RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing.

More than half of cases with suspected genetic disorders remain unsolved by genetic analysis using short-read sequencing such as exome sequencing (ES) and genome sequencing (GS). RNA sequencing (RNA-seq) and long-read sequencing (LRS) are useful for interpretation of candidate variants and detection of structural variants containing repeat sequences, respectively. Recently, adaptive sampling on nanopore sequencers enables target LRS more easily. Here, we present a Japanese girl with premature chromatid separation (PCS)/mosaic variegated aneuploidy (MVA) syndrome. ES detected a known pathogenic maternal heterozygous variant (c.1402-5A>G) in intron 10 of BUB1B (NM_001211.6), a known responsive gene for PCS/MVA syndrome with autosomal recessive inheritance. Minigene splicing assay revealed that almost all transcripts from the c.1402-5G allele have mis-splicing with 4-bp insertion. GS could not detect another pathogenic variant, while RNA-seq revealed abnormal reads in intron 2. To extensively explore variants in intron 2, we performed adaptive sampling and identified a paternal 3.0 kb insertion. Consensus sequence of 16 reads spanning the insertion showed that the insertion consists of Alu and SVA elements. Realignment of RNA-seq reads to the new reference sequence containing the insertion revealed that 16 reads have 5' splice site within the insertion and 3' splice site at exon 3, demonstrating causal relationship between the insertion and aberrant splicing. In addition, immunoblotting showed severely diminished BUB1B protein level in patient derived cells. These data suggest that detection of transcriptomic abnormalities by RNA-seq can be a clue for identifying pathogenic variants, and determination of insert sequences is one of merits of LRS.

[Allogeneic hematopoietic stem cell transplantation using myeloablative conditioning including total body irradiation for pediatric acute lymphoblastic leukemia: a single-center retrospective analysis].

This study aimed to investigate the clinical outcomes of hematopoietic stem cell transplantation (HSCT) with total body irradiation-based myeloablative conditioning (TBI-MAC) in pediatric patients with acute lymphoblastic leukemia (ALL). We retrospectively examined patients with ALL who underwent HSCT with TBI-MAC from January 2000 to August 2016 at our institute. We enrolled 67 patients with a median follow-up period of 8 years. The 5-year event-free survival (EFS) and overall survival (OS) were 51.2% and 59.6%, respectively. At the first complete remission, HSCT exhibited significantly superior EFS and OS in our patients than that in patients with other diseases. We encountered 57.9% of patients with at least one late complication. Major late complications were short stature (26.3%) and hypogonadism (18.4%). While late complications were observed in several recipients of HSCT, late complication-related deaths occurred in three patients. The TBI-MAC regimen led to favorable clinical outcomes in pediatric patients with ALL who underwent HSCT. Thus, proper evaluation and management of late complications are mandatory.