Journal of Clinical Medicine Article Utility of Transient Elastography for the Screening of Liver Disease in Patients with Alpha1-Antitrypsin Deficiency Mònica Pons 1,† , Alexa Núñez 2,3,† , Cristina Esquinas 2,3 , María Torres-Durán 4 , Juan Luis Rodríguez-Hermosa 5 , Myriam Calle 5 , Ramón Tubio-Pérez 4 , Irene Belmonte 2 , Francisco Rodríguez-Frías 6,7 , Esther Rodríguez 2 , Joan Genescà 1,7 , Marc Miravitlles 2,3,8, * and Miriam Barrecheguren 2 1 Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain; monica_xina@hotmail.com (M.P.); jgenesca@vhebron.net (J.G.) 2 Pneumology Department, Hospital Universitari Vall d’Hebron/Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain; alexagnd01@hotmail.com (A.N.); crise4@hotmail.com (C.E.); irbelmu@gmail.com (I.B.); estherod@vhebron.net (E.R.); mbarrech@vhebron.net (M.B.) 3 Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 4 Pneumology Department, University Hospital Complex of Vigo, Instituto de Investigación Biomédica Galicia Sur, 36213 Vigo, Spain; mtordur@yahoo.es (M.T.-D.); ramon.antonio.tubio.perez@sergas.es (R.T.-P.) 5 Pneumology Department, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC),  Hospital Clínico de San Carlos, Departamento de Medicina, Facultad de Medicina, Universidad Complutense  de Madrid, 28040 Madrid, Spain; jlrhermosa@yahoo.es (J.L.R.-H.); mcallerubio@gmail.com (M.C.) Citation: Pons, M.; Núñez, A.; 6 Department of Clinical Biochemistry, Hospital Universitari Vall d’Hebron, Vall d’Hebron Barcelona Hospital Esquinas, C.; Torres-Durán, M.; Campus, 08035 Barcelona, Spain; frarodri@gmail.com 7 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Rodríguez-Hermosa, J.L.; Calle, M.; Tubio-Pérez, R.; Belmonte, I.; 28029 Madrid, Spain 8 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain Rodríguez-Frías, F.; Rodríguez, E.; * Correspondence: marcm@separ.es et al. Utility of Transient † Both authors have contributed equally and should be considered first authors. Elastography for the Screening of Liver Disease in Patients with Abstract: Screening of liver disease in alpha-1 antitrypsin deficiency (AATD) is usually carried out Alpha1-Antitrypsin Deficiency. J. Clin. Med. 2021, 10, 1724. https://doi.org/ with liver enzymes, with low sensitivity. We conducted a multicenter cross-sectional study aiming to 10.3390/jcm10081724 describe the utility of transient elastography for the identification of liver disease in patients with AATD. A total of 148 AATD patients were included. Among these, 54.7% were Pi*ZZ and 45.3% Academic Editor: Francisco Dasí were heterozygous for the Z allele. Between 4.9% and 16.5% of patients had abnormal liver enzymes, without differences among genotypes. Liver stiffness measurement (LSM) was significantly higher in Received: 5 March 2021 Pi*ZZ individuals than in heterozygous Z (5.6 vs. 4.6 kPa; p = 0.001). In total, in 8 (5%) individuals Accepted: 13 April 2021 LSM was >7.5 kPa, considered significant liver fibrosis, and ≥10 kPa in 3 (1.9%) all being Pi*ZZ. Published: 16 April 2021 Elevated liver enzymes were more frequently observed in patients with LSM > 7.5 kPa, but in 5 out of 8 of these patients all liver enzymes were within normal range. In patients with AATD, the presence Publisher’s Note: MDPI stays neutral of abnormal liver enzymes is frequent; however, most of these patients do not present significant with regard to jurisdictional claims in liver fibrosis. Transient elastography can help to identify patients with liver fibrosis even with normal published maps and institutional affil- liver enzymes and should be performed in all Z-allele carriers to screen for liver disease. iations. Keywords: alpha1-antitrypsin deficiency; liver disease; transient elastography Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Alpha1-antitrypsin deficiency (AATD) is caused by a specific mutation of the SER- conditions of the Creative Commons PINA 1 gene which results in abnormal production and low circulating levels of alpha1- Attribution (CC BY) license (https:// antitrypsin (AAT). It is one of the most common genetic diseases in adulthood and is creativecommons.org/licenses/by/ associated with an increased risk of developing pulmonary emphysema and liver dis- 4.0/). ease [1,2]. J. Clin. Med. 2021, 10, 1724. https://doi.org/10.3390/jcm10081724 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 1724 2 of 13 AAT is a protein synthesized and secreted mainly by hepatocytes, the main function of which is to protect lung tissue from damage caused by proteolytic enzymes such as neutrophil elastase [2]. AAT is a highly polymorphic protein with more than 120 variants, including about 60 deficient alleles. The normal allele, present in more than 95% of normal subjects, is called M [1,2]. The most frequent deficient alleles are S and Z, and they are found in 10% and 2% of the Spanish population, respectively [3–6]. The Z variant presents an alteration in its tertiary structure that facilitates misfolding of the protein and gives rise to the spontaneous formation of polymers, leading to the accumulation of the protein in the