Porphyria is a rare group of genetic diseases involving the malfunction or reduced production of heme biosynthetic enzymes. This results in the accumulation of toxic precursors (substances that form the following substance during metabolic pathways) during metabolism, which if not cleared can lead to a variety of symptoms depending on the type of precursor. Typical symptoms of porphyria can include acute neural attacks, paralysis, high blood pressure, photosensitivity, inflammation, and severe seizures. The combined effect of the symptoms may even be fatal if left untreated, but there is still no effective cure that can completely eliminate the disease. Thus, a deeper understanding of the biological causes for porphyria will encourage the development of potential treatments or cures. This review aims to analyse the molecular basis of both acute intermittent porphyria and erythropoietic protoporphyria, as well as suggest possible methods of treatment.
Porphyria is a group of severe genetic diseases caused by the depletion of enzymes involved in heme biosynthesis (please see below). There are in total seven different porphyria diseases with varying combinations of symptoms and subsequent pathogenicity. According to the American Porphyria Foundation, the current prevalence of porphyria is estimated to be between 1 in 500 to 1 in 50000, depending on the geographic location and the type of porphyria1. Severe porphyria, such as Acute Intermittent Porphyria, has a death rate of around 20-30%8. Besides the pathogenicity itself, porphyria has even caused several incidences of suicide due to the patient\’s inability to withstand the severe symptoms. Despite its death rate and pathogenicity, there is currently no cure or prevention for porphyria. There are however, treatment methods and therapies that to some extent are effective at lowering the occurrence of symptoms.
This review will discuss the history, symptoms, current treatment, and the molecular basis of porphyria, focusing on acute intermittent porphyria and erythropoietic protoporphyria. Furthermore, this review will also provide possible directions for developing a porphyria cure.
Porphyria diseases have existed since the 1800s, with the first case documented in the 1890s. The term “porphyria” stems from the Greek word for purple, “porphura” 6, due to the purple skin that patients develop as a result of the disease. It has also been hypothesized that the legend of vampires originated from porphyria patients as they often display vampire-like characteristics such as photosensitivity6. This disease has been under constant investigation by scientists and its molecular basis is well understood. However, there is still no effective way to diagnose and treat porphyria.
Acute porphyria typically affects the nervous system and has a wide range of severity13. Symptoms include muscle weakness, paralysis, seizures, high blood pressure, abdominal pain and mental disorders13.
In cutaneous porphyria, symptoms are associated more with the skin. The most common symptom is sensitivity to light11. The patients of this disease will experience burning pain and swelling of the skin after exposure to light11.
Biological Mechanisms of Porphyria
Heme Function and Structure
Heme or haem is an important biochemical compound responsible for many biological pathways. Most notably, it is the essential oxygen-binding component of haemoglobin in red blood cells10. Other important functions include accepting and donating electrons during electron transfer between proteins as well as chemical catalysis10.
Chemically, heme is a transition metal complex consisting of a central iron ion bonded to a porphyrin complex (ring structured complexes which are formed as the end product in normal hemoglobin metabolism) and to one or two side chains. In hemoglobin, the iron ion will bind to diatomic gases such as oxygen10. The iron ion also acts as an electron acceptor during electron transfer.
Figure 1. Organic structure of heme group15.
Heme Biosynthesis and Porphyria
Heme biosynthesis usually requires the porphyrin to be synthesized first, before the incorporation of the iron ion10. Heme biosynthesis occurs in the mitochondria and moves to the cytoplasm. It requires eight different enzymes that work systematically to convert succinyl-CoA (a succinate bound to coenzyme A – a metabolite that is mainly used in the citric acid cycle of the cell) and glycine (an amino acid) into heme10. Different porphyria diseases are caused by the malfunction or depletion of one of these enzymes along this pathway10. Without functioning enzymes, certain precursors will build up without being converted into the next stage of synthesis. These precursors are usually toxic and will also cause heme storage deficiency, leading to porphyria10.
Figure 2. Summary of the heme biosynthetic pathway
Acute Intermittent Porphyria (AIP) (Acute Porphyria)
AIP is a disease that causes symptoms relating mostly to the peripheral and central nervous systems such as neurovisceral attacks and muscle weakness13. AIP occurs when the enzyme, Uroporphyrinogen I synthase, also called porphobilinogen deaminase (PBGD), becomes less active or dysfunctional13. In AIP patients, this enzyme is depleted and thus causes an accumulation of the toxic precursor ALA (Delta-aminolevulinic acid), which is responsible for acute neurological attacks and other AIP symptoms13.
ALA is structurally similar to GABA (Gamma-aminobutyric acid), a major inhibitory neurotransmitter in the body2, acting as a chemical messenger to prevent the transmission of electrical signals from one neuron to the next9. Neurotransmitters are usually highly regulated to ensure the right signal is passed. However, in the case of AIP, ALA can attach to GABA receptors and have similar inhibitory effects. Unlike GABA, ALA is not regulated by the nervous system and can freely attach to neurons2. Therefore, muscle weakness may be caused by uncontrolled attachment of ALA to target receptors on muscles at neuromuscular junctions2. Instead of the excitatory signal that causes muscle contraction, an inhibitory signal is transmitted, and the muscle cannot be stimulated. Other major areas of the nervous system may also be vulnerable to the random attachment of ALA as it accumulates in the body2. In addition to this, the resulting heme deficiency due to pathway disruption can also have a fatal effect. This is due to the metabolic significance of heme; its depletion could cause disruption of normal cellular activity which would in turn affect body metabolism.
