Exon trapping analysis of c.301-19G > A in intron 1 of the SHH gene in a patient with a microform of holoprosencephaly

It can be difficult to assess the clinical significance of novel genomic sequence variants which may potentially alter mRNA splicing. Segregation analysis is not helpful in isolated cases or small families. Bioinformatic tools can provide additional information, but direct analysis of mRNA from an appropriate tissue remains the preferred approach for analyzing the effect of a sequence variant on splicing. However, hundreds of disease-associated and developmental genes, including the Sonic Hedgehog homolog (SHH) gene, are not expressed in blood or fibroblasts postnatally. We identified a de novo nucleotide change, c.301-19G > A, in intron 1 of SHH in a four year old boy with a microform of holoprosencephaly. In silico analyses predicted unaltered splicing. We used a minigene approach to study the variant more closely. The genomic region of interest was inserted into an exon trapping vector to create an artificial pre-mRNA in transfected cells. We found virtually complete inactivation of the splice acceptor site in intron 1 in two different transfected cell lines. In light of the clinical context, the de novo nature of the substitution and the results of the exon trapping analyses, we conclude that the detected variant is pathogenic and that the recurrence risk for sibs is low. This case demonstrates that in the absence of a readily available mRNA source, exon trapping can be a robust and practical aid in clinical practice for assessing the effect of genomic variants on pre-mRNA splicing.