[Biochemical and molecular diagnosis of alpha 1 antitrypsin deficiency in a Tunisian family]. / Diagnostic biochimique et moléculaire du déficit en alpha 1 antitrypsine dans une famille tunisienne.

Our study investigated alpha 1 antitrypsin deficiency (AATD) diagnosis in a family originated from central Tunisia and showing a familial history of asthma. Biochemical and genetic diagnosis for AATD was performed according to current diagnostic standards. AAT level quantification in affected individuals showed plasma AAT levels consistent with intermediate AATD (ranged from 0.91 to 1.04 g/L). The molecular analysis was assessed using the genotyping of the most prevalent PI*S and PI*Z SERPINA1 mutations and the sequencing of AAT coding exons for rare AATD variants detection. No PI*S or PI*Z deficient variants were seen in this family. Sequencing results showed the inheritance of the deficient rare variant PI*M(wurzburg) (P369S) at the heterozygous state in the mother and two affected siblings. However, AATD status remains unexplained in the third affected case, with no mutations detected in the AAT coding exons.

Good and bad prognosis of alpha-1-antitrypsin deficiency in children: when to list for liver transplantation.

Alpha1-antitrypsin deficiency (alpha1-ATD) is a genetic disorder that may predispose to chronic liver disease. The clinical manifestations and prognosis of this disorder are variable. The aim of the study was to evaluate the clinical presentation and liver tests in two groups of children with alpha1-ATD: those with a good prognosis who survived long term with their native liver, and those with a bad one, requiring liver transplantation (OLT) or dying before OLT. We studied 59 children homozygous for alpha1-ATD admitted to our hospital with cholestasis or chronic hepatitis since infancy. Patients without liver transplantation were regarded to be the good prognosis group I (n=45). In contrast the 11 children who required liver transplantation and the three who died before OLT were the bad prognosis cohort (Group II, n=14). We analyzed the laboratory parameters of cholestasis, hepatitis, and liver insufficiency in both groups. In the group with a good prognosis, eight children still suffered from cholestasis at the ages of 9 to 14 years while nine had hepatitis at the ages of 9 to 14 years. We observed a temporarily increased international normalized ratio (1.2 to 1.5) in eight subjects at the ages of 1 month to 17 years, and slight hypoalbuminemia (30 to 35 mg/dL) in nine children at the ages of 1 month to 10 years. OLT was performed in 11 children at the ages of 10 to 17 years. Our center's experience suggested that in the PiZZ patients with portal hypertension, esophageal varices, or deterioration of hepatic function, liver transplantation should not be delayed.

Role of alpha-1-antichymotrypsin deficiency in promoting cirrhosis in two siblings with heterozygous alpha-1-antitrypsin deficiency phenotype SZ.

BACKGROUND: Alpha-1-antitrypsin (A1AT) deficiency is the most common inherited metabolic disorder with the potential to cause injury in the lung and liver. Recent reports suggested that alpha-1-antichymotrypsin (A1AC) deficiency may also be a possible cause of chronic liver disease. However, it has received little attention and is rarely investigated in the clinical setting. AIMS: To assess the role of A1AC deficiency in the pathogenesis of chronic liver disease in two siblings with heterozygous A1AT phenotype Pi SZ. PATIENTS: Two adult siblings with an A1AT Pi SZ phenotype and reduced levels of A1AC consistent with heterozygosity who developed cirrhosis and underwent liver transplantation. METHODS AND RESULTS: A1AT and A1AC levels in plasma measured by electroimmunoassay were 74 mg/dl and 90 mg/dl (140-470) and 0.12 mg/ml and 0.14 mg/ml (0.173-0.46), respectively. Immunohistochemistry revealed an apparent accumulation of both A1AT and A1AC in hepatocytes. A previously reported point mutation in exon III (Pro(229) to Ala substitution) of the A1AC gene was not detected by polymerase chain reaction amplification and a single strand conformation polymorphism analysis. CONCLUSIONS: Our report represents the first case of two siblings with A1CA phenotype Pi SZ who developed cirrhosis and underwent liver transplantation. Both siblings were heterozygous for A1AT and A1AC deficiency suggesting that combined deficiency of these two major serine protease inhibitors may enhance the risk of developing liver disease.

