow engraftment

Compare donor (D) and recipient (R) STR/SNP patterns at informative loci to monitor the transplant

Bone marrow engraftment: graft failure

Only recipient alleles present (no donor loci)

Bone marrow engraftment: mixed chimerism

Both donor and recipient alleles present

Bone marrow engraftment: full chimerism (goal)

Only donor alleles present; full complete transplant

Polymorphisms, full chimerism

SNPs can be used to identify a full transplant

Polymorphisms, overall frequency example

1/500 is the odds that a person has both alleles