Genetic Causes and Triggers
The PBGD (Porphobilinogen deaminase, also known as uroporphyrinogen I synthase) enzyme gene is responsible for the manufacture of PBGD and is roughly 10 kilobases in length2. Several different mutations that result in malfunctioning PBGD have been identified in this sequence. These mutations range from single base substitutions to insertion and deletion frameshift errors, forming premature stop codons2. As a result, PBGD activity in the body will decrease. AIP is an inheritable autosomal dominant genetic disease, but has a very low genetic penetrance as the human body contains two PBGD genes2. If one gene mutates, the other functioning gene can still produce sufficient PBGD enzymes to continue converting precursors down the biosynthetic pathway and suppress symptoms from occurring in patients2. However, symptoms may be triggered by many environmental factors such as excessive alcohol intake, smoking, and drug consumption10. One essential enzyme, Cytochrome P450 (CYP), is utilized in the liver for the metabolism of foreign drugs and chemicals and contains a heme group in its structure (Stein, Badminton and Rees 2016). Thus, intake of these substances will induce an increase in the rate of heme production in order to synthesise more CYP10. However, in patients with one mutated PBGD gene, the decreased activity of PBGD will not be able to sustain the increased demand for heme, and thus will result in heavy accumulation of precursors such as ALA10. AIP symptoms and acute attacks may begin occurring as the functional PBGD gene will no longer be enough to maintain ALA levels to a minimum.
Additionally, according to the study conducted by Goldberg (1954), it was found that anti-anxiety drugs such as barbiturates and female sex hormones also have a direct influence on triggering AIP5. Both substances may promote ALA synthase activity to convert succinyl coA and glycine into ALA (see Figure 1). Large amounts of ALA will begin to accumulate in the body and cause AIP symptoms. Thus, it was observed that many female patients began developing AIP during menstrual cycles due to higher levels of the progesterone hormone.
Detection and Treatment
Typical biochemistry tests can be conducted to detect affected patients. These tests include measurement of PBGD levels in blood cells and ALA precursor levels in urine13. However, these tests do not give a definite result of whether the patient is affected by AIP. Variations in enzymatic activity due to external factors can cause differences in precursor levels and there is no set threshold of chemical levels for correct diagnosis of an individual with AIP. Currently, a more reliable detection method associated with molecular genetics is under development. The new method involves the sequencing of the PBGD gene of an individual13 and comparing it with the sequence of a typical healthy gene. This can allow for an efficient identification of whether the patient is a carrier13. Early detection is crucial to control AIP symptoms in carriers.
Currently, there is no cure for porphyria but there are effective treatments that can shorten or reduce the severity of the symptoms. The most direct and commonly used method is to inject the patient with heme13. By increasing the concentration of heme in the body, the rate of heme biosynthesis and subsequence ALA accumulation will decrease13. The external factors that stimulate the occurrence of AIP symptoms need to be immediately identified and stopped. Earlier diagnosis will allow less porphyrins to accumulate which will lead to less severe symptoms.
Erythropoietic Protoporphyria (EPP) (Cutaneous Porphyria)
EPP is one of the most common forms of porphyria associated with children and can cause acute symptoms in the skin, including burning sensations, skin redness, increased skin fragility and scarring when exposed to light3,11. This phenomenon is called photosensitivity.
Biochemically, EPP is caused by the malfunctioning or depletion of the enzyme ferrochelatase, which is involved in the incorporation of the iron ion to form heme from protoporphyrin III3. Porphyrin precursor accumulation usually occurs in locations around the body that are mostly associated with heme, including the liver which uses heme for cytochrome P450 and hematopoietic tissue in bone marrow which uses heme for red blood cell production3. Protoporphyrin III is lipid-soluble, and binds to serum albumin carriers in the blood after accumulating in liver cells3. It is then circulated around the body and transported into tissues at exchange sites via lipid diffusion4. Some of the circulating protoporphyrin III will reach dermis tissues near the skin and attach to cell surface membranes or internal cell organelles3. When protoporphyrin III is stimulated by photons, its electrons will enter an excited state and be transferred to other molecules to form radicals that will react with oxygen to form oxidized products3. Oxidized products can include reactive oxygen species such as hydrogen peroxide, which may damage cell membranes and cause cell lysis. As a result, the skin will become more fragile and scarring may occur. Furthermore, protoporphyrin III may also attach to mast cells that reside near the dermis tissues3. Lysis of mast cells will cause the release of serotonin and histamine, chemicals associated with the inflammatory immune response3. The increased concentration of histamine in dermis tissue will result in heavy inflammation, causing burning sensations and skin irritation.