endoplasmic reticulum of the hepatocytes [7,8]. Liver damage is then caused by this protein accumulation, inducing apoptosis of the hepatocytes and a compensatory hepatocyte proliferation that eventually produces liver fibrosis that can evolve to cirrhosis or hepatocellular carcinoma [8,9]. Most patients with liver disease are homozygous for the deficient Z allele (Pi*ZZ), although different degrees of liver in- volvement have been described in heterozygotes (Pi*SZ and Pi*MZ), especially if associated with other co-factors such as alcohol consumption or metabolic syndrome [10,11]. Currently there is no non-invasive gold standard technique for the screening and early diagnosis of liver disease in patients with AATD [12]. In clinical practice, liver enzymes are routinely checked, while liver ultrasound is performed if necessary. However, it has been observed that transaminase levels have a low sensitivity to identify liver disease, and they correlate little with the degree of liver disease, especially in adulthood [13]. Serum biomarkers and image devices based on elastography technique have been developed to overcome this problem and to assess the presence of fibrosis in liver diseases of different etiologies [14,15]. Recently, there has been increasing interest in the use of elastographic methods, such as transient elastography, for screening liver disease in AATD patients [16–18]. However, the screening and management of asymptomatic liver disease in AATD can differ among centers due to a lack of consensus or guidelines. Therefore, the aim of our study was to describe the utility of transient elastography for the identification of liver disease in patients with AATD. 2. Materials and Methods This was a multicenter cross-sectional study including patients older than 18 years with mild, moderate, and severe AATD (Pi*MS, SS, MZ, SZ, ZZ, and rare variants) consecu- tively recruited from the outpatient Pneumology Clinics of three AATD reference centers in Spain (Vall d’Hebron University Hospital, Barcelona, University Hospital Complex of Vigo, and Hospital Clínico San Carlos, Madrid) from 1 April 2017 to 1 January 2020. As part of the assessment of patients with AATD, all of them were offered blood analysis, full lung function tests, and transient elastography, and the only exclusion criterion was to refuse to sign informed consent. The study was approved by the Vall d’He- bron Hospital Ethics Committee (Barcelona, Spain), number PR(AG)335/2016, and all patients provided written informed consent. 2.1. Variables During the first visit, a complete physical examination was performed in all patients with special interest in signs of chronic liver disease such as splenomegaly, jaundice, or palmar erythema. Sociodemographic and clinical characteristics were collected and other parameters such as body mass index (BMI), lung function tests (forced expiratory volume in the first second (FEV1), FEV1/forced ventilatory capacity (FVC), and carbon monoxide transfer coefficient (KCO)), comorbidities, treatments, and AAT augmentation therapy were reported. Diagnosis of chronic obstructive pulmonary disease (COPD) was established when the post-bronchodilator FEV1/FVC ratio was below 0.7. Blood samples were obtained for determination of liver function tests: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), international normalized ratio (INR), platelet count, J. Clin. Med. 2021, 10, 1724 3 of 13 and albumin. In addition, the Fibrosis-4 (FIB-4) score was calculated as age (years) × AST √ [IU/L]/(platelet count [109/L] × ALT [IU/L]) and AST-to-platelet ratio index (APRI) as (AST [IU/L]/40 IU/L)/platelet count [109/L] × 100. Patients were classified according to the previously established FIB-4 cut-offs of <1.45 with a high negative predictive value for ruling out advanced fibrosis and >3.25 with a high specificity and a 65% positive predictive value for ruling in advanced fibrosis [19]. For APRI, we used the cut-off <0.5 for excluding cirrhosis (high negative predictive value) and >1.0 as a high specific cut-off for predicting cirrhosis [20]. The Enhanced Liver Fibrosis (ELF) test (Siemens Healthcare Diagnostics, Vienna, Austria) was available as a biomarker of liver fibrosis in one of the centers. The ELF test is a panel of markers that consists of 3 components: Type III procollagen peptide, hyaluronic acid, and tissue inhibitor of metalloproteinase-1. We explored the manufacturer- recommended 9.8 cut-off to rule in advanced fibrosis [21]. 