Relationships between mutations and CD4 numbers


Student 3 and Student 4

Introduction
QUESTION: For our project, we decided to determine whether or not there was a difference in the amount of variability in rapid progressors
(decreasing T cell counts/CD4) versus non-progressors (increasing T cell counts/CD4).

The data were published by Markham et al. (1992) and the sequences made available on the BEDROCK web site (www.bioquest.org/bedrock.
Case It software, integrated with MEGA4, was used to analyze the sequences.

For this we examined five subjects who had a rapid and great decrease in CD4 counts. The subjects we chose included: subjects 1, 3, 4, 10 and 15.
Each one of these subjects had counts of less than 200 meaning they were no longer considered to have HIV, they were at the end of the visits
considered to be suffering from AIDS.

The other group we examined consisted of a group of four subjects who had an increase in CD4 counts. The subjects were subject 2, 6, 12 and 13.
This group of subjects had an increase of at least 100 or ended at above 800 for their CD4 count.

Hypothesis
We believe that the individuals who decrease rapidly with CD4 counts (T cells) will have more mutations in their amino acid sequence while those
who have increased CD4 counts will have fewer mutations in their amino acid sequences and have a similar phylogenic relationship.

Sequences: Increased CD4 Counts
immgr2-1.png
immgr2-2.png
THE INFORMATION BELOW IS COLLECTED FROM THE TWO DIAGRAMS SEEN ABOVE:
The top diagram is representative of the amino acid sequence of the subjects 2, 6, 12 and 13. From each subject the first two clones were obtain
and used to create the diagram based off of the first visit. Total number of mutations when comparing the first two clones from the selected subjects
is 36 amino acids difference.
The bottom diagram is representative of the amino acid sequence of the subjects 2, 6, 12 and 13. From each subject the first two clones were obtain
and used to create the diagram based off of the last visit. Total number of mutations when comparing the first two clones from the selected subjects
is 25 amino acids difference.

Sequences: Decrease CD4 Counts
imgr2-3.png
imgr2-4.png
THIS INFORMATION IS DESCRIBING THE TWO DIAGRAMS PICTURED ABOVE:
The top diagram is representative of the amino acid sequence of the subjects 1, 3, 4, 10 and 15. From each subject the first two clones were obtain
and used to create the diagram based off of the first visit. Total number of mutations when comparing the first two clones from the selected subjects
is 45 amino acids difference.
The bottom diagram is representative of the amino acid sequence of the subjects 1, 3, 4, 10 and 15. From each subject the first two clones were
obtain and used to create the diagram based off of the last visit. Total number of mutations when comparing the first two clones from the selected
subjects is 54 amino acids difference.

Phylogenic Tree: Subjects whose CD4 Levels Increased- Visit One
immgr2-5.png
This phylogenic tree represents subjects 2, 6, 12 and 13 during their first visit. From each of these subjects the first two clone sequences were
obtained. For example this is labeled S2V1-1 for subject two, visit one, clone one.

Phylogenic Tree: Subjects whose CD4 L Increased- Last Visit
immgr2-6.png
This phylogenic tree represents subjects 2, 6, 12 and 13 during their last visit. From each of these subjects the first two clone sequences were
obtained.

Phylogenic Tree: Subjects whose CD4 Levels Decreased- First Visit
immgr2-7.png
This phylogenic tree represents subjects 1, 3, 4, 10 and 15 during their first visit. From each of these subjects the first two clone sequences were
obtained.

Phylogenic Tree: Subjects whose CD4 Levels Decreased- Last Visit

immgr2-8.png
This phylogenic tree represents subjects 1, 3, 4, 10 and 15 during their last visit. From each of these subjects the first two clone sequences were
obtained.

Results and Interpretation

For the subjects who we hypothesized would have a lower mutation rate, those with increasing CD4 counts: subjects 2, 6, 12 and 13, we found that
there was a slight quantitative difference in the number of mutations when compared to the individuals who had lower CD4 counts: subjects 1, 3, 4,
10 and 15. For those subjects we hypothesized would have a lower quantitative number of mutations, those with increased CD4 (the
non-progressors), the average number of mutations was 30.5 amino acids. The group of subjects with a decrease in CD4 (the progressors), those
who progressed from HIV to AIDS having had a drastic drop in CD4 counts, had an average mutation rate of 49.5 amino acid mutations. For those
subjects whose CD4 counts decreased we expected that the decrease was most likely due to the changes and mutations of HIV. These results were
what we expected to find when we initially looked at our data. We believe that this data may verify our hypothesis that the rapid progressors had
greater variability in their protein sequences vs. the non-progressors but further information and a larger sample size is needed for the data to be
more accurate and significant.

As hypothesized, there was a difference in the number of overall mutations in the subjects of interest; those who increased and those who
decreased in their CD4 counts. The ever changing protein sequences may have resulted in the differences in the subject’s body thus leading to the
inability to create CD4. Although this may not be significant with respect to the variance in number of mutations it does show a possible avenue to
research further such as the specific regions in the amino acid sequence which may have greater influence on CD4 counts. Since we only looked at
the number of mutations at an amino acid level these numbers could be much different. If we would have looked at the nucleotide sequence the
numbers and mutations could be much greater and thus studied as an alternative to looking at amino acid sequence mutations.