Pi Phenotypes Research Articles

142 SCREENING FOR ALPHA-1 ANTITRYPSIN DEFICIENCY IN NEWBORN INFANTS IN SPAIN

Alpha-1 antitrypsin (AAT) deficiency is an hereditary metabolic disorder which predisposes affected individuals to juvenile liver disease and to pulmonary emphysema towards the third or fourth decade of life. Since prevention and treatment of this pulmonary complication is at present perfectly feasible, we have carried out a pilot study of neonatal screening for AAT deficiency in order to determine the incidence of this deficiency in Spain and to evaluate the parents' reactions on being told of this metabolic disorder. Fifteen thousand four hundred spanish newborn infants were screened for AAT deficiency. The study was carried out using the eluate from dried blood samples provided by the Cantabria University and the Basque Country Health Service Units for the detection of metabolic disorders. The screening test was based on a semiquantitative electroimmunoassay for AAT together with transferrin. For all cases suspected of AAT deficiency, the Pi phenotype was determined. With the screening test, 58 suspect cases were detected. Of these, the phenotype study showed 32 AAT deficient cases (8 Pi Z and 24 Pi SZ) representing a global incidence of 2.08±0.72 per thousand. In 4 Pi Z cases there was clinical and laboratory evidence of liver disease. The parents of the AAT-deficient children were informed of the potencially harmfull effect of tobacco smoke on the child. Their reaction towards this information was positive. Bearing in mind the high incidence of AAT-deficiency, and the existing preventive and therapeutic measures for pulmonary emphysema in AAT-deficient cases, mass-screening for this deficiency may be advisable. This screening could be considerably facilitated by recently developed genome analysis techniques.

48 ALPHA-1-PROTEINASE INHIBITOR (Pi) PHEHOTYPES IN CHILDREN WITH URINARY TRACT MALFORMATIONS

Pi phenotypes associated with decreased serum level of this protein are known to be more prevalent in subjects with a variety of diseases. The aim of this study was to reveal a possible linkage between deficient Pi phenotypes and urinary tract malformations. 374 pediatric patients were examined, including 72 with duplicated system, 97 with hydronephrosis, 23 with renal hypoplasia or aplasia, 179 with vesicoureteral reflux (VUR) and 3 with polycystic kidney disease. Pi typing was done by isoelectric focusing. Control group consisted of 1442 subjects examined in a population study. An increased frequency of heterozygous phenotyoes PiMZ, PiMS and PiMF was found in children with VUR grade 1 and 2 (PiMZ in 8.8%, PiMS in 5.295, PiMF in 2.6% of patients comparing with 3.5%, 3.3% and 0.4%, respectively, in the controls). These findings might suggest a certain role of moderately decreased antiproteinase activity in the appearance of low grade VUR (possibly via chronic inflammatory process).

Association of MS Pi Phenotype with Airway Hyperresponsiveness

Asthmatic families (AFs) and normal families (NFs) were studied to determine the relationship between bronchial hyperresponsiveness and alpha 1-antitrypsin protease inhibitor phenotype. We studied IgE levels, skin test scores, and methacholine sensitivity. In both the AF and NF groups, the subjects with the MS phenotype had significantly greater methacholine-induced bronchial hyperresponsiveness sensitivity than the MM and MZ subjects. These findings suggest that the S allele may be associated with bronchial hyperresponsiveness.

Heterozygous MZ Alpha-1-Antitrypsin Deficiency in Adults with Chronic Liver Disease

Pi phenotype was determined in 335 patients with liver diseases and compared with the results in 2830 healthy blood donors. Eleven of 335 patients had phenotype MZ (3.3%, compared with 2.9% in healthy blood donors (NS]. None of 53 patients with autoimmune chronic active hepatitis had the MZ phenotype, but it was found in 2 of 18 patients (11.1%) with cryptogenic cirrhosis, 3 of 78 (3.8%) with alcoholic liver cirrhosis, 2 of 36 (5.6%) with primary sclerosing cholangitis, and 1 of 26 (3.9%) with primary biliary cirrhosis. Altogether, 3 of 335 patients were homozygous for Pi ZZ and had cirrhosis. One of them (a male) developed a hepatoma and died. We conclude that the reported association between Pi MZ phenotype and chronic non-B active hepatitis does not seem to include patients with autoimmune chronic active hepatitis, whereas the possibility of an association between cryptogenic cirrhosis and the MZ phenotype cannot be excluded.

