A novel intronic mutation in SHOX causes short stature by disrupting a splice acceptor site: Direct demonstration of aberrant splicing by expression of a minigene in HEK-293T cells

SHOX, the short stature homeobox-containing gene, encodes a critical regulatory protein controlling long bone growth. We examined patients in one family, identified an intronic mutation, and expressed SHOX minigenes in HEK293T cells to characterize the effect on gene splicing. We identified a novel mutation at position - 3 (c.-432-3C > A;g.6120C > A) of the intron 1 splice acceptor site; three short (height Z-score - 2.4 to - 1.7) children were heterozygous and the father (height Z-score - 3.4) was homozygous. A wild-type minigene produced alternative transcripts; one utilized the normal splice site between intron 1 and exon 2, the other a cryptic splice site in exon 2. Mutant SHOX minigene generated only the smaller transcript. The exon 2 acceptor splice site is weak; an alternative transcript is normally produced using a downstream cryptic splice site. The c.-432-3C > A mutation causes further weakening, and the cryptic splice site is preferentially utilized, resulting in SHOX deficiency and short stature.