2.2. Liver Stiffness Measurement by Transient Elastography Liver stiffness measurements (LSM) were performed in a fasting state using a Fi- broscan 502 Touch (Echosens, Paris, France) using the M or XL probe as per device indi- cation. Quality criteria used in all centers were at least 10 valid measurements and an interquartile-to-median ratio ≤ 30%. The LSM technique was carried out in accordance with the European Association for Study of the Liver (EASL) clinical guidelines [22]. Results were expressed in kilopascals (kPa). Normal liver stiffness values are around 5 kPa. Transient elastography has good re-producibility and has good diagnostic perfor- mance for estimating liver fibrosis. However, the accuracy is not as good for detecting significant fibrosis compared to advanced fibrosis or cirrhosis [22,23]. Since there are no specific LSM cut-offs for AATD liver disease, a LSM > 7.5 kPa was used as suggestive of significant fibrosis and ≥10 kPa was suggestive of advanced fibrosis according to previ- ously established cut-offs in other liver diseases (mainly viral etiologies and alcoholic liver disease) [22,24]. The presence of steatosis was assessed by the controlled attenuation parameter (CAP) and results were expressed in decibel per meter (dB/m). The cut-off >268 dB/m was used as an indicator of moderate steatosis, and for severe steatosis the cut-off was >280 dB/m [25]. 2.3. Statistical Analysis Qualitative variables were described with absolute frequencies and percentages. The description of quantitative variables was performed using the mean, standard deviation (SD) or median, and interquartile range (IQR). The Kolmogorov–Smirnov test was used to assess the normality of distributions. Patient characteristics were compared according to genotypes and other clinical con- ditions. In the case of quantitative variables, the Student’s t-test for normally distributed variables or the Mann–Whitney U-test if normality was not assumed was used, while ANOVA tests were performed in the case of variables with more than 2 categories. The Chi-squared test (Fisher test for frequencies < 5) was used for the comparison of categorical variables. A linear relationship between quantitative variables, in particular between surrogates of liver disease (LSM, CAP and FIB-4) and spirometric markers of airflow ob- struction (FEV1(%) and FEV1/FVC), were analyzed using Spearman tests. For all the tests, p-values < 0.05 were considered statistically significant. The statistical package R Studio (V2.5.1) was used for the analyses. 3. Results 3.1. Demographic and Clinical Findings A total of 148 AATD patients were included from January 2017 to December 2019. Among these, 81 (54.7%) were homozygous Pi*ZZ and 67 (45.3%) were heterozygous for the Z allele (29 Pi*SZ, 35 Pi*MZ, 1 Pi*FZ, 1 Pi*PlowellZ, 1 Pi*MmaltonZ). J. Clin. Med. 2021, 10, 1724 4 of 13 The mean age was 52.5 and 57 years for heterozygous and Pi*ZZ, respectively, and 50% of the patients were male. Liver disease in infancy was reported as the cause of the diagnosis of AATD in 19.4% and 11.1% of heterozygous and homozygous patients, although there were no patients with an active diagnosis of liver disease at the time of the study. COPD was diagnosed in 22.7% of heterozygous subjects and up to 70% for Pi*ZZ patients. Consequently, the mean FEV1 (%) was significantly lower in Pi*ZZ compared with heterozygous (69% (SD: 30.5%) versus 92.9% (SD: 27.6%); p < 0.001). The baseline characteristics of the global population and the two genotype groups are shown in Table 1. Table 1. Baseline characteristics of the patients included by AAT genotype. Heterozygous Z ZZ (n = 81) p-Value (n = 67) Age 57.0 (14.4) 52.5 (14.5) 0.051 1 Sex, men 41 (50.6%) 34 (50.7%) 0.985 2 BMI 25.1 (3.9) 24.0 (7.0) 0.398 1 Smoking exposure: 0.010 2 Active 43 (53.1%) 22 (32.8%) Former smoker 7 (8.6%) 16 (23.9%) Never smoker 31 (38.3%) 29 (43.3%) Alcohol consumption 19 (23.5%) 19 (28.4%) 0.991 2 Diabetes mellitus 0 (0%) 2 (3.0%) 0.203 2 Hypertension 14 (17.5%) 16 (23.9%) 0.453 2 AAT levels, mg/dL 33.3 (61.9) 71.9 (20.8) <0.001 1 Reason for diagnosis: 0.002 2 Liver disease 9 (11.1%) 13 (19.4%) Lung disease 52 (64.2%) 23 (34.3%) Family study 17 (21.0%) 28 (41.8%) Other 3 (3.7%) 3 (4.5%) COPD 57 (70.4%) 15 (22.7%) <0.001 2 Asthma 5 (7.8%) 14 (21.2%) 0.056 2 Neonatal jaundice 6 (7.4%) 3 (4.5%) 0.513 2 FVC, L 3.6 (1.5) 3.9 (1.1) 0.197 1 FVC, % 90.0 (28.5) 99.8 (19.8) 0.033 1 FEV1, L 2.1 (1.2) 3.0 (1.2) <0.001 1 FEV1, % 69.0 (30.5) 92.9 (27.6) <0.001 1 FEV1/FVC 0.6 (0.2) 0.7 (0.2) 0.001 1 KCO, % 51.0 (32.5) 58.9 (36.7) 0.231 1 Footnote: BMI: Body mass index; COPD: Chronic obstructive pulmonary disease; FVC: Forced ventilatory capacity; FEV1: Forced expiratory volume in 1 s; KCO: Transfer coefficient of the lung for carbon monoxide; AAT: Alpha-1 antitrypsin. 1 Mann–Whitney U-test p-value, 2 Chi-squared p-value. 3.2. Clinical and Laboratory Signs of Liver Disease Thirty-two patients (21.6%) had abnormal liver enzymes. The distribution of values showed significant differences only in AST values, which were significantly higher in Pi*ZZ patients (29.2 UI/L (SD: 15.4) vs. 25.0 UI/L (SD: 8.0; p = 0.029). The most frequent pattern was an elevation in GGT (14.9% of patients). Pi*ZZ patients had a higher FIB-4 score compared to heterozygous Z (1.6 (SD: 0.8) vs. 1.2 (SD:0.5); p < 0.001). Only 5 patients J. Clin. Med. 2021, 10, 1724 5 of 13 had FIB-4 > 3.25 and all were Pi*ZZ. The APRI score was higher in Pi*ZZ patients than in heterozygous Z (0.35 (SD: 0.18) vs. 0.27 (SD: 0.09); p = 0.007), but most of the patients had APRI values < 0.5, excluding advanced fibrosis or cirrhosis, and only one Pi*ZZ patient had an APRI