Genetic Causes and Triggers
EPP is caused by mutations in the EPP gene located on chromosome 183. Both copies of the gene must be mutated to result in enzyme malfunction and ferrochelatase activity must be reduced by at least 30% to cause EPP3. Similar to AIP, EPP can be triggered by outside factors that stimulate the production of heme.
Detection and Treatment
EPP can be detected by observing photosensitivity symptoms in patients. This can be further confirmed by testing protoporphyrin III levels in feces, urine or blood3. Genetic sequencing and detection mentioned previously could also be used to detect mutations in the EPP gene.
Currently, there is no cure for EPP. However, symptoms will not occur if the patient’s lifestyle is controlled. This includes avoiding direct exposure to sunlight and using beta carotene medication to decrease the skin’s sensitivity to sunlight3.
Possible Treatments/Cures For Porphyria
Although diagnosis is crucial to porphyria control, further emphasis is placed on finding effective cures to the disease. As porphyria is genetic, the only way to completely cure the disease is to repair the gene itself. Methods such as somatic gene therapy, germline gene therapy, and CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 protein could potentially be utilized to cure porphyria in patients.
Somatic gene therapy is a new method in which a non-mutated version of the malfunctional gene can be artificially inserted into affected cells12. In the case of porphyria, a non-mutated wild type heme enzyme gene can be inserted into the affected cells. Once in the cell, homologous recombination during meiosis will occur, replacing the mutated gene with the new wild type gene7. If this was successful, the cell will now begin to produce the respective enzyme and the accumulated precursors can be used up7. However, unlike plant and bacteria cells, animal cells do not have cell walls. Thus, methods of DNA insertion such as transformation and gene guns cannot be utilized on animal cells. Instead, viruses can be artificially manufactured to inject the wild type gene into cells12. Another method of gene transport is by packaging the DNA into liposomes that can diffuse across the phospholipid bilayer (a bilayer that surrounds the internals of the cell; consisting of a phosphate group head bound to two fatty acid tails, forming a phospholipid) of the surface cell membrane12. The main issue with this technique, however, is that individual cells are targeted one at a time instead of all affected cells in the body at once. To completely fix the gene in the entire human body, all cells would need to be injected with the wild type gene which is currently impossible. Thus, somatic gene therapy is most effective in areas where heme production and subsequent porphyria-affected cells are most concentrated, such as in bone marrow or liver tissue.
Germline gene therapy methods improve upon this by targeting all somatic cells, but the effects are limited to only the immediate offspring of the patient12. Germline gene therapy uses similar methods to insert a wild type gene into the germline cells of an affected patient12. By doing so, the mutation can be fixed in the germ cells that are produced and the offspring of the patient will not inherit a malfunctional gene that may potentially cause porphyria. Germline gene therapy could be used in families that are known to inherit mutated porphyria genes and prevent the next generation from also contracting the disease.
The last potential method is the utilization of the CRISPR system, in order to edit the porphyria genes directly inside the cell. The system contains a nuclease enzyme, Cas9, which cuts the DNA at specific locations to precisely excise the gene. The exact location of the affected gene can be identified by taking a cell sample from the patient and purifying the DNA for positional cloning and Next generation sequencing (a method that is capable of quickly sequencing the entire human genome)12. Comparison of this mutated gene to wild type genes can identify any potential disease causing mutations12. Genome wide association studies can also be conducted to compare the mutant gene of porphyria patients. Once a potential nucleotide mutation is identified, a wild type gene can be artificially mutated in the exact same way and inserted into a somatic cell in vitro. The porphyrin precursor levels can be measured in the somatic cell after a certain time period compared to a normal somatic cell. If the precursor levels are too high, it may be possible that this particular mutation is causing the disease. A guide RNA can then be manufactured which will be complementary to that area on the gene where the mutation occurred. The guide RNA will lead CRISPR which is attached to Cas9, to the mutation site and Cas9 will precisely cut the mutated nucleotides, thus removing the mutation and nullifying its effects permanently12. However, similar to somatic gene therapy, this method is only effective in specific sites of the body and will not be sufficient to fix the mutation in all somatic cells.
In conclusion, these potential methods are either conceptual or still under experimental investigation and have many issues to be resolved before they can be used clinically. For example, viral delivery of genes may activate the immune system, which would destroy the viruses and cause the therapy to have no effects. Furthermore, the chances of precise homologous recombination is also low and could potentially still fail. Interestingly, however, experiments have been conducted by Yasuda Makiko in which liver-specific promoters and enhancers are delivered, which increase the expression of the second non-mutated PBGD gene in AIP affected mice14. The experiments did increase PBGD expression to a certain degree and may be awaiting for further investigation.
Despite understanding the biochemistry behind porphyria, there are only a limited number of studies and little research into finding cures. More emphasis must be placed on the importance of early diagnosis and treatment in order to reduce the severe and often deadly effects of this genetic disease.
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Zheng Gong is an Advanced Science student majoring in Biomedical Science, currently studying in The University of Queensland. He is very passionate about molecular biology and infectious diseases. Because of this, he really likes to read and write articles relating to these areas.