SIGNIFICANCE OF α1-PROTEINASE INHIBITOR DEFICIENCY IN ETIOPATHOGENESIS OF LIVER DISEASES IN CHILDREN

α1-proteinase inhibitor (α1PI) phenotypes were examined in 27 children with chronic persistent hepatitis (1st group) and in 70 newborns and infants with protracted icterus and neonatal hepatitis (2nd group). 1442 adults and children in population study served as controls (3rd group). Prevalence of phenotype PI MZ was equal to 7.41% in the 1st group, 4,29% in the 2nd group, and 3,47% in the 3rd group. Prevalence of phenotype PI MS in the groups studied was equal to 1,87%, 2,86%, and 3,33%, respectively, α1PI phenotype PI ZZ responsible for α1PI deficiency was not encountered in the 1st group. Its prevalence in the 2nd group reached 5,71% and in the 3rd group equaled to 0,07%. Thus, the cause of hepatitis in newborns and infants may be attributed to α1PI deficiency approximately in 6% of cases. Hepatitis related to α1PI deficiency was characterized by persistent jaundice, hepatomegaly, elevated serum level of conjugated and unconjugated bilirubin and hemorrhagic syndrome.

α1-PROTEINASE INHIBITOR PHENOTYPES IN PRETERM NEWBORNS

α- proteinase inhibitor (α1 PI) phenotypes were determined by isoelectric focusing in polyacrylamide gel in 305 preterm newborns and 109 their mothers, special attention being paid to the presence of Z allele responsible for deficiency of this protein. The newborns were divided in four groups according to anthropometric data: 1) appropriate for gestational age (AGA), 2) small for birth weight (SBW), 3) small for birth length (SBL), and 4) small for gestational age (SGA). Prevalence of Z allele (2,95%) in the total group of preterm newborns did not differ significantly from its prevalence in population study (1,7396). Z allele was more prevalent (5,4596) in preterm newborns with infections, birth defects (5,88%) and with combination of these pathological conditions (6,14%). Especially high prevalence (15%) of Z allele was encountered in SBW newborns with infections. We suggest that prematurity and α1PI deficiency (phenotypes PI MZ and PI ZZ) are high risk factors for neonatal morbidity.

Variation in alpha-1-antitrypsin phenotypes associated with penicillamine therapy

During a detailed study of alpha-1-antitrypsin (AAT) by isoelectric focusing, of 130 individuals of the PiZ phenotype and their families, an unusual alpha-1-antitrypsin protein pattern was identified which related directly to the effect of penicillamine therapy. The effect, believed to be mediated by reaction with the free cysteine of the alpha-1-antitrypsin molecule, was demonstrable by in vitro experiments. It was not apparently associated with loss of the anti-proteinase properties of alpha-1-antitrypsin and was not specific to the Z gene products. It is however, a source of possible confusion in the accurate assessment of Pi phenotypes.

Chromosome 14 markers in rheumatoid arthritis.

Phenotype frequencies for variants of the chromosome 14 markers, alpha 1 antitrypsin (protease inhibitor--Pi), and immunoglobulin heavy chain gene allotypes (Gm and Am) were examined in affected and unaffected members of multicase rheumatoid arthritis (RA) families and compared with published population data. Significantly higher frequencies of phenotypes containing Pi*Z and Pi*S were observed in unrelated index RA cases compared with UK population data. There was also a higher frequency of Pi*Z in family members without RA than in population controls but no such difference for the frequency of Pi*S. No difference in the frequency of PiM1M2 heterozygotes was seen between patients with RA and population controls. An examination of clinical data failed to show any relation between any particular feature of RA and positivity for Pi*Z or Pi*S. No significant differences in frequency of Gm phenotypes were observed between patients with RA and controls. Significant association was found, however, between Pi*Z and Gm phenotypes containing Gm(zax;g). These associations are interpreted as indicating linkage disequilibria between these alleles. No interactions between DR4 and either G1m(z), (a), or (x) allotypes were apparent in patients with RA. A significant association was seen in the index RA cases between DR4 and Pi phenotypes carrying Z or S alleles. Observations from this study provide evidence for the existence of a genetic component for RA susceptibility encoded on chromosome 14. An interactive effect of these genes with DR4 towards susceptibility appears likely.

Respiratory symptoms and pulmonary function in 871 miners according to Pi phenotype: a longitudinal study.