score > 1.0. The ELF score was obtained in 52 patients (27 Pi*ZZ and 25 Pi*Z patients). Pi*ZZ had significantly higher values compared to Pi*Z phenotypes (8.6 (SD: 0.8) vs. 8 (SD: 0.6); p = 0.007). Only 1 Pi*ZZ patient showed values above the cut-off of 9.8 (Table 2). Table 2. Results of blood analysis and transient elastography in patients with different AAT genotypes. Heterozygous Z ZZ (n = 81) p-Value (n = 67) Laboratory findings Platelet count, ×109 /L 222 (59) 239 (61) 0.074 1 INR 1.0 (0.2) 1.0 (0.1) 0.067 1 Bilirubin, mg/dL 0.8 (0.5) 0.7 (0.3) 0.158 AST, IU/L 29.2 (15.4) 25.0 (8.0) 0.029 1 AST > ULN 4 (4.9%) 4 (6%) 0.869 2 ALT, IU/L 26.6 (22.6) 26.1 (13.4) 0.967 1 ALT > ULN 6 (7.4%) 5 (7.5%) 0.952 2 ALP, IU/L 78.2 (29.6) 81.8 (21) 0.412 1 ALP > ULN 6 (7.4%) 2 (3%) 0.294 2 GGT, IU/L 36.2 (33.9) 31.1 (29.4) 0.336 1 GGT > ULN 13 (16.5%) 9 (13.6%) 0.637 2 Albumin, g/dL 4.3 (0.6) 4.4 (0.3) 0.044 1 Cholesterol, mg/dL 207 (35) 198 (36) 0.161 FIB-4 1.6 (0.8) 1.2 (0.5) <0.001 FIB-4 < 1.45 38 (47.5%) 51 (78.5%) <0.001 2 FIB-4 > 3.25 5 (6.2%) 0 0.065 2 APRI 0.35 (0.18) 0.27 (0.09) <0.001 1 APRI < 0.5 67 (83) 64 (91) 0.023 2 APRI > 1.0 1 (1.2) 0 0.956 ELF, n = 60 8.6 (0.8) 8 (0.6) 0.007 1 Transient elastography LSM 5.6 (2.4) 4.6 (1.2) 0.001 1 LSM > 7.5 kPa 8 (9.9%) 0 0.040 2 LSM ≥ 10 kPa 3 (3.7%) 0 CAP 256 (59) 253 (50) 0.252 1 CAP 268–280 dB/m 7 (8.6%) 4 (6%) 0.807 2 CAP > 280 dB/m 26 (32.1%) 21 (31.3%) Footnote: INR: International normalized ratio; ULN: Upper limit of normal; AST: Aspartate aminotransferase; ALT: Alanine amino- transferase; ALP: Alkaline phosphatase; GGT: Gamma-glutamyl transferase; FIB-4: Fibrosis 4; APRI: AST to platelet ratio index; ELF: Enhanced liver fibrosis; LSM: Liver stiffness measurement; CAP: Controlled attenuation parameter. 1 Mann–Whitney U-test p-value, 2 Chi-squared p-value. J. Clin. Med. 2021, 10, x FOR PEER REVIEW 6 of 13 3.3. Transient Elastography J. Clin. Med. 2021, 10, 1724 The mean LSM was significantly higher in Pi*ZZ individuals than in heterozygous Z 6 of 13 (5.6 (SD: 2.5) kPa vs. 4.6 (SD:1.2) kPa, respectively; p = 0.007). In total, LSM was >7.5 kPa in 8 (5%) individuals and ≥10 kPa in 3 (1.9%), all being Pi*ZZ (Figure 1). By lowering the cut-off of LSM to >7.1 kPa as suggested in other studies [11], we found 10 Pi*ZZ patients 3.3. Transient Elastography (12.3%) and 3 heterozygous patients (4.5%), two of whom were Pi*SZ patients with LSM Theand 7.3 kPa, mean oneLSM waswas significantly a Pi*MZ patient higher with LSM in Pi*ZZ 7.5 kPa.individuals than in heterozygous Z (5.6 (SD: Using the LSM > 8.45 kPa cut-off of the study by0.007). 2.5) kPa vs. 4.6 (SD:1.2) kPa, respectively; p = Clark Inet total, LSM al. [13], wewas >7.5have would kPa in 8 (5%) individuals and ≥ 10 kPa in 3 (1.9%), all being identified 4 Pi*ZZ patients (4.9%) suggestive of having F ≥ 3. Pi*ZZ (Figure 1). By lowering the cut-off of LSM to >7.1 kPa as suggested in other studies [11], we found 10 Almost one-third of the patients had severe steatosis according to CAP values > 280Pi*ZZ patients (12.3%) and no dB/m, with 3 heterozygous patients (4.5%), significant differences twohomozygous between of whom were andPi*SZ patients with heterozygous LSM patients. 7.3 kPa, and (Table 2). one was a Pi*MZ patient with LSM 7.5 kPa. Figure 1. Comparison of mean LSM values by phenotype. Figure 1. Comparison of mean LSM values by phenotype. Using the LSM > 8.45 kPa cut-off of the study by Clark et al. [13], we would have identified 3.4. 4 Pi*ZZ of Characteristics patients (4.9%) suggestive Pi*ZZ Patients According of having to LSM F ≥ 3. Values Almost Pi*ZZ individuals with LSM > 7.5 kPa were older and had to one-third of the patients had severe steatosis according CAP values a higher BMI.>Two-thirds 280 dB/m, with consumed alcohol, and all had COPD (versus 67% in patients with LSM ≤ 7.5 (Table no significant differences between homozygous and heterozygous patients. kPa; p2). = 0.097). 3.4. Characteristics of Pi*ZZ Patients According to LSM Values Elevated liver enzymes were more frequently observed in patients with LSM > 7.5 kPa. Pi*ZZ individuals Twenty-five percentwith LSM > with of patients 7.5 kPa LSMwere > 7.5older andelevated kPa had had a higher BMI. com- AST values Two- thirds consumed alcohol, and all had COPD (versus 67% in patients with LSM ≤ 7.5 pared to 2.7% in patients with LSM ≤ 7.5 kPa (p = 0.048), and 37.5% of patients with LSM kPa; p = 0.097). > 7.5 kPa had elevated GGT compared to 14.1% of patients with LSM ≤ 7.5 kPa (p = 0.120) Elevated liver enzymes were more frequently observed in patients with LSM > 7.5 kPa. (Table 3, Figure 2). Conversely, 11/61 patients (18%) had at least one elevated liver enzyme Twenty-five percent of patients with LSM > 7.5 kPa had elevated AST values compared to but with normal LSM values (LSM < 6 kPa). Correlations between LSM and liver enzymes 2.7% in patients with LSM ≤ 7.5 kPa (p = 0.048), and 37.5% of patients with LSM > 7.5 kPa were only significant, albeit weakly, between LSM and AST (0.311 (p < 0.001)), and LSM had elevated GGT compared to 14.1% of patients with LSM ≤ 7.5 kPa (p = 0.120) (Table 3, and GGT (0.389 (p < 0.001)). Figure 2). Conversely, 11/61 patients (18%) had at least one elevated liver enzyme but with normal LSM values (LSM < 6 kPa). Correlations between LSM and liver enzymes were only significant, albeit weakly, between LSM and AST (0.311 (p < 0.001)), and LSM and GGT (0.389 (p < 0.001)). J. Clin. Med. 2021, 10, 1724 7 of 13 Table 3. Comparison between Pi*ZZ individuals based on liver stiffness (LSM) and diagnosis of chronic obstructive pulmonary disease (COPD). LSM ≤ 7.5 LSM > 7.5 No COPD COPD p-Value p-Value (n = 73) (n = 8) (n = 24) (n = 57) Age 56.2 (14.5) 64.9 (11.4) 0.076 46.2 (14.5) 61.6 (11.7) <0.001 1 Sex, men 37 (50.7%) 4 (50%) 1.00 11 (45.8%) 30 (52.6%) 0.752 2 BMI 24.6 (3.4) 29.0 (5.3) 0.056 24.2 (3.7) 25.4 (3.9) 0.186 1 Smoking exposure: 0.527 <0.001 2 Active 37 (50.7%) 6 (75%) 7 (29.2%) 36 (63.2%) Former smoker 7 (9.6%) 0 (0%) 0 (0%) 7 (12.3%) Never smoker 29 (39.7%) 2 (25%) 17 (70.8%) 14 (24.6%) Alcohol consumption 16 (24.2%) 3 (60%) 0.115 5 (25%) 14 (27.2) 1.000 2 Hypertension 10 (13.9%) 4 (50%) 0.028 1 (4.2%) 13 (23.2%) 0.054 2 COPD 49 (67.1%) 8 (100%) 0.097 0 57 (100%) 0.001 2 Neonatal jaundice 6 (8.2%) 0 (0%) 1.000 4 (16.7%) 2 (3.5%) 0.060 2 FEV1, % 70.4 (30.7) 56.4 (27.3) 0.205 99.6 (13.1) 56.2 (26.3) <0.001 1 Laboratory findings: Platelet count, ×109 /L 224 (60) 202 (49) 0.267 210 (48) 226 (62) 0.214 1 INR 1.0 (0.2) 1.1 (0.1) 0.378 1.0 (0.1) 1.1 (0.2) 0.040 1 Bilirubin, mg/dL 0.8 (0.5) 0.6 (0.2) 0.262 1.0 (0.9) 0.7 (0.2) 0.143 1 AST, UI/L 27.2 (10.1) 47.6 (34.7) 0.141 27.2 (10.6) 30.0 (16.9) 0.375 1 AST > ULN * 2 (2.7%) 2 (25%) 0.048 1 (4.2%) 3 (5.3%) 0.675 2 ALT, UI/L 24.2 (14.2) 48.8 (55.8) 0.254 25.5 (14.4) 27.1 (25.3) 0.719 1 ALT > ULN * 4 (5.5%) 2 (25.0%) 0.108 3 (12.5%) 3 (5.3%) 0.226 2 ALP, UI/L 78.5 (30.9) 75.9 (14.8) 0.816 70.3 (31) 81.4 (28.7) 0.130 1 ALP > ULN * 6 (8.5%) 0 (0%) 1.000 2 (8.3%) 4 (7.0%) 1.000 2 GGT, UI/L 31.8 (19.3) 75.6 (84.1) <0.001 33.2 (22.2) 37.2 (37.7) 0.685 1 GGT > ULN * 10 (14.1%) 3 (37.5%) 0.120 5 (20.8%) 8 (14%) 0.589 2 Albumin, g/dL 4.3 (0.6) 4.4 (0.3) 0.615 4.5 (0.3) 4.2 (0.6) 0.004 1 Cholesterol, mg/dL 206 (35) 208 (39) 0.901 205 (39) 207 (34) 0.824 1 FIB-4 1.5 (0.8) 2.2 (0.7) 0.032 1.3 (0.8) 1.7 (0.8) 0.046 1 FIB-4 < 1.45: 37 (50.7%) 1 (12.5%) 0.059 15 (62.5%) 23 (40.4%) 0.077 2 FIB-4 > 3.25: 4 (5.5%) 1 (12.5%) 0.418 1 (4.2%) 4 (7%) 1.000 2 APRI 0.33 (0.1) 0.56 (0.3) <0.001 0.35 (0.17) 0.35 (0.19) 0.992 1 Transient elastography LSM 5.0 (1.1) 10.8 (4.6) 0.009 5.3 (1.1) 5.7 (2.8) 0.361 1 CAP 249 (56) 318 (48) 0.004 233 (56) 266 (58) 0.023 1 LSM > 7.5 kPa: 0 8 (100%) NA 0 8 (14.0%) 0.097 2 Footnote: BMI: Body mass index; COPD: Chronic obstructive pulmonary disease; FEV1: Forced expiratory volume in 1 s; AAT: Alpha- 1 antitrypsin; INR: International normalized ratio; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; GGT: Gamma-glutamyl transferase; ULN: Upper limit of normal; FIB-4: Fibrosis 4; APRI: AST to platelet ratio index; ELF: Enhanced liver fibrosis; LSM: Liver stiffness measurement; CAP: Controlled attenuation parameter. *: Upper limit of normal according to sex-specific cut-offs: For AST and ALT: >35 IU/L in female, >50 IU/L in male; for ALP: >120 IU/L for both genders; for GGT: >38 IU/L in females and >55 IU/L in males. 1 Mann–Whitney U-test p-value, 2 Chi-squared p-value. J.J.Clin. Clin.Med. Med.2021, 10,x1724 2021,10, FOR PEER REVIEW 8 8ofof13 13 Figure 2. All individuals from the cohort with liver enzymes above the highest level of normal based on LSM values. UPN: Upper limit of normal for GGT: >38 IU/L in females and >55 IU/L in males. Among the 8 patients with LSM > 7.5, 3 had GGT above the normal limit and 1 also Figure 2. All individuals from the cohort with liver enzymes above the highest level of normal based had a Figure FIB-4 score > 3.25from 2. values. All individuals (Figure 3). Thewith the cohort FIB-4 liver score (2.2 above enzymes (SD: 0.7) versus level the highest 1.5 (SD: 0.8); p = of normal on LSM UPN: Upper limit of normal for GGT: >38 IU/L in females and >55 IU/L in males. 0.032), as well as CAP measurement (317.9 (SD: 48) dB/m vs. 249.6 (SD: based on LSM values. UPN: Upper limit of normal for GGT: >38 IU/L in females and >55 IU/L in 56.5) dB/m; p= 0.004), were also males.Among thehigher in Pi*ZZ 8 patients withpatients LSM > 7.5, with LSMGGT 3 had > 7.5above kPa (Table 3). Severe the normal limitsteatosis, and 1 also with CAP > 280 dB/m, was present in 6 patients (75%) with LSM had a FIB-4 score > 3.25 (Figure 3). The FIB-4 score (2.2 (SD: 0.7) versus 1.5 (SD: > 7.5 kPa compared to 20 0.8); patients (27.4%) Among thewith 8 LSM patients< 7.5 withkPa LSM (p = > 0.041). 