DNA restriction-site polymorphisms associated with the alpha 1-antitrypsin gene.

Restriction-site variation in and around the alpha 1-antitrypsin gene has been studied using two genomic probes. With use of restriction enzymes SstI, MspI, and AvaII, three polymorphic sites have been described with a 4.6-kb probe in the 5' portion of the gene. With use of a 6.5-kb probe, polymorphisms in the coding and 3' regions of the gene have been detected with AvaII, MaeIII, and TaqI. All of these polymorphisms are of sufficiently high frequency to be useful in genetic mapping studies. The polymorphisms with AvaII and MaeIII (6.5-kb probe) are particularly useful for prenatal diagnosis. PI types and M subtypes tend to be associated with specific DNA haplotypes; there are two different types of DNA haplotypes associated with PI M1. The extent of linkage disequilibrium differs throughout the region of the alpha 1-antitrypsin gene.     

Deoxyribonucleic acid polymorphism of the apoprotein AI-CIII-AIV gene cluster and coronary heart disease in non-insulin-dependent diabetes.

The prevalence of an uncommon allelic variant (S2) of the apoprotein AI-CIII-AIV gene cluster was determined in non-insulin-dependent diabetics with or without evidence of coronary heart disease and in controls. Frequencies of the S2 allele were 14% for diabetics with coronary heart disease compared with 2% for non-diabetics with no clinical evidence of occlusive vascular disease. No subject with the S2 allele was detected among a further group of matched diabetics without clinical features of macrovascular disease. The results suggest that a genetic component contributes to the susceptibility to coronary heart disease in non-insulin-dependent diabetics. Whether the observed deoxyribonucleic acid variant is aetiological for atherosclerosis or in linkage disequilibrium with other atherogenic loci on chromosome 11 remains to be clarified.     

Identification of DNA polymorphisms associated with the V type alpha1-antitrypsin gene

alpha1-Antitrypsin (alpha1-AT) is a highly polymorphic protein. The V allele of alpha1-AT has been shown to be associated with focal glomerulosclerosis (FGS) in Negroid and mixed race South African patients. To identify mutations and polymorphisms in the gene for the V allele of alpha1-AT in five South African patients with FGS nephrotic syndrome DNA sequence analysis and restriction fragment length polymorphisms of the coding exons were carried out. Four of the patients were heterozygous for the BstEII RFLP in exon III [M1(Val213)(Ala213)] and one patient was a M1(Ala213) homozygote. The mutation for the V allele was identified in exon II as Gly-148 (GGG)-->Arg (AGG) and in all patients was associated with a silent mutation at position 158 (AAC-->AAT). The patient who was homozygous for (Ala213) also had a silent mutation at position 256 in exon III (GAT-->GAC) which was not present in any of the other four patients. Although the V allele of alpha1-AT is not associated with severe plasma deficiency, it may be in linkage disequilibrium with other genes on chromosome 14 that predispose to FGS. Furthermore, the associated silent mutation at position 158 and the Ala213 polymorphism are of interest, as these could represent an evolutionary intermediate between the M1(Ala213) and M1(Val213) subtypes.     

In vitro formation of triple-stranded DNA structures in the human alpha1-antitrypsin gene

We analyzed the possibility of triplex formation in the human alpha 1-antitrypsin gene. Conditions and nucleotide sequence dependence of triplex formation and thermostability of the product were determined.     

Cloning and characterization of an alpha 1-antitrypsin like gene 12 KB downstream of the genuine alpha 1-antitrypsin gene

Cosmid clones containing alpha 1-antitrypsin (alpha 1AT) gene sequences were observed to contain alpha 1AT-like sequences approximately 12 kb downstream of the authentic alpha 1AT gene. Restriction mapping suggested the alpha 1AT-like gene lacks promoter sequences. Cosmid clones from one library contained a truncated alpha 1AT-like gene with a deletion encompassing 1745 bp, including the whole exon IV and part of exon V. Sequencing of exon II of this truncated gene revealed a nucleotide homology of 76% but included critical mutations in the start codon (ATG - greater than ATA) and the 3' exon-intron junction. These results strongly suggest that the truncated alpha 1AT-like gene is a pseudogene, which is present at a frequency of 0.30 in the Dutch population.     

