5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia

Up to now alpha 1-antitrypsin (AAT) augmentation therapy has been approved only for commercial use in selected adults with severe AAT deficiency-related pulmonary emphysema (i.e. PI*ZZ genotypes as well as combinations of Z, rare and null alleles expressing AAT serum concentrations <11 μmol/L). However, the compassionate use of augmentation therapy in recent years has proven outstanding efficacy in small cohorts of patients suffering from uncommon AAT deficiency-related diseases other than pulmonary emphysema, such as fibromyalgia, systemic vasculitis, relapsing panniculitis and bronchial asthma. Moreover, a series of preclinical studies provide evidence of the efficacy of AAT augmentation therapy in several infectious diseases, diabetes mellitus and organ transplant rejection. These facts have generated an expanding number of medical applications and patents with claims for other indications of AAT besides pulmonary emphysema. The aim of the present study is to compile and analyze both clinical and histological features of the aforementioned published case studies and reports where AAT augmentation therapy was used for conditions other than pulmonary emphysema. Particularly, our research refers to ten case reports and two clinical trials on AAT augmentation therapy in patients with both AAT deficiency and, at least, one of the following diseases: fibromyalgia, vasculitis, panniculitis and bronchial asthma. In all the cases, AAT was successfully applied whereas previous maximal conventional therapies had failed. In conclusion, laboratory studies in animals and humans as well as larger clinical trials should be, thus, performed in order to determine both the strong clinical efficacy and security of AAT in the treatment of conditions other than pulmonary emphysema.     

Alpha 1 anti-trypsin: one protein, many functions

α-1 anti-trypsin (AAT) is the most abundant circulating serine protease inhibitor (serpin) and an acute phase reactant. Systemic deficiency in AAT (AATD) due to genetic mutations can result in liver failure and chronic lung disease such as emphysema. Considered the prototypic serpin, the emphysema observed in patients with AATD, consisting of progressive destruction of the alveoli and small airway structures, formed the basis of the protease/anti-protease imbalance theory of chronic obstructive pulmonary disease (COPD). Over the past decade, however, investigations of AATD have described multiple functions of AAT beyond those generally attributed to its antiprotease activity. Evidence now suggests that AAT plays an important role in modulating immunity, inflammation, proteostasis, apoptosis, and possibly cellular senescence programs. When integrated in vivo, these processes contribute to the lung maintenance program which preserves the lung despite a constant bombardment by damage associated molecular patterns (DAMPs) and/or pathogenassociated molecular patterns (PAMPs) initiated by cigarette smoke, pollutants, or infections. In this review, we discuss the clinical aspects of AATD as they pertain to emphysema; including similarities and differences to cigarette smoke-induced emphysema. Examining the lung maintenance program, we next consider the multiple mechanisms of airspace destruction and explore the role AATD contributes. Finally, we consider the data regarding treatment of AATD, including AAT supplementation and its current limitations, and suggest further avenues of research informed by the multiple functions of AAT.     

In silico analysis of alpha1-antitrypsin variants: The effects of a novel mutation

Alpha1-antitrypsin (AAT) is a highly polymorphic protein with more than 120 variants that are classified as normal (normal protein secretion), deficient (reduced circulating AAT level caused by defective secretion) or null (no protein secretion). Alpha1-antitrypsin deficiency, one of the most common genetic disorders, predisposes adults to pulmonary emphysema and, to a lesser extent, chronic liver disease and cirrhosis. In this report, we provide additional sequence data for alpha1-antitrypsin based on the characterization of a novel variant detected in a 53-year-old heterozygous patient with chronic obstructive pulmonary disease. The mutation occurred on a PI*M2 base allele and was characterized by a T → C transition at nt 97 in exon II that led to the replacement of phenylalanine by leucine (F33L). Since the mutation was found in the heterozygous state with the expression of a normally secreted variant (PI*M1) it was not possible to assess the pattern of F33L secretion. However, computational analyses based on evolutionary, structural and functional information indicated a reduction of 23 Å ³ in the side chain volume and the creation of a cavity in the protein hydrophobic core that likely disturbed the tridimensional structure and folding of AAT. The accuracy of the in silico prediction was confirmed by testing known mutations.     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.     

