An N-fold value of <0.6 was considered deleted. selleck Belinostat All PCRs were performed on a LightCycler 480 with the LightCycler 480 SYBR Green I Master kit (Roche Applied Science, Basel, Switzerland). PCR conditions and primer sequences are available on request. Experiments were done with triplicates for each data point. Microarray characterization of ABCB4 complete deletions The practical aspects of array-comparative genomic hybridization (array-CGH) are described in detail elsewhere.39 Briefly, array-CGH labeling and hybridization were performed on Agilent whole human genome 400K microarrays as recommended in the Agilent manual (Protocol v6.3, October 2010, Agilent Technologies, Palo Alto, CA, USA). Patients’ genomic DNA and six pooled normal control DNAs (reference) were labeled with Cy5-dUTP and Cy3-dUTP, respectively.

Arrays were scanned with an Agilent DNA Microarray Scanner (G2565BA). Log2 ratios were determined with Agilent Feature Extraction software (v 9.1.3.1). Results were visualized and analyzed with Agilent’s Genomic Workbench 5.0 software. DNA sequence information was referred to the public UCSC (University of California Santa Cruz) database (Human Genome Browser, February 2009 assembly: hg 19, National Center for Biotechnology Information (NCBI) Build 37). Fine characterization of ABCB4 intragenic deletion breakpoints Long-range PCR was performed with the Expand 20kb Plus PCR kit as recommended by the manufacturer (Roche Applied Science). The primer pairs and PCR conditions used to characterize the gross deletions are available upon request.

The PCR products were sequenced with the ABI BigDye terminator sequencing kit (Applied Biosystems) on an ABI Carfilzomib Prism 3130 automatic DNA sequencer (Applied Biosystems). Results In all, 102 index cases with LPAC and ICP/CIC were screened on the basis of their phenotype for mutations in the ABCB4 gene. A total of 32 ABCB4 heterozygous loss of function point or short insertion/deletion mutations (including non-sense, frameshift, splice, or previously reported/predicted to be deleterious missense mutations) were identified among the 102 tested index cases: in 37% (16/43) of LPAC patients and in 27% (16/59) of ICP/CIC patients. The 70 adult patients with no ABCB4 mutation found (27 with LPAC and 43 with ICP/CIC), were consequently screened for large rearrangements using MLPA. Four heterozygous deletions were identified with this approach in 7% (3/43) of the LPAC patients (BOC propositus of family 1, BEA propositus of family 2, and TIL) and in ~2% (1/59) of the ICP/CIC patients (ROC) (Figure 1). The four deletions were confirmed using real-time PCR-based gene dosage.