Primary structure of GPIbβ, PCR strategy, and DNA sequence analysis. (A) GPIbβ contains signal peptides (SP), a leucine-rich repeat (LRR), a transmembrane domain (TM), and a cytoplasmic tail. (B) DNA fragment amplified by the primers Ibb was cloned into the pCRII vector and sequenced. A single nucleotide substitution, an A to G transition at nucleotide 991, changed Tyr (TAC) to Cys (TGC) at residue 88 (left). A single nucleotide substitution, a G to C transversion at nucleotide 1050, changed Ala (GCC) to Pro (CCC) at residue 108 (right). Underlined nucleotides were changed. (C) Restriction analysis of the PCR-amplified GPIbβ gene. DNA fragments amplified using primers Ibb3 were digested with BsoFI or Hae III restriction enzymes, electrophoresed on 15% PAGE slab gels, and stained with ethidium bromide. The A to G transition at nucleotide 991 created a recognition site for BsoFI, generating a new 31-bp band (left). The G to C transversion at nucleotide 1050 abolished an Hae III recognition site, resulting in a new 54-bp band (right). Marker lane (M) is an Hae III digest of pBR322. MW, molecular marker; N, normal; F, father; M, mother; KT, elder sister; AK, patient; NK, younger sister.