Genetic Analysis and Prenatal Diagnosis of Acute Intermittent Porphyria Caused by Novel Classical Splicing Variant in the Insertion Region of 29-Residue Specific to Human HMBS Protein

Abstract Background: Acute intermittent porphyria (AIP; OMIM#176000) is caused by mutations in the hydroxymethylbilane synthetase (HMBS) gene, which encodes the third enzyme of the heme biosynthesis pathway. A 29-residue insert (residues 296-324) exists at the interface between domains 1 and 3 of human HMBS (hHMBS), and its function remains unknown. In this study, on the HMBS gene of an AIP pregnant woman from China, we predicted the damage to 29-residue insert regions of a new classical splicing variant, and made prenatal diagnosis of the fetus of this pregnant woman. Methods: We extracted peripheral blood from pregnant women and their husbands, as well as fetal amniotic fluid cells, and analyzed gene expression using prenatal whole exon sequencing and Sanger sequencing. RNA was extracted from peripheral blood of pregnant women and reverse transcribed into cDNA for alternative splicing verification. The protein structure of abnormal splicing caused by variant was predicted using I-TASSER and PyMOL software. Using AMBER14sb software, we simulated protein molecular dynamics. Results: Pregnant women and her fetuses have a classical splicing variant in the HMBS gene, c.912+1G>C. The husband of the pregnant woman did not detect the variant. There are no reports of this variant in the literature. Results of peripheral blood transcription tests in pregnant women showed that c.912+1G>C retained the intron 13 and caused the exon 13 skipping. Homology modeling and molecular dynamics analysis revealed that the variant affected the HMBS protein's secondary structure, which led to functional differences. Conclusion: A new classical splicing mutation with pathogenicity of HMBS gene c.912+1G>C was identified in this study, which expands the molecular heterogeneity of AIP and provides key information for genetic diagnosis.