7.5, 3 had GGT above the p = 0.032), as well as CAP measurement (317.9 (SD: 48) dB/m vs. 249.6 (SD: 56.5) dB/m; normal limit and 1 also p =The had APRI a FIB-4 0.004), were was score >higher also3.25 higherin in Pi*ZZ (Figure 3).patients Pi*ZZ The with FIB-4 patients LSM score with >7.5 (2.2 LSM kPa >(SD: 7.5 than 0.7) kPa in those versus (Table 3).1.5 with LSMp≤ = (SD: steatosis, Severe 0.8); 7.5 kPa CAP 0.032), with (0.56 >vs.280 as well 0.33, CAPp < 0.001). as dB/m, The APRI measurement was present 6had (317.9 in a(SD: patientssignificant 48) dB/m (75%) correlation with 249.6>with vs.LSM (SD: LSM 56.5) 7.5 kPa (rdB/m; = 0.353, compared p to = p 0.004), =200.030).were also higher in Pi*ZZ patients patients (27.4%) with LSM < 7.5 kPa (p = 0.041). with LSM > 7.5 kPa (Table 3). Severe steatosis, with CAP > 280 dB/m, was present in 6 patients (75%) with LSM > 7.5 kPa compared to 20 patients (27.4%) with LSM < 7.5 kPa (p = 0.041). The APRI was higher in Pi*ZZ patients with LSM >7.5 kPa than in those with LSM ≤ 7.5 kPa (0.56 vs. 0.33, p < 0.001). The APRI had a significant correlation with LSM (r = 0.353, p = 0.030). Figure 3. Relation between elevated GGT, FIB4, and LSM in Pi*ZZ patients. GGT: Gamma-glutamyl Figure 3. Relation transferase; between FIB-4: Fibrosiselevated 4; LSM: GGT, Liver FIB4, andmeasurement; stiffness LSM in Pi*ZZUPL: patients. Upper GGT: limitGamma-glu- of normal (accord- tamyl ing to sex-specific cut-offs: for GGT: >38 IU/L in females and >55 IU/L in males). limit of normal transferase; FIB-4: Fibrosis 4; LSM: Liver stiffness measurement; UPL: Upper (according to sex-specific cut-offs: for GGT: >38 IU/L in females and >55 IU/L in males). The APRI was higher in Pi*ZZ patients with LSM > 7.5 kPa than in those with 3.5. Comparison LSM ≤ 7.5 kPa between Pi*ZZ (0.56 vs. 0.33,Patients withThe p < 0.001). or without APRI hadCOPDa significant correlation with LSM Figure0.353, p = 0.030). (r =Fifty-seven 3. RelationPi*ZZ between patients (70.4%) elevated GGT, had FIB4,COPD. and LSM Pi*ZZ patients in Pi*ZZ with patients. COPD GGT: were older Gamma-glu- and more tamyl frequently transferase; had FIB-4: a history Fibrosis of smoking 4; LSM: compared Liver stiffness with non-COPD measurement; UPL: Upper individuals. As limit of normal 3.5. Comparison (according expected, they hadbetween to sex-specific worse Pi*ZZ cut-offs: Patients for GGT:with lung function with ora without >38 IU/L lower COPD in females FEV1 and (1.6>55 IU/L (SD: in males). 0.8) L vs. 3.5 (SD: 1) Fifty-seven L; p < 0.001) Pi*ZZ patients and KCO(%) (43.7% (SD:(70.4%) hadvs. 30.8%) COPD. Pi*ZZ 68% (SD: patients 30.4%); p =with COPD were older 0.003). 3.5. andComparison between more frequently Pi*ZZ had Patients a history with or without of smoking COPD compared with non-COPD individuals. As expected, they had Fifty-seven Pi*ZZworse lung (70.4%) patients functionhadwith a lower COPD. FEV1patients Pi*ZZ (1.6 (SD: 0.8)COPD with L vs. 3.5 (SD: were 1) L; older p < more and 0.001)frequently and KCO (%) had(43.7% a history(SD: of 30.8%) smoking vs.compared 68% (SD: 30.4%); p = 0.003).individuals. As with non-COPD Regarding the liver study, no differences were expected, they had worse lung function with a lower FEV1 (1.6 (SD: observed in transaminase 0.8) L vs. 3.5levels, (SD:but 1) the FIB-4 score was higher in COPD patients (1.7 (SD: 0.8) vs. L; p < 0.001) and KCO(%) (43.7% (SD: 30.8%) vs. 68% (SD: 30.4%); p = 0.003). 1.2 (SD: 0.8); p = 0.046). More J. Clin. Med. 2021, 10, 1724 9 of 13 individuals in the COPD group had a LSM > 7.5 kPa (14% vs. 0%; p = 0.097) and they also had higher CAP values (265.9 (SD: 58.3) dB/m vs. 233.5 (SD: 55.8) dB/m; p = 0.023) (Table 3). Significant, albeit weak, correlations were found between FIB-4 and FEV1 (mL) (r = −0.350, p = 0.002), and CAP and FEV1 (mL) and FEV1(%) (r = −0.391, p < 0.001 and r = −0.306, p = 0.006, respectively). No significant correlations were found between LSM or ELF and measures of airflow obstruction. 4. Discussion In our study population, we found that 10% of Pi*ZZ individuals had transient elastography results suggestive of liver fibrosis, but none of the heterozygous individuals reached the suggested threshold. Although individuals with higher LSM had higher transaminase levels and FIB-4 scores, normal levels of these biomarkers did not reliably rule out liver disease, since some of the patients with normal values had high LSM values. All patients with high LSM also had COPD. Transient elastography is a non-invasive tool that has proven to be useful in the diagnosis of liver fibrosis of different etiologies. More recently, its utility has also been explored in AATD-related liver disease with promising results [16–18,26]. Although dif- ferent cut-offs have been proposed, there is no validated cut-off of LSM for AATD liver disease. In a study including 94 Pi*ZZ patients with paired LSM and liver biopsies, Clark et al. [26] observed that cut-offs of 5.54 and 8.45 kPa had the highest accuracy for detecting significant fibrosis (≥F2) and advanced fibrosis (≥F3), respectively. However, these cut-offs had a low specificity and a low positive predictive value. Hamesch et al. [17] increased the cut-off for significant fibrosis to >7.1 kPa in order to increase the positive predictive value, confirming the presence of ≥F2 in 22 out of 23 patients with liver biopsies [27], while Guillaud et al. [16] suggested an LSM > 7.2 kPa for significant fibrosis and LSM > 14 kPa for cirrhosis. In another