Alpha-1 antitrypsin deficiency (AATD) is an inherited condition characterized by reduced levels of serum AAT due to mutations in the SERPINA1 (Serpin family A member 1) gene. The Pi*S (Glu264Val) is one of the most frequent deficient alleles of AATD, showing high incidence in the Iberian Peninsula. Herein, we describe two new alleles carrying an S mutation but producing a null phenotype: QOVigo and QOAachen. The new alleles were identified by sequencing the SERPINA1 gene in three patients who had lower AAT serum levels than expected for the initial genotype. These alleles are the result of combined mutations in cis in a PI*S allele. Sequencing detected the S mutation in cis with Tyr138Cys (S+Tyr138Cys) in two patients, whereas a third one had the S mutation in cis with Pro391Thr variant (S+Pro391Thr). When expressed in a cellular model, these variants caused strong AAT polymerization and very low AAT secretion to almost undetectable levels. The isoelectric focusing method for plasma AAT phenotyping did not show AAT protein encoded by the novel mutant alleles, behaving as null. We called these alleles PI*S-plus because the S variant was phased with another variant conferring more aggressive characteristics to the allele. The current data demonstrate that the clinical variability observed in AATD can be explained by additional genetic variation, such as dual cis-acting variants in the SERPINA1 gene. The possible existence of other unrevealed variants combined in the PI*S alleles should be considered to improve the genetic diagnosis of the patients.
PI*S alleles are considered as moderately deficient alleles related to alpha-1 antitrypsin (AAT) deficiency (AATD). In this work, we identified and characterize two new alleles carrying the S mutation, but they behaved as null alleles. This was caused by novel missense variants confirmed to be in in cis configuration with the S mutation, and demonstrated that they modify the properties of the PI*S allele in terms of higher intracellular polymerization and no extracellular secretion. We named these alleles as “PI*S-plus” alleles, as they have more pathogenic properties compared to typical PI*S alleles. That means that the genetic diagnosis of AATD, after identifying gene variants, should consider phasing these variants, especially when discrepancies between AAT level, genotype, and phenotype exist, to establish their definitive effect.
Alpha-1 antitrypsin (AAT) is a 52-kD glycoprotein encoded by the SERPINA1 gene, being the major plasma inhibitor of serine proteases in human plasma and an important immunomodulatory protein. AAT is primarily synthesized by hepatocytes and released into the blood circulation from the liver, reaching normal serum concentrations of ∼1.3–2.5 g/L. The protein exerts its major function in the lungs, where it provides essential protection against proteolytic attacks, mainly by neutrophil elastase and proteinase-3 (1).
AAT deficiency (AATD) is still a largely underrecognized genetic disorder. This hereditary condition is caused by mutations in the SERPINA1 gene, which cause a predisposition to lung and liver disease. The variants of SERPINA1 gene are classified as normal, deficient, dysfunctional, or null (2–4). Normal and dysfunctional variants are associated with normal levels of serum AAT, whereas deficient variants lead to a reduction of circulating AAT levels. Remarkably, null variants do not produce detectable AAT, mostly as a result of nonsense, frameshift, or splicing mutations. In addition, some null QO alleles could be caused by missense variants, such as QOCardiff (Asp256Val) or QOLudwigshafen (Ile92Asn) (5). The most common deficient alleles are Pi*Z (Glu342Lys) and PI*S (Glu264Val), with frequencies in subjects of European ancestry varying between 1% and 4% for Pi*Z, the highest frequency being in Latvia, and between 3% and 13% for PI*S, the highest being in Portugal and Spain (6). Normal AAT levels are associated with the wild-type Pi*M allele (frequency, 80–95%). Both PI*Z and PI*S mutations cause the misfolding and polymerization of the AAT protein within the rough endoplasmic reticulum (ER) of hepatocytes. Nonetheless, S proteins polymerize more slowly than Z proteins, resulting in less accumulation of AAT within hepatocytes and intermediate plasma deficiency (7). Thus, Z variant is associated with an 85–90% reduction of normal serum AAT, whereas S variant is associated with a 40% reduction. In addition, S variant does not lead to liver disease unless inherited with a rapidly polymerizing variant like Z (8, 9).
The PI*S allele has been found at a high frequency in the Iberian Peninsula (∼10%) (10, 11). PiMS, PiSS, and PiSZ genotypes are considered deficient genotypes expressing 80%, 60%, and 40% of serum AAT, respectively. Although the PiSZ genotype seems to be associated with an increased risk of pulmonary emphysema and liver disease, there is much less evidence that PiMS and PiSS genotypes predispose to lung or liver damage. Nevertheless, combination of the S mutation in the same allele with other rare variants can give rise to more pathogenic phenotypes (12, 13).
