Haemoglobin F

So, why is haemoglobin F so important when we are thinking about possible treatments for sickle cell disease? Well, let’s start at the beginning with haemoglobin itself and then move on to discuss haemoglobin F.

Haemoglobin is what gives blood its red colour, it is a protein molecule packed tightly inside the red blood cells, which circulate around the body in the bloodstream. The main function of haemoglobin and red blood cells is to pick up oxygen in the lungs, where it is extracted from the air we breathe, and carry it to the tissues of the body, where it is used to make energy and keep the body’s systems ticking over. The usual type of haemoglobin found in blood is called haemoglobin A (adult haemoglobin or Hb A for short). Haemoglobin S (sickle haemoglobin or Hb S) differs very slightly from Hb A in structure, as a result of which it has a tendency to crystallise within red cells which leads to all the problems we are familiar with in sickle cell disease. But, in terms of delivering oxygen to the tissues, Hb S is very similar to Hb A, and in fact, is somewhat better at this task that Hb A itself.

Haemoglobin F (foetal haemoglobin or Hb F) is also very slightly different from Hb A in structure and is the main haemoglobin found in the red blood cells of babies, as they are growing in their mother’s womb, and for a few months after birth. However, over the first 6-12 months of life Hb F gradually disappears from the blood and is replaced by Hb A, so that ultimately, in adults, only 1-2% of the total haemoglobin is Hb F, the rest is Hb A. This changeover from Hb F to Hb A is called the “gamma-beta switch”, because the gamma globin genes, which make Hb F, are switched off and the beta globin genes, which make Hb A, are switched on. Hb F is a perfectly good haemoglobin when it comes to transporting oxygen and is ideally suited for babies because it’s tendency to bind oxygen is stronger than that of Hb A, so in the womb, oxygen is effectively transferred from the mother’s Hb A to the baby’s Hb F.

The gamma-beta switch; before birth the baby's haemoglobin is made up of alpha chains and gamma chains = HB F and after birth by Hb A = alpha chains and beta chains.

The gamma-beta switch; before birth the baby’s haemoglobin is made up of alpha chains + gamma chains = Hb F and after birth by Hb A = alpha chains + beta chains.

Some adults have inherited rare alterations in their haemoglobin genes, which mean that the gamma-beta switch never really happens and they continue to have very high levels of Hb F throughout their lives. Critically, these individuals are perfectly well and suffer no ill effects from this – in other words it is completely possible to live a normal, healthy life with 100% Hb F.

An added advantage of Hb F, in patients with sickle cell disease, is that it does not interact with sickle haemoglobin, in fact it positively inhibits sickling. Hb F prevents the Hb S molecules from moving close to one another and thereby stops them from interacting with one another, the first step in the crystallisation process. For an individual with sickle cell disease the higher the level of Hb F, provided it is evenly distributed among all of their red cells, the fewer sickle cell problems they have.

This is a Kleihauer stain of red blood cells. Those that contain a lot of Hb F are dark staining, those that contain only a little are pale staining. To be effective in preventing the effects of sickle haemoglobin the Hb F needs to be present in high concentration in the majority of red blood cells.

This is a Kleihauer stain of red blood cells. Those that contain a lot of Hb F are dark staining, those that contain only a little are pale staining. To be effective in preventing the effects of sickle haemoglobin the Hb F needs to be present in high concentration in the majority of red blood cells.

Now, in sickle cell disease the problem lies in the beta globin genes, which normally make Hb A, but in this situation make Hb S instead. But the patient’s gamma globin genes, which make Hb F, are completly normal. Therefore, if we could reverse the gamma-beta switch, by inhibiting the damaged beta globin genes and re-activating the normal gamma globin genes, those individuals would stop making sickle haemoglobin and would replace it with Hb F. They would effectively be cured of their sickle cell disease. This is the “holy grail” of sickle cell research because it would be a treatment, which used the body’s normal genes, to bypass the problems created by the damaged beta globin genes.

Needless to say, the control of the gamma-beta switch is complicated and has taken many years to unravel. Understanding this involves some molecular biochemistry – so here goes. Only a few of our genes are active at any one time, the change from gamma to beta globin gene activity is just one example of this. The body therefore must have systems for switching genes on and off. Genes are normally regulated by other DNA sequences, called promoter regions. For the genes that control haemoglobin production the main promoter is called the LCR (the Locus Control Region). These promoters are in turn regulated by proteins called transcription factors, which bind directly to the DNA of the promoter, change the orientation of the chromosome so that the promoter region is brought into direct physical contact with the gene it regulates and increase the activity of an enzyme, RNA polymerase, which gets the gene working.