Three new alpha1-antitrypsin deficiency variants help to define a C-terminal region regulating conformational change and polymerization

Alpha1-antitrypsin (AAT) deficiency is a hereditary disorder associated with reduced AAT plasma levels, predisposing adults to pulmonary emphysema. The most common genetic AAT variants found in patients are the mildly deficient S and the severely deficient Z alleles, but several other pathogenic rare alleles have been reported. While the plasma AAT deficiency is a common trait of the disease, only a few AAT variants, including the prototypic Z AAT and some rare variants, form cytotoxic polymers in the endoplasmic reticulum of hepatocytes and predispose to liver disease. Here we report the identification of three new rare AAT variants associated to reduced plasma levels and characterize their molecular behaviour in cellular models. The variants, called Mpisa (Lys259Ile), Etaurisano (Lys368Glu) and Yorzinuovi (Pro391His), showed reduced secretion compared to control M AAT, and accumulated to different extents in the cells as ordered polymeric structures resembling those formed by the Z variant. Structural analysis of the mutations showed that they may facilitate polymerization both by loosening 'latch' interactions constraining the AAT reactive loop and through effects on core packing. In conclusion, the new AAT deficiency variants, besides increasing the risk of lung disease, may predispose to liver disease, particularly if associated with the common Z variant. The new mutations cluster structurally, thus defining a region of the AAT molecule critical for regulating its conformational state.     

Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.     

Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

Alpha‐1‐antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha‐1‐antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER‐associated degradation and autophagy. We hypothesized that alpha‐1‐antitrypsin (AAT)‐interacting proteins play critical roles in quality control of human AAT. Using co‐immunoprecipitation, we identified ERdj3, an ER‐resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin‐EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3‐mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD‐related liver disease. J. Cell. Biochem. 118: 3090–3101, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Segregation distortion of the alpha 1-antitrypsin Pi Z allele.

alpha 1-antitrypsin (alpha 1AT) of the Pi type Z is associated with two diseases: pulmonary emphysema and cirrhosis of the liver. We report 23 families with both parents heterozygous for the PiZ allele, characterized from our own analysis and from world literature sources. All families were identified through members expressing disease. From the extended pedigrees, 18 backcross families (parents with Pi types MM and MZ) were identified. Analysis of the backcross families reveals a significant increase in Pi MZ offspring (.73) among families where the male is heterozygous. The distortion is not detected among families where the female is heterozygous. Among the matings where both parents are heterozygous, we found 0.43 Pi ZZ from families where one or more members expressed hepatic cirrhosis, and 0.40 Pi ZZ for total families studied. This contrasts to the 0.25 Pi ZZ expected, but is consistent with the distortion observed in backcross matings. The implications of various statistical approaches are discussed, and we point out why our findings differ from previous reports. We suggest a possible biological explanation residing in the fertilization process.     

Rapid Genotyping of Alpha 1 Antitrypsin Deletion Mutation (PI*Mmalton) Using Bi-directional PCR Allele-specific Amplification

Alpha 1 antitrypsin deficiency (AATD) is a well recognized genetic risk factor for pulmonary disease and less common liver disease. The two most common deficiency alleles worldwide PI*S and PI*Z can be easily detected using several molecular methods. However, there are at least 30 other AATD variants, which are only detectable by alpha 1 antitrypsin (AAT) gene sequencing and, therefore, seem to be more under-recognized than the PI*S and PI*Z alleles. PI*Mmalton is the most frequent AATD variant in different regions of the Southern Mediterranean basin countries, where its prevalence seems to prevail over PI*S and PI*Z. In this work, we report the development of a simple PCR-based analysis designed for the detection of the PI*Mmalton deficiency alleles using two specific primers. A one-tube reaction enables the distinction between the different genotypes. This reliable, easy, fast, and low-cost technique might be useful for laboratories involved in the study of AATD-related diseases, especially those of the Southern Mediterranean basin area with modest budget or where sophisticated equipment is not available. This will allow larger targeted screening for PI*Mmalton in order to better understand this mutation epidemiology and its origin.     