study in 75 patients with AATD, the investigators offered a liver biopsy to all individuals with a LSM > 6 or altered liver enzymes in combination with an abnormal ultrasound. Among the 11 biopsies analyzed, they found that the LSM scores in patients with moderate or severe fibrosis were >8 kPa [18]. According to these results and the cut-offs previously established in other etiologies, we chose an arbitrary cut-off of LSM > 7.5 kPa as suggestive of significant fibrosis, and LSM ≥ 10 kPa as advanced fibrosis/cirrhosis. In our sample, there were two Pi*SZ patients with LSM = 7.3 kPa, one of whom was overweight and had diabetes mellitus and increased GGT values, and the other was a Pi*MZ patient with LSM = 7.5 kPa without other identified risk factors of liver disease. Since the etiology of liver disease has an impact on LSM and the data on AATD induced liver disease are limited [28], further studies are needed to validate the best LSM cut-off for screening of liver disease in AATD. Ten percent of Pi*ZZ patients in our cohort had LSM > 7.5 kPa, similar to the preva- lence of liver fibrosis reported in initial studies in AATD patients, which varied from 10–15% in clinical studies [29,30] to 37% in autopsy studies [31]. More recently, with the development of transient elastography, there has been growing interest in the early detec- tion of liver disease in AATD. The study by Guillaud et al. [16] described 5 patients (18%) with LSM suggestive of significant fibrosis and 2 patients (7%) with LSM suggestive of ad- vanced liver fibrosis/cirrhosis. Other studies have reported a higher prevalence; Hamesch et al. [17] described a prevalence of liver fibrosis of 23.6% among 403 Pi*ZZ individuals and observed that liver disease was 9 to 20 times more frequent in this population compared to non-AAT-deficient individuals. In a cohort of COPD Pi*ZZ patients referred for lung transplantation, Morer et al. [32] found that 13% of patients had significant fibrosis (F2) and 8% advanced fibrosis (≥F3). Similar to these numbers, 8 (14%) of our COPD Pi*ZZ patients had LSM > 7.5 kPa, while in 3 (5.7%) LSM was higher than 10 kPa, suggesting the presence of advanced fibrosis. In our cohort, Pi*MZ individuals had lower values of LSM compared to Pi*ZZ indi- viduals. The mean LSM was 4.7 kPa for the 34 Pi*MZ patients included. None of these patients had values above 7.5, and only one had LSM = 7.5 kPa. In this patient, other J. Clin. Med. 2021, 10, 1724 10 of 13 co-factors for liver disease such as obesity, alcohol consumption, or metabolic syndrome were not found. The incidence of liver disease could be higher in heterozygous Z than in the general population, although some authors have hypothesized that while the Pi*MZ genotype acts as a disease modifier, it is not sufficient per se to trigger clinically relevant liver impairment [33]. In a study that analyzed 1184 individuals with non-alcoholic fatty liver disease (NAFLD) and 2462 with chronic alcohol misuse, the Z variant increased the risk of patients with NAFLD to develop cirrhosis and was more frequently present in alcohol misusers with cirrhosis compared to those without significant liver injury [34]. In contrast, a recent analysis of data from the European alpha-1 liver cohort showed that 10% out 419 Pi*MZ had LSM values ≥ 7.1 kPa compared with 4% of non-Z carriers. After adjusting for potential confounders, Pi*MZ individuals still had significantly higher odds for LSM ≥ 7.1 kPa [12]. There is agreement that, in coexistence with other risk factors, and especially in the context of alcohol misuse or NAFLD, Z carriage is a strong risk factor for the development of cirrhosis [17,18] and may also lead to faster hepatic decompen- sations [35]. In our cohort, 60% of Pi*ZZ patients with LSM > 7.5 kPa had some alcohol consumption and had a higher BMI than those with LSM ≤ 7.5 kPa, and, therefore, these factors could have contributed to the progression of liver disease. Liver enzymes have often been used to screen liver disease in AATD in clinical practice [36]. In our cohort, elevated liver enzymes and FIB-4 were more frequently observed in patients with LSM > 7.5 kPa, but normal levels were also frequently present in patients with high LSM. In fact, liver enzyme alterations ranged from only 25% of cases for AST and ALT to 37.5% for GGT in Pi*ZZ patients with LSM > 7.5 kPa. Patients with fibrosis or even cirrhosis may present normal serum liver enzymes [11], and this has also been observed in Pi*ZZ individuals [13,17]. On the other hand, up to 10% of AATD patients with normal liver function tests and ultrasound may have increased LSM values [16]. Furthermore, an increase in ALT has a low sensitivity for identifying liver disease in AATD individuals [13,15]. In the European alpha-1 liver cohort, heterozygous Pi*MZ carriers also had higher serum transaminases compared to non-carriers, although this percentage varied from 5.4% to 28.6% and was higher in individuals older than 50 years [12]. The relationship between lung and liver disease in individuals with AATD is contro- versial. The first series