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

Abstract

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, 280-500 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.

Figure 1. Sequence readout by the resequencing “Jaundice Chip”

Panel A displays the hybridization cartridge assembled by Affymetrix, Inc., which contains a solid support featuring 25-mer probes designed to sequence 26,725 nucleotides (bases) of SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4. Panel B shows signals obtained from a high-resolution scanning after hybridization of a chip with amplicons from a subject. In the top portion of panel C, magnification of a region of the chip shows that the brightest signal for each set of four 25-mer probes for each column identifies a base. In the lower panel, identical base calls were made by standard capillary sequencing methodology.

Figure 2. Comparison of nucleotide calls made by the resequencing chip and by standard capillary sequencing

The top panel depicts the reference nucleotide sequence for SERPINA1 in red, the chip readout in black, and nucleotides identified by capillary sequencing. The T>C homozygous mutation identified by the chip (shaded in green) is reproduced by capillary sequencing. The same sequencing organization is presented for the other genes in the four lower panels. In JAG1, ATP8B1, and ABCB11, the chip produced heterozygous C/T calls (shown as a typical “y” symbol shaded in orange) that could be identified by red and blue peaks produced by capillary sequencing. In the lower panel, a homozygous A>T mutation in ABCB4 was identified by both methods. The mutations presented in this figure reflect the mutations described in patients #1,4,8,11 and 13 of Table 2.