Association of alpha(1)-antichymotrypsin deficiency with milder lung disease in patients with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is characterised by an excess of free proteinases that destroy lung tissue. Despite this, previous studies have shown that patients with CF with a mild deficiency variant of the proteinase inhibitor alpha(1)-antitrypsin have less, rather than more, severe pulmonary disease. Alpha(1)-antichymotrypsin is another important serine proteinase inhibitor that protects the lung against proteolytic attack, and point mutations in the alpha(1)-antichymotrypsin gene that result in plasma deficiency are associated with chronic obstructive pulmonary disease. METHODS: The effect of alpha(1)-antichymotrypsin deficiency and the -15 alpha(1)-antichymotrypsin signal peptide genotype on lung function was assessed in patients with CF. RESULTS: One hundred and fifty seven patients with CF were screened and 10 were identified with a plasma deficiency of alpha(1)-antichymotrypsin (plasma concentration <0.2 g/l). In a multivariate analysis these individuals had significantly less severe lung disease than those who had normal or raised levels of alpha(1)-antichymotrypsin: forced expiratory volume in one second (FEV(1)) 69.9% predicted versus 53. 2% predicted (p=0.04) and chest radiographic score of 7.2 versus 9.7 (p=0.03) for those with and without alpha(1)-antichymotrypsin deficiency, respectively. The -15 signal peptide genotype did not affect plasma levels, but the -15 Ala/Ala signal peptide genotype was over-represented in individuals with CF compared with healthy blood donor controls. CONCLUSION: These data indicate that deficiency of alpha(1)-antichymotrypsin is associated with less severe pulmonary disease in patients with CF, and support our previous observations that mild genetic deficiency of a proteinase inhibitor is associated with an improved outcome.

Genes, oxidative stress, and the risk of chronic obstructive pulmonary disease.

BACKGROUND: The first-pass metabolism of foreign compounds in the lung is an important protective mechanism against oxidative stress. We investigated whether polymorphisms in the gene for microsomal epoxide hydrolase (mEPHX), an enzyme involved in this protective process, had any bearing on individual susceptibility to the development of chronic obstructive pulmonary disease (COPD) and emphysema. METHODS: We designed PCR-based genotyping assays to detect variant forms of mEPHX that confer slow and fast activity. We used these assays to screen 203 blood-donor controls and groups of patients with asthma (n = 57), lung cancer (n = 50), COPD (n = 68), and emphysema (n = 94), who were attending specialised clinics in Edinburgh, UK. FINDINGS: The proportion of individuals with innate slow mEPHX activity (homozygotes) was significantly higher in both the COPD group and the emphysema group than in the control group (COPD 13 [19%] vs control 13 [6%]; emphysema 21 [22%] vs 13 [6%]). The odds ratios for homozygous slow activity versus all other phenotypes were 4.1 (95% CI 1.8-9.7) for COPD and 5.0 (2.3-10.9) for emphysema. INTERPRETATION: Genetic polymorphisms in xenobiotic enzymes may have a role in individual susceptibility to oxidant-related lung disease. Epoxide derivatives of cigarette-smoke components may be the cause of some of the lung damage characteristics of these diseases.

Serine protease inhibitors in patients with chronic viral hepatitis.

BACKGROUND/AIMS: This study aimed to determine whether deficiency of the major serine protease inhibitors (alpha1-antitrypsin (AAT) or alpha1-antichymotrypsin (ACT)) is associated with increased risk for chronic hepatitis B or C virus (HBV or HCV) infection. METHODS: We studied 709 adults with chronic liver disease who had undergone liver biopsy during the 14-year period 1978-92. Anti-HCV testing was carried out with second-generation ELISA and immunoblot assays (RIBA 2). HBV markers were tested with commercially available radioimmunoassays. ACT and AAT concentrations in plasma were measured with electroimmunoassay and immune nephelometry. Plasma samples were screened for the AAT PiZ deficiency with ELISA technique and phenotyped by isoelectric focusing. The 229Pro-->Ala mutation for ACT deficiency was identified by PCR techniques. RESULTS: Of the 709 patients, 132 (18.6%) were positive for anti-HCV according to RIBA 2. PiZ AAT deficiency was found in 44 (6.2%) of patients (one PiZZ, 38 PiMZ, and PiSZ), while subnormal ACT levels were found in 33 (4.6%) patients, frequencies that were higher than expected in the general population (p=0.0375 and p<0.0001, respectively). Of the PiZ-carriers, 8/44 (18%) were found to be anti-HCV positive according to RIBA 2, as compared to 123/662 (19%) non-PiZ-carriers (p>0.05). One of these patients had cirrhosis, four chronic active hepatitis, and three chronic persistent hepatitis. In contrast, 17/33 (51.5%) of the patients with subnormal ACT were anti-HCV positive (OR=5.2, CI=2.6-10.6; p<0.0001). No relationship was found between HBV infection and AAT deficiency or subnormal ACT levels. Only one patient with subnormal ACT levels was heterozygous for the 229Pro-->Ala mutation of ACT deficiency. There was no significant difference in the histological findings when the patients with subnormal ACT levels or PiZ allele were subgrouped according to HCV status. CONCLUSIONS: There is no overrepresentation of chronic HBV or HCV in heterozygous AAT deficiency, although an association with more severe liver disease in such patients cannot be excluded. In contrast, low plasma levels of ACT that may be acquired or hereditary, due to mutations other than 229Pro-->Ala, are frequent in HCV infection.