Pi phenotype was determined and alpha 1 protease inhibitor (alpha 1 Pi) was measured in 871 iron-ore miners examined twice at five years interval. A questionnaire on respiratory symptoms was administered and lung function tests: spirometry (VC, FEV1.0), measurements of the residual volume (RV/TLC) and of the CO diffusion (FuCO), were carried out by the same medical and technical team, using the same apparatus. There were no differences in age, length of employment or smoking habits among the three Pi Phenotype groups: M (90.03%), MS (7.1%), MZ (2.8%). The prevalence of clinical symptoms at the initial survey and the incidence of symptoms between the 2 surveys were not related to Pi Phenotype groups. However a significantly steeper decline of FEV1.0/VC was observed in the alpha 1 Pi partially deficient groups MS and MZ (-3.9%) compared with the non-deficient group (-1.8%). No significant difference was observed for VC, RV/TLC and FuCO. This finding suggests that an intermediate deficiency of alpha 1 Pi may be related to a slight but statistically significant impairment of a lung function in occupational conditions.

Application of immobilized pH gradient isoelectric focusing to forensic hemogenetics: a survey on a three year experience with the transferrin (TF) and alpha 1-antitrypsin (PI) systems.

The subtypes of transferrin (TF) and alpha 1-antitrypsin (PI), first discovered using isoelectric focusing, are now mostly determined in immobilized pH gradient gels. We report on our experience in the parentage expertise with both polymorphisms over a period of three years. The complexity of the technology was compensated by the fact that most subtypes of TF and PI could be more reliably recognized. The PI alleles PI*M1, M2, M3, S, F, T, and Z and TF alleles TF*C1, C2 and C3, and in addition four further rare TF alleles were observed. The allele frequencies from non-related individuals did not deviate from the Hardy-Weinberg equilibria and corresponded well to known frequencies from West Germany and other Caucasoid populations. With the TF system 36 accused men, and with the PI system 54 were excluded from paternity from a total of 344 (TF) respectively 347 (PI) cases. From the data presented here isoelectric focusing in immobilized pH gradient gels appears to be a major improvement over carrier ampholyte generated pH gradients in the distinction of TF and PI phenotypes.

Alpha 1-antitrypsin in acute anterior uveitis and rheumatic diseases.

To test the pathogenetic role of the phenotype MZ of alpha 1-antitrypsin/alpha 1-protease inhibitor (PI) in acute anterior uveitis (AAU) and in different rheumatic diseases we examined 360 unrelated patients including 93 with AAU alone, 24 patients with AAU and ankylosing spondylitis (AS), 21 patients with AAU and Reiter's disease (RD), 26 patients with AAU, AS, and RD 54 patients with AS alone, 16 patients with RD alone, 115 patients with rheumatoid arthritis (RA) alone, and 11 patients with psoriatic arthritis (PA) alone. Of the 164 AAU patients, 80 had a single attack, and 84 had repeated episodes. There were neither significant differences between different groups of the patients and 120 healthy controls nor between patients with AAU alone and patients with AAU and AS or RD in the frequencies of the PI phenotypes tested. The results indicate that the PI MZ type is not closely associated with AAU, AS, RD, RA and PA and that it does not play any role in determining whether AAU shows a pattern of a single attack or repeated episodes, and whether AAU occurs alone or together with AS or RD.

Rate of improvement of pulmonary function in sarcoidosis during treatment with corticosteroids.

Serial measurements of vital capacity were obtained in 11 patients with impaired pulmonary function due to sarcoidosis during 12 courses of corticosteroid treatment. Vital capacity improved promptly and approached a maximum value in about three weeks. A three week trial is probably sufficient to show whether or not corticosteroids are effective in a patient with sarcoidosis.

Distribution of alpha-1-antitrypsin (Pi) phenotypes in Denmark determined by separator isoelectric focusing in agarose gel.

The distribution of phenotypes of alpha 1-antitrypsin (Pi) in 909 unrelated Danes was determined by the use of separator isoelectric focusing in agarose gel. The frequencies calculated were: PiM1 = 0.728, PiM2 = 0.136, PiM3 = 0.082, PiZ = 0.023, PiS = 0.022, PiF = 0.006, Pivar = 0.003. The segregation of phenotypes in 39 families with 94 children is presented. The advantages and disadvantages of the method are discussed.

Determination of alpha-1-antitrypsin phenotypes and M subtypes by isoelectric focusing in immobilized pH gradients

A procedure to determine alpha-1-antitrypsin phenotypes (Pi types), including the six common PiM subtypes, is described. Phenotypes of the inhibitor, including the deficiency phenotype PiZ and the PiM subtypes, can be identified by isoelectric focusing in an immobilized pH gradient with a pH range of 4.2 to 5.0 or 4.0 to 5.0. This method gives clear separation of M, S, V, P and Z allele products. Up to 24 samples can be analyzed in a single overnight run using commercially available equipment and reagents.

A necropsy study of Pi phenotypes, emphysema, and smoking.

