Hereditary alpha-tryptasemia (HαT)By Jonathan J. Lyons, MDAffiliations: Translational Allergic Immunopathology Unit, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.Funding statement: This research was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, NIH. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.Quick facts about HαT:HαT is common and has been found to be present in approximately 5% of people in Western Europe and the United States.HαT is caused by extra inherited copies of the gene TPSAB1 that makes extra α-tryptase and leads to high tryptase in peripheral blood at baseline.If you have HαT, there is a 50% chance your children will inherit HαT.An estimated 90% of individuals in the general population with increased baseline serum tryptase (BST) levels have HαT.While certain tryptases stored inside mast cells is believed to contribute to allergy symptoms, the version of tryptase (pro-tryptases) that can be detected in people regularly at baseline – and that is elevated in HαT – has no known function in human health or disease.Many symptoms have been associated with HαT, but it is difficult to prove that symptoms are caused, or even contributed to, by HαT because it is so common.As many as 2 out of 3 individuals with HαT are believed to have few if any symptoms that would lead to them being diagnosed with this genetic trait.HαT has been shown to increase the frequency and/or severity of anaphylaxis in specific settings including following certain insect stings and in patients without identifiable causes for anaphylaxis (idiopathic) and/or the clonal mast cell disease systemic mastocytosis (SM).A small increase in the number of mast cells in bone marrow and the lining of the GI tract has been consistently shown among symptomatic individuals with HαT.Patients with SM are more likely to have HαT compared to the general population, but this may result from an increase in symptoms seen in patients that have both SM and HαT.HαT does not appear to be a risk factor for classical irritable bowel syndrome (IBS) but does appear to modify some of the symptoms in patients with this disorder.Hereditary alpha-tryptasemia (HαT) is a common genetic traitIn clinical genetics, a trait is a characteristic of an individual that may be inherited genetically, acquired from the environment, or result from an interaction between individual genetics and environmental exposures. Hereditary alpha-tryptasemia (HαT) is a common genetic trait that affects approximately 5% of populations in which it has been studied – that to date have been predominantly Caucasian. The genetic change responsible for HαT is one or more extra copies of the TPSAB1 gene encoding the protein α-tryptase. This kind of change is considered a structural variant as there is a change in the number of copies of a gene, rather than in the genetic sequence itself. HαT is inherited in a dominant manner, meaning that receiving one extra copy from one parent is sufficient to result in the clinical trait or phenotype – in this case, elevated basal serum tryptase (BST) levels generally greater than 8 ng/mL. Because the TPSAB1 gene is located on chromosome 16 (as opposed to the X or Y sex chromosomes), and because when one extra copy of the gene is present the trait is expressed, it is called an autosomal dominant. Interestingly, in the studies that have examined elevated BST in the general population (usually defined as greater than 11.4 ng/mL), the frequency of increased BST levels among Western populations have been found to be approximately 6%. Thus, best current estimates indicate that HαT likely accounts for a little over 90% of individuals with elevated BST in the general population. Based upon population prevalence data, significant kidney disease is estimated to account for another ~7% of people with elevated BST, and rare conditions including but not limited to clonal myeloid diseases such as mastocytosis – an acquired expansion of mast cells with unrestrained growth potential frequently associated with symptoms of immediate allergic reactions – account for the remainder of individuals.Baseline serum tryptase (BST) is determined by pro-tryptase release from mast cellsMast cells are born in the bone marrow, but leave as precursors and move into tissues, mainly the skin and lining of the gastrointestinal and respiratory tracts. Once they are in the tissues, they mature into bona fide mast cells. Not all mast cells are the same, or make all of the same proteins, but one of the most abundant proteins in a mast cell is an enzyme that cuts other proteins, or protease, called tryptase. In order for the tryptase enzyme to work properly, it must be assembled from 4 building blocks called pro-tryptases, into active “mature” tetramers (four-part proteins). Pro-tryptases are protein precursors that have no known biological function; these kinds of molecules that can ultimately be converted into active proteases are frequently called zymogens.Once mature tryptases are formed, they are stored in special compartments called secretory granules