Exploring The Cause Of The Disorder On The Far Side Of The Short-Read Sequencing

Short-read sequencing, such as whole exome sequencing and whole genome sequencing, can’t detect every case in disease diagnosis. More than 50% of cases that are demanded for research stays unsolved. In the research article Kawakami and colleagues 2023, delve into solving the unsolved the other part of the autosomal recessively inherited mosaic variegated aneuploidy (MVA) syndrome case which is finding a candidate variant that is joint to the maternal heterozygous variant found by exome sequencing.

Study Methodology
The researchers did standard karyotype analysis to the blood of 3 year old Japanese girl. Variant sequencing, urine derived cells isolation culture, PCR, sanger sequencing, RT-PCR, RNA sequencing, nanopore adaptive sequencing, minigene plasmid construction, cell culture, transfection and immunoblotting techniques were done.

Findings
By exome sequencing in intron 10 of BUB1B gene a heterozygous c.1402-5A>G variant was found. Sanger Sequencing showed that this variant was inherited from mother. RT-PCR and cloning PCR products showed that canonical splice acceptor site was disrupted and alternative splice acceptor site was built. To examine the mis-splicing event penetrance, the researchers studied mini gene splicing assay. Parental pEGFP plasmid showed intense GFP signal as wild type sample. In the mutant sample GFP nearly disappeared. Immunoblotting showed BUB1B-GFP fusion protein level was diminished.

To find another joint variant nanopore adaptive sequencing was done. This technique found almost 3-kb insertion in intron 2 of a repeat sequence. Consensus sequencing demonstrated that the insertion has Alu elements and SINE-VNTR-Alu elements. PCR showed that this insertion is transmitted by patients’s father.

Conclusion
Exome sequencing and genome sequencing can’t solve the disease cause completely. With the use of other techniques the disease causes can be more enlightened.

References:
Kawakami, R., Hiraide, T., Watanabe, K. et al. RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing. J Hum Genet 69, 91–99 (2024). https://doi.org/10.1038/s10038-023-01211-8