Mapping recombination hotspots in human phosphoglucomutase (PGM1).

Human phosphoglucomutase (PGM1) is a highly poly-morphic protein. Three mutations and four intragenic recombination events between the three mutation sites generate eight protein variants including the four universally common alleles, 1+, 1 -, 2+ and 2 -, and four others that are polymorphic in some Oriental populations, 3+, 3-, 7+ and 7-. The mutations 3/7, 2/1 and +/-are in exons 1A, 4 and 8, and are 40 and 18 kb apart, respectively. Using 12 polymorphic markers, including 2/1 and +/-, we have now obtained direct evidence for a high rate of intragenic recombination across this 58 kb region. From segregation analysis of PGM1 haplotypes in CEPH families, the recombination frequency was estimated to be 1.7%. We have also used a population genetics approach to map the patterns of linkage disequilibrium across the PGM1 gene in three diverse population samples (Caucasian, Chinese and Vietnamese). This has allowed us to compare indirect estimates of intragenic recombination with the meiotic data from family studies. Comprehensive pairwise allelic association analysis of the markers indicated the presence of two recombi-nation 'hotspots': one between exons 1A and 4 and the other in the region of exon 7. These locations are in keeping with the meiotic data and with the original hypothesis of intragenic recombination based on PGM1 isozyme analysis.

Alpha1-antitrypsin deficiency alleles and the Taq-I G-->A allele in cystic fibrosis lung disease.

Cystic fibrosis (CF) is characterized by progressive and ultimately fatal pulmonary disease although there are notable variations in clinical features. This heterogeneity is thought to lie outside the cystic fibrosis transmembrane regulator (CFTR) gene locus and may stem from deficiencies in the antiproteinase screen that protects the lung from proteolytic attack. One hundred and fifty seven patients were recruited from two UK CF centres. The serum concentrations of alpha1-antitrypsin, alpha1-antichymotrypsin and C-reactive protein (CRP) were determined and patients were screened for the common S and Z deficiency alleles of alpha1-antitrypsin and the G-->A mutation in the 3' noncoding region of the alpha1-antitrypsin gene (Taq-I G-->A allele). Alpha1-antitrypsin deficiency phenotypes were detected in 20 (16 MS, 1 S and 3 MZ) out of 147 unrelated tested CF patients and were, surprisingly, associated with significantly better lung function (adjusted mean forced expiratory volume in one second (FEV1) 62.5% of predicted for deficient group and 51.1% pred for normal alleles; p=0.043). The Taq-I G-->A allele was found in 21 out of 150 unrelated patients and had no significant effect on CF lung disease or on levels of alpha1-antitrypsin during the inflammatory response. We show here that, contrary to current thinking, common mutations of alpha1-antitrypsin that are associated with mild to moderate deficiency of the protein predict a subgroup of cystic fibrosis patients with less severe pulmonary disease. Moreover, the Taq-I G-->A allele has no effect on serum levels of alpha1-antitrypsin in the inflammatory response, which suggests that the previously reported association of the Taq-I G-->A allele with chronic obstructive pulmonary disease is not mediated by its effect on the serum level of alpha1-antitrypsin.

A phylogenetic approach to the identification of phosphoglucomutase genes.

The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes. We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM). Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms. In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3. Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database. Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium--and sugar-binding sites, were conserved in all 47 sequences. The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified. Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2. Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2. A third lineage may contain homologs to human PGM3. On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.

Molecular and cytological investigations of phosphoglucomutase (PGM1) in the K562 cell line.

Phosphoglucomutase 1 (PGM1) deficiency is a stable characteristic of the erythroleukaemic cell line, K562, whereas the activity of the isozymes of the other two PGM loci (PGM2 and PGM3) is slightly elevated. In this study the molecular basis of PGM1 deficiency was investigated by a combined approach utilising protein electrophoresis, immunodetection, cytogenetic techniques, and DNA and RNA analysis. Isoelectric focusing and activity staining confirmed that K562 has no detectable PGM1 activity. Immunoblot analysis of extracts, separated by isoelectric focusing, starch gel and SDS gel electrophoresis, using monospecific anti-PGM1 antibodies showed that K562 contained no detectable immunoreactive material. Karyotype analysis revealed the presence of two intact chromosomes 1 and a derivative chromosome 1, der(1)t(1;11), each of which carried a copy of the PGM1 gene as demonstrated by fluorescence in situ hybridization using a PGM1 cosmid as probe. Southern blot analysis using a PGM1 cDNA clone as probe suggested that the PGM1 genes had not been subject to any gross structural rearrangements. We were also able to determine that K562 is type PGM1 2+1+ by restriction endonuclease analysis of genomic DNA. Very low levels of PGM1 mRNA which appeared to be full length transcripts were detected in K562 using a reverse transcriptase PCR technique. We conclude that the most likely cause of PGM1 enzyme deficiency in K562 is abnormal regulation of transcription.