of patients with the deficiency suggested that lung and liver disease rarely coexisted in AATD, and liver disease was more frequently reported in AATD never smokers compared to smokers [37,38]. However, more recent studies using new diagnostic techniques have reported more frequent coexistence of the alterations in both organs [39]. In this line, all of our patients with elevated LSM also had COPD, although the correla- tion between lung function and LSM was not significant. Moreover, recruiting patients from respiratory departments may have influenced the high prevalence of COPD among patients with elevated LSM; although they were also older, with higher BMI and with a higher frequency of alcohol misuse compared with patients with normal LSM. Therefore, a clear relationship between elevated LSM and lung disease cannot be established from our results. Our study had some limitations. First, the identification of liver fibrosis was only made by transient elastography as we did not perform liver biopsies. However, as there are no specific treatments for AATD liver disease to date, the performance of an invasive diagnostic technique in otherwise asymptomatic patients may not be justified. Second, this was a cross-sectional study, and data on the evolution of LSM over time were not available. Third, the design of our study did not allow us to investigate a causal relationship between AATD and liver alterations. Our sample size was not big enough for a multivariate analysis adjusted for known confounders of increased liver fibrosis. However, the study had some strengths: We recruited individuals from three reference centers, and, considering that AATD is a rare disease, we reported information from a large series of patients with homozygous and heterozygous AATD. In conclusion, the results of this study support the assessment of liver disease in all AATD Pi*ZZ individuals and heterozygous Pi*Z individuals with additional liver risk J. Clin. Med. 2021, 10, 1724 11 of 13 factors. Transient elastography has been shown to be a valuable tool to screen for AATD liver disease, and collaboration between hepatologists and pneumologists is crucial for pro- viding the best care to AATD patients. Due to the poor correlation between liver enzymes and other serum biomarkers and the underlying liver disease, all Z-allele carriers, even those with normal serum biomarker values, should be screened with transient elastography. Since AATD is a rare disease, international collaboration in large registries is needed to investigate the best screening strategy for lung and liver disease [12,17,40]. Author Contributions: Conceptualization, M.P., A.N., C.E., M.M., and M.B.; methodology, M.P., A.N., and C.E.; formal analysis, C.E.; investigation, M.P., A.N., C.E., M.T.-D., J.L.R.-H., M.C., R.T.-P., I.B., F.R.-F., J.G., M.M., and M.B.; resources, M.T.-D., J.L.R.-H., M.C., R.T.-P., J.G., and M.M.; data curation, M.P., A.N., C.E., M.T.-D., J.L.R.-H. and E.R.; writing—original draft preparation, M.P., A.N., M.M., and M.B.; writing—review and editing, C.E., M.T.-D., J.L.R.-H., M.C., R.T.-P., I.B., F.R.- F., and J.G.; supervision, J.G., M.M., and M.B.; project administration, M.M.; funding acquisition, M.T.-D., J.L.R.-H., M.C., and M.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Grifols through an unrestricted grant from the Catalan Center for Research in Alpha-1 antitrypsin deficiency of the Vall d’Hebron Research Institute (VHIR) in the Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; from the Madrid Center for Research in Alpha-1 antitrypsin deficiency of the Hospital Clínico San Carlos, Madrid, Spain; from the Galicia Center for Research in Alpha-1 antitrypsin deficiency of the University Hospital Complex of Vigo, Spain; as well as a research grant from Fundació Catalana de Pneumologia (FUCAP). Institutional Review Board Statement: The study was approved by the Vall d’Hebron Hospital Ethics Committee (Barcelona, Spain), number PR(AG)335/2016 (28 October 2016). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data are available from the authors upon request. Acknowledgments: Mònica Pons was the recipient of a Rio Hortega contract in the 2018 Strategic Action Health Call from the Instituto de Salud Carlos III for the years 2019–2020. Alexa Núñez was the recipient of a Rio Hortega contract in the 2019 Strategic Action Health Call from the Instituto de Salud Carlos III for the years 2020–2022. We thank Ignacio Martín Granizo and Montserrat Figueira Alvarez for their collaboration in the elastography in the University Hospital Complex of Vigo. Conflicts of Interest: Myriam Calle received speaking or consulting fees from Boehringer Ingelheim, Grifols, Chiesi, CSL Behring, AstraZeneca, GlaxoSmithKline, Menarini, Gebro Pharma, Zambon, and Novartis. There is no real or perceived conflict of interest between these sources and the present paper. Juan Luis Rodríguez-Hermosa received speaking fees from Boehringer Ingelheim, GlaxoSmithKline, Grifols, CSL Behring, Zambon, and Gebro Pharma. 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