Research theme 2: New research into the pathogenesis of lung and liver disease in genotype ZZ alpha-1-antitrypsin deficiency

Lung disease:

Rationale: Alpha-1-antitrypsin (AAT) is the major serum anti-protease protein in humans. Its genetic deficiency (AATD) led in 1985 to the protease-antiprotease imbalance hypothesis to explain emphysema development. This hypothesis predicted that restoring the balance by intravenous AAT, isolated from healthy blood donors, would stop the progression of emphysema. To date, this has not been proven by an effect on lung function values (FEV1 or DLCO) in clinical trials. Therefore, we aim to investigate a new hypothesis. Based on our preliminary results we hypothesized that altered PBMC subpopulations and a consequent defect in the CX3CL1/CX3CR1 axis has a critical role in the pathogenesis of emphysema associated with inherited AAT deficiency. Furthermore, altered subsets of PBMC, like patrolling monocytes, may contribute to pulmonary microvascular endothelial cell (pMVEC) damage, which is enhanced in emphysema.

Objective: Primary: to identify in the study population the variability in the circulation of the number of patrolling monocytes and their expression of CX3CR1 in relation to emphysema. Secondary: to identify if the level of plasma endomicroparticles specific for pMVEC damage correlate with any of the results of the primary objective.

Study design: explorative case-control study

Study population:  28 patients in age range 30 – 75, known with the diagnosis of ZZ-AATD and 28 spouses serving as controls with normal AAT and with equal gender distribution between groups

Intervention (if applicable): not applicable

Main study parameters/endpoints: the number of patrolling monocytes in a peripheral blood sample and their expression level of CX3CR1 of study subjects and the relation to emphysema in ZZ-AATD  

Liver disease:

Background: PiZZ AATD displays an extraordinarily heterogeneous disease course since some individuals stay clinically unapparent, while others develop a severe lung or liver disease. The current clinical evidence suggests that both organ manifestations are largely independent and are likely to be triggered by a combination of unique host and environmental factors. However, the elucidation of this interesting biological phenomenon has been hampered by the lack of clinical data. In particular, the development of liver disease in PiZZ individuals has not been systematically evaluated.

The AATD deficiency can lead to neonatal cholestasis in about 10 % of the affected infants. About 30-50% of these will develop chronic progressive liver disease. The Z protein has different physicochemical features compared to the M variant, and will go through spontaneous polymerization. These polymerized macromolecules will aggregate in the endoplasmic reticulum of the hepatocytes . However, the macromolecules can leave the hepatocytes with the help of autophagy. Thus, medication that stimulates autophagy could be the proper treatment of choice for PiZZ persons.

The problem with this theory, however, is that clinical experience does not fully support it. First, only 30-50% of the infants with neonatal cholestasis will develop liver disease, at least under the first 2-3 decades of life, which is not more than 3-5 % of all homozygotes, while 95 % will not, although all of them have relatively similar portion of Z-AAT storing in hepatocytes. Second, PiZZ carriers producing Z-AAT protein have sequestered Z in other, extrahepatic tissues, as well, and therefore one could expect high susceptibility of PiZZ carriers to other pathologies. However, this is not a case.

One major sine-qua-non for the above pathogenetic theory is that eventually all PiZZ homozygous patients develop progressive liver disease. So far, this is not proven. On the contrary, the only prospectively and longitudinally followed cohort, those, by now roughly 80 persons who have been found by the Swedish mass-screening in the mid-70s, show no clinical signs of liver damage, although by now they are in their mid-forties.

Aim of the study: we recently found that SERPINA1 gene express not only full-length AAT protein but also short alternative transcripts, corresponding to C-terminal sequence of AAT. We developed specific antibody to detect the C-terminal peptide of AAT. Our preliminary data show that this peptide in present in the PiZZ cirrhosis liver as a cleavage product of AAT but shows no expression. Such peptide may increase Z  AAT polymer formation but also affect bile acid synthesis by inhibiting 7alpha- and 12alpha- hydroxylases. We previously found that mice injected with the C-36 peptide of AAT shows a decrease in 7alpha- and 12 alpha-hydroxylase mRNA. We predicted that this effect of C-36 peptide of AAT is dependent on peptide binding to the alpha1-fetoprotein transcription factor (FTF) and preventing binding of FTF to its target DNA recognition site, which results in suppression of 7alpha- and 12alpha-hydroxylase transcription. Hence, increased expression of SERPINA1 and levels of C-terminal hydrophobic peptide of AAT may contribute to the liver damage in PiZZ children.

Study design: We conduct a retrospective study using fixated, imbedded material from biopsies performed as an organic part of the routine clinical work-up, as well as follow-up of these children. We will include up to 20 patients each, belonging to one of the following three groups: a) neonatal cholestasis and early progressive liver disease; b) neonatal cholestasis and favorable outcome and c) no neonatal cholestasis and favorable outcome. Depending on the length and nature of the follow-up we want to investigate repeated biopsy specimens from the same patients

The biopsy specimens were taken as part of routine health-care, they have been processed and stored according to the bio-bank regulations.  The identity of the patients is known only by one of the investigators