Detection of known haemophilia B mutations and carrier testing by microarray

The molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (AS-APEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities. © 2005 Schattauer GmbH, Stuttgart.published_or_final_versio

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant.

Changes at the invariable donor splice site +1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6+1G>T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6+1G>T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30bp deleted transcript (8±2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2±0.04%) of protein with appreciable specific activity (48±16% of wt-FVII). Altogether these data indicate that the IVS6+1G>T mutation is compatible with the synthesis of functional FVII molecules (~0.01% of normal, 1pM), which could trigger coagulation. The low but detectable thrombin generation (352±55nM) measured in plasma from an IVS6+1G>T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition

“Compensatory” aberrant splicing supports residual expression levels in severe coagulation factor VII deficiency

INTRODUCTION The complete deficiency of factor VII (FVII), the protease triggering coagulation, is considered to be lethal. However, methodological limitations do not enable to investigate extremely low FVII expression levels. We investigated the FVII IVS6+1g/t change that, by altering the invariable +1 position of a donor splice site (5’ss), potentially represents a “”null mutation”. The IVS6+1g/t is frequent in Thailand FVII deficient patients and associated to life-threatening bleeding. METHODS FVII mRNA splicing and protein levels were investigated by expression in Hep3B and BHK cells of FVII minigenes, modified U1snRNAs and of FVII cDNA variants. RESULTS The IVS6+1g/t mutation mainly induced exon 6 skipping and, to a much lower extent, total and partial IVS6 retention, thus producing frame-shifts incompatible with correct FVII biosynthesis. We did not detect any trace of normal transcripts that would support a minimal FVII function in patients. Targeting the mutated 5’ss at the natural junction by modified U1-IVS6+1t did not rescue correct splicing to any extent. Intriguingly, capillary denaturing electrophoresis also identified traces of an in-frame 30bp deleted FVII mRNA resulting from the usage of an exonic cryptic 5’ss. Recognition of this cryptic site was improved (14% of the exon skipped transcript) by engineering U1snRNA. Upon in-vitro expression, the deleted FVII variant lacking 10 residues (Val158-Gln167) of the activation domain displayed, in fluorogenic functional assays in plasma systems, a low but detectable activity (0.05% of wt). CONCLUSION Re-programming mRNA processing by modified U1snRNAs able to induce cryptic 5’ss activation could represent a novel correction approach for null 5’ss mutations. Remarkably, compensatory aberrant splicing might explain residual FVII expression associated to a splicing mutation type that otherwise would result in a “null genetic condition”