A new alpha 1-antitrypsin mutation, Thr-Met 85, (PI Zbristol) associated with novel electrophoretic properties.

A new AAT allele (PI Zbristol) has been discovered in a woman with an obstetric history of three perinatal deaths from fulminant liver disease and no living offspring. She and her father were both PI M1Zbristol heterozygotes. The Zbristol protein is active as a proteinase inhibitor but appeared to be deficient in the plasma to about the same degree as the S protein in MS heterozygotes. It focuses on the basic side of Z and lacks the normal pattern of secondary isoforms associated with the commonly occurring AAT variants and migrates faster than normal on an SDS electrophoresis gel. The Zbristol mutation was found to be a C to T transition at codon 85 changing ACG (Thr) to ATG (Met). This disrupts the N-glycosylation site starting at Asn 83 preventing glycosylation at residue 83 in the PI Zbristol protein and explains the protein isoelectric focusing and SDS gel electrophoresis results. An analysis of haplotypes in the propositus and her father indicated that the Zbristol mutation occurred on the common M1(Val 213) genetic background. The new mutation also led to the generation of an NlaIII restriction endonuclease recognition site. Cell lines from two offspring tested for the presence of this NlaIII site revealed that one had the variant and the other did not. Thus, the relationship between Zbristol and fulminant liver disease in the offspring is unclear.

C-antineutrophil cytoplasmic antibody positivity in vasculitis patients is associated with the Z allele of alpha-1-antitrypsin, and P-antineutrophil cytoplasmic antibody positivity with the S allele.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) in vasculitis have either cANCA or pANCA patterns as defined by immunofluorescence. The target autoantigen of cANCA is usually proteinase 3 (PR3), whereas that of pANCA is usually myeloperoxidase (MPO). Alpha-1-antitrypsin (alpha 1AT) is the major physiological inhibitor of PR3, while MPO is an inhibitor of alpha 1AT. METHODS: To determine whether there was an association between ANCA positive vasculitis, ANCA pattern, and alpha 1AT deficiency alleles, we studied alpha 1AT phenotypes of 99 cANCA and 99 pANCA positive vasculitis patients by isoelectric focusing and immunoblotting, and compared them with 2310 controls from the same geographical area. RESULTS: C-ANCA patients showed an increased frequency of the Z allele (0.055 versus 0.018 in controls), conferring a relative risk of 3. They showed no increase in frequency of the S allele. P-ANCA patients showed an increased frequency of the S allele (0.091 versus 0.046 in controls) conferring a relative risk of 2. The frequency of the Z allele also appeared to be increased (0.030 versus 0.018 in controls), but this was not statistically significant. CONCLUSIONS: These findings demonstrate an association between ANCA-positive vasculitis and deficiency phenotypes of alpha 1AT, and suggest a role for alpha 1AT in the development of systemic vasculitis.

'SZ like' alpha 1-antitrypsin phenotypes in PI ZZ children with liver disease.

Using high resolution isoelectric focusing, alpha 1-antitrypsin phenotypes were studied in 106 individuals of the PI ZZ genotype including 71 with liver disease, 22 with chest disease and 13 healthy subjects. The resulting Z patterns were found to be highly variable. In the majority of cases (89/106) the maximum staining intensity was either in the most basic isoform or shared equally between two basic isoforms of the Z phenotype. However, in 17 cases there was a marked intensification of the more acidic isoforms resulting in a pattern which closely resembled the SZ phenotype. This 'SZ like' pattern occurred more frequently in the liver group (16/71) than the chest group (0/22) or healthy (1/13) controls. One possible consequence of the 'SZ like' pattern is confusion with the genuine SZ phenotype leading to misclassification. If this were so, there could be an erroneous exaggeration of the actual incidence of childhood liver disease associated with PI SZ.

