Two McLeod patients with novel mutations in XK

Abstract

McLeod syndrome (MLS) is a rare, X-linked, late-onset, disease involving hematological, brain, and neuromuscular systems, caused by mutations in XK that result in either defective XK or complete loss of XK protein. Acanthocytosis of erythrocytes is a typical feature. We report novel mutations in two patients who exhibited typical clinical characteristics of MLS. The coding and flanking intronic regions of XK were amplified by PCR, sequenced, and compared with the normal XK sequence. XK protein, and its complexed partner protein, Kell, were assessed by Western blot analysis. Patient 1 was found to have a single base insertion, 605insA at 175Ile creating a frame shift within the coding sequence of XK. Patient 2 had a single base substitution in the 3' splice sequence of intron 2 (IVS2-2a>g). In both cases mutations resulted in the absence of XK protein.

Fig. 1

A: Reverse sequencing result of partial exon 3 off XK of patient 1 DNA showing a single nucleotide insertion at nt 605 (605insA) which changes the Ile at amino acid 175. The inserted nucleotide, “A” is Written in red. B: Reverse sequencing result of the boundary region of intron 2 and exon 3 of XK of patient 2 DNA showing the IVS2–A>G at the Splice acceptor consensus sequence, …ag, at 3′ of the intron 2. The mutated nucleotide, “G”, is written in red.

Fig. 2

A: Western blot of patients 1 and 2 red cell ghosts probed with anti-XK antibodies under non-reducing and reducing conditions with short and long exposure. Left panel (non-reducing conditions) showing absence of Kell/XK complex in samples of both patient 1 (lane 2) and patient 2 (lane 3). Normal red cell ghosts were analyzed similarly for comparison (lane 1 in both conditions). The over exposed panel also does not show presence of Kell/XK complex in either sample. Right panel (reducing conditions) showing absence of XK in patient 1 (lane 2) and patient 2 (lane 3). The same blot was exposed for longer time to confirm absence of XK. The protein of about 150 kDa in size marked as asterisk is present only in patient samples and is unknown. B: Western blot results of patients 1 and 2 red cell ghosts probed with anti-Kell antibodies under non-reducing and reducing conditions with short and long exposure. Lane 1 normal red cells: lane 2, patient 1 red cells. Lane 3, patient 3, red cells. Left panel (non-reducing conditions), showing absence of Kell/XK complex in lanes 2 and 3 indicating absence of XK. Right panel (reducing conditions) showing reduced amount of Kell in lanes 2 and 3 of long exposure.