Heterozygosity for the splicing donor mutation as shown by Bsp HI analysis and subcloning. (A) Restriction digestion of genomic segments 1P/In-1a by Bsp HI. Lanes 1 and 2, normal; lanes 3 and 4, Rh_null (YT). (−), uncut; and (+),Bsp HI cut. Note that no digestion is seen in controls. For Rh_null (YT), the size of the uncleaved fragment from the Rh50(836A) allele and the two smaller fragments expected of the Rh50(836G) allele is indicated. Shown schematically at the bottom are the wild-type or mutant exon 1/intron 1 boundary (triangles) and recognition sequence of Bsp HI. (B) Sequencing profiles of the subcloned fragments 1P/In-1a from Rh_null (YT). Two types of the inserts that carry either the G or A residue at +1 position of intron 1 (IVS) were identified, confirming the proband to be heterozygous for the splice donor mutation.