along with other molecules that lead to symptoms of immediate allergic reactions, such as histamine. Because the contents of these granules are powerful, they are highly regulated and require significant stimulation to be released under normal conditions. During severe systemic immediate allergic reactions (i.e., anaphylaxis) due to mast cell secretory granule release – something also commonly referred to as mast cell activation or degranulation – serum tryptase levels can be used to confirm mast cells as the cause of the reaction. However, tryptase does not always increase during an allergic reaction, even when we believe that mast cells are the cause. More research is needed to understand why tryptases don’t always increase, and to identify new tests that can help in diagnosing patients during allergic reactions of all kinds.Perhaps because mast cells make so much tryptase – though we don’t know for certain why – mast cells also constantly release moderate amounts of pro-tryptases into the area surrounding them. From this area pro-tryptases pass into the peripheral blood where they can be measured. In everyone, including individuals with the clonal mast cell disorder mastocytosis, the level of serum tryptase that can be measured in the peripheral blood at baseline (meaning not during an immediate systemic allergic reaction) is determined by the amount of pro-tryptase constantly released by mast cells. In HαT extra copies of the TPSAB1 gene result in increased production of α-tryptase and elevated BST. While in certain circumstances HαT is associated with more severe allergic reactions (discussed below), increases in BST measured in peripheral blood are not evidence of mast cells releasing granule contents and mature tryptases are not detectable in BST samples from individuals with HαT or even those with systemic mastocytosis.Symptoms associated with HαTThe two initial studies describing families with HαT published in 2014 and 2016, identified multisystem complaints or symptoms among family members with HαT that were coinherited with elevated BST. In some cases, similar symptoms were also present in other family members who did not have HαT but in those individuals, symptoms were less severe. Symptoms included: skin flushing, itching and in some cases recurrent hives were present; abdominal pain, bloating and other irritable bowel syndrome (IBS)-like symptoms; anaphylaxis to several causes, most notably to stinging insects (e.g., yellow jackets, wasps, hornets, and honeybees); connective tissue abnormalities including joint hypermobility and retained primary teeth; and symptoms suggestive of autonomic nerve dysfunction such as inappropriate changes in blood pressure or heart rate.Tryptase is principally made by cells responsible for allergic symptoms – primarily tissue mast cells, and to a lesser extent cells called basophils that circulate in peripheral blood. Because of this, tryptases are rarely measured or studied in patients who do not present with allergic symptoms, diseases or reactions. So far, all published studies involving individuals with HαT have also been from institutions which focus on genetic and/or mast cell-related disorders, creating both detection and referral biases inherent in many of symptoms that have been associated with HαT.Only one small group of individuals within a larger study has queried symptoms from unselected, ostensibly healthy, adults. Among the nine individuals that were identified with HαT agnostically, one-third (3/9) were found to have minimal symptoms, one-third (3/9) had a few symptoms, and the remaining one-third (3/9) reported lots of symptoms. These symptoms were similar to the group of symptoms that have been reported in multiple studies and included: anaphylaxis in particular in response to stinging insects, connective tissue abnormalities such as joint hypermobility and retained primary dentition, symptoms of autonomic dysfunction, eosinophilic gastrointestinal disease, food intolerances, chronic pain and fatigue. Studies are ongoing in order to test these prior associations. Of these, only systemic reactions to stinging insects, skin flushing and itching, IBS-like abdominal complaints, retained primary teeth, and symptoms of autonomic dysfunction – measured using a scoring system – were significantly increased among individuals with HaT.HαT is associated with more severe anaphylaxis and mast cell mediator symptoms in certain clonal and non-clonal mast cell-associated disorders In the first study to describe the genetic cause for HαT, systemic immediate allergic reactions, or anaphylaxis to stinging insects (most commonly Hymenoptera species) were found to be nearly ten times more common among individuals prospectively identified with HαT. This finding prompted an follow-up NIH-led collaboration to study two large groups of patients with Hymenoptera venom-triggered anaphylaxis (HVA) from Italy and Slovenia. The frequency of HαT among venom allergic individuals presenting with severe anaphylaxis was found to be approximately double, compared