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Keywords: Genetic testing; Genotype-phenotype correlations; Massively parallel sequencing; Mendelian disorder; Diagnostics

T. Togawa 1; S. Ito 1; K. Ohashi 2; K. Ito 1; T. Endo 3; T. Sugiura 4; S. Saitoh 1

1) Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 2) Department of Pediatrics, Kainan hospital, Yatomi, Japan; 3) Department of Pediatrics, Komaki prefecutual hospital, Komaki, Japan; 4) Department of Pediatrics, Sugiura Kodomo Clinic, Hekinan, Japan

Alagille syndrome (ALGS; OMIM 118450) is an autosomal-dominant multisystem disorder affecting the liver, heart, face, eyes, skeleton, and other organs. The prevalence is estimated to be approximately 1 in 30,000 live birth. ALGS is caused by a mutation in one of two genes in the Notch signaling pathway, JAG1 or, rarely, NOTCH2. JAG1 is located in 20p12 and consists of 26 exons. A mutation in JAG1 can be identified in approximately 90% of clinically diagnosed patients with ALGS. To date, over 500 JAG1 mutations have been reported, and approximately 10% of the mutations were gross deletions. Here, we present a patient with clinically diagnosed ALGS and with novel heterozygous double gross deletions in JAG1. The patient was a Japanese male from nonconsanguineous parents. He represented intrahepatic cholestasis and was introduced to our institution at 3 years in order to test molecular genetic analyses. His clinical features showed as follows; intrahepatic cholestasis with elevated serum liver enzyme, characteristic facial features including a high broad forehead and a triangular face, and dysplastic kidney. These features fulfilled clinical diagnostic criteria of ALGS. We carried out our targeted next-generation sequencing using the AmpliSeq and Ion Torrent Personal Genome Machine (ThermoFisher Scientific) system as we previously established. Sequenced reads were analyzed through Ion Reporter software (IR, ThermoFisher Scientific). Consequently, the IR called 2 heterozygous gross deletions in JAG1; 1616bp deletion in exon10 and 659bp deletion in exon 25 encompassing to exon 26. To eliminate false-positive mutations, we ascertained those by multiplex ligation-dependent probe amplification (MLPA) analysis using the SALSA MLPA probemix P184-C2 JAG1 (MRC-Holland), and we successfully validated those two regions. Next, we validated the origin of the two deletions with the same method using gDNA from his parents. The examination revealed that his mother carried the both deletions. Thus, we confirmed that the double gross deletions were on the same allele which was transmitted from his mother. After the molecular testing, we carefully checked his mother, and she showed slightly elevated liver enzyme, agenesis of one kidney, and hypertension. These were compatible with clinical aspects of ALGS. In conclusion, this is the first report that a patient with clinically diagnosed ALGS had doubly mutated allele, including 2 gross deletions, in JAG1.