Mednotes

L04 Blood AAT

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aliasesL04 Blood AAT

11 min read

Overview

This lecture covers the acute phase response and one of its key protein mediators, alpha-1-antitrypsin (AAT), then moves through the molecular genetics of AAT deficiency (AATD), how it is diagnosed and treated, and finally the gene-environment interaction between AATD genotype and smoking, including how to counsel a patient with a predisposing genotype.

Acute phase response and inflammation mediators

  • Acute inflammation involves stimuli, vascular changes, and cellular events (leukocyte recruitment and activation), with leukocyte effector mechanisms carrying potential for damage to normal tissue.
  • Mediators of acute inflammation are grouped by source:
    • Cell-derived: preformed mediators in secretory granules (histamine, serotonin, from mast cells/basophils/platelets); newly synthesised mediators (prostaglandins, leukotrienes, platelet-activating factor, reactive oxygen species, nitric oxide, cytokines, neuropeptides, from various leukocytes).
    • Plasma protein-derived: complement activation (producing C3a, C5a anaphylatoxins and the C5b-9 membrane attack complex); Factor XII (Hageman factor) activation, which triggers both the kinin system (bradykinin) and the coagulation/fibrinolysis system.
  • Proteins of the Acute phase response: coagulation proteins (e.g. fibrinogen), complement, kinins, lysosomal proteases (e.g. elastase of neutrophils and monocytes), alpha-1-antitrypsin, and C-reactive protein (CRP).
  • C-Reactive Protein (CRP) is a sensitive diagnostic marker of general inflammation (infection and chronic disease), functions in the innate immune system, and rises and falls more rapidly than ESR (erythrocyte sedimentation rate). High-sensitivity CRP (hs-CRP) also indicates increased risk of heart disease.
  • Phagocytosis proceeds in three steps: (1) recognition and attachment, where microbes bind phagocyte receptors; (2) engulfment, where the phagocyte membrane closes around the microbe, forming a phagosome that fuses with a lysosome to form a phagolysosome; (3) killing and degradation, where microbes are killed by reactive oxygen species (via phagocyte oxidase) and nitric oxide (via iNOS converting arginine to NO), then degraded by lysosomal enzymes.

Neutrophil elastase, AAT structure, and mechanism of inhibition

  • Neutrophils release elastase, which can damage elastin in the alveoli of the lungs; this underlies AATD-related emphysema, which is predominantly basilar and panacinar, with destruction and enlargement of alveolar airspaces and destruction of alveolar septa on light microscopy.
  • Alpha-1-antitrypsin (AAT):
    • Produced in hepatocytes and diffuses into tissues.
    • A protease inhibitor (PI), specifically a SERine Protease INhibitor (a SERPIN).
    • Inhibits trypsin in vitro, but elastase in vivo.
  • Mechanism of inhibition (suicide inhibitor):
    1. AAT binds 1:1 with elastase.
    2. AAT presents an exposed reactive loop that acts as ‘bait’ for the protease.
    3. The protease attacks the loop; AAT is irreversibly cleaved, entrapping and inactivating the protease (“taking the bait” then “entrapment and destruction”).
    4. Both the elastase and AAT are subsequently degraded.
  • The AAT polypeptide fold has an N-terminus, C-terminus, and the exposed reactive loop; key residues are Met 358 and position 342, the latter being the site of the Z-mutation (Glu→Lys, i.e. p.E342K).

