Figure 4.

Identification of a novel 4.4-kb deletion in kindred W. A, Simplified map of the 20q13.3 region and the localization of the two known microdeletions. Sequence of the depicted region is included in AL050327 (clone 907D15) and AL139349 (clone 261P9). Exons are depicted as blackened rectangles, and direct repeats are shown as striped arrowheads. The 3-kb and 4.4-kb deleted regions lie between the thin and thick brackets, respectively. The locations of recognition sites for the restriction endonucleases used in the Southern blot analysis are shown under the gene map. The location of the 1.6-kb probe is marked by the gray bar, and primers A, B, C, D, E, and F, which were used for PCR analyses, are indicated by arrowed lines; cen = centromeric; tel = telomeric; N = NESP55; AS = antisense; X = XLα_s; A/B = exon A/B. B, Southern blot analysis of genomic DNA from an unaffected (“U”) subject, W-III/4, and an obligate carrier (“OC”), W-II/5, which was digested with three different restriction endonucleases (AflII, BclI, and BgIII). Hybridization with the 1.6-kb ³²P-labeled probe (nucleotides 8299–9921 of AL139349) showed the expected wild-type bands of 7.9 kb, 9.8 kb, and 9.2 kb, respectively. Additional bands (arrowheads) in the obligate carrier (6.8 kb, 11.1 kb, and 7 kb, respectively) are compatible with a 4.4-kb deletion removing one AflII site, two BclI sites, and one BglII site—all of which are centromeric of STX16 exon 2. C, Confirmation and exact localization of the deletion by PCR. Amplification across the predicted location of the deletion, performed using one pair of primers (A [forward] and B [reverse]), yielded a 5.9-kb wild-type fragment in the unaffected subject, the affected individual, and the obligate carrier; both the affected individual and the obligate carrier also showed a shorter fragment (of 1.5 kb). Direct nucleotide sequence analysis of the short amplicon revealed the lack of 4,368 bp extending from nucleotide 704 to nucleotide 5072 of AL139349. D, Amplification of the STX16 region with four different primers, located at nucleotides 404 (primer C [forward]), 1020 (primer D [reverse]), 4938 (primer E [forward]), and 5190 (primer F [reverse]) of AL139349. The sizes of expected wild-type PCR products are 616 bp (primers C and D), 252 bp (primers E and F), and 4.8 kb (primers C and F [not shown]). Obligate carriers and affected individuals show an additional 422-bp amplicon (arrow [primers C and F]). Results are shown for each individual in the family (blackened symbols represent affected individuals, striped symbols represent obligate carriers, and unblackened symbols represent unaffected individuals).