Pi phenotypes, determined post mortem by isoelectric focusing and immunofixation, emphysema, assessed from inflation fixed specimens and smoking history were correlated in 186 hospital necropsies. It was possible to determine Pi phenotype in 98.4% of the specimens. Phenotype M occurred in 87.5%, MZ in a 8.1%, MS in 2.1% and FM in 0.5%. An expected association was found between smoking and emphysema but not between the Pi phenotypes and emphysema. Thus, while smoking is a major aetiological factor, the Pi MZ heterozygous state does not seem to predispose to structural emphysema. However, the small number of cases did not allow an estimation of the risk of smoking in Pi MZ heterozygous persons compared to those with the normal Pi phenotype.

Correlation between alpha-1-antitrypsin (AAT) immunohistochemistry in the liver Pi phenotype and AAT concentration in the serum

Correlation between alpha-1-antitrypsin (AAT) immunohistochemistry in the liver Pi phenotype and AAT concentration in the serum

Esterase-26 (ES-26): characterization and genetic location on chromosome 3 of an eserine-sensitive esterase of the house mouse (Mus musculus).

Genetic variation of a codominantly inherited esterase, designated ES-26, has been discovered in the house mouse using isoelectric focusing in polyacrylamide gels. The ES-26A phenotype (pI 8.2) was found in C57BL/10Sn. A/J showed the ES-26B phenotype (pI 7.8-7.9). A third phenotype, ES-26C (double-banded: pI's 8.1 and 8.3), was observed in SJL/J. ES-26 was detected only in liver, stomach, and small intestine. The enzyme was shown to be controlled by the presumed structural locus Es-26, located on chromosome 3. From a four-point cross, the gene order Car-2--6.2 +/- 2.7--Es-16--21.0 +/- 4.5--Es-26--13.6 +/- 3.8--Amy-1 was established.

Lymphocyte cytotoxicity to autologous hepatocytes in alpha 1-antitrypsin deficiency.

To investigate the possible role of cell mediated cytotoxicity in the pathogenesis or perpetuation of liver damage associated with alpha 1-antitrypsin (AAT) deficiency, peripheral blood lymphocytes (PBL) from 13 children with PiZZ phenotype who had had liver disease in infancy were incubated with autologous hepatocytes in a microcytotoxicity assay. There was a clear trend for cytotoxicity to increase with age and thus significantly increased cytotoxicity was found in four cases older than 2 years, while intermediate values were recorded in three children between 6 months and 2 years and normal values in children younger than 6 months. Fractionation of PBL into T and non-T-enriched subsets showed that both were involved in the cytotoxic reaction. A purified liver membrane lipoprotein preparation (LSP) inhibited both T and non-T-cell cytotoxicity suggesting that LSP is a major target antigen in this system. Control experiments performed in infants with neonatal hepatitis syndrome, but with normal Pi phenotype, showed consistently increased cytotoxicity values. Cell-mediated immune reactions directed against autologous hepatocytes develop late in the course of the liver disease associated with AAT deficiency. While this reaction cannot be involved in the pathogenesis of the initial liver lesion, it may contribute to perpetuation of liver damage.

Alpha-1-antitrypsin types and rheumatoid arthritis.

Frequencies of alpha-1-antitrypsin (Pi) phenotypes were studied in 100 female and 100 male Swedish patients with classical rheumatoid arthritis and compared with the population frequencies. A significant increase of rare Pi types (MS, MZ, MF and SZ) was found among the patients. However, the increase concerned mainly Z heterozygotes and was more strongly pronounced in male patients. The M-subtypes showed no association with rheumatoid arthritis. Previous investigations of Pi types in rheumatoid arthritis have shown somewhat variable results. The results so far indicate, however, that an association between the Z allele and rheumatoid arthritis is likely to exist, while the evidence for a relationship between rheumatoid arthritis and other Pi alleles is considerably weaker.

Re-evaluation of the proposed interrelationship between thyroxine-binding globulin (TBG) and α 1-antitrypsin (PI)

Human plasma samples of known thyroxine-binding globulin (TBG) and α 1-antitrypsin (PI) phenotypes have been radiolabelled with 125I-thyroxine and subjected to isoelectric focusing. The TBG phenotypes were identified by autoradiography which was followed by protein staining to identify the PI phenotypes. The variant banding patterns of both TBG and PI were clearly independent and failed to show any interrelationship on either agarose or polyacrylamide gels. However, autoradiography of agarose gels after fixing in sulphosalicylic acid and trichloracetic acid solution produced banding patterns identical to the PI phenotypes obtained by protein staining. This unexpected finding results from the binding of 125I-thyroxine to PI during the acid-fixing process in agarose gels.