Abstract

Patients and Methods

For this single‐center retrospective study, informed consent for genetic testing, biobanking, and survival analysis was obtained from all patients included. This study was approved a priori by the ethics committee of the Medical University of Innsbruck (study number UN4218/AN3478).

Results

In this cohort of 540 patients with cirrhosis, heterozygous Z allele carriers and patients with SERPINA1 genotype MM had comparable liver function at baseline and a significantly higher median MELD‐Na and Model for End‐Stage Liver Disease (MELD) score at the end of follow‐up (Fig. ​(Fig.1A,B;1A,B; Supporting Table 1). Age and underlying etiology of liver disease were not significantly different between the group of MM and MZ patients (Tables ​(Tables11 and ​and2).2). Patient stratification by MELD‐Na quartiles showed a gradually increasing prevalence of risk allele carriers (MZ) in subgroups with higher MELD‐Na scores (Fig. ​(Fig.1C).1C). When individual markers of liver function were compared, albumin and fibrinogen were significantly lower and international normalized ratio (INR) was significantly higher in the MZ group (Table ​(Table2).2). These findings show that across etiologies MZ is associated with more advanced liver disease at the end of follow‐up.

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Figure 1

(A) MELD‐Na and (B) MELD scores of patients with MM or MZ SERPINA1 genotype at the time of follow‐up. (C) Comparison of SERPINA1 genotype frequencies in patients grouped according to MELD‐Na score quartiles. The overall degree of association between increasing MELD‐Na score and MZ prevalence was statistically significant (P = 0.01, 1‐sided Mantel‐Haenszel test for linear‐by‐linear association).

When patients were grouped by transplant status, the prevalence of MZ was highest among liver transplant recipients. Heterozygosity for the Z allele was less prevalent in nontransplanted patients with cirrhosis than in liver transplant patients, but still significantly more common than in a control group of liver donors, representing a control group from the same geographical region (8.8% versus 3%; P = 0.01). This difference was not found for the S allele, which had a similar prevalence among the 3 groups (Fig. ​(Fig.2).2). Taken together, these findings demonstrate that the Z but not the S allele is more prevalent among patients with end‐stage liver disease and liver transplant recipients than in liver donors.

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Figure 2

Comparison of SERPINA1 genotype frequencies in patients with liver cirrhosis, liver transplant recipients, and controls (healthy liver donors).

In search for specific decompensating events in association with PI*MZ, the prevalence of ascites, hepatic encephalopathy, variceal bleeding, and hepatocellular carcinoma was compared between groups of patients with cirrhosis and different SERPINA1 genotypes. This analysis revealed that at the end of follow‐up, hepatic encephalopathy and ascites were significantly more common in MZ patients. No statistically significant differences in variceal bleeding or HCC were observed (Table ​(Table33).

Next, the diagnostic utility of A1AT concentration in serum was assessed for the identification of MZ patients at baseline. As shown in Fig. ​Fig.3,3, mean serum A1AT concentration was significantly lower in the MZ patient group when compared with MM patients (108.0 versus 174.0 mg/dL; P < 0.001). Although A1AT is negatively correlated with MELD‐Na score (ρ = –0.13; P = 0.009; Fig. ​Fig.4),4), binary logistic regression analysis showed that the plasma concentration of A1AT is a MELD‐Na independent predictor of SERPINA1 genotype (P < 0.001). The optimal diagnostic test performance was achieved with a cutoff of 137 mg/dL A1AT concentration (Supporting Fig. 1). An A1AT concentration ≤137 mg/dL had a sensitivity of 84% (95% confidence interval [CI], 70.9%‐92.8%) and a specificity of 86.9% (95% CI, 83.4%‐89.9%) for the diagnosis of risk allele carrier status. The corresponding negative predictive value is 97.7%, and the positive predictive value is 43.2%.

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Figure 3

A1AT plasma concentrations of patients with SERPINA1 genotype MM and MZ at the time of diagnosis of cirrhosis.

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Figure 4

Correlation matrix among A1AT plasma concentration, markers of liver disease, inflammation, and iron metabolism. Tile colors code for direction and magnitude of the correlation. The first 2 decimal digits of ρ are displayed. Only significant correlations are shown.

In search for laboratory parameters associated with A1AT, a significant negative correlation with MELD‐Na and INR as surrogates of liver function was confirmed. In addition, A1AT and C‐reactive protein (CRP) as an indicator of inflammation were positively correlated. A1AT is negatively correlated with transferrin (Tf) saturation and positively with Tf (ρ = –0.28, P < 0.001; ρ = 0.17, P < 0.001; Fig. ​Fig.4),4), but no significant correlation with the acute‐phase reactant ferritin was found.

To study the impact of the MZ genotype on survival, a Kaplan‐Meier curve analysis was carried out. As shown in Fig. ​Fig.5A,5A, the proportion of patients surviving without liver transplantation was lower in the group of Z allele carriers at all time points, when compared with MM patients. However, this difference did not reach statistical significance (P = 0.07). To further stratify survival analysis for established risk factors for liver transplantation and death, patient subgroups with MELD‐Na score ≥15, Tf saturation ≥50%, or Tf ≤ 180 mg/dL were analyzed independently. As shown in Fig. ​Fig.5B‐D,5B‐D, MZ was associated with increased risk of liver transplantation/mortality as compared with MM when stratified for each risk factor. Significant differences were noted in patients with a Tf saturation ≥50% and in patients with Tf ≤ 180 mg/dL. Multivariate Cox regression analysis further showed that in the subgroup of patients with high Tf saturation, the SERPINA1 genotype was a MELD‐Na independent predictor of transplantation/survival (hazard ratio, 1.32; 95% CI, 1.03‐1.70; P = 0.03; Supporting Table 2).

