Azim Jamani

Heteroduplex analysis detects frameshift and point mutations in patients with acute intermittent porphyria

We used heteroduplex analysis to screen for mutations in the porphobilinogen deaminase gene in 21 patients with acute intermittent porphyria (AIP). Unique banding patterns were investigated by direct sequencing of polymerase chain reaction products and, when indicated, sequencing of cloned DNA containing the exon of interest. Two frameshift mutations were found, a 2-bp deletion in exon 5 and a 1-bp insertion in exon 7. Both mutations generate a premature stop codon. Two point mutations, in exons 10 and 14, were also observed. The C→T mutation in exon 10 codes for an Arg173 to Trp substitution, while a G→A mutation in exon 14 changes Trp283 into a premature stop codon. This study extends the spectrum of mutations that cause AIP and demonstrates the utility of heteroduplex analysis as a screening technique.

Hereditary coproporphyria: Exon screening by heteroduplex analysis detects three novel mutations in the coproporphyrinogen oxidase gene

Hereditary coproporphyria is a dominantly inherited disorder of porphyrin metabolism caused by a partial deficiency of coproporphyrinogen oxidase, the sixth enzyme in the heme synthetic pathway. We investigated the molecular basis of hereditary coproporphyria in three unrelated patients, amplifying each exon of the coproporphyrinogen oxidase gene and performing heteroduplex analysis to look for mutations. Unique heteroduplex patterns were noted in exons 2, 3, and 6. Sequencing revealed different mutations in each patient: a G→A point mutation encoding a glutamic acid to lysine substitution at codon 101 (E101K), a C→T point mutation encoding a proline to serine substitution at codon 149 (P149S), and a one base‐pair insertion in exon 6 (968insT). No other mutations were found on sequencing the remaining exons and their intron‐exon junctions. The two point mutations affect amino acids that are conserved in all species studied to date. The one base‐pair insertion in exon 6 is the first frameshift mutation to be described in the coproporphyrinogen oxidase gene. This study adds three new mutations to those that have been previously reported, and all have been restricted to single families. These results indicate that hereditary coproporphyria is a genetically heterogeneous disease. Hum Mutat 10:196–200, 1997.

Low concentration galactose determination in plasma adapted to the cobas-bio

Galactose elimination at blood concentrations lower than 2.22 mmol/L has been advocated as a measure of functional liver blood flow. We have adapted an assay employing galactose dehydrogenase (EC 1.1.1.48) to the Cobas-Bio to measure low galactose concentrations in plasma. The collection of blood in sodium fluoride/potassium oxalate anticoagulant tubes eliminated the necessity for the plasma deproteinization step required in similar, manual methods. The between run CVs for plasma samples spiked with galactose to concentrations of 0.13-0.5 mmol/L were 3.6% and 3.2%, respectively. Our automated assay was more precise and had a greater range of linearity than a manual galactose oxidase (EC 1.1.3.9) method set up in our laboratory (0.04-1.10 mmol/L as compared to 0.06-0.56 mmol/L). The total assay time was 20 min.