Factor IX polypyrimidine tract mutation analysis using mRNA from peripheral blood leukocytes

Abstract

To cite this article: Van de Water NS, Tan T, May S, Browett PJ, Harper P. Factor IX polypyrimidine tract mutation analysis using mRNA from peripheral blood leukocytes. J Thromb Haemost 2004; 2: 2073-5.The production of illegitimately transcribed mRNA from peripheral blood leukocytes and its use for mutational analysis of many tissue-specific genes has been known for 15 years [1]. Two recent publications in this journal by Green et al. and Cutler et al. [2,3] have, however, highlighted the difficulty in using ÔillegitimateÕ transcripts of factor FIX mRNA isolated from white blood cells for sequence analysis. Both groups suggested that it was not possible to obtain full-length FIX transcript from peripheral lymphocytes. Green et al.[2] also postulated that because only exons g-h could be amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), this truncated transcript produced a novel serine protease which was unrelated to blood coagulation. RNA degradation in yeast proceeds predominantly via a mechanism involving decapping and 5¢-3¢ exonuclease activity. In humans this mechanism also plays a major role in RNA degradation and, in addition, a 3¢-5¢ pathway involving decapping and deadenylation is also evident [4]. The predominance of the 5¢-3¢ mechanism would explain why the 3¢ regions of transcripts are the most well preserved in cell extracts and why, when isolating low copy number transcripts, the 3¢ end is often the only portion seen by RT-PCR analysis. It is therefore possible that normal full-length FIX mRNA is produced but is quickly degraded because of the low copy number of FIX transcripts in peripheral blood cells and the inherent susceptibility of mRNA to degradation. By careful RNA preparation and nested PCR it is possible to detect FIX mRNA containing exons other than g-h. We present here a case analysis which describes a hemophilia B patient with a mutation within the polypyrimidine tract of intron b which results in exon skipping and the production of FIX mRNA with exon b spliced to exon d.The patient has mild hemophilia B with a FIX level of 2%. On examination of his genomic DNA by sequencing his FIX gene exon by exon the only defect observed was a four-base deletion of TTCT at nucleotide 6659-6662 within the polypyrimidine tract of intron b (Fig. 1a,b).RNA was extracted from 10 mL of peripheral blood collected into citrate, phosphate, dextrose (CPD) anticoagulant. The red cells were initially lyzed using NH 4 Cl/KHCO 3 lysis buffer and the leukocyte cell pellet RNA was extracted by the method of Chomczynski and Sacchi [5]. cDNA was then produced with random primers and SuperScript II (Invitrogen, Auckland, New Zeland). Thirty cycles of PCR using this cDNA as template and primers located in exons b and d at positions 6342 and 10464 were followed by a further 30 cycles using 5 lL of first-round reaction mix as template and nested primers at positions 6358 and 10424. The final RT-PCR product from normal FIX mRNA was expected to be 212 bp.We extracted RNA from our patient with hemophil...

Novel FBN1 gene mutation and maternal germinal mosaicism as the cause of neonatal form of Marfan syndrome

“…This region is important to initially recognize ASS through U2AF protein complex binding [Zorio and Blumenthal, ]. It has been shown several times that its deletions or even single point substitutions can hamper normal splicing of the affected gene, most likely due to restraint binding of U2AF larger subunit [Van de Water et al, ; Carboni et al, ; Dardis et al, ]. It would correspond with Sroogle predictions showing a marked decrease of PPT quality in terms of score and percentile between a set of more than 50,000 constitutive exons.…”

Insight into molecular changes of the FIX protein in a series of Italian patients with haemophilia B

“…It has been suggested that a missense mutation might introduce a cryptic splice site [13], or interfere with the exonic splicing enhancer motifs [14,15]. Because of the difficulty in recovering illegitimately transcribed mRNA from peripheral blood leucocytes [16–18], we were unable to ascertain whether the absence of the protein in patients who presented FIX:Ag level below 1% was due to an incorrect mRNA splice or if an altered and/or unstable protein had been transcribed. Therefore, we excluded these patients from the protein model inspection.…”

Hemophilia B: molecular pathogenesis and mutation analysis

“…It appears challenging in some instances to obtain full-length F9 cDNA from peripheral leukocytes. Sarkar et al analyzed F9 mRNA by using single-strand conformation polymorphism, but did not highlight difficulties in obtaining mRNA [32], and van der Water et al [33] sequenced leukocyte exon 2-4 transcript in wild-type and mutant to identify an intron 2 splice mutation. Green et al could only obtain a partial transcript of exons 7-8 from leukocytes, in contrast to liver-derived F9 mRNA, where the entire transcript was present [34]; similarly, Cutler et al [35] reported leukocyte transcripts from exons 1-2 and 7-8, but not from central F9 exons.…”