Novel resequencing chip customized to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis

Background & Aims: Inherited syndromes of intrahepatic cholestasis commonly result from mutations in the genes SERPINA1 (alpha(1)-antitrypsin deficiency), JAG1 (Alagille syndrome), ATP8B1 (progressive familial intrahepatic cholestasis type 1 [PFIC1]), ABCB11 (PFIC2), and ABCB4 (PFIC3). However, the large gene sizes and lack of mutational hotspots make it difficult to survey for disease-causing mutations in clinical practice. Here, we aimed to develop a technological tool that reads out the nucleotide sequence of these genes rapidly and accurately. Methods: 25-mer nucleotide probes were designed to identify each base for all exons, 10 bases of intronic sequence bordering exons, 280500 bases upstream from the first exon for each gene, and 350 bases of the second intron of the JAG1 gene and tiled using the Affymetrix resequencing platform. We then developed high-fidelity polymerase chain reactions to produce amplicons using 1 mL of blood from each subject; amplicons were hybridized to the chip, and nucleotide calls were validated by standard capillary sequencing methods. Results: Hybridization of amplicons with the chip produced a high nucleotide sequence readout for all 5 genes in a single assay, with an automated call rate of 93.5% (range, 90.3%-95.7%). The accuracy of nucleotide calls was 99.99% when compared with capillary sequencing. Testing the chip on subjects with cholestatic syndromes identified disease-causing mutations in SERPINA1, JAG1, ATP8B1, ABCB11, or ABCB4. Conclusions: The resequencing chip efficiently reads SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 with a high call rate and accuracy in one assay and identifies disease-causing mutations.