In this study, we describe two new AATD alleles that combine the S variant with other amino acid variants in cis, unexpectedly producing AAT null phenotypes. These “PI*S-plus” alleles, in contrast to the majority of null alleles, produce AAT protein, but this protein is strongly retained intracellularly so that it cannot be detected in the serum of patients.
Following our genetic diagnosis algorithm for AATD (Figure 1), three cases showed discrepancies between AAT serum levels, phenotype pattern, and genotype. The AAT serum concentration was determined by immune nephelometry and protein phenotype by isoelectric focusing (IEF). The clinical data of these patients are shown in Table 1. Signed informed consent was obtained from all subjects, and the Ethics Committee of the Instituto de Salud Carlos III approved the study.
|Case||Sex||Age (yr)||Indication for AAT Screening||Smoking Status||Pack-Years||Diagnosis||Serum AAT (Expected) (mg/dl)†||Protein Phenotype||Genotype|
|1||Male||58||Lung disease||Former||40||COPD||54 (70–105)||SS||S/S+p.Tyr138Cys|
|2||Male||28||Elevated liver enzymes||Never||0||—||74 (103–200)||MM||M/S+p.Tyr138Cys|
|3||Female||62||Lung disease||Former||90||COPD, BE||71 (103–200)||MM||M/S+p.Pro391Thr|
DNA was isolated from whole peripheral blood or dried blood spot samples using Qiamp DNA blood mini kit (Qiagen) and standard methods. Genotyping of S and Z variants was performed with AAT Pi*S and Pi*Z kit (Roche Diagnostics). Sanger sequencing of coding exons of SERPINA1 gene (NG_008290.1, NM_000295.4) was performed as previously described (14, 15). Haplotypes were deduced by familial segregation analysis or allele-specific oligonucleotide PCR (ASO-PCR) and amplicon sequencing. In this last case, a 2,605-bp DNA fragment downstream of the no-S/S site was selectively amplified from the S allele using the allele-specific primer, 5′-GGGAAACTACAGCACCTGGT-3′, and the conserved primer, 5′-TGGGAGGGATTTACAGTCACA-3′.
We used databases to investigate the population frequency of the newly identified variants: the Short Genetic Variations Database (https://www.ncbi.nlm.nih.gov/snp); the Exome Aggregation Consortium (http://exac.broadinstitute.org/); and the Genome Aggregation Database (gnomAD; https://gnomad.broadinstitute.org/). The effect of the amino acid substitutions was predicted with the following in silico tools: SIFT (Sorting Intolerant From Tolerant; https://sift.bii.a-star.edu.sg/); PoplyPhen-2 (http://genetics.bwh.harvard.edu/pph2/); Condel (https://bbglab.irbbarcelona.org/fannsdb/); and CADD (Combined Annotation Dependent Depletion) Phred predictions through Ensembl Variant Effect Predictor (https://www.ensembl.org/tools/vep).
For in vitro expression of AAT, we used the vector, pCMV6 (OriGene), with SERPINA1 M1 (Val213) cDNA. The S mutation (Glu264Val), Tyr138Cys, or Pro391Thr variants were introduced by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies), and the primers are described in Table E1 in the data supplement. HEK293T cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% FBS (Sigma-Aldrich). Transfections were performed by DNA transfection reagent (Biotool) and 4 μg of expression plasmid in serum-free Opti-MEM culture medium (Gibco). At 48 hours after transfection, cells and conditioned media were collected.
Transfected HEK293T cells were fixed on slices with 95% ethanol and stained with hematoxylin and eosin and periodic acid–Schiff (PAS) staining. The accumulation of AAT aggregates was recognized as bright magenta globules. Quantification of PAS+ cells was performed by counting the percentage of PAS+ in at least three different fields (100–300 cells).
Transfected HEK293T cells and cell media were collected for the detection of retained and secreted AAT, respectively. Cell pellets were lysed with radioimmunoprecipitation assay buffer, and cell lysates and insoluble elements were obtained. Then, proteins retained in the insoluble fraction were obtained after sonication. Acrylamide SDS-PAGE (10%) or 8% acrylamide nondenaturing PAGE was performed for detection of monomeric or polymeric AAT, respectively. Blots were probed with 1:2,000 anti-AAT B9 (sc-59438; Santa Cruz Biotechnology) and 1:5,000 anti–β-actin (AC-74; Sigma-Aldrich), followed by 1:5,000 chicken anti-mouse IgG–horseradish peroxidase (sc-2954; Santa Cruz Biotechnology). Membranes were visualized after labeling with Immobilon horseradish peroxidase substrate (Millipore).
