The patient is a Caucasian 3-years old male child, late-preterm born (36 weeks) from vaginal delivery, after a pregnancy complicated by placental detachment. Birth weight was 2,490 g (26  centile). He was the first child of an unrelated couple. Family history was negative for cardiac or hepatic disorders. The main stages of psychomotor development were delayed (sitting position at 8 months with hypotonia; walking at 18 months; speaking at 3 years). At 20 months of age a systolic murmur was found at the cardiac auscultation and heart ultrasound was performed, showing a mild stenosis of the pulmonary branches. Screening for metabolic diseases was negative, except for the finding of hypertransaminasemia. Because of dysmorphic facial features, delayed neurological development and elevated liver enzymes, a genetic condition was suspected and the patient was referred to the Clinical Genetics Unit of the Giovanni XXIII Children's Hospital in Bari.
At referral, height, weight and head circumference were normal (>50  centile). He featured prominent frontal bossing, saddle nose with a bulbous tip, 2/VI systolic cardiac murmur, severe psychomotor retardation suggesting an autistic phenotype. His stools were hypocholic with remains of undigested food. Fundus oculi and brain resonance were normal. Karyotype and FRAXA analysis resulted negative. After patient's parents signed the informed consent, gene sequencing of JAG1 (NM_000214) was performed by Next Generation Sequencing. Target enrichment was done by TruSeq custom amplicon (Illumina, San Diego, CA, United States) according to the manufacturer's instructions. Template library was prepared and was sequenced using MiseqIllumina platform (Illumina, San Diego, CA, United States). Annotation and filtering of variants were performed with Illumina Variant Studio version 2.0, following recommended settings. To evaluate the completeness of the method for the screening of the targeted gene, the sequencing coverage of each amplicon was analyzed in detail using Integrative Genome Viewer version 2.3 (Broad Institute, Cambridge, MA). Variants and region with a depth coverage below 30x were confirmed by Sanger sequencing. The heterozygous sequence variant c.2026delT; p.Cys676AlafsTer67 in exon 16 was identified and confirmed using Sanger sequencing (Figure 1), also because the coverage in exon 16 was very low (<30 x). Primers sequences for PCR amplification of the exon 16 were designed using Primer3 software: Forward primer CCTGTCGTGAATGGTCCTG, Reverse primer CCAGGCCCAGAGAAATATCA. The variant was absent in both parents, arisen as a de novo variant, which determines the formation of a stop codon. Variant was checked for previously reported causative mutations in published works and mutation databases: Human Gene Mutation Database (HGMD) and Leiden Open Variation Database (LOVD) and it has never been described before. Moreover, variant was searched indbSNP, 1,000 Genomes and ExACdatbases, to exclude common single nucleotide polymorphism.
The child started symptomatic therapy with ursodeoxycholic acid and multidisciplinary follow-up, in particular rehabilitative psychomotor follow-up.