Combined effects of Human Immunodeficiency Virus with Mycobacterium tuberculosis and Dementia

Report By:

Brittany Gottfried, Stephanie Grimes, Jacob Kim, Rachel Miro, Ashton Paris, Zain Sharif

BACKGROUND

Human Immunodeficiency virus (HIV) is an infectious virus that commonly causes Acquired Immune Deficiency Syndrome (AIDS) and causes death in 60% of hosts. The main effect of HIV comes from their attack on CD4 cells or T cells. HIV takes over CD4 cells and uses them to replicate itself, quickly multiplying to millions of virus particles.

HIV uses CD4 cells for their replication machinery -- destroying them in the process

HIV uses CD4 cells for their replication machinery — destroying them in the process

 T cells’ job is to fight off infection and foreign particles. HIV destroys so many T cells that HIV infected people are much more susceptible to many other infections and diseases acting as opportunistic pathogens.

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Infection with Mycobacterium tuberculosis (TB) is the leading cause of death in Human Immunodeficiency Virus (HIV) infected patients, yet there is still not an accurate way of diagnosing TB while involved in a co-infection with HIV. Approximately 20% of cases go undiagnosed until death. The first paper compared individuals that were infected with HIV alone to those co-infected with TB and HIV to see if they could identify a genetic signature that would allow a more accurate diagnosis of co-infected patients. HIV patients are more susceptible to TB because of their weakened immune system. Furthermore, once infected with HIV, the virus can awaken latent TB that the host was never aware of having. Another emerging problem is multi-drug resistant strains of TB (MDR-TB) that are becoming especially common in highly infected TB countries in Africa. Because of this, research for diagnostic and prognostic tools is highly needed. Infection with M. tuberculosis induces changes in the host immune system by interaction with the messenger-RNA. One paper published by the National Center for Biotechnology Information (NCBI) analyzes a 251 – gene genetic signature found in patients with HIV + TB that can accurately diagnose the co-infection 87% of the time.

Scientists at the National NeuroAIDS Tissue Consortium tested different levels of gene expression in the brain to determine different pathways that occur in neurocognitive disorders that occur in HIV. They used 24 human subjects and placed them into four groups: uninfected controls, HIV infected subjects with no neurocognitive impairment, HIV  infected subjects with neurocognitive impairment but no HIV encephalitis, and infected with neurocognitive impairment and HIV encephalitis. They obtained RNA from the neocortex, white matter, and neostriatum areas of the patient’s brains. They found that the HIV load in brain tissue with encephalitis was significantly higher than in the other groups and that about 1900 genes were regulated (including interferon response genes, antigen presentation, complement  components and CD163 antigen). With encephalitis, the frontal neocortex had strongly down regulated neuronal pathways. Expression was significantly different in all the other groups of patients, who had low HIV loads, weak brain responses, etc. These findings suggest that different pathophysiological pathways occur in different types of HIV associated neurocognitive impairment.

METHODOLOGY

Students wanted to investigate some form of cancer that had importance to the members on the team. Students originally searched NCBI Geo Datasets for information on chronic myeloid leukemia and acute myeloid leukemia. After submitting a proposal investigating the differences as compared to CD34+ cells, students determined that they could not create a gene list since there was no common variable between the two types— one investigated tissue types while the other investigated temporal effects. Students redirected their project towards HIV, but this time, students set the condition (HIV) as the control and looked for two different illnesses. After going through multiple papers, students decided to focus on comorbidity of dementia and TB with HIV, thinking there may be a common pathway between the two that caused such high mortality rates.

 

Students derived this information from NCBI, in which students selected groups A and B for either project— HIV + TB and HIV in one experiment and HIV associated Dementia (HAD) and HIV in the other experiment. After selecting groups A and B, students queried group A and B, downloaded the profile data as a text file, and then uploaded to DAVID to perform a functional analysis.

Genes identified from the first paper found on NCBI

Genes identified from the first paper found on NCBI, comparing genes from HIV patients (left) to those co-infected with HIV and TB (right)

 

Genes sets identified from paper 2 found on NCBI, comparing HIV patients to patients with both HIV and Dementia

Genes sets identified from paper 2 found on NCBI, comparing HIV patients (left) to patients with both HIV and Dementia (right)

Running a Query on the genes in NCBI to get our final gene list

Running a Query on the genes in NCBI to get our final gene list

After working with BioArray Tools unsuccessfully for hours, students went to DAVID Bioinformatics Database to yield gene lists; previously, only a list of experiment IDs would pull up from BioArray Tools. While HIV/TB derived gene lists from Illumina sequencing and HIV/Dementia derived gene lists from Affymetrix, both gene lists were converted to a DAVID format and yielded a gene list of 19 genes each.

Left: Genes identified as unique for HIV + TB co-infection Right: Genes idenitified as unique for HIV patients with Dementia

Left: Genes identified as unique for HIV + TB co-infection
Right: Genes idenitified as unique for HIV patients with Dementia

Gene list from HIV + TB patients, converted from Illumina into DAVID format

Gene list from HIV + TB patients, converted from Illumina into DAVID format

 

Gene list from HIV patients affected by dementia. Converted from affymetrix into DAVID format

Gene list from HIV patients affected by dementia. Converted from affymetrix into DAVID format

 

ANALYSIS

After looking through the gene lists, students determined that there was no gene overlap. Genes were so distinct that even class or function did not overlap in TB or dementia, leading us to believe that co-morbidity alters gene function and expression significantly. Students also derived the cluster map for each experiment from NCBI. Students determined that HIV/TB, investigated in peripheral blood mononuclear cells, have fewer branch points and more similarly acting genes. Surprisingly, HIV/TB also demonstrated more varied expression of genes as opposed to the either high or low expression of HIV/HAD. HIV/HAD with many more branch points and showed more varied gene function.

