A 3′ RACE-PCR Method of Identification of Unknown MLL Gene Partner and Break-Point at RNA Level.
Authors: Formisano-Tréziny Christine , Hermitte Fabienne , Quilichini Benoît , Chambost Hervé , Michel Gérard , Gabert Jean 
Published: April 2020

Abstract
Context: Chromosomal translocations involving the MLL gene at 11q23 are particularly common in infant acute leukaemia (AL) (80% of all cases) and are also associated with secondary leukemias. Monitoring minimal residual disease (MRD) is important in predicting efficacy of treatment and detecting early relapse. In case of MLL translocation, detection of MLL fusion transcript by real time PCR is a powerful technique to investigate MRD. Currently approximately 50 different fusion partners of the MLL gene have been characterized at the molecular level and at least 36 remain to be identified. In addition, MLL chromosomal translocations are routinely investigated by FISH. This cytogenetics method allows determination of the presence of a chromosomal translocation involving the MLL gene and the chromosomal location of the partner gene. In this context, only the most frequent and well characterised MLL partner genes are identified, then the corresponding fusion transcripts are analysed by real time PCR.
Rational: Here we describe a useful and simple method of identifying any unknown MLL gene partner and its specific break-point at RNA level.
Methods: This method is based on 3′ RACE-PCR follow-up by cloning and sequencing of resulting PCR products. Briefly, a reverse transcription is performed on total RNA with an anchor random primer. The resulting cDNA is subjected to a long range PCR (PCRI) allowing amplification of normal MLL transcripts and MLL fusion transcripts. Primers used are: a sense primer specific to an MLL region upstream of the break-point cluster region, and an antisense primer specific to the anchor of the random primer used at the reverse transcription step. PCRI products are amplified again with nested primers (PCRII), and PCRII products are cloned. Recombinant clones are screened by 3 different PCR in order to differentiate clones corresponding to the normal MLL transcript from those corresponding to the MLL fusion transcript. Finally clones of interest are sequenced.
Results:. A first case of an infant (AL) was investigated in our laboratory. We identified an infrequent t(11;19)(q23;p13) involving the MYO1F gene, associated with exon 10 deletion of the non translocated MLL transcript. This infant AL case is now routinely followed up with a specific real time PCR assay which we developed. Currently a study of a second case with an uncharacterised MLL translocation involving chr. 7 is in progress.
Conclusion: This method is especially convenient because of its simplicity, speed, and universality. Furthermore, this technique not only allows identification of the MLL fusion transcript sequence in order to choose a specific real time PCR assay, but it also permits immediate lay out of the plasmid which can be used as real time PCR calibrator. Thus, this method can be introduced in molecular therapy management of MLL rearranged patients with rare partner gene or molecular break-point.