Genetically engineered cell lines for α1-antitrypsin expression

Objectives

To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination.

Results

α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines. Cell clones obtained had the AAT gene integrated at the AAVS1 locus and secreted approx. 0.04 g/l recombinant AAT into the medium. Moreover, the secreted AAT showed an inhibitory activity that is comparable to plasma AAT.

Conclusions

CRISPR/Cas9-mediated engineering of human cells is a promising alternative for generating isogenic cell lines with consistent AAT production. This work sheds new light on the generation of therapeutic liver stem cells for AATD.

     

Alpha 1-antitrypsin deficiency in childhood

Alpha-1-antitrypsin is a blood glycoprotein synthesized in the liver. It is a protease inhibitor of the serpine group and has a specific action for elastase. Alpha-1-antitrypsin electrophoresis shows about 20 phenotypes, the normal one being PiM. The allele PiZ is usually responsible for liver or lung disease in children or adults, respectively. Eleven per cent of PiZZ infants present with prolonged neonatal cholestasis. Twenty-five of 45 PiZZ infants with prolonged neonatal cholestasis presented with later cirrhosis. Persistence of jaundice beyond the sixth month of age, early development of splenomegaly, persistence of hard hepatomegaly and liver function abnormalities, and early portal fibrosis have a poor prognostic significance. The most severe course occurs in infants with an early histologic pattern of paucity of interlobular bile ducts. Portal hypertension was present in 19 of 25 children presenting with cirrhosis; 8 of 25 PiZZ children with cirrhosis died during childhood.     

Alpha(1)-antitrypsin deficiency,liver disease and emphysema

alpha(1)-Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of alpha(1)-antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the protein's tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of alpha(1)-antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z alpha(1)-antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of alpha(1)-antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition.     

Cytidine-5'-monophosphate-N-acetylneuraminic acid. Asialoglycoprotein sialic acid transferase activity in liver and serum of patients with juvenile hepatic cirrhosis and alpha-1-antitrypsin deficiency.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.     

Immunocytochemical localization of oncodevelopmental proteins in human germ cell and hepatic tumors.

In a combined tissue and serum study alpha-1-antitrypsin (AAT) and alpha-fetoprotein are demonstrated in parallel within tumor tissue inclusions in both endodermal sinus (yolk sac) tumors and malignant hepatomas, and AAT is demonstrated as a marker in both neoplastic and preneoplastic liver lesions occurring in oral contraceptive users, all in association with normal serum AAT phenotype. The tumor inclusions in the first two instances differ immunocytochemically from AAT liver cell globules found in inherited AAT deficiency, which are unreactive for alpha-fetoprotein. It is concluded that unlike the molecular basis of storage associated with AAT phenotypic variation, the tumor inclusions reflect a separate, nongenetic mechanism of AAT storage, which may be epigenetic in nature. AAT and alpha-fetoprotein both are synthesized normally in yolk sac and fetal liver, a parallelism which disappears soon after birth. The reexpression of both proteins in two distinct tumor types arising from endodermal origins (yolk sac and liver), suggests that these markers may represent reemerging fetal gene products, a phenomenon previously proposed only for alpha-fetoprotein, a prototypic "oncofetal antigen."     

Exploring the optimum approach to the use of CT densitometry in a randomised placebo-controlled study of augmentation therapy in alpha 1-antitrypsin deficiency

Background

Computed tomography (CT) lung densitometry has been demonstrated to be the most sensitive and specific outcome measure for the assessment of emphysema-modifying therapy, but the optimum densitometric index has yet to be determined and targeted sampling may be more sensitive than whole lung assessment. The EXAcerbations and CT scan as Lung Endpoints (EXACTLE) trial aimed to clarify the optimum approach to the use of CT densitometry data for the assessment of alpha 1-antitrypsin (AAT) augmentation therapy on the progression of emphysema in AAT deficiency (AATD).

