Novel BCAP31 alternative splicing transcripts in patient
The novel variant (NM_001256447.2:c.92G>A) sits at the 2nd anticodon that encodes the 31th amino acid of BCAP31 which is highly conserved in vertebrates. Moreover, the variant is part of a consensus 5’ donor splice site sequence at the end of the exon. We evaluated the wildtype and variant allele sequence by the Human Splicing Finder (HSF) (Desmet et al., 2009) to see whether the variant would cause any changes to the splice site. The HSF predicts that the wild type G allele position is a donor splice recognition site (underlined G in CTCCTAAAAG gtatggccta), but that the mutant A allele will alter the donor site recognition, thereby disrupting proper splicing.
To confirm the hypothesis that the A variant in the patient’sBCAP31 gene would lead to improper splicing, we examined the transcript pattern of white blood cells (WBCs) in the patient and her parents by RT-PCR followed by Sanger sequencing (Fig 3). The results showed that there were no wildtype transcript in the patient’s WBCs, but that two novel transcripts were found. The major novel transcript is devoid of exon 2 (NM_001256447.2:r.54_190del, p.Ser2AlafsTer42) while the minor transcript has a 110nt insertion in intron 2 (NM_001256447.2:r.190_191ins[g>a;190+1_190+110], p.Arg31LysfsTer11). The major transcript leads to a frameshift transcript utilizing a new start codon site in exon 3.
In summary, a de novo heterozygous variant in the patient results in a novel frameshift transcript that replaces the wildtype form of BCAP31. This variant is deemed Pathogenic (ACMG criteria PS2, PS3, PM2, PP3, PP4), according to de novo variant (PS2), RT-PCR study (PS3), absent from controls (PM2), deleterious effect on the gene product (PP3), and clinical phenotype (PP4) based on the criteria of ACMG/AMP 2015 guideline (Li & Wang, 2017).