The entire coding sequence of SERPINA1 gene was analyzed in three patients who had lower serum levels of AAT than expected for the initial phenotype and genotype (PI*S and PI*Z alleles; Table 1 and Figure 2). Two novel, previously undescribed missense variants were identified: p.Tyr138Cys in patients 1 and 2, and p.Pro391Thr in patient 3. Both were confirmed to be the in-cis configuration with S mutation, either by means of familial segregation or allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The clinical characteristics and final genotype of these patients are shown in Table 1. Descriptions of these new PI*S-plus alleles is summarized in Table 2.
|PI*S-plus||Allele Name||Additional Variant||Phenotype||Reference|
|cDNA (NM_000295.4)||Mature Protein|
Two unrelated patients carried this variant in heterozygosity. Patient 1 was a 58 year-old male former smoker (40 pack-years) with chronic obstructive pulmonary disease (COPD) with lung function of forced vital capacity of 113%, forced expiratory volume in 1 second (FEV1) of 41%, and FEV1/forced vital capacity of 28%. The last evaluation by computed tomography of the lung revealed lung emphysema with residual fibrotic lesions. Evaluation of hepatic disease with an abdominal ultrasound showed a liver with normal structure and morphology, and a cholelithiasis was noted. Fibroscan analysis showed no signs of liver fibrosis (fibroscan value [FS], 4.3 KPa; interquartile range [IQR], 0.3 KPa), but a severe steatosis (controlled attenuation parameter [CAP], 286 dB/m; IQR, 16). The patient was initially diagnosed as PiSS genotype, with an AAT serum concentration of 54 mg/dl.
Patient 2 was a 28-year-old male never-smoker without evidence of pulmonary disease, but with signs of hepatic pathology mirrored by elevated hepatic enzymes, which prompted us to test AAT levels (aspartate aminotransferase [AST], 26; alanine aminotransferase [ALT], 46; γ-glutamyl transferase [GGT], 197). Fibroscan analysis showed no fibrosis (FS, 4.3 kPa). His initial genetic diagnosis was PiMS genotype, with an AAT serum concentration of 74 mg/dl. Protein phenotype was evaluated by IEF, showing a phenotype compatible with PiMM genotype.
Sequence analysis in both patients revealed an A > G transition in exon 2 (NM_000295:c.485A > G) producing an amino acid change of tyrosine to cysteine at codon 138 (p.Tyr138Cys) in the mature protein. Family screening in both cases showed that this variant segregates with the S mutation.
Patient 3 was a 62-year-old female former smoker of five cigarettes per day for 5 years, who presented with COPD and bronchiectasis, with an FEV1 of 62%. She had no evidence of significant liver fibrosis (FS, 5.3–4.8 kPa), but had some degree of steatosis (CAP, 253–302), with normal liver enzymes (AST, 23–21; ALT, 17–19; GGT, 15–12). Allele-specific genotyping (PI*S and PI*Z alleles) revealed the presence of heterozygous S mutation, and the patient genotype was therefore initially reported as PiMS. The AAT serum concentration of 71 mg/dl was found to be lower than expected based on Pi*MS genotype. DNA sequencing showed that the patient carried the S mutation and a C > A transversion at the end of exon 5 (NM_000295:c.1243C > A), producing a substitution of proline to threonine (Pro391Thr) in the mature protein. Selective amplification of the S allele followed by sequencing confirmed that the new variant was phased in the S allele (Figure E1). Protein phenotyping by IEF of patient serum revealed an unexpected Pi*M phenotype (Figure 2).
To investigate the putative pathogenic impact of these variants on the gene and protein sequence, we analyzed their allele frequency, amino acid conservation, and localization on protein tertiary structure. Neither Tyr138Cys nor Pro391Thr substitutions were found described in any of the assessed genetic databases (Short Genetic Variations Database, Exome Aggregation Consortium, or Genome Aggregation Database). Both variants are classified as deleterious by SIFT (score: 0) and Condel (score: 0.945) algorithms, as probably damaging by Polyphen (score: 1), and likely benign by CADD (Phred-like scaled CADD scores are 25 and 27.5 for Tyr138Cys and Pro391Thr, respectively). Accordingly, Tyr138Cys and Pro391Thr involve the replacement of a highly conserved residue in AAT protein (Figure 3A). The Pro391 is located in the C-terminal region, very close to the S mutation in the tertiary structure of the protein. The Tyr138 localizes at the end of α-helix(E) at the opposite side of the protein, where the S mutation (Glu264Val) is located.
