A healthy control sample yielded no PCR product, the 17-DMAG cost wild-type allele being too large (~27kb) to be amplified. Sequencing the PCR product revealed a simple deletion of 24720bp (Supplementary Figure 1). This deletion was also detected in three other affected individuals with available DNA samples (Figure 2: individuals II:4, II:7, and II:10 of family 1). Figure 4 Illustration of two possible mechanisms underlying the complex rearrangement detected in patient BEA. (a) In the SRS model,42, 43 the newly synthesized leading strand (C) misaligned with the lagging template strand (B) through inverted short repeats (the … Discussion In this study, we showed for the first time that a significant subset of patients with symptomatic cholestasis/cholelithiasis has underlying ABCB4 deletions.

Partial or complete heterozygous ABCB4 deletions were found in 7% of the patients with LPAC and in ~2% of the patients with CIC. A large family of 12 affected patients with severe LPAC and cholecystectomy (family 1) was notably reported (Table 1; Figure 2). Recent gene dosage methodologies have allowed identification of these ABCB4 deletions. Our observations urge to systematically test the patients with LPAC for the presence of ABCB4 deletions. MLPA, a sensitive, rapid, and cost-effective approach seems particularly adapted to routine diagnosis in molecular genetics. We developed a molecular algorithm tailored to ABCB4 routine analysis that includes ABCB4 gene dosage by MLPA, in case of ABCB4 negative sequencing in patients with suggestive phenotype.

MLPA allows a fast and inexpensive first-line screening for both the partial and complete ABCB4 deletions, complementary to a high-resolution technique such as array-CGH that can be used to characterize larger deletions. Real-time PCR-based gene dosage is useful for deletion’s confirmation, particularly Drug_discovery for the samples with ratio profiles considered as doubtful, that is, between 0.6 and 0.85 (patient ROC; Figure 1). The two intragenic deletion breakpoints were cloned at the nucleotide level and no recurrent breakpoints were found. That no significant sequence similarity was found between the centromeric and telomeric breakpoints of both the deletions, effectively excluded homologous recombination as the underlying mutational mechanisms in both the cases. However, the presence of microhomology in each of the aberrant junctions is consistent with both the microhomology-dependent replication-based recombination (MMRDR) and non-homologous end-joining (NHEJ) mechanisms.