Jacqueline Makowski, Alexis Shawver, Connor Anderson, Alexandra Fulton, Megan Sheldon and Victoria Soewarna

Advisor: Dr. Harry Kestler

The Gene Frequency of the CCR5 Delta 32 Allele from a Population in Northeastern

Ohio

HIV Entry

Deletion of 32 base pairs in the CCR5 sequence

Stemmed from Eastern Europe during the time of the Black Plague

Black Plague lasted over 500 years

This gave the human body a chance to mutate and adapt to the plague

Most commonly found in Caucasians

Has not been studied in other races

Delta 32

Procedure

1. Map Primers for CCR5 gene

2. Determine what primers would visually indicate Delta 32

3. Develop protocol for DNA sample collection

4. Test subject approval ( & College Internal Review Board approval)

5. Presenting study to test subject

6. Collect samples (labeled randomly)

7. Extract DNA, PCR, Run a gel

• Analyze and Record Data

8. Repeat process with improvements

• Fix mistakes (contamination, etc.)

9. Continue to test and retest samples 10. Analyze results

CCr5 Sequence map

CCR5 Sequence Around Delta32 MutationPrimers F7 & B8 will generate a 140 base pair PCR product for wild-

type and a 108 base pair product Δ32 allele. PCR conditions: (5 minutes at 95° (once) 1 minute at 95° (thirty times) 1 minute at 60°2 minutes at 72°10 minutes at 72°,then, stops at 4°)

using Pure Taq Ready to go PCR beads.

Amino acids come in groups of three called codons

GCA ATC GTA

32 is not divisible by three

Everything after the 32 base pair deletion mutation consequently changes

For example:

THE CAT RAN

Remove the “C” in “CAT”

THE ATR AN

The effects of Δ32

Protocol

1. Participants were given a sterile swab

2. Inside of cheek swabbed for one minute

3. Swab placed in a tube containing a lysing agent to extract

4. Tubes were randomly labeled

5. PCR was performed on the samples

6. PCR samples were analyzed by agarose gel electrophoresis

Agarose gel

+/

Δ 3

2

+/

Δ 3

2

+/

Δ 3

2

Results

• 45 out of the 50 samples are homozygous wild-type• Normal progressor

• 5 out of the 50 samples are heterozygous for Δ32• Long term non-progressor

• 0 out of the 50 samples are homozygous for Δ32• Less likely to contract HIV

Conclusion

• Five out of fifty people were found to be heterozygous for the delta 32 deletion mutation

• Five alleles out of one hundred alleles or a gene frequency of 5% was found within the Early College Population

Future Plans

• Continue testing for the Δ32 deletion mutation at LCCC

• Clone the Δ32 mutation into a plasmid vector to transform hematopoietic stem cells

• This could be used for gene therapy to potentially cause all new blood cells to be Δ32.