Next, we investigated the intracellular accumulation and secretion of AAT in HEK293T cells transfected with plasmids expressing SERPINA1 wild-type (WT) or S (Glu264Val), Tyr138Cys, combined S+Tyr138Cys, Pro391Thr, and combined S+Pro391Thr mutants. As illustrated in Figure 4A, cells expressing WT-AAT are negative for PAS staining, whereas cells expressing S-AAT showed PAS positivity, which is characteristic of intracellular accumulation of AAT. Both Tyr138Cys and Pro391Thr AAT-expressing cells showed PAS+ intracellular aggregates, independently of whether the variant was alone or in a combination with the S mutation. In both cases, Tyr138Cys and Pro391Thr, an increase in the number of PAS+ cells was observed when these variants were combined with the S variant (Figure 4B), suggesting the synthesis of a more polymerogenic AAT when both variants are present.
We also assessed the presence of AAT polymers in the insoluble fraction of cells expressing these variants by Western blot analysis (Figures 5A and 5B). We detected AAT polymers in cells expressing the S variant, but not in cells transfected with WT-AAT or empty vector. In accordance with PAS staining, Tyr138Cys- and Pro391Thr-expressing cells, either alone or in combination with the S mutant, formed polymers of AAT. Again, when Pro391Thr and S mutation were expressed together, we observed an enhanced polymerization of AAT protein. The proportion of AAT in the insoluble fraction of double mutant S+Tyr138Cys increased from 25% (shown by Tyr138Cys alone) to 79% (Figure 5B). In the case of Pro391Thr, the percentage of AAT protein in the insoluble fraction was 82%, and increased to 98% when the combination of S+Pro391Thr was expressed (Figure 5B).
The analysis of the secretion of AAT into the culture medium by Western blot revealed the expected result, that WT-AAT was efficiently secreted into the medium, whereas S-AAT showed lower secretion. No AAT was detected for the empty vector. The Tyr138Cys AAT was partially secreted into the medium, whereas combined S+Tyr138Cys and Pro391Thr or combined S+Pro391Thr AAT were almost undetectable, suggesting strong intracellular retention.
The most important finding of the present study is the discovery of two novel pathogenic genetic variants of SERPINA1 gene (Tyr138Cys and Pro391Thr), which appeared in in cis configuration with S mutation (PI*S-plus alleles) and modified the properties of the PI*S allele in terms of higher intracellular retention and almost no extracellular secretion. When analyzed in a clinical laboratory, plasma samples from patients carrying these combinations showed that these new PI*S-plus mutant alleles had features of null-phenotype behavior.
The PAS staining and Western blot analysis of AAT in cells expressing the Tyr138Cys variant in combination with the S mutation led us to demonstrate an increase of AAT polymerization and a reduction in AAT secretion to almost undetectable levels. This was in accordance with the low levels of serum AAT observed in the two patients who presented the S+Tyr138Cys allele (54 and 74 mg/dl for those initially labeled as PiSS and PiMS genotypes, respectively). In addition, elevated liver enzymes in the serum of the patient carrying an M/S+Tyr138Cys genotype could be related with an increased polymerization of the AAT protein within the ER of hepatocytes. However, the characteristics of these polymers are still unknown and should be further studied. Because they are the result of a combination of the S mutant with another missense mutation, it is possible that they have different polymerization, degradation, or stability properties compared with the Z polymers.
The other variant, Pro391Thr, also formed polymers in our cellular model, and the protein was not secreted into the media. This in vitro effect was in line with the low levels of serum AAT observed in the patient (71 mg/dl, initially diagnosed as MS genotype). These results, together with the lack of bands for the S allele in the IEF, lead us to classify this variant as a null allele. Codon 391 has been described previously by Brodbeck and Brown (16) as essential for a normal secretion of AAT. They expressed truncated forms of AAT in COS1 cells, and demonstrated that proteins with less than 391 amino acids were retained in the ER and were not transported to the Golgi apparatus, and consequently not secreted. They suggested an important role of Pro391 in AAT secretion, either by allowing proper folding or as part of a transport signal in the C-terminal region.