Alpha 1-antitrypsin Null(isola di procida): an alpha 1-antitrypsin deficiency allele caused by deletion of all alpha 1-antitrypsin coding exons.

alpha 1-Antitrypsin (alpha 1AT) deficiency, a common hereditary disorder responsible for emphysema in Caucasians of northern European descent, is caused by single base substitutions, deletions, or additions in the seven exons (IA-IC and II-V), of the 12.2-kb alpha 1AT gene located on chromosome 14 at q31-32.3. Of the five known representatives of the "null" group of alpha 1AT-deficiency alleles (alpha 1AT genes incapable of producing alpha 1AT protein detectable in serum) evaluated at the gene level, all result from mutations causing the formation of stop codons in coding exons of the alpha 1AT gene. The present study identifies an alpha 1AT allele (referred to as "Null(isola di procida")) caused by complete deletion of the alpha 1AT coding exons. The Null(isola di procida) allele was identified in an individual with heterozygous inheritance of M(procida) (an allele associated with alpha 1AT deficiency) and a null allele. Although results of karyotypic analysis were normal, quantification of the copies of alpha 1AT genes in this individual revealed that the index case had only half the normal copies of alpha 1AT genes. Cloning and mapping of the Null(isola di procida) gene demonstrated a deletion of a 17-kb fragment that included exons II-V of the alpha 1AT structural gene. As a consequence of the deletion, the normal noncoding exons (IA-IC) were followed by exons II-V of the downstream alpha 1AT-like gene. Sequence analysis of the deletion demonstrated a 7-bp repeat sequence (GAGGACA) both 5' to the deletion and at the 3' end of the deletion, a 4-bp palindromic sequence (ACAG vs. CTGT) bracketing the deletion, and a novel inserted 4-bp sequence (CCTG) at the breakpoint, suggesting that the mechanism of the deletion may have been "slipped mispairing."     

Disruption of the murine alpha1-antitrypsin/PI2 gene.

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.     

Association between an alpha(2) macroglobulin DNA polymorphism and late-onset Alzheimer's disease.

An association between a five-base-pair deletion/insertion DNA polymorphism at the alpha(2) macroglobulin gene (A2M) and late-onset Alzheimer's disease (LOAD) has been recently described. We developed a PCR assay to analyze this polymorphism in 190 LOAD patients (older than 65 years) and 400 controls from Spain. Controls were stratified into three groups: <65 years (n = 200), 65 to 80 years (n = 100), and 81 years or older (n = 100). We found a significantly higher frequency of carriers of the D allele in patients older than 81 years compared to controls older than 81 years (p = 0.0012). In addition, the frequency of the D allele was significantly lower in controls older than 81 years compared to controls younger than 65 (p = 0.048). Our work suggests that the D allele confers an age-dependent increased risk to develop late-onset Alzheimer's disease.     

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.     

Aggrecanase-1 (ADAMTS-4) interacts with alpha1-antitrypsin

In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1 (ADAMTS--4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY 48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha1-antitrypsin, a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha1-antitrypsin were expressed in mammalian cells and used in co-immunoprecipitation experiments, which showed that full-length aggrecanase-1 and alpha1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of alpha1-antitrypsin against elastase, and alpha1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha1-antitrypsin remains unclear.     

Genetic variants of serum alpha1-antitrypsin (Pi types) in Portuguese.

The results of Pi typing on 330 Portuguese from the area of Lisbon are reported. We found six phenotypes and four alleles out of the 24 described in the literature. The allele PiM is the most frequent as in other populations, PiS shows a high frequency (0.1152), and PiF is absent, which agrees satisfactorily with former studies carried out in Spain. These results are compared with others and the entity of the Iberian population is evoked.     

Cell-specific involvement of HNF-1beta in alpha(1)-antitrypsin gene expression in human respiratory epithelial cells

The synergistic action of hepatocyte nuclear factor (HNF)-1alpha and HNF-4 plays an important role in expression of the alpha(1)-antitrypsin (alpha(1)-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the alpha(1)-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that alpha(1)-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1alpha and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that alpha(1)-AT gene expression in H441 cells is predominantly driven by HNF-1beta, even though HNF-1beta has no effect on alpha(1)-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of alpha(1)-AT and HNF-1beta was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on alpha(1)-AT gene expression in H441 cells, even when it is cotransfected with HNF-1beta or HNF-1alpha. HNF-3 by itself has little effect on alpha(1)-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1beta in alpha(1)-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.