to the general population or those who had milder reactions. Importantly, the overall number of venom allergic patients with HαT was comparable to the general population. This suggested that HαT is unlikely to be a risk factor for venom allergy, but in venom allergic patients, roughly doubles the chance of having a bad systemic reaction. Initial reports describing HαT also reported that up to 20% of people with this trait in symptomatic families had a history of anaphylaxis. These reactions occurred not only following insect stings, but also were triggered by foods, radiocontrast media, allergy shots, and in some cases happened spontaneously (an event called idiopathic anaphylaxis). In order to determine whether HαT might increase the rate of anaphylaxis, the same NIH-led team examined the frequency of HαT among groups of patients with systemic mastocytosis (SM) and idiopathic anaphylaxis. In both groups HαT was found to be approximately 3-times more common than the general population. A subsequent large European study also found that HαT was about 3-times more common among patients with SM compared to the general population. Importantly, in both the NIH and European studies, the frequency of anaphylaxis in patients with SM and HαT was also approximately doubled when compared to patients with SM alone. The increased number of SM patients with HαT compared to the general population is likely due in part to a detection bias, where individuals with both HαT and SM are more likely to be highly symptomatic, and thus more likely to come to medical attention. However, a number of studies have now demonstrated moderate increases in mast cell numbers in the bone marrow and GI lining – called epithelium – of symptomatic individuals with HαT when compared to healthy volunteers or patients with similar symptoms who do not have HαT. These findings suggest that having extra α-tryptase may somehow affect mast cell growth or survival in some way. Potential mechanism(s) underlying these findings are an active area of research. Despite these interesting research findings, and in part because SM is quite rare, simply having HαT is not associated with a clinically relevant increase in the risk for developing SM. In addition to anaphylaxis, HαT has been associated with more severe mast cell mediator-associated symptoms in European SM patients based upon a validated questionnaire-based scoring system. Interestingly, symptom severity demonstrated a gene-dosage effect – where increasing TPSAB1 copy number was associated with more symptoms and higher symptom scores. While one small study in the UK did not observe a similar gene-dosage effect among allergy patients in a clinical practice, potentially due to the small number of patients with more than one extra TPSAB1 gene copy in this study, a similar gene-dosage effect on symptom severity was also reported in the first and largest study of symptomatic individuals with HαT that has been published to date.Gastrointestinal complaints and immune cell differences are distinct from IBS in HαT patients with GIWhile irritable bowel syndrome (IBS)-like symptoms have been frequently reported among symptomatic individuals with HαT, a recent study of a well-characterized large group of patients with IBS failed to demonstrate any increase in the frequency of individuals with HαT, arguing strongly again HαT being a risk factor for classical IBS. In this same study, the authors went on to show that among individuals with GI symptoms and HαT an unusual kind of inflammation called pyroptosis was seen in the lining of the gastrointestinal system at a comparable amount to what is typically seen in patients with the gluten-associated inflammatory disorder called Celiac disease and seen in asymptomatic patients with a treated inflammatory bowel disease (IBD) called Crohn’s disease. These inflammatory changes were associated with immunologic changes both in the lining of the GI system as well as similar changes that could be detected in peripheral blood, but the significance of these immunologic differences remain uncharacterized. One interesting change was the finding of increased numbers of mast cells, which confirmed a prior publication reporting a similar finding. How HαT or increased α-tryptase expression might lead to any of these findings remains speculative.Possible mechanisms explaining findings among symptomatic individuals with HαTHow α-tryptase may contribute to symptoms associated with HαT remains unclear. One possible contributing explanation that has recently been proposed relates to the composition of the mature four-part tryptase tetramers. It has been known for some time that while mature tryptases that contain four β-tryptase are enzymatically active proteases, mature tryptases that contain four α-tryptase subunits have almost no ability to cut other proteins. However, it was recently discovered that some mature tryptases contain both α-tryptase and β-tryptase subunits, so-named heterotetramers. A surprising aspect of these newly identified heterotetramers