Moreover, the substitution of Pro391 by a His residue has been described as pathogenic. Jardi and collaborators (17) described the PI*YBarcelona allele, which combines a Pro391His variant with an Asp256Val change on a normal M1(Val213) background. The patient who presented this allele in homozygous form had severe COPD, serum AAT levels of 16 mg/dl, and a PiZZ phenotype pattern, obtained by IEF. Similarly, Fra and coworkers (18) characterized the PI*YOrzinuovi allele, which is the result of a Pro391His substitution on a normal M1(Val213) background. The latter was identified in heterozygous form in a patient with a history of mild hypertransaminasemia and an AAT plasma level of 68 mg/dl. Expression of the variant in cellular models demonstrated an accumulation of AAT polymers and a severe secretion defect. In this case, substitution of Pro391 by His seems to reduce dramatically the secretion of AAT, but does not lead to a null phenotype. Although the newly identified Pro391Thr variant seems to be enough to cause a lack of secretion in HEK293T cells by itself, the combination in the same allele with the S mutation may contribute to the null state. In fact, Pro391 and Glu264 are very close in the tertiary structure, and the accumulation of both changes could produce conformationally unstable protein.
The combination of the S mutation with other rare missense variants in the same allele has been reported previously in AATD (Table 2). The allele, QOLaPalma, was previously described in a patient with AAT serum concentration of 8.5 mg/dl carrying a PiZ/QOLaPalma genotype (12). Similarly to the present study, the QOLaPalma allele is the result of the S variant in cis with the ΔPhe52 variant, and also gives rise to an AAT null phenotype (12). The PI*SDonosti allele was described by our group in two patients with PiSS and PiSZ phenotypes (13), and subsequently was reported in two additional cases with PiMS and PiSS phenotypes (19). This allele combines the S variant with a Ser14Phe substitution, and it is associated with polymerization and reduced secretion of AAT. Moreover, an in cis PiSZ allele has recently been reported in a woman with emphysema and very low serum AAT (20 mg/dl) (20). Thus, the novel alleles reported here expand the list of pathogenic variants identified in a PI*S background.
Importantly, at least some of the cis-acting variants in PI*S alleles, which we call Pi*S-plus, make them behave as null alleles in the sense that they are absent in protein phenotyping by IEF. However, in contrast to QO alleles that are caused by mutations that prevent the expression of AAT, these new alleles express an AAT protein that is strongly retained in cells, and phenotypically can be described as null. These PI*S-plus alleles, in contrast to the majority of QO alleles, might have increased risk of hepatic damage, as protein is highly accumulated in cells. This might be particularly important in cases homozygous for these PI*S-plus alleles, and should be further studied. In addition, because the protein is not secreted, these PI*S-plus alleles might also be associated with higher risk of developing lung disease.
The detection of rare variants in combination with the S variant in the same allele is of importance because they can confer new properties to the AAT protein and modify clinical consequences. The existence of different PI*S-plus alleles could, at least in part, explain the phenotypical variability observed among patients with AATD. The incidence of these PI*S-plus alleles is still to be estimated, although, given the rarity of these variants, the presence of other missense mutations in cis with PI*S allele will probably explain only a minor fraction of AATD phenotypical variability. Other genetic and nongenetic factors are likely to play a role as well.
In conclusion, we describe two new null PI*S-plus alleles causing AATD: QOVigo (S+Tyr138Cys) and QOAachen (S+Pro391Thr). Both alleles are the result of a rare missense variant in cis with the S variant. This study highlights the additional effect that rare variants may have when combined with more common mutations associated with AATD, thus contributing to the clinical variability of this genetic condition. Therefore, when phenotyping or allele-specific genotyping (PI*S and PI*Z) is inconclusive, a complete sequencing of the SERPINA1 gene with phase information and allelic segregation of variants is highly recommended for an accurate diagnosis and, consequently, an improved clinical management of the patients.
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*These authors contributed equally to this work.
Supported by Institute of Health Carlos III grant AESI PI17CIII/00042, and by Deutsche Forschungsgemeinschaft (DFG) grant STR 1095/6-1 (Heisenberg Professorship) (P.S.) and DFG consortium grant SFB/TRR57 “Liver fibrosis” (P.S.).
Author Contributions: N.M. and G.G.-M. performed experiments, analyzed the data, and drafted the manuscript. J.A.P. performed experiments, interpreted the data, and drafted the manuscript. B.B. performed the experiments. R.S. collected the patient data and interpreted the results. I. Belmonte and F.R.-F. performed isoelectric focusing analysis and interpreted the results. M.T.-D., F.J.M., J.M.H.-P., and P.S. reviewed the patient clinical data, interpreted the results, and helped in drafting the manuscript. I. Blanco participated in collection of the samples, interpretation of the results, and drafting and critically revising the manuscript. S.J. participated in the protein expression analysis, interpretation of the results, and drafting the manuscript. B.M.-D. designed and coordinated the study, and drafted the manuscript. All authors read and approved the final version of the manuscript.
This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1165/rcmb.2020-0021OC on June 9, 2020