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Figure 5

Kaplan‐Meier analysis: (A) transplant‐free survival in all patients stratified by SERPINA1 genotype. Transplant‐free survival in the subgroup of patients with (B) Tf saturation ≥ 50%, (C) with Tf of ≤180 mg/dL, and (D) with MELD‐Na ≥15 at baseline.

To assess the effect of the SERPINA1 genotype on each outcome of the combined endpoint transplantation/mortality separately, a competitive risk analysis was carried out. The majority of patients underwent liver transplantation, and only a small proportion of patients died. As a result, for the entire cohort and all subgroups analyzed (patients with Tf saturation ≥50%, Tf of ≤180 mg/dL or MELD‐Na ≥15 at baseline), the likelihood for receiving a donor organ was higher in patients with the MZ genotype at all time points, whereas mortality did not differ between subgroups (Supporting Fig. 2).

Discussion

In this cohort of patients with cirrhosis, we found that MZ patients have significantly higher MELD‐Na scores and impaired hepatic synthetic function without differences in kidney function. These findings support the conclusion that the MZ genotype is a risk factor for more advanced disease in patients with cirrhosis.

Hepatocellular accumulation of misfolded A1AT Z protein polymers is an established risk factor for the development of cirrhosis in adults homozygous for the Z allele.15, 16 In contrast, the risk conferred by the MZ genotype is controversial.11, 17 It has been proposed that endoplasmic reticulum (ER)–stress response and ER‐associated degradation can protect affected cells from potential damage and remove the mutant protein. When only a single allele is affected as in MZ patients, these protective mechanisms are more likely to fully compensate for the genetic defect.16, 18 In accordance with this hypothesis, one can speculate that additional hepatocellular stressors such as alcohol, fatty liver, or chronic hepatitis virus infection are more likely to overcharge the cellular ER‐stress response in MZ patients and cause cell death.

Findings from our cohort also demonstrate that higher MELD‐Na scores and more advanced liver disease are reflected in a higher prevalence of patients with the MZ genotype, but not the genotype MS, among patients with liver cirrhosis and liver transplant recipients when compared with liver donors. The prevalence of MZ in the donor group matched the expected frequency of the general Austrian population (2.9%).19

The higher liver transplantation rate among MZ patients was also paralleled by more frequent hepatic decompensation with ascites and hepatic encephalopathy but not due to variceal hemorrhage or occurrence of hepatocellular carcinoma. Epidemiological studies suggest the ZZ patients have an increased risk for the development of hepatocellular carcinoma.15 Obvious differences between previous studies and the present cohort are that liver disease in MZ patients may not just be a mild variant of ZZ A1ATD and the increased risk reported in patients with the A1AT phenotype PI*ZZ could be accounted for by the increased prevalence of cirrhosis in these patients. PI*ZZ therefore is a risk factor for HCC when compared with a control group without cirrhosis, but patients with established cirrhosis heterozygous for the Z allele do not have a higher HCC risk than patients with cirrhosis and SERPINA1 genotype MM.

The risk for reaching the combined endpoint death or liver transplantation was not significantly different for patients with cirrhosis with genotype MZ and MM. In contrast, in the subgroup of patients with low Tf (≤180 mg/dL) or elevated Tf saturation (≥50%), MZ patients had significantly worse outcomes compared with MM controls. This observation further supports previous studies showing an association between A1ATD and iron metabolism.20, 21, 22, 23 The functional link between iron and A1AT in cirrhosis is also reflected in the results of our correlation analysis. Impaired hepatic synthetic function is expected to reduce the concentration of A1AT and Tf, whereas inflammation is expected to increase A1AT but reduce Tf. The observed negative correlation between A1AT and Tf suggests that in cirrhosis, inflammation rather than liver function determines the functional interaction of A1AT and serum iron parameters.

The prognostic implication of the Z allele renders diagnosis of this risk factor important. In contrast to previous studies, we have used genotyping instead of isoelectric focusing for the diagnosis of the SERPINA genotype in unselected patients with cirrhosis. Although median A1AT concentration was significantly lower in MZ patients, the positive predictive value was only 43%. This highlights that quantitative analysis of A1AT concentration in plasma is insufficient to screen for the risk allele. Accordingly, Cox regression analysis did not show that A1AT concentration is a MELD‐Na–independent significant predictor of clinically relevant endpoints. In contrast, MZ is an independent risk factor for liver disease severity, liver transplantation and death in patients with cirrhosis, and elevated Tf saturation.

According to current guidelines, only patients with unexplained chronic liver disease should be tested. Our results indicate that in all patients with cirrhosis, regardless of the underlying etiology Z allele carriers are at higher risk.24 Considering the low positive predictive value of A1AT serum concentration, for accurate risk assessment and prognostication genotyping for the Z allele should be considered the first‐line test in all patients with cirrhosis. Currently, early liver transplantation is the only causal treatment for A1ATD, but targeted therapies are under development, which include chemical chaperones, autophagy stimulating agents, and silencing of A1AT production in the liver (reviewed in Ref. 16).

In conclusion, SERPINA1 genotyping for the Z allele when cirrhosis is diagnosed can assist risk assessment. PI*MZ patients with low Tf concentration or elevated Tf saturation are especially more likely to develop encephalopathy or ascites.

Supporting information

Notes

Stefan Schneeberger consults for Astrellas and Chiesi; is on the speaker's bureau for Astellas, Genzyme, and MSD; and receives grants/contracts (unrestricted) from Sandoz and Chiesi.

The study was funded by the Medical University of Innsbruck. Benedikt Schaefer received a grant from the Verein zur Foerderung der Wissenschaft in Gastroenterologie and Hepatologie. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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