is that there are specific proteins including stimulating receptors on mast cells and the cells that line blood vessels (i.e., endothelial cells) that can only be cut by heterotetramers when tested in the laboratory. Some of these receptors may contribute to mast cell activation to certain stimuli such as vibration or make blood vessels leakier in response to mast cell degranulation, and in turn may contribute to some of the symptoms associated with HαT. However, mature tryptases are principally released all at once following mast cell activation and are not observable in carefully handled and preserved tissue sections or baseline peripheral blood samples, making this explanation for many of the chronic symptoms somewhat unsatisfactory. It is also difficult to explain the increased numbers of mast cells that multiple studies have reported in bone marrow and in the GI epithelium based upon these findings, or the increased prevalence among SM patients – which may not entirely be explained by the aforementioned detection bias. Additional mechanism(s) potentially underlying all of these findings are active areas of research.How is HαT diagnosed?The first step in diagnosing HαT involves having your physician measure a total serum tryptase level. In the large majority of individuals with HαT, serum tryptase levels are over 8 ng/mL. Most genetic sequencing techniques available for clinical use are unable to determine the number of α– and β-tryptase encoding sequences anyone has. However, a specialized test that uses a technology called droplet digital polymerase chain reaction (ddPCR) is available for clinical use from a Clinical Laboratory Improvement Amendments (CLIA)-certified clinical laboratory: https://genebygene.com/tryptase/ The assay run for clinical use is the same assay that was developed by Lyons et al. in 2016. Tryptase genotyping by ddPCR should be considered in symptomatic individuals with elevated basal serum tryptase levels, and is currently the only test available to confirm the diagnosis of HαT. Correct interpretation of tryptase genotyping can be a challenge even for doctors. α-Tryptase and β-tryptase sequences are encoded by the tryptase genes TPSAB1 and TPSB2 within the human tryptase locus near the end of the short arms of chromosome 16. While β-tryptase sequences can be present at either gene, only TPSAB1 is known to contain the sequences needed to make α-tryptase. Because these genes are so close together physically on chromosome 16, they are nearly always inherited as a pair, or haplotype. Each person received one haplotype from each parent. The three most common haplotypes are: αα, ββ, or αβ. Because each person inherits two of these haplotypes, the resulting tryptase genotypes that make-up approximately 95% of population are: 4β (ββ/ββ), 1α3β (ββ/αβ), or 2α2β (αβ/αβ). HαT is diagnosed when there are extra inherited copies of TPSAB1 encoding α-tryptase on a αβ haplotype, making the most common HαT-associated haplotype ααβ. Because the other inherited haplotype can be either ββ or αβ, the two most common HαT-associated genotypes are 2α3β (ααβ/ββ) and 3α2β (ααβ/αβ), respectively. The most common misunderstanding is that a 2α3β genotype results from increased β-tryptase encoding sequence. While variation in the number of β-tryptase encoding sequences has been observed, this is uncommon, and has not been associated with inherited increases in serum tryptase levels. While one extra germline TPSAB1 copy – frequently referred to as a duplication – accounts for the majority (approximately 80-90%) of individuals with HαT, as many as four extra copies of TPSAB1 encoding α-tryptase have been reported to occur.I have been diagnosed with HαT, now what?How an increase in the number of genes making α-tryptase could contribute to the symptoms that have been associated with HαT, or how this relatively common genetic trait can make certain mast cell-associated symptoms worse – as seen among some individuals with systemic mastocytosis – remains unproven. Because of this, there are currently no specific treatments for HαT. Rather, clinical approaches to treatment initially used in symptomatic individuals with HαT are often the same approaches currently recommended for mast cell-associated disorders such as mastocytosis and mast cell activation syndrome – both of which may be present in individuals with HαT. While no prospective trials have been performed to date in symptomatic individuals with HαT, there are two retrospective reports describing symptoms in patients with HαT treated with the anti-IgE monoclonal antibody omalizumab. These reports have suggested that as anticipated, some symptoms including skin itch and asthma-related complaints improved in these patients. However, other symptoms including certain GI complaints and connective tissue findings did not. Future prospective trials that incorporate tryptase genotyping and/or target HαT-associated symptoms specifically are needed to study targeted therapies in these individuals. 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