Design and validation of a colorimetric test for the genetic diagnosis of hemochromatosis using α-phosphorothioate nucleotides.

Hereditary hemochromatosis is an autosomal recessive disease highly prevalent in Northern Europe. Here we describe the performance of a genetic test for two mutations of the HFE gene (C282Y and H63D). It is based on a solid-phase PCR coupled with an α-phosphorothioate-mediated primer extension, conferring resistance to hydrolysis by ExoIII. Next, Elisa-like detection allows a colorimetric reading of the genetic test. We performed 322 tests (212 on the C282Y mutation, 110 on the H63D mutation) and compared the results with the RFLP method. Using OD ranges giving the minimum of uncertainty, the tests lead to high specificity and sensitivity, and they address the detection of mutated or normal bases in the HFE gene or the deduced phenotype (safe or ill), with positive predictive values or negative ones greater than 0.96. This method is therefore proposed as a primary test or as a confirming test.

Single-tube genotyping using a solid-phase method that combines alpha-phosphorothioate-mediated primer extension and ExoIII: proof of concept with the F508del cystic fibrosis diagnosis.

Detection of single nucleotide polymorphisms (SNPs) and of mutations is of importance in the field of genetics, biomedical research and in vitro diagnosis. We report here a genotyping procedure that can be virtually applied to any locus within a genome: it uses alpha-phosphorothioate deoxynucleotides in a primer-extension step followed by an ExoIII treatment. Non-extended primers are hydrolyzed whereas extended primers resist this treatment, indicating which nucleotide has been incorporated, i.e. the genotype of the locus. A 3-bp deletion in the CFTR gene (F508del, the most prevalent mutation involved in cystic fibrosis) was used as a model, in a single-tube procedure for each nucleotide to be tested. Human genomic DNA samples were correctly genotyped in less than 3h by a solid-phase PCR followed by primer extension, ExoIII treatment and an ELISA-like detection method. The same principle (primer extension with alpha-phosphorothioate deoxynucleotide, ExoIII treatment) should also be combined with other detection systems such as gel or capillary electrophoresis, mass spectrometry or DNA chips.

PLP1 splicing abnormalities identified in Pelizaeus-Merzbacher disease and SPG2 fibroblasts are associated with different types of mutations.

The proteolipid protein 1 (PLP1) gene encodes the two major proteins of the central nervous system (CNS) myelin: PLP and DM20. PLP1 gene mutations are associated with a large spectrum of X-linked dysmyelinating disorders ranging from hypomyelinating leukodystrophy, Pelizaeus-Merzbacher disease (PMD), to spastic paraplegia (SPG2) according to the nature of the mutation. Genetic heterogeneity exists and mutations in the gap-junction alpha 12 (GJA12) gene have been related to PMD. About 20% of patients with the PMD phenotype remain without mutation in these two genes and are classified as affected by Pelizaeus-Merzbacher-like disease (PMLD). To study PLP1 splicing abnormalities, we analyzed PLP/DM20 transcripts from nerves and/or skin cultured fibroblasts of 14 PMD/SPG2 patients carrying different PLP1 mutations and 20 PMLD patients. We found that various types of PLP1 mutations result in missplicing, including one considered as a missense in exon 2 and a nucleotide substitution in intron 3 outside the classical donor and acceptor splicing sites. Moreover, we demonstrated for two patients that the fibroblast transcript pattern was in accordance with the one observed in the corresponding CNS/peripheral nervous system (PNS) tissues. Finally, we observed no abnormal splicing in fibroblasts of 20 PMLD patients tested; suggesting that PLP1 gene splicing abnormalities, potentially caused by undetected intronic mutations, are either not involved or are very rarely implicated in the PMLD phenotype. These results confirm that fibroblasts are reliable, accessible cells useful in detecting PLP1 transcript abnormalities, better characterizing the functional consequences of PLP1 mutations for genotype-phenotype correlation, characterizing new PLP1 splicing regulatory elements, and identifying PLP1 mutations undetected by conventional PLP1 screening.