Evaluation of "at risk" alpha 1-antitrypsin genotype SZ with synthetic oligonucleotide gene probes.

Abstract

Alpha 1-antitrypsin (alAT), a 52,000-mol-wt serum glycoprotein produced by hepatocytes and mononuclear phagocytes, functions as the major inhibitor of neutrophil elastase. The alAT haplotype S is associated with childhood liver disease and/or adult emphysema when inherited with the Z haplotype to give the phenotype SZ. To accurately identify the SZ phenotype at the level of genomic DNA, four 32P-labeled 19-mer synthetic oligonucleotide probes were prepared; two to identify the M and S difference in exon III, and two to identify the M and Z difference in exon V. These probes were hybridized with various cloned DNAs and genomic DNAs cut with the restriction endonucleases BglI and EcoRI; the genomic DNAs represented all six possible phenotype combinations of the M, S, and Z haplotypes (MM, MS, MZ, SS, ZZ, and SZ). Using the four probes to evaluate 42 samples of genomic DNA, the "at risk" SZ and ZZ phenotypes were correctly identified in all cases, as were the "not at risk" phenotypes SS, MS, MM, and MZ, demonstrating that both exon III and exon V directed probes are necessary to properly identify all of the major "at risk" alAT genes. However, when used to evaluate a very rare family carrying a null allele, these four oligonucleotide probes misidentified the "at risk" null-null and S null phenotypes as "not at risk" MM and SM combinations. These observations indicate that oligonucleotide gene probes yielded reliable and accurate assessment of "at risk" alAT genotypes in almost all situations, but in the context of prenatal diagnosis and genetic counseling this approach must be used with caution and in combination with family studies so as not to misidentify rare genotypes that may be associated with a risk for disease.

Z-type alpha 1-antitrypsin is less competent than M1-type alpha 1-antitrypsin as an inhibitor of neutrophil elastase.

“…The primary structure of the polypeptide differs from the MI(Val213) at two residues (Val23 to Ala213; Glu342 to Lys342) and from MI (Ala213) at one residue (Glu342 to Lys342) (48, [50][51][52]. Like the Ml molecule, the active site of the Z-type a 1AT is centered at Met35t-Ser359.…”

Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals

“…A 19-mer oligonucleotide with the normal sequence (5' TACCCTA-GAACCTCCTGT 3', probe A), a 19-mer oligonucleotide with the adenosine to guanosine substitution (5' TACCCTA-GAGCCTCCTGT 3', probe B), and an 8-mer primer oligonucleotide (5' ACAGGAGG 3') were synthesized .s described above. High specific-radioactivity probes used in the allelic-specific hybridization experiments were synthesized by primer extension reactions (23,24).…”

Use of DNA probes for diagnosis and prevention of inherited disorders

“…As shown recently for phenylketonuria, the incidence of the most frequent mutant allele, which has been recently characterized at the DNA level, exceeds 30%, and no more than four mutant alleles account for over 80% of all mutations found in the population [ 102,1031. For alpha-I -antitrypsin deficiency the situation is similar [ 104,105]. Here, sequencing of the mutant alleles has allowed us to synthesize specific oligonucleotide probes, which can be routinely employed to diagnose the respective mutations in genomic DNA.…”