The classical human phosphoglucomutase (PGM1) isozyme polymorphism is generated by intragenic recombination.

The molecular basis of the classical human phosphoglucomutase 1 (PGM1) isozyme polymorphism has been established. In 1964, when this genetic polymorphism was first described, two common allelozymes PGM1 and PGM1 2 were identified by starch gel electrophoresis. The PGM1 2 isozyme showed a greater anodal electrophoretic mobility than PGM1 1. Subsequently, it was found that each of these allelozymes could be split, by isoelectric focusing, into two subtypes; the acidic isozymes were given the suffix + and the basic isozymes were given the suffix -. Hence, four genetically distinct isozymes 1+, 1-, 2+, and 2- were identified. We have now analyzed the whole of the coding region of the human PGM1 gene by DNA sequencing in individuals of known PGM1 protein phenotype. Only two mutations have been found, both C to T transitions, at nt 723 and 1320. The mutation at position 723, which changes the amino acid sequence from Arg to Cys at residue 220, showed complete association with the PGM1 2/1 protein polymorphism: DNA from individuals showing the PGM1 1 isozyme carried the Arg codon CGT, whereas individuals showing the PGM1 2 isozyme carried the Cys codon TGT. Similarly, the mutation at position 1320, which leads to a Tyr to His substitution at residue 419, showed complete association with the PGM1+/- protein polymorphism: individuals with the + isozyme carried the Tyr codon TAT, whereas individuals with the - isozyme carried the His codon CAT. The charge changes predicted by these amino acid substitutions are entirely consistent with the charge intervals calculated from the isoelectric profiles of these four PGM1 isozymes. We therefore conclude that the mutations are solely responsible for the classical PGM1 protein polymorphism. Thus, our findings strongly support the view that only two point mutations are involved in the generation of the four common alleles and that one allele must have arisen by homologous intragenic recombination between these mutation sites.

Prenatal diagnosis of alpha-1-antitrypsin deficiency by PCR of linked polymorphisms: a study of 17 cases.

We describe our experiences of prenatal diagnosis of alpha-1-antitrypsin deficiency using closely linked polymorphisms detected by Southern blotting and the polymerase chain reaction. Prenatal diagnosis was carried out for 17 pregnancies from 15 couples.

Phosphoglucomutase 1: complete human and rabbit mRNA sequences and direct mapping of this highly polymorphic marker on human chromosome 1.

A cDNA clone encoding the mRNA for the highly polymorphic human enzyme phosphoglucomutase 1 (PGM1; EC 5.4.2.2) has been isolated and characterized. This was achieved indirectly by first isolating a rabbit cDNA from an expression library using anti-rabbit PGM antibodies. A comparison of the nucleotide sequences shows that the homologies between human and rabbit PGM1 mRNAs are 92% and 97% for the coding nucleotide sequence and the amino acid sequence, respectively. The derived rabbit amino acid sequence is in complete agreement with the published protein sequence for rabbit muscle PGM. A physical localization of the human PGM1 gene to chromosome 1p31 has been determined by in situ hybridization. Analysis of DNA from a wide variety of vertebrates indicates a high level of PGM1 sequence conservation during evolution.

Genetic studies on a new deficiency gene (PI*Ztun) at the PI locus.

During a study of the alpha 1 antitrypsin (AAT) protein and its locus (PI) by high resolution isoelectric focusing and direct molecular analysis of 106 PIZ probands and their families, a new allele (Ztun) was identified that resembles Z in many of its properties. Two sibs, both compound heterozygotes for Ztun and Z, showed similar evidence of mild liver involvement that was indistinguishable from that associated with classical ZZ homozygotes. The Ztun protein appeared to be deficient in the plasma to about the same degree as the Z protein. Allele specific oligonucleotide analysis of amplified genomic DNA indicated that the new allele is the result of a mutation in exon V that is identical to the classical G----A transition at codon 342 that results in the Glu----Lys substitution characteristic of the Z allele. An analysis of DNA haplotypes constructed from polymorphic restriction enzyme recognition sites in and around the PI locus confirmed that Ztun probably represents a new mutation at codon 342 that has occurred on an M2-like genetic background.

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.