Course No. 7 “Correlation of Disease Genes to Phenotypes”


Problem 2

This course focuses on the correlation of a disease gene to the phenotype. The following module demonstrates how NCBI resources such as literature, expression and structure information can help provide potential functional information for disease genes.

Mutations in the HBB gene are associated with the sickle cell anemia disease. A laboratory working on sickle cell anemia wants to elucidate the biochemical and structural basis for the function of the mutant protein.

In this exercise, we have the following goals:
1. Determining what is known about the HBB gene and protein (using Entrez Gene).
2. Determining identified
SNPs and their locations in the HBB gene (using dbSNP).
3. Learning more about the sickle cell anemia disease and its genetic testing (using
OMIM and Gene Tests)
4. Elucidating the biochemical and structural basis for the function of the wild type and the mutant protein, if possible.

Step 1. Determining what is known about the HBB gene and protein (using Entrez Gene):

Search for 'HBB" in Entrez Gene. One entry is for the human HBB gene. Retrieve the entry by clicking on the HBB link.

What is the location and orientation of the HBB gene on the human genome? List the genes adjacent to it. How many alternatively spliced products have been annotated for the HBB gene when the RefSeq mRNA entries were reviewed? List some of the HBB gene aliases. What are the phenotypes associated with the mutations in the HBB gene?

What is the name and function of the protein encoded by the HBB gene? Beta globin is a subunit of which protein? Name other subunit(s) in that protein.

Step 2. Determining other identified SNPs and their locations in the HBB gene:

From the Links menu on the top right hand side of the page, click on the "SNP:GeneView" to access a list of the known SNPs (reported in dbSNP). Select the “Clinical Source” box and click Refresh.  By default, the SNPs in the coding region of a gene are reported. Additional SNPs such as in the upstream region or the introns can be viewed by clicking on the "in gene region" button. Currently, how many coding SNPs are placed on the beta hemoglobin transcript NM_000518? We will concentrate on the Glu7Val mutant in the following analysis.

Step 3. Learning more about sickle cell anemia disease and its genetic testing:

Click on the OMIM link next to the one of the SNPs in the Clinically Associated column.  As mentioned in the OMIM report, the allelic variants are listed for the mature beta hemoglobin protein which lacks an initiator methionine. Hence, the allelic variants in the OMIM report are off by one amino acid compared to the precursor protein in NP_000509.

Open the Table of Contents menu and Click on the Allelic Variants “View List” to get information about the mutant proteins. Is the Glu6Val variant mentioned in the list? (It is the variant number 0243). Which phenotype does it cause? What is the name of the mutant hemoglobin (hemoglobin S).

Go back to the OMIM HBB page.  Click on the GeneTests link Additional Links menu at the bottom of the page.  Now refer to the Reviews section for Sickle Cell Disease.  Mutation analysis is available for which of the HBB alleles? List one explanation for the sickle cell anemia phenotype caused by the Glu7Val mutant beta hemoglobin.

Step 4. Elucidating the biochemical and structural basis for the function of the wild type and mutant proteins, if possible:

A. Information about the wild type protein:

Go back to Entrez gene report. Based on the RefSeq summary, the PubMed articles, and Gene Ontology section, describe the biochemical functions of beta hemoglobin.

Let us first take a look at the structure of the wild type protein. Click on the NP_000509 protein link listed in the mRNA and protein section. Click on Protein 3D Structure link. Click on the first link for 2M6Z. Note on this page that the chains A and C in the structure represent alpha chains, and B and D represent beta chains.  View the deoxyhemoglobin tetramer structure by clicking on the "Structure View” button. Identify the 6th glutamate residue in chain B.

B. To view structure of the mutant, hemoglobin S:

In the MMDB page, click on the Structure Home link in the top blue bar and search for the structure of the mutant by typing "Deoxyhemoglobin S" in the search box.  Two entries, 1HBS and 2HBS, are retrieved. Click on the structure link for 2HBS. 

C. To show the side chains of the mutant residue and view its interaction with another hemoglobin molecule:

Click on the asymetric unit radio button.  Download the structure 2HBS by clicking on “View Structure”. For easier viewing, remove the helix and strand objects using Style--Edit global style, and unclick the boxes next to the Helix objects and Strand objects. Highlight valine 6 from the H chain (one of the beta chains). To show the side chains of the residue, use the Structure window--Style--Annotate--new. Give a name to this annotation such as "valine" and then click on Edit Style. Change the protein backbone Rendering to "Space Fill", Color Scheme to "charge" or "hydrophobicity". Repeat these steps for the Protein Sidechains row and click the Protein Sidechains on. To show the amino acid number, choose the Labels panel, and change the Protein Backbone spacing to 1. Click on the Done, OK then Done buttons. The valine interacts with a pocket between the two helices on another tetramer. Identify the residues from other molecules within 4 angstroms of the valine, use Show/Hide--Select by distance--other molecules. To unclick the highlighted residues, click on the white portion of the sequence window.

You can now easily explain why the Glu7Val mutant has an altered function.

The HBB gene encodes beta hemoglobin which is a part of hemoglobin along with alpha hemoglobin. Hemoglobin is a tetramer consisting of 2 beta and 2 alpha chains. Mutation of the 7th negatively charged amino acid, glutamic acid, to hydrophobic valine leads to polymerization of hemoglobin forming a sickle fiber that changes the shape of red blood cells leading to sickle cell anemia.


Questions, Comments:  Medha Bhagwat, PhD