SERPINA1 genetics and AATD phenotypes

  • AATD (“Alpha-1”) arises from genetic variation in the SERPINA1 gene.
  • Common Protease Inhibitor (PI/Pi) alleles:
    • PI*M: normal; common subtypes designated M1, M2, M3…
    • PI*Z (p.E342K): the most common deficiency allele.
    • PI*S (p.E264V): common in some populations (e.g. Italy).
    • PI*null.
  • Table 3 (SERPINA1 mutation examples) lists many named variants by nucleotide and protein nomenclature (e.g. S-wrexham, Z-procida, S-iiyama, S-mineral springs, Z-lisbon, Null-ludwigshafen, Z-devon/Z-newport, Null-granite falls, Null-west, Null-bellingham, F, P-duarte/P-lowell/Null-cardiff, S, Null-hong kong 1, Null-bethesda, W); the classic Z allele is c.1096G>A(b), p.Glu342Lys.
  • Protein phenotypes and risk:
    • ZZ: very high risk of emphysema/COPD before age 45 and of liver disease (cirrhosis, neonatal hepatitis); prevalence 1:1600-1:6000 in Europeans/Caucasians, 0.8% in Maori; ZZ individuals have only ~15% of normal AAT.
    • SZ: increased risk (some SZ individuals overlap the risk threshold).
    • Znull: increased risk (high).
    • MM: no increased risk.
    • MZ: possible risk (per newer studies); ~10% of the European/Caucasian population is MZ.
  • Inheritance: autosomal recessive, but both alleles are expressed (co-dominant) when inherited together (e.g. SZ). There are two main founding mutations in Europeans/Caucasians and one founder mutation in Maori. There is a strong genotype-phenotype correlation for disease (e.g. emphysema).
  • AATD increases risk of COPD/emphysema, and the variant protein can accumulate in the liver causing cirrhosis. AATD is under-diagnosed or misdiagnosed as asthma.
  • Epidemiology: not limited to people of European descent; also seen in African, Middle Eastern, South Asian, and Maori (around 1%) populations in NZ. Worldwide there are an estimated 116 million carriers and 3.4 million people with a deficiency or risk genotype.
  • The 11 µM serum AAT threshold: individuals with null-null and ZZ phenotypes have insufficient AAT to protect the lung and are at high risk; MM, MS, SS, and MZ phenotypes generally have enough AAT to provide an adequate anti-neutrophil-elastase screen for the lower respiratory tract; some SZ individuals straddle the 11 µM threshold and are therefore at risk.

Diagnosis of AATD

  • Phenotype testing: AAT activity via inhibition assay, and AAT protein concentration via immunodiffusion/immunoelectrophoresis; this is confounded by the acute phase response (levels rise with inflammation), so CRP is tested first to check for confounding.
  • Protein isoform variant detection by isoelectric focusing (IEF): proteins are separated on a gel with an immobile pH gradient and migrate to their isoelectric point (no net charge), where they stop and are “focused”; AAT variants have different isoelectric points and are readily separated this way. Each allele typically shows two bands (incomplete glycosylation), e.g. MM shows two bands high on the gel (~pH 4-4.5), while MZ, SS, MS, SZ, ZZ show band pairs positioned differently depending on the S/Z isoform charge.
  • Genotype testing: specific mutation testing (described in the lecture as a future direction).
  • Current NZ test (Registration Code SPEP), “A-1 antitrypsin (Plasma/Serum)”, external price $103.80 (excl. GST). Indications include: familial AAT deficiency; suspected familial chronic obstructive lung disease; and unexplained necrotising panniculitis, liver disease, bronchiectasis, ANCA vasculitis, or COPD in a patient aged 30-40. AAT deficiency increases trypsin/elastase activity, leading to early-onset emphysema in adults and, in neonates, neonatal hepatitis progressing to cirrhosis.

Treatment and management of AATD

  • Manage as COPD, with monitoring of the liver (cirrhosis risk).
  • Reduce environmental damage: stop smoking, reduce infection risk.
  • Genetic counselling, given autosomal recessive inheritance.
  • Exercise.
  • If emphysema is declining, consider IV AAT augmentation (weekly infusion) for non-smokers.
  • Late stage disease: liver or lung transplant.

