Fifty nanograms of genomic DNA from each patient with LCA were combined into a “patient DNA pool” and 50 ng of genomic DNA from each control individual were combined into a “control DNA pool.” A series of oligonucleotide primers were designed to amplify the NPHP5 coding sequence in fragments that were 200 to 300 base pairs in length. Each primer pair was used to amplify the corresponding candidate gene segment from both the patient DNA pool and the control DNA pool. Polymerase chain reaction (PCR) amplification was confirmed by agarose gel electrophoresis with ethidium bromide visualization and the products were quantified using densitometry of the agarose gels and comparison with standard DNA samples of known concentration. All PCR products amplified from the pooled DNA from patients with LCA were combined in an equimolar fashion into 1 sample and the PCR products amplified from the pooled control subjects' DNA were combined into a second sample. The amplified DNA segments from patients and controls were separately ligated to emulsion PCR adapters (Roche, Branford, Connecticut) as directed by the manufacturer. The ligated fragments were hybridized to sequencing beads in an amplification mixture containing DNA polymerase, deoxynucleoside triphosphates, and buffer. The mixtures were mixed with oil, emulsified, and thermocycled for 50 cycles. The DNA-coated beads were recovered from the emulsions and subjected to pyrophosphate sequencing on a Roche 454 instrument using titanium chemistry. The beads corresponding to the samples from patients with LCA were analyzed on one-half of a picotiter plate and the beads corresponding to the control individuals were analyzed on the other half of the plate during a single run of the sequencing instrument. Roche GSAmplicon software was used to analyze the standard flowgram files and detect the presence of variations from the specified reference sequences of each amplimer. Variations that were significantly more common in patients than controls were confirmed using automated DNA sequencing with dye termination chemistry on an ABI 3730 sequencer (Applied Biosystems, Carlsbad, California). Six members of 1 family—the proband, both parents, and 3 unaffected siblings—were genotyped for 16 short tandem repeat polymorphisms, as previously described.19 Five non–chromosome 3 markers (D6S1611, D1051174, D195902, D115956, and D55820) were used to confirm the biological relationships within the family while 11 chromosome 3 markers (D3S1568, D3S3664, D3S3513, D3S3620, D3S3720, D3S4497, D3S3709, D3S3576, D3S1269, D3S1267, and D3S3558) were used to investigate the possibility of uniparental isodisomy.