6884_6885 insT ATRX mutation in a patient with ATMDS. (A) Sequences from patient no. 12, exon 33 (5′ to 3′, running left to right). The top line is a sequence from a normal subclone, the middle line represents one of the 10% of subclones bearing the thymidine insertion 6884_6885insT (arrowhead) that results in a frame shift, and the lower line is unfractionated marrow DNA, where the inserted thymidine (red, with arrow) can be seen as a secondary peak under the 3′ cytidine (blue); this is followed by a series of secondary peaks (denoted in red letters under primary base calls) as a result of the frame shift. (B) Hinf I digest from patient no. 12. The predicted normal exon 33 amplicon digestion fragments are 218-bp and 21-bp long. The T insertion at 6884_6885insT expands the amplicon from 239 bp to 240 bp and creates a new restriction site, leading to 3 digestion fragments of lengths 115 bp, 104 bp, and 21 bp. A 2-log DNA ladder (New England Biolabs) is at far left. Lane 2 is a digest of a subclone containing 6884_6885insT, and has the predicted 115-bp and 104-bp fragments. Lane 3 is a digest of a subclone from patient no. 12 where sequencing did not show the mutation, and only the 218-bp fragment can be seen, similar to lane 4, a wild-type control with only the 218-bp fragment. Lane 1 is a digest of amplified unfractionated marrow DNA from patient no. 12, and contains both the normal 218-bp fragment as well as faint bands corresponding to the 115-bp and 104-bp fragments from the minor population of mutant DNA.