All individuals with inherited AnWj-negative phenotype are homozygous for the same deletion in MAL, encompassing exons 3 and 4. (A) Portion of chromosome 2 sequence, spanning MAL exons 2 to 4, from next-generation sequencing (NGS) targeted–panel sequencing of 3 representative individuals. Wild-type sequence is shown in the upper panel, whereas individual P2 (middle) shows no sequencing reads mapping to exons 3 and 4, with reads spanning the deleted area mapping to intron 2 (boxed in purple) and in the region downstream from exon 4 (boxed in green). The bottom panel shows a deletion heterozygote (P2_F4), with only ∼50% of expected reads mapping to MAL exons 3 and 4, and the end of sequencing reads spanning the deleted area is clearly visible, mapping in the same region as with the homozygous deletion sample above. (B) Gene schematic showing deletion break points. Wild-type MAL (top) consists of 4 coding exons (blue cylinders), whereas the AnWj-negative individuals lack exons 3 and 4 and parts of the adjacent introns (gray cylinders). Deletion (6646 bp) represented by dashed red line. (C) Details of portions of MAL intron 2 (upper left) and 3' region downstream from MAL (lower right) sequence alignment in representative AnWj-negative individual (P2) compared with wild-type control. Sanger sequencing of P2 across the deletion break points is shown in bottom panel; sequence boxed in purple derives from intron 2, whereas sequence boxed in green derives from the 3' region downstream from exon 4. Position of deletion is indicated by red triangle. Break points confirmed by Sanger and/or NGS sequencing to be identical in all AnWj-negative samples (Table 1).