Gene-environment interaction: smoking and AATD prognosis

  • Smoking, and the level of smoking, strongly affects prognosis in AATD.
  • PI*ZZ individuals who smoke have a severe reduction in life expectancy, roughly halved compared with non-smoking PiZ individuals.
  • Survival curve data (Kaplan-Meier style, cumulative probability of survival vs age) shows: smoking PiZ men/women decline earliest and steepest (survival falls sharply from the 40s, approaching 0 by 70-80y); non-smoking PiZ men/women decline later (50s-60s); the general Swedish population (men and women) declines latest (70s-90s). This demonstrates that smoking greatly worsens survival specifically in the PiZZ genotype relative to both non-smoking PiZ individuals and the general population.

Smoking cessation is the single most important modifiable factor for a patient with a high-risk AATD genotype (e.g. ZZ or SZ), given the marked survival difference between smoking and non-smoking PiZ individuals.

  • Behavioural evidence (Carpenter et al., 199 US smokers surveyed 3 months after receiving AAT genotype results, minimal-contact intervention of reading materials plus phone support): severely AAT-deficient smokers (n=17) had a 59% 24-hour quit attempt rate vs 26% in those testing normal; carriers had a 34% quit attempt rate. Severely deficient smokers were more likely to seek treatment information, use pharmacotherapy, and report a >50% reduction in smoking (59% vs 15% in controls). There was no significant group difference in 3-month abstinence. More recent studies critique the quality of this type of intervention.
  • Strategy for advising a patient with a predisposing genotype: draw on best-practice behaviour modification, available resources and personnel (GP, Quitline, specialists such as Genetic Health Services), and clear explanation of the genetic basis of their increased risk (communicating genetic risk).

Genetic testing and referral pathways (NZ)

  • Scenarios that may be managed without a genetics referral include: cystic fibrosis screening with a family history of CF; alpha-1-antitrypsin testing; haemochromatosis (HFE) testing; thalassaemia testing; hyperlipidaemia testing; carrier testing for a common recessive condition in a family; and initial assessment of Marfan syndrome. Most other genetic tests require a genetics or subspecialist review.
  • Tests GPs can order directly: karyotype, cystic fibrosis common mutation screen, alpha-1-antitrypsin, haemochromatosis (HFE) testing, thalassaemia molecular testing, and Factor V Leiden.
  • Subspecialists are strongly encouraged to discuss non-routine genetic testing with their local genetics service to ensure the most clinically relevant and cost-effective testing strategy.

Self-test

  1. Define alpha-1-antitrypsin (AAT) and state where it is produced and what type of enzyme inhibitor it is.
  2. Describe the steps of the AAT “suicide inhibitor” mechanism of elastase inhibition.
  3. What protein marker is commonly used as a general measure of inflammation, and how does it differ from ESR in its kinetics?
  4. Distinguish cell-derived from plasma protein-derived mediators of acute inflammation, giving an example of each.
  5. Describe the three steps of phagocytosis, including the mechanisms of microbial killing.
  6. List the common PI/Pi alleles of SERPINA1 and state which is the most common deficiency allele.
  7. Distinguish the risk associated with the ZZ, SZ, MZ, and MM AAT phenotypes, including approximate serum AAT level relative to the 11 µM threshold.
  8. Explain why AATD is inherited as autosomal recessive but described as co-dominant.
  9. Describe the two main methods used to diagnose AATD (protein-level and isoform-level), and explain why CRP is tested first.
  10. What is the pathophysiology by which the Z variant of AAT causes disease in both the liver and the lung?
  11. A 38-year-old man with the SZ genotype smokes. Using the survival curve data and the Carpenter et al. findings, explain how you would counsel him about smoking cessation.
  12. What group in NZ is noted as being at particular risk of AATD besides those of European descent, and what is the approximate carrier/prevalence figure given?
  13. Integrative: trace the pathway from neutrophil elastase release, through AAT’s normal inhibitory role, to how a Z-mutant AAT genotype combined with smoking increases the risk of both emphysema and reduced life expectancy.

Answers

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