Methods

Patients with AATD (n = 77) were randomised to weekly infusions of 60 mg/kg human AAT (Prolastin^®) or placebo over 2 to 2.5 years. Lung volume was included as a covariate in an endpoint analysis and a comparison was made of different CT densitometric indices (15th percentile lung density [PD15], mean lung density [MLD] and voxel index at a threshold of -910 [VI-910] and -950 [VI-950] Hounsfield Units) obtained from whole lung scans at baseline and at 24 to 30 months. Targeted regional sampling was compared with whole lung assessment.

Results

Whole lung analysis of the total change (baseline to last CT scan) compared with placebo indicated a concordant trend that was suggestive of a treatment effect for all densitometric indices (MLD [1.402 g/L, p = 0.204]; VI-910 [-0.611, p = 0.389]; VI-950 [-0.432, p = 0.452]) and that was significant using PD15 (1.472 g/L, p = 0.049). Assessment of the progression of emphysema in the apical, middle and basal regions of the lung by measurement with PD15 showed that this treatment effect was more evident when the basal third was sampled (1.722 g/L, p = 0.040). A comparison between different densitometric indices indicated that the influence of inspiratory variability between scans was greatest for PD15, but when adjustment for lung volume was made this index was the most sensitive measure of emphysema progression.

Conclusion

PD15 is the most sensitive index of emphysema progression and of treatment modification. Targeted sampling may be more sensitive than whole lung analysis.

Trial registration

Registered in ClinicalTrials.gov as ''Antitrypsin (AAT) to Treat Emphysema in AAT-Deficient Patients''; ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT00263887","term_id":"NCT00263887"}}NCT00263887.     

Do supervised weekly exercise programs maintain functional exercise capacity and quality of life, twelve months after pulmonary rehabilitation in COPD?

Background

Individuals with severe Z α1-antitrypsin (AAT) deficiency have a considerably increased risk of developing chronic obstructive lung disease (COPD). It has been hypothesized that compensatory increases in levels of other protease inhibitors mitigate the effects of this AAT deficiency. We analysed plasma levels of AAT, α1-antichymotrypsin (ACT) and secretory leukocyte protease inhibitor (SLPI) in healthy (asymptomatic) and COPD subjects with and without AAT deficiency.

Methods

Studied groups included: 71 asymptomatic AAT-deficient subjects (ZZ, n = 48 and SZ, n = 23, age 31 ± 0.5) identified during Swedish neonatal screening for AAT deficiency between 1972 and 1974; age-matched controls (MM, n = 57, age 30.7 ± 0.6); older asymptomatic ZZ (n = 10); healthy MM (n = 20, age 53 ± 9.6); and COPD patients (ZZ, n = 10, age 47.4 ± 11 and MM, n = 10, age 59.4 ± 6.7). Plasma levels of SLPI, AAT and ACT were analysed using ELISA and immunoelectrophoresis.

Results

No significant difference was found in plasma ACT and SLPI levels between the healthy MM and the ZZ or SZ subjects in the studied groups. Independent of the genetic variant, subjects with COPD (n = 19) had elevated plasma levels of SLPI and ACT relative to controls (n = 153) (49.5 ± 7.2 vs 40.7 ± 9.1 ng/ml, p < 0.001 and 0.52 ± 0.19 vs 0.40 ± 0.1 mg/ml, p < 0.05, respectively).

Conclusion

Our findings show that plasma levels of ACT and SLPI are not elevated in subjects with genetic AAT deficiency compared MM controls and do not appear to compensate for the deficiency of plasma AAT.     

α-Antitrypsin Deficiency and Neonatal Hepatitis

Five out of 28 infants investigated in a regional survey of neonatal hepatitis were found to have genetically-determined deficiency of α₁-antitrypsin (ZZ phenotype). The clinical course and pathological changes varied considerably. All five infants had an acute hepatitis-like illness, and although this subsided cirrhosis later developed in three cases. The remaining two infants had minimal abnormalities of the liver function tests at 12 and 18 months of age, and one had increased hepatic fibrosis. Australia antigen was found in the serum of three infants, and Australia antigen or antibody in one or both parents of these and of one further case whose serum was negative. It is suggested that the association of neonatal hepatitis with α₁-antitrypsin deficiency may be commoner than previously realized and that Australia antigen acts as a trigger factor in these cases.