DNA was isolated from blood samples using standard protocols. For diagnostic tests of the ABCA4 gene (the adenosine triphosphate–binding cassette, subfamily A, member 4 gene), preliminary analysis for known mutations and polymorphisms was performed by an outside laboratory using the ABCA4 chip, as described previously.9 Mutations identified in this manner were confirmed by sequencing relevant exons in the laboratory according to our clinical protocol following CLIA guidelines. If fewer than 2 causative mutations were identified using the ABCA4 chip, and for analysis of all other genes, sequencing was performed by amplification of all exons and at least 20 base pairs of flanking intronic sequence using primers described previously.6,10- 16 In cases of predictive or carrier testing in which the familial mutation was known, only the exon in question was analyzed. Amplicons were sequenced in both directions using polymerase chain reaction primers and a cycle-sequencing reaction, and were separated using a genetic analyzer (ABI Prism 3100; Applied Biosystems, Foster City, Calif), as described earlier.17 Current technology can detect 1 or a few nucleotide substitutions, minor deletions,and minor insertions in the coding region. Large deletions, large insertions,and mutations in noncoding regions are types of mutations that cannot be detected by current methods and, thus, would not be identified. To determine if a newly identified sequence change was pathogenic, DNA, when available, from the patient's parents or affected blood relative(s) was screened for the change in question.Sequence changes, novel and previously published, were compared with DNA analysis of at least 100 chromosomes from control subjects of similar ethnicity18- 21 (also A.J.K. and R.A., unpublished data, 2006).