Absence of the A1252G mutation in alpha 1-antichymotrypsin in a North American population suffering from dementia.

Associations have been reported between polymorphisms in the gene for alpha 1-antichymotrypsin (ACT) and both Alzheimer's disease (AD) and cerebrovascular disease. An A-to-G substitution at nucleotide position 1,252 of ACT that produces a methionine to valine substitution at codon 389 has been found previously in four of 32 individuals with cerebrovascular disease from a Japanese population. We genotyped 194 individuals [59 controls, 35 with non-AD-type dementia (primarily vascular) and 100 with Alzheimer's-type dementia] for this polymorphism and found none that carry this polymorphism. Therefore, the allelic association of the A1252G mutation of ACT with cerebrovascular disease may be confined to the Japanese population and is not generalizable to other populations.

The search for susceptibility genes of COPD.

Environmental factors, such as cigarette smoking, outdoor and indoor pollution, and childhood respiratory infections, are believed to play a major role as risk factors for developing chronic obstructive pulmonary disease (COPD). The only confirmed genetic risk factor for COPD is the inherited deficiency of alpha 1-proteinase inhibitor. However, the evidence of familial clustering of lung function and COPD occurrence and the development of COPD among susceptible smokers, at variance with the so-called resistant smokers, would suggest that the weight of genetic risk factors is greater than recognized. In this paper the role of candidate genes for increasing the risk of COPD (such as alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, cystic fibrosis transmembrane conductance regulator, and others) is reviewed.

The molecular basis of alpha 1-antichymotrypsin deficiency in a heterozygote with liver and lung disease.

Alpha 1-antichymotrypsin (alpha 1-ACT) is a serine proteinase inhibitor (serpin) with cathepsin G, mast cell chymase and chymotrypsin as target enzymes. We present the case of a middle-aged man with low plasma levels of alpha 1-ACT, asthma with progression to emphysema, and chronic HCV positive liver disease with selective accumulation of alpha 1-ACT in hepatocytes. This secretory defect is analogous to that seen in Pi Z alpha 1-antitrypsin deficiency. The molecular basis of alpha 1-ACT deficiency in this patient has been characterized by direct sequencing of the alpha 1-ACT genes from the patient and his father. A C-->G transversion in exon III causing a 229Pro-->Ala substitution is proposed to cause a conformational change resulting in abnormal transport through the RER. This mutation was found in one of 20 additional tested patients with chronic obstructive lung disease, but in no control. Two additional polymorphisms of the gene have been identified in unrelated healthy individuals with normal plasma alpha 1-ACT levels. The alpha 1-ACT deficiency state may predispose to obstructive lung disease and influence the course of liver disease. Identification of a specific mutation allows identification of heterozygotes for this deficiency allowing future evaluation of its clinical significance.

Hepatic endoplasmic reticulum storage diseases.

Endoplasmic Reticulum Storage Diseases (ERSD) represent a novel group of inborn errors of metabolism affecting secretory proteins and resulting in hepatocytic storage and plasma deficiency of the corresponding protein. The hepatocellular storage is due to a molecular abnormality hindering the translocation of the abnormal protein from the rough (RER) to the smooth endoplasmic reticulum (SER). The molecular abnormality is genetically determined; hence it is hereditary, congenital, familial and permanent. The storage is selective and exclusive for the mutant protein and predisposes to the development of chronic cryptogenic liver disease. ERSD include alpha-1-antitrypsin deficiency, fibrinogen storage and alpha-1-antichymotrypsin deficiency. Basically, the diagnosis of ERSD is a morphological one: immunohistochemistry and electron microscopy are essential tools for their identification.

Heterozygous alpha 1-antichymotrypsin deficiency may be associated with cold urticaria.

