A novel homozygous splice-site mutation in ALPL (c.863–2 A > G) causes severe perinatal hypophosphatasia in an Iranian family

Hypophosphatasia (HPP; OMIM: 241500) is a rare inherited disorder of bone and mineral metabolism caused by mutations in the ALPL gene, which encodes the tissue-nonspecific alkaline phosphatase (TNSALP). The clinical spectrum is highly variable, with infantile forms often being severe or lethal. This study aimed to identify the underlying genetic cause of severe skeletal abnormalities observed in an aborted fetus from a consanguineous Iranian family with a history of similar adverse pregnancy outcomes. Whole Exome Sequencing (WES) was performed on genomic DNA extracted from the aborted fetus’s tissue. The identified candidate variant was validated in the fetus and segregated in the parents using Polymerase Chain Reaction (PCR) and Sanger sequencing. A comprehensive suite of in silico tools—categorized into splicing impact prediction (SpliceAI, Human Splicing Finder), general deleteriousness (DANN, GenoCanyon), and evolutionary conservation analysis (PhyloP, PhastCons)—was used to predict the pathogenic impact of the variant on splicing and protein function. WES identified a novel homozygous variant in the ALPL gene: NM_000478.6:c.863–2 A > G. This variant is located at the canonical acceptor splice site of intron 8. Sanger sequencing confirmed the homozygous state in the fetus and the heterozygous carrier state in both consanguineous parents. The variant was absent from major population databases, including gnomAD and the Iranome database. In silico analyses predicted that this variant abolishes the canonical splice site, likely leading to the skipping of exon 9. According to ACMG/AMP guidelines, the variant was classified as “Likely Pathogenic”. We report a novel homozygous splice-site mutation, c.863–2 A > G, in the ALPL gene as the definitive cause of severe perinatal HPP in this family. This finding expands the mutational spectrum of ALPL and underscores the importance of this specific splice site for correct TNSALP synthesis. The identification of this variant facilitates accurate genetic counseling and prenatal diagnosis for future pregnancies in this family.