A whole series of transcription factors have been shown to be of importance in controlling the gamma globin gene, which makes Hb F, and they have rather strange names, such as BCL11A, KLF1, SOX6, and FOP etc. An important one seems to be BCL11A. Genetically engineered mice, which lack a functional gene for BCL11A, have persistently high levels of Hb F in adult life and normal mice, manipulated by a viral mediated knockdown of the BCL11A protein, also show enhanced Hb F production. “Knockdown” is a technical term meaning that the  mouse is prevented from making BCL11A by toxic RNA fragments introduced into mouse cells by a virus. BCL11A therefore seems to be crucial in suppressing the gamma globin genes and inhibiting Hb F production in adult life.

This complicated diagram illustrates the various transcription factors which may influence the gamma-beta switch. The blunt end arrows indicate inhibition and the pointed arrows indicate stimulation of the different genes.

This complicated diagram illustrates the various transcription factors which may influence the gamma-beta switch. The blunt end arrows indicate factors which inhibit and the pointed arrows indicate factors which stimulate  the different genes.

These findings have recently been confirmed in human cells. If you grow human red cell precursors in a test tube and treat them with the same viral mediated knockdown of BCL11A production, then those red cell precursors increase the amount of Hb F they synthesise from 1% to more than 30% of their total haemoglobin.

Of course, this is a long way from having a safe drug to do the same thing in patients with sickle cell disease. Not least, with any drug designed to interact with DNA or change gene activity, it is very important to be sure that the actions of the drug are limited to the gene or transcription factor that you are interested in, and that there are no unexpected or undesirable side-effects on other DNA sequences or genes. But it is a start.

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About rogerjamos

I am a consultant haematologist who has worked in Hackney, London, UK with patients who have sickle cell disease for many years. Knowledge is power; the hope is that this blog will empower patients by putting them in touch with contemporary research into sickle cell disease and facilitating informed discussion on the issues raised. Dr Roger Amos MA, MD, FRCPath
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5 Responses to Haemoglobin F

  1. Bala says:

    Amazing article/blog, like it very much, how faraway are we from clinical trails in humans?

    • rogerjamos says:

      I think we are some way from clinical trials which use drugs to manipulate the transcription factors I described. This is because the multiple transcription factors involved and their interactions have only recently been understood in detail. BCLIIA seems to be the most favoured factor to be targeted but I am not aware of any active trials at the moment. Is anyone else?

      Other agents which induce Hb F, acting by less specific, epigenetic mechanisms, have of course been trialled, but only one, hydroxycarbamide, made it through into clinical practice. They include 5-azacytidine, and it’s analogue decitabine, which together with hydroxycarbamide, are thought to work by encouraging DNA hypomethylation and gene activation, and butyrate, which inhibits histone deacetylation. 5-azacytidine and decitabine are too toxic for clinical use and butyrate is limited by difficulties in administering sufficient amounts of the drug.

  2. Lisa Rose says:

    Thank you for bringing light to hgF and how it factors into SCD. This has been a question on everyone’s mind for years, yet little info has been presented to really explain it. I have some f/u questions for all of the nonmedical people reading:
    1) Does the percentage of hgF refer to the cellular level or the system level?
    2) Does every bone marrow “factory” produce the same levels of hgF?
    3) Does the uneven distribution conversation refer to the cell itself or throughout the bloodstream?

    • rogerjamos says:

      Thank you for your kind words. I will try and answer the questions you pose.
      1) When people talk about the percentage of Hb F they mean the amount of Hb F in the blood as a whole, expressed as a percentage of the total amount of haemoglobin. Typically a normal adult would have say 90% Hb A and tiny amounts of Hb F and Hb A2, another minor haemoglobin; someone who is a carrier for sickle cell would have 45% Hb A and usually slightly less Hb S, say 38%, whereas someone with sickle cell disease would have close to 100% Hb S. The percentage of Hb F does not refer to the relative amounts of Hb F and Hb A inside each red cell.

      2) When red blood cells are produced in the bone marrow they gradually accumulate more and more haemoglobin inside them until, when they leave the bone marrow and enter the blood stream, that is virtually all that they contain. In a normal adult most of the red cells, when they are released, will contain mainly Hb A with tiny amounts of Hb F and Hb A2, but a small number will contain much higher concentrations of Hb F, these are the so called F-cells. What determines the reactivation of the Hb F genes in a small minority of red cells as they develop I do not know, but this is clearly a very important process to try to understand. Presumably, the control mechanisms involved are one, or several, of those discussed in the blogs. For some reason, in these red cells, the gamma-beta switch, which is normally complete by 6-9 months of age is partially reversed. It is of course very difficult to isolate these developing red cells and investigate what is going on inside them individually.

      3) So, in any one red blood cell in the circulation, there will either be a very small amount of Hb F or, in a minority, a much larger amount, if it is an F-cell. This distribution is fixed for the lifespan of the red cell. As they circulate in the blood stream these two types of red cell both look the same but you can distinguish them in the laboratory down the microscope by using a Kleihauer stain. This stain colours the F-cells a dark red colour whereas the other red cells are very pale. An example of a Kleihauer stain is in one of the blogs.

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