Proteins of the serpin family (serine protease inhibitor) control key steps in the inflammatory, coagulation and complement systems. C1-inhibitor deficiency predisposes to hereditary angioneurotic oedema, and other serpins control proteolytic enzymes that may cause complement activation or the forming of oedema. We investigated whether deficiency of proteins of the serpin family may predispose to cold urticaria and therefore screened 7 male patients with severe cold urticaria for the presence of deficiency alleles of some of the members of the serpin antiprotease family. There were no findings of C1-inhibitor, alpha 1-antitrypsin, alpha 2-antiplasmin, antithrombin III, tissue plasminogen activator inhibitor or thyroxine binding protein deficiency. The prevalence of heterozygous alpha 1-antichymotrypsin deficiency was significantly higher than expected (prevalence ratio 25.8 (95% confidence interval 6.0-112), p < 0.0001). This finding is in concert with previous studies that have shown lower mean levels of alpha 1-antichymotrypsin among patients with cold urticaria and suggests that heterozygous deficiency of this antiprotease, which controls neutrophil cathepsin G and mast cell chymase may predispose to cold urticaria. The present series is, however, small and the results need confirmation in larger materials.

Deletion (14) (q24.3q32.1): evidence for a distinct clinical phenotype.

We report on a 4-year-old girl with distinctive facial features (redundant skin, bushy eyebrows, narrow palpebral fissures, short, upturned nose, epicanthal folds, and a long upper lip with well-defined philtrum) who has an interstitial deletion of chromosome 14 including band 14q31, designated as 46,XX,del(14)(pter-->q24.3::q32.1-->qter). Comparison with previously reported patients with deletions of 14q involving band 14q31 suggests that there is a distinctive clinical phenotype associated with this deletion. Our patient had dental abnormalities (3 maxillary and 3 mandibular incisors) not described in the other patients.

Improved phenotyping of alpha 1-antichymotrypsin (ACT) by isoelectric focusing and immunoprinting: first demonstration of a deficient protein variant in the ACT system.

Genetic variation of human alpha 1-antichymotrypsin (ACT) was investigated in sera using thin-layer polyacrylamide gel isoelectric focusing (pH range 4.0-6.5) followed by immunoprinting with a monospecific anti-human ACT antibody. Sialidase-treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant "ACT Bochum" was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.

Partial deficiency of alpha 1-antichymotrypsin is associated with chronic cryptogenic liver disease.

alpha 1-Antichymotrypsin (ACT) and alpha 1-antitrypsin (AAT) are two closely related antineutrophil proteinase inhibitors. Whereas AAT deficiency is clearly linked to liver disease, an association between liver disease and partial ACT deficiency has not been established. In a previous study we noted an increased prevalence of liver abnormalities among subjects with heterozygous ACT deficiency. To study a possible association between partial ACT deficiency and liver disease, we screened 316 consecutive patients with biopsy-verified liver disease for partial ACT deficiency and compared the prevalence with that of an unselected adult population in a case-control study. In all, 9 of 316 patients had partial ACT deficiency, which is more than expected (prevalence ratio (PR), 2.46 (1.15-5.27), P less than 0.05). The prevalence of partial ACT deficiency was highest in the chronic active hepatitis (5 of 40; PR, 12.0 (5.33-27.0] and the cryptogenic cirrhosis (3 of 24; PR, 12.0 (4.38-32.9] subgroups. In the chronic active hepatitis subgroup two patients (PR, 8.16 (2.25-29.5] were ACT deficiency heterozygotes, thus partly explaining the high prevalence of partial ACT deficiency among patients with chronic liver disease. The majority (6 of 9) of the patients with partial ACT deficiency lacked autoimmune and viral markers and were thus cryptogenic. The present findings show that partial ACT deficiency and chronic cryptogenic liver disease are associated. To some extent (the true magnitude of which is at present unknown) partial ACT deficiency is caused by a rare trait, heterozygous ACT deficiency, which in parallel with heterozygous alpha 1-antitrypsin deficiency (PiMZ) also seems to be associated with chronic cryptogenic liver disease.

The isoelectric focusing pattern of desialylated alpha 1-antichymotrypsin of heterozygous alpha 1-antichymotrypsin deficiency and of acute phase plasma.

