The heterozygous ~11 kb
deletion is present in all affected members of the 0124 kindred. (A) Primers were designed to bind upstream of the ~11 kb deletion (primer 1), within the deletion (primer 2), and downstream of the deletion (primer 3). In the presence of a normal allele, primers 1 and 2 result in a 373 bp PCR product. Primers 1 and 3 do not form a product in the absence of the ~11 kb deletion but form a 298 bp PCR product when the deletion is present. (B) In the five affected members of kindred 0124 (III-5, IV-4, V-6, III-3, and IV-2), PCR using these primers resulted in two fragments of the expected sizes. In the three unaffected members (III-4, V-1, and IV-3), PCR produced only one fragment. (C) Sanger sequencing of the PCR product from affected member III-5 using primer 3 confirmed the presence of the ~11 kb deletion. The sequencing trace transitions from coordinate chr5:112,034,825 to chr5:112,045,846 (GRCh37/hg19), a span of 11,020 bp. (D) Sequencing the same PCR product using primer 2 showed the presence of a normal allele and the absence of the gap with a transition from coordinate chr5:112,034,825 to chr5:112,034,826.