Cluster heat map of HIV + TB co-infection

Cluster heat map of HIV + TB co-infection

Cluster heat map of HIV patients with Dementia

Cluster heat map of HIV patients with Dementia

Students submitted converted list to DAVID as a gene list and created three lists, HIV/TB, HIV/Dementia, and combined. From the HIV/TB gene list, five gene records with KEGG Pathway were found.  Out of five records, both ‘Fc fragment of IgG, low affinity IIa, receptor (CD32)’ and ‘actin related protein 2/3 complex, subunit 1B, 41kDa’ were found in ‘Fc gamma R-mediated phagocytosis’ KEGG pathway. The analysis tool found gene ‘SNW domain containing 1’ in KEGG Pathway of ‘Spliceosome’ and ‘Notch signaling pathway’. Also, ‘Chemokine (C-X-C motif) receptor 3’ gene was found in KEGG Pathway of ‘Cytokine-cytokine receptor interaction’ and ‘Chemokine signaling pathway’. ‘Glutamate-ammonia ligase (glutamine synthetase)’ gene was found in multiple KEGG Pathways – ‘Alanine’, ‘aspartate and glutamate metabolism’, ‘Arginine and proline metabolism’, and ‘Nitrogen metabolism’.

Pathways altered in HIV + TB patients found on DAVID bioinformatics database

Pathways altered in HIV + TB patients found on DAVID bioinformatics database

5 pathways found to be significantly altered in HIV + TB patients -- explaining the high rate of comorbidity

5 pathways found to be significantly altered in HIV + TB patients — explaining the high rate of comorbidity

Fc gamma R-mediated phagocytosis KEGG pathway -- two critical genes found to be altered in HIV + TB patients are involved in this pathway and indicated via red arrows

Fc gamma R-mediated phagocytosis KEGG pathway — two critical genes found to be altered in HIV + TB patients are involved in this pathway and indicated via red arrows

 

Two gene records with KEGG Pathway were found from HIV/Dementia gene list. ‘NADH-ubiquinone oxidoreductase chain 6’ gene were spotted in ‘oxidative phosphorylation’ and ‘Parkinson’s disease’ KEGG Pathway. Similarly, ‘Phosphodiesterase 5A, cGMP-specific’ gene was found in ‘Purine metabolism’ KEGG Pathway.

Two pathways were found to be altered in HIV patients that also suffered from dementia

Two pathways were found to be altered in HIV patients that also suffered from dementia

 

KEGG pathway for Parkinson's disease, where NADH-ubiquinone oxidoreductase chain 6 gene was found. This gene was also a significant one found in HIV patients suffering from dementia

KEGG pathway for Parkinson’s disease, where NADH-ubiquinone oxidoreductase chain 6 gene was found. This gene was also a significant one found in HIV patients suffering from dementia

KEGG pathway for purine metabolism where phosphodiesterase 5A, cGMP-specific gene was found to be important

KEGG pathway for purine metabolism where phosphodiesterase 5A, cGMP-specific gene was found to be important

Students then combined the two sets of genes in DAVID bioinformatics database and created a functional annotation chart. This chart clusters all significant genes from both HIV + TB infected patients and HIV patients with Dementia and puts them into functional categories. From the 38 identified significant genes (19 from HIV + TB patients and 19 from HIV + HAD patients), 49 functionalities were found.

Functional annotation chart for the combined list of genes

Functional annotation chart for the combined list of genes

As shown in the chart above, 19 functionalities of the genes deal with alternative splicing and 19 are involved with a splice variant. This means that 38 out of 49 identified genes from the combined list of HIV + TB patients and HIV + HAD patients have a significant role in the splicesome. The splicesome is a protein that cuts mRNA to ensure that the correct proteins are produced in the body. This chart shows that although there is no specific gene overlap, HIV + TB patients and HIV + HAD patients both suffer from splicing variations which effect the entire workings of the human body. Focusing on these results new treatments could possibly be devised to correct any problems with splicing found in these patients.

 

KEGG pathway of the splicesome

KEGG pathway of the splicesome

 

CONCLUSION

This research was conducted on the basis of comorbidity between tuberculosis and dementia with HIV. Analysis of these data sets resulted in no gene overlap. This result suggests that having HIV with tuberculosis or HIV with dementia causes significant alteration of specific genes. Although none of the specific genes overlapped, functional analysis did suggest an overlap of 38 genes that deal with splicing. Therefore, there was some overlap of genes with similar functions, but no overlap of specific genes. Because of the high comorbidity of TB and dementia with HIV, research was also conducted to find other papers that determined a link between TB and dementia. Unfortunately, no link has been identified.

REFERENCES

  • Dawany N, Showe LC, Kossenkov AV, Chang C et al. Identification of a 251 gene expression signature that can accurately detect M. tuberculosis in patients with and without HIV co-infection. PLoS One 2014;9(2):e89925. PMID: 24587128
  • Gelman BB, Chen T, Lisinicchia JG, Soukup VM et al. The National NeuroAIDS Tissue Consortium brain gene array: two types of HIV-associated neurocognitive impairment. PLoS One 2012;7(9):e46178. PMID: 23049970
  • Will, Cindy and Luhrmann, Reinhard. Splicesome Structure and function. Cold Spring Harbor Perspectives in Biology 2010. 
  • Smith, Issar. Mycobacterium tuberculosis pathogenesis and Molecular Determinants of Virulence. American society for microbiology. 2003.
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Posted on October 31, 2014, in Microarray data analysis. Bookmark the permalink. Leave a comment.

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