In an attempt to find a molecular marker for the putative abnormal allele in heterozygous alpha 1-antichymotrypsin (ACT) deficiency (a rare trait associated with early emphysema, childhood asthma and chronic "cryptogenic" liver disease) the isoelectric focusing pattern of neuraminidase treated plasma samples from subjects of ACT deficiency families as well as acute phase plasma were compared. There was no difference in the isoform pattern of plasma from ACT deficiency heterozygotes, normal subjects or patients with acute phase response. However, in acute phase plasma there was a disproportional increase in two isoforms, one of which conceivably may be used to mark the early phase of the acute phase response.

Heterozygous alpha 1-antichymotrypsin and PiZ alpha 1-antitrypsin deficiency. Prevalence and clinical spectrum in asthmatic children.

In a case-control study we compared the prevalence of heterozygous deficiency of two closely related anti-neutrophil protease inhibitors, alpha 1-antitrypsin and alpha 1-antichymotrypsin, in 172 consecutive children with asthma. In a cohort study the clinical spectrum and severity were compared. On the basis of family studies 5/172 (2.9%) were classified as heterozygotes for alpha 1-antichymotrypsin deficiency, a high prevalence compared with that of an unselected adult population (prevalence ratio 4.5 (1.7-11.9), P less than 0.005). This finding suggests that the carrier state of this rare allele (prevalence 0.64%) may predispose to asthma in children. Among these heterozygous patients the prevalence of positive RAST tests for foodstuffs was significantly increased (prevalence ratio 4.8 (1.7-13.2), P less than 0.005) and 2/5 manifested food allergy with Quincke oedema. Either the PiMZ or SZ phenotype of alpha 1-antitrypsin deficiency was found in 12 (7.0%) of the 172 patients, a prevalence similar to that of a normal population (prevalence ratio 1.3 (0.67-2.6), P = 0.44). However, the asthma was more severe among the Z allele carriers, judged by the number of hospital admissions, compared with the non-Z asthmatic children (mean 2.92 vs. 1.72, P less than 0.05). The results indicate that heterozygous deficiency of protease inhibitors directed against neutrophil proteases may affect the severity and clinical spectrum of childhood asthma, and to some degree be predisposing.

Pulmonary function in middle-aged women with heterozygous deficiency of the serine protease inhibitor alpha 1-antichymotrypsin.

Heterozygous alpha 1-antichymotrypsin (ACT) deficiency is inherited in an autosomal dominant mode independently of alpha 1-antitrypsin with a gene frequency (q) of 0.003. In a previous study, a high prevalence of enlarged residual volumes in subjects with the trait were noted. Neutrophil cathepsin G, the target proteinase of ACT, enhances the elastolytic action of elastase. Thus, hypothetically, subjects with the trait may have increased risk for developing pulmonary emphysema. To test whether heterozygous ACT deficiency predisposes to lung disease, plasma ACT concentrations were determined in a cohort of 1,872 middle-aged women. Women with subnormal levels were studied with respect to heredity, airway symptoms, and lung function. Twelve women (0.64% of the cohort) were classified as heterozygotes after family studies and were compared with control subjects, matched for age, weight, sex, and smoking status. There were no significant differences in airway symptoms between heterozygotes and control subjects. However, the prevalence of ex-smokers was significantly higher among heterozygotes than among the screened population as a whole (prevalence ratio, 2.18; 95% confidence interval, 1.004-4.72). There were no differences between the heterozygotes and the control subjects in the basal spirometry. However, after bronchodilation, five of the 12 heterozygotes manifested residual volumes greater than 2.5 standard deviations above normal mean compared with one of 24 control subjects (p = 0.012). The present investigation thus confirms our previous findings of an increased prevalence of enlarged residual volumes in heterozygous ACT deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocyte inclusions of alpha 1-antichymotrypsin in a patient with partial deficiency of alpha 1-antichymotrypsin and chronic liver disease.

We present a case of chronic liver disease with selective and exclusive hepatocyte endoplasmic reticulum storage of alpha 1-antichymotrypsin in the form of granules, detected by specific immunohistochemistry at the light microscopy level and corresponding to material found in dilated endoplasmic reticulum of hepatocytes by electron microscopy. The patient had intermediate deficiency of alpha 1-antichymotrypsin. Thus, the hepatocyte accumulation of alpha 1-antichymotrypsin may indicate the presence of an export block resembling that of a closely-related protein, namely PiZ alpha 1-antitrypsin. It is proposed that hepatocyte storage of alpha 1-antichymotrypsin may be an expression of an inborn error of metabolism bearing the characteristics of endoplasmic reticulum storage diseases such as PiZ alpha 1-antitrypsin deficiency and hereditary hypofibrinogenaemia.